7.3.2.1 Solvent Extraction

The process of extracting oil from oil-containing materials using a suitable solvent is called solvent extraction. It is well suited for lipid recovery from materials with low oil content, and it produces oil cake with low residual oil content (<1% by weight) (Erickson et al., 1984; Hamm and Hamilton, 2000). Algae cell walls are made of multiple layers and they are more recalcitrant than those of other microorganisms (Sander and Murthy, 2009). Some species having an additional trilaminar sheath (TLS) containing an algaenan component are resistant to degradation (Allard et al., 2002; Versteegh and Blokker, 2004).

The solvent extraction process occurs when a solvent comes into contact with microalgae to release lipids, solvate the lipids in solvent, and separate the oil from miscella by distillation of the solvent. The drawbacks of solvent-based oil extraction are that (1) the solvent is highly inflammable, (2) the energy requirements are high, and (3) the process requires high capital investments.

A solvent extraction plant consists of extractors, desolventizers, evaporators, strip­ping towers, and condensers in the extractor. Solvent is sprayed over the oil-bearing materials and solvent penetrates the biomass, targeting the soluble compounds. The mixture of algae lipid dissolved in the solvent is called a miscella and is sent to col­lection tanks. The algal oil is separated from the solvent using evaporators and strip­ping towers. The oil coming out of these units is first cooled, and then filtered and sent to storage tanks. The oil cake coming out of the extractor unit may contain some residual amounts of solvent. The residual solvent present in the oil cake is removed in the desolventizer unit, and recovered solvent can be reused in the extractor.

The various organic chemical solvents employed for oil extraction include ben­zene, hexane, cyclohexane, acetone, chloroform, ethanol (96%), and hexane-ethanol (96%) combinations. It is possible to extract up to 98% quantitative of purified fatty acids (Richmond, 2004). Among these solvents, hexane is most commonly used in the food industry. Hexane meets many of the requirements of an ideal oil solvent (Johnson and Lusas, 1983) due to it having a high extraction efficiency, a low viscos­ity, and a low boiling point, and being a nonpolar solvent, is easily miscible with oil and inexpensive.

At present, new players Aurora Algae and AlgaeBio (both producing biomass in autotrophic open ponds) have not yet progressed to commercialization with final products on the shelf. Aurora Algae’s crude algal oil prototype contains 65% EPA and is intended for use in the pharmaceutical, animal feed, as well as heath food and beverage sectors (Aurora Algae Online, 2011). These prototypes have been dis­tributed to potential customers and, according to Van Der Meulen, Aurora Algae has already “signed multiple letters of intent with key players across the industry” (Watson, 2011b). Bob Thompson, chairman of AlgaeBio, believes that they have a competitive economic advantage in terms of production costs. Between their patent, proprietary information, and intellectual property, they can “produce a wide array of high-value, algae-based products at a fraction of the cost” compared with their competitors (Watson, 2011b). There is still a lack of information available on the potential products in the pipeline.

The University of Almeria (Spain) has developed an outdoor tubular photobioreac­tor process for producing “high-purity” 96% EPA from Phaeodactylum tricornutum. The total cost of production of the esterified oil occurs at US$4,602 kg-1, with an estimated yield of 430 kg yr-1 (Molina-Grima, 2003). Some 60% of this cost is attributable to the recovery process, and the remaining 40% accounts for biomass production costs. The total cost still needs to be reduced by 80% to be economi­cally feasible. The most common lipid extraction methods include oil press, solvent extraction, super-critical fluid extraction, and ultrasound (Harun et al., 2010). Solvent extraction is the most common method employed in the recovery of fatty acids from microalgae (Belarbi et al., 2000).

Solazyme-Roquette has created “high-lipid algal flour” (Daniells, 2011), intended for use as a main ingredient alternative to make healthier processed foods such as chocolate milk (4.5% algal flour), frozen desserts, and even low-calorie salad dress­ings. Household names such as Unilever, Nestle, and Abbott Laboratories are a few companies jumping onto the “omega-3 bandwagon.” A fast-moving consumer goods company, Unilever has invested in a multimillion-dollar deal with Solazyme Inc. to potentially replace palm oil with algal oil as a sustainable alternative in products such as food, soaps, and lotions (Sonne, 2010).

PUFAs in general aid in the prevention and treatment of scaly dermatitis and skin dehydration (Kim et al., 2008). Ethanolic or supercritical CO2 extracts are gaining commercial recognition in lipid-based creams and lotions as a result of their nourish­ing and protective effects on the skin. In progressing skin care research, glycol — and phospholipids should be given special attention (Pulz and Gross, 2004). Novel and innovative cost-effective technologies are the way to satisfy the growing demands of the health-conscious consumer.

Genome analysis is available for only four unicellular algae: Chlamydomonas reinhardtii, Cyanidoschyzon merolae, Ostreococcus tauri, and Thalassiosira pseudonana (Misumi et al., 2008). Genetic modification (GM) of microalgae holds promise as a strategy to attain higher lipid yields while concurrently generating value-added products (Jin et al., 2003; Leon and Fernandez, 2007; Gressel, 2008). Although several hundred strains of microalgae have been cultured, detailed inves­tigation of cellular physiology and biochemistry is limited to fewer than thirty species. Fewer still are the algal strains that have been studied at the genomic level. Genetic transformation of microalgae has been constrained by the presence of rigid cell walls (Rosenberg et al., 2008). However, using a plethora of techniques such as bombardment, electroporation, and treatment with silicon whiskers and glass beads, several species have been modified genetically (Leon and Fernandez, 2007), including Amphidinium sp., Anabaena sp., Chlamydomonas sp., Chlorella ellipsoidea, C. kessleri, C. reinhardtii, C. sacchrophila, C. sorokiniana, C. vulgaris, Cyclotella cryptica, Cylindrotheca fusiformis, Dunaliella salina, Euglena gracilis, Haematococcus pluvialis, Navicula saprophila, Phaeodactylum tricornutum, Porphyridium sp., Symbiodinium microadriaticum, Synechocystis sp., Thalassiosira weisflogii, and Volvox carteri. The red alga Cyanidoschyzon merolae and the euglenoid Euglena gracilis have also been genetically transformed (Rosenberg et al., 2008). We agree with Pienkos et al. (2011), who suggest that through genetic engineering a few “designer algal strains” that have all the properties needed for large-scale biotechnology should be developed, and more research must be carried out in parallel with natural strains to fully understand their physiological function­ing. Such modifications can impart properties to improve yield. For instance, Li and Tsai (2008) demonstrated that the microalga Nannochloropsis oculata, which was codon-optimized to produce bovine lactoferricin (LFB) fused with a red fluorescent protein (DsRed), has bactericidal defense against V. parahaemolyticus infection in the shrimp digestive tract.

The utility of engineered microalgae for augmented lipid biosynthesis, conver­sion from autotrophy to heterotrophy, enhancing photosynthetic conversion effi­ciency and expression of recombinant proteins is gaining prominence (Rosenberg et al., 2008). While it is possible to enhance lipid synthesis through cloning acetyl — CoA carboxylase (ACC) genes in yeast, fungi, bacteria, and a few higher plants, there was no change in lipid content of a similarly engineered diatom Cyclotella cryptica (Dunahay et al., 1995; Dunahay et al., 1996). Three possible strategies exist for enhanced lipid production: biochemical engineering (BE), genetic engineering (GE), and transcription factor engineering (TFE). BE approaches are currently the most widely established in microalgal lipid production (Courchesne et al., 2009).

Radakovits et al. (2010) discussed the potential of manipulating the central car­bon metabolism in eukaryotic microalgae through genetic engineering to enhance lipid production. They suggested that it should be possible to increase production of not only carbon storage compounds, such as TAGs and starch, but also designer hydrocarbons that may be used directly as fuels.

Another possibility is to engineer the light-harvesting antennae in autotrophic algae. Smaller antennae lead to greater photosynthetic efficiency (Mitra and Melis, 2008); mutating genes that control antennae biogenesis is a possible mechanism for enhancing photosynthetic efficiency (Scott et al., 2010). Possibilities exist to improve solar energy conversion efficiency from the present the 1-4% to 8-12% to realize fully the potential of microalgal co-production systems in Chlamydomonas perigranulata, C. reinhardtii, Chlorella vulgaris, Cyclotella sp., Dunaliella salina, Scenedesmus obliquus, and Synechocystis PCC 6714 (Stephens et al., 2010).

A mechanistic model developed by Flynn et al. (2010) explores cellular chlorophyll and photosynthetic efficiency to optimize commercial algal biomass production. The model predicts that genetically modified strains with a large antenna size, indi­cated by a low Chl:C ratio, are more suitable for commercial biofuel production than strains selected from nature. However, for the generation of hydrogen and hydrocarbons as biofuels, smaller light-collecting antennae seem to be more effi­cient in Botryococcus braunii (Eroglu and Melis, 2010). Three races (Race A, B, and L) of the strain Botryococcus braunii are recognized (Banerjee et al., 2002); these races are regarded as a potential source of renewable fuel with yields of hydro­carbons reaching up to 75% of algal dry mass. A Botyrococcus Squalene Synthase (BSS) gene from a Race B variant of B. braunii has been sequenced, amplified as a 1,403-bp fragment, and expressed as a heterologous protein in E. coli BL21 cells. Following Isopropyl-P-D-thiogalactoside (IPTG) induction, recombinant squalene synthase activity was detected, suggesting that a key hydrocarbon synthesis gene from a commercial alga can be isolated and cloned into a heterologous expression system. This opens the door for large-scale hydrocarbon synthesis in more amenable systems such as E. coli and may help reduce the problems associated with the vis­cous nature of Botyrococcus cultures (Banerjee et al., 2002).

Open systems include natural waters, lakes, and dams where the growth of the microalgae of interest either occurs naturally or is encouraged through addition of nutrients. Harvesting is carried out in situ; for example, Spirulina is harvested com­mercially from Lake Texcoco in Mexico. Although naturally harvested microalgae incur very little cost in cultivation, the productivity and product quality (biologically and toxicologically) cannot be assured (Lee, 2001).

5.3.1.1 Circular Ponds

The first mass culture of microalgae was carried out in circular ponds (Lee, 2001). They are generally simple, round, concrete ponds or dams, mixed by a rotating cir­cular arm fixed in the center of the pond, or by manual stirring. The size of the pond is limited by the strain of the water resistance against the rotating motor. The largest reported pond is 50 m in diameter (Lee, 2001). They are commonly used in Japan, Taiwan, Indonesia, and Ukraine for Chlorella cultivation (Lee, 2001; Pulz, 2001).

In most studies performed to consider the environmental burden of microalgal biodiesel, the key contributors to the algal cultivation process have been identi­fied across raceway ponds and closed photobioreactors. Across all reactor configu­rations, the major contributions to environmental burden in terms of net energy ratio, abiotic depletion, and GHGs were incurred from the energy requirement for mass transfer and mixing in the reactor, as well as the energy requirement asso­ciated with the provision of combined nitrogen for cell growth (Lardon et al., 2009; Batan et al., 2010; Stephenson et al., 2010; Richardson et al., 2012b). These requirements are sensitive to algal biomass concentration, lipid content, and algal productivity. Algal productivity and concentration were the most influential (e. g., Stephenson et al., 2010; Razon and Tan, 2011), owing to their impact on system volume, influencing both energy input for mixing and pumping, and the amounts of nutrients required, as previously demonstrated in other microbial systems (Harding et al., 2008; Harding et al., 2012).

Studies by Jorquera et al. (2010), Stephenson et al. (2010), and Richardson et al. (2012b) compared selected types of reactors. Jorquera et al. (2010) compared horizontal tubular reactors, flat-plate reactors, and raceway ponds using a basis of 100 tons algal biomass per year. The closed photobioreactors provided higher biomass concentrations, and higher volumetric and areal productivities than ponds. Correlated with this, the raceway ponds had an increased land requirement. However, their energetic requirements were significantly lower than the closed photobioreac­tors. Under their operating conditions, the NER of the horizontal tubular reactor illustrated that it was not feasible in terms of either oil or biomass production (NER of 0.07 and 0.20, respectively). The NER of the flat-plate reactor was 54% of that for the open raceway for oil production (NER of 1.65 and 3.05, respectively) and for production of algal biomass (NER of 4.51 and 8.34, respectively).

Stephenson et al. (2010) compared the performance of the integrated algal biodiesel process, from cradle to combustion, using the open raceway and tubular airlift photobioreactor and a two-stage cultivation method to maximize lipid for­mation under nitrogen starvation in the second stage. In their system, the reactor was the dominant contributor to energy consumption. The design of the tubular airlift photobioreactor required energy input an order of magnitude greater than the raceway on an energy equivalence basis, despite its higher productivity. While 85% of the energy requirement of the tubular reactor was attributed to operation and the remainder to reactor manufacture, the latter exceeded the total GWP of the raceway system.

Similarly, Richardson et al. (2012b) compared the performance of the raceway, horizontal tubular reactor, and airlift tubular reactor as a component of the algal bio­refinery producing biodiesel and biogas. Their comparison was made using literature data for Phaeodactylum tricornutum, extensively studied in the Aquatic Species Programme (Sheehan et al., 1998). Considering an integrated biorefinery system with combined biogas production, the relative NER values for these photobioreac­tors compared to the raceway were 64% and 8%, respectively. Under the operating conditions selected, the NER of the airlift reactor was unacceptable, owing to the low productivity achieved relative to the energy input. The airlift reactor can be operated at much reduced gas flow rates and concomitant reduction in energy input without compromising productivity (data not shown), indicating the need to make these comparisons using optimized performance data relevant to commercial-scale operation. In all cases, the reactor energy requirement dominated that of the process, with that of the horizontal tubular reactor being some twofold that of the raceway per unit biodiesel. Owing to the lower biomass and oil concentrations achieved in the raceway reactor, this advantage of reduced reactor energy was partially offset by the greater pumping energy required for the larger volume processed from the raceway (2.2-fold); the pumping energy within the raceway biorefinery is a quarter of the reactor energy requirement. Extending the analysis beyond energy, the acidification and eutrophication impacts of the horizontal tubular reactor were 61% and 73%, respectively, of the raceway system under the standard operating conditions selected. The GWP was negative for the raceway system compared to a positive value of 60% for the horizontal tubular reactor.

Recognizing the increased energy requirement of traditional photobioreactors for mixing and mass transfer as well as manufacture, Batan et al. (2010) assessed the sparged polyethylene photobioreactor bags. While they report positive NER values for these systems, agreement in the literature with respect to the feasibility of the airlift system for biofuel production has not been found and further assessment is required.

Razon and Tan (2011) assessed the combined production of biodiesel and biogas using Haematococcuspluvialis. While low biomass concentrations and productivity resulted in a negative NER, the energy requirement of the flat-plate bioreactor used for intermittent inoculum supply exceeded that of the raceway system used for large — scale production by some twofold, thus confirming the much higher energy demand per unit biofuel in closed reactor systems.

Stephenson et al. (2010) disaggregated the contributions to the reactor energy and GWP in the tubular airlift reactor and raceway pond. These are shown relatively in Figure 9.2 (see color insert), where the total fossil energy requirements estimated for cultivation in the tubular airlift reactor and raceway under standard conditions were approximately 230 and 29 GJ per tonne biodiesel formed, respectively. The corresponding GWPs were 13,550 and 1,900 kg CO2 per tonne biodiesel, respec­tively. In addition to the magnitude, the relative values illustrate that electrical energy for mixing and mass transfer dominates the reactor energy and related GWP in the tubular reactor. The lower mixing energy in the raceway results in the energy for com­bined nitrogen provision being significant. Furthermore, the much larger raceways required, owing to lower productivities achieved, lead to the construction compo­nents making a more dominant contribution, especially the PVC liners of the ponds.

■ N fertilizer

■ P fertilizer

■ Electrical power ■ Perspex tubing

■ N fertilizer

■ P fertilizer

■ Electrical power

■ Concrete walls

■ Steel paddle wheel

■ PVC lining

■ Water supply & treatment

FIGURE 9.2 (See color insert.) The relative contribution of fossil energy (left) and GWP (right) to the total requirements for microalgal biodiesel production using a tubular airlift reactor (upper) and a raceway (lower). From the LCA of C. vulgaris conducted by Stephenson et al. (2010) under standard conditions. The total fossil energy requirements of 230 and 29 GJ and GWP of 13,550 and 1,900 kg CO2 per tonne biodiesel formed were estimated for the tubular reactor and raceway, respectively.

Research efforts into using microalgae for CO2 sequestration, biodiesel produc­tion, and other VAP syntheses will continue to power several of the assets inherent in these photosynthetic organisms. The high lipid content of microalgae has been taken as the major screening criteria for selecting and exploiting such species for biodiesel production, but has not been evaluated critically. The species that have been exploited for biodiesel production are very few (Griffiths and Harrison, 2009). The majority of the research work has focused on increasing lipid content and bio­mass productivity, whereas the studies related to chemical conversion of lipid to biodiesel, quality improvement, and cost reduction of the process are progressing at a slow pace (Krohn et al., 2011). Taking into consideration the current scenario, there is a need to look into the complete fatty acid profile of microalgal lipids in addition

TABLE 11.4

Biotechnological Application of Some Microalgae Species for Food-Based Applications

Source: Adapted from Pulz and Gross (2004); and Raja et al. (2008).

to the qualitative and quantitative profiling of triacylglycerides and free fatty acids (Ramos et al., 2009; Liu et al., 2010). These factors primarily influence the quality of biodiesel produced. Once the right microalgae species have been selected consider­ing all physico-chemical properties, culture conditions can be optimized to obtain higher biomass productivity (Rodolfi et al., 2008) in an economical way in a raceway pond and/or closed photobioreactor system. In addition, an understanding of micro­algal behavior at the molecular level during the process of CO2 tolerance and uptake for intracellular lipid enhancement is a must.

Although CO2 sequestration by microalgae into biomass and triacylglycerol stor­age plays a critical role in an organism’s ability to withstand stress, information concerning the enzymes of CO2 uptake and tolerance, triacylglycerol synthesis, their regulation by nutrients, physiological conditions, their mechanisms of action along with the roles of specific isoforms has been limited by the lack of studies on pro — teomics and genomics (detailed protein and gene profiling) of microalgae for CO2 sequestration and biodiesel production.

The exploration of the vast biodiversity of microalgae in natural habitats for selection of suitable strains for CO2 sequestration and VAPs is possible. Potential
microalgae tolerating high CO2 concentrations can be isolated from relevant sources such as lakes, ponds, etc. near thermal power plants. The microalgal strains that can tolerate high CO2 concentrations and also synthesize food/feed and biofuel precur­sors need to be developed by exploring the microbial diversity. It should be possible to control the composition of food and biofuel precursors by suitably manipulating stress conditions such as light, temperature, and nutrients. The high performance of cultivation systems (open-pond and/or closed photobioreactor system) for microal­gae with high biomass productivity and energy efficiency should possibly be devel­oped through a fundamental understanding of culture behavior as well as gas-liquid mass transfer, reactor hydrodynamics, shear stress profiles, light penetration, pho­toperiod, etc.

Therefore, future research in this area is required to provide new insights into novel ways to use microalgae in economically viable value-added production processes along with their integration with CO2 sequestration.

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Filtration is the most competitive method compared to other harvesting tech­niques. It is most appropriately used for relatively large sized (>70 pm) algae such as filamentous species or agglomerates. Diatomaceous earth or cellulose can be used to increase filtration efficiency (Brennan and Owende, 2010). However, con­ventional filtration operated under pressure or suction is not suitable for smaller sized algae such as Chlorella, Dunaliella, and Scenedesmus. Membrane micro­filtration and ultrafiltration are alternative filtration methods. The disadvantage of these processes is their high cost due to the frequent replacement of membranes and pumping costs (Pittman et al., 2011). There are many different types of filtra­tion processes, such as dead-end filtration, microfiltration, ultrafiltration, pressure filtration, vacuum filtration, and tangential flow filtration (TFF). Mohn (1980) stud­ied different pressure and vacuum filtration units for maximum dewatering of algae and warned against the use of these filters for harvesting Coelastrum probosci — deum as he did not find it appropriate. However, he demonstrated that filtration processes can achieve a concentration factor of 245 times the original concentra­tion for C. proboscideum to produce a slurry of 27% solids. Recent studies reveal that TFF and pressure filtration can be considered energy-efficient dewatering pro­cesses as they consume minimal amounts of energy considering the output and initial amounts of feedstock (Danquah et al., 2009). Simple filters can be used with centrifugation to achieve better results. Mohn (1980) and Danquah et al. (2009) have presented data on concentration factors and energy consumption of all filtra­tion units. Large-scale recovery of microalgae using this technique is not recom­mended due to continuous fouling and the subsequent need to replace membranes. Few researchers have tried polymer membrane for continuous recovery. However, the performance of these membranes depends on several factors, such as hydro­dynamic condition, concentration, and properties of microalgae. Although this method appears as an attractive dewatering method, the significant operating cost requirements cannot be overlooked.

PRODUCTS, AND APPLICATIONS

At present, the most significant product of microalgal biotechnology in terms of produc­tion amounts and economic value is microalgal biomass (Figure 10.1; see color insert). Microalgal biomass has been widely used in the fuel and energy sectors; however, the nutritional value of algal biomass has endorsed its use as a high-protein supplement in human nutrition, aquaculture, and as a nutraceutical (Del Campo et al., 2007).

The algal biomass market size is estimated at around 10,000 tonnes y-1 (dry weight) (Becker, 2007), with an annual turnover of over US$1.25 million (Milledge, 2011). Despite being a biodiverse resource, microalgae remain understudied in terms of their morphology and physiology. Much of the literature regarding the systematics and taxonomy of microalgae focuses on biotechnologically relevant species. More than 200,000 species are known to exist; however, only 10 to 20 species (Table 10.1) have been exploited worldwide for biomass, pigments, antioxidants, and special products (toxins and isotopes) for various product applications (Borowitzka, 1992; Radmer, 1996; Olaizola, 2003).

This chapter addresses in detail various types of high-value products derived from algal biomass, their respective applications, production systems, and market positions.

The assimilation of nitrogen and phosphorus into algal and bacterial biomass is seen as advantageous due to the recycling potential of the nutrients via biomass treatment. Unicellular microalgae are found to be the most efficient and most pre­dominant in wastewater treatment ponds (Pittman et al., 2011). The use of combined algae-bacteria cultures increases the nitrogen accumulation efficiency; for exam­ple, in the treatment of acetonitrile, 53% ammonia was assimilated into biomass as compared to only 26% in a bacterial system under the same conditions. Under optimal conditions, 100% removal can be achieved (Su et al., 2011). The increased removal efficiency of nutrients may be attributed to the algal requirement of high amounts of nitrogen and phosphorus for the production of proteins, nucleic acids, and phospholipids, which account for 45% to 60% of the algal dry weight (Munoz and Guieysse, 2006). Su et al. (2011) demonstrated COD, ammonia, and phosphate removal efficiencies of up to 98%, 100%, and 72.6%, respectively, for the treatment of municipal wastewater. Nutrient removal efficiencies depend on the cultural con­ditions as mentioned previously and the nutrient loading rate. Boelee et al. (2011) showed a linear increase in nitrate and phosphate uptake with increasing loading rate up to 1.0 g m-2d-1 and 0.13 g m-2d-1, respectively, from municipal wastewater. Wang et al. (2011) showed an ammonia removal rate of 90%, irrespective of the initial con­centration used. Furthermore, total nitrogen and phosphorus was found to be greatly reduced from piggery wastewater. Nutrient removal efficiencies ranging from 91% to 96% ammonia and 72% to 87% phosphate, depending on the season and depth of the culture, were observed by Olguin (2003).

The quantitative determination of biomass by gravimetric analysis is easy, cost effective, and the equipment required for this purpose is not very expensive. However, this technique determines the weight of the whole biomass and does not discriminate individual cells in the media. In general, wet or dry weights of the biomass can be measured, although wet weight determination is not very accurate. The microalgal cells are harvested by centrifugation and the pellet washed twice with isotonic ammonium formate (NH4HCO2, 2%) to remove suspended residual salts (Valenzuela-Espinoza et al., 2002). The advantage of using ammonium formate is that it does not leave any residue as it decomposes to volatile compounds during the drying process. An empty watch glass is weighed and the microalgal sample pellet transferred to the watch glass. The biomass is dried in an oven at 60°C for 12 hours. After drying, the weight of the watch glass plus the dry biomass is deter­mined, and the net dry cell weight (DCW) is calculated using Equation (4.1):

DCW (mg L-1) = [{Watch glass (mg) + Dry biomass (mg)}

— Watch glass (mg)]/Volume (L) (4.1)