In general, algal oils contain a higher degree of polyunsaturated fatty acids (PUFA) (i. e., more than four double bonds) than vegetable oil (Belarbi et al., 2000; Harwood and Guschina, 2009) and higher free fatty acid content (>2%). ASTM D6751 (United States) and EN 14214 (European Union) provide the specifications for pure biodiesel (B100, Table 7.1) and are used in many parts of world for comparing the fuel proper­ties of biodiesel. The biodiesel standards developed in many countries are based on the availability of region-specific biodiesel feedstocks. The specifications developed for ensuring biodiesel fuel quality are frequently subjected to modifications, and biodiesel-producing countries are required to update their specifications according to changes in ASTM — or EN-based biodiesel standards.

The lipid composition of algal oil is different from plant oils/animal fats, and it varies with species and growing conditions (Mutanda et al., 2011). Major fuel and chemical properties considered for the selection of an alternate diesel fuel are

kinematic viscosity (KV), higher heating value (HHV), Cetane Number (CN), den­sity, flashpoint, cold flow properties (cloud and pour points), carbon residue, oxi­dation stability, ash content, ignition quality, acid value (AV), saponification value (SV), and iodine value (IV). These properties can be compared with well-established international fuel standards for ensuring fuel quality for diesel engine applications. Properties such as SV, IV, and CN are considered more important for assessing alternate diesel fuels because they give basic information about the ignition quality of fuel, the presence of unsaturated fatty acids (UFAs), and the ignition properties of FAMEs, respectively. The higher iodine values of algal oil indicate the presence of higher UFAs, and heating these UFAs may be lead to the formation of deposits due to the polymerization of glycerides at high temperatures (Mittelbach, 1996; Ramos et al., 2009). The algal oils/FAMEs containing higher degrees of unsaturation are not recommended for biodiesel. The values of the SV, IV, and CN can be easily calcu­lated from the lipid profiles of algal oil using equations developed by Krisnangkura (1986) and Kalayasiri et al. (1996).

Based on the lipid profiles of identified strains/species grown on a laboratory scale, they can be easily screened for suitability in biofuel production. Based on cor­relations developed between fatty acid compositions and the fuel properties of oils, the fuel quality of biodiesel derived from selected algal oils can be predicted through the lipid composition. Hence, it is necessary to determine the fatty acid profiles of extracted algal lipids for suitability in biodiesel production and assess the fuel qual­ity. It is essential for biodiesel derived from microalgal oil to meet ASTM (2008) or EN (2003) biodiesel standards for ensuring fuel quality.

Microalgae are responsible for the production of a range of lipids, with contents varying from 1% to 70% of dry weight, and reaching up to 90% (Metting, 1996, Spolaore et al., 2006). However, the most significant contribution to the overall microalgal market by algae is their ability to synthesize PUFAs (polyunsatu­rated fatty acids). Potential applications of various microalgal PUFAs are given in Table 10.6.

The omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are of particular interest as they cannot be efficiently synthesized by humans, and instead must be consumed in their diet (Simopoulos, 1999). Over the years and even today, omega-3 EPA and DHA are regarded as common constituents of fish oil. Table 10.6 presents microalgal producers of interesting PUFAs, and presently DHA is the only algal PUFA that is commercially available (Spolaore et al., 2006).

TABLE 10.6

Essential Microalgal PUFAs

Potential Application Microalgal Producer

Infant formula Arthrospira

(full-term infants) (Spirulina) sp.

Nutraceuticals

Microalgae produce tri — and di-glycerols, phospho- and glycol-lipids, and hydrocar­bons (Chisti, 2007). Although claims regarding yield per acre are often exaggerated, third-generation microalgal biomass could yield 58,700 L biodiesel ha-1y-1, or even 90,000 L ha-1y-1, comparable to 53,200 L ha-1y-1 (Weyer et al., 2010), an order of magnitude greater than the yields from first-generation biofuel crops (Chisti, 2007).

The mode of cultivation of algae is reflected in biomass and lipid yield (Table 13.3). Lipids as a percent of dry cell weight ranged between 1.9 and 75, and Botryococcus braunii yielded the highest percent (Malcata, 2010). Pienkos et al. (2011) summarized lipids (% of DW) in the range of 9.8% in cyanobacteria, 22.7% to 37.8% in diatoms, 25.5% to 45.7% in green microalgae, and 27.1% to 44.6% in other eukaryotic algae. Lipid production in autotrophic algae ranged from 0 to 2500 mg L-1d-1, and the high­est was in Chlorella protothecoides (Chen et al., 2011). Areal production ranged from 0.57 to 38 g m-2d-1, and Dunaliella salina was the most productive (Mata et al., 2010).

Heterotrophy promotes faster growth and lipid accumulation. Compared to pho­totrophic cultures, cultures of Chlorella protothecoides grown heterotrophically had higher values of biomass productivity (1.7 to 7.4 g L-1d-1) and lipid productiv­ity (732.7 to 3,701.1 mg L-1d-1), with lipid as percent dry cell weight ranging from 43% to 57.8% DW. C. protothecoides, when grown under heterotrophic conditions, yielded 55% lipid per cell dry weight (Xu et al., 2006). In mixotrophic cultures of C. protothecoides using glucose/acetate, higher levels of biomass (4.76 ± 1.50 g L-1d-1), biomass productivity (1.59 ± 0.50 g L-1d-1), and lipid productivity (0.25 g L-1d-1) were obtained; but because the cost of the raw materials was unacceptable, glycerol and acetate were used as carbon sources (Heredia-Arroyo et al., 2010). With glyc­erol, the corresponding values were 3.97, 0.93, and 0.19 g L-1d-1 (Heredia-Arroyo et al., 2010). However, in phototrophic cultures of C. protothecoides, corresponding

TABLE 13.3 Summary of Inter-specific and Intra-specific Variations in Microalgal Lipid Criteria Example Min. Max. Remarks3 Ref. Lipid Chlorella protothecoides 43 57.8 Heterotrophic cultivation Chen et al., 2011 (% of cell dry weight) 11 23 Chen et al., 2011 32 species 5 67.8 Chen et al., 2011 44 species 5 63 Mata et al., 2010 Several species 5 63 Chisti, 2007; Khan et al., 2009; Harun et al., 2010; Pitman et al., 2011; Chen et al., 2011 Scenedesmus dimorphus 6 Gouveia and Oliveira, 2009 Botryococcus braunii 25 75 Chisti, 2007 Schizochrtrium sp. 77 Chisti, 2007 Scenedesmus obliquus 11 55 Gouveia and Oliveira, 2009 Chlorella vulgaris 14 55 Gouveia and Oliveira, 2009 Chlorella protothecoides 23 55 Gouveia and Oliveira, 2009 Neochloris oleoabundans 35 65 Gouveia and Oliveira, 2009 Nannochloropsis oculata 31 Log phase Chiu et al., 2009 Nannochloropsis oculata 40 Early stationary phase Nannochloropsis oculata 50 Stationary phase Nannochloropsis sp. 22 60 Rodolphi et al., 2009 Lipid production Chlorella protothecoides 733 3701.1 Heterotrophic cultivation Chen et al., 2011 (mg L-1d-1) Chlorella protothecoides 0 5.4 Chen et al., 2011 32 species 0 178.8 Chen et al., 2011 Chlorella protothecoides 0 1214 Huerlimann et al., 2010 a All values are for autotrophic cultivation unless specified otherwise.

TABLE 13.3 (Continued) Summary of Inter-specific and Intra-specific Variations in Microalgal Lipid Criteria Example Min. Max. Remarks3 Ref. 27 species 0 133 Huerlimann et al., 2011 Several species 3 2500 Chisti, 2007; Khan et al.. 2009; Harun et al„ 2010; Pitman et al., 2011; Chen et al., 2011 Nannochloropsis sp. 30 86.3 Chiu et al., 2009 44 species 10.3 142 Mata et al„ 2010 pg lipid cell-1 4 species 0 29.108 Huerlimann et al., 2010 All values are for autotrophic cultivation unless specified otherwise.

values were 0.002 to 0.02 g L-1d-1 biomass, 0.2 to 5.4 mg L-1d-1 lipid, and 11% to 23% lipid dry cell weight (Chen et al., 2011). Twenty-one other phototrophic spe­cies had a range of biomass production rates from 0.02 to 0.53 g L-1d-1, 0.2 to 178.8 mg L-1d-1 lipid, and 5.1% to 67.8% lipid dry cell weight. Calculation of lipids on a cell basis also varied from 0.068 to 29.11 pg cell-1 (Huerlimann et al., 2010).

The data on lipid variations given by Chisti (2007), Khan et al. (2009), Harun et al. (2010), Basova (2005), Huerlimann et al. (2010), Mata et al. (2010), Malcata

(2010) , and Chen (2011) summarized in Table 13.3 show that wide inter — and intra­specific variations in lipid levels as percent dry cell weight exist. For example, the lowest (6%) was in Scenedesmus dimorphus (Gouveia and Oliveira, 2009), com­pared to 75% in Botryococcus braunii (Chisti, 2007) and 77% in Schizochrtrium sp. (Chisti, 2007). Gouveia and Oliveira (2009) reported a wide range of values within the same species: 11% to 55% in Scenedesmus obliquus, 14% to 56% in Chlorella vulgaris, 23% to 55% in Chlorella protothecoides, and 35% to 65% in Neochloris oleoabundans. Chisti (2007) reported 25% to 75% in Botryococcus braunii. These variations could be attributed to variations in the physiological state of the cells; marked differences in the lipid were noticed in cultures har­vested in logarithmic, late logarithmic, and stationary phases of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. (Huerlimann et al., 2010). Results of Chiu et al. (2009) corroborate that lipids vary with the phase of growth of the alga Nannochloropsis oculata; lipids were 30.8% in log phase cultures, 39.7% in early stationary phase, and 50.4% in stationary phase cells (Chiu et al., 2009). In Nannochloropsis sp., the lipid as percent dry cell weight ranged from

21.6 to 60 (Rodolfi et al., 2008; Chiu et al., 2009), and their production rates correspond to 30 mg L-1d-1 and 86.3 mg L-1d-1, respectively. Sturm and Lamer

(2011) , based on energy evaluation from wastewater algal biomass production, concluded that if the lipid in dry biomass from the field is less than 10%, com­pared to 50% to 60% in laboratory-scale reactors, it would be better to use the biomass as a combustible source of viable energy.

Mixing is of paramount importance in microalgal culture systems as it is directly linked to other key parameters such as light provision, gas transfer, and nutrient provision. Good mixing keeps the cells in suspension, eliminates thermal stratifica­tion, determines the light-dark regime by moving cells through an optical gradient, ensures efficient distribution of nutrients, improves gas exchange, reduces mutual shading at the center of the reactor, and decreases photo-inhibition at the surface (Ugwu et al., 2008). Mixing affects the mass transfer rates of dissolved nutrients and gases by reducing the boundary layer between the surface of cells and gas particles and the bulk liquid (Grobbelaar, 2009). The synergistic effect on several parameters at once means that mixing efficiency has a strong effect on growth rate.

One of the major differences between open and closed reactors is the degree of tur­bulence achieved. Higher turbulences are more easily achieved in closed PBRs with narrow tubes or plates. Mixing in open ponds is typically provided by a paddlewheel or rotating arm. In closed reactors, mixing can be achieved mechanically (by pumping or stirring) or by aeration via a variety of gas transfer systems (e. g., bubble diffusers, pipes, blades, propellers, jet aerators, or aspirators). Stirring is efficient but incurs high mechanical stress. Mixing by gas injection is relatively gentle and efficient, but may require energy intensive gas pressurization. Gas introduced into reactors can serve a number of purposes, including supply of nutrients, control of pH, stripping of O2, and mixing. Bubbling of CO2 into the bottom of reactors is generally favored, although it achieves only moderate transfer efficiencies (13% to 20%) due to loss of CO2 to the atmosphere, fouling of diffusers, and poor mass transfer (Kumar et al., 2010).

Mixing must be almost continuous to prevent settling of biomass (Molina Grima, 1999) and can represent the major energy input into reactor maintenance. High rates of mixing can also impose shear stress on microalgal cells, particularly in filamen­tous species or those with delicate morphology (Greenwell et al., 2010). Mixing rates are therefore a trade-off among enhanced growth rate, cell damage, and energy requirement.

Microalgae are well recognized for their potential to contribute as an important energy source as well as to provide a renewable feedstock for commodity organic products. Their role as a renewable energy source has promise for both the pro­duction of liquid fuels, including diesel, gasoline, and jet fuels, and electricity generation. Routes to these energy sources include accumulation of algal oil for transesterification to biodiesel, fermentation to alcohols for inclusion in gasoline, production of hydrogen, anaerobic digestion to biogas, thermal processing to bio-oil, co-combustion, and gasification (Chisti and Yan, 2011).

The technical feasibility of algal biomass as a source of bioenergy has been demon­strated for a number of the products described above. Further evidence of the commer­cial interest in these products is demonstrated by both the R&D investment of leading energy companies, including Exxon, BP, Chevron, Shell, and Neste Oil (Norsker et al., 2011), as well as a number of start-up companies attempting commercialization of algal fuels (Table 9.1). However, it is recognized that these products remain expensive in comparison to petroleum-based products. Further, it is essential to understand the environmental benefits of these process routes objectively. Optimization of the product spectrum in terms of economic and environmental sustainability is best informed by a rigorous analytical approach that informs the key process targets for their improvement.

This chapter focuses on the economic and environmental impacts of the production of algal oil and biodiesel as primary products, owing to the comprehensive analysis of these routes. Processing of algal biomass to secondary products in a biorefinery approach is included to explore the potential of algal energy more completely. The alternative bioenergy products are not considered comprehensively here.

TABLE 9.1

Start-Up Companies for Algal Biofuels

Source: From Chisti and Yan (2011).

11.4.1 ^-Carotene

The biomass of certain microalgae could find application as food supplements due to their nutritional content other than proteins. The P-carotene content of Dunaliella salina, a halotolerant green microalga, can reach up to 14% of dry weight (Metting, 1996). P-Carotene, a component of the photosynthetic reaction center, accumulates as lipid globules in interthylakoid spaces of chloroplasts of alga (Vorst et al., 1994). It contributes to light harvesting and protects the alga from oxidative damage dur­ing excessive irradiance by quenching the triplet-state chlorophyll or by reacting with singlet oxygen (1O2), thus preventing the formation of reactive oxygen species (Demming-Adams and Adams, 2002; Del Campo et al., 2007; Raja et al., 2007; Telfer, 2002). The beneficial effects of P-carotene on human health are attributed to its antioxidant properties (Guerin et al., 2003; Higuera-Ciapara et al., 2006; Hussein et al., 2006), and several studies have indicated that adequate intake of carotenoids has the ability to prevent degenerative diseases (Astorg, 1997; Demming-Adams and Adams, 2002; Krinsky and Johnson, 2005). P-Carotene also has the ability to act as provitamin A (Garcia-Gonzalez et. al., 2005; Gouveia and Empis, 2003). Because of these properties, P-carotene has found applications as a food supplement and colorant.

The extent of P-carotene accumulation in Dunaliella biomass is a function of high salinity, temperature stress, high light intensity, and nitrogen limitation. Being an extremophile, by virtue of its ability to grow at high salinity, it is possible to grow Dunaliella biomass in open-pond cultivation systems in photo-autotrophic mode. The production ponds are typically located in areas that could provide high solar irradi — ance, warm temperatures, and hypersaline waters (Ben-Amotz, 1999). Commercial cultivation facilities of Dunaliella are located in Australia, Israel, China, and the United States (Del Campo et al., 2007), with global production estimates at about

I, 200 MT y-1 (Pulz and Gross, 2004). The open-pond cultivation systems used are either very large ponds (without mixing) of up to 250 ha or paddle-mixed raceway ponds of about 3,000 m2 surface area (Del Campo, 2007). Commercial producers are offering Dunaliella biomass directly as a powder for application as an ingredient in human dietary supplements and functional foods (Spolaore et al., 2006).

Downstream processing of Dunaliella biomass is carried out to extract P-carotene for use as a natural food colorant and food supplement. The natural P-carotene from Dunaliella must compete with cheaper synthetic P-carotene in the marketplace. Synthetic P-carotene is dominated by all-trans-P-carotene (Von Laar et al., 1996), whereas natural P-carotene from Dunaliella contains more than 50% 9-cis-P-carotene (Johnson et al., 1996). Therefore, although more expensive, natural P-carotene provides the natural isomers in their natural ratio (Guerin et al., 2003; Garcia-Gonzalez et al., 2005; Spolaore et al., 2006), and the natural isomer of P-carotene is accepted as superior to the synthetic all-trans-isomer (Radmer, 1996; Vilchez et al., 1997; Lorenz and Cysewski, 2000; Becker, 2004; Spolaore et al., 2006). Although not yet cost compared to synthetic P-carotene, production of natu­ral P-carotene from Dunaliella has been reported as an economically viable and growing industry (Singh et al., 2005; Chisti, 2006). The algal meal of Dunaliella after extraction of P-carotene is reported to contain about 40% protein and therefore could find application in fish and poultry feed (Iwamoto, 2004).

The term harvesting refers to the concentration of dilute microalgal culture sus­pension to slurry or paste containing 5% to 25%, or more, total suspended solids (TSS). This slurry can be obtained in either a one-step or two-step harvesting pro­cess. Subsequent processing of the algal paste depends on the concentration of the algal paste. Increased product concentration decreases the cost of extraction and purification, as well as the effective unit cost of biomass. Concentration of algal paste significantly influences downstream processes, including drying. Microalgae are particles that have a colloidal character in suspension. Electric repulsive inter­action between algal cells and cell interaction with the surrounding water provide stability to the algal suspension. Algal cells are usually characterized as negatively charged surfaces where the intensity of charge is a function of the species, ionic strength, and pH of the cultivation media (Taylor et al., 1998). These surface charges are helpful in the growth culture because they assist in keeping the cells in the water column so that they do not settle to the bottom of the pond, particularly in regions of the pond where the water velocity is low. However, the charges pose a challenge to the dewatering process because they eliminate the option of using a simple settling tank (or pond) for harvesting.

Harvesting and dewatering processes can be divided into two categories, namely

(1) those in which the dewatering is performed directly on the algae culture, and

(2) those involving agglomeration of the algae into macroscopic masses to facilitate the dewatering process. The former, which include centrifuges and membrane filters, avoid the complications and costs associated with the addition of coagulation and flocculation chemicals. Processes like flocculation, flotation and gravity sedimenta­tion have acceptable energy requirements but have a fairly wide range of costs asso­ciated with motors and controls.

Potential by-products from the production of algal biodiesel include the capture of carbon, the generation of biogas through anaerobic digestion, the generation of bioethanol through fermentation of carbohydrates, the production of feeds for aqua­culture, the production of animal feeds rich in protein and carbohydrates (~60% protein), and the production of a high-grade, protein-rich material (~90% protein). In the case of fermentation or digestion of the algal cell debris, the recycling of N and P media components can be used to decrease media costs.

Williams and Laurens (2010) considered the scenarios of biogas formation, preparation of animal feed, and preparation of high-grade, protein-rich material. They highlighted the uncertainty and process specificity of costing these options and noted the requirement for improved knowledge in this area. The 60% protein feed was valued at US$750 tonne-1 based on FAO (Food and Agriculture Organization) statistics. Relating it to the value of soya meal containing 45% protein, a value of US$500 tonne-1 was proposed. In comparison, the high-grade, protein-rich material was valued at US$900 tonne-1. Biodiesel was estimated at a value of US$125 bbl-1. Using this estimate, positive scenarios were found at protein feed values of US$350 tonne-1 and above, and protein-rich extracts at US$600 tonne-1 and above. Under the conditions used, anaerobic digestion was not cost effective; however, the high energy requirement to maintain the digester temperature was a result of the temperate environment, and revised analysis is required for warmer climates where the performance of biodigesters is well documented.

The three general types of maturation ponds employed in wastewater treatment are facultative ponds, anaerobic ponds, and the most common, waste stabilization ponds. Aerobic ponds, also known as high-rate ponds, are shallow and completely oxygen­ated (Oswald, 1978). High-rate algal ponds (HRAPs) were developed beginning in the 1950s as an alternative to unmixed oxidation ponds for BOD, suspended solids, and pathogen removal (Rawat et al., 2011). They constitute a low-cost, low-maintenance technology for the remediation of various types of effluents (De Godos et al., 2010). HRAPs exhibit better performance when compared to anaerobic, aerobic, and facul­tative ponds using the same influent. The co-habitation of photosynthetic algae and heterotrophic bacteria is referred to as HRAP symbiosis. HRAPs have been used for the treatment of a variety of wastewaters, including domestic wastewater, piggery and animal wastewaters, agricultural runoff, and mine drainage and zinc refinery wastewater (Rawat et al., 2011). The utilization of microalgae for the assimilation of nitrogen and phosphorus at low concentrations presents a sustainable alternative to the use of existing treatment systems, as the nitrogen and phosphorus can be recov­ered from the algal biomass for reuse (Boelee et al., 2011). HRAPs are designed to promote algal growth, and the technology generally consists of mechanically mixed shallow raceway ponds (Olguin, 2003; Garcia et al., 2006). A large paddlewheel vane pump is used to create a channel velocity sufficient for gentle mixing. The ponds are generally 2 to 3 m wide, 0.1 to 0.4 m deep, and range from 1,000 to 5,000 m2 in area, depending on the scale of application (Garcia et al., 2006; De Godos et al., 2009; Rawat et al., 2011). The hydraulic retention time of such systems is generally in the range of 4 to 10 days, depending on climatic conditions. Continuous mixing is pro­vided to keep the cells in suspension and reduce the shading effect, thereby exposing the algae to light periodically, even in denser cultures. The most common design that has proven successful on a large scale is the single-loop paddlewheel mixed. Due to the energy cost dependence on velocity, most ponds have been operated at velocities from 10 to 30 cm s-1 (Olguin, 2003; Rawat et al., 2011). The mode of action of the

HRAP occurs directly via growth of algae and harvesting of biomass and indirectly by ammonia-nitrate volatilization and orthophosphate precipitation via a change in pH. Algal photosynthesis thus controls the efficiency of nitrate and phosphate removal (Olguin, 2003). Algal photosynthesis provides oxygen for the decomposi­tion of organic matter by aerobic heterotrophic bacteria, allowing for a reduction in organic matter coupled with the removal of nitrogen and phosphorus due to uptake by the algae (Garcia et al., 2006). The biomass produced as a result can be harvested and used for the production of biofuels via various pathways (Park et al., 2011b).

These systems are simple to operate when compared to conventional tech­nologies, thus making them ideal for use by small rural communities (Garcia et al., 2006). HRAPs have been successfully used in the remediation of pig­gery effluent and also the effluent from aquaculture systems (Olguin, 2003). The combination of wastewater treatment and biofuel production is receiving much more interest than previously, owing to the advantageous implications of such a combination. However, fundamental large-scale research must be undertaken in order to optimize algal production and maintain high-quality effluent standards (Park et al., 2011a).

Microalgal cells must be preserved after sampling, most preferably in Lugol’s iodine solution before counting, to arrest the movement of some live cells around the counting chambers, for example, flagellates and diatoms, and therefore hampering accurate enumeration. Other methods of preservation, such as the use of aldehydes,

saline ethanol, and freezing, can also be adopted for a wide range of phytoplankton. Microalgal preservation is recommended so as to prevent cell disintegration and to avoid any changes in cell population size due to zooplankton grazing (Hotzel and Croome, 1999).