A hydrocyclone is constructed of a cylindrical section joined to a conical section. Feed is injected tangentially at high speed into the upper cylindrical section, which develops a strong swiveling fluid motion. Fluid containing fine particles is discharged through overflow pipe, while the remaining suspension containing course particles discharges though the underflow orifice at the cone tip. Application of hydrocyclone for algae harvesting was studied by Mohn (1980). It was reported that only Coelastrum algae that grow in large aggregates are harvested by this method. The solid concentration of harvested algae slurry was low, with incomplete solid-liquid separation.

Living organisms can be divided into two large groups, autotrophs and heterotrophs, according to the type of carbon source they utilize. Autotrophic organisms have the capability to convert physical (light) and chemical (CO2 and H2O) sources of energy into carbohydrates, which further form the base for the construction of all other carbon-containing biomolecules (Yoo et al., 2011). Mostly, the external energy is stored as a reduced form (carbohydrates) that is compatible with the needs of the cell. Autotrophic organisms are relatively self-sufficient and self-sustainable because they obtain their energy from sunlight (Nelson et al., 1994; Eberhard et al., 2008; Nelson and Yocum, 2006; Krause and Weis, 1991). On the contrary, het­erotrophic organisms utilize organic carbon produced by autotrophs as energy sources for their metabolic functions because they cannot utilize atmospheric CO2 as a carbon source. Oxidative assimilation of carbon begins with a phosphorylation of glucose/hexose, yielding phosphorylated glucose, which is readily available for storage, cell synthesis, and respiration (Figure 8.5). Nutritional modes significantly influence the carbon assimilation and lipid pro­ductivity of the microalgae (Xu et al., 2006). Three types of nutritional modes—autotrophic,

I

Triacylglycerides

heterotrophic, and mixotrophic—are reported to produce algal fuel in the presence of light. In addition, the dark hetrotrophic nutrition mechanism is also found to be capable of lipid bio­synthesis by microalgae under specific conditions.

Other than direct photolysis, photosynthetic hydrogen can be produced through the use of green algae that can directly produce hydrogen under the condition of sulfur depri­vation (Manis and Banerjee, 2008). Deprivation of sulfur nutrients in the growth medium causes a reversible inhibition in the activity of oxygenic photosynthesis in green algae
(Melis et al., 2000). Protein biosynthesis is impeded in the absence of sulfur, and the green algae are unable to perform the required turnover of the D1/32-kD reaction center protein of PSII (known as the psbA chloroplast gene product) in the thylakoid membrane of algae (Wykoff et al., 1998). Under sulfur deprivation, the photochemical activity of PSII declines, and the absolute activity of photosynthesis becomes less than that of respiration. As a result, the rates of photosynthetic oxygen evolution drop below those of oxygen consumption by respiration (Melis et al., 2000). Such imbalance in the photosynthesis-respiration relationship by sulfur deprivation resulted in net consumption of oxygen by the cells, causing anaerobic conditions in the growth medium. Consequently, an anaerobic condition prevails in the sealed light-dependent algal cultures. With energy derived from light under deprivation of sulfur, the anaerobic algal cultures would elicit the [Fe]-hydrogenase pathway of electron transport in the chloroplast to photosynthetically produce hydrogen (Melis, 2002).

In essence, hydrogen can be produced under sulfur deprivation by circumventing the sensitivity of the [Fe]-hydrogenase to molecular oxygen through a temporal separation of the reactions of oxygen and hydrogen photoproduction. In the course of such a hydrogen pro­duction condition (sulfur-deprivation), algal cells consumed significant amounts of internal starch and protein (Zhang et al., 2002). Such catabolic reactions apparently indirectly sustain the hydrogen production process.

Hydrogen production via indirect photolysis by algae is deemed feasible if photon conver­sion efficiency can be improved for large-scale applications. Algal bioreactors can provide an engineering approach to regulate light inputs to the culture to improve the photon conversion efficiency of algal cell. The improvement of photosynthesis efficiency is too difficult to achieve for conventional crop plants (Hankamer et al., 2007). Recent research has reported a substan­tial increase in light utilization efficiency of up to 15%, compared with the previous utilization of around 5% (Tetali et al., 2007; Laurinavichene et al., 2008). Some researchers claimed that efficiency between 10% and 13% is attainable by engineering the microorganisms to better utilize the solar energy (Turner et al., 2008). However, improvements must be made to opti­mize the solar conversion efficiency of the algae under mass culture conditions. Optical short­comings associated with the chlorophyll antenna size and the light-saturation drawback of photosynthesis need to be addressed before high photosynthetic solar conversion efficiencies in mass culture can be achieved (Melis et al., 1999). Additional challenges that must be tackled include finding ways to recycle photobioreactor components and minimize the chemical cost of the nutrients to support algal growth, since these two items constitute 80-85% of the overall cost of commercial hydrogen production (Melis, 2002).

Photoproduction of hydrogen at a rate of about 12.5 ml H2/h per gram cell dry weight was reported in a study on indirect biophotolysis with cyanobacterium anabaena variabilis (Markov et al., 1997). In another study on indirect biophotolysis with cyanobacterium gloeocapsa alpicola, it was found that maintaining the culture at pH value between 6.8 and 8.3 yielded optimal hydrogen production (Troshina et al., 2002). Increasing the temperature from 30°C to 40°C resulted in twofold increase in the hydrogen production. The hydrogen production rate through indirect biophotolysis is comparable to hydrogenase-based hydrogen production by green algae.

Currently, less than 10% of the algae photosynthetic capacity was utilized for biohydrogen production. Research is underway to further improve algal photosynthetic capacity using a molecular engineering approach. Mutant algae with less chlorophyll could be manipulated for large-scale commercial applications that disperse more light to deeper algae layers in the bioreactor (Hankamer et al., 2007; Beer et al., 2009). Hence, sunlight is made available for more algal cells to generate hydrogen, thus improving the production rate. With technology ad­vancement, biohydrogen production via algae bioreactors will offer a sustainable alternative energy resource in future.

Commercial fertilizers, used for long periods, have adverse effects on soil productivity and environmental quality, so interest in environmentally friendly, sustainable agricultural practices has been on the rise. In developing and implementing sustainable agriculture tech­niques, biofertilization is of great importance to alleviate deterioration of natural ecosystems and to reduce the impact of environmental pollution while integrating nutrient supply into agriculture. Biofertilizers include mainly nitrogen-fixing, phosphate-solubilizing, and plant growth-promoting microorganisms, as in the case of microalgae.

Marine algae and algae-derived products have been widely used as nutrient supplements and as biostimulants or biofertilizers to increase plant growth and yield. The regulatory sub­stances cytokinins, auxins, gibberellins, and betaines in algae can induce plant growth (Valente, Gouveia et al., 2006), but their roles as macro — and micronutrients also make them valuable components of biofertilizers. A few commercial products based on marine algae are ready available for use in agriculture, but ongoing research has featured several alga species in terms of ascertaining their effects on plant growth. For instance, recent work with Laminaria digitata indicated that this marine macroalga (traditionally used as soil amendment in many parts of the world) improves seed germination and rooting in terrestrial plants (Thorsen, Woodward et al., 2010).

Several pieces of evidence confirmed that microalgae are beneficial in plant cultivation by producing growth-promoting regulators, vitamins, amino acids, polypeptides, and antibacterial and antifungal substances that exert phytopathogen biocontrol as well as poly­mers, especially exopolysaccharides that improve both plant growth and productivity (de Mule, de Caire et al., 1999). Other indirect growth-promotion effects may be claimed, such as enhancing the water-holding capacity of soils or substrates, improving availability of plant nutrients, and producing antifungal and antibacterial compounds (Schwartz and Krienitz,

2005) . In hydroponic cultivation, microalgae present a few extra benefits: The oxygen pro­duced by photosynthesis avoids anaerobiosis in the root system while releasing such growth-hormones as auxins, cytokins, gibberelins, abscisic acid, and ethylene (Schwartz and Krienitz, 2005). Equally important and promising is the high N:P ratio exhibited by microalgae, which is an extra indicator of its potential as fertilizer.

10.5.1 Animal Feed

The moisture content of fresh marine algae is quite high and can account for up to 94% of their biomass. However, marine algae contain such nutritional elements as proteins, lipids, carbohydrates, vitamins, and minerals that are in high demand for animal feed (Zubia et al., 2008). In particular, the ash content is high compared to that of vegetables (Murata and Nakazoe, 2001) and includes both macrominerals and trace elements.

Fish feeding represents over 50% of the whole operating costs in intensive aquaculture, with protein being the most expensive dietary source (Lovell, 2003). Nowadays 24% of the fish harvested by fisheries worldwide is used to produce fish meal and fish oil, thus putting high pressure on fisheries that aquaculture has attempted to alleviate. This demand promoted extensive efforts to find alternative sources of protein sources for aquatic feed; unfortunately, plants are poor protein sources in fish diets owing to their deficiency in certain essential amino acids, their content of antinutritional compounds, and taste problems.

Conversely, microalgae have been traditionally used to enrich zooplankton, which will in turn be used to feed fish and other larvae. In addition to providing proteins contain essential amino acids, they carry such other key nutrients as vitamins, essential PUFAs, pigments, and sterols, which may then be transferred upward through the food chain (Guedes and Malcata, 2012).

On the other hand, contamination by bacteria that attack fish can potentially devastate aquaculture farms. Microalgal fatty acids longer than 10 carbon atoms can induce lysis of bacterial protoplasts; said ability depends on composition, concentration, and degree of unsaturation of free lipids (Guedes et al., 2011b). The contents of carotenoids are important in aquaculture as well. In fact, artificial diets that lack natural pigments preclude such organisms as salmon or trout to acquire their characteristic red color (muscle), which, in nature, is a result of ingesting microalgae containing red pigments; without such a color, a lower market value will result (Guedes and Malcata, 2012).

The use of a heterogeneous catalyst (base or acid) for biodiesel production has been recently and extensively explored. The main advantage of a heterogeneous catalyst over a homogeneous catalyst is that the former can be recycled and regenerated for repeated reac­tion cycles, resulting in minimum catalyst loss and improvement of the economic feasibility of biodiesel production. Furthermore, the catalyst can easily be separated at the end of the reaction using filtration; therefore, product contamination is reduced and the number of water washing cycles (purification) is minimized (Lam and Lee, 2012).

Heterogeneous catalysts can generally be divided into two categories:

1. Catalysts with basic sites, such as CaO, MgO, ZnO, and waste materials impregnated with KOH or NaOH. This type of catalyst can catalyze transesterification under mild reaction conditions with a high yield of biodiesel usually attained. Nevertheless, due to the basic properties of the catalyst, it is highly sensitive to the FFA content in the oil and results in soap formation instead of biodiesel. Leaching of active sites from the catalyst is another limitation that can cause product contamination and catalyst deactivation.

2. Catalysts with acidic sites, such as SO42~/ZrO2, SO42~/TiO2, SO42~/SnO2, zeolites, sulfonic ion-exchange resins, and sulfonated carbon-based catalysts. These catalysts are insensitive to the FFA content in oil and are able to perform esterification and transesterification simultaneously. However, the reaction rate is exceptionally slow; hence, extreme reaction conditions such as high temperature (more than 100 °C) with high alcohol-to-oil molar ratio (more than 12:1) are necessary to accelerate the overall reaction rate.

To date, the application of heterogeneous catalysts to algal biodiesel conversion is still scarcely reported in literature, primarily because algal lipids are a relatively new feedstock that is not commercially available in the market. In a recent study carried out by Umdu et al. (2009), CaO supported with Al2O3 was used as a heterogeneous base catalyst in transesterification of Nannochloropsis oculata lipids (Umdu et al., 2009). The highest algal bio­diesel yield attained was 97.5% under the following reaction conditions: reaction temperature of 50 °C, methanol-to-lipid molar ratio of 30:1, catalyst loading of 2 wt%, and reaction time of 4 h. The long reaction time was required mainly because of the initial three immiscible phases (lipid-alcohol-catalyst) that increase mass-transfer limitations in the system. When pure CaO was used as catalyst in the transesterification of algal lipids, insignificant biodiesel yield was recorded, but when CaO supported with Al2O3 (ratio 8:1 w/w) was used instead, signifi­cantly better results were achieved due to the increasing basic density and basic strength of the catalyst. Other heterogeneous catalysts such as Mg-Zr and hierarchical zeolites have also been investigated for transesterification of algal lipids; however, unsatisfactory biodiesel yield (less than 30%) was attained (Carrero et al., 2011; Krohn et al., 2011).

The use of a large variety of impact assessment methodologies can be potentially problem­atic if one wants to compare LCA studies. For instance, the comparison of the results of the "eutrophication" impact is not possible between the study of Kadam (2002) and the study of Brentner et al. (2011). In the first case, the methodology used is CML and the eutrophication impact is expressed in phosphate equivalent, whereas in the second publication the method­ology used is TRACI and the impact is expressed in nitrogen equivalent. Moreover, some studies do not precisely state the impact assessment methodology (Clarens et al., 2010, 2011).

A comparison of the LCA results of bioenergy production from microalgae with results for fossil fuels and other biofuels should be included. The strengths and weaknesses of this new kind of bioenergy production compared to fossil fuel or classical bioenergy production from biomass must be identified. Assessed impacts should include climate change and an energy balance, but impacts that have reduced the interest in first-generation biofuel (such as land use change occupation or impacts linked with the nitrogen flows or the use of chemical prod­ucts) and have motivated the abandonment of fossil fuel (ozone layer depletion or abiotic re­source depletion) should also be presented. A focus should also be made on the quantity and the quality of required water, since evaporation or water spray to cool the process could lead to drastic water consumption (Bechet et al., 2010, 2011).

13.5 CONCLUSION

This chapter presents a critical review of 15 publications about LCA and bioenergy produc­tion from microalgae. The review illustrated the variability of assumptions made about technological and environmental performance of the different processes involved in the pro­duction and transformation of algal biomass. The main conclusion of this analysis is that there is real difficulty in comparing the environmental burdens of the proposed setups, and there is now a need for clear guidelines to ensure that each new LCA study will consolidate the cur­rent knowledge. This is of key importance, since the objective of LCA works will more and more often consist of guiding the design of new biofuel production systems and prove that they lead to actual progress in terms of environmental impact. In this spirit, there is a clear gain for the LCA community to accept a set of rules and guidelines to make any new analysis comparable to the existing ones.

As a consequence, we have proposed some guidelines for the LCA to allow a clearer and sounder comparison between processes and to better estimate the potential and challenges of microalgae for biofuel production.

Acknowledgments

This chapter presents research results supported by the ANR-08-BIOE-011 Symbiose project. Authors P. Collet, A. Helias, and L. Lardon are members of the Environmental Life Cycle and Sustainability Assessment research group (ELSA; www. elsa-lca. org). They thank all the other members of ELSA for their advice.

Under phototrophic growth conditions, microalgae absorb solar energy and assimilate car­bon dioxide from the air and nutrients from aquatic habitats. However, commercial produc­tion must replicate and optimize the ideal conditions of natural growth. The choice of the reactor is one of the main factors that influence the productivity of microalgal biomass.

Open tanks come in different forms, such as raceway, shallow big, or circular (Masojidek and Torzillo, 2008). Circular ponds with a centrally pivoted rotating agitator are the oldest large-scale algal culture systems and are based on similar ponds used in wastewater treat­ment. The design of these systems limits pond size to about 10,000 m2 because relatively even mixing by the rotating arm is no longer possible in larger ponds. Raceway tanks are the most widely used artificial systems of microalgal cultivation. They are typically constructed of a closed loop and have oval-shaped recirculation channels. They are usually between 0.2 and 0.5 m deep, and they are stirred with a paddlewheel to ensure the homogenization of culture in order to stabilize the algal growth and productivity. Raceways may be constructed of concrete, glass fiber, or membrane (Brennan and Owende, 2010).

Compared to closed tanks, the raceway is the cheapest method of large-scale microalgal production (Chisti, 2008). These tanks require only low power and are easy to maintain and clean (Ugwu et al., 2008).

The construction of open tanks is low cost and they are easy to operate; however, it is dif­ficult to control contamination, and only highly selective species are not contaminated by other microalgae and microorganisms. Environmental variations have a direct influence, and the maintenance of cell density is low due to shadowing of the cells (Amaro et al.,

2011) . Light intensity, temperature, pH, and dissolved oxygen concentration may limit the growth parameters of open tanks (Harun et al., 2010).

Open photobioreactors have lower yields than closed systems due to loss by evaporation, temperature fluctuations, nutrient limitation, light limitation, and inefficient homogenization (Brennan and Owende, 2010). The amount of evaporated water can be periodically or contin­uously added to the raceway. The amount of evaporated water in raceways depends on the temperature, wind velocity, solar radiation, and pressure of water vapor. Water can also be lost during harvesting; however, recycling of the medium reduces this problem, and nutrients from the culture medium can be reutilized (Handler et al., 2012).

Open ponds are the microalgal cultivation systems that have been studied for the longest time. These reactors are used on an industrial scale by companies such as Sosa Texcoco, Cyanotech, Earthrise Farm, Parry Nutraceuticals, Japan Spirulina, Far East Microalga, Taiwan Chlorella, Microbio Resource, Betatene, and Western Biotechnology (Spoalore et al., 2006). Earthrise Farm began cultivation on a large scale in 1976 with Spirulina. Currently the company produces Spirulina and Spirulina-based products. The cultures are grown in 30 open ponds that are 5,000 square meters in size, each one mixed by a 50-foot paddlewheel (Earthrise, 2012).

Since 1981 Parry nutraceuticals has produced Spirulina in powder form, capsules, pills and tablets, and extracts astaxanthin from Haematococcus pluvialis. The company is located in South India (Oonaiyur), and the crops are grown in open ponds, covering an area of 130 acres (Parry Nutraceuticals, 2012). Cyanotech, located in Kailua Kona, Hawaii, on the Pacific Ocean, develops and markets astaxanthin from Haematococcus in gel capsules and Spirulina in tablet form in an area of 90 acres. Since 1984, Spirulina has been cultivated in open ponds, with the medium supplemented with water from the Pacific Ocean and agitation by paddlewheels (Cyanotech, 2012).

In Brazil, since 1998 the Laboratory of Biochemical Engineering (LEB) at the Federal University of Rio Grande (FURG) has been developing a project that studies the cultivation of Spirulina on a pilot scale in an open pond (Figure 1.1) on the edge of Mangueira Lagoon (Morais et al., 2009), for addition to meals for children. Products that are easy to prepare, con­serve, and distribute have been developed. These products include instant noodles, flan, powdered mixture for cakes, cookies, chocolate powder, instant soup, isotonic sports drinks, gelatin powder, and cereal bars (Costa and Morais, 2011).

The LEB, along with the President Medici Power Plant (UTPM), operated by the Society of Thermal Electricity Generation (CGTEE) since January 2005, has carried out the cultivation of microalgae for the biofixation of CO2 that is emitted in the combustion of coal at UTPM in an open pond (Figure 1.2) (Morais and Costa, 2007).

In southern Brazil, the company Olson Microalgae began commercial production of Spirulina capsules as a nutritional supplement in 2012, with an annual target of 6,000 kg in open ponds (Figure 1.3).

The Company Ouro Fino produces biomass and protein from microalgae for human and animal feed using the culture medium vinasse, cane husks, and carbon dioxide generated from the alcohol industry (Figure 1.4).

Several studies of open systems have taken place. According to Lee (2001), only species with high resistance are grown in open systems, such as Dunaliella (resistant to high salin­ity), Spirulina (grown in high alkalinity), and Chlorella (grown with high nutrient concentrations).

The type of light source is known to be a critical factor affecting the growth of microalgae due mainly to the difference in the coverage of wavelength range (Terry, 1986). In addition to the type of light source, the light intensity is also very important for microalgae growth (Grobbelaar et al., 1996; Sanchez et al., 2008; Ugwu et al., 2008; Yoon et al., 2008). In general, the effect of light intensity on the photoautotrophic growth of microalgae could be classified into several phases, such as the light-limitation phase, the light-saturation phase, and the light- inhibition phase (Ogbonna and Tanaka, 2000). To maximize biomass productivity, the satura­tion light intensity needs to be distributed throughout the entire microalgae cultivation system. However, this is impossible in practical cultivation systems, since the light distribution inside the photobioreactor normally decreases significantly along with the distance due to the light shading effects (see Figure 2.1), especially when the cell concentration gets very high or when

FIGURE 2.1 Effect of light intensity on specific growth rate of microalgae under phototrophic cultivation (Ogbonna and Tanaka, 2000).

significant biofilm formation on the surface of the reactor vessel occurs (Chen et al., 2008). Im­proving the mixing of the cells can reduce the effects of light shading or photoinhibition at dif­ferent zones of the photobioreactor. Some literature describes the effect of light intensity on the lipid content of microalgae. Lv et al. (2010) demonstrated that in comparing low and high light intensity (i. e., light-limitation and light-saturation conditions), using a light intensity of 60 gmol/m2/s led to an increase in biomass concentration and lipid content of Chlorella vulgaris, along with changes in pH, NADPH, and Mg2+ concentration (Lv et al., 2010).

The most important parameter considered for the development and utilization of a specific type of reactor for microalgae cultivation is the light diffusion. The productivity of photo­autotrophic cultures is primarily limited by the supply of light and suffers from low energy-conversion efficiencies caused by inhomogeneous distribution of light inside the cultures (Grobbelaar, 2000). At culture surfaces, light intensities are high, but absorption and scattering result in decreasing light intensities and complex photosynthetic productivity profiles inside the cultures (Ogbonna and Tanaka, 2000). High light intensities at culture
surfaces may cause photoinhibition, and the efficiency of light energy conversion into biomass (photosynthetic efficiency) is low. An overdose of excitation energy can lead to pro­duction of toxic species (e. g., singlet oxygen) and to photosynthesis damage (Janssen, 2002)

By minimizing depth, volume is reduced or area is increased, light diffusion is maximized, and so is cell concentration. From common types of photobioreactors, light paths in open ponds are usually 10-30 cm depth, in tubular reactors ranges from 1-5 cm, and in flat panel reactors from 2-5 cm.

The light regimen itself is influenced by incident light intensity, reactor design and dimension, cell density, pigmentation of the cells, mixing pattern, and more. In outdoor photobioreactors the light regimen is also influenced by geographical location, time of day, and weather conditions. Nowadays, open paddlewheel-mixed pond is the most commonly used photobioreactor.

Some studies discuss the effect of mixing and productivity due to the "flashing-light" effect: A few milliseconds’ flashes of high light intensity followed by a several-fold longer period of darkness do not reduce culture productivity from those under constant illumination (Kok, 1953). This effect is not observed in ponds, where the light/dark period is longer. For example, although light/dark cycles of 94/94 ms were sufficiently short to increase the pho­tosynthesis efficiency in cultures of Dunaliella tertiolecta, light/dark cycles of 3/3 s were too long and the PE decreased in comparison to continuously illuminated cultures (Janssen et al.,

2001) . This refers to the theory of photosynthesis, in which carbon fixation is not dependent on the presence of light because sufficient energy has been absorbed.

The competitiveness of using heterotrophic algae over photoautotrophic ones for oil production rests largely with the high yield and productivity of biomass as well as of oil in heterotrophic cultivation modes. The high cell density of heterotrophic algae can be achieved by the employment of fed-batch, continuous, and cell-recycle culture strategies that are widely used in the fermentation of bacteria or yeasts.