Filtration is a physical separation process in which the particles in suspension are retained using a filter. The filters are highly efficient and safe in the solid-liquid separation process (Pires et al., 2012). The filtration is a separation method that is suitable for large microalgae

such as Spirulina but unsatisfactory for smaller cells such as Chlorella and Scenedesmus (Ho et al., 2011).

Filtration provides easy operation and construction, low investment, and insignificant abrasion. The filters can be operated under pressure or vacuum (Harun et al., 2010). The main limitation of filtration is the reduction of the permeation flow during the process; this is due to adsorption and concentration of the compounds on the membrane surface (Rossi et al., 2008).

3.4.1 Optimization of Light Conversion Efficiency (LHCB)

Optimization of light conversion efficiency (LCE) is another way to make microalgae-based biofuels cost-effective. LCE is defined by Ghirardi et al. (2009) as the "fraction of the energy content of the incident solar spectrum that is converted into chemical energy by the organ­ism." It has been known that sunlight intensities are much higher than those required to saturate photosynthesis. To avoid overexcitation of the photosystem, plants and green microorganisms deal with excess light by dissipating heat and emitting fluorescence. As a consequence, the realistic LCE converts solar energy to biomass is much lower than the theoretical calculation (Dismukes et al., 2008; Melis, 2009; Wijffels and Barbosa, 2010).

Another energy issue dealing with light efficiency is uneven distribution of light in a high — density cultivation system. For cells directly exposed to sunlight, up to 80% of the absorbed photons could be wasted due to dissipation of excitation by nonphotochemical quenching and photoinhibition of photosynthesis (Melis, 1999; Melis et al., 1999). On the other hand, cells underneath the culture are shaded from sunlight and have reduced photosynthesis rates.

To improve solar illumination distribution of the microalgal culture, mutants with reduced light-harvesting chlorophyll antenna sizes that would allow for efficient utilization of light energy, and therefore would increase productivity, have been proposed. The rationale of this approach is to minimize light absorption by cells on the surface and to permit greater sunlight penetrance into the deeper layers of the culture. This concept was experimentally validated by isolation and characterization of truncated light-harvesting chlorophyll antenna size (tla) mutants (Lee et al., 2002; Polle et al., 2000; Polle et al., 2003). Reduction of photosystem chlorophyll antenna size in tla mutants has been demonstrated to improve solar energy conversion efficiency and productivity. The notion has also been verified independently by an RNAi approach. Reduction of the light harvest complex I(LHCI) and LHCII antenna complex system by knocking down light harvest complex B major proteins results in improved photon capture efficiency, enhanced growth rate, and reduced photoinhibition (Mussgnug et al., 2007).

In summary, accumulated experimental evidence indicates optimization of light-capture efficiency by genetic engineering can be very useful to improve culture productivity. Designs integrating growth optimization and fuel production will be important to making microalgae-based fuel cost effective.

Microalgae are a potential source of fermentable substrate. According to the conditions of cultivation, microalgal biomass can provide high levels of carbon compounds. These com­pounds are available directly for fermentation or after pre-treatment and may be used for eth­anol production.

Biogas is the product of the anaerobic digestion of organic matter and can be obtained from domestic sewage, animal waste, solid waste, or aquatic biomass, such as macro- and microalgae (Omer and Fadalla, 2003; Gunaseelan, 1997). The type of digestion using microalgal biomass processes can eliminate the biomass harvesting and drying and the asso­ciated costs (Vonshak, 1997).

The fatty acids that microalgae produce can be converted into biodiesel, which is a renew­able, biodegradable, nontoxic, and environmentally friendly fuel. Biodiesel has the advantage that it emits 78% less carbon dioxide when burned, 98% less sulfur, and 50% of particulate matter emissions (Brown and Zeiler, 1993).

Another promising biofuel is hydrogen. Photobiological hydrogen production can be in­creased according to the carbon content in the biomass. The microalgae are candidates for such a process because they produce hydrogen under certain conditions and can be grown in closed systems, allowing the capture of hydrogen gas (Benemann, 1997). This biomass can be burned to produce energy because the calorific value of these microorganisms is greater than that of some charcoals.

Botryococcus is a colonial microalga that is widespread in fresh and brackish waters of all con­tinents. It is characterized by its slow growth and by containing up to 50% by weight of hydro­carbons. B. braunii is classified into A, B, and L races, mainly based on the difference between the hydrocarbons produced (Metzger and Largeau, 2005). Banerjee et al. (2002) differentiate the races as follows: Race A produces C25 to C31 odd-numbered n-alkadienes and alkatrienes; B race pro­duces polymethylated unsaturated triterpenes, called botryococcenes (CnH2n-10, n = 30-37); and L race produces a single tetraterpene hydrocarbon C40H78 known as lycopadiene.

The cells of B. braunii are embedded in a communal extracellular matrix (or "cup"), which is impregnated with oils and cellular exudates (Banerjee et al., 2002). B. braunii is capable of synthesizing exopolyssaccharides, as reported by Casadevall et al. in 1985. Higher growth and production of EPS, which ranges from 250 g m-3 for A and B races to 1 kg m-3 for the L race, occur when nitrate is the nitrogen source instead of urea or ammonium salts (Banerjee et al., 2002). Phosphorus and nitrogen are also important factors in accumulation of hydrocarbons by the microorganism (Jun et al., 2003).

The metabolic energy devoted to produce such large amounts of hydrocarbons makes this species noncompetitive in open mass cultures, since strains not so burdened can grow much faster and soon dominate an outdoor pond culture (Benemann et al., 2002). B. braunii has been reported to convert 3% of the solar energy to hydrocarbons (Gudin and Chaumont, 1984). Being synthesized by a photosynthetic organism, hydrocarbons from algae can be burned without contributing to the accumulation of CO2 in the atmosphere.

Dayananda et al. (2007) cultivated Botryococcus braunii strain SAG 30.81 in shake flasks and obtained a maximum cell concentration of 0.65 g L-1 under 16:8 light:dark cycle. Experiments with different strains of B. Braunii indicate that the biomass yield is inversely proportional to lipid accumulation. The maximum biomass yield achieved was 2 g L-1 (with 40% of lipids) and the lower was 0.2 g L-1 (with 60% of lipids). Outdoor experiments with this microalga achieved a high biomass yield of 1.8 gL-1 but a very low lipid accumulation. It was also showed by Dayananda and collaborators that exopolyssaccharides production by Botryococcus braunii SAG 30.81 is not affected by light regimen in MBM media, different from lipids and proteins pro­duction. Sydney et al. (2011) carried experiments with this same strain under 12 h light: dark cycle in 5% CO2 enriched air and achieved a high biomass production of3.11gL-1 with 33% lipids in 15 days. Carbon dioxide fixation rate was calculated as near 500 mg L-1 day-1. B. braunii biomass composition also included 39% proteins, 2.4% carbohydrates, 13% pigments, and 7.5% ash.

Marukami and Ikenouochi (1997) achieved a carbon dioxide fixation greater than 1 gram per liter by Botryococcus braunii cultivated for hydrocarbon accumulation.

The light spectrum and intensity are factors that directly affect the performance of phototrophic microalgal growth, both indoors and outdoors. In outdoor cultures, sunlight is the major energy source, whereas innovations in artificial lighting, such as light-emitting diodes (LED) and optical fiber, are interesting for indoor cultivation systems. In indoor cul­tures, the biggest challenge is the high cost of artificial lighting (Chen et al., 2011).

Regardless of the light source, its usage by microalgae occurs in the same way. In a photo­synthetic system, 8 photons of radiation are required to fix one CO2 molecule in the form of carbohydrate; this results in the maximum photosynthetic efficiency (Chini-Zittelli et al., 2006).

Multiproteic complexes, also called photosystems, catalyze the conversion reaction of light energy captured by excited molecules of chlorophyll into the form of usable energy. A photosystem consists of a center of photochemical reaction consisting of a protein com­plex, and molecules of chlorophyll that enable the conversion of light energy into chemical energy. This photosystem also has an antenna complex consisting of pigment molecules that capture light energy and feed the reaction center. The antenna complex is important for the capture of light. In chloroplasts, it consists of a cluster of hundreds of chlorophyll molecules held together by proteins that keep them firmly together on the thylakoid mem­brane (Alberts et al., 2008).

When a chlorophyll molecule from the antenna complex is excited, the energy is rapidly transmitted from one molecule to another through a resonance energy transfer process until it reaches a special pair of chlorophyll molecules from the center of the photochemical reaction. Each antenna complex acts like a funnel collecting light energy and directing it to a specific site where it can be used effectively (Alberts et al., 2008). One strategy to optimize the utili­zation of light is to reduce the size of the antenna, which makes the cells less opaque and facilitates the transmission of light (Chen et al., 2011).

Several studies have been developed to improve the efficiency of light utilization and re­duce the costs of systems with artificial lighting. The advantage of cultivation in a laboratory is that is uses fluorescent tubes. Although they consume high amounts of energy, that usage can be reduced by more than 50% with the use of LEDs. Many cultures use only solar energy as a light source, which has no cost. However, the performance of outdoor systems is lower than indoor ones, and they require large areas of land (Chen et al., 2011).

Raceway ponds are a modified version of the open pond system that has a different flow pattern compared to that of the simple pond. In raceways, the water flow direction is con­trolled by the rotation speed of paddlewheels, in contrast to only coaxial mixing in conven­tional open ponds. Therefore, in the raceway systems, the microalgae, water, and nutrients are continuously circulated around a racetrack, following the same direction as a paddlewheel. In this way, the circulation rate around the racetrack can be adjusted by the paddle speed. With paddlewheels providing the driving force for liquid flow, the microalgae are kept suspended in the water and are circulated back to the surface on a regular frequency.

Despite their diversified appearance, the most common raceway cultivators are driven by paddlewheels and are usually operated at a water depth of 15-20 cm. The raceways are usu­ally operated in a continuous mode with constant feeding of CO2 and nutrients into the sys­tem while the microalgae culture is removed at the end of the racetrack. This operation is quite similar to that of plug-flow reactors (PFRs) used in the chemical industry.

The same drawbacks observed in the operation of open ponds are also found in raceways. Furthermore, the requirement of large areas for microalgae cultivation is considered the bar­rier for commercialization of microalgae processes. Nevertheless, control of environmental factors (such as mixing) in raceways is easier than in conventional open ponds, making the use of raceways for the cultivation of microalgae more attractive.

Flotation is a separation process in which air or gas bubbles are directed at the solid par­ticles and then drive these particles to the liquid surface. Flotation is more beneficial and efficient for removing cells than sedimentation. Flotation can capture particles smaller than 500 pm in diameter (Chen et al., 2011).

According to the bubble size used in the process, the application can be divided into dissolved air flotation and dispersed flotation. In dissolved air flotation, the application of reduced pressure produces bubbles of 10-100 pm. This process is influenced by the tank pres­sure, rate of recycling, hydraulic retention time, and particle flotation rate (Uduman et al.,

2010) . In dispersed air flotation, bubbles of 700-1,500 pm are formed by the high-speed mechanical stirrer with an air injection system (Rubio et al., 2002).

To enhance the economics of microalgae-based biofuels, utilization of every ingredient of the raw biomass is important (Georgianna and Mayfield, 2012; Sheehan et al., 1998). Whereas the majority of fuels derived from microalgae have been focused on storage oils, the extracted oil accounts for only 37.9% of the energy and 27.4% of the initial fixed carbon (Lardon et al.,

2009) . The remaining carbon is stored in the leftover oil cakes composed of abundant proteins and carbohydrates. Hence, recycling these nutrient elements may help increase biomass margins of microalgae-based fuels (Lardon et al., 2009). Recycling algal waste by anaerobic digestion has been proposed to support the microalgae production process (Ras et al., 2011; Zamalloa et al., 2012).

Several innovative metabolic engineering strategies have been proposed recently to reduce the energy debt and increase the margins of microalgae-based fuels. One of the approaches is to establish an integrated system that takes advantage of the amenable genetic modification capability of the Escherichia coli (E. coli) system. Although microalgae can grow photosynthet­ically to accumulate biomass for biodiesel purposes, the leftover paste can be utilized for alcohol-fuel production by feeding it into an engineered bacterial system. Huo et al. accom­plished this by genetically engineering an E. coli strain that is capable of converting the back­bone and side chains of amino acids in pretreated biomass into two-, four — and five-carbon alcohol fuels, ammonia, and other chemicals (Huo et al., 2011). In a small-scale experiment, the authors successfully converted hydrolyzed microalgal protein biomass into alcohol fuels. This demonstration supports the potential of using microalgal biomass as a feedstock for protein-based biorefinaries.

The greatest issue in agriculture nowadays is the availability of chemical fertilizers at af­fordable costs. Nitrogen fixation has been acknowledged as the limiting factor in food pro­duction. The concept of using cyanobacteria to fix nitrogen is based on the ability of these microalgae to grow in soil.

The microalgae Nostoc, Anabaena, Oscillatoria, Cylindrospermun, and Mastigocladus Tolypothrix form heterocysts and can fix nitrogen aerobically. Nonheterocyst-forming fila­mentous microalgae, such as Oscillatoria and Phormidium, can fix nitrogen in the absence of oxygen and in the presence of nitrogen and carbon dioxide. Filamentous forms without heterocysts, such as Trichodesmium, may fix nitrogen aerobically (Richmond, 1990).

The heterocysts, which are specialized in aerobic nitrogen fixation, are the site of the en­zyme nitrogenase, which catalyzes the conversion of nitrogen into ammonia. Nitrogen-fixing cyanobacteria were isolated in soils from various cities in South Asia, India, and Africa. In that study, 33% of 2,213 soil samples collected in India contained cyanobacteria. Microalgae such as Nostoc, Anabaena, Calothrix, Aulosira, and Plectonema were found in soils in India, while Halosiphon, Scytonema and Cylindrospermum were observed in the other regions (Richmond, 1990).

1.2 CONCLUSION

Open ponds are the most widely used reactors in the world for large-scale microalgal cul­tures. This is due to the low construction cost, low power demand, appropriate scale-up, and their easy cleaning process compared to closed photobioreactors. The cultures that are grown in open ponds can be protected from adverse environmental conditions (rainfall, tempera­ture, and luminosity) through the use of a greenhouse. Microalgae that grow in extreme conditions, such as an alkaline medium and high salinity, should be adopted in order to achieve axenic cultures. The obtained microalgal biomass can be used in the production of food, drugs, biopigments, biopolymers, biofuels, and biofertilizers.

Spirulina are multicellular ilamentous cyanobacteria actually belonging to two separate genera: Spirulina and Arthrospira. These encompass about 15 species (Habib et al., 2008). This microorganism grows in water, reproduces by binary fission, and can be harvested and processed easily, having significantly high macro — and micronutrient contents. Their main photosynthetic pigments are chlorophyll and phycocyanin. The helical shape of the filaments (or trichomes) is characteristic of the genus and is maintained only in a liquid environment or culture medium.

Spirulina is found in soil, marshes, freshwater, brackish water, seawater, and thermal springs. Alkaline, saline water (>30 g/L) with high pH (8.5-11.0) favors good production of Spirulina, especially where there is a high level of solar radiation. It predominates in higher pH and water conductivity. Like most cyanobacteria, Spirulina is an obligate photoautotroph,

i. e., it cannot grow in the dark on media containing only organic carbon compounds. It reduces carbon dioxide in the light and assimilates mainly nitrates.

Spirulina contains unusually high amounts of protein, between 55% and 70% by dry weight, depending on the source. It has a high amount of polyunsaturated fatty acids (PUFAs), 30% of its 5-6% total lipids, and is a good source of vitamins (B1, B2, B3, B6, B9, B12, C, D, E). Spirulina is a rich source of potassium and also contains calcium, chromium, copper, iron, magnesium, manganese, phosphorus, selenium, sodium, and zinc. These bacteria also contain chlorophyll a and carotenoids.

The optimum pH of the Spirulina sp. culture is between 8.5 and 9.5 (Watanabe et al., 1995). Cyanobacteria possess a CO2-concentating mechanism that involves active CO2 uptake and HCO — transport. In experiments conducted by Morais and Costa (2007), carbon fixation in terms of biomass by Spirulina platensis was estimated in 413 mg L-1 d-1, near those achieved by Sydney et al. (2011).