**2. Photosynthetic pigments**

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**356**

**Figure 2.**

*growth [4].*

**Figure 1.**

*The microalga* Chlamydomonas reinhardtii's *cell structure [3].*

Algae have six types of life cycles viz. haplontic, diplontic, isomorphic, heteromorphic, haplobiontic, and diplobiontic cycles; the exposition of these algal life cycles is discussed elsewhere [5]. The microscopic algae are the microphytes or microalgae and are typically found in freshwater and marine ecosystems at the benthic depths and in the water column. They are reported to be the chief converters of water and carbon dioxide to biomass and oxygen (see Eq. (1)) as they receive radiation from sunlight, and are therefore referred to as primary producers. Microalgae

*Schematic of a prokaryotic cell with an indication of some of the methods used to probe cellular activity or* 

Pigments are chemical compounds that reflect and transmit only certain wavelengths of visible light. This makes them appear as the colors perceived. More important than their reflection of light is the ability of pigments to absorb light of certain wavelengths. A photosynthetic pigment (accessory pigment; chloroplast pigment; antenna pigment) is a pigment that is present in chloroplasts of algae and other photosynthetic organisms and captures the light energy necessary for photosynthesis. The reaction of each pigment is associated with only a narrow range of the spectrum, and it is necessary to produce several kinds of pigments with different colors to capture more of the sun's energy. Five important pigments found in algae are (i) chlorophyll (ii) xanthophyll (iii) fucoxanthin (iv) phycocyanin and (v) phycoerythrin [6].

### **2.1 Chlorophyll**

Algae and plants have chloroplasts in which the light-capturing chlorophyll is located, while in cyanobacteria the main light-capturing complex protein molecular assemblies are the phycobilisomes, which are located on the surface of thylakoid membranes [7]. Both chlorophyll and phycobilisomes absorb light most strongly between the high-frequency, high-energy wavelengths of 450 and 495 nm, which happen to be the blue region of the electromagnetic spectrum. Also, the photosynthetic pigments absorb the low-frequency, low-energy wavelengths between 620 and 750 nm, which is the red region of the electromagnetic spectrum. The chlorophyll pigment comes in different forms, and the structure of each type of Chlorophyll pigment is anchored on a chlorin ring with a magnesium ion at the centre. The side chain of each chlorophyll pigment type is different and they are so identified (see **Figure 3** and **Tables 1** and **2**) [7, 8].

Chlorophyll a with the molecular formula C55H72O5N4Mg is the most common type of Chlorophyll. It is a green pigment with a chlorin ring having magnesium at the centre (see **Figure 3**). Chlorin is a tetrapyrrole pigment, which is partially hydrogenated porphyrin. The ring-shaped molecule is stable with electrons freely migrating around it to establish resonance structures [9]. It also has side chains and a hydrocarbon trail and contains only –CH3 groups as side chains. The long hydrophobic tail anchors the molecule to other hydrophobic proteins on the surface of the thylakoid membrane. The chemical structural layout of chlorophyll shows a porphyrin ring attached to a protein backbone (see **Figure 3**). By substituting functional groups at positions C2, C3, C7, C8, and the C17-C18 bond, one can identify the structure of the desired chlorophyll (see **Tables 1** and **2**). Chlorophyll captures and absorbs blue, violet, and red light from the spectrum to transmit or reflect green, which is the color that the green algae exhibit [9, 10]. Oxygenic photosynthesis uses chlorophyll a to furnish electrons in the electron-transport chain. Photosystems I and II harbor many pigments that help to capture light energy.

#### **Figure 3.**

*Chlorophyll - a porphyrin ring structure attached to a protein backbone. The porphyrin is built up of pyrrole molecules – 5 membered aromatic rings which are made of four carbons and one nitrogen atom. This ring system acts as a polydentate ligand and has a magnesium cation at its Centre [8].*


#### **Table 1.**

*Chemical structure of chlorophyll.*


#### **Table 2.**

*Chlorophyll structural formulae.*

A unique pair of pigment molecules are located at the reaction site of each photosystem. For photosystem I the unique pair is referred to as P700, while for photosystem II it is identified as P680. These reaction sites receive resonance energy released from chlorophyll a to sustain the redox reactions [10].

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*Microalgae: The Multifaceted Biomass of the 21st Century*

Chlorophyll b is found only in the green algae and in plants, and it absorbs most effectively at 470 nm (blue) but also at 430 nm and 640 nm. Molecular formula - C55H70O6N4Mg. It is an accessory photosynthetic pigment. The molecular structure consists of a chlorin ring with Mg centre. It also has side chains and a phytol tail. Pyrrole ring II contains an aldehyde group (− CHO). Chlorophyll b absorbs energy that chlorophyll a does not absorb. It has a light-harvesting antenna in

Xanthophyll is one of the two major groups of the carotenoids group. Generally, it is a C40 terpenoid compound formed by condensation of isoprene units. Xanthophyll, with the formula C40H56O2, contains oxygen atoms in the form of hydroxyl groups or as epoxides. Xanthophyll acts as an accessory lightharvesting pigment. They have a critical structural and functional role in the photosynthesis of algae and plants. They also serve to absorb and dissipate excess light energy or work as antioxidants. Xanthophyll may be involved in inhibiting

Fucoxanthin, with the formula C42H58O6, is a xanthophyll carotenoid, being an accessory pigment that drives limited photosynthetic reactions in brown algae (phaeophytes) and other stramenopiles. It renders the brown or olive-green color to these seaweeds. Fucoxanthin captures the red light of the spectrum for photosynthetic activities. Some edible brown algae produce this pigment in abundance, and typical candidates in this category include *Sargassum incisifolium*, *Sargassum fulvellum, Undaria pinnatifida, Laminalia japonica,* and others. The alga *Sargassum incisifolium* has been used as a source of Fucoxanthin as a nutraceutical for its antiobesity effects and as much as 0.45 mg/g has been reported [12, 13]. Another rich source of Fucoxanthin is the South African brown alga *Zonaria subarticulata* and extracts as high as 0.50 mg/g have been reported, leading to preparations such as FucoThin™ [13]. The concentration of Fucoxanthin in any algal species may depend on geographical location, seasonal variations, life-cycle, and other factors.

Phycocyanin is a protein-pigment complex found in cyanobacteria as an accessory pigment to phycobilisomes. As a phycobiliprotein, phycocyanin is identified by the color it bears as blue phycocyanin. Depending on the cyanobacterial species, this can be phycocyanin, showing maximum absorbance at 620 nm and identified as C-PC, and allophycocyanin with maximum absorbance at 650 nm and identified as A-PC. From the red microalgae, phycocyanin is identified as R-PC [13]. The molecular structure of phycocyanin changes with the pH of the medium, exhibiting the (αβ)3 trimeric structure at pH 7. However, at the pH range of 5–6, the much more available phycocyanin, C-PC, assumes the hexameric structural conformation (αβ)6. Phycocyanin boosts the human and animal immune systems and protects against certain diseases. It exhibits hepatoprotection, cytoprotection, and neuroprotection. Persons undergoing chemotherapy and radiation for cancer are placed on Phycocyanin from spirulina as a dietary supplement to ease negative symptoms during treatment as well as rejuvenate post-treatment. Phycocyanin is used in the

*DOI: http://dx.doi.org/10.5772/intechopen.94090*

Photosystem I [11].

lipid peroxidation [12].

**2.4 Phycocyanin (PC)**

food industry as a food additive [12, 14].

**2.3 Fucoxanthin**

**2.2 Xanthophyll**

*Microalgae: The Multifaceted Biomass of the 21st Century DOI: http://dx.doi.org/10.5772/intechopen.94090*

Chlorophyll b is found only in the green algae and in plants, and it absorbs most effectively at 470 nm (blue) but also at 430 nm and 640 nm. Molecular formula - C55H70O6N4Mg. It is an accessory photosynthetic pigment. The molecular structure consists of a chlorin ring with Mg centre. It also has side chains and a phytol tail. Pyrrole ring II contains an aldehyde group (− CHO). Chlorophyll b absorbs energy that chlorophyll a does not absorb. It has a light-harvesting antenna in Photosystem I [11].

## **2.2 Xanthophyll**

*Biotechnological Applications of Biomass*

**Chlorophyll**

*system acts as a polydentate ligand and has a magnesium cation at its Centre [8].*

C17 group -CH2CH2COO-Phytyl

*Chemical structure of chlorophyll.*

*Chlorophyll structural formulae.*

Molecular Formula

**Figure 3.**

C17-C18 bond

**Table 1.**

**358**

**Table 2.**

A unique pair of pigment molecules are located at the reaction site of each photosystem. For photosystem I the unique pair is referred to as P700, while for photosystem II it is identified as P680. These reaction sites receive resonance energy

C17 group −CH2CH2COO − Phytyl −CH2CH2COO − Phytyl C17-C18 bond Single (Chlorin) Single (chlorin) Occurrence Cyanobacteria Cyanobacteria

**a b c1 c2**

*Chlorophyll - a porphyrin ring structure attached to a protein backbone. The porphyrin is built up of pyrrole molecules – 5 membered aromatic rings which are made of four carbons and one nitrogen atom. This ring* 

C2 group -CH3 -CH3 -CH3 -CH3 C3 group -CH=CH2 -CH=CH2 -CH=CH2 -CH=CH3 C7 group -CH3 -CHO -CH3 -CH3 C8 group -CH2CH3 -CH2CH3 -CH2CH3 -CH2CH3

> -CH2CH2COO-Phytyl

Single (chlorin) Single (chlorin) Double

**Chlorophyll**

Molecular formula C54H70O6N4Mg C55H70O6N4Mg C2 group -CH3 -CHO C3 group -CHO -CH=CH2 C7 group -CH3 -CH3 C8 group -CH2CH3 -CH2CH3

Occurrence Universal Plants Algae Algae

**d f**

C55H72O5N4Mg C55H70O6N4Mg C35H30O5N4Mg C35H28O5N4Mg


Double (porphyrin)

(porphyrin)

released from chlorophyll a to sustain the redox reactions [10].

Xanthophyll is one of the two major groups of the carotenoids group. Generally, it is a C40 terpenoid compound formed by condensation of isoprene units. Xanthophyll, with the formula C40H56O2, contains oxygen atoms in the form of hydroxyl groups or as epoxides. Xanthophyll acts as an accessory lightharvesting pigment. They have a critical structural and functional role in the photosynthesis of algae and plants. They also serve to absorb and dissipate excess light energy or work as antioxidants. Xanthophyll may be involved in inhibiting lipid peroxidation [12].

### **2.3 Fucoxanthin**

Fucoxanthin, with the formula C42H58O6, is a xanthophyll carotenoid, being an accessory pigment that drives limited photosynthetic reactions in brown algae (phaeophytes) and other stramenopiles. It renders the brown or olive-green color to these seaweeds. Fucoxanthin captures the red light of the spectrum for photosynthetic activities. Some edible brown algae produce this pigment in abundance, and typical candidates in this category include *Sargassum incisifolium*, *Sargassum fulvellum, Undaria pinnatifida, Laminalia japonica,* and others. The alga *Sargassum incisifolium* has been used as a source of Fucoxanthin as a nutraceutical for its antiobesity effects and as much as 0.45 mg/g has been reported [12, 13]. Another rich source of Fucoxanthin is the South African brown alga *Zonaria subarticulata* and extracts as high as 0.50 mg/g have been reported, leading to preparations such as FucoThin™ [13]. The concentration of Fucoxanthin in any algal species may depend on geographical location, seasonal variations, life-cycle, and other factors.

#### **2.4 Phycocyanin (PC)**

Phycocyanin is a protein-pigment complex found in cyanobacteria as an accessory pigment to phycobilisomes. As a phycobiliprotein, phycocyanin is identified by the color it bears as blue phycocyanin. Depending on the cyanobacterial species, this can be phycocyanin, showing maximum absorbance at 620 nm and identified as C-PC, and allophycocyanin with maximum absorbance at 650 nm and identified as A-PC. From the red microalgae, phycocyanin is identified as R-PC [13]. The molecular structure of phycocyanin changes with the pH of the medium, exhibiting the (αβ)3 trimeric structure at pH 7. However, at the pH range of 5–6, the much more available phycocyanin, C-PC, assumes the hexameric structural conformation (αβ)6. Phycocyanin boosts the human and animal immune systems and protects against certain diseases. It exhibits hepatoprotection, cytoprotection, and neuroprotection. Persons undergoing chemotherapy and radiation for cancer are placed on Phycocyanin from spirulina as a dietary supplement to ease negative symptoms during treatment as well as rejuvenate post-treatment. Phycocyanin is used in the food industry as a food additive [12, 14].

#### **2.5 Phycoerythrin (PE)**

Phycoerythrin is an accessory pigment to the main chlorophyll pigment complex found in red algae and cryptophytes; it is part of a covalently bonded phycobilin chromophore in the family of phycobilins, typical of which is phycoerythrobilin, the phycoerythrin acceptor chromophore. Phycoerythrin is made up of (αβ) monomers aggregates. Except for phycoerythrin 545 (PE545), these monomer aggregates are assembled into (αβ)3 trimers or (αβ)6 hexamers with 3 or 32 symmetry and enclosing central channel [13, 14]. In red algae, they are attached to the stroma of thylakoid membranes of chloroplasts, whereas in cryptophytes, phycobilisomes are reduced and housed inside the lumen of thylakoids. Phycoerythrin captures light energy from the electromagnetic radiation and directs it to the reaction site through the phycobiliproteins, phycocyanin, and through A-PC. Each trimer and hexamer in the phycobilisome (PBS) has a minimum of one linker protein at the central channel. The α and β chains in B-phycoerythrin (B-PE) and R-phycoerythrin (R-PE) from the red algae also have γ sub-units conferring both link and light-capturing capabilities due to the presence of chromophores [14] (**Figure 4**).

The chloroplast of algal cell contains the water-soluble phycobilin pigments and while the same phycobilin pigments are found in the phycocyanin and phycoerythrin of Cyanobacteria and the red algae, the Rhodophyta. The algal chlorophyll has a structural difference from Bacteriochlorophylls (Bchl) of cyanobacteria, the latter having one of the porphyrin rings saturated, and absorbing longer wavelengths of light as opposed to chlorophylls. *Rhodopseudomonas viridis* has its bacteriochlorophyll b absorb 960 nm wavelength of light [15].

#### **2.6 The Chromophore**

The colors of pigments are the reflections of the electromagnetic spectrum from the pigments. A portion of the pigment molecule causes the formation of the color perceived, and this moiety is referred to as **chromophore**. A chromophore has two energy levels referred to as orbitals, and the difference in their energies lie within the visible spectrum of electromagnetic radiation. Thus, a photon of incident light can excite an electron from its ground-state orbital to the excited state. Chromophores are generally either **conjugated pi-systems** or transition **metal complexes** [16]. For **conjugated pi-systems,** electrons are excited between **pi-**orbitals distributed over alternating single and double bonds. Where conjugated systems are less than eight conjugated double bonds, absorbance occurs only in the ultraviolet region and is visible to the human eye. But blue or green compounds essentially do not rely on conjugated pi-bonds alone. Typical chromophores in this category are the azo compounds, lycopene, β-carotene, anthocyanin, and retinenes. **Metal complex chromophores** have transition metals whose d-orbitals are incomplete but are shared with the ligands. Chromophores in this category are the chlorophylls, hemoglobin, and hemocyanin [17].

In general, chromophores comprise four pyrrole rings; identified as (i) open-chain pyrroles with no transition metal involved – typically, carotenoids, phycobilins, and phytochromes, (ii) pyrroles arranged as a porphyrin ring with a central transition metal atom – typically, chlorophylls and bacteriochlorophylls (C55H74MgN4O6). Chlorophyll absorbs all other visible components of light except green, which is the color the human eye sees of plants in their leaves. Various chlorophylls and accessory pigments (as discussed in sections 2.1–2.5) have characteristic *absorption spectra*; and the *action spectrum* that drives photosynthetic

**361**

hydrates [18].

**Figure 4.**

*[12–14].*

*Microalgae: The Multifaceted Biomass of the 21st Century*

reactions relates proportionately to the different wavelengths of light (see **Figure 5**).

On absorbing light energy, one or more of the following effects happen in a pigment: (i) light energy is transformed to heat energy, or (ii) there is fluorescence, signifying that light energy is re-emitted at longer wavelengths, or (iii) the quantum of **energy is passed** from an excited **molecule of chlorophyll to another molecule** in a process called **exciton** transfer, or (iv) the reaction centre (RC) chlorophyll absorbs the energy and gives up an excited electron to an electron acceptor and (v) the RC chlorophyll is unstable and wants to replace its missing electron, which creates concentration gradients, leading to the production of ATP and NADPH, which are fed into the Calvin cycle (see **Figure 6**) to produce carbo-

*The structure of the pigments: (a) xanthophyll (b) Fucoxanthin (c) Phycocyanin and (d) Phycoerythrin* 

*DOI: http://dx.doi.org/10.5772/intechopen.94090*

*Microalgae: The Multifaceted Biomass of the 21st Century DOI: http://dx.doi.org/10.5772/intechopen.94090*

*Biotechnological Applications of Biomass*

presence of chromophores [14] (**Figure 4**).

phyll b absorb 960 nm wavelength of light [15].

chlorophylls, hemoglobin, and hemocyanin [17].

**2.6 The Chromophore**

Phycoerythrin is an accessory pigment to the main chlorophyll pigment complex found in red algae and cryptophytes; it is part of a covalently bonded phycobilin chromophore in the family of phycobilins, typical of which is phycoerythrobilin, the phycoerythrin acceptor chromophore. Phycoerythrin is made up of (αβ) monomers aggregates. Except for phycoerythrin 545 (PE545), these monomer aggregates are assembled into (αβ)3 trimers or (αβ)6 hexamers with 3 or 32 symmetry and enclosing central channel [13, 14]. In red algae, they are attached to the stroma of thylakoid membranes of chloroplasts, whereas in cryptophytes, phycobilisomes are reduced and housed inside the lumen of thylakoids. Phycoerythrin captures light energy from the electromagnetic radiation and directs it to the reaction site through the phycobiliproteins, phycocyanin, and through A-PC. Each trimer and hexamer in the phycobilisome (PBS) has a minimum of one linker protein at the central channel. The α and β chains in B-phycoerythrin (B-PE) and R-phycoerythrin (R-PE) from the red algae also have γ sub-units conferring both link and light-capturing capabilities due to the

The chloroplast of algal cell contains the water-soluble phycobilin pigments and while the same phycobilin pigments are found in the phycocyanin and phycoerythrin of Cyanobacteria and the red algae, the Rhodophyta. The algal chlorophyll has a structural difference from Bacteriochlorophylls (Bchl) of cyanobacteria, the latter having one of the porphyrin rings saturated, and absorbing longer wavelengths of light as opposed to chlorophylls. *Rhodopseudomonas viridis* has its bacteriochloro-

The colors of pigments are the reflections of the electromagnetic spectrum from the pigments. A portion of the pigment molecule causes the formation of the color perceived, and this moiety is referred to as **chromophore**. A chromophore has two energy levels referred to as orbitals, and the difference in their energies lie within the visible spectrum of electromagnetic radiation. Thus, a photon of incident light can excite an electron from its ground-state orbital to the excited state. Chromophores are generally either **conjugated pi-systems** or transition **metal complexes** [16]. For **conjugated pi-systems,** electrons are excited between **pi-**orbitals distributed over alternating single and double bonds. Where conjugated systems are less than eight conjugated double bonds, absorbance occurs only in the ultraviolet region and is visible to the human eye. But blue or green compounds essentially do not rely on conjugated pi-bonds alone. Typical chromophores in this category are the azo compounds, lycopene, β-carotene, anthocyanin, and retinenes. **Metal complex chromophores** have transition metals whose d-orbitals are incomplete but are shared with the ligands. Chromophores in this category are the

In general, chromophores comprise four pyrrole rings; identified as (i) open-chain pyrroles with no transition metal involved – typically, carotenoids, phycobilins, and phytochromes, (ii) pyrroles arranged as a porphyrin ring with a central transition metal atom – typically, chlorophylls and bacteriochlorophylls (C55H74MgN4O6). Chlorophyll absorbs all other visible components of light except green, which is the color the human eye sees of plants in their leaves. Various chlorophylls and accessory pigments (as discussed in sections 2.1–2.5) have

characteristic *absorption spectra*; and the *action spectrum* that drives photosynthetic

**2.5 Phycoerythrin (PE)**

**360**

**Figure 4.** *The structure of the pigments: (a) xanthophyll (b) Fucoxanthin (c) Phycocyanin and (d) Phycoerythrin [12–14].*

reactions relates proportionately to the different wavelengths of light (see **Figure 5**). On absorbing light energy, one or more of the following effects happen in a pigment: (i) light energy is transformed to heat energy, or (ii) there is fluorescence, signifying that light energy is re-emitted at longer wavelengths, or (iii) the quantum of **energy is passed** from an excited **molecule of chlorophyll to another molecule** in a process called **exciton** transfer, or (iv) the reaction centre (RC) chlorophyll absorbs the energy and gives up an excited electron to an electron acceptor and (v) the RC chlorophyll is unstable and wants to replace its missing electron, which creates concentration gradients, leading to the production of ATP and NADPH, which are fed into the Calvin cycle (see **Figure 6**) to produce carbohydrates [18].

**Figure 5.** *Relative absorbance of photosynthetic pigments as a function of the wavelength of light [19].*
