Haematococcus is a green algae (Chlorophyta), mobile, single-celled, and capable of synthe­sizing and accumulating the pigment astaxanthin in response to environmental conditions, reaching from 1.5% up to 6% by weight astaxanthin (Vanessa Ghiggi, 2007). The astaxanthin produced by Haematococcus pluvialis is about 70% monoester, 25% diesters, and 5% free (Lorenz and Cysewski, 2000).

These algae, however, have some undesirable characteristics compared to other microalgae grown successfully on a commercial scale. The biggest concern is mainly related to a relatively slow growth rate, allowing easy contamination. Therefore, many studies have sought to improve the low rate of growth of vegetative cells, which is, exceptionally, 1.20 div/day (Gonzales et al., 2009).

Alternatively, its mixotrophic (Guerin et al., 2003; Gonzales et al., 2009) and heterotrophic (Hata et al., 2001) metabolism, using acetate as carbon source, has also been studied and documented; however, these conditions have not been applied to commercial-scale cultures and are not interesting in terms of carbon fixation.

In comparison to photoautotrophy, heterotrophic growth mode offers substantial advan­tages, e. g., elimination of the light requirement, ease of control for monoculture, high cell density, and great biomass productivity (Chen, 1996). Lab-scale heterotrophic production of algae has been reported in recent decades, either in shaking flasks or in small-volume fermen­ters (Cheng et al., 2009; Liang et al., 2009; Liu et al., 2010, 2011b; Yan et al., 2011). Liang et al (2009) examined the growth of Chlorella vulgaris under both phototrophic and heterotrophic conditions and indicated heterotrophic C. vulgaris had around threefold higher biomass yield than a phototrophic one. Liu et al (2011b) investigated the growth of Chlorella zofingiensis; the alga achieved 10.1 g L-1 of cell density under heterotrophic conditions compared to 1.9 g L-1 under phototrophic conditions. Chlorella protothecoides, another well-studied green alga, was reported to achieve as high as up to 17 g L-1 of cell density in heterotrophic batch cultures (Cheng et al., 2009). This may be further improved through using culture techniques such as fed-batch, chemostat, and cell recycling, which have been widely used for fermentation of bacteria or yeasts. For example, the fed-batch C. protothecoides achieved a high cell density of 97 g L-1 in a 5-L fermenter (Yan et al., 2011), much higher than that obtained in photoau­totrophic culture systems (open ponds or photobioreactors) and close to the yeast yield

Stoichiometric Reactions.

Glycolytic pathway

Glc + ATP => G6P + ADP + H 1

G6P <=> F6P 2

F6P + ATP => 2GAP + ADP + H 3

2GAP + H2O => F6P + Pi 4

GAP + NAD + Pi + ADP <=> G3P + ATP + NADH + H 5

G3P <=> PEP + H2O 6

PEP + ADP => Pyr + ATP 7

Pyr + NAD + CoA => AcCoA + NADH + CO2 + H 8

PEP + CO2 + ADP => OAA + ATP 9

Stoichiometric Reactions—Cont’d Tricarboxylic acid cycle

OAA + AcCOA + H2O <=> ICT + CoA + H 10

ICT + NAD <=> AKG + NADH + CO2 11

AKG + CoA + NAD => Suc + NADH + CO2 + H 12

Suc + ADP + P; + FAD <=> Fum + FADH2 +ATP + CoA 13

Fum <=> Mal 14

Fum + NAD + H2O <=> OAA + NADH + H 15

Pentose phosphate pathway

G6P + 2NADP + H2O => Ru5P + CO2 + 2NADPH + 2H 16

Ru5P <=> R5P 17

Ru5P <=> X5P 18

R5P + X5P <=> S7P + GAP 19

S7P + GAP <=> F6P + E4P 20

X5P + E4P <=> F6P + GAP 21

Utilization of nitrogen

AKG + NADPH + Gln => 2Glu + NADP 22

Glu + NH3 + ATP => Gln + ADP + Pi 23

(Li et al., 2007b; Kurosawa et al., 2010; Zhang et al., 2011). Although the growth and biomass production of algae are species/strain dependent and may vary greatly, the overall bio­mass yield and productivity of heterotrophic algae are significantly higher than those of phototrophic ones, as illustrated by Figures 6.2a and 6.2b.

Heterotrophic culture of algae offers not only high cell density but also high level of oils. The lipid contents of alga cultured heterotrophically were shown in Table 6.3. The lipid content varies from 4.8% to 60% of dry weight, depending on the algal species/strains and culture con­ditions. Commonly, stresses such as high light intensity and/or nitrogen starvation are re­quired to induce intracellular oil accumulation of algae under photoautotrophic conditions. These stresses, however, are unfavorable for algal growth and biomass production, causing the contradiction between growth and oil synthesis. In contrast, the heterotrophic algae are able to accumulate oil while simultaneously building up biomass; for example, the intracellular oil content of C. zofingiensis increased from 0.25 to 0.5 g g-1 (on a dry-weight basis) when the cell density increased from 5 to 42 g L-1 (Liu et al., 2010). The accumulated oil contains mainly neu­tral lipids, in particular triacylglycerol (TAG). The TAG may account for up to 80% of neutral lipids or 71% of total lipids (Liu et al., 2011b). TAG is regarded as superior to polar lipids (phos­pholipids and glycolipids) for biodiesel production due to its higher content of fatty acids. Taking into account the rapid growth and abundance of oils, heterotrophic algae usually allow

a high volumetric oil productivity (Figures 6.2c and 6.2d), e. g., 7.3 g L-1 day-1 in the case of C. protothecoides under fed-batch culture conditions (Yan et al., 2011). The fatty acid character­istics of oils, e. g., carbon chain length and unsaturation degree, largely determine the properties of biodiesel such as cetane number, viscosity, cold flow, and oxidative stability (Knothe, 2005). Although the fatty acid species of algae grown heterotrophically may show few differences in comparison to photoautotrophy, the proportions of individual fatty acid vary greatly. Liu et al. (2011b) investigated the fatty acid profiles of C. zofingiensis and indicated that heterotrophic cells contained low levels of C16:0, C16:3, C18:0, and C18:3 but much higher content of C18:1 than autotrophic cells. The proportion of C18:1 is regarded as an important factor for bio­diesel quality because it can provide a compromise solution between oxidative stability and low-temperature properties (Knothe, 2009). The higher the C18:1 content, the better the biodie­sel quality. The biodiesel derived from heterotrophic algae was analyzed with respect to the key properties (e. g., energy density, viscosity, flash point, cold filter plugging point, and acid value), and the results showed that most properties complied with the specifications established by the American Society for Testing and Materials (Xu et al., 2006).

In addition to the lab-scale cultures, many attempts have been made to develop industrial — scale processes for the heterotrophic cultivation of algae. The heterotrophic Chlorella cultures have long been initiated in Japan and Taiwan in the late 1970s; Chlorella species were cultured in stainless steel tanks using glucose and/or acetate as carbon and energy sources, with an annual production of 1,100 tons biomass (Lin, 2005). Thereafter, large-scale heterotrophic cultivation of several other algal strains were reported, for example, Tetraselmis suecica in 50,000-L fermenters (Day et al., 1991), Crypthecodinium cohnii with a capacity of 150,000 L (Radmer and Fisher, 1996), and Spongiococcum exetriccium fed-batch cultured in 450-L fermen­ters (Hilaly et al., 1994), though these cultures were used not for oils but for high-value prod­ucts. Recently, a scale-up heterotrophic cultivation of C. protothecoides was reported for oil production in 11,000-L fermenters, where the daily biomass production of 20 kg and oil pro­duction of 8.8 kg were achieved (Li et al., 2007a).

Because of the elimination of light requirements and sophisticated fermentation systems that have developed, the scale-up of heterotrophic cultures for high cell density and oil yield is rel­atively easier to achieve than that of autotrophic cultures. The production of heterotrophic algal cultures, however, is restricted, due largely to (1) the limited number of available heterotrophic species, (2) possible contamination by bacteria or fungi, (3) inhibition of growth by soluble or­ganic substrates (e. g., sugar) at high concentrations, and (4) the relatively high cost of organic carbon sources. The first limitation might be overcome by performing extensive screening an­alyses. For example, Vazhappilly and Chen (1998) intensively studied the heterotrophic poten­tial of 20 algal strains and suggested that 6 of them showed good heterotrophic growth. As the screening expands, increasing algal species/strains will be identified with heterotrophic poten­tial. In some cases, the obligate photoautotrophic algae can be metabolic engineered to grow heterotrophically. Zaslavskaia et al (2001) reported that a genetically modified Phaeodactylum tricornutum, through introducing a gene encoding a glucose transporter, was capable of thriv­ing on exogenous glucose in the absence of light, suggesting an alternative approach to increas­ing the available number of heterotrophically grown algae. The second problem is due mainly to the relatively slow growth of algae compared with other microorganisms such as bacteria or yeast that grow fast and finally dominate the cultures. Rigorous sterilization and aseptic oper­ation are necessary and considered to be effective to circumvent such possible contamination. Growth inhibition is a common problem occurring in batch cultures, which has restricted the use of batch cultures in commercial production processes. The growth inhibition may be attrib­uted to the high initial concentration of substrates (e. g., sugars) or the possible buildup of cer­tain inhibitory substances produced by algae during culture periods. For example, the sugar concentration of over 20 g L-1 was reported to inhibit the growth of C. zofingiensis (Liu et al., 2010, 2012a). Advances in heterotrophic culture systems may eliminate or reduce the growth-inhibition problems, where fed-batch, chemostat, and cell recycle have been intensively investigated (Wen and Chen, 2002a; De la Hoz Siegler et al., 2011; Liu et al., 2012a). The organic carbon sources—in particular, glucose—account for the major cost of a culture medium and contribute to the relatively high cost of heterotrophic production, which makes the algal oils from heterotrophic cultures less economically viable than those from autotrophic cultures. Cheap alternatives are sought with the goal of bringing down production costs, e. g., waste mo­lasses (Yan et al., 2011; Liu et al., 2012a), carbohydrate hydrolysate (Cheng et al., 2009; Gao et al.,

2010) , and biodiesel byproduct glycerol (O’Grady and Morgan, 2011).

Vertical tubular photobioreactors are made up of transparent vertical tubing to allow light penetration (Richmond, 2004). The bottom of the reactor is attached with a sparger to convert the sparged gas into tiny bubbles. This enables mixing and mass transfer of CO2 and removes the O2 produced during photosynthesis. Based on the mode of flow, these vertical tubular photobioreactors can be classified as bubble column and airlift reactors (Ramanathan et al., 2011). Ramanathan and his co-workers (2011) cultivated marine microalgae, that is, Nanochloropsis occulata and Chaetoceros calcitrans, in tubular photobioreactors. The study resulted in higher biomass productivity due to the large illuminating surface area of the photobioreactor.

Production of ethanol from algal biomass is chiefly obtained via fermentation of its starch, sugar, and cellulose. In the case of microalgae, carbohydrate contents amount to 70-72% (Branyikova, Marsalkova et al., 2011), with starch dominating (i. e., up to 60% dry weight, depending on culture condition) (Dragone, Fernandes et al., 2011). Conversely, the most abundant sugars in brown macroalgae are alginate, mannitol, and glucan, i. e., glucose poly­mers in the form of laminarin or cellulose (Wargacki, Leonard et al., 2012).

In the case of microalgae, production of ethanol starting from the microalgal oilcake after biodiesel production is to be taken into consideration. By the end of production of ethanol, the waste can be in turn recycled, and the CO2 generated can be fed to phototrophic microalgae culture, while nonfermentable cellulose can be further processed as an animal feed supple­ment (Suali and Sarbatly, 2012).

Finally, the nonfermentable (or residual) slurry, composed mainly of proteins, lipids, and organic acids or alkali, can be used as feedstock for methane production by up to 10%; alter­natively, the cells may be ruptured to release their proteins or enzymes as useful byproducts (Suali and Sarbatly, 2012).

Hydrothermal upgradation (HTU) is a process for the conversion of complex organic mate­rials such as waste biomass into crude oil and other value-added chemicals. Hydrothermal liquefaction involves the reaction of biomass in water at high temperature and pressure, with or without the presence of a catalyst. The products include a biocrude, an aqueous fraction, a gaseous fraction, and unconverted organic and inorganic content. The hydrothermal processing of biomass was investigated by Shell research in the 1980s (Ruyter et al., 1987) and is the basis of the HTU process (Goudriaan et al., 2000). Hydrothermal technologies are broadly defined as chemical and physical transformations in high-temperature (200-600°C), high-pressure (5-40 MPa) liquid or supercritical water. Hydrothermal processing of lignocellulosic biomass has received extensive interest over the last two decades for both the production of liquid fuels (subcritical conditions) and for gasification (supercrit­ical conditions) and is extensively reviewed by Peterson et al. (Peterson et al., 2008).

According to the LCA method, once the FU, the perimeter of the study, and the system have been defined, each process included in the perimeter has to be characterized in terms of technical inputs and outputs, energy and resource consumption, and emissions into the environment. Because of the lack of industrial data on microalgae culture or transformation, the inventory data compiled in the selected studies often rely on extrapolation from lab-scale results, adaptation from similar processes used in different conditions or with different feedstock, or modeling.

Temperature is one of the major factors that regulate cell morphology and physiology as well as the byproducts of the microalgal biomass. A high temperature generally accelerates the metabolism of microalgae and a low temperature can inhibit growth (Munoz and Guieysse, 2006).

The optimum temperature for growth varies among species of microalgae (Ono and Cuello, 2003). High temperatures during the day have a favorable effect on growth rates due to photosynthesis. High temperatures at night are not desired in microalgal cultivation due to the increased respiration rate; they result in a high expenditure of cellular energy and consequent reduction of cellular concentration.

The temperature also influences other factors that are important for cultivation, such as the ionic balance of water, pH, and solubility of O2 and CO2. Different species of microalgae are affected by temperature at different levels (Park et al., 2011). In the case of combustion gases emitted in power plants, the gas temperatures reach 120 °C. In this case, the rate of CO2 biofixation may depend on the installation of a heat exchange system or the use of thermo­philic species. The solubility of O2 and CO2 increases the temperature and results in the fix­ation of high concentrations of O2 by oxigenase of RuBisCO. Thus the affinity for RuBisCo by CO2 decreases with increasing temperature (Kumar et al., 2011).

The temperature of cultivation in the photobioreactor is determined by the air temperature, the duration of solar radiation, and the relative humidity of air. The depth and the surface of the culture and the material of construction of the photobioractor are factors that stabilize the temperature of the culture. Mechanisms of temperature control cause significant changes in the design of a photobioreactor. With no temperature control, a closed photobioreactor can reach values of 10-30 °C above ambient temperature. Some mechanisms of temperature control in closed photobioreactors include immersion of the culture in water, spraying with water, shading, or incorporating a heat exchanger with the photobioreactor (Wang et al.,

2012) . In raceway-type photobioreactors, the temperature is generally greenhouse controlled. At low temperatures the greenhouse is kept closed, maintaining the temperature. On hot days the sides of the greenhouse can be erected, thus reducing the temperature in the inner area where the raceways are located.

Flat panel photobioreactors feature important advantages for mass production of photoauto­trophic microorganisms. The simple flat plate photobioreactor consists of vertically translu­cent flat plates, which are illuminated on both sides and stirred by aeration (Figure 2.3). This simple building methodology for glass flat plate reactors provides the opportunity to easily construct reactors with any desired light path. Light is evenly emitted from a flat transparent surface screen or from lamps above the culture. The plate surface is usually made of glass or optical light film, and the circulation is achieved by the same means of rising air bubbles, as

with the tubular systems. However, flat plate systems may also experience problems with relatively high space requirements, high light energy requirements, difficulties in cleaning, and possible low efficiency in terms of mass production per unit of space (Slegers et al.,

2011) . The productivity of flat-plate photobioreactors is highly dependent on the space requirements between the panels and the areal productivity constraint for outdoor applica­tion. On the other hand, if the flat plate systems are to be operated indoors, then some crucial factors would be involved, including distance of light sources from panels, temperature effects, illumination of one or both panel sides, light path, and so on. Scale-up of the flat plate system is potentially difficult due to the increase of hydrostatic pressure with the increase of volume. In general, the structure of flat plate systems cannot tolerate very high pressure. Moreover, the hydrodynamic stress on microalgae cells may affect the microalgae growth. In addition, the biomass productivity in parallel flat panels is strongly influenced by shading and light penetration between the panels (Posada et al., 2012). To further reduce the equip­ment cost, a novel design of a vertical flat panel photobioreactor, consisting of a transparent bag (i. e., plastic) located on a rigid frame, has been proposed and could greatly enhance the economic feasibility (Tredici and Rodolf, 2004).

Algae can be dewatered and harvested by pressure filtration using either plate-and-frame filter presses or pressure vessels containing filter elements. In plate-and-frame filter press filtration, dewatering is achieved by forcing the fluid from the algal suspension under high pressure. The press consists of a series of rectangular plates with recesses on both sides, which are supported face to face in a vertical position on a frame with a fixed and movable head. A filter cloth is hung or fitted over each plate. The plates are held together with sufficient force to seal them so as to withstand the pressure applied during the filtration process.

In the operation, fluid containing algal suspension is pumped into the space between the plates, and pressure is applied and maintained for several hours, forcing the liquid through the filter cloth and plate outlet ports. The plates are then separated and the dewatered algal cake is harvested. The filtration cycle involves filling the press, maintain the press under pres­sure, opening the press, washing and discharging the cake, and closing the press. Chemical conditioners such as polyelectrolytes may be used to increase the solids content of the cake.

In filtration by pressure vessel containing filter elements, a number of designs have been devised, such as rotary-drum pressure filters, cylindrical-element filters, vertical tank vertical leaf filters, horizontal tank vertical leaf filters, and horizontal leaf filters. A comparison of the use of different pressure filters for Coelastrum harvesting has been investigated (Mohn, 1980). Five different pressure filters—chamber filter press, belt press, pressure-suction filter, cylin — dric sieve, and filter basket—were operated. Solids concentrations in the range of 5% to 27% were measured for the harvested algae. Chamber filter press, cylindric sieve, and filter basket were recommended for algae filtration with respect to energy consideration, reliability, and concentrating capability. A belt filter press was not recommended because of low-density al­gal cake if filtration was carried out without prior coagulants dosing to the feed. A pressure — suction filter was also not recommended because of low filtration ratio, high investment costs, and unclear operational expenses.

One of the big challenges of microalgae culture is the search for alternative (and cheap) culture media. Some microalgal species can accumulate up to 70% of lipids but only when cultured in a specific balanced medium, as mentioned by Chisti (2007).

Medium costs are difficult to estimate because much depends on the species of microalga to be cultured. In the literature, medium cost is described as between US$0.27 and $0.588 per kg algal biomass (Molina-Grima et al., 2003; Tapie and Bernard, 1988). Such high cost, the major drawback in biofuel production processes involving microalgae, makes these processes unfeasible. (Just the biomass production step represents almost 40% of the price of the final product.) The necessity to exploit inexpensive and abundantly produced nutritional sources to substitute artificial media is clear.

In this context, the patented technology developed by the company Ourofino Agronegcicio, in partnership with the Laboratory of Biotechnological Processes (Federal University of Parana, Brazil), is a very interesting and economical alternative for the production of biofuels from high-lipid-content microalgal biomass cultured in wastewater from ethanol distilleries. (The present technology was patented: PI0705520-0.)