7. Secondary metabolite production

In the mixotrophic growth mode, certain molecules are accumulated, and there is a need to elucidate which metabolite is able to accumulate under specific growth conditions, but in general mixotrophic growth, it seems to be an efficient way for secondary metabolite accumulation [15, 22].

carotenogenesis, mechanism such as esterification of astaxanthin and adonixanthin

Red marine microalga has proven their ability to produce pigments and hydrocolloids, due to their diversity, and perhaps produce a diversity of high valuable compounds. Fine chemicals are used as cosmetics, nutraceutics, and therapeutic agents; some are used in the food industry, diagnostic, biomedical research, and biosensor. Carbon source such as glucose, sucrose, glycerol, or acetate in the culture medium can help for accumulation of β-carotene and zeaxanthin by red

Microalgae produce a wide range of antioxidants, some of them involved in the scavenging machinery of photosynthesis, respiration, and oxidative protection mechanisms. Pigments (carotenoids, chlorophylls, phycobiliproteins) play an important role in the photosynthetic mechanism in tocopherols including α, δ, and γ tocopherols, and pigments are accumulated as secondary metabolites at different amounts. Tocopherols have been found in Nannochloropsis oculata,Tetraselmis suecica, Spirulina maxima, Chaetoceros sp., Synechococcus, and Porphyridium cruentum. These compounds are formed depending several factors, such as microalga specie, growth phase, nutrient availability, light supply, and oxygen concentration, but also their production is affected by the processes of extraction

Lipid metabolism can be induced by a nitrogen-limiting condition; nitrogen obtained from amino acid catabolism is assimilated via the glutamate-glutamine pathway; then, it is stored as an amino acid. The excess of carbon obtained from photosynthesis or glycolysis is redistributed into carbon-containing compounds. Carbon enters lipid metabolism via gamma-aminobutyrate pathway, glycolysis, and the tricarboxylic acid cycle [35]; malonyl-CoA is formed via acetyl-CoA from respiration; then, lipogenesis proceeds [15]. Supplementing microalgae cultures with an organic carbon source increases the productivity of biomass, lipid, and carbohydrates, enhancing the production of biodiesel, ethanol, starch, and polyunsaturated fatty acids. However, organic carbon source addition has limitations, for example,

the cost and the bacterial contamination during cultivation. Progress on

biorefineries has been focused on mixotrophic cultivation to enhance either secondary metabolite accumulation or fine chemicals [36]. Triacylglycerol content in Neochloris oleoabundans, Dunaliella sp., and Botryococcus braunii is more abundant when glycerol was used as organic carbon source than with autotrophic cultures. Profile of free fatty acids is also different. Saturated free fatty acids increase significantly in the presence of glycerol, but unsaturated free fatty acids decrease in general [37]. Biomass productivity and also the lipid productivity increased with the addition of acetate, glucose, and glycerol; although lipid content is smaller than other cultures, light supply also affected the content of lipids [38]. In contrast, lipid concentration in Chlorella protothecoides was as high as 55%, four times those obtained in autotrophic growing cells. Microalgae metabolic pathways for lipid accumulation are influenced by nitrogen-limiting conditions and carbon metabolism, where distribution pathways contribute to lipid biosynthesis [39, 40]. Biomass is considered as a renewable fuel source and does not affect the overall balance of CO2 in the atmosphere. Algal biofuel production coupled to a biomass power plant waste can serve as a cost-effective process to enhance microalgae biomass and

in Scenedesmus sp. [30].

Microalgae Cultivation for Secondary Metabolite Production

DOI: http://dx.doi.org/10.5772/intechopen.88531

microalga [31].

7.3 Antioxidants

and purification [32–34].

7.4 Biofuels

201

#### 7.1 Fine chemicals

Several high valuable products have been described to be produced by photosynthetic microorganisms: antitumor agent from Amphidinium sp., food supplements from Dunaliella and Isochrysis galbana, antioxidants from Phaeodactylum tricornutum, and elastase inhibitor from Oscillatoria agardhii [17]. Algae biomass can accumulate or produce (i) bioenergy-based products, such as ethanol, methanol, biodiesel, biohydrogen, biogas, and long-chain hydrocarbons; (ii) staple food and vitamins such as yellow-white proteins, β-carotene, and phycobiliproteins, such as phycocyanin; (iii) polyunsaturated fatty acids, such as linolenic acid and arachidonic acid, that is, omega-3 fatty acids [23, 24]; (iv) base compounds for cosmetic industry and plant growth regulators; and (v) compounds with anticancer, antimicrobial, and antiviral activities.

Spirulina platensis showed higher antioxidant activity than other microalgae tested; Nostoc muscorum and Oscillatoria sp., moreover, have an important increment of phycobiliproteins by increasing nitrogen to the culture medium. It produces an important increment of the antioxidant activity in aqueous extracts of these microalgae. These extracts exhibit anticancer activity as well; in the extracts phenolic compounds, terpenoids, and alkaloids have been detected which can be responsible for several biomedical activities [24]. Water extracts of S. platensis have shown vulvovaginal antifungal activity on Candida and antifungal activity on several strains of Candida sp.; this can be the basis for therapeutic treatments, where secondary effects seem to be absent [25].

#### 7.2 Pigments

Dietary supplements have been produced from biomass of microalgae; they include pigments and colorants from Haematococcus pluvialis, Chlorella sp., Dunaliella, red algae, cyanobacteria, and S. platensis [23]. A profile of natural pigments in dietary supplements of Spirulina including 51 pigments has been found in commercial products [26]. Pre-column reaction with DPPH radical followed by fast UHPLC-PDA separation revealed different classes of pigments grouped among carotenes, xanthophylls, and chlorophylls. Diadinoxanthin, alloxanthin, canthaxanthin, diatoxanthin, zeaxanthin, and echinenone were found in powder and tablets as minor components, in addition to β-carotene as a major component of dietary supplements [26]. Astaxanthin from H. pluvialis, c-phycocyanin from Limnothrix sp., and phycoerythrin from Phormidium have been produced [27–29], respectively.

Production of pigments is affected by the amount of light supplied, and in combination with mixotrophic growth mode, phycocyanin, chlorophyll-a, and carotenoid concentrations, increased as light intensity increased, the concentration increased at least 30% in S. platensis [4, 11]. Production of chlorophylls and carotenoids increases 1.5 fold in Chlorella vulgaris in stirred tank photobioreactor [4]. Carotenoid accumulation and composition seem to be induced by light intensity, nitrogen starving, and salt stress. Higher light and salt stresses active synergistically Microalgae Cultivation for Secondary Metabolite Production DOI: http://dx.doi.org/10.5772/intechopen.88531

carotenogenesis, mechanism such as esterification of astaxanthin and adonixanthin in Scenedesmus sp. [30].

Red marine microalga has proven their ability to produce pigments and hydrocolloids, due to their diversity, and perhaps produce a diversity of high valuable compounds. Fine chemicals are used as cosmetics, nutraceutics, and therapeutic agents; some are used in the food industry, diagnostic, biomedical research, and biosensor. Carbon source such as glucose, sucrose, glycerol, or acetate in the culture medium can help for accumulation of β-carotene and zeaxanthin by red microalga [31].

## 7.3 Antioxidants

7. Secondary metabolite production

Microalgae - From Physiology to Application

secondary metabolite accumulation [15, 22].

antimicrobial, and antiviral activities.

secondary effects seem to be absent [25].

7.2 Pigments

respectively.

200

7.1 Fine chemicals

In the mixotrophic growth mode, certain molecules are accumulated, and there is a need to elucidate which metabolite is able to accumulate under specific growth conditions, but in general mixotrophic growth, it seems to be an efficient way for

Several high valuable products have been described to be produced by photosynthetic microorganisms: antitumor agent from Amphidinium sp., food supplements from Dunaliella and Isochrysis galbana, antioxidants from Phaeodactylum tricornutum, and elastase inhibitor from Oscillatoria agardhii [17]. Algae biomass can accumulate or produce (i) bioenergy-based products, such as ethanol, methanol, biodiesel, biohydrogen, biogas, and long-chain hydrocarbons; (ii) staple food and vitamins such as yellow-white proteins, β-carotene, and phycobiliproteins, such as

Spirulina platensis showed higher antioxidant activity than other microalgae tested; Nostoc muscorum and Oscillatoria sp., moreover, have an important increment of phycobiliproteins by increasing nitrogen to the culture medium. It produces an important increment of the antioxidant activity in aqueous extracts of these microalgae. These extracts exhibit anticancer activity as well; in the extracts phenolic compounds, terpenoids, and alkaloids have been detected which can be responsible for several biomedical activities [24]. Water extracts of S. platensis have shown vulvovaginal antifungal activity on Candida and antifungal activity on several strains of Candida sp.; this can be the basis for therapeutic treatments, where

Dietary supplements have been produced from biomass of microalgae; they include pigments and colorants from Haematococcus pluvialis, Chlorella sp., Dunaliella, red algae, cyanobacteria, and S. platensis [23]. A profile of natural pigments in dietary supplements of Spirulina including 51 pigments has been found in commercial products [26]. Pre-column reaction with DPPH radical followed by fast UHPLC-PDA separation revealed different classes of pigments grouped among carotenes, xanthophylls, and chlorophylls. Diadinoxanthin, alloxanthin, canthaxanthin, diatoxanthin, zeaxanthin, and echinenone were found in powder and tablets as minor components, in addition to β-carotene as a major component of dietary supplements [26]. Astaxanthin from H. pluvialis, c-phycocyanin from Limnothrix sp., and phycoerythrin from Phormidium have been produced [27–29],

Production of pigments is affected by the amount of light supplied, and in combination with mixotrophic growth mode, phycocyanin, chlorophyll-a, and carotenoid concentrations, increased as light intensity increased, the concentration increased at least 30% in S. platensis [4, 11]. Production of chlorophylls and carotenoids increases 1.5 fold in Chlorella vulgaris in stirred tank photobioreactor [4]. Carotenoid accumulation and composition seem to be induced by light intensity, nitrogen starving, and salt stress. Higher light and salt stresses active synergistically

phycocyanin; (iii) polyunsaturated fatty acids, such as linolenic acid and arachidonic acid, that is, omega-3 fatty acids [23, 24]; (iv) base compounds for cosmetic industry and plant growth regulators; and (v) compounds with anticancer,

Microalgae produce a wide range of antioxidants, some of them involved in the scavenging machinery of photosynthesis, respiration, and oxidative protection mechanisms. Pigments (carotenoids, chlorophylls, phycobiliproteins) play an important role in the photosynthetic mechanism in tocopherols including α, δ, and γ tocopherols, and pigments are accumulated as secondary metabolites at different amounts. Tocopherols have been found in Nannochloropsis oculata,Tetraselmis suecica, Spirulina maxima, Chaetoceros sp., Synechococcus, and Porphyridium cruentum. These compounds are formed depending several factors, such as microalga specie, growth phase, nutrient availability, light supply, and oxygen concentration, but also their production is affected by the processes of extraction and purification [32–34].

## 7.4 Biofuels

Lipid metabolism can be induced by a nitrogen-limiting condition; nitrogen obtained from amino acid catabolism is assimilated via the glutamate-glutamine pathway; then, it is stored as an amino acid. The excess of carbon obtained from photosynthesis or glycolysis is redistributed into carbon-containing compounds. Carbon enters lipid metabolism via gamma-aminobutyrate pathway, glycolysis, and the tricarboxylic acid cycle [35]; malonyl-CoA is formed via acetyl-CoA from respiration; then, lipogenesis proceeds [15]. Supplementing microalgae cultures with an organic carbon source increases the productivity of biomass, lipid, and carbohydrates, enhancing the production of biodiesel, ethanol, starch, and polyunsaturated fatty acids. However, organic carbon source addition has limitations, for example, the cost and the bacterial contamination during cultivation. Progress on biorefineries has been focused on mixotrophic cultivation to enhance either secondary metabolite accumulation or fine chemicals [36]. Triacylglycerol content in Neochloris oleoabundans, Dunaliella sp., and Botryococcus braunii is more abundant when glycerol was used as organic carbon source than with autotrophic cultures. Profile of free fatty acids is also different. Saturated free fatty acids increase significantly in the presence of glycerol, but unsaturated free fatty acids decrease in general [37]. Biomass productivity and also the lipid productivity increased with the addition of acetate, glucose, and glycerol; although lipid content is smaller than other cultures, light supply also affected the content of lipids [38]. In contrast, lipid concentration in Chlorella protothecoides was as high as 55%, four times those obtained in autotrophic growing cells. Microalgae metabolic pathways for lipid accumulation are influenced by nitrogen-limiting conditions and carbon metabolism, where distribution pathways contribute to lipid biosynthesis [39, 40]. Biomass is considered as a renewable fuel source and does not affect the overall balance of CO2 in the atmosphere. Algal biofuel production coupled to a biomass power plant waste can serve as a cost-effective process to enhance microalgae biomass and

biofuel productivity by sequestration of the CO2 produced in the power plant [35]. Productivity of biodiesel from oily plant crops, in terms of produced oil by surface production, varies from 27.57 to 972 L per ha, whereas that from microalgae cultivation is 7688–23,067 L per ha [23].

Mixotrophy is coupled with three metabolic mechanisms, glycolysis, Calvin-Benson-Bassham, and the tricarboxylic acid cycle, where ATP is formed in the tricarboxylic acid cycle helping to drive electron flux on the light reactions of the photosynthesis to generate NADH, which is needed in the tricarboxylic acid cycle. These mechanisms are focal point to perform metabolic engineering, which open new routes to enhance the synthesis of fine chemicals by microalgae [47].

Microalgae Cultivation for Secondary Metabolite Production

DOI: http://dx.doi.org/10.5772/intechopen.88531

In the past, microalgae cultures were used as components of aquaculture feeds and human food supplements. Recently, new alternatives have been opened for the production of fine chemicals and biofuels. However, production costs have been a concern; several efforts have been made to reduce processing costs to construct a profitable process. In this context, Allen et al. propose an integration of biology, ecology, and engineering topics for a sustainable biofuel and bioproduct production

The potential markets of value-added products from microalgae are nutraceuticals for human applications and nutraceutical with applications for animal and fish feed, bulk chemicals, and biofuels, with commercial costs of 100 €/kg biomass, 5–<sup>20</sup> €/kg biomass, <sup>&</sup>lt;<sup>5</sup> €/kg biomass, and <sup>&</sup>lt;0.4 €/kg biomass, with a

volume market of 60 million, 3–4 billion, <sup>&</sup>gt;50 billion, and <sup>&</sup>gt;1 trillion €,

High value-added products such as antiviral, anticancer, and antioxidants are target products to be obtained from microalgae, since it is an alternative process that can be continuously cultivated of axenic cultures in a closed photobioreactor adapted with a special light source of irradiation, such as fiber-optic or halogen lamps. In this case, biomass increases as long as microalgae receive light and the broth hydrodynamic allows enough movement to reach the illuminated surface (see Table 1), in continuous cultivation. Once the light limitation occurred and due to the effect of washing out, biomass starts to decrease to a new dilution rate. When an organic carbon source has a positive effect on the growth, continuous cultivation can be used as well, to produce an increment in biomass density (Table 1) and secondary metabolite formation as well, producing an increment of biomass and in the metabolites. Productivity also has a substantial increment at same light intensity

obtained in semicontinuous cultivation with a biomass of 5.31 g L<sup>1</sup> and productivity of 1.32 g L<sup>1</sup> d<sup>1</sup> [50]. Therefore, semicontinuous cultivation seems to be a good

Secondary metabolite production can be effectively improved, by three advan-

tages,(i) using a continuous process (up- and downstream processes),

) <sup>Δ</sup>XAD (g L<sup>1</sup> <sup>h</sup><sup>1</sup> <sup>10</sup><sup>3</sup>

3.22 0.050 1.74 0.079 2.76 5.85 0.092 3.21 0.136 4.75 11.11 0.175 6.11 0.241 8.41 18.98 0.301 10.81 0.405 14.13

Biomass concentration and productivity in continuous culture, in autotrophic and mixotrophic conditions

). Productivity and biomass concentration have been

) <sup>Δ</sup>XMD (g L<sup>1</sup> <sup>h</sup><sup>1</sup> <sup>10</sup><sup>3</sup>

)

) ΔXM (g L<sup>1</sup>

8. Concluding remarks

from microalgae [48].

respectively [49].

and same dilution rate (D, h<sup>1</sup>

) ΔXA (g L<sup>1</sup>

strategy as well.

Io (J cm<sup>2</sup> h<sup>1</sup>

Modified from Ref. [51].

).

Table 1.

203

(D = 0.03 h<sup>1</sup>

Biofuels derived from algal biomass depend on algal species: for biodiesel, Cladophora fracta, C. protothecoides, and B. braunii; for biohydrogen, C. protothecoides, S. platensis, and Chlamydomonas reinhardtii; for bioethanol Palmaria, Porphyra, Ascophyllum, Ulva lactuca,Tetraselmis sp., and Chlorococum sp.; and for biogas C. reinhardtii, Chlorella kessleri, and Spirogyra neglecta [23, 41–44]. To produce biodiesel from microalgae, it is very important to select strains with oil content over 50% to improve biodiesel yield. With respect to oil content, microalgae can be divided into low, medium, and high oil content strains [45].

#### 7.5 Proteins

Soluble proteins have been used as nutritional supplements and personal care products or insoluble proteins for animal feeds [36]. Protein production has been reported in S. platensis using beet vinasse-supplemented culture media, in tubular photobioreactor biomass, which reached to 6.5 g L<sup>1</sup> and 168 mg L<sup>1</sup> d<sup>1</sup> of protein productivity. Continuous cultivation was also suitable for protein production from S. platensis using a medium supplemented with beet vinasse [46].

Incorporation of carbon from an organic carbon source, the type of carbon source, the amount supplemented to the culture, and the specie of microalgae are important for lipid accumulation in the cells of microalgae. The content of protein mostly increases by the addition of an organic carbon source, but lipid content decreases, although productivity of biomass, protein, and lipids increases substantially in the presence of organic carbon source [38].

Figure 2 represents the secondary metabolite production along with biomass that should be included in balance equations. The main components are carbon, hydrogen, oxygen, and nitrogen; in other words, a secondary metabolite can be a fraction of the total biomass, and it can be defined as ΘΔX, where is the fraction corresponding to the secondary metabolite produced, for chlorophyll accumulation, and it depends on the availability of carbon and nitrogen sources [40].

#### Figure 2.

Drawing describing the production of secondary metabolites under mixotrophy. P, metabolite from autotrophy and heterotrophy; A, biomass from autotrophy; H, biomass from heterotrophy. Modified from Ref. [5].

Mixotrophy is coupled with three metabolic mechanisms, glycolysis, Calvin-Benson-Bassham, and the tricarboxylic acid cycle, where ATP is formed in the tricarboxylic acid cycle helping to drive electron flux on the light reactions of the photosynthesis to generate NADH, which is needed in the tricarboxylic acid cycle. These mechanisms are focal point to perform metabolic engineering, which open new routes to enhance the synthesis of fine chemicals by microalgae [47].
