A cross-flow ultrafiltration system was adopted for treatment of algae pond effluents to produce thickened algae for animal feed. Up to 20 times of the concentration of the algae had been collected with very high-quality filtered effluent. The main disadvantage of this system is the high energy requirements, which rendered this process uneconomical.

5.3.3.1 Magnetic Filtration

Magnetic filtration was initially used in wastewater treatment for removal of suspended solids and heavy metals (Bitton et al., 1974; Okuda et al., 1975). Magnetic separation using suspended magnetic particles (such as Fe3O4 magnetite) was subsequently used in algae re­moval (Yadidia et al., 1977). Algal cells and the magnetic particles were coagulated, and the fluid was passed through a filter screen encompassed by magnetic field to retain the magnetic precipitates. Algae removal efficiency of between 55% and 94% by a commercial magnetic filter dosed with alum coagulant was reported (Bitton et al., 1974). Higher algae removal (>90%) was achieved using 5-13 mg/L Iron (III) Chloride as primary coagulant and 500-1,200 mg/L magnetite as magnetic particles for pond algal harvesting (Yadidia et al., 1977).

Pyrolysis is a physical-chemical process in which biomass is heated to between 400°C and 800°C, resulting in the production of a solid phase rich in carbon and a volatile phase

composed of gases and condensable organic vapors (Mesa-Perez et al., 2005). These organic vapors condensate in two different phases: bio-oil and acid extract (Beenackers and Bridgwater, 1989).

Through pyrolysis, carbon-carbon bonds are broken, forming carbon-oxygen bonds. It is a redox process in which part of the biomass is reduced to carbon (coal) while the other part is oxidized and hydrolyzed yielding phenols, carbohydrate, aldehydes, ketones, and carboxylic acids, which combine to form more complex molecules such as esters and polymers (Rocha et al., 2004).

Due to the extreme conditions to which biomass is submitted, many simultaneous reac­tions occur, resulting in gaseous, liquid, and solid products:

1. Gas phase. Consists primarily of low-weight products that have moderate vapor pressure at room temperature and do not vaporize at pyrolysis temperature.

2. Liquid phase. Further subdivided into two other phases determined by density differences:

• Bio-oil, which is a mixture of many compounds with high molecular weight that became vapors at pyrolysis temperature but condense at room temperature.

• Acid extract (or aqueous extract), which consists of an aqueous phase with numerous soluble and/or suspended substances.

3. Solid phase. Also known as biochar, the solid phase is composed of an extremely porous matrix, very similar to charcoal (DalmasNeto, 2012).

Pyrolysis conditions can be manipulated to produce preferably one phase or the other. Residence time is one of the factors that most influence the final result. To produce incondensable gases, high residence time at high temperature is generally used; higher yields of solids are generally achieved by very high residence time at low temper­atures (allowing polymerization reactions) (Sanchez, 2003). For preferential production of the liquid phase, fast pyrolysis is often chosen. Table 7.1 summarizes the conditions and main effects of residence time and temperature in gaseous, liquid, and solid product generation. Other pyrolysis technologies and their characteristics are presented in Table 7.2.

Due to its tendency to preferentially form bio-oil, coupled with high-speed reaction and greater productivity, fast pyrolysis is the best model for the production of biofuels from algae.

Pulsed electric field (PEF) processing is a method for processing cells by means of brief pulses of a strong electric field (Guderjan et al., 2007). Algal biomass is placed between two electrodes and the pulsed electric field is applied. The electric field enlarges the pores of the cell membranes and expels its contents (Guderjan et al., 2004).

8.Є.2.8 Enzymatic Treatment

Enzymatic extraction uses enzymes to degrade the cell walls, with water acting as the solvent (Mercer and Armenta, 2011). This makes the fraction of oil much easier. The combi­nation of "sono-enzymatic treatment" causes faster extraction and higher oil yields compared to individual ultrasonication and enzymatic extractions alone (Fajardo et al., 2007). The draw­backs associated with the process are lack of commercial feasibility and inapplicability for mass cultures (Halim et al., 2011).

Sterols occur naturally in plants and animals; the most familiar type of the latter is cholesterol, which is vital to cellular functioning due to its role in the fluidity of the cell membrane, besides serving as a secondary messenger in developmental signaling. Further­more, cholesterol is a precursor of fat-soluble vitamins and steroid hormones.

The content and type of sterols vary with the alga species: green algae contain 28-isofucocholesterol, cholesterol, 24-methylene-cholesterol, and p-sitosterol, whereas brown algae contain fucosterol, cholesterol and brassicasterol; red algae contain desmosterol, cholesterol, sitosterol, fucosterol, and chalinasterol. The predominant sterol in brown algae, fucosterol, accounts for 83-97% of the total sterol content, whereas desmosterol, in red algae, accounts for 87-93% (Sanchez-Machado, Lopez-Hernandez et al., 2004; Kumar, Ganesan et al., 2008).

The rapid commercial expansion of the algal biofuel industry is an excellent example of sustainable product development with dramatic future potential for contributions to fuel sup­plies, yet many questions regarding algae production remain unanswered. The state of knowledge regarding the potential environmental impact of the production of algae and algae-derived biofuels continues to be incomplete, fragmented, and largely obscured by proprietary concerns. However, this knowledge is changing rapidly, facilitated by research and industry and driven by economics. Commercialization of the production of algae — derived biofuels as part of the overall biofuel industry will have a profound future impact on society. Waste products that are currently discharged into the environment as contami­nants will be utilized to produce much-needed renewable energy sources. Now is the time to initiate the development of an algae industry evaluation methodology that allows for the advancement of knowledge and evaluation tools for authorities to best understand the potential implications (Menetrez, 2012).

The process of generating biofuel from algae involves the growth, concentration, separa­tion, and conversion of microalgae biomass, some of which can be genetically altered. After separating the desired biofuel product or products from the microalgae biomass, a significant portion of byproduct remains. It is important that the remaining byproducts have a useful and safe purpose for the economic feasibility and environmental sustainability of the process. Post-extraction byproducts must be used efficiently and completely. Since no biofuel is car­bon-neutral in the current scenario, significant fossil-fuel input is needed for growing, processing, and extracting the oil, which might offset the positive aspects of the algal biofuel (U. S. DOE, 2006).

Microalgae hold great promise as starting materials for biofuel production, but signifi­cant challenges exist for the developing industry. Present economy-of-scale differences between the algal oil industry and the petrochemical industry are immense and will require significant investment in the form of government-funded incentives for liquid fuels de­rived from microalgae. The present microalgae manufacturing industry is very small at only 5000 tons y-1 (Pulz and Gross, 2004). It is almost completely devoted to synthesizing high-value nutraceutical products and is not extensively engaged in the mass production of high oil-containing microalgae. In addition, microalgae require significantly higher levels of nitrogen than terrestrial plants to achieve effective growth rates, which increases the cost of production (Brezinski, 2004).

Moreover, microalgae do not achieve concentrations at maturity in their natural aqueous growth media >1 wt%. Therefore, to become a significant industrial commodity in terms of cost and scale, growth and harvesting technologies need to be developed that can econom­ically provide higher concentrations required for industrial-scale processing operations. Actual extraction processes for lipids and lipid-derived materials require considerable improvement. Essentially, any process suitable for commercial consideration must not dry algae by evaporating water. The energy input to evaporate water is significant, and the heat energy input required will, with very few exceptions, be greater than any energy output that can be obtained by combustion of the dried material (Heilmann et al., 2011).

Although algal biofuels possess great potential, profitable production is quite challeng­ing. Much of this challenge is rooted in the thermodynamic constraints associated with producing fuels with high energy, low entropy, and high exergy from dispersed materials (Beal et al., 2012).

One of the difficulties with using conventional gasification technologies for converting high-moisture biomass such as algae is the low thermal efficiency that results from the need to vaporize the water in and with the feedstock. Thus, conventional biomass gasification pro­cesses require a dry feedstock. Of course, energy is still required to do this drying prior to gasification, and the energy needed here offsets some of the energy gained by producing the gaseous product. Gasifying wet biomass in supercritical water is a means of circumventing this energy penalty (Guan et al., 2012).

In addition to these holistic challenges, some of the other challenges with respect to the reactors and catalyst are as follows: Due to the high pressures needed for processing, special reactor and separator designs are required. The process has to be designed in a way that can handle solids loading in excess of 15-20 wt% and handle feedstock with impurities. Proper heat-recovery systems have to be designed because the reactors operate at very high temper­atures in pressurized conditions. Feeding at high pressures into the reactor is always a chal­lenge and is a major problem in small-scale plants. In the case of heterogeneous catalysts, the catalyst has to be robust and should not deactivate easily due to the formation of coke. If ho­mogeneous catalysts are used, they must be recovered at the end of the process and reused again. Another most important phenomenon that occurs is the wall effect, which can cause serious problems after scaling up if not understood at lab-scale level (Peterson et al., 2008).

With respect to the conversion methods, effective heat and mass transfer is required for the proper conversion of feedstock into the desired products. This requires advanced design of reactors used for conversion and preparation of hybrid catalysts.

Acknowledgments

The authors thank the Director, Indian Institute of Petroleum, Dehradun, for his constant encouragement and sup­port. RS thanks the Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing a senior research fellowship (SRF). The authors also thank the CSIR for financial support.

One of the statements of the LCA methodology is to link every economic and environmen­tal flow to the reference flow of the FU. However, several processes implied in the production of the FU can lead to the production of several products. Two approaches are possible to han­dle the multifunctionality of the system: allocation or substitution. The allocation approach consists of distributing the environmental burden of the upstream between all the coproducts of the multi-output process. This distribution should be based on the most sensitive criterion, e. g., mass, economic value, or energy content of the products. The perimeter expansion (or sub­stitution) option consists of adding the coproduct to the FU. The ISO norm for LCA stipulates that perimeter expansion should be preferred when possible. When substitution is not pos­sible, energy allocation should be preferred for processes leading to the production of energy.

Among the 15 publications, 3 publications (Kadam, 2002; Clarens et al., 2010; Jorquera et al., 2010) analyze systems without coproducts. Table 13.8 presents the various coproducts and the choices between allocation and substitution. Several processes can lead to coproducts:

• The oil extraction process leads to the production of an extraction residue (oilcake); only Lardon et al. (2009) chose to use an energy-based allocation at this level. Other authors (Stephenson et al., 2010; Brentner et al., 2011; Campbell et al., 2011; Clarens et al., 2011) chose to directly treat the oilcake by anaerobic digestion. Oilcakes can also replace other products: aquaculture or livestock food, carbohydrates’ source for bioethanol production.

• Oil esterification produces methylester and glycerol; here economic and energy allocation are often used. In case of substitution, glycerol is mainly used as a source of heat.

• Anaerobic digestion produces biogas and solid and liquid digestates; these digestates can be considered waste (and hence cannot support a part of the environmental burden of the process), fertilizer, or soil conditioner. The liquid digestate can be recirculated to the culturing device and hence substituted to a fraction of the mineral fertilizer required for the algae. The produced biogas is transformed into heat used on site to heat the digesters and/ or converted into electricity. Electricity is also consumed on site, and the surplus is injected into the network (Stephenson et al., 2010; Clarens et al., 2011).

In the 1950s, the increase in world population and the prediction of insufficient protein supplement for humans led to the search for alternative and unconventional sources of nutrients. Microalgae emerged as candidates for this purpose. Research has been directed to­ward the development of functional products—food additives such as vitamins, antioxidants, highly digestible proteins, and essential fatty acids. Microalgae can supply several of these nutrients, and they have potential health benefits (Cavani et al., 2009; Petracci et al., 2009).

Microalgae are currently used in the form of tablets, capsules, or liquids. These microor­ganisms can be incorporated into pastas, cookies, food, candy, gum, and beverages (Liang et al., 2004). Due to their varying chemical properties, microalgae can be applied as a nutri­tional supplement or as a source of natural proteins, dyes, antioxidants and polyunsaturated fatty acids (Spolaore et al., 2006; Soletto et al., 2005).

The Laboratory of Biochemical Engineering (LEB) at the Federal University of Rio Grande (FURG) in southern Brazil has developed research projects since 1998 that study the cultiva­tion of Spirulina on a pilot scale on the banks of the Mangueira Lagoon, as additives to meals for children of the region. Products that are easy to prepare, store, and distribute and that are highly nutritious and accepted by the consumer have been developed here.

These products include instant noodles, pudding, powdered mixture for cake, cookies, chocolate milk powder, instant soup, isotonic drinks, powdered gelatin, and cereal bars.

These products will be prepared at the Center for Enrichment of Foods with Spirulina (CEAS) located at the university. In Camaqua (Brazil), the company Olson produces organic Spirulina capsules for importation.

4.1.1 Carbon Dioxide’s Role in Photobioreactors

An important issue in most photobioreactors and the first step in CO2 fixation is the diffusion of CO2 from the gas phase to the aqueous phase. The solubility of CO2 in the culture media de­pends on depth of the pond, the mixing velocity, the productivity of the system, the alkalinity, and the outgassing. It has been reported (Becker, 1994) that only 13-20% of the supplied CO2 was absorbed in raceway ponds when CO2 gas was bubbled into the culture fluid as a carbon source. Binaghi et al. (2003) achieved a maximum value of 38% efficiency of carbon utilization in Spirulina cultivation. Gas-liquid contact time and gas-liquid interfacial area are, therefore, two key factors to enhance the gas-liquid mass transfer. In addition, high oxygen tension is problematic, since it promotes CO2 outgassing and competes with CO2 for the CO2-fixing enzyme (RuBisCO).

The capacity for carbon dioxide storage in a growth medium is important because it deter­mines the amount of CO2 that may be used for medium saturation, leading to high growth rates and in-process economics. Since CO2 reacts with water, producing carbonic acid and its anions, chemical equilibrium will have a significant impact on the amount of carbon dioxide stored. pH is the major determinant of the relative concentrations of the carbonaceous system in water and affects the availability of carbon for algal photosynthesis in intensive cultures (Azov, 1982).

The absorption of CO2 into alkaline waters may be accelerated by one of two major uncatalyzed reaction paths: the hydration of CO2 and subsequent acid-base reaction to form bicarbonate ion, and the direct reaction of CO2 with the hydroxyl ion to form bicarbonate. The rate of the former reaction is faster at pH values below 8, whereas the latter dominates above pH 10. Between pH 8 and 10, both are important.

Microalgae can fixate carbon dioxide from different sources, including CO2 from the atmo­sphere, from industrial exhaust gases (e. g. furnaces flue gases), and in form of soluble carbon­ates. Traditionally, microalgae are cultivated in open or closed reactors and aerated with air or air enriched with CO2. Industrial exhaust gases contain up to 15% of carbon dioxide in their composition, being a rich (and cheap) source of carbon for microalgae growth.

In microalgae cultivation, high concentrations of CO2 are not usually used because it may result in decreasing the pH, since unutilized CO2 will be converted to HCO3 . Shiraiwa et al. (1991) and Aizawa and Miyachi (1986) reported that an increase in CO2 concentration of sev­eral percent resulted in the loss of a carbon concentration mechanism (CCM), and any further increase was always disadvantageous to cell growth. Most processes use air enriched with CO2 (2-5% CO2 final concentration), but some studies using high CO2-resistant strains are being described in scientific literature.

If there is not enough CO2 gas supply, algae will utilize (bi)carbonate to maintain its growth. When algae use CO2 from bicarbonate, an increase of pH is observed (a growth in­dicator), even reaching growth-inhibition pH values. To overcome pH fluctuation, the CO2 gas injection should be controlled in such a way that photosynthesis rates are balanced with enough and continuous availability of dissolved carbon. Interesting studies about isolation and selection of strains with high CO2 absorption capacity, which is an important step no matter the process in development, are available in scientific literature. Maintaining constant CO2-free concentration in the media will keep carbon uptake constant.

The ability to accumulate DIC has been shown to occur in many algae and cyanobacteria (Williams and Colman, 1995). Whereas CO2 can diffuse into algal cells and is the substrate for carbon fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase (RubiscO), it forms a small proportion of the total available inorganic carbon. The largest proportion of total DIC available to microalgae consists of ionic HCO3 , which has a low capacity for diffusion across cell membranes (Young et al., 2001). A number of eukaryotic microalgae have devel­oped mechanisms that permit the use of HCO3 for photosynthesis (Miller and Canvin, 1985). Access to the larger pool of HCO3 is assumed to involve one or both of two basic processes:

1. In some green algae, the use of HCO^ has been correlated with the presence of external carbonic anhydrase (CA) activity (Aizawa and Miyachi, 1986). In these cases external CA is thought to facilitate the use of HCO^ by maintaining equilibrium between HCO^ and CO2, and thereby maintaining the supply of CO2 to a specific transporter (Aizawa and Miyachi, 1986).

2. Direct HCO^ transport via a transmembrane bicarbonate transporter, which has been demonstrated even in cells that have external CA activity (Williams and Turpin, 1987). The involvement of transmembrane ATPase proteins was also reported in DIC uptake by chlorophytes (Ramazanov et al., 1995).

Continuous discharge of solids as a slurry is possible with the nozzle-type disc centrifuge. The shape of the bowl is modified so that the slurry space has a conical section that provides sufficient storage volume and affords a good flow profile for the ejected cake (Shelef et al., 1984). The bowl walls slope toward a peripheral zone containing evenly spaced nozzles. The number and size of the nozzles are optimized to avoid cake buildup and to obtain reasonable concentrated algal biomass.

The application of a nozzle-type disc centrifuge for algae harvesting was suggested by Golueke and Oswald (1965). The influence of nozzle diameter on flow rate, algae removal efficiency, and resultant slurry concentration was looked into. Through comparison with other algae harvesting methods, it was concluded that the nozzle-type centrifuge seemed promising, albeit it is less attractive because of power requirements and capitalization costs. In other studies, the centrifuge appeared to be more effective to harvest Scenedesmus than Coelastrum (Mohn and Soeder, 1978; Mohn, 1980). By returning the centrifuge underflow to the feed, the solids content of the algae suspension (0.1%) can be concentrated by a factor of 15-150%. The reliability of this device can be ensured as long as the clogging of the nozzles is avoided.

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).