The transesterification process consists of the reaction of triglyceride molecules with alco­hol in the presence of a catalyst to produce glycerol and mono-alkyl fatty acid esters (Harrison et al., 2012). Biodiesel is typically transesterified using methanol, and therefore the fatty acid

alkyl esters that are produced are fatty acid methyl esters (FAME). The fatty acids are reacted with methanol to form diacyl glycerides, monacyl glycerides, and finally, fatty acid methyl esters (FAMEs) (Gong and Jiong, 2011). In this process glycerol is formed as byproduct (Figure 8.10). The transesterification process reduces the viscosity of the FAME compared to the parent oil, whereas the fatty acid composition will not be altered. FAMEs are the most prevalent alkyl esters in the current biodiesel market because of the price and availability of methanol compared to other alcohols (Knothe et al., 1997). Alcohols are the key substrates in transesterification. The commonly used alcohols are methanol, ethanol, propanol, butanol, and amyl alcohol, but methanol is widely applied in the transesterification of microalgae oils because of its low cost and physical and chemical advantages. Acid, base, or enzyme catalyzed processes may be applied in transesterification reactions (Canakci and Gerpen, 1999). The nature of the catalyst (acid/base/enzyme) influences the type of reaction. Transesterification can also be performed in the absence of catalysts using a supercritical methanol process that occurs at high temperatures (200-350 °C) and pressures (20-50 MPa). The transesterification reaction proceeds in shorter times (<5 min). Currently, this method is applied for the conversion of vegetable oils and animal fats rather than for microalgae oils (Gong and Jiong, 2011; Kusdiana and Saka, 2001).

Carotenoids are the most widespread pigments in nature and they appear in all algae, higher plants, and many photosynthetic bacteria. Their role is to protect from light radiation in the red, orange, or yellow wavelengths. Chemically speaking, carotenoids are tetraterpenes, whereas carotenes are hydrocarbons and xanthophylls contain one or more ox­ygen molecules (Lobban and Harrison, 1994). All xanthophylls synthesized by higher plants, e. g. violaxanthin, antheraxanthin, zeaxanthin, neoxanthin, and lutein, can also be synthesized by green algae. However, these possess additional xanthophylls, that is, loroxanthin, astaxanthin, and canthaxanthin. Diatoxanthin, diadinoxanthin, and fucoxanthin can also be produced by brown algae or diatoms (Guedes et al., 2011c). In general, green algae contain p-carotene, lutein, violaxanthin, neoxanthin, and zeaxanthin, whereas red species contain mainly a — and p-carotene, lutein, and zeaxanthin. p-carotene, violaxanthin, and fucoxanthin are present chiefly in brown species (Haugan and Liaaen-Jensen, 1994).

Extraction of carotenoids from algae has been boosted in recent years in the alimentary and aquaculture fields (Lamers, Janssen et al., 2008), driven by consumers’ environmental and health awareness and commercial feasibility. The major large-scale applications are food and health. Carotenoids’ antioxidant properties have been shown to play a role in preventing pathologies linked to oxidative stress (Yan, Chuda et al., 1999).

Recall that most oxidation reactions in foods are deleterious, e. g., degradation of vitamins, pigments, and lipids, with consequent loss of nutritional value and development of off — flavors (Bannister, O’Neill et al., 1985; Fennema, 1996). On the other hand, carotenoids are particularly strong dyes, even at ppm levels. Specifically, canthaxanthin, astaxanthin, and lutein have been in regular use as pigments and accordingly have been included as ingredi­ents of feed for salmonid fish and trout as well as poultry, to enhance the reddish color of fish meat or the yellowish color of egg yolk (Lorenz and Cysewski, 2000; Plaza, Herrero et al.,

2009) . Furthermore, p-carotene has experienced an increasing demand as pro-vitamin A (ret­inol) in multivitamin preparations. It is usually included in the formulation of healthy foods under antioxidant claims (Krinsky and Johnson, 2005; Spolaore, Joannis-Cassan et al., 2006). Some carotenoids are part of vitamins, which have diverse biochemical functions, including hormones, antioxidants, mediators of cell signaling, and regulators of cell and tissue growth and differentiation (Holdt and Kraan, 2011).

In humans, oxidation reactions driven by reactive oxygen species can lead to protein damage as well as DNA decay or mutation; these may, in turn, lead to several syndromes, such as cardiovascular diseases, some kinds of cancer, and degenerative diseases, besides aging in general (Kohen and Nyska, 2002). As potential biological antioxidants, carotenoids have the ability to stimulate the immune system and may be involved in as many as 60 life — threatening diseases, including various forms of cancer, coronary heart diseases, premature aging, and arthritis (Mojaat, Pruvost et al., 2008). Carotenoids exhibit hypolipidemic and hypocholesterolemic effects as well (Guedes et al., 2011c). A summary of these bioactivities is provided in Table 10.5.

Concerning carotenoid extraction, methodologies such as solvent extraction, supercritical extraction, or expanded bed absorption chromatography can be applied, as described by Liam et al. (Liam, Anika et al., 2012).

Ever since the positive prospects of cultivating algal cells for biofuel production began being extensively deliberated in the literature (Chisti, 2007; Singh et al., 2011; Singh and Gu, 2010; Wijffels and Barbosa, 2010), recent active research and development have further propelled this industry a step closer to scaling up and commercialization. However, the issue of energy balance in the entire system boundary of algal biofuels is not clearly addressed, mainly due to limited availability of commercial cultivation plants for technical assessment. Based on several life-cycle assessments (LCAs) of algal biofuel production, most of the studies unfortunately revealed a negative energy balance in their assessments, especially when algae were cultivated in closed photobioreactors (Jorquera et al., 2010; Razon and Tan, 2011; Stephenson et al., 2010). Although some important parameters (biomass yield, lipid pro­ductivity, specific growth rate) assumed in the LCA studies were predominantly based on findings from laboratory scale and might be irrelevant for large-scale production, it gives a baseline to visualize and to verify energy balance-related problems in the algal biofuel production system. As a result, several precautionary steps could be suggested to further improve the energy conversion efficiency of algal biofuel production before commencing the commercialization stage.

Energy-efficiency ratio (EER) is usually used as an indicator to address the energy con­version efficiency for the entire biofuel production process. The EER is defined as the ratio of total energy output to total energy input, where a ratio higher than 1 designates net positive energy generated, and vice versa (Lam and Lee, 2012; Lam et al., 2009). Table 12.1 shows a comparative study on EER for biodiesel derived from various energy feedstocks such as oil palm, jatropha, rapeseed, sunflower, and algae. The values presented in the table are a rough indicator because all the LCA studies were conducted based on different assumptions and system boundaries. From the information presented in the table, it can be observed that biodiesel derived from oil-bearing crops is much more energy efficient than biodiesel derived from algae. All the EER values for biodiesel derived from oil-bearing crops are more than 1, whereas algal-derived biodiesel has an EER value as low as 0.07. These quantitative results showed that the cultivation of algae for biodiesel production does not necessarily produce a positive energy output but, worse still, could pose a critical risk of unsustainable biodiesel production. In addition, several issues such as reusability of water to recultivate algae, the possibility of using contaminated wastewater as a nutrient source, and the extraction and transesterification conversion efficiency have not been clearly accounted for in those LCA studies. If these factors are taken into consideration, the EER value is expected to decrease significantly.

However, there are exceptional cases where the EER values are positive, such as those studies performed by Lardon et al. (2009), Batan et al. (2010), Jorquera et al. (2010), Sander and Murthy (2010), and Clarens et al. (2010). These studies highlighted the importance of choosing suitable cultivation methods (e. g., nutrient deficiency to increase lipid productivity), nutrient sources (e. g., wastewater), open pond/photobioreactor design, and downstream biomass

processing options that can enhance the EER value of algal biodiesel. In the following sections, several energy-related problems in producing algal biofuels are comprehensively elaborated to propose possible strategies to commercialize this renewable fuel.

Most of the studies include impact assessments in their results. Only Yang et al. (2011) limit their publication to the inventory step, and Jorquera et al. (2010) only assess the energy bal­ance. All the other publications assess the potential reduction of greenhouse gas emissions in addition to the energy balance. However, only three studies estimate other environmental impacts, as defined by the LCA ISO norm: abiotic depletion, potential acidification, eutrophi­cation, ozone depletion, human toxicity, marine toxicity, photochemical oxidation, ionizing radiation, land use, freshwater toxicity, and terrestrial toxicity. In most of the studies, climate change is assessed with the characterization factors given by the IPCC (IPCC, 2006) for a tem­poral horizon of 100 years. Brentner et al. (2011) and Campbell et al. (2011) use different char­acterization factors, and Khoo et al. (2011) do not present the used methodology to assess climate change. Table 13.9 illustrates the divergence of characterization factors among the dif­ferent methods.

As explained in the previous sections, perimeters, modeling assumptions, and impact assessment methods can differ significantly among the publications. This results in a large

variability of the results related to the global-warming potential (GWP) and the energy return on investment (EROI) and hampers the capacity to compare results. However, we gathered results for these two indicators within the selected studies. Table 13.10 pre­sents the GWP of the various publications, and Figure 13.5 illustrates the relationship between EROI and GWP. Coproduct management has an important influence on the climate-change results. In some studies, climate change impact is negative, which means that the considered system fixes more greenhouse gases than it emits. In Batan et al. (2010), the negative score is due to the substitution of algal oilcakes to soybean oilcakes used to feed livestock. In Sander and Murthy (2010), it corresponds to the substitution of algal oilcakes to maize for the production of bioethanol. Finally, in Campbell et al. (2011), it corresponds to the electricity production from biogas produced by anaerobic digestion of the algal oilcakes.

When only the NER is considered to determine the EROI, favorable values are determined by most of the studies. However, when CER is taken into account, the EROI is limited (1.8 for the best case, 0.96 for the less favorable). It can also be observed that poor EROI (between 0 and 1) corresponds to high GWP.

Microalgae have three main pigments: chlorophyll that absorbs blue light; red carotenoids that absorb blue and green light; and phycobilins that absorb green, yellow, and orange light. These pigments have been used as natural colorants in food products. In many countries biodyes have replaced artificial dyes, which are currently prohibited.

p-carotene is a carotenoid found in all higher plants and algae. p-carotene acts as pro­vitamin A and may be used as a natural food color. Phycolibins are water-soluble pigments and are found only in red algae or cyanobacterias. Most members of cyanophyceae contain blue pigment (phycocyanin), although several species may also contain erythrin. Phycoery- thrin and phycocyanin can be used as natural pigments in food, medicine, and cosmetics, avoiding the use of artificial pigments that are carcinogenic (Richmond, 1990).

Carbon fixation by microalgae is in vogue. In the last decade, more than 4,000 papers were published globally on this subject. Table 4.1 presents some rates of carbon dioxide described in the literature.

Among all species of microalgae, four are most common industrially: Spirulina, Chlorella, Dunaliella, and Haematococcus. Despite not being used industrially, Botryococcus is also largely studied due to its potential use as a source of hydrocarbons. These microalgae’s potential for carbon fixation is discussed next.

Electrical approaches to algae dewatering include exploiting electrophoresis, electrofloc­culation, and electroflotation. An obvious consideration, because algae normally carry a negative charge, is electrophoresis. In a water solution, however, both electrophoresis and electroflocculation can occur under the same set of circumstances. If a tray of algae in its growth medium were exposed to an electric field by placing metallic electrodes on two sides of the tray and energizing them with a DC voltage, algae concentrations would occur at both electrodes (electrophoresis) and at the bottom of the tray (electroflocculation). A study fo­cused on assessment of the factors influencing electrophoresis and electroflocculation of algae in its growth medium was conducted (Pearsall et al., 2011). The reported experiments show that electrophoresis does occur but is complicated by the effects of the fluid motion. It appears that the coupling of the algal cell and the fluid can be sufficiently strong that fluid motion effects can influence or dominate behavior. Electroflocculation appears to be a robust process (Poelman et al., 1997; Alfafara et al., 2002; Azarian et al., 2007). It does, however, inherently leave electrically induced trace metal flocculants in the dewatered algae.

As mentioned in Section 5.3.5.2, fine gas bubbles formed during the electrolysis, causing the algal particles to float to the surface, where they are skimmed off. An efficient bench-scale electroflotation system for algae flocculation was reported by using the magnesium hydrox­ide formed in the electrolysis to enable precipitation and, consequently, flocculation (Contreras et al., 1981). Laboratory — and field-scale electroflotation units for algae removal from wastewater oxidation pond effluent were operated (Sandbank et al., 1974; Schwartzburd, 1978; Kumar et al., 1981). A 2 m2 pilot-scale unit was tested for clarification of high-rate oxidation pond effluent (Shelef et al., 1984). For satisfactory algae separation, electroflotation is to be followed by or be operated concurrently with alum flocculation (Sandbank et al., 1974).

A wide range of microalgae species were harvested by electroflotation with up to 5% solids in the harvested algae. Decantation after one day further increased the solids concentration to 7-8% (Sandbank, 1979). The energy needs of the electroflotation process are generally high, but for small units (<5 m2 area) electricflotation operating costs are less than those of dissolved-air flotation units (Svarovsky, l979).

8.4.1 CO2

CO2 fixation by microalgae through a photoautotrophic mechanism for harnessing liquid fuel is considered a reliable and sustainable approach for the neutralization of CO2 (Graham and Wilcox, 2000; Takagi et al., 2000; Ge et al., 2011; Wang et al., 2008; Yoo et al., 2010). Microalgae are considered as more photosynthetically efficient than terrestrial plants at fixing CO2 (Chiu et al., 2008; Indra et al., 2010). Microalgae also have the functional ability to fix CO2 from the atmosphere and industrial emissions (Brennan and Owende, 2010; Venkata Subhash et al., 2013). In the process of fixation, microalgae use CO2 as an inorganic carbon source, while water acts as an electron donor for the storage of reserve food material such as carbo­hydrates, which are further transformed to lipids under certain stress conditions. Many microalgae species are able to utilize carbonates such as Na2CO3 and NaHCO3 for cell growth (Wang et al., 2008). Most algae and cyanobacteria have different CO2-concentrating mecha­nisms (CCM) and act as enhancers for higher growth (Ramanan et al., 2010). CCM is activated only at low carbon levels and further depends on the strain, pH, light availability, and so on. The expression of the enzyme carbonic anhydrase (CA) has been associated with the induc­tion of the CCM. Chlorella sp., Spirulina sp., and Dunaliella sp. have been studied for CO2 se­questration. CO2 tolerance of Dunaliella sp. has been examined and used in the industrial production of beta-carotene (Graham and Wilcox, 2000). In Chlorella sp., growth was reported at 20% CO2 concentration (Hanagata et al., 1992). Scenedesmus obliquus and Spirulina showed good CO2 fixation rates when cultivated at 30 °C (Wang, et al., 2008). Mixotrophic cultivation of microalgae (mixed) by supplementing CO2 externally at different concentrations in domes­tic sewage showed enhanced biomass growth and lipid productivities (Prathima Devi and Venkata Mohan, 2012). The study documented functional advantages of the mixotrophic mode of nutrition. Photoautotrophic microalgae cultivation facilitates harnessing of renewable fuel in conjunction with CO2 fixation in a unified and sustainable approach. How­ever, algal cells cannot efficiently trap atmospheric CO2 to support the rapid growth needed for commercial operations (Duan and Savage, 2010).

The costs of an algal hydrogen production facility and its operation are important factors to be considered for practical large-scale applications. Detailed cost analyses must be conducted on minimizing the materials and operating costs as well as optimizing the yield and gas collection. Although there are many reports in the literature about biohydrogen production, only a handful of them deal with economic analyses of biohydrogen production. Critical parameters in the cost analyses include the light environment, the climate and land space, reactor construction materials, the mechanism of culture mixing, reactor maintenance, and long-term operational stability with maximal gas production (Melis, 2002).

Benemann (1997) estimated an initial cost for an indirect algal biophotolysis system consisting of open ponds (140 ha) and photobioreactors (14 ha). The plant was assumed to generate 1.2 million GJ per year at 90% plant capacity, with estimated total capital costs for the system at US$43 million and annual operating costs at US$12 million. Overall total hydrogen production costs were estimated at US$10/GJ. The capital costs were almost 90% of total costs at a 25% annual capital charge (Akkerman et al., 2003). The algal ponds were estimated at a cost of US$6 per square meter (sq m), whereas the photobioreactors, with assumed costs of US$100 per sq m, were the major capital and operating cost factors. The costs of gas handling were not estimated but were presumed a significant cost factor.

An initial cost for a large-scale (>100 ha) single-stage algal or cyanobacterial biophotolysis process in a near-horizontal tubular reactor system was analyzed (Tredici and Zittelli, 1998). The main objective of the analysis was to determine whether the proposed photobioreactor design could meet the cost requirements for hydrogen production through single-stage biophotolysis. The tubular photobioreactor offers superior features for biohydrogen produc­tion due to the internal gas exchange and the effective water-spray cooling. Based on 10% solar energy conversion efficiency, the costs of the tubular photobioreactor were estimated at US$50 per sq m. The analysis did not include costs for gas handling and assumed a rela­tively low annual capital charge at 17%. The capital fixed costs were estimated at 80% of total costs, with the tubular material for the photobioreactor the major cost. The hydrogen produc­tion costs were estimated at US$15/GJ, which are comparable to the costs projected for hydrogen produced in a two-stage process from biomass residues projected at €19/GJ (Tredici and Zittelli, 1998).

An estimated 80 kilograms of hydrogen can be produced commercially per acre of cultivation area per day, assuming that the entire capacity of the photosynthesis of the al­gae could be diverted toward hydrogen production (Melis and Happe, 2001). Based on a realistic 50% capacity, the cost of producing hydrogen comes close to US$2.80 a kilogram. The authors maintained that the biohydrogen thus generated could compete with gasoline at this price, assuming one kilogram of hydrogen is equivalent to a gallon of gasoline. Currently, less than 10% of algae photosynthetic capacity is utilized for biohydrogen production.

A scale-up modular pilot photobioreactor was operated on site over a six-month period for assessment of economic and reactor performance (Melis, 2002). From the distribution among the various cost inventories derived from the field operation, the costs of materials and nu­trients turned out to be the major expenses (84%). A construction cost of US$0.75 per sq m was

established, which was considerably lower than the range of US$20-100 per sq m commonly quoted (Zaborsky, 1998). Although the economic analysis probably reflects a simplified, strip — down, bare design, it does provide an indication of the relative cost of the various components such as materials, nutrients, labor, water use, land lease, power, and others that are necessary and sufficient to assemble a commercially viable photobioreactor. The analysis also indicated that, to substantially lower the cost of the overall operation, effort should be directed toward the recycling and reuse of photobioreactor construction materials and growth nutrients.

These economic analyses indicated that photobiohydrogens could be produced at a cost between US$10 and US$20 per GJ (Akkerman et al., 2003). This is a reasonable maximal cost target for renewable hydrogen fuel, according to Benemann (2000). It should be noted that the economic analyses were based on optimistic assumptions and are highly presumptive and were intended predominantly to ascertain the major cost drivers for photobiological hydro­gen production. At present, biologically produced hydrogen is more costly than other fuel alternatives. Before economic barriers can be meaningfully addressed, many technical and engineering challenges have to be tackled. Nevertheless, these economic analyses provide an indicator that the development of low-cost photobioreactors and the optimization of photosynthetic efficiency are the major R&D challenges.

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).