Renewable and Sustainable Energy Reviews 58 (2016) 832–841

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Renewable and Sustainable Energy Reviews

journal homepage: www.elsevier.com/locate/rser

Concepts and studies on lipid and pigments of microalgae: A review

Emmanuel B. D’Alessandro, Nelson R. Antoniosi Filho n
LAMES, Chemistry Institute, Federal University of Goiás, Goiânia, Brazil

art ic l e i nf o a b s t r a c t

Article history: This review describes compounds produced microalgae, such as biodiesel, lipids, fatty acids (FA), tria-
Received 11 December 2015 cylglycerides (TAG), and pigments (phycobilins, chlorophylls, and carotenoids). We discuss the factors
Accepted 17 December 2015 inducing the accumulation of these metabolites and their economic importance. We focused on cell wall
breaking methods of microalgae used to produce biodiesel. A special approach was made to extremo-
Keywords: phile microalgae used in biodiesel production. The type of methodology used in the cultivation and the
Biodiesel use of extremophiles microalgae can permit feasible biodiesel production.
Carotenoids & 2016 Elsevier Ltd. All rights reserved.
Stress
Extremophile
Lipid metabolism

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
2. Lipids of microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
3. Fatty acids in microalgae. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
4. Factors influencing the increase of lipids in microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834
5. Pigments in microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 835
5.1. Phycobilins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 835
5.2. Chlorophyll. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 835
5.3. Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 835
6. Market for pigment production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 836
7. Cell membrane of microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
8. Extremophile microalgae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837
9. Metabolism of fatty acids and acylglycerides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838
10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 838
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 839

1. Introduction such as oxi-compounds. Accordingly, several crops have been used

for biofuel production, like sugarcane (Saccharum officinarum
The dwindling oil reserves, rising levels of carbon dioxide, and Linn.), sugar beet (Beta vulgaris Linn.), cassava (Manihot esculenta
global warming increased governmental interest in renewable Crantz), soybean (Glycine max Linn (Merrill), canole (Brassica napus
energy, such as biofuels. Plants rich in lipids or carbohydrates are Linn var. oleifera Moench), and palm (Elaeis guineensis Jacq.) [1].
used to produce, respectively, derivates of fatty acids and alcohols, Biodiesel is the main biofuel derived from oils and vegetable or
animal fats. Biodiesel is usually produced by transesterification in
the presence of methanol or ethanol and alkaline catalysis,
n
Correspondence to: Laboratory of Extraction and Separation Methods (LAMES), resulting in a mixture of alkyl esters from fatty acids [2,3]. The
Chemistry Institute, Federal University of Goiás, Campus Samambaia, Goiânia,
transesterification method was developed in 1937 by George
Goiás, Brazil.
E-mail address: [email protected] (N.R. Antoniosi Filho). Chavanne, who patented the "Procedure for the transformation of

http://dx.doi.org/10.1016/j.rser.2015.12.162
1364-0321/& 2016 Elsevier Ltd. All rights reserved.
 E.B. D’Alessandro, N.R. Antoniosi Filho / Renewable and Sustainable Energy Reviews 58 (2016) 832–841 833

vegetable oils for their uses as fuels" [4]. Biodiesel production is Table 1
mainly based on grease raw materials, such as palm oil, canola oil, Lipid content in different cyanobacteria and microalgae.
animal tallow, and soybean oil [5]. However, the prices of these
Division Lipid content (%) Reference
commodities are regulated internationally and affect biodiesel
price [6]. For example, about 80% of biodiesel cost vary with the Cyanophyta
price of grease raw materials [7], suggesting that new greases Lyngbya birgei 127 2.8 [22]
sources are needed in order to reduce the biodiesel price. Spirulina maxima 6
7 [10]
Spirulina platensis 8.5 7 2 [22]
As a result, there is growing interest in using microalgae for Spirulina platensis 4–9 [10]
biodiesel production, especially because some produce about 150 Synechocystis pevalekii 97 2 [22]
times more oil per hectare than soybean [8]. Soybean is the main Chlorophyta
oilseed used for biodiesel production in Brazil. However, turning Ankistrodesmus falcatus 1.58 [23]
Ankistrodesmus gracilis 7.9–20.5 [24]
biodiesel production from microalgae into an economically viable
Auxenochlorella protothecoides 39.3 7 0.8 [25]
activity is still a challenge [9]. Auxenochlorella protothecoides 32.9 7 0.6 [25]
Producing biofuel from microalgae has many advantages [10], Botryococcus braunii 14.0–28.6 [26]
such as: (1) high growth rate; (2) lower demand for water than Botryococcus braunii 25–75 [19]
commercial crops; (3) High efficiency in CO2 mitigation; (4) smal- Chlamydomonas rheinhardii 21 [10]
Chlorella pyrenoidosa 2 [10]
ler areas for cultivation. However, there are also disadvantages, Chlorella sorokiniana 19 [27]
such as: (1) dependence of light incidence and penetration into Chlorella spp. 10.5 [23]
the aquatic environment to ensure high biomass production Chlorella vulgaris 12.0 7 0.56 [28]
(2) difficulty of developing simple and inexpensive procedures to Chlorella vulgaris 267 0.5 [25]
Chlorella vulgaris 14
22 [10]
convert lipids into biodiesel.
Chlorococcum infusionum 11.3 7 1 [22]
Additionally, there are other drawbacks related to the selection Dunaliella bioculata 8 [10]
of species with appropriate production time and yield of biomass, Dunaliella salina 6 [10]
lipids, and pigments. Ideal species would also have biomass easily Scenedesmus acuminatus 1.58 [23]
separable from the culture medium, adapted to low-cost cultiva- Scenedesmus dimorphus 16–40 [10]
Scenedesmus obliquus 12–14 [10]
tion conditions, and resistant to invasive organisms. Scenedesmus obliquus 6.187 0.20 [29]
Thus, closed systems (photobioreactors) and open ponds Scenedesmus quadricauda 1.9 [10]
(raceways) are used for microalgae cultivation on a large scale and Spirogyra sp. 11-21 [10]
both have advantages and disadvantages. The cost to build and Spirogyra orientalis 217 2.5 [22]
Heterokontophyta
operate a closed system is higher than ponds. Conversely, it
Navicula minima 16.2 7 0.6 [22]
requires less light and area, besides having volumetric productivity Nitzschia spp 3.68 [23]
about 13 times higher than raceways [11]. Phaeodactylum tricornutum 9.40 7 1.77 [28]
Key aspects of lipid metabolism in microalgae have been ana- Euglenophyta
lyzed to improve biodiesel production. For example: (1) stress may Euglena acus 5.78 [23]
Euglena gracilis 14–20 [10]
increase lipid content [12]; (2) manipulating the growing medium Rhodophyta
can increase biomass productivity [13]; (3) some microalgae can Catenella repens 87 1.5 [22]
survive in extreme environments, which facilitates production in Ceramium manorensis 87 1.9 [22]
raceway and decreases contamination [14]; (4) the lipid metabo- Geledium pusillum 9.7 7 2.8 [22]
Dinophyta
lism is known at the molecular level ([15]; and 5) some microalgae
Gymnodinium sp. 29.6 [30]
produce valuable pigments, such as astaxanthin, lutein, and β-
carotene, which can prevent and treat diseases [16].
Thus, the cultivation of microalgae for biodiesel production The main Chlorophyta studied for biodiesel production are
involves not only the selection and production of species, assess- unicellular, such as Auxenochlorella protothecoides, Chlorella vul-
ment of types and amount of lipids, but also the potential market garis, Chlamydomonas rheinhardii, and Dunaliella salina. Since
for co-products. growth rate of these species are faster, lipid productivity is higher
than in other divisions. But the divisions Euglenophyta and
Dinophyta have potential to be used for biodiesel production, since
2. Lipids of microalgae their lipid amount is greater than some Chlorophyta (Table 1).
Cyanophyta are also evaluated for biodiesel production, due to
Lipids can be classified into two groups: polar and neutral. They their high cell growth rate. However, some produce toxic and
are insoluble in water but soluble in most organic solvents. Polar
bioaccumulative substances, such as microcystin, which is carci-
lipids include phospholipids and glycolipids, while neutral lipids
nogen [20].
include acylglicerids (tri, di- and monoglycerides) and free fatty
It is also important to assess the types of fatty acids in micro-
acids. Microalgae use neutral lipids as energy source and polar
algae, since they influence biodiesel quality, especially oxidative
lipids to form cell membranes. Nonetheless, microalgae also have
stability, cold filter plugging point, and contents of mono-, di- and
fatty-acid free components that are not converted into biodiesel,
triglycerides [21].
such as steroids and pigments [17]. As a result, higher production
of pigment implies lower production of fatty acids. Therefore,
although some microalgae produce high lipid content [18,19], this
not necessary means a high biodiesel production. Accordingly, the 3. Fatty acids in microalgae
percentage of lipid content per dry weight of microalgae may
range from 1.5% to 75% (Table 1), and a given microalga can pro- Several authors have evaluated fatty acid composition in
duce different amounts of lipids depending on the culture medium microalgae [18,30–37]. Fatty acids are carboxylic acids with 4-36
and procedure. For example, the lipid content of Chlorella vulgaris carbons chains. Fatty acids without unsaturations are called satu-
ranged from 12% to 26% and Botryococcus braunii ranges from 14% rated (SFA), those with only one unsaturated bond are called
to 75%. monounsaturated (MUFA), those with two are di-unsaturated
834 E.B. D’Alessandro, N.R. Antoniosi Filho / Renewable and Sustainable Energy Reviews 58 (2016) 832–841

Table 2 Table 5
Main saturated fatty acids (SFA) in microalgae. Main tri-unsaturated fatty acids (TUFA) in microalgae.

Fatty acids Common name Representation Fatty acids Common name Representations

Butanoic Butyric C4:0 cis-6, cis-9, cis-12 γ-linolenic C18:3 c6 c9 c12 or C

Hexanoic Caproic C6:0 octadecatrienoic 18:3ω6
Octanoic Caprylic C8:0 cis-9, cis-12, cis-15 α-linolenic (Ln) C18:3 c9 c12 c15 or
Decanoic Capric C10:0 octadecatrienoic C18:3ω3
Undecanoic Undecylic C11:0 cis-5, cis-8, cis-11eicosatrienoic – C20:3 c5 c8 c11 or C20:3ω9
Dodecanoic Lauric C12:0 cis-8, cis-11, cis-14 Di-homo-γ- C20:3 c8 c11 c14 or
Tridecanoic Tridecylic C13:0 eicosatrienoic linolenic C20:3ω6
Tetradecanoic Mirystic C14:0 cis-11, cis-14, cis-17 Di-homo-α- C20:3 c11 c14 c17 or
Pentadecanoic Pentadecylic C15:0 eicosatrienoic linolenic 20:3ω3
Hexadecanoic Palmitic C16:0 cis-13, cis-16, cis-19 – C22:3 c13,16,19 or C22:3ω3
Heptadecanoic Margaric C17:0 docosatrienoic
Octadecanoic Estearic C18:0
Nonadecanoic Nonadecylic C19:0 Adapted from Costa [39].
Eicosanoic Arachidic C20:0
Heneicosanoic Heneicosylic C21:0
single bonds, without unsaturations, is expressed by C14:0 [40].
Docosanoic Behenic C22:0
Tricosanoic Tricosylic C23:0 Also, a fatty acid can be classified as omega-x depending on the
Tetracosanoic Lignoceric C24:0 position of the first double bond in relation to the terminal methyl
Hexacosanoic Cerotic C26:0 end of the carbon chain. For example, the linoleic acid (C18:2 c9,
Octacosanoic Montanic C28:0 c12) is a ω6 acid, since the unsaturations at the position 12 is
Adapted from Costa [39].
6 carbons away from the terminal methyl group, which is the
eighteenth carbon in the chain.
The composition of fatty acids is key to biodiesel production,
Table 3
Main monounsaturated fatty acids (MUFA) in microalgae. since it directly influences biodiesel quality. Thus, large amounts of
polyunsaturated fatty acids may positively impact outflow prop-
Fatty acids Common name Representations erty, especially in cold weather, but may negatively affect oxidative
stability [41]. But antioxidants can correct this latter problem [42].
cis-9-tetradecenoic Myristoleic C14:1c9 or C14:1ω5
cis-9-hexadecenoic Palmitoleic C16:1c9 or C16:1ω7
Conversely, large amounts of saturated fatty acids have excellent
cis-6-octadecenoic Petroselinic C18:1c6 or C18:1ω12 combustion properties, but can cause problems in cold outflow
cis-8-octadecenoic – C18:1c8 or C18:1ω10 [43]. Thus, the European standard EN 14214 states that the content
cis-9-octadecenoic Oleic C18:1c9 or C18:1ω9 of linolenic acid, and consequently tri-unsaturated fatty acids,
cis-10-octadecenoic – C18:1c10 or C18:1 ω8
must be at most 12%, and at most 1% of polyunsaturated acids.
cis-11-octadecenoic Vaccenic C18:1c11 or C 18:1ω7
cis-12-octadecenoic – C18:1c12 or C 18:1ω6
cis-13-octadecenoic – C18:1c13 or C 18:1ω6
cis-5-eicosenoic – C20:1c5 or C 20:1ω15 4. Factors influencing the increase of lipids in microalgae
cis-9-eicosenoic Gadoleic C20:1c9 or C 20:1ω11
cis-11-eicosenoic Gondoic C20:1c11 or C 20:1ω9
cis-11-docosenoic Cetoleic C22:1c11or C 22:1ω11 Microalgae store acylglicerids during the day (photosynthesis)
cis-13-docosenoic Erucic C22:1c13 or C 22:1ω9 and consume them at night (respiration) to support maintenance
cis-15-tetracosenoic Nervonic C24:1c15 or C 24:1ω9 and reproduction via cell division [44]. Several factors influence
stress-induced lipid accumulation, such as: different nutrient
Adapted from Costa [39].
concentration (e.g., nitrogen, phosphorus, iron), high salinity, high
temperature, light intensity, and alternative sources of organic
Table 4
carbon. However, stress in microalgae cultivation increases lipid
Main di-unsaturated fatty acids (DUFA) in microalgae.
accumulation but reduce growth rate, affecting lipid productivity
Fatty acids Common name Representations [45].
Lei et al. [46] used BBM medium for inducing stress in H. plu-
cis-7, cis-10-hexadecadienoic – C16:2 c7, c10 or C16:2ω6
vialis and found that nitrogen depletion, increase in iron con-
cis-9, cis-12-octadecadienoic Linoleic (L) C18:2 c9, c12 or C18:2ω6
cis-8, cis-11-eicosadienoic – C20:2 c8, c11 or C20:2ω9
centration (450 mM of FeSO4), increase in salinity (45 mM sodium),
cis-11, cis-14-eicosadienoic – C20:2 c11, c14 or C20:2ω6 low temperature (4 °C), and high temperature (42 °C) increased
cis-13, cis-16 -docosadienoic – C22:2 c13, c16 or C22:2ω6 the percentage of fatty acids by 32, 9, 22, 1, and 23%, respectively.
Despite high salinity had increased fatty acids concentration by
Adapted from Costa [39]
22%, marine microalgae seem not appropriate for biodiesel pro-
duction, because microalgae biomass can contain high con-
(DUFA), three are tri-unsaturated (TUFA), and more than three are
centrations of sodium and other inorganic elements that would
polyunsaturated (PUFA). Although, some studies call PUFA those
contaminate biodiesel [47].
with three or more unsaturations (e.g., Li et al. [38]). Most fatty Zhang and Hong [48] cultivated Chlorella sp. and Scenedesmus
acids of microalgae (Tables 2–6; adapted from Costa [39]) vary sp. in media with more and less nutrients. Lipid accumulation was
from C14:0 to C22:6, being majority monounsaturated and satu- higher in media with less nutrients for both microalgae, since
rated. The most common are C16:0, C18:0, C16:1, C18:1, C16:2, nutrient deficiency influence lipid accumulation. Wan et al. [49]
C18:2, and C18:3. The number of double bonds in fatty acid chains cultivated Nannochloropsis oceanica f/2 media and diluted the
generally is about two or three, rarely exceeding six. medium with three different ratios in the stationary phase (1/10,
The nomenclature of fatty acids is given according to the 1/5, and 2/5). They found that lipid percentage was 42.7–64.3%,
number of carbon atoms and unsaturated bonds. For example, higher than that of Van Vooren et al. [50], who only induced
oleic acid has a 18 carbon chain and one unsaturated bond is nitrogen stress without diluting the fresh medium. Grama et al.
expressed by C18:1 cis9 or C18:1 c9. Myristic has 14 carbons and [51] found an increase in lipid productivity of Acutodesmus sp.
 E.B. D’Alessandro, N.R. Antoniosi Filho / Renewable and Sustainable Energy Reviews 58 (2016) 832–841 835

Table 6
Main polyunsaturated fatty acids (PUFA) in microalgae.

Fatty acids Common name Representations

cis-6, cis-9, cis-12, cis-15 octadecatetraenoic Stearidonic C18:4 c6 c9 c12 c15 or C18:4ω3
cis-5, cis-8, cis-11, cis-14 eicosatetraenoic Arachidonic C20:4 c5 c8 c11 c14 or C20:4ω6
cis-8, cis-11, cis-14, cis-17 eicosatetraenoic – C20:4 c8 c11 c14 c17 or C20:4ω3
cis-7, cis-10, cis-13, cis-16 docosatetraenoic Adrenic C22:4 c7 c10 c13 c16 or C22:4ω6
cis-10, cis-13, cis-16, cis-19 docosatetraenoic – C22:4 c10 c13 c16 c19 or C22:4ω3
cis-5, cis-8, cis-11, cis-14, cis-17 eicosapentaenoic EPA C20:5 c5 c8 c11 c14 c17 or C20:5ω3
cis-4, cis-7, cis-10, cis-13, cis-16 docosapentaenoic Docosapentaenoic C22:5 c4 c7 c10 c13 c16 or C22:5ω6
cis-7, cis-10, cis-13, cis-16, cis-19 docosapentaenoic DPA C22:5 c7 c10 c13 c16 c19 or C22:5ω3
cis-4, cis-7, cis-10, cis-13, cis-16, cis-19 docosahexaenoic DHA C22:6 c4 c7 c10 c13 c16 c19 or C22:6ω3

Adapted from Costa [39].

Fig. 1. Phycobilin structures.

with increasing light intensity and concluded that the best con- number of patents, the range of red algae and cyanobacteria stu-
dition was 600 mE m2/s with lipid productivity of 85 mg/L/day. died for this purpose is still small.
Zhang et al. [52] found that Chlorella vulgaris produced more
lipid in photoautotrophic cultures mixed with bacteria than in 5.2. Chlorophyll
pure autotrophic and heterotrophic cultures. Previous studies have
also shown that mixed photoautotrophic cultures are more ben- Chlorophylls are greenish and fat-soluble pigments with a
eficial and low cost, eliminating the need to add organic carbon porphyrin ring [55]. They are responsible for converting solar
sources. Therefore, mixed photoautotrophic cultures are more energy into chemical energy in photosynthesis. Most microalgae
economically viable to produce biodiesel than autotrophic or have chlorophyll a, but some may have chlorophyll b and c (Fig. 2),
heterotrophic cultures. such as Dinophyta [58]. Microalgae contain from 0.5 to 1.0% of
chlorophyll per dry weight [57].
Chlorophyllin is a derivate of chlorophyll in which the mag-
nesium is replaced by sodium or copper and phytol chains are lost
5. Pigments in microalgae
[59]. Chlorophyllins have been used to control body odor of ger-
iatric patients [60], and dietary supplement [61]. Several studies
Natural pigments have an important role in the photosynthetic
found that chlorophyll and chlorophyllin have also antimutagenic
metabolism and pigmentation in algae. Also, they have beneficial
and anticarcinogenic action [62–65].
biological activities, such as antioxidant, anticancer, anti-inflam-
matory, anti-obesity, anti-angiogenic, and neuroprotective [53,54].
5.3. Carotenoids
At least three classes of pigments occur in microalgae: phycobilins,
chlorophylls, and carotenoids.
Carotenoids (Fig. 3) are fat-soluble substances with colors
varying form brown, red, orange to yellow. They perform two key
5.1. Phycobilins roles in photosynthesis: 1) absorb light in regions of the visible
spectrum, in which chlorophyll does not absorb efficiently; 2)
Phycobilins (or phycobiliproteins; Fig. 1) are soluble in water photoprotect the photosynthetic systems. Photoprotection
and relatively easy to isolate and purify, because they comprise a mechanisms remove the most energetic states of chlorophyll,
large portion of the total cell protein. Phycobilins are accessory for resulting from the excessive absorption of light radiation. This
collecting light during photosynthesis. For example, at four phy- hinders the formation of reactive oxygen species (ROS), makes
cobilin classes are produced in red algae (Rhodophyta and Glau- carotenoids good antioxidants [66]. The main carotenoids of
cophytas): allophycocyanin (green-bluish), phycocyanin (blue), microalgae are: β-carotene, lycopene, astaxanthin, zeaxanthin,
phycoerythrin (purple), and phycoerythrocyanin (orange) [55,56]. violaxanthin, and lutein. Of these, β-carotene, lutein, and astax-
Phycobilins are widely used in industry and immunology anthin are the most studied ones [67].
laboratories, due to their absorption properties. They are often β-carotene is a orange-yellowish pigment with growing
employed in molecular biology as fluorescent markers, used in demand, being used as colorant for food or nutritional supple-
immunoassays and as fluorescent dyes for microscopy [57]. There ment, because it is a precursor of vitamin A (retinol) [68,69].
are at least 297 patent records for these substances, mostly based Demand for natural carotenoids is increasing, because the natural
on applications, such as fluorescent markers. The United States of form has properties that the artificial one does not. For example,
America have most patents related to fluorescent applications, Jayappriyan et al. [70] found that β-carotene from Dunaliella salina
while Japan has patents relating to production, purification, and causes a higher apoptosis rate in prostate cancer cells than the
therapeutic and diagnostic purposes [56]. Despite the large synthetic version.
836 E.B. D’Alessandro, N.R. Antoniosi Filho / Renewable and Sustainable Energy Reviews 58 (2016) 832–841

Fig. 2. Structure of chlorophylls.

more successful than D. salina. However, the cultivation of H.

pluvialis is more complex than D. salina, and productivity is
inferior. Moreover, extraction, purification, and concentration are
key aspects of production cost. Since the cost of the final product is
also depends on productivity and growth period, cultivating H.
pluvialis is not yet economically viable [58]. Thus, to turn pro-
duction viable, stress must be induced to increase carotenoid
concentration [84], besides improving technology [85]. Increasing
secondary carotenoids in this microalgae means improving cell
survivability under oxidative stress generated by certain light
conditions, UV-B and nutrients [86].
Lutein is yellowish at low concentrations and red-orangish at
high concentrations. It protects tissues from free radicals. This
pigment protects photoreceptors by filtering blue light (500 nm).
Such radiation is harmful to the eye macula, but lutein can reduce
by 40% light incidence that damages the retina [87]. Lutein can
also prevent atherosclerosis, cataracts, diabetic retinopathy, and
retinal degeneration, [88,89]. One of the most studied microalgae
Fig. 3. Carotenoid structures.
for lutein production is Muriellopsis sp. Lutein content of this
species can vary from 0.4% to 0.6% per dry biomass [90]. Therefore,
More than 600 types of carotenoids are known [71], but a
it is a key commercial source of lutein [91].
typical human diet only contains 40. Of these, about 20 have been
Several microalgae have been studied for lutein production,
identified in human blood and tissues. About 90% of carotenoids in
such as Muriellopsis sp. [90], Chlorella zofingensis [92], Chlorella
the diet and human body are composed of β-carotene, α-carotene,
protothecoides [93], Scenedesmus almeriensi [94], but few were
lycopene, lutein, and cryptoxanthin.
grown on a large scale (raceway or photobioreactors). Only Mur-
Alencar et al. [72] quantified β-carotene in the commercial
iellopsis sp. and Scenedesmus almeriensis were grown for biomass
cyanobacteria Spirulina and Spirulina platensis cultivated in the
production [95], i.e., in large-scale outdoor systems and for a
laboratory. They found that those grown in the laboratory had 14 reasonable period with continuous and feasible operation.
times more β-carotene, providing suggestions for culture stan- The main source for lutein are the petals of marigold (Tagetes
dardization and the management of production line. The primary patula) [96]. Del Campo et al. [67] found that Muriellopsis culti-
industrial source of β-carotene is the microalgae Dunaliella salina, vated on a large scale (55-L photobioreactor) produced 180 mg/
which produces β-carotene above 14% dry weight [57]. This is 10 m2/day of lutein. If we extrapolate the calculation for one year, the
to 100 times the amount found in carrots, which is the second system is capable of producing more than 65 g lutein or more than
source of this pigment. The microalgae Dunaliella has long been 14 kg/m2 of dry biomass. According to these same authors, this
recognized as an efficient producer of this carotenoid. More amount cannot be achieved in marigold culture, especially con-
information on Dunaliella and β-carotene can be found in Ben- sidering that only petals are used.
Amotz and Avron [73], Lers et al. [74], Ben-Amotz [75], García-
González et al. [76], Raja et al.[77], Davidi et al. [78], and in the
patent of Ben-Amotz and Mori [79]. 6. Market for pigment production
Astaxanthin has a rosy color, is insoluble in water, and a
byproduct of β-carotene, since under stress β-carotene is con- Spolaore [57] estimated that the prices of natural phycobilin
verted into astaxanthin through β-carotene oxygenase (CRTO) and products range from US$ 3 to US$ 25/mg, but can reach U$ 1500/
β-carotene hydroxylase (CRTR-B). The pigment is then accumu- mg for specific pigments (e.g., antibodies and fluorescent mole-
lated in lipid vesicles outside the chloroplast [80]. Astaxanthin cules). Bermejo Román et al. [97] estimated the production cost of
sequester free radicals by removing reactive oxygen and has β-phycoerythrin from Porphyridium cruentum in US $ 56,566.00/kg
higher antioxidant activity than other carotenoids. Because of this, of dry biomass. Brennan and Owende [98] estimate the production
it is used in disease treatment such as, atherosclerosis and heart cost of phycobilin from Spirulina sp. in € 11/mg (about U$ 16.61/
disease [53], diabetes [81], skin conditions [82], chronic inflam- mg, at 2009 values of 1 €¼ U $ 1.51).
matory diseases, and in the prevention of some cancers [83]. The global market for carotenoids was US$ 1.2 billion in 2010
Haematococcus pluvialis can produce and accumulate astax- and is expected to increase to $ 1.4 billion by 2018, with compound
anthin up to concentrations of 1–8% per dry weight. This con- annual growth rate (CAGR) of 2.3%. The highest market share is β-
centration within the cell would make cultivation of H. pluvialis carotene [99]. The market value of astaxanthin produced from
 E.B. D’Alessandro, N.R. Antoniosi Filho / Renewable and Sustainable Energy Reviews 58 (2016) 832–841 837

microalgae is US $ 2500/kg to $ 3000/kg, at production cost of H2O2 alone and with H2O2 þFeSO4. Lipid extraction in the first
microalgae of US$ 5 to US$ 20/kg dry weight [100]. The artificial treatment increased from 6.9 to 9.2% and from 6.9 to 17.4% in the
form costs around US$ 1000/kg. However, Li et al. [85] surveyed second treatment. Authors also optimized the time that algal
the costs and concluded that China can sell astaxanthin at a lower biomass was exposed to peroxide at 4 minutes, since peroxide
price (US$ 718/kg) using low-cost photobioreactors, two phases in starts degrading lipids after that time.
raceways (ponds), and low cost of labor and reagents. Thus, it is important that the cell membrane of microalgae used
Thus, various carotenoids have high commercial value and can for biodiesel production and other byproducts (e.g., pigments) be
be produced from microalgae together with biodiesel, specially for rigid to allow movement and isolation of biomass in the culture.
their high demand as nutraceutical. Thus, antioxidant pigments, However, cell membrane cannot be too rigid, because it could
known as nutraceuticals, are currently the most marketed pro- make component extraction unviable.
ducts from microalgae, renovating the interest in increasing the
concentration of these substances in microalgae.
Stressful conditions in cultivation (e.g., changes in nutrient 8. Extremophile microalgae
levels or light intensity), induce microalgae to produce ROS as a
signaling molecule for control processes, such as cell death pro- Important parameters to select microalgae for biodiesel pro-
gramming, response to abiotic stress, pathogen defense, and sys- duction include lipid content and production, biomass productiv-
temic signaling. ROS are reduced forms of atmospheric oxygen ity and potential of adaptability to different growth conditions.
(O2), produced from O2 excitement to form singlet oxygen (1O2) or Additionally, the same microalgae species can produce different
from the transfer of one, two, or three electrons to O2, forming a fatty acids depending on the environment in which it is grown. As
superoxide radical (O
2 ), hydrogen peroxide (H2O2), or hydroxyl a result, recent studies have attempted to increase biomass pro-
radical (  OH), respectively. Therefore, they can oxidize various ductivity or change the profile and content of fatty acids of
cellular components and cause oxidative cell destruction [101]. microalgae by using species collected in extreme environments, or
Thus, microalgae produce ROS as a defense mechanism to stress, collected in conventional environments, but grown in extreme
producing antioxidant enzymes as by-products, such as super- ones, such as thermal, acidic, alkaline, salty, or eutrophic waters.
oxide dismutase, catalase, peroxidase, and glutathione reductase, For example, Teoh et al. [115] compared the fatty acid profile of
as well as non-enzymatic antioxidant molecules, such as phy- Chlamydomonas from the Antarctica, temperate and tropical
tochelatins, polysaccharides, polyphenols, and pigments [102,103]. environments. The percentage of saturated fatty acids of Antarctic
However, few studies relate ROS to pigment induction in micro- Chlamydomonas increased from 21.1% when grown at 4 °C to 32.6%
algae, despite the pathways for pigment production being known. when grown at 20 °C, while that of temperate Chlamydomonas
increased from 48.7% at 4 °C to 51.6% at 25 °C. Finally, the content
of saturated fatty acids of tropical Chlamydomonas increased from
7. Cell membrane of microalgae 28.7% when grown at 18 °C to 69.8% at 33 °C. Thus, the percentage
of saturated fatty acids of microalgae from warmer environments
Several steps are involved in converting microalgae products or cultured in high temperatures may increase. This suggests that
(e.g., pigments and fatty acids) into biodiesel, specially breaking microalgae reduces the production of unsaturated fatty acids,
microalgae cell, since it influences lipid productivity and pigments. which maintains the cellular content in the liquid state at low
Studies of cell disruption have advanced in recent years [104–106]. temperatures, changing to produce saturated fatty acids that
For example, the efficiency of cell disruption methods depends on provide more energy. Accordingly, An et al. [116] found genes in
microalgae species and is related to cell membrane composition microalgae more sensitive at low temperatures, and associated
and morphology [107]. Moreover, culture medium influences cell unsaturated fatty acids production to cell protection, preventing
membrane rigidity [9]. Thus, the costs of breakage and extraction freezing. Also, Ghozzi et al. [117] collected microalgae and cyano-
can be significantly reduced by using the proper medium. bacteria from different geothermal sources in Tunisia, in which
Solvents are key components of lipid extraction, since several temperatures ranged from 40 to 61 °C. They found 14 taxa of
studies found a variation in lipid content extracted from the same cyanobacteria, but only three microalgae above 60 °C, suggesting
microalgae using different solvents and extraction forms [108– that cyanobacteria are more adapted to high temperatures than
112]. In turn, lipid content is influenced not only by the solubility microalgae.
of fatty acids, but also by solvent ability to permeate and break cell Onay et al. [118] collected Micractinium sp., H. tetrachotoma, and
membrane. Scenedesmus sp. in geothermal waters (43–44 °C) in Turkey. They
There are two methods for breaking cell membrane [107]: cultivated these species at three temperatures (25, 37, and 50 °C),
(1) the mechanical one, subdivided into grinding, high-speed and found that biomass productivity of all decay at 50 °C. Con-
homogenization, high pressure homogenization, ultrasonication, versely, biomass productivity of Micractinium sp. increased at
microwave, and pulsed electric field; and (2) non-mechanical, 37 °C, but lipid content was greater at 25 °C than 37 °C (22.7 and
subdivided into chemical and enzymatic. The mechanical method 10.7%, respectively). These results were similar to Smith-Bädorf
is the best one for industry, but its energy consumption is high. et al. [119]. Therefore, microalgae from warm environments may
Conversely, the treatment time of the non-mechanical method is be resistant to high temperatures, but generally biomass and fatty
longer, affecting product quality, although consuming less energy acid productivity is greater under conventional conditions (20–
and disrupting cell membrane uniformly. Furthermore, the non- 30 °C).
mechanical method is more difficult to control than mechanical Thus, microalgae usually produce large amounts of poly-
methods. unsaturated fatty acids, which are useless for biodiesel production,
The laser is a new mechanical method of cell disruption of because they reduce biofuel oxidative stability. As a result, using
microalgae, whereas the hydrogen peroxide (H2O2) is a new non- species adapted to or cultivated in high temperatures can provide
mechanic one. McMillan et al. [113] tested five methods for fatty acids profile more suitable for biodiesel production, i.e. with
breaking cells of N. oculata: microwave, heated bath, blender, lower proportion of unsaturated fatty acids.
ultrasound, and laser (1064 nm). Laser was the best one, with a Ruiz-Dominguez et al. [14] isolated Coccomyxa sp. from acidic
mean of 96.5% breakage, while ultrasound was less efficient, waters of Rio Tinto (Huelva, Spain) to produce lipids and anti-
averaging 67.6% breakage. Steriti et al. [114] treated C. vulgaris with oxidants. This river has an acidic pH (1.7–3.1) and high
838 E.B. D’Alessandro, N.R. Antoniosi Filho / Renewable and Sustainable Energy Reviews 58 (2016) 832–841

concentrations of potentially toxic metals. Authors concluded that The de novo lipid synthesis pathway in microalgae has been
Coccomuxa sp. acidophile can be produced in highly acidic cultures studied lately. Lipids can be produced in three locations: fatty
(pHr2.5), allowing outdoor cultivation with low bacterial acids synthesized in the chloroplast and triacylglyceride (TAG) in
contamination. the endoplasmic reticulum (ER), and thylakoids [44,134]. In the de
Skorupa et al. [120] analyzed the effects of both visible and UV novo lipid synthesis pathway, acetyl-CoA (CoA) is converted into
lights on cellular events of Cyanidioschyzon, a red algae that grow malonyl-CoA by acetyl-CoA-carboxylase (ACCase) combined with
in acidic (pH 0.2–4.0) and hot (38–56 °C) environments. They HCO3-, which is the first step in fatty-acid biosynthesis in various
found that both light wavelengths can be photoinhibitor of organisms. The second step is the conversion of malonyl-CoA into
growth. Thus, wavelength and light intensity must be carefully malonyl-ACP by malonyl-CoA:ACP transacylase (MAT). Posteriorly,
chosen in microalgae cultivation, since they can strongly affect malonyl-ACP is converted into 3-ketoacyl-ACP by 3-ketoacyl-ACP
biomass and lipid productivity. For example, Ho et al. [121] found synthase (KAS), starting the fatty-acid cycle. Finalized by acyl-ACP
that the appropriate light intensity for Scenedesmus obliquus cul- fatty-acid thioesterase (FAT), free fatty acids are exported to the
ture was 420 mmol/m2/s. Conversely, Chen et al. [122] cultivated cytosol. In the chloroplast, acyl-ACP and glycerol 3-phosphate
Chlorella sp. and developed a lumostatic strategy, based on the (G3P) form the diacylglicerids (DAG). Then, the TAGs are expor-
distribution of light detected by image analysis and chlorophyll ted to the cytosol through thylakoids. In the cytosol, the dihy-
content, which increases biomass productivity by 74.3% compared droxyacetone phosphate is converted into G3P by G3P dehy-
to constant light intensity. drogenase (G3PDH). Then, G3P along with acyl-CoA catalyze the
Eutrophic waters with high concentrations of toxic metals are formation of TAG by various enzymes produced by the ER, such as
extreme environments for some microalgae. For example, Park GPAT (glycerol 3-phosphate acyltransferase), LPAAT (lysopho-
et al. [123] cultivated microalgae in eutrophic water from waste- sphatidic acid acyltransferase) LPAT (lysophosphatidylcholine
water treatment effluents. The major difference between acyltransferase), and DAGAT (diacylglycerol acyltransferase).
eutrophic waters and regular culture media is the high con- The enzymes involved in lipid metabolism may be significantly
centration of nitrogen, phosphorus, and toxic metals [124]. Most altered by gene expression levels, because they depend on envir-
nitrogen content in sewage is in form of ammonia, which in high onmental conditions. For example, the expression of the FAD gene
concentrations can inhibit algal growth [125,126]. Sewage waters in H. pluvialis was about six times higher in the low temperature
can still contain metals that inhibit algal growth, such as cadmium, (4 °C) than control (22 °C). This difference in gene expression
zinc, iron, nickel, and copper [127]. Furthermore, such metals may correlated with increasing C18:3ω3 content (1.83 to 2.66 mg/g dry
influence catalysis during biodiesel production or remain in the weight) [46]. Thus, low temperatures can induce the expression of
FAD, raising the content of linolenic acid. Similarly, [116] cultured
final product, producing toxic fumes during biofuel combustion.
Chlamydomonas sp. from Antarctica and found that gene expres-
Some aerial microalgae are also extremophiles, because they
sion of Δ9ACPCiFAD, ω3CiFAD1 and ω3CiFAD2 were more sensi-
can grow on the surface or inside a variety of terrestrial habitats,
tive at 0 °C than 5, 10, and 15 °C, indicating that these genes may
such as rocks, soil, and vascular plants [128,129]. Microalgae are
be involved in a defense mechanism at 0 °C. However, Δ6CiFAD
more exposed to difficult and rapid climate change than aquatic
was more sensitive at 15 °C than at low temperatures, suggesting
ones and produce several carotenoids to adapt to these environ-
that it may be a defense mechanism above 10 °C. The highest fatty
mental conditions (e.g., canthaxanthin, β-carotene, astaxanthin,
acid content in Chlamydomonas sp. at all temperatures was C18:3
and lutein [130,131]). The main stress sources used in carotenoid
(28.2% in mean).
production in aerial microalgae is increasing light intensity and
A way to increase TAG content is to block the metabolic path-
nitrogen deficiency. Aburai et al. [132] found that the color of
ways involved in the production of energetic compounds, such as
cellular content of aerial microalgae changed from green to red
starch [44]. Recently, [135] found a positive correlation between
when cultivated under high light intensity, indicating high pro-
the increase in the enzyme G3PDH and TAG concentration in in
duction of free-ester carotenoids (astaxanthin and adonixanthin),
Phaeodactylum tricornutum growing in nitrogen poor environ-
as well as lutein, canthaxanthin, and β-carotene.
ments. Therefore, to make biodiesel production from microalgae
Thus, culture conditions directly affect not only the levels and
economically feasible it is key to understand the expression pat-
types of pigments, but also fatty acids synthesized by microalgae.
tern of genes involved in lipid metabolism to improve the pro-
Accordingly, pigment production must be reduced to increase fatty
duction and accumulation of FAs and TAGs.
acids production for biodiesel production. Furthermore, lipid
production, especially those with lower unsaturation, should be
stimulated by means of nitrogen depletion [15] and increased 10. Conclusions
water temperature [38].
Microalgae can be used to produce fatty acids and biodiesel. Its
cultivation should consider the following characteristics: (1) avoid
9. Metabolism of fatty acids and acylglycerides using marine microalgae, because they contaminate biodiesel with
sodium and other inorganic compounds; (2) prefer extremophile,
Most of what we known about lipid metabolism in photo- since they are less susceptible to contamination by other micro-
synthetic organisms is based on Arabidopsis [133]. Consequently, organisms (3) microalgae should preferably be able to develop in
Lei et al. [46] used Arabidopsis gene sequences to understand lipid extreme culture media with nutritional disposal, to reduce
metabolism of the microalgae H. pluvialis. It is known that spending with reagents; (4) microalgae must produce large
microalgae accumulate lipids when cell division and growth sta- amounts of biomass in a short term, both at small and large scales;
bilize. For example, Wan et al. [49] found that growth rate (5) species must also withstand stress that induce fatty acid pro-
decreased with increasing lipid accumulation, and Mansour et al. duction, such as temperature or light changes and nutrient
[30] shown that higher lipid accumulation occurred in the linear depletion; (6) cell membrane should have little adherence to the
phase of Gymnodinium sp. cultures, but higher lipid yield took culture system; (7) must cell membrane should allow permeation
place in the stationary phase, due to increased cell density. This of solvents or mechanical action to ease the extraction of fatty acid
relationship is economically important for biodiesel production and other components; (8) should have adequate percentage of
from microalgae. saturated and unsaturated fatty acids; (9) should the microalgae
 E.B. D’Alessandro, N.R. Antoniosi Filho / Renewable and Sustainable Energy Reviews 58 (2016) 832–841 839

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