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

8.6.1.1 Expeller Press Method

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

Algal proteins may play both structural and nutritional roles, so their extraction from spent biomass is of potentially commercial interest. One application is for animal feed due their richness in essential amino acids (Williams and Laurens, 2010). The nonprotein nitrogen consists of amino acids, peptides, amines, and nucleotides and accounts for 10-20% of the total nitrogen in algae (Arasaki and Arasaki, 1983).

Recently, a few studies have been reported with respect to the organic solvent extractions due to the experience of remaining toxic residues with the target compounds, so enzyme — assisted extractions have attracted particular interest. Mechanical techniques such as ultra­sound sonication and pulverizing the lyophilized materials by grinding might also be helpful. Namely, bioactive peptides can be obtained in three ways: (1) hydrolysis by digestive enzymes from animals; (2) hydrolysis by proteolytic enzymes, harvested by microorganisms or plants; and (3) hydrolysis by proteolytic microorganisms during fermentation (Samarakoon and Jeon, 2012).

To understand the reactivity of wet algal biomass, it is necessary to understand the reac­tivity of model compounds (components of algal biomass). Experiments with wet algal bio­mass are very useful for understanding how the yields and comparison of different product fractions (e. g., crude bio-oil, aqueous phase products, gaseous products, and solid products) vary with hydrothermal processing conditions. Such data can be used to develop phenome­nal kinetics models that have utility for process design and optimization. Such data provide little insight into the details of the chemistry that occurs. However, to elucidate some of these details, several studies have been carried out with simpler organic molecules (phytol, ethyloleate, phenylalanine, and a model phospholipid) that mimic the structural features and functional groups present in microalgae and/or crude algal bio-oil from hydrothermal liquefaction (Savage et al., 2012a).

Changi et al. examined the behavior of phytol, an acyclic diterpene C20-alcohol and a model compound for algal biomass, in high-temperature water (HTW) at 240°C, 270°C, 300°C, and 350°C. Under these conditions, the major products include neophytadiene, isophytol, and phytone. The minor products include pristene, phytene, phytane, and dihydrophytol. Neophytadiene is likely formed via dehydration of phytol, whereas isophytol can be obtained via an allylic rearrangement. Phytol disappearance follows first-order kinetics with activation energy of 145 ± 20 kJ mol-1 and a pre-exponential factor of 109.94 ±0.12 s-1. Delplot analysis discriminated between primary and nonprimary products and led to a potential set of reaction pathways. A kinetics model based on the proposed path­ways was consistent with the experimental data (Changi et al., 2012).

Formic acid, acetic acid, lactic acid, glycolic acid, 2-hydroxybutyric acid, succinic acid, malic acid, mannuronic acid, and guluronic acid were obtained by the hydrothermal treat­ment of alginate. The total yield of the organic acids was 46% at maximum yield 350°C, 40 MPa, and 0.7 s reaction time (Aida et al., 2012). The formation of organic acids suggests that the carboxyl group structure of the alginate is preserved during the hydrothermal de­composition of the alginate. The formation of dicarboxylic acids is evidence that oxidation reactions occur during the hydrothermal treatment, introducing carboxyl groups into the de­composition products. The product distribution indicates that both acid and base catalyzed reactions occur during the hydrothermal treatment of alginate. Hydrothermal treatment of uronic acid, glucuronic acid, gave the same organic acids as those obtained from hydrother­mal treatment of alginate (Aida et al., 2012).

CO2 emissions inevitably occur in ORW because of the poor efficiency of the injection sys­tem and because of the natural outgassing from the growth medium. Only four publications

(Kadam, 2002; Stephenson et al., 2010; Campbell et al., 2011; Collet et al., 2011) take into account these losses, with respective emissions of CO2 equal to 0.07%, 30%, 10%, and 10%. A few studies only consider emissions of other gases. Campbell et al. (2011) consider that 0.11% of the nitrogen is volatilized without specifying the forms of the emissions. According to Hou et al. (2011), 0.5% is volatilized as NH3. Finally, Batan et al. (2010) mention NH3 volatilization without quantification.

An alternative to gravity sedimentation is a process called flotation, which is particularly effective for very thin algae suspension. Whereas gravitational separation works best with heavy algae suspension, flotation is used when suspended particles have a settling velocity so low that they are not able to settle in sedimentation tanks. Flotation is simply gravity thick­ening upside down. Instead of waiting for the sludge particles to settle to the bottom of the tank, liquid-solid separation is brought about by introducing fine air bubbles at the bottom of a flotation tank. The bubbles attach themselves to the particulate matter, and their combined buoyancy encourages the particles to rise to the surface. Once the particles have been floated to the surface, a layer of thickened slurry will be formed and can be collected by a skimming operation. The air-to-solids ratio is probably the most important factor affecting performance of the flotation thickener, which is defined as the weight ratio of air available for flotation to the solids to be floated in the feed stream.

Limited algae biomass is harvested by flotation processes unless coagulant in optimal dose is injected to the algae suspension (Bare et al., 1975). Different coagulants have been used in flotation systems. Chemicals such as aluminum and ferric salts as well as polymers are used to facilitate the flotation. The overall objective is to increase allowable solids loading, percent­age of floated solids, and clarity of the effluent.

The principal advantage of flotation over sedimentation is that very small or light algal particles that settle slowly can be harvested in a much shorter time. Flotation systems also offer higher solids concentrations and lower initial equipment cost. There are three basic variations of the flotation thickening system: dissolved-air flotation, electroflotation (also called electrolytic flotation), and dispersed-air flotation.

Based on observation of partial natural flotation of algae (van Vuuren and van Duuren, 1965), a full-scale flotation project was carried out (van Vuuren et al., 1965). Subsequently, work on the flocculation-flotation process for clarifying algae pond effluents was conducted (Bare et al., 1975; Moraine et al., 1980; Sandbank et al., 1974).

Other than algae, flotation of other microorganisms (bacteria) was suggested as a classi­fication and separation process. Gaudin et al. (1962) found that E. coli may be floated success­fully with 4% sodium chloride. Quaternary ammonium salts were used as surface-active agents for effective bacterial flotation (Grieves and Wang, 1966).

S. Venkata Mohan, M. Prathima Devi,

G. Venkata Subhash, Rashmi Chandra

Bioengineering and Environmental Center, CSIR-Indian Institute of Chemical
Technology, Hyderabad, India

7.3 INTRODUCTION

Continuous use of petroleum-derived fuels is recognized as unsustainable due to their de­pleting supplies and their contribution to the accumulation of greenhouse gases (GHG) in the environment. Biologically produced fuels have been identified as potential alternative energy sources (Posten and Schaubb, 2009; Smith et al., 2009; Rojan et al., 2011; Venkata Mohan et al.,

2011) that can mitigate GHG emissions (Hossain et al., 2008). Biofuels are being promoted as one of the most promising routes to lower CO2 emissions and to reduce the world’s depen­dency on fossil fuels (Groom et al., 2008; Smith et al., 2009). Biofuel production from renew­able sources is widely considered as one of the most sustainable alternatives to petroleum sourced fuels and a viable means for environmental and economic sustainability (Dragone et al., 2010).

Crop-based terrestrial sources of biomass face problems associated with a finite area of land available for its cultivation. In this context, algae draw much attention as an alter­native source of biomass that is capable of generating fuel. Compared to crop-based coun­terparts, algae have rapid growth rates. It is estimated that algae could yield 61,000 liters per hectare (L/ha), compared with 200 L/ha to 450 L/ha from crops such as soya and ca­nola (Duan and Savage, 2010). Algae are a known rapidly growing species of which the carbon-fixing rates are much higher than those of terrestrial plants. Microalgae commonly double their biomass within 24 hours (h), and this duration during the exponential growth phase can be as short as 3.5 h (Harrison et al., 2012; Chisti 2007). The prominence of algae — based biofuels evolved due to their domestic origin, carbon neutrality, renewability, abun­dant availability, higher combustion efficiency, and higher biodegradability (Zhang et al., 2003). Different algal species showed varied lipid content (Prymnesium paryum, 22-38%;

Chlamydomonas rheinhardii, 21%; Chlorella vulgaris, 22%; Spirogyra sp., 11-21%; Scenedesmus obliquus, 12-12%; Scenedesmus dimorphous, 16.40%; Porphyridium cruentum, 4-14%; Synchoccus sp., 11%; Dunaliella bioculata, 8%; Tetraselmis maculate, 3%; based on dry biomass) (Becker, 1994; 2004).

Photosynthesis has been recognized as an efficient carbon sequestration mechanism. Microalgae can sequester atmospheric CO2 (Chisti, 2007) and utilize carbon as well as in­organic nutrients present in wastewater for their growth and survivability (Venkata Mohan et al., 2011). During photosynthesis, microalgae capture atmospheric CO2, resulting in the synthesis of carbohydrates. Creating stress on microalgae at this stage causes the photosynthetic mechanism to switch from enhancing the biomass to accu­mulating lipids. The intracellular lipid granules stored under stress conditions act as precursors for fatty acid biosynthesis. The triglyceride composition of algae upon transesterification with an alcohol can produce algae-derived biodiesel (alkyl esters). Depending on the species, growing conditions, and growth stages, microalgae have been shown to produce various types of lipids including triacylglycerides, phospholipids, gly — colipids, and betaine lipids (Greenwell et al., 2010). Microalgae-derived lipids and biomass can be converted into alcohols, methyl esters, and alkanes for use in spark-ignited engines, compression ignition engines, and aircraft gas turbine engines (Harrison et al., 2012). Under specific cultivation conditions, algal oil content can exceed 50% by weight of dry biomass (Chisti, 2007). According to an estimate, the productivity of algae-derived biofuels is predicted to be on the order of 5,000 gallons/acre/year, which is approximately two or­ders of magnitude greater than the yield from terrestrial oil seed crops such as soybeans (Demirbas, 2007; Weyer et al., 2009).

Biofixation/sequestration of CO2 using photosynthetic microalgae is one potential op­tion for harnessing renewable energy. Cultivation of algae for biodiesel production is con­sidered more beneficial to the environment than the cultivation of oil crops (Chisti, 2007) because the productivity of algae-derived oils is much higher than the best oil-producing crops (Abou-Shanab et al., 2010). Compared to fossil-driven fuels, microalgae-based biofuels are renewable, biodegradable, and eco-friendly (Ma and Hanna, 1999; Knothe, 2006; Vicente et al., 2010). The cultivation of algae doesn’t require arable land, since they can be grown in artificial ponds, on land that’s unsuitable for agriculture, on surfaces of lakes or coastal waterways, or in vats on wasteland (Duan and Savage, 2010). Algal-based fuel addresses the major constraints posed by the first — and second-generation biofuels due to its fast growing nature and capability to produce several times higher biomass com­pared to terrestrial crops and trees, requires low and marginal land and other resources, produces higher lipid and carbohydrate, and so on (Singh et al., 2011). Production of biofuels from microalgae is gaining acceptance because of its economic feasibility and en­vironmental sustainability compared to agro-based fuels. Microalgae-derived biofuels have the potential for scalability (Harrison et al., 2012). Algae-derived biodiesel is currently being promoted as a third-generation biofuel feedstock since algae doesn’t compete with food crops and can be cultivated on nonarable land (Dragone et al., 2010). In writing this chapter, a comprehensive attempt was made to summarize the basic and applied aspects of algal-based fuel by synthesizing the contemporary literature in conjunction with recent developments.

The conversion of microalgae oil to biodiesel by direct transesterification involves both extraction and esterification in a single step (Sathish and Sims, 2012; Ehimen et al., 2010). The algae biomass can be effectively converted to fatty acid methyl esters through this process in relatively less time. Minimization of solvents and requirement of less time for reactions are the advantages of this method; the lipid productivity and success rate of the reactions are the associated drawbacks.

8.7.1 Acid-Catalyzed Transesterification

The acid-catalyzed transesterification process involves an acid catalyst (H2SO4/HQ) to undergo the reaction. These reactions are usually performed at high alcohol-to-oil-molar ra­tios, low to moderate temperatures and pressures, and high acid-catalyst concentrations (Zhang et al., 2003). Compared to base catalysts, acid catalysts are less susceptible to the pres­ence of free fatty acids in the source feedstock (Helwani et al., 2009), but the reaction rates of
converting triglycerides to methyl esters are too slow (Gerpen, 2005). Repeated application of catalyst in the reactions increases the acid value of the microalgae oil.

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.