Heterotrophism is a mode of nutrition whereby microalgae utilize external substrates as sole carbon sources for their growth and lipid accumulation. The circumstances in which microalgae use organic molecules as primary energy and carbon sources is called heterotrophic nutritional mode (Kaplan et al., 1986). In heterotrophic nutrition, the simpler carbohydrates enter the cell and are subsequently converted to lipids and participate in other metabolic pathways such as respiration (Figure 8.7). Heterotrophic nutrition takes place both in the presence and absence of light. In photoheterotrophic nutrition, light acts as an energy source, but the source of carbon remains organic only. Heterotrophic growth in the dark condition is supported by a carbon source replacing the light energy. This unique ability is shared by several species of microalgae (Perez-Garcia et al., 2011). Glucose is the simpler carbon source for heterotrophic microalgae. Higher rates of growth and respiration are obtained with glucose than with any other substrate, such as sugars, alcohols, sugar phosphates, organic acids, and monohydric alcohols. This oxidative assimilation takes place in algae apparently through two pathways; i. e., the Embdenn Meyerhoff pathway (EMP) and the pentose phos­phate pathway (PPP) (Neilson and Lewin, 1974).

Carbon metabolism in heterotrophic growth of microalgae under dark condition occurs via a PPP pathway, whereas the EMP pathway is the main glycolytic process in light conditions (Lloyd, 1974; Neilson and Lewin, 1974; Yang et al., 2000; Hong and Lee, 2007). Both pathways are carried out in the cytosol and are functional in microalgae. However, the PPP pathway might have a higher flux rate than the other, depending on the carbon source and the presence of light (Perez-Garcia et al., 2011). Light is not required for the transport of glucose inside the

cell during dark heterotrophic operation. Glucose transport system in the algal cell become inefficient in the presence of light, because of higher availability of photosynthates inside the cell due to photosynthesis and down-regulation of hexose transport protein. The carbon is obtained from outside the cell and converted to the acetyl-CoA via pyruvate, which further converts to malonyl-CoA and subsequently enters the lipid biosynthetic pathway (Figure 8.7). In heterotrophic nutrition mode, because of abundant glucose availability, respiration and other metabolic processes do not compete with the lipid biosynthesis, unlike autotrophic mode. Moreover, microalgae can utilize organic carbon under dark conditions because of the ability of light-independent glucose uptake. Hence, the lipid productivity is high in het­erotrophic nutrition mode (Abeliovich and Weisman, 1978).

Heterotrophically it is possible to obtain high densities of microalgal biomass that provide an economically feasible method for large-scale mass production (Chen, 1996; Chen and Johns, 1996; Lee, 2004; Behrens, 2005; Perez-Garcia et al., 2011). Photoheterotrophic nutri­tional mode avoids the limitations of light dependency, which is the major obstruction to gaining high cell density in large-scale photobioreactors (Huang et al., 2010). Chlorella protothecoides showed higher lipid content (40%) during heterotrophic growth (Xu et al., 2006). Higher lipid productivity (3,700 mg/L/d) was also reported by using an improved fed-batch culture strategy in heterotrophic nutritional mode, where the lipid productivity was 20 times higher than that obtained under photoautotrophic cultivation (Xiong et al., in 2008). The major advantage of heterotrophic nutritional mode is the facilitation of wastewater treatment along with lipid productivity, which gives an edge to its application in the present state of increasing pollution loads. Moreover, cost effectiveness, relative simplicity of opera­tion, and easy maintenance are the main attractions of the heterotrophic growth approach (Perez-Garcia et al., 2011). However, heterotrophic systems suffer from contamination prob­lems (Abeliovich and Weisman, 1978; Olguin et al., 2012).

Dark or heterotrophic fermentation by anaerobes as well as some microalgae, such as green algae on carbohydrate-rich substrates, can produce hydrogen in an anaerobic environment without the need of light energy (Zhang et al., 2006; 2007a, b, c; 2008a, b, c, d; Show et al., 2007; 2010; Lee et al., 2011). The possibility with dark fermentative hydrogen production from algal biomass remains that hydrogen was produced by heterotrophic bacterial satellites present in the algal biomass slurries (Carver et al., 2011; Lakaniemi et al., 2011). It has been well established that methane is generated in conventional anaerobic fermentation in two distinct stages: acidification and methane production. Each stage is carried out by specific microorganisms through syntrophic interactions. Hydrogen is produced in the first-stage

acidogenesis as an intermediate metabolite, which in turn is used as an electron donor by methanogens at the second-stage methanogenesis.

Formation of molecular hydrogen in dark fermentation is generally accom­plished through two pathways in the presence of specific coenzymes (Show et al.,

2011) . One pathway is by a formic acid decomposition route; the other pathway is the reoxidization of nicotinamide adenine dinucleotide (NADH) route represented by NADH + H+ + 2Fd2+! 2H+ + NAD+ + 2Fd+ and 2Fd2+ + 2H+! 2Fd+ + H2 under the mediation of hydrogenase. The Embden-Meyerhof, or glycolytic, pathway is un­doubtedly the most common route for glucose degradation to pyruvate, which functions in the presence or absence of oxygen (Prescott et al., 2002). In this pathway, glucose is converted into pyruvate associated with the conversion of NADH from NAD+ via an­aerobic glycolysis represented by C6H12O6 + 2NAD+! 2CH3COCOOH + 2NADH + 2H+. Electron transfer via pyruvate-ferredoxin oxidoreductase or NADH-ferredoxin oxidore- ductase and hydrogenase could be affected by the corresponding NADH and acetyl-CoA levels or prevailing environmental conditions. Thus, the oxidation-reduction state has to be balanced through the NADH utilization to form several reduced compounds, i. e., lactate, ethanol, and butanol, resulting in a lowered hydrogen yield.

Theoretically, it is possible to harvest hydrogen at the acidogenesis stage of anaerobic fer­mentation if only acidogens are left to produce hydrogen gas and other metabolites and the final methanogenesis stage and other hydrogen-consuming biochemical reactions are inhibited during the dark fermentation. However, inhibition of hydrogen-consuming micro­organisms in complex microbial consortia decomposing algal biomass for hydrogen produc­tion poses a challenging task. It has been reported that the hydrogen produced from green algae C. vulgaris and D. tertiolecta biomass by anaerobic enriched cultures containing BESA was subsequently consumed by nonmethanogenic microorganisms (Lakaniemi et al.,

2011) . Similar hydrogen utilization was also reported from the work on hydrogen production by anaerobic sludge fed with lipid-extracted Scenedesmus algal biomass (Yang et al., 2010).

During the dark fermentation, carbohydrates are converted into hydrogen gas and volatile fatty acids and alcohols, which are organic pollutants and energy carriers. For the purpose of energy production and protection of the water bodies, a second-stage process is necessary to recover the energy residues remaining in the effluent in the form of fatty acids and alcohols. Thus the fermentative reactor becomes part of a process wherein the effluent post-treatment process and hydrogen utilization should also be included. A possible second-stage process is photofermentation, anaerobic digestion, or microbial fuel cells, which have been assessed in a recent review (Show et al., 2012).

For hydrogen produced from dark fermentation to be used alone in an internal combustion engine or a fuel cell, some issues such as biohydrogen purification, storage, and transport are to be addressed. Unlike a biophotolysis process that produces only hydrogen, the gaseous product of dark fermentation is a mixture of primary hydrogen (generally less than 70%) and CO2 but may also contain other gases such as CH4, H2S, ammonia, and/or moisture. Pu­rification of the hydrogen is essential before the hydrogen utilization can be practical (Show et al., 2011; 2012). Nevertheless, hydrogen production by dark fermentation is an attractive process in the sense that it does not demand large land space and is not affected by weather conditions (solar radiation is not a requirement). Also, among the biohydrogen production processes, dark fermentation is deemed to be more favorable. Hydrogen is yielded at a high rate, and various organic compounds and wastewaters are enriched with carbohydrates as the substrate results in low-cost hydrogen production (Hallenbeck and Ghosh, 2009). Hence, the feasibility of the technology yields a growing commercial value.

Due to the dramatic increase in primary energy consumption and the increasingly strict environmental issues triggered by fossil-fuel sources, it is our firm belief that development of algal biofuel is urged. As discussed in this chapter, the main challenges pertaining to algal biofuel viability entail lower environmental impact beyond a number of associated benefits (namely, CO2 reduction and wastewater treatment), which may contribute to ensuring eco­nomic competitiveness.

However, associated with biofuel production is the spent biomass that is produced, with huge potential in terms of applications—from secondary biofuels through feed formulations and fine chemicals to bioremediation purposes. Therefore, biofuel production using spent biomass entails a strong economic interest, as thoroughly discussed in this chapter.

For competitiveness in this algae-based scenario, industry should follow an integral upgrade approach via implementation of an algal-based biorefinery, thus maximizing the economic return on all components of algal biomass, aiming at the point of zero residues.

A careful analysis of the current state of the art indicates that it is difficult to develop algal biofuel to the point where it can fully replace fossil fuels, in either developing or developed economies. Governments should indeed adopt an affirmative action by enforcing carbon taxes to limit use of fossil fuels as well as subsidizing investment, funding R&D efforts, and promoting consumption of renewable energies. Multilateral alternative energy develop­ments will probably be necessary to fully address the CO2 emission objectives of the Copenhagen Agreement and the Kyoto Protocol—and extensive cultivation of algae could play a central role in that process.

Acknowledgments

This work received partial funding from project MICROPHYTE (ref. PTDC/EBB-EBI/102728/2008), coordinated by author F. Xavier Malcata and under the auspices of ESF (III Quadro Comunitario de Apoio) and the Portuguese State.

A postdoctoral fellowship (ref. SFRH/BPD/72777/2010), supervised by author F. Xavier Malcata and cosupervised by author Isabel Sousa-Pinto, was granted to author A. Catarina Guedes, also under the auspices of ESF. A Ph. D. fellowship (ref. SFRH/BD/62121/2009), further supervised by author F. Xavier Malcata and cosupervised by author Isabel Sousa-Pinto, was granted to author Helena M. Amaro, again under the auspices of ESF.

In situ transesterification, better known as reactive extraction, has been developed with the purpose of simplifying the biodiesel production process by allowing extraction and transesterification to occur in a single step, in which oil-bearing seeds or algal biomass are in direct contact with the chemical solvent in the presence of a catalyst (acid or base). Through intensive research in recent years, the optimum conditions for in situ transesterification have become well established for different edible and nonedible oil feedstock, such as jatropha (Shuit et al., 2010), soybeans (Haas and Scott, 2007), and castor (Hincapie et al., 2011). However, the main constraint in commercializing this technology is the requirement of a high volume of chemical solvent, and the process is limited to homogeneous catalyst usage only.

In situ transesterification of algal biomass has been explored to attain high biodiesel con­version, including optimization of the alcohol-to-lipid molar ratio, reaction temperature, catalyst loading, and the effect of the use of a cosolvent, microwave, and ultrasonication. In a study performed by Ehimen et al. (2010), dried Chlorella biomass was subjected to in situ transesterification, attaining 90% of biodiesel yield at a reaction temperature of 60 °C, a methanol-to-lipid molar ratio of 315:1, a H2SO4 concentration of 0.04 mol, and a reaction time of 4 h.

To further reduce methanol consumption for the in situ transesterification, adding a cosolvent to the reaction mixture is suggested to increase the solubility of the algal lipids in methanol, creating a single phase reaction that could subsequently improve the reaction mass transfer rate. A yield of approximately 95% Chlorella pyrenoidosa biodiesel was attained when hexane was used as a cosolvent (hexane-to-lipid molar ratio of 76:1). The methanol-to — lipid molar ratio was significantly reduced to 165:1 and the total reaction time was shortened to 2 h at a reaction temperature of 90 °C and a catalyst loading of 0.5 M H2SO4. Nevertheless, the presence of water in the reaction media could impede the in situ transesterification and cause negligible biodiesel conversion (Ehimen et al., 2010). Thus, extensive drying of algal biomass is absolutely necessary to facilitate biodiesel conversion by avoiding the occurrence of any side reactions and to simplify the subsequent refining processes (Lam and Lee, 2012).

Other technologies that could further improve the reaction conditions for in situ transes­terification of algal biomass are microwave irradiation (Patil et al., 2011a; Patil et al., 2012), ultrasonication (Koberg et al., 2011), and supercritical alcohol (Levine et al., 2010; Patil et al., 2011b). However, these technologies are still far from commercialization due to safety — and health-related problems.

F. G. Aden, J. M. Fernandez, E. Molina-Grima

Department of Chemical Engineering, University of Almeria, Almeria, Spain

13.2 INTRODUCTION

Microalgae have been proposed as the potential source for a wide range of products, rang­ing from fine chemicals and pharmaceuticals to nutraceuticals and additives, foods, and feeds and as a biofuel source as well as playing a role in wastewater treatment (Borowitzka, 1999; Richmond, 2000). However, of all these products and roles, only a few are performed on an industrial scale. Microalgae are produced as a source of certain carotenoids, such as p-carotene and astaxanthin; microalgae biomass is also produced as food in nutraceutial applications and as feed for aquaculture. The amount of microalgae produced worldwide for these markets is around 5 kt/year. The price of microalgae biomass ranges from €10-300/kg, and the size of these markets is from 10-50 kt/year (Pulz and Gross, 2004). The development of new applications for microalgae biomass can increase the present pro­duction capacity. Thus, large-scale markets such as energy or commodities have the potential to absorb enormous amounts of microalgae biomass—up to 104 kt/year—but the price of biomass in these markets is far lower, from €0.01-0.50/kg. For this reason, microalgae bio­mass production costs must likewise be reduced to comply with these markets (Chisti, 2007).

Even though biomass production is normally performed under continuous operation in order to maximize the system yield, some products can be produced by varying operation modes from discontinuous to continuous-discontinuous combinations, as is the case with astaxanthin. Whatever the final use of the microalgae biomass and whichever production mode is used, the steps required to produce it are the same. The culture medium has to be prepared and introduced into the photobioreactors, where the biomass is produced, then it has to be harvested and stabilized. Alternatively, it can be processed to create products according to adequate downstream schemes (see Figure 14.1). Each one of these steps requires

FIGURE 14.1 General scheme of microalgae biomass production systems. Major inputs are nutrients, water, and CO2 in addition to energy. Processes can be built to produce stabilized biomass or final products according to an adequate downstream process.

materials and energy input. In addition, waste released in each step has to be treated. Differ­ent possibilities exist for each of the necessary steps, the overall yield and cost of the finished product being a function of the final scheme used. For example, a culture medium might be prepared using fine chemicals, fertilizers, or wastes—the resultant costs using wastes being less but the final biomass quality produced significantly diminished.

In this chapter, the cost of producing microalgae biomass is reviewed for various applica­tions using various schemes. Analysis is performed based on (1) the product obtained, (2) the overall scheme of the process, and (3) the production capacity. In each case, the major factors determining total production costs are identified and strategies are discussed to reduce those costs.

Separating algae from its medium and/or algal biomass concentrating is known as harvesting. Selection of harvesting technologies depends principally on the type of algae (Chen et al., 2011). Harvesting large algae, namely macroalgae, employs laborious work in­volving simple operations, whereas minute microalgae are normally harvested using me­chanical means. Macroalgae grow either in fluid suspension or on a solid medium fed with substrate. To harvest macroalgae grown on solid substrate, they must be detached di­rectly from the medium. The modus operandi in harvesting suspended macroalgae is relatively simple and laborious. The harvesting can be accomplished by nets raised from the pond bot­tom and pulled over a petrol-driven rotary cutter mounted on the harvesting boat. The macroalgae are cut, collected, and transported to land and dried under the sun. Nets are nor­mally harvested three to four times, but the yield reduces progressively over each harvest.

Current work on algae for the commercial production of biofuel mainly focuses on microalgae. The inclination toward microalgae is due largely to its simpler cell structure, rapid growth rate, and high lipid content. However, the most rapidly growing algal species are frequently minute and often motile unicells; these are the most difficult algae to harvest. Processes for biodiesel production from microalgae engage a production unit whereby microalgae is cultivated, separated from the growing medium and thickened, and put through subsequent downstream processing such as dewatering, drying, and lipid extrac­tion. Extracted lipids are processed for biodiesel or other biofuels in similar methods to existing technologies used for other biofuel feedstock.

For microalgae grown in an aqueous medium, thickening of loose algae suspension until a thick algae slurry or cake forms is a vital stage of harvesting. In other words, the water content of algae suspension must be reduced as far as possible to enable practical harvesting and downstream processes. Algae are technically harvested based on the principles of solid — liquid separation processes. The harvesting process may include one or more of the stages of thickening, dewatering, and drying (Figure 5.1). The most common harvesting processes are screening, coagulation, flocculation, flotation, sedimentation, filtration, and centrifuga­tion. Other harvesting techniques such as electrophoresis, electroflotation, and ultrasound are used to lesser extents (Chen et al., 2011). In essence, the choice of technology for algae harvesting must be energy-efficient and relatively inexpensive for viable biofuel production.

4.2.1 Screening

Screening is the first unit operation used in most wastewater treatment plants as well as algae harvesting. The principle of screening involves introducing algae biomass onto a screen of given aperture size. The efficiency of the screening operation depends on the spacing between screen openings and algal particle size. For algae harvesting, microstrainers and vibrating screens are common screening devices.

Continuous cultivation with cell recycling, denoted as perfusion culture, is a culture technique combining the advantages of both fed-batch and continuous culture systems, namely, avoiding the substrate inhibition and the inhibition caused by toxic metabolites produced by accumulated algal cells while maintaining high cell density and productivity

(Chen and Johns, 1995; Wen and Chen, 2002a). As illustrated by Figure 6.5b, in a perfusion culture system the algal cells are retained by a retention device, whereas the spent medium (cell-free) was removed from the bioreactor; at the same time, fresh medium was fed into the bioreactor to maintain sufficient nutrient supply. Wen and Chen (2002a) used the perfusion culture system to investigate the heterotrophic production of N. laevis. By employing an ex­ponential feeding of glucose and manipulating the rates of glucose feeding and spent me­dium perfusion, the optimal glucose concentration in the feed was determined to be 50 g L-1 (Figures 6.7a and 6.7b). With the feeding of optimized glucose concentration (S0 = 50 g L-1), a high cell density of 40 g L-1 was achieved in the perfusion culture of N. laevis (Figure 6.7c). Together with the relatively simple setup and operation as well as high biomass

yield coefficient based on glucose, the perfusion culture system potentially may be used to grow algae for heterotrophic production of bio-oils.

A modified perfusion culture system that introduces cell bleeding during perfusion oper­ation was also developed for heterotrophic production of algae (Figure 6.5c; Wen and Chen, 2001b). This system could potentially improve the biomass productivity but at the same time lower the cell density, e. g., from 40 g L-1 to less than 20 g L-1 (Wen and Chen, 2001b; Wen and Chen, 2002a).

It is worth mentioning that different algal species/strains may favor different culture systems to achieve maximized cell density, biomass productivity, and oil productivity. An experimental optimization is required for a selected algal strain to demonstrate which culture system is best for the heterotrophic production of oils. Regardless of the algal strain selected and culture system used, the key to optimizing a production system rests with the cost balance of output and input from a cost-effectiveness point of view.

A coiled transparent and flexible tube of small diameter with separate or attached degassing unit is the basis for the helical type of bioreactor. A centrifugal pump is used to drive the culture through a long tube to the degassing unit. CO2 gas mixture and feed can be circulated from either direction, but injection from the bottom gives better photosynthetic efficiency (Morita et al., 2001). A degasser facilitates removal of photosynthetically produced oxygen and residual gas of the injected gas stream. This system facilitates better CO2 transfer from gas phase to liquid phase due to a large CO2 absorbing pathway (Watanabe et al., 1995). The energy required by the centrifugal pump in recirculating the culture and associated shear stress limits this reactor’s commercial use (Briassoulis et al., 2010). Fouling on the inside of the reactor is another disadvantage of this system.

The group of phycocolloid polymers, commonly termed hydrocolloids because they are soluble in water, includes alginates, carrageenans, and agars—and red and brown macroalgae have long been used for the production of such compounds (Carlsson, 2007). These polymers are either located in the cell walls or within the cells where they serve as storage materials (Tseng, 2001).

Hydrocolloids account for the major industrial products derived from algae (Radmer, 1996; Pulz and Gross, 2004). They possess several useful properties for the food industry in thickening agents, forming gels and water-soluble films that are commonly applied to sta­bilize such products as ice cream, toothpaste, and mayonnaise (Tseng, 2001), thus taking ad­vantage of their forming a gel upon cooling (Carlsson, 2007). Each major subgroup is described in further detail in the follow subsections.

Conventional hydrothermal treatment processes are divided into three categories: batch — type reactor, semibatch reactor, and continuous reactor.

In a batch reactor, water and reactant are sealed in the same reactor. The reactor is heated from outside or inside. Due to the easy handling and operation of a batch reactor, many re­sults and analysis data in various operation conditions have been reported. But productivity in a batch system does not meet commercial demand. Steel batch autoclaves are used in most cases. Steel autoclaves have the disadvantage of heating slowly, and thus some time is re­quired to reach reaction temperature (Manarungson et al., 1990). Other reactor types include capillaries and tubular steel reactors. Quartz capillaries have also been used as batch microreactors.

In a semibatch reactor, a reactor is filled with reactant and hot compressed water is introduced to the reactant separately. Temperature control of the slurry and flow rate control of the hot water are simple, and moreover product is obtained continuously. However, reactants have to be refilled in the reactor for continuous production. Sakaki et al. developed a semibatch system (Sakaki et al., 1998), but productivity was still very low.

There are two methods in a continuous system; one is a separate type, and the other is a slurry type. Feeding of solid feedstock into a high-pressure reactor is the biggest challenge to the operation of the separate process. On the other hand, a commercial high-pressure slurry pump is available for continuous feeding of high-concentration slurry (Kobayashi et al., 2011). For continuous operation, tubular steel reactors are often used. Other types of reactors, such as the stirred tank reactor, can be used in principle, but to date this configuration has not yet been applied (Navarro et al., 2009).