Biofuel derived from algae is currently a hotly debated topic because its production is one of the more costly processes, which can dictate the sustainability of algae-based biofuel products. There are two major energy and cost constraints to bulk production of microalgae for biofuels: expensive culture systems with high capital costs and high energy requirements for mixing and gas exchange, and the cost of harvesting in achieving feasible algal solids concentration.

Because of the dilute algal suspension, the cost of harvesting microalgal biomass accounts for a significant portion of the overall production cost of microalgal biofuels. Certainly, energy-efficient and cost-effective harvesting are two major challenges in the commercializa­tion of biofuels from algae (Dismukes et al., 2008; Reijnders, 2008). The algae must be concen­trated by removing water in an economically viable fashion before further processing such as drying and oil extraction. The lack of cost-effective methodologies for harvesting has been one of the major hurdles for the economic production of algal biofuels, along with challenges associated with variability of microalgae species (e. g., cell size, robustness, surface charge, culture medium constituent, and desired end-product) (Cooney et al., 2009). An effective microalgae separation process should be workable for all microalgae strains, yield a product with a high dry biomass weight, and require moderate cost of operation, energy, and maintenance.

Microalgae harvesting can be a considerable problem because of the small size (3-30 micrometers in diameter) and the stable suspended state of unicellular algal cells. Since the mass fractions in a culture broth are low (typically less than 0.5 kg/m3 dry biomass in some commercial production systems), large volumes of culture need to be processed to order to recover biomass in a feasible quantity (Cooney et al., 2009; Ramanan et al., 2010). In addi­tion, microalgae harvesting is a major bottleneck to microalgae bioprocess engineering owing to its high operating cost, thus reducing the cost of microalgae harvesting is vital. If microalgae can be concentrated about 30-50 times by coagulation-flocculation and gravity sedimentation prior to dewatering, the energy demand for microalgae harvesting could be significantly reduced (Jorquera et al., 2010).

In comparing algae removal using filtration, flotation, centrifugation, precipitation, ion exchange, passage through a charged zone, and ultrasonic vibration, it was concluded that only centrifugation and precipitation can be economically feasible, with centrifugation being marginal (Golueke and Oswald, 1965). In another study examining three different tech­niques of harvesting microalgae involving centrifugation, chemical flocculation followed by flotation, and continuous filtration with a fine-weave belt filter, it was reported that centrifu­gation gave good recovery and a thickened slurry but required high capital investment and energy inputs (Sim et al., 1988). Dissolved-air flotation was more economical, but, if the recovered algae were to be incorporated into animal feed, the use of coagulants such as alum could have undesirable effects on the growth rate of the animals. This problem could be overcome by the use of nontoxic coagulants. The continuous filtration process had significant advantages in terms of energy efficiency, economics, and chemical-free operation. The only drawback of this process was that the efficiency depended on the size and morphology of the algae.

Most of the algae-harvesting techniques present several disadvantages, not only because of the high costs of operation but also due to the frequently low separation efficiencies and the intolerable product quality. Algae separation processes such as sedimentation, centrifu­gation, and filtration involve the use of equipment that could result in deterioration in algal quality due to cell rupture that causes leakage of cell content. Furthermore, in the case of flocculation, the high concentration of metal salts, which is normally used as the coagulant, can have a negative effect on the quality of the final product, as discussed previously (Kim et al., 2005).

High production yields of microalgae have called forth interest due to economic and sci­entific factors, but it is still unclear whether the production of biodiesel is environmentally sustainable and which transformation steps need further adjustment and optimization. A comparative life-cycle assessment (LCA) of a virtual facility has been undertaken to assess the energetic balance and the potential environmental impacts of the whole process chain, from biomass production to biodiesel combustion (Lardon et al., 2009). The outcome vali­dated the potential of microalgae as an energy source but highlighted the imperative neces­sity of decreasing the energy and fertilizer requirements.

From another comparative LCA study to compare biodiesel production from algae with canola and ultra-low sulfur diesel with respect to greenhouse gas emissions and costs, it was concluded that the need for a high production rate is a vital key to make algal biodiesel economically attractive (Campbell et al., 2011). In a separate study, it was concluded that the potential greenhouse gas emissions from microalgae operational activities are likely to be outweighed by the emission reductions associated with the production efficiency and seques­tration potential of microalgae (Williams and Laurens, 2010).

Some commercial interests in large-scale algal-cultivation systems are looking to tie into existing infrastructures, such as coal-fired power plants or sewage treatment facilities. Wastes generated from those infrastructures, such as flue gas (carbon dioxide) and wastewater nu­trients (nitrogen, phosphorous and other micronutrients), can be converted into raw material resources for algal cultivation. While use of carbon dioxide for algal photosynthesis would help attain carbon sequestration, uptake of waste nutrients for algal growth would eliminate use of fertilizers derived from fossil-fuel energy, thus mitigating emissions.

In essence, algal biofuel is currently more expensive than other fuel options, but it is likely to play a major role in the economy in the long run if technology improvements succeed in bringing down costs. The main challenges are to decrease the energy and fertilizer requirements and to accomplish high production rates in order to make algal bio­diesel economically attractive. The potential of anaerobic digestion of waste oilcakes from oil extraction as a way to reduce external energy demand and to recycle part of the mineral fertilizers is to be further explored (Lardon et al., 2009). Algal biofuel production employing renewable substrates may be a potential answer to overcome some of the economic constraints. There is scope to use certain wastewater effluents containing waste nutrients as cultivation broth. Therefore, production as well as unit energy cost of algal biofuel would be reduced.

A rigorous techno-economic analysis is necessary to draw a clearer prospect comparison between algal biofuel and the various other conventional fossil fuels. In addition to benefits that can be quantified from the use of biofuel for clean energy production, intangible benefits such as flue gas carbon dioxide sequestration, uptake of waste nutrients in place of fertilizers, and biogas energy produced from anaerobic digestion of oilcake should also be considered. These benefits would render a potential for claims of certified emission reductions (CERs) under the Kyoto Protocol for reducing emissions that can be estimated through a holistic LCA of algal biofuel production. The potential for claims of CERs to generate revenue and to finance algal biofuel projects under the Kyoto Protocol for reducing emissions of green­house gases appears to be promising. In view of the prospects of technology development and global carbon trading, it may not be an unreasonable expectation that, in the future, algal biofuel will experience a global shift toward employment of energy-efficient algae biofuel production while mitigating greenhouse gas emissions.

5.4 CONCLUSIONS

Algal biofuel is believed to be one of the biofuels for the future in view of its potential to replace depleting fossil fuels. The future role of algal biofuel as a clean fuel producing near­zero emissions and as an energy carrier is increasingly recognized worldwide. Because energy-efficient and cost-effective harvesting are two major hurdles in the commercialization of biofuels from algae, research addressing these challenges should be intensified. Knowl­edge exchange and cooperation between expert groups of various disciplines should be strengthened in order to leapfrog technological development for algal biofuel.

Cultivation of microalgae influences both biomass growth and lipid productivity. Cultur­ing of algae requires the input of light as an energy source for photosynthesis with a sufficient supply of macronutrients (nitrogen and phosphate) and micronutrients (sulphur, potassium, magnesium) in dissolved form (Mata et al., 2010). The main options for algae cultivation on a commercial scale are open-ponds or closed systems called photobioreactors (Chisti, 2007; Robert et al., 2012). There are also hybrid configurations that include a mix of the two growth options. Innovations in algae production allow it to become more productive while consum­ing resources that would otherwise be considered waste (Campbell, 2008).

і і л

A. Catarina Guedes, Helena M. Amaro ’ ,
Isabel Sousa-Pinto1’3 , F. Xavier Malcata1,4

1CIIMAR/CIMAR — Interdisciplinary Centre of Marine and Environmental Research,
University of Porto, Porto, Portugal
2ICBAS — Institute of Biomedical Sciences Abel Salazar, Porto, Portugal
^Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal
^Department of Chemical Engineering, University of Porto, Porto, Portugal

10.1 INTRODUCTION

Over the past 50 years, the world population more than doubled. This fact, coupled with an extension of life expectancies and rising standards of living, has led to a dramatic increase in primary energy consumption, chiefly from fossil sources (Jones and Mayfield, 2012).

A suitable alternative is to produce biofuel from photosynthetic organisms, that is, higher plants, algae, and cyanobacteria, which can use sunlight and carbon dioxide to produce a va­riety of organic molecules, namely carbohydrates, proteins, and lipids. These biomolecules can then be used to generate biomass rich in fuel-like metabolites that can then be extracted (Yang, Guo et al., 2011; Jones and Mayfield, 2012). However, the problem remains: What to do with the spent biomass? In particular, third-generation biofuels based on micro — and macroalgae offer an excellent possibility to displace fossil fuels; it is even believed that ances­tors of marine microorganisms were responsible for the formation of petroleum in the first place (Goh and Lee, 2010).

Macroalgae (or seaweeds) are multicellular organisms that take many forms and sizes. They are classified into three broad groups based on their pigmentation: brown algae (Phaeophyceae), red algae (Rhodophyta), and green algae (Chlorophyta). In contrast, microalgae are microscopic organisms, which, beyond Rhodophyta and Chlorophyta, may belong to another three specific groups of unicellular organisms: blue-green algae (Cyanobateria), diatoms (Bacillariophyta), and dinoflagellates (Dinophyceae). These species are commonly referred to as phytoplankton (Garson, 1993; Samarakoon and Jeon, 2012).

Despite looking similar to land plants, microalgae miss the lignin cross-linking in their cellulose structures because their growth in aquatic environments does not require strong supports (John, Anisha et al., 2011). On the other hand, macroalgae contain significant amounts of sugars (at least 50%) suitable for fermentation (Wi, Kim et al., 2009). In certain marine algae (e. g., red algae), the carbohydrate content is strongly influenced by the presence of agar, a polymer of galactose and galactopyranose. Recent research has attempted to develop methods of saccharification to release galactose from agar and to release glucose from cellulose so as to increase fermentation yields in terms of bioethanol (Jones and Mayfield, 2012). Other studies have shown that red algae such as Gelidium amansii and brown algae such as Saccharina japonica are both potential sources of biohydrogen via anaerobic fermentation (Jones and Mayfield, 2012). Unfortunately, harmful algal blooms in lakes, ponds, and oceans may result in drastic effects on those ecosystems, so removal of those algae for biogas production is welcome (Du, Li et al., 2011).

Microalgae are ubiquitous microorganisms that are characterized by a remarkable metabolic plasticity; they may indeed be cultivated in brackish and wastewaters that provide suitable nutrients (e. g., NH4, NO3, and PO4-) at the expense of only sunlight and atmospheric carbon dioxide (CO2). On the other hand, metabolic engineering has been taken advantage of to produce molecular hydrogen or to improve the lipid content as storage products (Amaro et al., 2011).

Overall, economic analyses have consistently indicated that algal-based biofuel feasibility hinges on the possibility of production coproducts with a market value from the spent biomass (Stephens, Ross et al., 2010). A wide range of fine chemicals may indeed be extracted from said biomass, depending on the species at stake (Raja, Hemaiswarya et al., 2008); these hold added value sufficiently high to contribute to the economic feasibility of biofuel manu­facture. Such bioproducts include sugars for production of bioethanol and biomethane, both via fermentation of biomass; intermediate value products, e. g., proteins for animal feedstock; and high-value products such as active principles bearing antimicrobial, antioxidant, antitumoral, and anti-inflammatory features for pharmaceutical purposes. Finally, biomass may be pyrolyzed to produce sequestered carbon in the form of biochar, which holds value as a soil enhancer (Kruse and Hankamer, 2010). A general overview of applications of spent biomass is given in Figure 10.1.

When discussing the upgrade of spent biomass, one should take into account the process that originated it or the target metabolite from which the biofuel is obtained. For example, if the objective is to produce biohydrogen, the spent biomass consists of essentially intact cells, whereas when accumulated lipids are required of biodiesel, the spent biomass takes the form of oilcake. Compounds such as carbohydrates, hydrocarbons, and the biomass itself may still be transformed into secondary biofuels such as ethanol, oil, biochar, and syngas, as shown in Figure 10.2. On the other hand, the spent biomass from production of a biofuel may be used to high value-added products via extraction (see Table 10.1). Therefore, this chapter is organized according to two perspectives: spent biomass used for further biofuel production and spent biomass as a source of value-added products, namely as fine chemicals or feed or even in bioremediation

Seaweeds or macroalgae belong to the lower plants, meaning that they do not have roots, stems, and leaves. Instead they are composed of a thallus (leaf-like structure) and sometimes a stem and a foot. Macroalgae represent a diverse group of eukaryotic, photosynthetic marine organisms. Unlike microalgae, which are unicellular, the macroalgal species are multicellular and possess plant-like characteristics. They are typically composed of a blade or lamina, the stipe, and a holdfast for anchoring the entire structure to hard substrates in marine environ­ments. The general features of these structures are very diverse in the various taxa comprising macroalgae. There are forms of which the primary feature comprises long blades, forms that are branched, and others that are leafy and that form mats. Moreover, some forms possess air bladders that act as flotation devices that enable some species to stand upright or occur free — floating on ocean surfaces. They are often fast growing and can reach sizes of up to 60 m in length (McHugh, 2003). They are classified into three broad groups based on the composition of photosynthetic pigmentation: (1) brown seaweed (Phaeophyceae), (2) red seaweed (Rhodophyceae), and (3) green seaweed (Chlorophyceae). Seaweeds are mainly utilized for the production of food and the extraction of hydrocolloids.

Algal biofuel technology is currently still in an early stage of development and therefore economically unfavorable for scaling-up purposes. Thus, analyzing the detailed economic breakdown of the multistage processing of algal biofuels will certainly open up a new direc­tion in identifying, evaluating, and verifying the actual problems that result in the high pro­duction cost of algal biofuels. A detailed discussion of the economic breakdown follows:

• Capital cost. The capital cost is the main cost driver in the entire system boundary of algal biofuels. A study performed by Davis et al. (2011) revealed that the capital cost of algae cultivated in an open pond and in a closed photobioreactor contributed approximately 91.0% and 94.7% of the total production costs, respectively. The total capital cost for algae cultivated in a closed photobioreactor was 153.8% higher than an open pond system, indicating the high investment risk in scaling up a closed photobioreactor for algal biomass production. Furthermore, the closed photobioreactor manufacturing cost contributes a large portion to the total capital cost at 52.7%, or 12.7 times higher than the open pond manufacturing cost. A similar result was also reported by Acien et al. (2012); in an actual algal biomass production plant, the capital cost contributed 87.2% of the total production cost, whereas 34% of the total capital cost was utilized to purchase equipment such as closed photobioreactors, a freeze dryer, and

a decanter (Acien et al., 2012). The closed photobioreactor manufacturing cost was lower compared to the study by Davis et al. (2011), which accounted only 16.1% of the total capital cost, but if other associated expenses such as installation costs, instrumentation and control, piping, engineering, and supervision were included, the cost to set up the closed photobioreactor cultivation system would reach up to 45% of the total capital cost. Based on the data presented, reduction of the associated equipment cost for algal cultivation systems by simplifying the overall designs and materials used, but allowing high productivity of algal biomass, is deemed necessary.

• Operating cost. The total operating cost for algal biomass production cultivated in a closed photobioreactor was dominated by labor cost (88.3%), followed by power consumption and water cost (9.2%), and finally nutrient and CO2 cost (2.5%) (Acien et al., 2012). In this regard, it is obvious that reducing the amount of labor (e. g., one worker/hectare or less) could significantly help reduce the overall operating cost. Reducing the amount of labor can be accomplished by introducing extensive automation into the entire algal biomass production plant, from cultivation farm to final biofuel production process (Acien et al., 2012). On the contrary, the raceway pond required 32.7% lower operating costs than the closed photobioreactor (Davis et al., 2011), primarily due to the ease of operating the open pond system and, hence, less power consumption. The high power consumption in the closed photobioreactor cultivation system (usually referred as an airlift tubular photobioreactor) is caused primarily by the use of heavy-duty pumps to circulate and to provide sufficient mixing of the algae (Lam and Lee, 2012). Hence, extensive research efforts to design an innovative closed photobioreactor with less power consumption that has the potential

to be easily scaled up are necessary to move the algal biofuels industry to the next level. Water consumption cost is another important issue that should not be ignored. Although the total water cost is lower compared to the power consumption cost, incessant waste of water could cause an enormous water footprint in algal biofuel

production and lead to irreversible consequences for regional water resources (Subhadra, 2011). Several precautionary steps should be taken because the evaporation rate for the open pond system is exceptionally high (~0.3 cm/day), resulting in a massive waste of water in this cultivation system. The water consumed in the open pond system was approximately 3.3-6.7 times higher than in the photobioreactor (Davis et al., 2011; Delrue et al., 2012), where continuously pumping fresh water into the system could inevitably increase the overall operating cost, especially for long-term operation.

Coproducts. Valuable coproducts such as carbohydrates and proteins remain in the algal biomass after lipid extraction. These products could be further utilized to increase the revenue of algal biomass. Unfortunately, in some recent techno-economic studies, the coproducts did not bring a significant return to reduce the production cost of algal biofuels (Davis et al., 2011; Sun et al., 2011). For example, when biogas production facilities (e. g., using residue of algal biomass for biomethane production) was incorporated into the algal biodiesel production plant, the total coproduct sales revenue could reduce the operating cost by only 12.7-18.2% (Davis et al., 2011). However, the contributions from coproducts, especially those that have higher economic value, such as bio-butanol, should not be totally ignored, because the process for producing them could be further improved in the near future as technology develops (Davis et al., 2011).

12.5 CONCLUSION

Cultivating algae as a sustainable source of biomass for biofuel production illustrates a new trend in the renewable energy industries. The advantages and promises of algal biofuels are alleged to bring a revolutionary breakthrough in balancing the global fuel demand with better environmental protection. However, producing algal biofuels requires a large cultiva­tion system and substantial energy requirements, which subsequently induce a negative im­pact in commercializing these renewable fuels. Several technical challenges, such as cultivation method, harvesting and drying processes, and biofuels conversion technologies using algal biomass, are still in the infancy phase, and extensive ventures in research and de­velopment are urgently needed to address the commercial feasibility of this renewable energy source. From the techno-economic point of view, algal biofuels are currently considerably more expensive than fossil fuels; thus political support is desirable to strengthen the eco­nomic viability of algal biofuels and to be able to compete in the global fuel market. Sustained support from technology developers, politicians, and policymakers, as well as acceptance from the public, are the driving forces to materialize this commercially viable biofuel source as a solution to future energy concerns.

Acknowledgment

The authors would like to acknowledge the funding given by the Universiti Sains Malaysia (Research University Grant No.814146, Postgraduate Research Grant Scheme No. 8044031, and USM Vice-Chancellor’s Award) for the preparation of this chapter.

The light spectrum and intensity are factors that directly affect the performance of phototrophic microalgal growth, both indoors and outdoors. In outdoor cultures, sunlight is the major energy source, whereas innovations in artificial lighting, such as light-emitting diodes (LED) and optical fiber, are interesting for indoor cultivation systems. In indoor cul­tures, the biggest challenge is the high cost of artificial lighting (Chen et al., 2011).

Regardless of the light source, its usage by microalgae occurs in the same way. In a photo­synthetic system, 8 photons of radiation are required to fix one CO2 molecule in the form of carbohydrate; this results in the maximum photosynthetic efficiency (Chini-Zittelli et al., 2006).

Multiproteic complexes, also called photosystems, catalyze the conversion reaction of light energy captured by excited molecules of chlorophyll into the form of usable energy. A photosystem consists of a center of photochemical reaction consisting of a protein com­plex, and molecules of chlorophyll that enable the conversion of light energy into chemical energy. This photosystem also has an antenna complex consisting of pigment molecules that capture light energy and feed the reaction center. The antenna complex is important for the capture of light. In chloroplasts, it consists of a cluster of hundreds of chlorophyll molecules held together by proteins that keep them firmly together on the thylakoid mem­brane (Alberts et al., 2008).

When a chlorophyll molecule from the antenna complex is excited, the energy is rapidly transmitted from one molecule to another through a resonance energy transfer process until it reaches a special pair of chlorophyll molecules from the center of the photochemical reaction. Each antenna complex acts like a funnel collecting light energy and directing it to a specific site where it can be used effectively (Alberts et al., 2008). One strategy to optimize the utili­zation of light is to reduce the size of the antenna, which makes the cells less opaque and facilitates the transmission of light (Chen et al., 2011).

Several studies have been developed to improve the efficiency of light utilization and re­duce the costs of systems with artificial lighting. The advantage of cultivation in a laboratory is that is uses fluorescent tubes. Although they consume high amounts of energy, that usage can be reduced by more than 50% with the use of LEDs. Many cultures use only solar energy as a light source, which has no cost. However, the performance of outdoor systems is lower than indoor ones, and they require large areas of land (Chen et al., 2011).

Raceway ponds are a modified version of the open pond system that has a different flow pattern compared to that of the simple pond. In raceways, the water flow direction is con­trolled by the rotation speed of paddlewheels, in contrast to only coaxial mixing in conven­tional open ponds. Therefore, in the raceway systems, the microalgae, water, and nutrients are continuously circulated around a racetrack, following the same direction as a paddlewheel. In this way, the circulation rate around the racetrack can be adjusted by the paddle speed. With paddlewheels providing the driving force for liquid flow, the microalgae are kept suspended in the water and are circulated back to the surface on a regular frequency.

Despite their diversified appearance, the most common raceway cultivators are driven by paddlewheels and are usually operated at a water depth of 15-20 cm. The raceways are usu­ally operated in a continuous mode with constant feeding of CO2 and nutrients into the sys­tem while the microalgae culture is removed at the end of the racetrack. This operation is quite similar to that of plug-flow reactors (PFRs) used in the chemical industry.

The same drawbacks observed in the operation of open ponds are also found in raceways. Furthermore, the requirement of large areas for microalgae cultivation is considered the bar­rier for commercialization of microalgae processes. Nevertheless, control of environmental factors (such as mixing) in raceways is easier than in conventional open ponds, making the use of raceways for the cultivation of microalgae more attractive.

Microstrainers consist of a rotary drum covered by a straining fabric, stainless steel or poly­ester. The partially submerged drum rotates slowly in a trough of suspended algal particles. The screen is fine mesh that captures only fairly large particles such as algae. As the mesh moves to the top, water spray dislodges the drained particles. When a microstrainer is used to harvest algae, the concentration of harvested algae is still low. Smaller algae can still pass through the screen and are thus not harvested.

Unit costs of microstraining range between $5 and $15 per 106 liters, depending on algae size and scale of operation (Benemann et al., 1980). For larger algae, even lower costs may be achieved. Favorable features of microstraining include simple function and construction, simple operation, low investment, neg1igable wear and tear due to absence of fast-moving mechanical parts, low energy consumption, and high filtration ratios.

Problems encountered with microstrainers include low harvesting efficiency and difficulty in handling particles fluctuations. These problems may be overcome in part by varying the drum rotation speed (Reynolds et al., 1975). Another problem associated with microstraining is the buildup of bacterial and algae biofilm slime on the fabric or mesh. Ultraviolet irradia­tion, in addition to periodic fabric or mesh cleaning, may help inhibit this biomass growth.

Microstrainers have been widely used in the removal of particles from sewage effluents and in removal of algae from the water supply (Berry, 1961). Successful removal of Micractinium from algae ponds has been reported under a condition that growth of unicellular strains of Scenedesmus and Chlorella does not overcompete the algae to cause deterioration of algae removal (van Vuuren and van Duuren, 1965). Thickening of Coelastrum proboscideum to about 1.5% suspended solids by microstrainers was reported when operating at a cost of about DM 0.02/m3 and power consumption of 0.2 kWh/m3 (Mohn, 1980). Some success in clarifying high rate pond effluent with continuous backwashing in microstrainers was achieved (Koopman et al., 1978; Shelef et al., 1980). However, the success was confined to ef­fluent dominated by algae species such as Micractinium and Scenedesmus, since the smallest mesh available at that time was of 23 pm openings. Greater success has been reported in clar­ifying stabilization lagoon effluent in reducing suspended solids from up to 80 mg/L to 20 mg/L or less by rotary microstrainers mounted with screens as fine as 1 pm (Wettman and Cravens, 1980).

In a study using microstrainers fitted with 6 pm and 1 pm meshes in clarifying algae pond effluents, the Francea Micractinium algae were completely retained by the 6 pm screen, whereas the Chlorella algae passed through the 1 pm screen (Shelef et al., 1980). The distinction in algae retention on the screens was evidently due to the difference in size of the algae in each pond. It was noted that although the size of the Chlorella algae were larger than 1 pm, they were not retained by the microstrainers. A possible reason could be due to the poor

quality control of mesh size. Continuous operation may overcome part of the problem by building up and maintaining an algal biofilm base layer that serves as a biological fine screen.

Although heterotrophy of algae shows its potential for oil production, the overall produc­tion cost of heterotrophic oils remains relatively high, restricting the commercialization of heterotrophic algal oils. From an estimation of Yan et al. (2011) using heterotrophic C. protothecoides for oil production, the unit production cost of algal oils was still much higher than that of plant oils. Glucose represents a major share of the cost of heterotrophic oil pro­duction. Using alternative low-cost carbon sources may represent a promising approach to bring down the cost of heterotrophic algal oils. Recently, it has been reported that low-cost sugars were used to grow algae for heterotrophic oil production, e. g., hydrolyzed carbohy­drates (Xu et al., 2006; Cheng et al., 2009; Gao et al., 2010) and waste molasses (Yan et al., 2011; Liu et al., 2012a). All these reports suggested the potential of producing algal oils for less cost, such that the algal oils from C. protothecoides based on waste molasses cost approx­imately half those based on glucose (Yan et al., 2011).

The heterotrophic utilization of sugars for biomass by algae remains at a relatively low level, namely, below 0.5 (Cheng et al., 2009; Liu et al., 2010; Yan et al., 2011), which means that more than 50% of sugars were wasted in the form of CO2. To increase the sugar-to- biomass conversion, a photosynthesis-fermentation mode was proposed and resulted in a high sugar-to-biomass conversion of 0.62 (Xiong et al., 2010b). The increased sugar — conversion efficiency may be attributed to the refixation of partial CO2 released from sugar catabolism by the enzyme RuBisCO, which maintained its carboxylation activity in the fer­mentation stage (Xiong et al., 2010b). In a fermentation system, productivity is greatly related to the medium nutrients as well as fermentation parameters. The manipulation of these factors to achieve a maximized output/input ratio may have great potential for improving production economics of heterotrophic algal oils.

Heterotrophic algal biomass contains not only oils but also substantial amounts of proteins and carbohydrates as well as high-value components such as pigments and vitamins. From a biorefinery’s point of view, the residual biomass after oil extraction can be potentially used as food additives, nutraceuticals, and animal feed (Figure 6.8). Also, carbohydrates may be uti­lized for producing the bio-gas methane by anaerobic digestion. The integrated production of oils and other value-added production, coupled with the possible recycling of water and nu­trients, remains a potential strategy to reduce the production cost of algal oils.

Strain improvement by genetic engineering is another feasible and complementary approach to enhancing algal productivity and improving the economics of algal oil produc­tion. Introduction of a bacterial hemoglobin in various hosts has been shown to contribute to growth improvement in oxygen-limited conditions (Zhang et al., 2007). This strategy is par­ticularly suitable for heterotrophic growth of algae to achieve the ultrahigh cell density that may be restricted by the lowered dissolved oxygen associated with cell mass buildup. The­oretically, enhanced oil content can be achieved by the direct genetic engineering of oil bio­synthetic pathways, e. g., overexpression of the genes involved in fatty acid/lipid synthesis (Madoka et al., 2002; Lardizabal et al 2008); the manipulation of transcriptional factors

related to lipid biosynthesis regulation (Courchesne et al., 2009); or the blocking of compet­ing metabolic pathways that share the common carbon precursors such as starch synthesis (Li et al., 2010). Genetic engineering can also be employed to alter fatty acid compositions of oils for improving biofuel quality, e. g., heterologous expression of thioesterases to accumu­late shorter-chain-length fatty acids (Radakovits et al., 2011) or inactivation of the A12 desaturase gene to produce more oleic acid (Graef et al., 2009). In addition, genetic engineer­ing may confer on algae the possibly improved characteristics of tolerance of temperature, salinity, and pH, which will allow cost reduction in algal biomass production and be ben­eficial for growing selected algae under extreme conditions that limit the proliferation of invasive species. Although genetic engineering of algal oils is currently restricted to certain model algae such as Chlamydomonas, the rapid advances in the development of genetic ma­nipulation tools, plus the better understanding of lipid biosynthesis and regulation, will be extended to industrially important algal species for improving the economics of algal oil production.

6.5 CONCLUSIONS

Heterotrophic production has substantial advantages, including rapid growth, ultrahigh cell density, high oil content, and substantial oil productivity. These merits allow significantly lower downstream process costs, though so far the overall oil production from heterotrophic algae is considered not as economically viable as phototrophic production of algal oils. The relatively high cost of heterotrophic algal oils is mainly attributed to the use of expensive or­ganic carbon—in particular, glucose. Advances in the exploration of using low-cost raw ma­terials such as hydrolyzed carbohydrates and waste sugars have enabled potential cost reductions in heterotrophic production of algal oils. Finding ways to further improve the production economics still remains the major challenge ahead for commercialization of heterotrophic algal oils, which will depend to a large extend on significant advancements in culture systems, biorefinery-based integrated production, and algal strain improvement. Breakthroughs and innovations occurring in these areas will greatly expand production capacity and lower production costs, driving heterotrophic algae from today’s high-value market into the low-cost commodity product pipelines.

Thanks to the increasing interest of using Chlorella biomass as the feedstock for oils, great achievements have been made in heterotrophic culture systems and production models for the algae of this genus, allowing ultrahigh cell density comparable to oleaginous yeasts. To this end, sequencing both the genomes and transcriptomes of several typical Chlo — rella strains is currently underway, which will benefit the development of a new molecular toolbox to successfully manipulate Chlorella for more economically feasible industrial production.

Acknowledgments

This study was partially supported by a grant from the 985 Project of Peking University and by the State Oceanic Administration of China.

The bead-beating method involves the application of beads for the disruption of the algal cell wall. Continuous exposure of biomass to beads leads to cell-wall rupture, resulting in the release of intracellular contents into the solvent medium. Similar to expeller pressing, this method can also be applied for both disruption and extraction. The influence of bead beating on cell-wall disruption was evaluated for the strains Botrycoccus braunii, Chlorella vulgaris, and Scenedesmus sp. using a bead beater (bead diameter of 0.1 mm) (Lee et al., 2010). The method showed a lipid productivity of 28.1%.

Though the disruption of algae cell walls prior to extraction requires an additional step, which is the selection of a cost-effective method, it helps to enhance lipid production efficien­cies. The methods discussed here are economical and applicable to mass cultures compared to few other techniques, such as microwaves, sonication, and autoclaving.