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.

A process of flocculation followed by gravity sedimentation for algae separation has been studied (Golueke and Oswald, 1965). Treating high rate oxidation pond effluent, the process achieved up to 85% of the algal biomass using alum as a coagulant. The process was found reliable, and various algae species could be separated to achieve an algae slurry of 1.5% solids content. A comparison of the flocculation-sedimentation process with the flocculation — flotation method indicated that the latter exhibited very clear optima operating conditions for algae separation (Friedman et al., 1977; Moraine et al., 1980).

Algae are diverse group of organisms that inhabit a vast range of ecosystems, from the ex­tremely cold (Antarctic) to extremely hot (desert) regions of the Earth (Guschina and Harwood, 2006; Round, 1984). Algae account for more than half the primary productivity at the base of the food chain (Hoek et al., 1995). Lipid metabolism (the biosynthetic pathways of fatty acids and triacylglycerol, or TAG synthesis), particularly in algae, has been less stud­ied than in higher plants (Fan et al., 2011). Based on the sequence homology and some shared biochemical characteristics of a number of genes and/or enzymes isolated from algae and higher plants that are involved in lipid metabolism, it is generally believed that the basic path­ways of fatty acid and TAG biosynthesis in algae are directly analogous to higher plants (Fan et al., 2011). The de novo synthesis of fatty acids in algae occurs primarily in the thylakoid and stromal region of the chloroplast (Liu and Benning, 2012). Algae fix CO2 during the day via photophosphorylation (thylakoid) and produce carbohydrate during the Calvin cycle (stroma), which converts into various products, including TAGs, depending on the species of algae or specific conditions pertaining to cytoplasm and plastid (Liu and Benning,

2012) . Microalgae are proficient at surviving and functioning under phototrophic or hetero­trophic conditions or both. A schematic illustration of algal-based lipid biosynthesis by a pho­toautotrophic mechanism is given in Figure 8.1. The biosynthetic pathway of lipid in algae occurs through four steps: carbohydrates accumulating inside the cell, formation of acetyl — CoA followed by malony-CoA, synthesis of palmitic acid, and finally, synthesis of higher fatty acid by chain elongation.

Base-catalyzed transesterification of microalgae oil is used most frequently and involves the presence of a base catalyst (hydroxides/carbonates) to precede the reaction (Meher et al., 2006; Vargha and Truter, 2005). In the reaction, the triglycerides are readily transesterified batchwise in the presence of the catalyst at an atmospheric pressure and tem­perature of 60-70 °C in the presence of excess methanol (Srivastava and Prasad, 2000). The main drawback with the process is the formation of soap at high free fatty acid concentrations (Furuta et al., 2004). Prior removal of free fatty acid and water from algae oils is a prerequisite for the reaction (Demirbas, 2008).

8.7.2 Enzyme-Catalyzed Transesterification

The reaction in an enzyme-catalyzed transesterification process is catalyzed by the enzyme lipase, whereby total triacylglycerides (both extracellular and intracellular) can be converted to biodiesel (Bisen et al., 2010). The conversion process requires complex processing instru­ments, and the costliness of the enzymes makes the process limiting. Immobilization was employed to overcome the limitations. However, the low feasibility of the process makes the reaction complex (Helwani et al., 2009; Watanabe et al., 2001).

10.3.5.1 Iodine

Marine algae are known for their high mineral content, so they have been used as feed and food supplements. In fact, they have 10-100 times the mineral content of traditional vege­tables (Arasaki and Arasaki, 1983; Nishizawa, 2002), with ash reaching levels of up to 55% on a dry-weight basis, whereas sweet corn has a content of 2.6% and spinach an excep­tionally high mineral content of 20% (Rupierez, Ahrazem et al., 2002). The mineral composi­tion varies according to phylum as well as such other factors as seasonal, environmental, geographical, and physiological variations.

The mineral iodine deserves particular attention because its concentration may reach quite high levels in certain brown algae—say, 1.2% of dry weight. For instance, Saccharina japonica (kombu) is an excellent source of iodine, so it has been used for centuries in China as a dietary iodine supplement to prevent goiter; most of it is dried and eaten directly in soups, salads, and tea or used to make secondary products with various seasonings (Lobban and Harrison, 1994). Furthermore, kelp was used as raw material for extraction of iodine in Ireland during the 17th century (Morrissey, Kraan et al., 2001). Nevertheless, excessive iodine intake in sen­sitive persons can trigger hyperactivity of the thyroid gland, similar to the myxoedema reac­tion (Holdt and Kraan, 2011), so brown alga consumption has to be limited. The main methods of extracting iodine from seaweed, such as incineration, blowout, ion exchange, and activated carbon adsorption, have been fully discussed and compared in terms of advan­tages and shortcomings by Jinggang et al. (Wang, Feng et al., 2008).

The raceway pond system is currently the most economically feasible cultivation method for mass production of algal biomass, primarily due to its relatively low capital cost and ease of operation. The pond usually consists of a closed-loop recirculation channel (oval in shape) where mixing and circulation are provided by paddlewheels to avoid algal biomass sedi­mentation. The CO2 source is sparged at the bottom of the raceway pond, as shown in Figure 12.1 (Chisti, 2007; Greenwell et al., 2010; Stephenson et al., 2010). Some raceway ponds incorporate artificial light in the system; however, this design is not practical and is economically infeasible for commercial production (Singh et al., 2011).

FIGURE 12.1 Raceway pond for algal cultivation. (Modified from Brennan and Owende, 2010.)

Raceway ponds are normally constructed with either concrete or compacted earth and lined with white plastic bags. The depth of the pond is usually 0.2-0.5 m to ensure that algae receive adequate exposure to sunlight (Brennan and Owende, 2010; Chisti, 2007). Under this cultivation system, the recorded algal biomass productivity and yield were 0.05-0.1 g/L/day and 0.3-0.5 g/L, respectively (Pulz, 2001); but are highly dependent on algal strains, cultivation conditions, and local weather.

Although raceway ponds have the advantages of low energy input and low operating cost, this system still suffers several limitations, such as massive loss of water due to high evap­oration rate and being easily contaminated by undesired microorganisms (e. g., bacteria, fun­gus, and protozoa) that could annihilate the entire algal population (Schenk et al., 2008). Hence, regular cleaning and maintenance are required in the raceway pond to ensure that the algae are growing under optimal conditions. In this regard, high lipid content and bio­mass productivity of algae for biofuel production are not the only factors to be considered, but other considerations such as fast growth rate, ease of cultivation, and ability to survive under extreme environmental conditions are equally important to ensure the existence of monoculture in a raceway pond. Chlorella, for example, can grow well in a nutrient-rich medium, Spirulina grows favorably at high pH and bicarbonate concentration, and D. salina is well adapted to a highly saline medium (Borowitzka, 1999; Brennan and Owende, 2010).

Inventory data of microalgal-based energy production systems are based on models or extrapolation of lab-scale or pilot-scale data. This is a clear source of uncertainty and variabil­ity between studies. Consequently, it is important that each new study clearly sources its data and provides detailed inventory data for each process of the production. Hence, a mass and energy balance of each process should be provided, with specific attention to the flow of fossil and biogenic carbon.

13.6.2.1 Input

It is a common practice when performing an LCA of a first — or second-generation biofuel to exclude infrastructures. Indeed, in these systems, it has been shown that their impact was negligible, and the inventory of every element of the infrastructure could be a tedious task. On the contrary, algal biomass production requires the construction of culture facilities, either raceways or photobioreactors. These two options differ from each other by the type of infrastructure they require, and they also differ from a usual crop by the need for a heavy culture infrastructure. As a consequence, LCAs of algae-based systems that exclude the infrastructure do not allow a fair comparison between options for algae culture and between algal-based and terrestrial plant-based biofuels.

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.