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.

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.

Biofuel derived from algae is currently a hotly debated topic because its production is one of the more costly processes, which can dictate the sustainability of algae-based biofuel products. There are two major energy and cost constraints to bulk production of microalgae for biofuels: expensive culture systems with high capital costs and high energy requirements for mixing and gas exchange, and the cost of harvesting in achieving feasible algal solids concentration.

Because of the dilute algal suspension, the cost of harvesting microalgal biomass accounts for a significant portion of the overall production cost of microalgal biofuels. Certainly, energy-efficient and cost-effective harvesting are two major challenges in the commercializa­tion of biofuels from algae (Dismukes et al., 2008; Reijnders, 2008). The algae must be concen­trated by removing water in an economically viable fashion before further processing such as drying and oil extraction. The lack of cost-effective methodologies for harvesting has been one of the major hurdles for the economic production of algal biofuels, along with challenges associated with variability of microalgae species (e. g., cell size, robustness, surface charge, culture medium constituent, and desired end-product) (Cooney et al., 2009). An effective microalgae separation process should be workable for all microalgae strains, yield a product with a high dry biomass weight, and require moderate cost of operation, energy, and maintenance.

Microalgae harvesting can be a considerable problem because of the small size (3-30 micrometers in diameter) and the stable suspended state of unicellular algal cells. Since the mass fractions in a culture broth are low (typically less than 0.5 kg/m3 dry biomass in some commercial production systems), large volumes of culture need to be processed to order to recover biomass in a feasible quantity (Cooney et al., 2009; Ramanan et al., 2010). In addi­tion, microalgae harvesting is a major bottleneck to microalgae bioprocess engineering owing to its high operating cost, thus reducing the cost of microalgae harvesting is vital. If microalgae can be concentrated about 30-50 times by coagulation-flocculation and gravity sedimentation prior to dewatering, the energy demand for microalgae harvesting could be significantly reduced (Jorquera et al., 2010).

In comparing algae removal using filtration, flotation, centrifugation, precipitation, ion exchange, passage through a charged zone, and ultrasonic vibration, it was concluded that only centrifugation and precipitation can be economically feasible, with centrifugation being marginal (Golueke and Oswald, 1965). In another study examining three different tech­niques of harvesting microalgae involving centrifugation, chemical flocculation followed by flotation, and continuous filtration with a fine-weave belt filter, it was reported that centrifu­gation gave good recovery and a thickened slurry but required high capital investment and energy inputs (Sim et al., 1988). Dissolved-air flotation was more economical, but, if the recovered algae were to be incorporated into animal feed, the use of coagulants such as alum could have undesirable effects on the growth rate of the animals. This problem could be overcome by the use of nontoxic coagulants. The continuous filtration process had significant advantages in terms of energy efficiency, economics, and chemical-free operation. The only drawback of this process was that the efficiency depended on the size and morphology of the algae.

Most of the algae-harvesting techniques present several disadvantages, not only because of the high costs of operation but also due to the frequently low separation efficiencies and the intolerable product quality. Algae separation processes such as sedimentation, centrifu­gation, and filtration involve the use of equipment that could result in deterioration in algal quality due to cell rupture that causes leakage of cell content. Furthermore, in the case of flocculation, the high concentration of metal salts, which is normally used as the coagulant, can have a negative effect on the quality of the final product, as discussed previously (Kim et al., 2005).

High production yields of microalgae have called forth interest due to economic and sci­entific factors, but it is still unclear whether the production of biodiesel is environmentally sustainable and which transformation steps need further adjustment and optimization. A comparative life-cycle assessment (LCA) of a virtual facility has been undertaken to assess the energetic balance and the potential environmental impacts of the whole process chain, from biomass production to biodiesel combustion (Lardon et al., 2009). The outcome vali­dated the potential of microalgae as an energy source but highlighted the imperative neces­sity of decreasing the energy and fertilizer requirements.

From another comparative LCA study to compare biodiesel production from algae with canola and ultra-low sulfur diesel with respect to greenhouse gas emissions and costs, it was concluded that the need for a high production rate is a vital key to make algal biodiesel economically attractive (Campbell et al., 2011). In a separate study, it was concluded that the potential greenhouse gas emissions from microalgae operational activities are likely to be outweighed by the emission reductions associated with the production efficiency and seques­tration potential of microalgae (Williams and Laurens, 2010).

Some commercial interests in large-scale algal-cultivation systems are looking to tie into existing infrastructures, such as coal-fired power plants or sewage treatment facilities. Wastes generated from those infrastructures, such as flue gas (carbon dioxide) and wastewater nu­trients (nitrogen, phosphorous and other micronutrients), can be converted into raw material resources for algal cultivation. While use of carbon dioxide for algal photosynthesis would help attain carbon sequestration, uptake of waste nutrients for algal growth would eliminate use of fertilizers derived from fossil-fuel energy, thus mitigating emissions.

In essence, algal biofuel is currently more expensive than other fuel options, but it is likely to play a major role in the economy in the long run if technology improvements succeed in bringing down costs. The main challenges are to decrease the energy and fertilizer requirements and to accomplish high production rates in order to make algal bio­diesel economically attractive. The potential of anaerobic digestion of waste oilcakes from oil extraction as a way to reduce external energy demand and to recycle part of the mineral fertilizers is to be further explored (Lardon et al., 2009). Algal biofuel production employing renewable substrates may be a potential answer to overcome some of the economic constraints. There is scope to use certain wastewater effluents containing waste nutrients as cultivation broth. Therefore, production as well as unit energy cost of algal biofuel would be reduced.

A rigorous techno-economic analysis is necessary to draw a clearer prospect comparison between algal biofuel and the various other conventional fossil fuels. In addition to benefits that can be quantified from the use of biofuel for clean energy production, intangible benefits such as flue gas carbon dioxide sequestration, uptake of waste nutrients in place of fertilizers, and biogas energy produced from anaerobic digestion of oilcake should also be considered. These benefits would render a potential for claims of certified emission reductions (CERs) under the Kyoto Protocol for reducing emissions that can be estimated through a holistic LCA of algal biofuel production. The potential for claims of CERs to generate revenue and to finance algal biofuel projects under the Kyoto Protocol for reducing emissions of green­house gases appears to be promising. In view of the prospects of technology development and global carbon trading, it may not be an unreasonable expectation that, in the future, algal biofuel will experience a global shift toward employment of energy-efficient algae biofuel production while mitigating greenhouse gas emissions.

5.4 CONCLUSIONS

Algal biofuel is believed to be one of the biofuels for the future in view of its potential to replace depleting fossil fuels. The future role of algal biofuel as a clean fuel producing near­zero emissions and as an energy carrier is increasingly recognized worldwide. Because energy-efficient and cost-effective harvesting are two major hurdles in the commercialization of biofuels from algae, research addressing these challenges should be intensified. Knowl­edge exchange and cooperation between expert groups of various disciplines should be strengthened in order to leapfrog technological development for algal biofuel.

Cultivation of microalgae influences both biomass growth and lipid productivity. Cultur­ing of algae requires the input of light as an energy source for photosynthesis with a sufficient supply of macronutrients (nitrogen and phosphate) and micronutrients (sulphur, potassium, magnesium) in dissolved form (Mata et al., 2010). The main options for algae cultivation on a commercial scale are open-ponds or closed systems called photobioreactors (Chisti, 2007; Robert et al., 2012). There are also hybrid configurations that include a mix of the two growth options. Innovations in algae production allow it to become more productive while consum­ing resources that would otherwise be considered waste (Campbell, 2008).