Successful application of ultrasound technique to harvest microalgae has been reported in a laboratory-scale study (Bosma et al., 2003). An algae separation process based on acousti­cally induced aggregation followed by enhanced sedimentation was carried out. The effi­ciency of algae harvesting and the concentration factor of the feed algal biomass concentration were optimized. Efficiency of the separation process was modeled with a sat­isfactory R-squared value of 0.88. The study found that feed-flow rate and algal biomass con­centration would significantly influence the process efficiency. Efficiencies higher than 90% were recorded at high biomass concentrations and flow rates between 4 and 6 L/day. As much as 92% of the algae biomass could be harvested, and a concentration factor of 11 could be achieved at these settings. Attempts to harvest at higher efficiency were unfruitful due to small size and low particle density of the microalgae. Feed-flow rate, biomass concentration, and ratio between harvest and feed flows had a significant effect on the concentration factor. Highest concentration factors up to 20 could be reached at low biomass concentrations and low harvest flow rates.

The study claimed that on lab or pilot scale, ultrasonic harvesting has the advantage that, in addition to small footprint, the process can be operated continuously without evoking hydrodynamic shear stress on algal cells, thus maintaining integrity of the algae. Moreover, the system can function as a biofilter when the algae excrete a soluble, high-value product. However, comparing ultrasonic with other harvesting processes, the authors pointed out that on an industrial scale, microalgae-harvesting centrifuges can be better used over the ultra­sound aggregation-sedimentation process because of lower energy requirements, better algae separation efficiencies, and higher concentration factors.

Use of ultrasound to improve the removal by coagulation of Microcystis aeruginosa, a common species of toxic algae, was investigated (Zhang et al., 2009). The results show that sonication significantly enhances the reduction of algae cells, solution UV254, and chlorophyll-a without increasing the concentration of aqueous microcystins. The main mech­anism involved the destruction during ultrasonic irradiation of gas vacuoles inside algae cells that acted as "nuclei" for acoustic cavitation and collapse during the "bubble crush" period, resulting in the settlement of cyanobacteria. The investigation revealed that coagulation effi­ciency depended strongly on the coagulant dose and sonication conditions. With a coagulant dose of 0.5 mg/L and ultrasonic irradiation for 5 seconds, algae removal efficiency increased from 35% to 67%. Optimal sonication time was determined at 5 seconds, since further sonication would only marginally enhance the coagulation efficiency. The most effective sonication intensity was found to be at 47.2 W/cm2, and the highest removal of the algae was recorded at 93.5%. Supported with experimentation on reservoir water, the authors recommended that this method could be successfully applied to natural water containing multiple species of algae.

Microalgae have the capability to grow in nutrient-rich environments and accumulate nutrients and metals from wastewater (de-Bashan and Bashan, 2010; Hoffmann, 1998; Mallick, 2002). This makes heterotrophic cultivation of microalgae one of the viable options for lipid biosynthesis. Algae-based biodiesel production is considered both economically and environmentally sustainable when wastewater is used as substrate (Brune et al., 2009; Chisti, 2007; Huntley and Redalje, 2007; Stephens et al., 2010; Venkata Mohan et al., 2011; Prathima Devi et al., 2012). Use of algae as a biocatalyst was generally documented for wastewater treat­ment in conventional oxidation ponds, raceway ponds, and suspended algal ponds to remove high concentrations of nutrients, especially for polishing purposes. Algae-based treatment systems are efficient in removing nutrients from wastewater compared to chemical-based treatments (Hoffmann, 1998; Martinez et al., 2000; Ruiz-Marin et al., 2010; Zhang et al., 2008) and are environmentally amenable and provide efficient recycling of nutrients (Munoz and Guieysse, 2006; Wilkie and Mulbry, 2002). Usually Chlorella sp. and Scenedesmus sp. are predominantly observed in the oxidation ponds (Bhatnagar et al., 2010; Ruiz-Marin et al., 2010; Shi et al., 2007; Wang et al., 2010; Masseret et al., 2000). Especially for industrial wastewater treatment, the algae-based remediation process was used as a tertiary unit oper­ation for the removal of heavy metal and organic toxins rather than nutrients (Ahluwalia and Goyal, 2007; de-Bashan and Bashan, 2010). Microalgae cultivation with wastewater treatment is a potential option for environmental sustainability and carbon neutrality.

Five characteristically different ecological water bodies (mixotrophic) were evaluated to assess the biodiesel production capability of their native microalgae (mixed) (Venkata Mohan et al., 2011). The lipid yield varied between 4-26%, mostly depending on the nature and func­tion of the water body. Algal fuel showed reasonably good fuel properties, with higher sat­urated fatty acids. Algal diversity profiling depicted the presence of high-lipid-accumulating species. The dominance of mixotrophic microalgae (Chlorella, Scenedesmus, and Euglena) and facultative heterotrophs (centric and pinnate diatoms), along with a few photoautotrophs (Spirogyra), were observed. Euglena can act as both autotrophs (day) and heterotrophs (nights). Scenedesmus sp. is generally involved in the natural purification process, and its dom­inance in all the cultures is a positive sign for wastewater treatment. Chlorella, Euglena, and diatoms are also known to have the capability to use organic carbon present in the wastewater along with atmospheric CO2. Ecological water bodies can be considered potential reservoirs for bioenergy production, conjugating with the natural purification process.

Carpet mill effluent documented as a potential source for algal biomass production asso­ciated with biodiesel production (Chinnasamy et al., 2010). Two agroindustrial co-products, dry-grind ethanol thin stillage and soy-whey, were studied as nutrient feedstock for mixotrophic/heterotrophic microalgal cultivation for fuel production (Debjani et al., 2012). Scenedesmus sp. cultivated in artificial wastewater showed about 12% lipid accumulation along with 33% protein and 27% carbohydrates (Voltolina et al., 1999). Botryococcus braunii grown in secondarily treated sewage as tertiary treatment documented good treatment effi­ciency along with 17% lipid accumulation (Orpez et al., 2009). Chlorella sp. grown in attached mode with dairy manure wastewater showed high biomass growth as well as fatty acid yield (Johnson and Wen, 2010). Cultivation of Scenedesmus sp. in fermented swine wastewater yielded lipids and other value-added products in association with nutrient removal (Kim et al., 2007). Nitrogen and phosphorus assimilation associated with lipid production was studied with freshwater microalgae using industrial wastewater (Li et al., 2012).

The functional role of macro/micronutrients—carbon, nitrogen, phosphorus, and potassium—on heterotrophic cultivation of microalgae (mixed) in domestic wastewater was studied on biomass growth and lipid productivity employing sequential growth and starvation phases (Prathima Devi et al., 2012). Nutrient limitation during the starvation phase showed a positive influence on lipid productivity. Nutrient-deprived conditions caused a decrease in the cellular thylakoid membrane content by activating the acyl hydrolase and stimulating the hydrolysis of phospholipids. All these changes increase the intracellular content of fatty acid acyl-CoA. Nitrogen limitation can also activate diacylglycerol acyl transferase, which converts acyl-CoA to TAG (Takagi et al., 2000). Lipid composition of the microalgae oil varied in accor­dance with the nutrients supplemented (Prathima Devi et al., 2012). Efficient removal of nutri­ents (nitrates and phosphates) and carbon (as COD) was also noticed. Microalgae diversity visualized the presence of potential lipid-accumulating species, such as Cosmarium quadrifarium, Pediasatrum boryanum, Cyclotella bodanica, Scenedesmus sp., and Cosmarium depressum. Acid-rich effluents from a fermentative hydrogen-producing reactor were evaluated as potential substrate for lipid accumulation by heterotrophic microalgae cultivation with simultaneous treatment (Venkata Mohan and Prathima Devi, 2012). Microalgae can grow heterotrophically by utilizing volatile fatty acids (VFA), resulting in lipid accumulation. Acetate can be easily assimilated by the algal cell as part of the acetyl-coenzyme A (acetyl-CoA) metabolism in a single-step reaction catalyzed by acetyl-CoA synthetase (Boyle and Morgan, 2009; Chandra et al., 2012). Similar to acetate consumption, butyrate is broken down and gets converted to acetate and then enters the TCA cycle to stimulate the synthesis of glucose. TAG accumulation in response to environmen­tal stress likely occurs as a means of providing an energy deposit that can be readily catabolized in response to a more favorable environment, to allow rapid growth (Prathima Devi et al., 2012). Integration of microalgae cultivation with biohydrogen production showed lipid productivity for biodiesel production along with additional treatment (Chandra and Venkata Mohan, 2011).

Given the delicate oxygen-sensitive hydrogenase and the prevailing oxidative environ­mental conditions, questions have been asked as to whether algal hydrogen production via direct photolysis can ever be utilized to generate hydrogen for practical applications. A practical approach to overcome the oxygen sensitivity of hydrogenases needs to be devel­oped to motivate research on applied algal hydrogen production systems. To this end, it is critical to develop novel methods to separate oxygen from the biochemical activities, thus en­abling hydrogen production for extended periods. Advancement in molecular bioengineer­ing also indicates that genetic engineering might offer a feasible approach to developing oxygen-tolerant algal mutant.

Although indirect photolysis hydrogen production technology has significant promise, some crucial challenges are to be addressed. Given that hydrogen production by sulfur deprivation is time limited, a major challenge is to maintain stable hydrogen production for practical uses. Hydrogenase is too oxygen-labile for sustainable hydrogen production. Light — dependent hydrogen production ceases within a few days, since photosynthetically produced oxygen inhibits or inactivates hydrogenases. Substantial rates of hydrogen production were steadily sustained initially for about 60 h in the light, but the yield begins to level off gradually thereafter (Zhang et al., 2002). After about 100 h of sulfur deprivation, the algae need to go back to normal photosynthesis in order to be rejuvenated by replenishing endogenous substrate (Ghirardi et al., 2000). Improvements must be made to maintain the process continuity for com­mercial applications.

Although it has been established that hydrogen can be produced from endogenous substrate catabolism, the mechanisms this entails are yet fully understood. Rates of water ox­idation by the photosynthetic systems can be determined precisely, but the electron transport by endogenous substrate catabolism and NADPH-PQOR activity are hard to measure (Melis, 2002). Research on hydrogen production from anaerobically incubated and DCMU-inhibited chloroplasts suggests that sizable rates of hydrogen production can be detected only in the initial incubation period (Florin et al., 2001). In other words, hydrogen production via endog­enous substrate catabolism is not a sustainable process (Zhang et al., 2002). This observation may suggest a limitation in the capacity of the electron transport and the attendant NADPH — PQOR activity. Nevertheless, the prospect of hydrogen production with endogenous sub­strate catabolism is important and should warrant further research to fully tap its potential. Application of molecular bioengineering might help increase the capacity of this important process.

Low hydrogen yield and production rate are two major challenges for practical application of biohydrogen production. Genetic manipulation or modification of the hydrogen — producing microorganisms probably will play a vital role in tackling the problem of low yields (Beer et al., 2009). In a recent development, metabolic engineering has received increas­ing attention in improving biohydrogen production. Improvements in hydrogen yields by existing pathways have been attempted by increasing the flux through gene knockouts of competing pathways or increased homologous expression of enzymes involved in the hydrogen-generating pathways (Hallenbeck and Ghosh, 2009).

Several algal strains related to biohydrogen production have been isolated and manipu­lated; for example, C. reinhardtii strain can increase hydrogen production under high starch conditions (Hankamer et al., 2007). Mutant algae with less chlorophyll were manipulated for large-scale commercial applications that allow more sunlight penetrating into deeper algae layers beneath the water surface in the bioreactor. Hence, sunlight is made available for more algal cells to generate hydrogen, thus improving the production rate. Whereas metabolic flux analysis has been used to guide a priori most suitable genetic modifications oriented to a hy­drogen yield increase for a fermentative hydrogen production process (Show et al., 2012), the flux balance analysis may also offer a useful tool to provide valuable information for optimi­zation and design of the photosynthetic hydrogen production process.

It has been reported that a truncated chlorophyll antenna size of the photosystems in the chloroplast of the microalgae could alleviate the optical shortcomings and light-saturation ef­fect associated with a fully pigmented chlorophyll antenna (Melis, 2002). With the genetically manipulated algal cells, the drawback of overabsorption of photons by the photosystems can be minimized. A truncated chlorophyll antenna will reduce the loss of energy by the cells, and it will also dampen down photoinhibition of photosynthesis at the surface of the culture. Moreover, a truncated chlorophyll antenna size will alleviate the problem of light attenuation and mutual cell shading by permitting a more consistent illumination to the entire algal bio­mass. Such altered optical properties of the cells would result in much greater photosynthetic productivity and better solar utilization efficiency in the culture.

Experiments have shown that a smaller chlorophyll antenna size would bring about a higher light intensity for the saturation of photosynthesis in individual algal cells but with an associated threefold improved productivity of the culture (Neidhardt et al., 1998; Melis et al., 1999; Nakajima and Ueda, 1999). Excitation pressure was used as a bioengineering tool in the work to culture green algae with a truncated chlorophyll antenna size. The stud­ies concluded that green algae with a truncated chlorophyll antenna size are essential in augmenting photosynthetic efficiencies and the hydrogen yield under mass culture conditions. Manipulation of the chlorophyll antenna size in response to light is essentially an inherent reaction of the chloroplasts, since they are inversely related to the incident light. In principle, it is possible to genetically manipulate the relevant regulatory mecha­nism in the photosystems and, in transforming green algae, to direct the chloroplast bio­synthetic and assembly activities toward a permanently truncated chlorophyll antenna size (Melis, 2002).

At this moment, the acceptability of genetically modified microorganisms is another challenge, due to the possible risk of horizontal transference of genetic material. However, this can be ruled out by chromosomal integration and the elimination of plasmids containing antibiotic markers with available molecular tools (Datsenko and Wanner, 2000). Moreover, the improvement of hydrogen production by gene manipulation is mainly focused on the disruption of endogenous genes and not introducing new activities in the microorganisms.

Hydrogen production from water photolysis has the potential to be the cleanest and most direct energy conversion process. Direct biophotolysis, albeit limited by its low hydrogen production, provides a feasible scheme for hydrogen production from water and sunlight. Technology advancement and innovations in enzymes, electron carriers, biomaterials, and genetic engineering may lead to a practical water photolysis system that overcomes the intrinsic oxygen inhibition shortcoming. Hydrogen production via indirect biophotolysis remains far behind the productivity rates of other biofuels. The low energy productivity of biohydrogen can be improved if the energy stored in fermentative products, such as acetic acid, is reused. Mutant algae could be cultured to produce the maximum amount of hydrogen from endogenous carbohydrates via dark fermentation, and then use the residual acetate for accumulation of endogenous carbon reserve in photosynthesis. Alternatively, a microbial electrolysis cell can be incorporated into the system (Figure 9.2) to convert the organic acids generated from the dark fermentation into hydrogen under light-independent process. The hydrogen yield and productivity rate can therefore be significantly improved to make highly compact energy generators for a future hydrogen economy.

In essence, the future of algal hydrogen production depends not only on research advances such as improvement in efficiency through genetically engineering microorganisms and/or the development of bioreactors but also on economic considerations, social acceptance, and the development of hydrogen energy systems.

9.2 CONCLUSIONS

Biohydrogen is believed to be one of the biofuels of the future, combining its ability to potentially reduce our dependence on fossil fuels and to contribute to lowering greenhouse gas emissions from the energy and transportation sectors. The future role of hydrogen as a clean fuel for fuel cells producing near-zero emissions and as an intermediate energy carrier for storage and transport of renewable energy is increasingly recognized worldwide. The role of biohydrogen in a future hydrogen economy, however, remains to be seen. Nevertheless, it is clear that the advent of hydrogen as a renewable energy source will have important economic implications, provided that scientific and technological challenges are overcome. The R&D in the field of algal hydrogen production will therefore be intensified.

Microalgae are microscopic photosynthetic organisms that are found in both marine and freshwater environments. Their photosynthetic mechanism is similar to that of land-based plants, but due to a simple cellular structure and submerged in an aqueous environment where they have efficient access to water, CO2, and other nutrients, they are generally more efficient in converting solar energy into biomass. These organisms constitute a polyphyletic and highly diverse group of prokaryotic (two divisions) and eukaryotic (nine divisions) or­ganisms. The classification into divisions is based on various properties such as pigmentation, the chemical nature of the photosynthetic storage product, the organization of photosynthetic membranes, and other morphological features. The most frequently used microalgae are Cyanophyceae (blue-green algae), Chlorophyceae (green algae), Bacillariophyceae (including the diatoms), and Chrysophyceae (including golden algae). Many microalgae species are able to switch from phototrophic to heterotrophic growth. As heterotrophs, the algae rely on glucose or other utilizable carbon sources for carbon metabolism and energy. Some algae can also grow mixotrophically (Carlsson et al., 2007).

Microalgae have the following advantages over crops as a source of biomass. They are more effective biological systems for converting sun power into organic compounds; microalgae, like bryophytes, have no complex reproductive system; it is possible to induce in many microalgae species generation of valuable proteins, hydrocarbons, lipids, and pig­ments in extremely high concentrations; they are organisms that have a simple cycle of cell pressure; and they can be grown in various water areas (Vonshak, 1990).

Figure 12.2 shows the projected techno-economic performance of algal biofuels at the cur­rent phase as well as the projected outcome for the future. From the figure, it can be observed that current algal biofuel production is still in its infancy stage for commercialization, predominantly due to the high investment cost and premature technology. However, with emerging technologies and innovation through progressive R&D efforts, the way will be paved to develop truly sustainable algal biofuels with affordable production costs in the near future. All the technical problems surrounding the supply chain of algal biofuels, such as nutrient source, cultivation system, harvesting and drying processes, and biofuel-conversion technology, must be resolved to attain its commercialization threshold and economic viability. Thus, current techno-economic assessments of algal biofuels should be highly

interdisciplinary, with a flexible modeling and analysis framework that can completely address the multiple pathways, coupled with various integration systems for algal biofuel production.

The production cost of algal biofuels is defined as the sum of capital and operating costs minus the revenues derived from all coproduct, as shown in Eq. 12.1 (Sun et al., 2011). Capital cost is usually related to one-time expenses such as the cost of land, buildings (e. g., indoor cultivation systems, offices, and laboratories), equipment (photobioreactors, dryer, filter press), and infrastructure (piping and pumps), whereas operating cost is associated with day-to-day expenses such as power (e. g., to operate photobioreactors), raw materials (nutrients and water), labor costs, and maintenance fees (Sun et al., 2011).

The coproduct that could possibly be derived from algal biomass residue after lipid extraction is carbohydrate, which can be an excellent substrate for anaerobic digestion or fermentation process to produce biomethane or bioethanol, respectively. The biomethane and/or bioethanol produced can be diverted to use at an algal cultivation farm to reduce the associated energy burden, or it can be sold as energy fuel. However, additional capital and operating cost will be required to build the facilities to convert carbohydrates to biomethane/bioethanol. In the worst-case scenario, the coproduct processing facilities may consume substantial amounts of cost and energy due to the low conversion efficiency of biomethane or bioethanol, making it commercially impractical to integrate the coproducts into the supply chain of algal biodiesel. On the other hand, coproducts that have high market value, such as phytonutrients and proteins, could be the alternative options instead of carbohydrates.

Certain strains of microalgae biomass are accepted as functional food for human and an­imals. This is because they contain active compounds such as omega-3 fatty acids (eicosapentanoic acid-EPA, decosahexaenoic acid-DHA), chlorophylls, carotenoids, phycobiliproteins, and the like. Although the production of strains containing these com­pounds can be performed in open reactors, it is preferable to carry out the process in closed photobioreactors to ensure good manufacturing practices, as imposed by the food and phar­maceutical industry.

Marine strains such as Pavlova viridis (Hu et al., 2008), Nannochloropsis sp. (Chini Zittelli et al., 1999) and Phaeodactylum tricornutum (Acien et al., 2000) have proven useful for the out­door production of omega-3 fatty acid-rich biomass in closed photobioreactors. However, its production cost is higher than omega-3 produced heterotrophically or obtained from fish oil.

For this reason, a cost-effective system based on autotrophic growth is not yet available. Concerning freshwater strains, Chlorella biomass is accepted for human consumption and is produced by more than 70 companies. The Taiwan Chlorella Manufacturing Co. Ltd. (Taipei, Taiwan) is the largest producer, with 400 t/year, although there is also significant production in Klotze, Germany, with 130 t/year using tubular photobioreactors. Annual world sales of Chlorella are in excess of $38,000 million (Spolaore et al., 2006).

Chlorella biomass has related health benefits, such as being an active inmunostimulator and reducer of blood lipids, among other things, in addition to its taste, flavor, and coloring prop­erties. There is also a demand for microalgae biomass production of monoalgal strains by the feed market. Thus, microalgae can be incorporated into fish, pet, and farm animal feed. In 1999, the production of microalgae for aquaculture reached 1,000 t/year (62% for molluscs, 21% for shrimps, and 16% for fish) (Spolaore et al., 2006). The importance of algae in this do­main is not surprising, given that microalgae are the natural food source for these animals. The main microalgae applications in aquaculture are associated with nutrition, either being used fresh (as the sole component or as a basic nutrient food additive) or for coloring the flesh of salmonids and for inducing other biological activities.

The production cost of monoalgal microalgae biomass in closed photobioreactors has re­cently been analyzed (Acien et al., 2012a) (see Figure 14.5); a pilot-scale facility of 0.04 Ha consisting of 10 tubular photobioreactors, each one 3 m3, operated in continuous mode at an average dilution rate of 0.34 L/day year round. Fertilizers are used to prepare the culture medium, which is filtered and ozonized to sterilize it. The strain Scenedesmus almeriensis is cultivated under controlled pH (by injecting pure CO2) and temperature excess is avoided by passing cool water from heat exchangers located inside the reactors. S. almeriensis has proven to be a source of lutein, with an average percentage of this carotenoid in the biomass of 1% (dw) all through the year (Sanchez et al., 2008).

Biomass productivity throughout the year ranges from 0.3 to 0.7 g/L. The biomass is harvested and concentrated by centrifugation daily in continuous mode, then is freeze-dried to obtain dry biomass; a production capacity of 3.8 t/year has been reported. Analysis of the facility’s production cost shows depreciation (42.6%) and labor (51.6%) as being the main fac­tors contributing to the final biomass cost of up to $89/kg (see Figure 14.6). As expected for the production of microalgae biomass using closed photobioreactors for the production step, in addition to centrifugation for harvesting and freeze drying for stabilization, these are the

FIGURE 14.5 Block diagram of the process for the production of Scenedesmus almeriensis dry biomass in tubu­lar photobioreactors. (Adapted from Acien et al., 2012a.)

FIGURE 14.6 Analysis of production costs related to the production of Scenedesmus almeriensis using tubular reactors scaled up to 3.8 t/year. (Adapted from Acien et al., 2012a.)

major contributions to the depreciation cost. Closed photobioreactors make up 47% of the de­preciation cost, whereas harvesting represents 45%. Although raw material and utility costs are much lower than depreciation and labor costs, the contributions of CO2 and power costs are highly relevant. The cost of CO2 represents 71% of raw material cost, whereas power rep­resents 98% of the utility cost. These reported data have been obtained and verified over two years of operation, making their robustness higher than other cost analyses performed using laboratory data.

From these results it can concluded that, to reduce the production cost, it is necessary to reduce labor by implementing extensive automation in addition to reducing the depreciation cost by simplifying the equipment used and increasing production capacity. Indeed, by in­creasing the production capacity up to 200 t/year, by adequate scale-up of the process, by reducing manpower to 1 person/ha, and by avoiding the use of expensive equipment such as freeze dryers and sterilization units, the production cost can be reduced to $16/kg versus $89/kg at small scale (Acien et al., 2012a). This reduction to less than 20% of the initial value demonstrates that the production capacity increase has a great effect on the reduction of the production cost. Therefore, the obtained value is similar to that reported (Norsker et al., 2010), indicating a production cost of $5.4/kg when producing microalgae biomass in tubular photobioreactors at scales up to 100 ha.

It has also been reported that the production cost in tubular photobioreactors is lower than that obtained using flat panels or open raceways. This is due to the lower productivity and
higher harvesting costs in these reactors compared to tubular photobioreactors (Posten, 2009), production costs being as high as $6.4/kg and $7.7/kg when using flat panels and open race­ways, respectively (Norsker et al., 2010). These studies demonstrate that biomass productivity is a key factor determining the total production cost, in addition to the unitary cost of the re­actor and unitary harvesting cost. Moreover, production costs can be lower using tubular photobioreactors instead of open raceways in spite of lower costs for the latter; this is because of the higher productivity achieved in tubular photobioreactors and lower volume to be processed in the harvesting step.

Use of chemicals to induce coagulation-flocculation of algal cells is a routine upstream treat­ment in various algae-harvesting technologies such as sedimentation (Friedman et al., 1977; Mohn, 1980), flotation (Moraine et al., 1980), filtration (Danquah et al., 2009) and centrifuga­tion (Golueke and Oswald, 1965; Moraine et al., 1980). Coagulation-flocculation causes algal cells to become aggregated into larger clumps, which are more easily filtered and/or settle more rapidly to facilitate harvesting. Chemicals that were used as algal coagulants can be broadly grouped into two categories: inorganic and long-chain organic coagulants.

Inorganic coagulants include metal ions as Al+3 and Fe+3, which form polyhydroxy com­plexes at appropriate pH. Hydrated lime is a common coagulant inducer used in water and wastewater treatment. Its use would raise the pH to the point at which a milk-like inor­ganic compound, magnesium hydroxide, is formed and acts as a coagulant (Folkman and Wachs, 1973; Friedman et al., 1977). Aluminum sulphate (commonly called alum, with the chemical formula Al2(SO4)3 • 18 H2O) or other salts of aluminum, common coagulants used in water treatment, have also been used as coagulants in algae harvesting (Golueke and Oswald, 1965; McGarry, 1970; Moraine et al., 1980). Ferric sulfate was found to be inferior in comparison with alum with respect to the optimal dose, pH, and the quality of the harvested algal paste (Bare et al., 1975; Moraine et al., 1980).

Satisfactory treatment of algal pond effluent has been achieved by lime addition (Folkman and Wachs, 1973; Friedman et al., 1977). However, satisfactory lime treatment was limited to algal cultures containing magnesium above 10 mg/L, and the quality of the harvested prod­uct was significantly affected due to excessive calcium content of up to 25% by weight.

Common flocculation theory states that alkaline flocculants neutralize the repelling surface charge of algal cells, allowing them to coalesce into a floc. Based on such electrostatic

flocculation theory, the more cells to be flocculated, the more coagulant would be needed in a linear stoichiometric fashion, rendering flocculation overly expensive. Contrary to this theory of electrostatic flocculation, a study found that the amount of alkaline coagulant needed is a function of the logarithm of cell density, with dense cultures requiring an order of magnitude less base than dilute suspensions, with flocculation occurring at a lower pH (Schlesinger et al., 2012). Various other theories abound that flocculation can be due to multivalent cross-linking or coprecipitation with phosphate or with magnesium and calcium. However, the study revealed that monovalent bases that cannot cross-link or precipitate phosphate work with the same log-linear stoichiometry as the divalent bases, obviating those theories and leaving electrostatic flocculation as the only tenable theory of flocculation with the materials used.

Long-chain organic coagulants or polyelectrolytes could exist as anionic, cationic, and non­ionic synthetic or natural polymeric substances (Stumm and Morgan, 1981). In examining various organic polymers as algal coagulants, it was reported that only the cationic polyelec­trolytes were found to be efficient coagulants (Tenney et al., 1969; Tilton et al., 1972; Moraine et al., 1980). Organic cationic polyelectrolytes at low dosages (1-10 mg/L) can induce efficient flocculation of freshwater microalgae (Bilanovic et al., 1988). Effective flocculation was attained at salinity levels lower than 5 g/L. However, the high salinity of the marine environ­ment was found to inhibit flocculation with polyelectrolytes. The reduced effectiveness of cationic polymers to induce microalgae flocculation in high-salinity medium is primarily attributed to the effect of medium ionic strength on the configuration and dimension of the polymer, as indicated by changes in the intrinsic viscosity. At high ionic strength, the polymer shrinks to its smallest dimensions and fails to bridge between algal cells.

Studies also revealed that while anionic polyelectrolytes enhanced lime flocculation, most polyelectrolytes can be used in conjunction with alum or ferric sulfate as coagulant aids to strengthen the flocs, thus enhancing algae harvesting (Friedman et al., 1977). When used as coagulant aids, the polyelectrolytes can be applied at reduced dosages than they would have been used alone. This helps save chemical costs.

Algal coagulation-flocculation mechanisms based on the use of polymeric coagulants were postulated (Tenney et al., 1969; Tilton et al., 1972). Adsorption and the bridging model were hypothesized, and parameters affecting the process were investigated. It was reported that higher molecular weight cationic polyelectrolytes are superior in flocculating algal particles than their lower molecular weight counterparts. Optimal dose decreased with increasing mo­lecular weight. However, very high molecular weight polymers may reverse the algal surface charge, thus stabilizing the suspension (Tilton et al., 1972). The study also pointed out that for a given level of algal flocculation, variations in algal concentrations would affect the polyelec­trolyte dosage needed, and the relationship between algal concentration and polyelectrolyte dosage can be established based on stoichiometry (Tenney et al., 1969).

A commercial product called chitosan, commonly used for water purification, can also be used as a coagulant but is far more expensive. To create chitosan, the shells of crustaceans are ground into powder and processed to acquire chitin, a polysaccharide found in the shells, from which chitosan is derived via deacetylation. Flocculation of three freshwater algae, Spirulina, Oscillatoria, and Chlorella, and one brackish alga, Synechocystis, using chitosan was examined (Divakaran and Pillai, 2002). With suspension in the pH range of 4 to 9 and chlorophyll-a concentrations in the range of 80 to 800 mg/m3, the chitosan-aided flocculation achieved a clarified water turbidity of 10 to 100 NTU units. The chitosan was found to be effective in separating the algae by flocculation and settling. It was found that the flocculation efficiency is very sensitive to pH, with optimal pH 7.0 for maximum flocculation of fresh­water algal species. The optimal chitosan concentration for maximum flocculation depended on the concentration of algae. Flocculation and settling rates were faster when higher than optimal concentrations of chitosan were used. The settled algal cells were intact and live and could not be redispersed by mechanical agitation. The clarified water may be recycled for fresh cultivation of algae. Studies of harvesting microalgae with chitosan flocculation were also reported (Lavoie and de la Noue, 1983; Morales et al., 1985).

In addition to the type of coagulant, the composition of the algal medium can also influence the optimum flocculation dosage. For lime treatment whereby magnesium hydroxide pre­cipitate is functioning as a coagulant, as discussed earlier, it was found that the higher the dissolved organic substances in the algal suspension, the higher was the concentration of magnesium hydroxide required for good algal flocculation (Folkman and Wachs, 1973). In­hibition of flocculation caused by the presence of dissolved organic matter was also observed in other investigations (Hoyer and Bernhardt, 1980; Narkis and Rebhun, 1981). Conversely, it was found in another study that algal exocellular organic substances reduced the optimal coagulant dose during the early declining growth phase of algal culture but increased the dose during the late growth stages (Tenney et al., 1969). The authors attributed the increased optimal dose to the development of the organic substances into protective colloid.

There are many variables that could affect algal coagulation-flocculation in a collective and complicated manner, rendering predictions for operational conditions almost impos­sible. Other than algal type, the optimal coagulant dosages can be dictated by the concen­trations of phosphate, alkalinity, ammonia, dissolved organic matter, and temperature of the algal medium (Moraine et al., 1980). In practice, optimal coagulant dosages are determined using bench-scale jar tests to simulate the complex coagulation-flocculation process.

Harvesting by chemical flocculation is a method that is often too expensive for large operations. The main disadvantage of this separation method is that the additional chemicals are difficult to remove from the separated algae, probably making it inefficient and uneco­nomical for commercial use, though it may be practical for personal use. The cost to remove these chemicals may be too expensive to be commercially viable. One way to solve this prob­lem is to interrupt the carbon dioxide supply to the algal system, which would cause algae to flocculate on its own—namely, autoflocculation. In some cases this phenomenon is associated with elevated pH due to photosynthetic carbon dioxide consumption corresponding to pre­cipitation of inorganic precipitates (mainly calcium phosphate), which cause the flocculation (Sukenik and Shelef, 1984). In addition to this coprecipitative autoflocculation, the formation of algal aggregates can also be due to excreted organic macromolecules (Benemann et al., 1980), inhibited release of microalgae daughter cells (Malis-Arad et al., 1980), and aggregation between microalgae and bacteria (Kogura et al., 1981).

A fungi pelletization-assisted bioflocculation process for algae harvesting and wastewater treatment was developed (Zhou et al., 2012). Microalga Chlorella vulgaris UMN235 and two locally isolated fungal species, Aspergillus sp. UMN F01 and UMN F02, were used to study the effect of various cultural conditions on pelletization for fungi-algae complex. The results showed that pH was the key factor affecting formation of fungi-algae pellets, and pH could be controlled by adjusting glucose concentration and the number of added fungal spores.

The best pelletization occurred when adding 20 g/L glucose and approximately 1.2 x 108/L spores in BG-11 medium, under which almost all of algal cells were captured onto the pellets with shorter retention time. The fungi-algae pellets can be easily harvested by simple filtration due to their large size (2-5 mm). The filtered fungi-algae pellets were reused as immobilized cells for wastewater treatment. It was claimed that the technology developed is highly promising compared with current algae harvesting and biological wastewater treat­ment technologies in the literature.

Downstream processes of C. protothecoides cultures include biomass harvest and drying, cell disruption, oil extraction, and transesterification for biodiesel. Various harvesting methods are applied to Chlorella cultures, including flocculation, flotation, filtration, gravity sedimen­tation, and centrifugation (Lin, 2005; Wiley et al., 2009; Papazi et al., 2010; Lee et al., 2012). The harvest efficiency rests not only with harvesting methods used but also algal species, cul­ture ages, and cell densities. Usually, a harvesting method is not used alone but is coupled with one or more other methods to achieve the highest harvesting efficiency, e. g., a preceding treatment of flocculation was used to improve the performance of flotation, filtration, sedi­mentation, or centrifugation (Sim et al., 1988; Liu et al., 1999; Wiley et al., 2009). A drying

process following biomass harvest may be needed, depending on whether drying or wet bio­mass is used for oil extraction. The harvest and drying processes may contribute 20-30% of the total cost of photoautotrophic algal biomass production (Molina Grima et al., 2003). Although the high cell density associated with heterotrophic algae can reduce the cost contribution,

finding ways to improve the cost-effectiveness of the harvest and drying steps still represents a big challenge for the Chlorella industry if it is to expand from the current high-value, low — quantity specialty products market to a low-value, high-volume commodity products market.

Chlorella has a tough, rigid cell wall, and thus disruption of the cell wall is required for the facilitation of oil extraction. Various disruption methods, e. g., mechanical crushing, ultra­sonic treatment, and enzymatic degradation, can be employed for cell-wall disruption. The oils released after cell disruption are suitable for extraction using organic solvents. Supercrit­ical CO2 is another way for efficiently extracting algal oils, but it is expensive and energy in­tensive, which restricts the commercialization of this technology (Herrero et al., 2010).

The extracted algal oils are suitable for biodiesel conversion through transesterification. Transesterification is a catalytic reaction of oils with a short-chain alcohol (typically methanol or ethanol) to form fatty acid esters. The reaction is reversible; as such, a large excess of alcohol is used in industrial processes to ensure the direction of fatty acid esters. Methanol is the pre­ferred alcohol for industrial use because of its low cost. Commonly, a catalyst is required to facilitate the transesterification, including acids, alkalis, and enzymes. Acid transesteri­fication is considered suitable for the conversion of oils with high free fatty acids but with low reaction rate (Gerpen, 2005). In contrast, alkali catalyzes a much higher transesterification rate, thought it is unfavorable for free fatty acids (Fukuda et al., 2001). As a result, alkalis are preferred catalysts for industrial production of biodiesel, and acid pretreatment is usually employed when the oils contain a high content of free fatty acids. The use of lipases for transesterification has also attracted much attention because it produces a high-purity prod­uct and enables easy separation of biodiesel from the byproduct glycerol (Ranganathan et al.,

2008) . However, the cost of the enzyme is still relatively high and remains a barrier to its industrial implementation.

The disruption of algae cells prior to extraction is of particular importance because the con­tents of the extracted lipids are determined according to the disruption method and device employed. The selection of appropriate device for disruption is the key factor for enhancing the lipid extraction efficiency (Lee et al., 2010). The following are the methods commonly used for the disruption of algae cells.

8.6.1.1 Expeller Press Method

Expeller pressing (also called oil pressing) is a mechanical method applied for the disrup­tion of algae cell membranes by squeezing the cells under high pressure (Mercer and Armenta, 2011). Expeller pressing can also be used as an extraction technique because it
can recover nearly 75% of the oil from algae cells in a single step. The advantages of this method include elimination of a solvent requirement and easy operation, the drawback associated with it is the requirement of a large amount of biomass.

Agar is a mixture of polysaccharides, typically extracted from the cell walls of red algae; it is composed of agarose and agropectin and exhibits structural and functional properties sim­ilar to those of carrageenans. Like carrageenans, agar is also extracted with hot water. The genera Gelidium and Gracilaria are the major commercial sources of agar (Carlsson, 2007). Like carrageenans, agar is used as stabilizers for emulsions and suspensions and as gelling agents. Approximately 90% of all agar produced worldwide is intended for food applications; the remainder is used in the manufacture of capsules for medical applications and as a medium for cell cultures (Carlsson, 2007). Agar affects absorption of ultraviolet radiation (Murata and Nakazoe, 2001) and exhibits a few bioactivities as well (see Table 10.2).