Batan et al. (2010) reported that the net energy ratio of biodiesel produced from microalgae (Nannochloropsis species) was found to be 0.93 MJ (MJ = megajoule) of energy needed to produce 1 MJ of energy. The major advantage of using microalgae-derived biodiesel is the reduction in CO2 equivalent emissions amounting to 75 g MJ-1 of energy produced. Liu et al. (2012) also report that biodiesel from algae has a positive energy impact and estimate 1.4 MJ of energy production per megajoule consumption of energy. In addition, there will be reduction of 0.19 kg CO2-equivalents per kilometer of travel by transport.

Johnson and Wen (2009) adopted two methods for the production of biodiesel from a heterotrophic microalga, Schizochytrium limacinum. The first method adopted was direct transesterification of algal biomass using wet and dry biomass separately. The second method consisted of two steps involving extraction of oil from algae, followed by transesterification of wet and dry biomass. When the direct transesterification method was used, the yield of biodiesel obtained was greater than 66% for wet as well as dry biomass. However, the fatty acid methyl ester (FAME) content in the wet biomass was found to be very low (7.76%) in comparison with that from dry biomass (63.47%). A 57% crude biodiesel yield was obtained through the two-step method with a FAME content of 66.37%. Using the wet biomass, the FAME content of biodiesel was 52.66%. The one-stage direct transesterification method used various solvents (e. g., chloroform, hexane, and petroleum ether) to treat the algal biomass. However, a comparatively higher content of FAME was observed when only chloroform was used as the solvent. It has been found that direct transesterification is preferable instead of the conventional steps involved in the production of biodiesel from microalgae (i. e., extraction of oil from microalgae and transesterification of the expelled oil) as the production cost of the fuel will be reduced. However, the drying of algal biomass was found to be a prerequisite in order to obtain a high yield and conversion of biodiesel when using direct transesterification of the microalgae. To prevent oxidation of the unsaturated FAME in biodiesel, Johnson and Wen (2009) added 100 ppm butylated hydroxytoluene to the biodiesel. However, the fuel did not meet the European standards (EN 14103) specifications, which specify that the ester content in biodiesel must be at least 96.5% (Sarin et al., 2009).

Vijayaraghavan and Hemanathan (2009) reported on the production of biodiesel from freshwater algae. A high lipid content of 45 ± 4% was obtained from the micro­algae and was used for the synthesis of biodiesel by transesterification using metha­nol as a reactant and KOH as a catalyst. Upon transesterification, the fuel properties of biodiesel were determined. The acid value of the biodiesel (0.40 mg KOH g-1) was within ASTM (American Society for Testing & Materials) D 6751 specifications for biodiesel. The values of the other important parameters (i. e., density, ash, flash point, pour point, calorific value, cetane number, water content, and copper strip corro­sion) were characterized. It was found that some of the parameters (i. e., density, ash, flash point, and water content) did not meet the ASTM specifications. The density of biodiesel was found to be low (801 kg m-3), whereas the Indian specifications have a range of 860 to 900 kg m-3. The ash content of the biodiesel was 0.21 mass%, in contrast to the 0.01% specified by Indian standards. Similarly, the flash point also had a low value of 98°C instead of the minimum value of 120°C. The water content (<0.02 vol.%) was also slightly higher than specifications (<0.03 vol.%). However, other parameters (i. e., pour point, calorific value, and cetane number) were found to be within Indian specifications.

An in-situ transesterification method has been adopted by Velasquez-Orta et al. (2012) for the transesterification of microalgae, Chlorella vulgaris. The in situ transesterification of this microalga was performed by combining the two steps of lipid extraction and transesterification into a single step. Although the reaction ran to com­pletion in less time (75 min) using NaOH as the catalyst, a low conversion of FAME (77.6 ± 2.3 wt%) was obtained that does not meet the specifications of the European Union (EN 14103), which specifies that the ester content must be at least 96.5% (Sarin et al., 2009). However, when an acid catalyst (sulfuric acid) was used, a high FAME yield of 96.8 ± 6.3 wt% was obtained although a longer reaction time (20 h) was required. Also, a high methanol ratio (600:1) was employed, which will escalate the production cost of biodiesel. Tran et al. (2012) produced biodiesel from Chlorella vul­garis (ESP-31) using an enzyme (Burkholderia lipase) as a heterogeneous catalyst. The biodiesel was synthesized in two ways: (1) transesterification of microalgal oil, and (2) direct transesterification of the microalgae after disruption of its cells by sonica- tion. A moderate conversion (72.1%) of the microalgae to biodiesel was obtained with the first method, whereas a high conversion (97.25%) was obtained using the second method. The immobilized enzyme was reused for six runs without any significant loss in catalytic activity. Being catalyzed by an enzyme, the catalyst was found to func­tion even in the presence of water (>71.39 wt%). However, a higher molar ratio (67.93, methanol to oil) was needed to achieve an ester conversion of greater than 96 wt% of oil that will escalate the production cost of the biodiesel.

The fuel properties of the biodiesel synthesized from the microalgal oil derived from Chlorella protothecoides has been investigated by Chen et al. (2012). The microalgal oil methyl ester (MOME) with a high ester content (97.7%) demonstrated development of biodiesel of high fuel quality with a cold filter plugging point of -13°C, which is an indication that the fuel may be used even under extremely cold conditions. The vis­cosity of the MOME was 4.43 mm2 s-1 at 40°C, which is within the specifications for biodiesel specified by the ASTM. However, the oxidation stability of the fuel was low (4.5 h), which was due to high amounts of unsaturated fatty acid content in the MOME (i. e., 90.7 wt%). The induction time as per the Indian and European specifications is at least 6 h (Sarin et al., 2009). Hence, it had been recommended that the MOME should use up to 20 vol% blended with mineral diesel. A higher blend of biodiesel in mineral diesel will require the addition of antioxidants so that the fuel does not get oxidized rapidly and remains within specifications. Siegler et al. (2012) extracted oil from the microalgae Auxenochlorella protothecoides and found it to be a potential source for biodiesel production. The degree of unsaturation (DU) in the microalgal oil, which is a measure of the unsaturated fatty acid content, was determined to be 137. Using the DU, the cold filter plugging point value of the biodiesel was expected to be -12°C, which can support the use of fuel even in cold climatic conditions.

Lardon et al. (2009) observed that despite biodiesel derived from microalgae having immense potential to provide an alternative source of fuel, energy and fertilizer consumption should be reduced for its economic viability. Using Chlorella vulgaris as a model species, it has been found that a substantial portion of energy consumption amounting to 70% and 90% of the total energy is used for lipid extrac­tion when using wet and dry biomass, respectively. Hence, technologies must be developed for economical extraction of oil from microalgal cells. Rosch et al. (2012) advocate the reuse of residual algal biomass after oil extraction for the supply of nutrients, which according to estimates may vary from 0.23 to 1.55 kg nitrogen and 29 to 145 g phosphorous (depending on the cultivation conditions of microalgae) for the production of 1 L biodiesel.

Algal biomass is the main product in microalgal technology and has various applica­tions. The final biomass product is usually green or orange in color (Pulz and Gross, 2004). Most commercial fertilizers are derived from petroleum; however, rising fuel prices influence the cost price of commercial fertilizers derived from petroleum. A cost-effective alternative would be the use of algal biomass as organic fertilizers (http://www. algaewheel. com).

Microalgae have been used in the agriculture industry as biofertilizers and as soil conditioners (Metting, 1996). Employing microalgae as biofertilizers and soil conditioners is a common agricultural practice in Asian countries such as China and India, where they provide more than 20 kg nitrogen ha-1y-1. Nitrogen-fixing cyanobacteria such as Anabaena, Nostoc, Aulosira Tolypothrix, and Scytonema are used in rice cultivation. Mucilage-producing species of the genus Chlamydomonas have been used as soil conditioners to control soil erosion of pivot-irrigated soils in North America (Metting, 1996). The rationale behind using microalgae as biofertilizers is that they have the ability to increase the water-binding capacity and mineral composition of the soil (Pulz and Gross, 2004). This market generates a turnover of US$5 billion y-1 (Pulz and Gross, 2004).

Tubular reactors are characterized by very high area-to-volume ratios (dependent on tube diameter) but poor mass transfer, leading to O2 build-up and CO2 depletion (resulting in photorespiration, oxidative damage, and pH gradients) over the length of closed tubing. Tubes are generally manufactured from polyethylene or glass. The most important criteria for construction material are transparency, to allow good light penetration, and low cost (Ugwu et al., 2008). Additional challenges include photo-inhibition, temperature control, and fouling due to cells adhering to the inside of tube walls, leading to decreased light penetration (Ugwu et al., 2008). Narrow — diameter tubes can present a challenge to clean.

Most tubular reactors can be categorized into one of three types:

1. Vertical airlifts or bubble columns consisting of a clear vertical tube mixed by gas sparging from the bottom

2. Horizontal tubular systems with clear, thin-diameter tubing lying or stacked horizontally, usually connected to a gas transfer system

3. Helical tubular reactors consisting of thin, flexible tubing coiled around a circular framework

Airlift and bubble column reactors (Figures 5.2a and b) are examples of vertical tubular reactors. Air, or air enriched with CO2, is bubbled into the bottom, providing efficient mixing and gas transfer throughout the reactor. The simplest form of bubble column reactor is a hanging polyethylene bag, and these have frequently been used as a low-cost option. Plastic bags have a high transparency, good sterility at start-up (due to the high temperatures used in plastic extrusion), and are readily replace­able. Concentrations three times that of open ponds were obtained by culturing Porphyridium in 25 L hanging bags (Cohen and Arad, 1989). Other researchers have also found that 40 to 50 L bags are practical (Trotta, 1981; Martinez-Jeronimo and Espinosa-Chavez, 1994).

Although cultivation in plastic bags is simple, cheap, and widely employed, par­ticularly in the production of microalgae as feed for aquaculture hatcheries, scale-up is limited by the fragility of cheap plastic and light penetration, as increases in bag volume lead to decreased productivity due to mutual shading (Martinez-Jeronimo and Espinosa-Chavez, 1994). Rigid vertical tubes have also been frequently used (Carvalho et al., 2006). In an airlift reactor, an inner tube called the riser directs air bubbles up the center of the reactor and then down the outer region, called the down­comer (Figure 5.2b). This provides effective, gentle mixing and produces regular light-dark cycles.

Vertical reactors are compact, low cost, and easy to clean and keep sterile (Ugwu et al., 2008); however, their size is limited by the surface-to-volume ratio. The scale — up of any tank, container, or hanging bag becomes limited by light penetration at a

Schematic diagram of (a) vertical bubble column and (b) airlift reactor.

volume of between 50 and 100 L. Underwater lighting has been considered, either in the form of submersible lamps, or optical fibers redirecting sunlight, but these add to the cost and energy footprint of the system (Pulz, 2001). In addition, the vertical angle of reactors is not ideal for capturing incident sunlight (Carvalho et al., 2006).

Horizontal tubular reactors are designed to optimize light capture by increasing the angle to sunlight (Figure 5.3). Internal tube diameters range between 1.2 and 13 cm (Lee, 2001). The thinner the tubing used in the solar collector, the more efficient the light capture but the lower the culture volume per length of tubing. Thin tubing is also particularly susceptible to overheating, and temperature regulation mechanisms must be installed—for example, evaporative water cooling (often requiring large volumes of water), immersing the tubes in water, or shading them by covering or overlap­ping the tubes. The length of the closed tubing is constrained by O2 build-up; there­fore, reactors are usually modular, with parallel sets of shorter tubes interconnected, rather than a single long tube. Gas transfer takes place either at tube connections or in a dedicated gas exchange unit, where aeration and mixing are provided either by pump or airlift (Ugwu et al., 2008). There have been successful runs of horizontal tubular reactors with volumes of up to 8,000 to 10,000 L (Torzillo et al., 1986). One of the major disadvantages of horizontal reactors is the large land area required for horizontal tubing. The increased productivity with respect to open ponds may not be cost effective if a greater land area is necessary (Carvalho et al., 2006).

Helical tubular reactors (Figure 5.4) are a promising alternative to horizontal tubular reactors as they reduce the land area required. By expanding vertically,

the areal footprint of the reactor is smaller, but the angle to sunlight is also reduced. Placing a light in the center of the coil can improve light penetration. A conical, instead of helical, framework has also been suggested, as it improves the spatial distribution of tubes for sunlight capture (Morita et al., 2000). However, scale-up is then limited as the angle and height of the coil are defined. One of the most effective designs is the Biocoil developed by Robinson (1987). It consists of a set of polyethyl­ene plastic tubes (2.4 to 5 cm in diameter) wound helically around an open circular framework. Parallel bands of tubes connect to a gas exchange tower. A centrifugal pump is used to move culture to the top of the coil, which may not be suitable for all species due to shear stress in sensitive cells and pump fouling in filamentous species. A heat exchanger or evaporative cooling provides temperature control. The system provides good mixing; minimal cell adhesion to the inside of tubes and scale-up is easy, involving the addition of more parallel coils. Several marine species and Spirulina have been successfully cultivated for more than 4 months in a 700 L Biocoil (Borowitzka, 1999; Carvalho et al., 2006).

An а-shaped reactor, presenting an interesting alternative tube layout, was designed by Lee et al. (1995) (Figure 5.5). The symmetrical design uses airlift pumps to aerate and mix the culture in vertical tubes at either end. This then flows down tubes at a 45° angle, thus maximizing the angle of tubes to sunlight while saving space. As flow through the entire system is in the same direction, with two CO2 injection points, relatively high liquid flow and mass transfer rates can be main­tained with relatively low air supply rates (Lee et al., 1995).

In a number of studies, it has been assumed that the biodiesel production from algal oil should be conducted using the same approach as for vegetable oil. This requires the drying of the algal biomass to 90% solids by mass (Sazdanoff 2006; Lardon et al., 2009; Batan et al., 2010; Yang et al., 2011). Typically, belt or drum drying is used, with energy provided by natural gas. This may represent the major or a significant portion of the energy demand of the process. Sander and Murthy (2010) estimated that 89% of the energy requirement of their “well-to-pump,” raceway — based algal biodiesel system was required for drying the intermediate product prior to conversion to biodiesel, using a natural gas powered dryer. Razon and Tan (2011) attributed 48% of their energy requirement (310 MJ per tonne biodiesel) to drying, following production of 1 kg FAME and 1.5 m3 biogas using a raceway pond. Drying of the biomass is only feasible where natural resources (e. g., solar drying) can be used while preventing lipid oxidation (Lardon et al., 2009).

Furthermore, the requirement of cell disruption for product recovery has also been considered. Razon and Tan (2011) demonstrated that the energy input to the bead mill for disruption of Haematococcus pluvialis formed the greatest contribution to the process energy required (>30%). Stephenson et al. (2010) estimated cell dis­ruption by high-pressure homogenization to account for approximately 5 GJ tonne-1 biodiesel formed and 320 kg CO2 eq tonne-1 biodiesel in GWP. The need for cell disruption is a function of both the flowsheet selected and the algal species.

In a comparison of the hypothetical wet and dry processing routes for the trans­esterification to biodiesel, the benefit of developing an effective wet processing route for which a discrete cell disruption step and rigorous drying are not essential is clearly demonstrated (Lardon et al., 2009). The energy requirement was reduced to 70% to 75% of the dry processing route while the energy recoverable through further processing of the oil cake increased by 67% to 115%. Overall, the additional energy recoverable was 0.6 to 1.1 MJ per 1 MJ biodiesel. Direct esterification in the presence of water has been demonstrated under analytical conditions (Griffiths et al., 2010) and requires further optimization for large-scale use.

The perceptual structure of the physical and chemical characteristics of wastewater can change substantially with changes in stream habitats and their individual patterns. Wastewater physical characteristics include

1. Color: The physical appearance, qualitative significant color of wastewater depends on holding times in tanks, and varies from a light brown to light gray color. The color is known to turn dark gray or black in the event of wastewater going stale. A color change of wastewater is due to fermenta­tion of the various chemical compounds produced, in particular hydrogen sulfide and ferrous sulfide. Color can be measured by comparisons using standard methods.

2. Odor: The offensive odor in wastewater is mainly due to dissolved impuri­ties and a number of odor compounds produced by living enriched microbes and decaying aquatic organisms when under anaerobic conditions. The principal odor-producing compound is hydrogen sulfide, produced as gas by bacterial decomposition under anaerobic conditions.

3. Solids contents: The total solids are made up of both dissolved and sus­pended material that remains as residue in wastewater (Metcalf and Eddy, 1987) upon evaporation at 103°C to 105°C.

4. Temperature: The wastewater temperature will vary season to season and with the geographical location, from 10°C to 21°C (Muttamara, 1996). Temperature plays a major role in wastewater treatment, and its variation may cause changes as a result of the chemical and biological reactions of planktonic organisms. Wastewater contains bacteria and fungi that may have a substantial influence on the physical characteristics of the wastewa­ter, especially when in abundance due to abnormal temperatures. Turbidity and color are indirectly related to temperature because most of the chemical reaction products, including equilibrium of wastewater coagulation, can change with temperature. Temperature is also very important in the deter­mination of various parameters, such as changes in pH often occurring in regions with low acid neutralizing capacity, conductivity, different satura­tion levels of gases, various forms of alkalinity, etc.

The electrolytic coagulation (EC) process has recently been adapted by wastewater treatment plants for final polishing and removal of algae from partly treated waste­water. Active polyvalent metal anodes (usually iron or aluminum) are used to gener­ate ionic flocculants such as Al3+ and Fe3+ ions. The latter agglomerate algae to form flocs due to the net negative charge and colloidal behavior of algal cells (Gao et al., 2010). The entire coagulation process involves the formation of coagulants by dissolution of the reactive anode, destabilization of colloidal suspensions, and aggregation of destabilized suspensions, resulting in the formation of algal flocs. The EC process is a more efficient chemical flocculation technique compared to conventional processes of direct interaction of the aluminum sulfate with algal suspensions (Aragon et al., 1992). The flocculated biomass is removed from water, either by sedimentation or flotation and skimming. For the latter to be effective, inactive metal cathodes are used to generate micro-gas (mainly hydrogen) bubbles that get entrapped in algae flocs and float them to the surface. Complete biomass removal from algal cultures having cell densities of 0.55 x 109 to 1.55 x 109 cells mL-1 has been reported using this process (Gao et al., 2010). However, the process may not be very effective for very dilute algal solutions because at low concentra­tions of total suspended solids (representing algal cells), the amount of colloids pres­ent in the culture solution may not be sufficient for significant amounts of settleable solids (Azarian et al., 2007).

Carotenoid production usually occurs in open pond raceway systems and in pho­tobioreactors, depending on the robustness of the algal strain toward contamina­tion and the purity requirements or application of the final product. Carotenoids such as astaxanthin, lutein, and zeaxanthin are produced in photobioreactors (Dufosse et al., 2005; Milledge, 2011) due to the sensitivity of the algal strains to contamination. Algal strains that have an ability to grow in harsh environments are usually cultivated in open pond raceway systems. As an example, Dunaliella salina, for the production of P-carotene, can grow in high-salinity environments (Dufosse et al., 2005).

Biomass harvesting methods also depend on the algal strain cultivated. The pre­ferred methods of harvesting biomass for carotenoid production are centrifugation, sedimentation, and filtration (Dufosse et al., 2005; Weiss et al., 2008). Subsequent to centrifugation, the hard cell walls are broken and then extraction of the carotenoid occurs (Dufosse et al., 2005).

10.2.1.1 Foresight

Carotenoid production has established itself as the most successful area of micro­algal biotechnology; and with the increasing market demands for these natural pigments, the future of microalgal carotenoid production appears promising (Del Campo et al., 2007). The ability of microalgae to be genetically modified opens doors for enhancing specific carotenoid production through metabolic engineering. However, this approach might not be welcomed by the food and aquaculture indus­tries due to the controversy surrounding genetically modified products. The market demand for carotenoids is expected to increase even further with the discovery that carotenoids exhibit tumor-suppressing activity (Schmidt-Dannert et al., 2000). Carotenoid exploitation is restricted to only a few algal species; more algal strains have yet to be screened.

10.2.2 Phycobiliproteins

Phycobiliproteins are photosynthetic accessory pigments produced by microalgae. These pigments are responsible for improving the efficiency of light energy utilization (Pulz and Gross, 2004). Phycobiliproteins are deeply colored (red or blue), water — soluble complex proteins and have a broad spectrum of potential applications as natural coloring agents in the food and feed, pharmaceutical, and cosmetics industries. Among the cyanobacteria and red algae, there are four main classes of phycobilipro — teins that are synthesized (Table 10.4): allophycocyanin (APC, bluish-green), phycocy — anin (PC, blue), phycoerythrin (PE, purple), and phycoerythrocyanin (PEC, orange).

These phycobiliproteins are geometrically incorporated into structures called phycobilisomes, which are located on the outer surface of the thylakoid membranes.

Ravi V. Durvasula and Durvasula V. Subba Rao

Center for Global Health, Department of Internal Medicine University of New Mexico School of Medicine and The Raymond G. Murphy VA Medical Center Albuquerque, New Mexico, USA

Vadrevu S. Rao

Department of Mathematics

Jawaharlal Nehru Technological University Hyderabad Kukatpally Campus, Hyderabad, India

CONTENTS

13.1 Introduction…………………………………………………………………………………………….. 201

13.2 Cultivation………………………………………………………………………………………………. 203

13.3 Native Strains, Consortia of Species, and Extremophiles……………………….. 206

13.4 Variations in Algal Production: Crucial but Ignored……………………………….. 208

13.5 Lipid Variations: Physiological State………………………………………………………. 210

13.6 Biochemical Manipulation: Higher Yields……………………………………………… 213

13.7 Harvesting………………………………………………………………………………………………. 214

13.8 Genetic Modification of Algae……………………………………………………………….. 216

13.9 Summary………………………………………………………………………………………………… 217

Acknowledgments…………………………………………………………………………………………….. 220

References…………………………………………………………………………………………………………. 220

13.1 INTRODUCTION

As the global use of energy is projected to increase fivefold by 2100, several countries are investing in microalgal biotechnology as a source of renewable energy to enhance their energy security. Although microalgae are a source of high-value chemicals such as nutraceuticals and pharmaceuticals, with a gold-rush mentality many entrepreneurs focus primarily on biofuel as an end-product utilizing a few selected “traditional” algal species not native to the region, and extrapolate results obtained from controlled laboratory culture to large-scale outdoor production systems. For optimization of harvesting algal biomass, it would be crucial to know that wide intra — and interspe­cific variations in the biochemical constituents of microalgae exist, depending on their growth conditions. For example, in eight algal species, the percent lipid per dry weight ranged from 5 to 63, lipid production 10.3 to 90 mg L-1d-1, biomass 0.003 to

2.5 g L-1d-1, and biomass production on an areal basis from 0.91 to 38 g m-2d-1. Also, the commercially important carotene content in Dunaliella strain B32 and strain I3 isolated from the Bay of Bengal varied from 0.68 pg carotene per cell to 17.54 pg carotene per cell. As microalgae are renewable, sustainable, and affordable, their potential to produce biofuels and bioactive compounds is great. However, we argue that (1) improvements in strain selection, particularly the extremophile microalgae that have the required properties for large-scale biotechnology; (2) biochemical mod­ification; (3) utility of engineered “designer algal strains”; (4) optimization of growth, biomass production, and harvesting; and (5) enhancement of extraction of biofuel and conversion to co-products would all be necessary to make microalgal biotechnology an economically viable enterprise. A robust bio-economy built on a platform of inno­vative microalgal technologies is recommended.

Photosynthetic microalgae have been cultivated (Miquel, 1893) and utilized to support the production of animal life in the sea (Allen and Nelson, 1910). The most common “traditional” species used for biotechnology, usually isolated from temper­ate waters, include Botryococcus braunii, Chaetoceros calcitrans, Chlamydomonas reinhardtii, Chlorella vulgaris, Chroomonas sp., Dunaliella bardawil, D. salina, D. tertiolecta, Haematococcus pluvialis, Isochrysis galbana, Nannochloropsis oculata, Neochloris oleoabundans, Phaeodactylum tricornutum, Rhodomonas sp., Scenedesmus obliquus, Skeletonema costatum, Spirulina maxima, and Tetraselmis chuii. Usual practice involves the purchase of a few “traditional” species from a culture center for large-scale propagation, although quite a few researchers are look­ing at isolating species adapted to local environments.

In addition to utilizing algae as biofeed, there is a global surge in microalgal biotechnology activities for commercial applications such as biofuel, bioactive com­pounds, and bioremediation. From virtually none in 1990, the total number of publi­cations on microalgal biotechnology leapt to 153 by June 2011; of these, 103 were on microalgal biofuel. This surge coincides with the 1991 Gulf War, when the mind-set of several countries changed to reduce their dependence on imported crude oil and to enhance their energy security. The annual worldwide consumption of motor fuel is 320 billion gallons, of which United States accounted for 44% (http://eia. doe. gov/ pub/internationjal/iea 2005/table35.xls). At the current rate of usage, the global use of energy will increase fivefold by 2100 (Huesmann, 2000), prompting major invest­ments in renewable energy. Since 2007, the United States alone has injected more than $1 billion into algae-to-energy research and development.

Microalgal biotechnology has received global attention and the attributive advantages include (1) cultivability on nonarable land, (2) bioremediation of wastewater by growing photosynthetic algal biomass, (3) ease of access to metabolic products that are stored intracellularly, (4) production of biofuel and value-added co-products, and (5) carbon sequestration, a result of the accelerated growth of microalgae for biofuel production. Photosynthetic production of algal biomass can be enhanced by an extraneous enrichment with CO2; industrial effluents containing CO2 can be utilized to sustain high algal productivity (Raven, 2009; Benemann, 1993). This could help a nation lower its emissions of greenhouse gases and could be used for carbon tax credit. The International Energy Agency (IEA) estimated that biofuels contribute to approximately 2% of global transport fuel today but could increase to 27% by the year 2050. They project that if biofuel production is sustained, it could displace enough petroleum to avoid the equivalent of 2.1 Gt y-1 CO2 emission— comparable to the net CO2 absorbed by the oceans calculated by Fairley (2011).

The algae-to-biotechnology framework has five stages—that is, algal cultiva­tion, biomass harvesting, algal oil extraction, oil residue conversion, and by-product distribution—and each has several composite processes (Natural Resources Defense Council, 2009). Given the vast potential of microalgal biotechnology, many entre­preneurs focus largely on algal biomass as a source of biofuel rather than high-value chemicals such as nutraceuticals and pharmaceuticals. For example, by the end of this decade, the projected worldwide market value of carotenoids alone will be US$1,000 million (Del Campo et al., 2007). Some of the co-products fetch higher prices; for example, astaxanthin is about 3,000 times more expensive than the $1,000-per-ton crude oil (Cysewski and Lorenz, 2004). Although the payoffs for entre­preneurs are attractive, building biotech businesses based on a new, unproven technol­ogy poses more formidable challenges. Continuous production of vast quantities of algal biomass under optimal conditions is crucial in sustaining economically viable biofuel technology. Although fifty algal biofuel companies exist (http://aquaticbiofuel. com/2008/12/05/2008-the-year-of algae-investments/.), production on a commercial scale at competitive prices has not yet taken place (Pienkos and Darzins, 2009; St. John, 2009). One of the biggest challenges to commercial algal operations is to trans­late laboratory conditions to large scale, and most companies operate in “stealth” mode (Natural Resources Defense Council, 2009). To make it cost effective, Wijffels (2007) suggested that production costs must be reduced up to two orders of magni­tude. When operating an algal biofuel production facility, plans should be in place to tackle unforeseen exigencies such as weather changes, and crashing of algal populations that could disrupt production and cause huge losses. As microalgae are renewable, sustainable, and affordable, their potential to produce biofuels is great if the current practices are cost competitive with petroleum diesel. Improvements in harvesting practices, extraction of biofuels, and conversion to co-products could bring down the production costs. Here we discuss the need to optimize various ele­ments such as algal strains, cultivation, production costs, lipid variations, harvesting biomass, and genetic modification of microalgae to make microalgal biotechnology economically viable.

Melinda J. Griffiths

Centre for Bioprocess Engineering Research University of Cape Town, South Africa

CONTENTS

5.1 Introduction………………………………………………………………………………………………… 51

5.2 Growth Requirements and Design Parameters……………………………………………. 52

5.2.1 Light……………………………………………………………………………………………….. 52

5.2.2 Temperature…………………………………………………………………………………… 55

5.2.3 Nutrient Provision…………………………………………………………………………… 56

5.2.4 Mixing…………………………………………………………………………………………….. 57

5.3 Cultivation Systems……………………………………………………………………………………. 58

5.3.1 Open Systems…………………………………………………………………………………. 58

5.3.1.1 Natural Waters…………………………………………………………………. 59

5.3.1.2 Circular Ponds………………………………………………………………….. 59

5.3.1.3 Raceway Ponds……………………………………………………………… 59

5.3.2 Closed Systems…………………………………………………………………………….. 61

5.3.2.1 Tubular Reactors……………………………………………………………. 62

5.3.2.2 Flat-Plate Reactors…………………………………………………………. 65

5.3.3 Alternative Designs……………………………………………………………………….. 66

5.3.3.1 Stirred Tank Fermenter…………………………………………………….. 66

5.3.3.2 Wave/Oscillatory Flow Reactors……………………………………. 66

5.3.3.3 Hybrid Production Systems…………………………………………….. 68

5.4 Comparison of Reactor Types……………………………………………………………………. 68

5.4.1 The Open versus Closed System Debate………………………………………… 68

5.5 Conclusion………………………………………………………………………………………………….. 72

References…………………………………………………………………………………………………………… 72

5.1 INTRODUCTION

One of the most important factors in achieving economically and environmentally feasible commercial-scale production of microalgae is the development of cost — effective, sustainable culture systems (Borowitzka, 1999; Richmond, 2000). The design of the cultivation system influences the environmental conditions experienced by the cells, which in turn determine the productivity (Greenwell et al., 2010).

Improving productivity is key to achieving economic viability in large-scale, outdoor cultures (Lee, 2001).

Microalgal cultivation has been carried out in a variety of vessels, ranging from natural open lakes and ponds to highly complex and controlled photobioreactors (PBRs). Typically, the term photobioreactor has been used to refer to closed systems exclusively; however, by definition, open systems are also PBRs. A bioreactor is a container in which living organisms are cultivated and carry out biological conver­sions (e. g., biomass or product formation) (Grobbelaar, 2009). A PBR is a reactor in which organisms that obtain energy from light, such as algae, plants, and certain microbial cells (phototrophs), are used to carry out reactions (Mata et al., 2010). Each type has advantages and disadvantages, but the overall goals are similar. In the design of commercial algae cultivation systems, the aim is to achieve:

• Optimal volumetric and/or areal productivity

• Efficient conversion of light energy to product

• Consistency and reliability of production

• Cost effectiveness

Effective reactor design requires knowledge of both algal physiology and reac­tor engineering, such as aspects of hydrodynamics and mass transfer (Ugwu et al., 2008). Section 5.2 outlines the key requirements for algal growth and how these relate to design considerations of the cultivation system. Section 5.3 describes the range of open and closed systems that have been used for microalgal cultivation. These are compared with respect to a range of attributes in Section 5.4.

The major cost attributed to the production of biodiesel is the dewatering and drying step, which consumes 9 to 16 GJ of energy per ton of biodiesel produced (Chowdhury et al., 2012). The dewatering and drying step can be negated if the microalgal oil is subjected to pyrolysis for the synthesis of bio-oil as a biofuel. The thermochemical method adopted for the preparation of fuel from microalgae is through pyrolysis, where the organic compound is thermally decomposed at a high temperature in the absence of oxygen. Zou et al. (2009) produced bio-oil by thermochemical catalytic liquefaction of Dunaliella tertiolecta. A yield of 97.05% was obtained. The reaction conditions were optimized and found to be H2SO4 (2.4 wt%); reaction temperature, 170°C; and reaction time, 33 min. A high-quality bio-oil was produced that possessed significant ester content. The bio-oil also possessed a low ash content of 0.4% to 0.7%. However, the product had a low pH value (3.8 to 4.0) and thus necessitates storage in acid-resistant bottles (e. g., polypropylene or stainless steel). Thermochemical treat­ment resulted in a high calorific value of 28.42 MJ kg-1. The bio-oil also had a low nitrogen content compared to bio-oils produced by methods such as pyrolysis or direct liquefaction. A high oxygen content was observed, thus providing the requirement for deoxygenation of the bio-oil. The composition of microalgae bio-oil obtained through thermochemical catalytic liquefaction consists of several methyl and ethyl esters, which result from esterification between organic acids and the glycol solvent, and is similar to that of biodiesel. Campanella et al. (2012) performed thermolysis of microalgae (consisting of mixed wild culture with Scenedesmus sp. as the principal constituent) and duckweed (primarily Wolffia and Spirodela species) in a fixed-bed reactor using CO2 as a sweep gas for the synthesis of bio-oil and called it “bioleum.” The thermolysis of microalgae gave a higher bioleum yield in comparison to that from the duckweed. This is attributed to the difference in composition of the two feed­stocks. The fuel properties of the bioleum were found comparable to heavy petroleum crude oil. The use of microalgal oil over lignocellulosic materials for pyrolysis has advantages in the form of lower oxygen concentration and a higher heating value for the former. The heating rate during the thermolysis of microalgae was found to be an important parameter in the formation of the bioleum, where the slow ther­molysis did not produce a liquid fuel that could be used as fuel. Maddi et al. (2011) reported that the pyrolysis products of algal (primarily consisting of Lyngbya sp. and Cladophora sp.) and lignocellulosic biomass (corncobs, woodchips, and rice husk) gave a similar yield of bio-oil. Other compounds that formed along with the bio-oil were bio-char, gases, and ash. The calorific value of lignocellulosic bio-char (except rice husk) was higher than that of algae-derived bio-char. This has been attributed to the higher carbon content in the lignocellulosic biomass. The difference in composi­tion of the bio-oil in the two feedstocks was the presence of nitrogenous compounds in the algal bio-oils. This is assumed to have occurred through degradation of the proteins present in the algae and may decrease its fuel value.

Chakraborty et al. (2012) synthesized bio-oil from Chlorella sorokiniana in a two — step sequential hydrothermal liquefaction technique to produce bio-oil and valuable co-products. The bio-oil consisted of 76% carbon, 12% hydrogen, 11% oxygen, 0.78% nitrogen, and 0.16% sulfur. The low nitrogen content avoids the denitrogenation step involved in the production of bio-oil. High oxygen content in the bio-oil necessi­tates further processing viz. hydrogenation to improve its quality. The yield of the bio-oil obtained by this method consisted of 24% of the dry weight, and optimum polysaccharide extraction occurred at 160°C. The advantage of the two-step sequen­tial hydrothermal liquefaction technique over the direct hydrothermal liquefaction technique was a low formation of bio-char in the former (i. e., 7.6% in comparison to 20.8% in the latter). Li et al. (2012) synthesized bio-oil from the marine brown microalgae, Sargassum patens C. Agardh, via hydrothermal liquefaction within a modified reactor. A comparatively moderate yield of 32.1 ± 0.2 wt% bio-oil was obtained in 15 min at 340°C. The feedstock used had a concentration of 15 g biomass per 150 mL water. The bio-oil obtained had a heating value of 27.1 MJ kg-1. The major constituent of the bio-oil was carbon (64.64%), followed by oxygen (22.04%), hydrogen (7.35%), nitrogen (2.45%), and sulfur (0.67%). The characterization of the bio-oil by infrared spectroscopy showed a diverse group of compounds consisting of fats, alkanes, alkenes, alcohols, ketones, aldehydes, carboxylic acids, phenol, esters, ethers, aromatic compounds, nitrogenous compounds, and water. A high concentra­tion of water may be the reason for the low calorific value of the bio-oil produced from the microalgae. Pie et al. (2012) carried out the co-liquefaction of a Spirulina and high-density polyethylene (HDPE) mixture in sub — and super-critical ethanol at a reaction temperature of 340°C to obtain bio-oil. The bio-oil thus produced was similar to that obtained from the pure HDPE derived bio-oil. The benefit of the co­liquefaction process of Spirulina and HDPE was the synthesis of bio-oil that pos­sessed a high calorific value (48.35 MJ kg-1) due to higher “carbon” and “hydrogen” contents and a lower oxygen content. The samples analyzed by gas chromatograph — mass spectroscopy (GC-MS) showed different compositions for bio-oil derived from Spirulina, HDPE, and Spirulina-HDPE mixture. While the bio-oil derived from Spirulina consisted of oxygen-containing compounds along with fatty acids, fatty acid esters, and ketones as prominent compounds, the bio-oil derived from pure

HDPE consisted of a wide spectrum of hydrocarbons, including saturated and unsat­urated aliphatic hydrocarbons. The bio-oil component obtained from the mixture of Spirulina and HDPE possessed more hydrocarbons and less oxygen-containing com­pounds. Hence, the product of the co-liquefaction of Spirulina and HDPE was similar in nature to that of pure HDPE liquefaction with a lower reaction temperature needed for thermal degradation of the feedstock. Hu et al. (2012) utilized the microwave — assisted pyrolysis of Chlorella vulgaris for the production of bio-oil with a yield of 35.83 and 74.93 wt% using microwave powers of 1,500 and 2,250 W, respectively. It was found that using activated carbon as a catalyst could enhance the bio-fuel yield to 87.47%. The calorific value of the microalgae was determined to be low (21.88 MJ kg-1).

Tabernero et al. (2012) evaluated the industrial potential for production of bio­diesel from Chlorella protothecoides. It has been estimated that supercritical fluid extraction (supercritical CO2) for biomass covering a surface area of 7,500 m2 could generate 10,000 tonnes biodiesel per year in a 150-m3 bioreactor. Lohrey and Kochergin (2012), in an attempt to minimize the energy consumption of algal bio­fuels, suggested locating a biodiesel plant close to a sugar mill plant to complement one another. It has been estimated that a cane sugar mill that discards 15% excess bagasse of 10,000 tonnes-per-day capacity can support a 530-ha algae farm to pro­duce 5.8 million L biodiesel per year and will also reduce CO2 emissions from the mills by 15%. The input in parameters of CO2, energy, and water are estimated at 2.5 kg kg-1, 3.4 kW-h kg-1, and 1.9 L kg-1, respectively, of algae dry weight.

The fatty acid composition of feedstock plays a significant role in the quality of the biodiesel produced. The European Standard (EN 14214) has limited the linolenic acid (C18:3) content, to not more than 12%. Wu et al. (2012) studied Chlamydomonas sp. as a potential feedstock for the synthesis of biodiesel. It was found that Chlamydomonas sp. possessed linolenic acid less than 12% and an oleic acid (a monounsaturated fatty acid) constituent of 31.6%. The almost equal compositions of saturated and unsaturated fatty acids in Chlamydomonas sp. are desirable for a trade-off between the oxidation stability and low-temperature prop­erty of the biodiesel. The FAME (fatty acid methyl ester) content in biodiesel was found to be 25% of total volatile suspended solids from microalgae cultivated using municipal wastewater (Li et al., 2011). Although the ester content in the biodiesel was low, the utilization of microalgae for the production of lipids coupled with wastewater treatment has environmental and economic significance. Upon increas­ing the ester content in the biodiesel by improving the technology, the process will become far more attractive. Lam and Lee (2012) are of the opinion that biodiesel production will be the ideal product with microalgae as feedstock. To ensure cost effectiveness, the residual biomass after lipid extraction can contain high concentra­tions of carbohydrates, which should be further utilized for bio-oil and bio-ethanol production. Table 8.1 depicts the ester content and calorific value of the biofuels (biodiesel and bio-oil).

A unique method of thermal analysis to differentiate the oleaginous and non­oleaginous microorganisms (fungi, algae, and yeasts) was developed by Kang et al. (2011). Along with the synthesis of biodiesel, algal biomass residue can be used for other purposes. A linear relationship was observed between exothermic heat and

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TABLE 8.1 Comparison of the FAME Ester Content and Calorific Value of the Biodiesel and Bio-oil from Microalgae Microalgae Method for Extraction of Oil Process Product FAME Content HHV/Calorific Value Ref. Unidentified Using hexane as solvent Transesteri fication Biodiesel Not reported 40 MJ kg-1 11 Chlorella vulgaris (ESP 31) Sonication to disrupt the cell wall Transesteri fication Biodiesel 72.1% (from extracted — 13 Chlorella sorokiniaria of microalgae and then vigorously mixing the disrupted cell with biphasic solvent of chloroform & methanol Two-step sequential hydrothermal Bio-oil microalgal oil) 97.25% (upon direct disruption of algal biomass) 40.8 MJ kg-1 22 Sargassum patens C. Agardh liquefaction Hydrothermal liquefaction _ Bio-oil _ 27.1 MJ kg-1 23 Sp і m І і nafH D P E Co-liquefaction — Bio-oil — 48.35 MJ kg-1 24 Chlorella vulgaris Microwave-assisted pyrolysis — Bio-oil — 21.88 MJkg-la 25 Low calorific value reported that of microalgae Chlorella vulgaris.

the total lipid content in the tested microorganisms. The exothermic heat per dry sample mass (kJ g-1) in the temperature range from 280°C to 360°C differentiated the oleaginous from the non-oleaginous microorganisms. It was found that the heat evolved from the oleaginous microorganisms was larger than that from the non­oleaginous microorganisms in the specified temperature range. The sharpness of the exothermic peak was also more distinct in the oleaginous microorganisms. Kim et al. (2011) utilized the residual biomass of Nannochloris oculata as a biosorbent for the removal of chromium from aqueous solutions. The biological route can also be adopted for biodiesel synthesis. It is anticipated that biodiesel production will increase in the coming years and there will be large amounts of residual biomass that can be used for the treatment of wastewater. The process for the synthesis of biodiesel and bio-oil from microalgae can be depicted through a flowchart (Figure 8.1).

Microalgae

/

Cultivation
k

Harvesting

М/

De-watering and concentrating microalgae

Extraction of oil/lipids
l/

Crude lipids (Neutral lipids & Pigments)
k

Neutral lipids (triglycerides, free fatty acids, hydrocarbons,
sterols, wax and sterol esters, and free alcohols)

Separation

Triglycerides and free fatty acids

Pyrolysis/thermochemical catalytic liquefaction

4/

Biodiesel

FIGURE 8.1 Steps in the production of bio-oil and biodiesel from microalgae.