**6. Classification of microalgae**

Microalgae were among the first life forms on earth [34]. As prefix "micro" mean small, so microalgae are very small in their size (in size of micrometers).Microalgae known as unicellular organisms because it has one cell. Microalgae can make their own energy and this energy is stored in the cell. They are capable of fixing large amounts of carbon dioxide (CO2) while contributing to approximately 40 percent to 50 percent of the oxygen in the atmosphere thereby helping to support the majority of life on our planet.

Microalgae are highly productive on a global scale, with cell doublings of 1-4 per day. While microalgae make up only 0.2 percent of global biomass generated through photosynthesis, they account for approximately 50 percent of the total global fixed organic carbon [36]. Microalgae, like terrestrial plants, grow and multiply through photosynthesis, a process whereby light energy is converted into chemical energy by ―fixing atmospheric CO2.



**Table 3.** Lipid content and productivities of different microalgae species [111]

Microalgae, like terrestrial plants, grow and multiply through photosynthesis, a process

**Lipid productivity (mg/L/day)**

**Volumetric productivity of biomass (g/L/day)**

**Areal productivity of biomass (g/m2 /day)**

whereby light energy is converted into chemical energy by ―fixing atmospheric CO2.

**Ankistrodesmus sp.** 24.0–31.0 – – 11.5–17.4 **Botryococcus braunii** 25.0–75.0 – 0.02 3.0

**Chaetoceros calcitrans** 14.6–16.4/39.8 17.6 0.04 – **Chlorella emersonii** 25.0–63.0 10.3–50.0 0.036–0.041 0.91–0.97 **Chlorella protothecoides** 14.6–57.8 1214 2.00–7.70 – **Chlorella sorokiniana** 19.0–22.0 44.7 0.23–1.47 – **Chlorella vulgaris** 5.0–58.0 11.2–40.0 0.02–0.20 0.57–0.95 **Chlorella sp.** 10.0–48.0 42.1 0.02–2.5 1.61–16.47/25 **Chlorella pyrenoidosa** 2.0 – 2.90–3.64 72.5/130 **Chlorella** 18.0–57.0 18.7 – 3.50–13.90 **Chlorococcum sp.** 19.3 53.7 0.28 – **Crypthecodinium cohnii** 20.0–51.1 – 10 – **Dunaliella salina** 6.0–25.0 116.0 0.22–0.34 1.6–3.5/20–38 **Dunaliella primolecta** 23.1 – 0.09 14 **Dunaliella tertiolecta** 16.7–71.0 – 0.12 – **Dunaliella sp.** 17.5–67.0 33.5 – – **Ellipsoidion sp.** 27.4 47.3 0.17 – **Euglena gracilis** 14.0–20.0 – 7.70 – **Haematococcus pluvialis** 25.0 – 0.05 – 0.06 10.2–36.4 **Isochrysis galbana** 7.0–40.0 – 0.32–1.60 – **Isochrysis sp.** 7.1–33 37.8 0.08–0.17 – **Monodus subterraneus** 16.0 30.4 0.19 – **Monallanthus salina** 20.0–22.0 – 0.08 12 **Nannochloris sp.** 20.0–56.0 60.9–76.5 0.17–0.51 – **Nannochloropsis oculata.** 22.7–29.7 84.0–142.0 0.37–0.48 – **Nannochloropsis sp.** 12.0–53.0 37.6–90.0 0.17–1.43 1.9–5.3 **Neochloris oleoabundans** 29.0–65.0 90.0–134.0 – – **Nitzschia sp.** 16.0–47.0 8.8 – 21.6 **Oocystis pusilla** 10.5 – – 40.6–45.8

**Lipid content (% dry weight biomass)**

**Chaetoceros muelleri** 33.6 21.80 0.07

**Marine and freshwater microalgae species**

110 Biofuels - Status and Perspective

**Figure 1.** (a). Scanning electron micrograph of a microalgae (Chlorella)[47]; (b).Cyanobacteria range from simple uni‐ cellular organisms to colonies [122]

Over 40,000 separate species of algae have been identified, and that number almost certainly represents a small fraction of the true population (perhaps as high as 10,000,000 different species [55]. Because of the diverse nature of algae, it has been difficult to settle on a universally accepted classification system. For example, some experts will exclude cyanobacteria because of their simple cellular structure relative to other classes of algae. Others will focus on a separation of unicellular (microalgae) and multicellular (macroalgae).

Much of the classification of algae depends upon photosynthetic pigments, whole organism morphology, cellular anatomy and ultrastructure, and metabolism and physiology. The biological divisions that encompass the various classes of algae are;


Of these classes, those that produce significant amounts of lipids are considered to be of interest for the production of Biofuels. Macroalgae typically require deep bodies of water for growth, and generally are viewed to lack the potential to make a significant contribution to the world's future liquid transportation fuel needs. Notwithstanding this view macroalgae production is increasing and there is interest in the EU and Japan in its use as a feedstock for methane production by anaerobic digestion and ethanol production by saccharification and fermenta‐ tion.

Most of the algae known to produce more than 20% of their biomass as lipids fall into the divisions *Cryptophyta*, *Chlorophyta*, and *Chromophtya*. Cryptomonads are biflagellate unicellu‐ lar algae carrying the photosynthetic pigments chlorophyll a and c, α-carotene and β-carotene giving them the colours green, olive, brown, yellow, red, or blue. They are found in waters ranging from fresh to hypersaline, sometimes in great abundance. *Rhodomonas salina* (also known as *Chroomonas salina)* is a cryptomonad known to produce lipids at high levels.

Chlorophyta or green algae range from unicellular forms to large seaweeds. Their photosyn‐ thetic pigments are similar to those in higher plants and include chlorophyll a and b, α-, β-, and γ-carotene, and various xanthophylls. Their cell walls contain cellulose and they often use starch as an energy reserve (attributes of potential feedstocks for ethanol production). *Chlamydomonas reinhardtii,* a chlorophyte, was selected as a model system for the study of plants, and is one of the few algae whose entire gene sequence is known. *C. reinhardtii* can grow autotrophically on a simple medium of inorganic salts and in the presence light and CO2, but can also grow heterotrophically in total darkness using acetate as a carbon source and O2.

Several Chlorophytes are known to produce high levels of lipids including *Botryococcus braunii, Chlorella vulgaris, Neochloris oleoabundans,* and *Nannochloris sp.*The chromophyta contain chlorophyll a and b, α-, β-, and γ-carotenes, zeaxanthin and several other xanthophylls. They comprise many different classes of algae including the *Chrysophyceae* (golden-brown algae), *Bacillariophycease* (diatoms), *Xanthophyceae* (yellow-green algae), *Eustigmatophyceae*, and *Prymnesiophyceae*. Examples of each of these classes are known to produce high levels of lipids including *Ochromonas danica, Phaeodactylum tricornutum, Nitzschia palea, Monallantus salina, Nannochloropsis sp.,* and *Isochrysis sp.*

Unlike the other divisions of algae, cyanobacteria or blue green algae is prokaryotic, that is, they lack nuclei and are members of the bacterial kingdom. They contain many different photosynthetic pigments including chlorophyll a and d, phycobilins, β-carotene, zeaxanthin, and other xanthophylls, and phycobilins. Although a *Nostoc* commune has been shown to produce triacylglycerides, cyanobacteria rarely produce more than 20% of their cell weight as lipids, but they will be included in this discussion because they have been shown to accumulate high levels of glycogen (as much as 60% of dry weight) as a storage material, and it is possible to divert the carbon flux from carbohydrate production to lipid production. In addition, cyanobacteria have long-established commercial production methods (mainly for food supplements and nutraceuticals) and genetic techniques have been developed for many different strains.

According to size, color/ pigment, shape, lifecycle and their cellular structure, microalgae are classified in four classes as abundant in below table 4.

### **6.1. Diatoms (Bacillariophyceae)**

of their simple cellular structure relative to other classes of algae. Others will focus on a

Much of the classification of algae depends upon photosynthetic pigments, whole organism morphology, cellular anatomy and ultrastructure, and metabolism and physiology. The

Of these classes, those that produce significant amounts of lipids are considered to be of interest for the production of Biofuels. Macroalgae typically require deep bodies of water for growth, and generally are viewed to lack the potential to make a significant contribution to the world's future liquid transportation fuel needs. Notwithstanding this view macroalgae production is increasing and there is interest in the EU and Japan in its use as a feedstock for methane production by anaerobic digestion and ethanol production by saccharification and fermenta‐

Most of the algae known to produce more than 20% of their biomass as lipids fall into the divisions *Cryptophyta*, *Chlorophyta*, and *Chromophtya*. Cryptomonads are biflagellate unicellu‐ lar algae carrying the photosynthetic pigments chlorophyll a and c, α-carotene and β-carotene giving them the colours green, olive, brown, yellow, red, or blue. They are found in waters ranging from fresh to hypersaline, sometimes in great abundance. *Rhodomonas salina* (also known as *Chroomonas salina)* is a cryptomonad known to produce lipids at high levels.

Chlorophyta or green algae range from unicellular forms to large seaweeds. Their photosyn‐ thetic pigments are similar to those in higher plants and include chlorophyll a and b, α-, β-, and γ-carotene, and various xanthophylls. Their cell walls contain cellulose and they often use starch as an energy reserve (attributes of potential feedstocks for ethanol production). *Chlamydomonas reinhardtii,* a chlorophyte, was selected as a model system for the study of plants, and is one of the few algae whose entire gene sequence is known. *C. reinhardtii* can grow autotrophically on a simple medium of inorganic salts and in the presence light and CO2, but can also grow heterotrophically in total darkness using acetate as a carbon source and O2.

separation of unicellular (microalgae) and multicellular (macroalgae).

biological divisions that encompass the various classes of algae are;

**•** Cyanophyta (cyanobacteria)

**•** Rhodophyta (red algae)

**•** Cryptophyta (cryptomonads)

**•** Pyrrophyta (dinoflaggellates), and

**•** Chromophyta (heterokonts)

**•** Chlorophyta (green algae)

**•** Chloroarachniophyta

**•** Prochlorphyta

112 Biofuels - Status and Perspective

**•** Glaucophyta

**•** Euglenophyta

tion.

Diatoms (Bacillariophyceae) are a type of algae. Mainly diatoms are unicellular but have different in shape such as stars, zigzag, ribbons, fans, spheres, elliptical and triangles when they exists as colonies. Carbon is stored in the form of oil in Diatom. This oil and water current helps them to move within the water to find their food and nutrient. Diatom cells have a unique feature is that, they are enclosed within a cell wall made of silica which is called a frustules. This silica is used to protect the cell.


**Table 4.** Microalgae Classification

**Figure 2.** Microalgal Biodiversity (diatoms and green algae) [105]

### **6.2. Green Algae (Chlorophyceae)**

Green Algae (Chlorophyceae) can be unicellular or colonial, generally it found quite abundant in fresh water. They have flagella (tails) attached to each cell, they use these flagella to swim. They include some of the most common species, as well as many members that are important both ecologically and scientifically. There are approximately 350 genera and 2650 living species of chlorophyceans. They come in a wide variety of shapes and forms, including free-swimming unicellular species, colonies, non-flagellate unicells, filaments, and more. They also reproduce in a variety of ways, though all have a haploid life-cycle, in which only the zygote cell is diploid. The zygote will often serve as a resting spore, able to lie dormant though potentially damaging environmental changes such as desiccation.

The Chlorophyceae includes three major groups distinguished primarily by basic differences in the arrangement of their flagellae:


### **6.3. Blue Algae (Cyanophyceae)**

Blue Algae (Cyanophyceae) grow in both fresh and salt waters of dams, rivers, creeks, reservoir, lakes. Blue Algae are a type of bacteria but due to some ways it act like plant by using to manufacture carbohydrates from carbon dioxide and water and release oxygen, through a process of photosynthesis [105].

**Figure 3.** (a) Green Algae [112] Figure (b) Blue Green Algae [112]

### **6.4. Golden Algae (Chrysophyceae)**

**Figure 2.** Microalgal Biodiversity (diatoms and green algae) [105]

Green Algae (Chlorophyceae) can be unicellular or colonial, generally it found quite abundant in fresh water. They have flagella (tails) attached to each cell, they use these flagella to swim. They include some of the most common species, as well as many members that are important both ecologically and scientifically. There are approximately 350 genera and 2650 living species of chlorophyceans. They come in a wide variety of shapes and forms, including free-swimming unicellular species, colonies, non-flagellate unicells, filaments, and more. They also reproduce in a variety of ways, though all have a haploid life-cycle, in which only the zygote cell is diploid. The zygote will often serve as a resting spore, able to lie dormant though potentially damaging

The Chlorophyceae includes three major groups distinguished primarily by basic differences

**•** *Volvocales, Chaetophorales, & Chlorococcales* - together make up more than half of all chloro‐ phyceans. Members of these orders have an offset flagellar arrangement (1 o'clock-7 o'clock).

**•** *Chlorellales* - Members of this order have opposed flagellae (12 o'clock-6 o'clock), though some have only vestigial flagellae and so have not been definitively associated with this

**•** *Oedogoniales* - Members of this smallest group have a complex multiflagellate crown on their swimming spores. All are filamentous, oogamous, and have net-like chloroplasts.

Blue Algae (Cyanophyceae) grow in both fresh and salt waters of dams, rivers, creeks, reservoir, lakes. Blue Algae are a type of bacteria but due to some ways it act like plant by using to manufacture carbohydrates from carbon dioxide and water and release oxygen,

group. Similarities with members of the Chlorococcales make distinctions difficult.

**6.2. Green Algae (Chlorophyceae)**

114 Biofuels - Status and Perspective

environmental changes such as desiccation.

in the arrangement of their flagellae:

**6.3. Blue Algae (Cyanophyceae)**

through a process of photosynthesis [105].

Golden Algae (Chrysophyceae), similar to diatoms in pigment and biochemical composition, are mostly found in fresh water. A single species "Prymnesium parvum" are referred as Golden The chrysophyceans (golden algae) are heterokontophyte algae with golden chloro‐ plasts. Many chrysophycean algae are unicellular, but colonial or simple multicellular species are also known. The chrysophycean algae are basically autotrophic but there are many mixotrophic & colorless heterotrophic species. Heterotrophic chrysophyceans such as *Spumella and Paraphysomonas* play an important role as lower consumers. The chrysophycean algae mainly inhabit in freshwater, but some species (especially heterotrophs) are common in marine.

**Figure 4.** a: Dinobryon b: Uroglena [82]

The cells are naked or covered by scales, lorica or cell wall. The flagellate cell usually possesses two heterodynamic flagella but posterior (a) flagellum is sometimes reduced. Tubular mastigonemes on anterior (b) flagellum possess lateral filaments. Mixotrophic and heterotro‐ phic species engulf particles (e.g. bacteria) through splitted R2 microtubules. Because major photosynthetic carotenoid is fucoxanthin, chrysophycean chloroplasts are golden-yellow in color. Asexual reproduction by means of binary fission, sporogenesis etc. Sexual reproduction has been reported in some species. The chrysophycean algae produce cysts surrounded by siliceous wall, statospore via sexual or asexual reproduction. Statospores form microfossils to be used for paleoenvironmental reconstruction.
