Microalgae

Microalgae covers all unicellular and simple multi-cellular microorganisms, including both prokaryotic microalgae and eukaryotic microalgae (Scott et al., 2010). Algae can either be autotrophic or heterotrophic; the former require only inorganic compounds such as CO2, salts and a light energy source for growth; while the latter are non-photosynthetic therefore require an external source of organic compounds as well as nutrients as an energy source. For autotrophic algae, photosynthesis is a key component of their survival, whereby they convert solar radiation and CO₂ absorbed by chloroplasts into adenosine triphosphate (ATP) and O₂

the usable energy currency at cellular level, which is then used in respiration to produce energy to support growth (Falkowski and Raven, 1997).

Microalgae, just like plants, are photosynthetic microorganisms which convert sunlight, CO2 and water to biomass, potential biofuels, foods, feeds and high-value bioactives

(Borowitzka, 1999; Banerjee et al., 2002; Walter et al., 2005; Spolaore et al., 2006; Chisti, 2007). Microalgae are responsible for over 50% of primary photosynthetic productivity on earth and are budding sunlight factories for a wide range of potentially useful products, but are scarcely used commercially (Gavrilescu and Chisti, 2005; Wijffels, 2007). The large-scale cultivation of microalgae and the use of its biomass for the production of useful products were first considered seriously in Germany during World War II (Becker, 1994).

Microalgae have the potential to develop biotechnology in a number of areas including nutrition, aquaculture, pharmaceuticals, and biofuels. Microalgae produce many valuable substances such as vitamins and color pigments, essential fatty acids, amino acids, and even antibiotics and pharmaceutically-active substances, such as high-quality food, food

supplements or alternatives for synthetic substances in the cosmetics and chemical industry.

Microalgae has the wide range of benefits in producing valuable chemicals or healthy foods, vitamins and as feedstock for animals on land and in aquaculture (Pulz and Gross, 2004;

Spolaore et al., 2006; Raja et al., 2008), consume waste and the metallic pollutants in

wastewater (Perales-Vela et al., 2006; Jácome-Pilco et al., 2008) and produce biodiesel (Chisti, 2007; Hossain et al., 2008). Microalgae convert CO2 into biomass and use CO2 efficiently.

Therefore, microalgae are cultivated at large-scale outdoor for the purpose of industrialization.

In the reason of depleting supplies and the contribution of petroleum or natural fossil fuels to the accumulation of CO2 in the environment, continued use of these fuels is now widely recognized as unsustainable. Renewable, carbon neutral, transport fuels are necessary for environmental and economic sustainability. Biodiesel (monoalkyl esters) is nontoxic and less emissions of CO2, sulfur oxides (SOX)and nitrogen oxides (NOX), and it is biodegradable and renewable as well as environmentally safe (Ma and Hanna, 1999). Biodiesel derived from oil crops is a potential renewable and carbon neutral alternative to petroleum fuels.

Unfortunately, biodiesel from oil crops, waste cooking oil and animal fat cannot realistically satisfy even a small fraction of the existing demand for transport fuels. Microalgae appear to be the only source of renewable biodiesel that is capable of meeting the global demand for transport fuels. Like plants, microalgae use sunlight to produce oils but they do so more

efficiently than crop plants. Oil productivity of many microalgae greatly exceeds the oil productivity of the best producing oil crops (Chisti, 2007). Table 1 shows comparison of microalgae with other biodiesel feedstocks. Moreover, microalgal biomass can be used to produce biofuel by pyrolysis, direct combustion or thermal chemical liquefaction (Mata et al., 2010). The lipid fraction of microalgal biomass can be extracted and transesterified for

biodiesel production (Li et. al, 2008; Brennan and Owende, 2010; Lee et al., 2010).

Although algae have been commercially cultivated for over 50 years, metabolic engineering now seems necessary in order to achieve their full processing capabilities.

Recently, the development of a number of transgenic algal strains boasting recombinant protein expression, engineered photosynthesis, and enhanced metabolism encourage the prospects of designer microalgae (Rosenberg et al., 2008).

1.2.1 Chlorella

Chlorella species are encountered in all water habitats exhibiting a cosmopolitan

occurrence, having been isolated from widely differing fresh, as well as marine, water habitats.

The species of the genus Chlorella have simple life cycles and nutritional requirements.

Classification is complex because Chlorella species cannot be readily discerned on the basis of morphological features, the taxonomy of Euchlorella, which comprises the most common species is, therefore, incomplete. It has indeed been proposed to use physiological and

biochemical rather than morphological criteria, for species identification. On the basis of their external morphlogy, Chlorella species could nevertheless be placed in four general groups: (1) spherical cells (ratio of the two axes equals one); (2) ellipsoidal cells (ratio of the longest axis to the shortest axis being 1.45 to 1.60); (3) spherical or ellipsoidal cells; (4) globular to subspherical cells (Richmond, 1986). In reproduction, which is exclusively asexual, each mature cell divides usually producing four or eight (and more rarely, 16) autospores, which are freed by rupture or dissolution of the parental walls.

1.2.2 Microalgal Physiology

Autotrophic organisms obtain their energy through the absorption of light energy for the reduction of CO₂ by the oxidation of substrate, mainly water, with the release of O₂.

Photoautotrophic organisms only require inorganic mineral ions and obligate photoautotrophs

are those that cannot grow in the dark. By far, most algae belong to this category, although many require minimal quantities of organic compounds for growth, such as vitamins.

For high rates of autotrophic production, supply of CO2 and HCO3

is most important.

Contrary to land plants, atmospheric CO2 cannot satisfy the C-requirements of high yielding autotrophic algal production systems. The CO2-H2CO3- HCO3-CO3

system is the most important buffer generally present in culture broth and it is the best means available to control and maintain specific pH levels that are optimal for mass-cultivated species. The

bicarbonate-carbonate buffer system can provide CO2 for photosynthesis. The buffer system reactions imply that during photosynthetic CO2 fixation, OH^- accumulates in the growth solution leading to a gradual rise in pH. pH-static control via direct CO2 sparging into the culture media is the best and most convenient method for pH control and at the same time supplying CO₂ for high yield in mass algal cultures. Since active photosynthesis results in an increase in pH, the opposite is true for CO₂ release during respiration. The overall influence is little since as a general rule dark respiration is less than 10% of total photosynthetic

production (Grobbelaar and Soeder, 1985).

After carbon, nitrogen is the most important nutrient contributing to the biomass produced. The nitrogen content of the biomass can range from 1% to more than 10% and it not only varies between different groups but within a particular species, depending on the supply and availability. Typical responses to nitrogen limitation is discoloration of the cells and accumulation of organic carbon compound such as polysaccharides and certain oils (Becker, 1994). Nitrogen is mostly supplied as nitrate (NO3

-), but often ammonia (NH4+

) and urea are also used, with similar growth rates recorded. Ammonia nitrogen is often the

preferred N-source for microorganisms and the assimilation of either NO3

or NH4+

is related to the pH of the growth media. When ammonia is used as the sole source of N, the pH could drop significantly during active growth, due to the release of H⁺ ions. An increase in pH occurs when deciding whether to supply either nitrate or ammonia, is that the latter could be lost form the growth media due to volatilization, particularly when the pH increases.

Sulfur is generally present in small quantity in all plant cells but is probably not a limiting factor for many algae under normal conditions. Sulfur is incorporated into numerous organic compounds and sulfates are present in the vacuoles. As compared with other

macronutrients sulfur uptake and metabolism in algae have been studied only scarcely. In fact, major studies on sulfur assimilation by algae were done more than 20 years ago and present research in this field equals nearly nothing. Uptake sulfur by both Chlorella pyrenoidosa and

Scenedesmus sp. is stimulated by light (Kylin, 1961; Tseng et al., 1971). As with N

assimilation, light could be acting by providing energy via photophosphorylation, reductant, or C skeletons. A large part of the sulfur in most algae is incorporated into protein. Two sulfur-containing amino acids, cysteine and methionine, are important in maintaining the three-dimensional configuration of proteins through sulfur bridges. Incorporation of sulfur from sulfate in the medium into normal cells of Scenedesmus was enhanced by light relatively most in the case of lipid S and least in the inorganic sulfate fraction; the effects of light were, generally, increased by the presence of CO2 and nitrogen salts.