After the extraction process, the algal lipids are ready to be converted into biodiesel through transesterification, whereby the lipids react with a short-chain alcohol (e. g., metha­nol) in the presence of a catalyst (Sharma and Singh, 2009). Since the reaction is reversible, an alcohol-to-oil molar ratio of more than 3 is usually used to push the reaction toward the product side at reflux temperature (60-70 °C) using a homogeneous base catalyst (e. g., KOH and NaOH) to accelerate the reaction. At the end of the reaction, two obvious layers will be observed due to gravity separation: The top layer is biodiesel (the main product), whereas the bottom layer is glycerol (byproduct). Subsequently, biodiesel is subjected to sev­eral purification steps, such as water washing and evaporation, to produce pure biodiesel of high quality. In a recent optimization study, more than 90% of algal biodiesel (Chlorella vulgaris) yield was attained at a reaction temperature of 43 °C, with a methanol-to-oil molar ratio of 14, 0.42 wt% of NaOH, and a reaction time of 90 minutes (Plata et al., 2010).

Nevertheless, it is important to note that the presence of a high free fatty acid (FFA) content in algal lipids (more than 0.5% w/w) may prevent the use of a homogeneous base catalyst for the transesterification reaction (Ehimen et al., 2010; Zhu et al., 2008). This is because FFA will react with the base catalyst to form soap, resulting in a low biodiesel yield and causing sig­nificant difficulty in product separation and purification. An alternative acid catalyst (e. g., sulfuric acid, H2SO4) will be a better choice because it is not sensitive to the FFA content in the oil and thus esterification (FFA is converted to alkyl esters) and transesterification can occur simultaneously. Nevertheless, the acid catalyst has a significant drawback from a commercialization aspect for the following reasons: (1) the reaction is extremely slow and a high concentration of catalyst is required to accelerate the reaction, (2) reuse and recycling of the catalyst are not possible, and (3) strong acidic properties of the catalyst will cause serious corrosion on valves, pipelines, and reactor walls (Lam et al., 2010).

13.6.3.1 Climate Change and Consideration of Biogenic Carbon

An important point in the assessment of greenhouse gases is the consideration of the fix­ation of CO2 during photosynthesis in the cultivation step and the emissions of CO2 during the combustion step (if this last step is included in the perimeter of the study). In the publi­cations of Batan et al. (2010) and Clarens et al. (2010), the fixed CO2 is negatively counted in the global balance of the greenhouse gas (respectively, -75.3 g CO2 eq MJ-1 and -69.4 g CO2 eq MJ-1). But this CO2 is then emitted to the atmosphere during the combustion step. This emission is considered by Batan et al. (2010) but not by Clarens et al. (2010), so in this last case the production of bioenergy from microalgae is a sink of carbon, and the greenhouse balance is widely underestimated. In most of the LCA studies, fixation and then emission of biogenic carbon in the atmosphere are considered neutral processes from a "climate-change" point of view. Consequently, most of the authors do not count the fixation of the CO2 during the cultivation step nor the emission during the combustion step. We recommend dedicating specific attention to this point to guarantee a sound carbon balance.

Biotechnology is a major interdisciplinary science, combining biology, chemistry, and engineering and incorporating and integrating knowledge from the areas of microbiology, genetics, chemistry, biochemistry, and biochemical engineering. The key word in this context

is biotransformation.

The application of biotechnology to marine organisms and processes is an area of signif­icant industrial importance with ramifications in many areas, including human health, the environment, energy, food, chemicals, materials, and bioindicators. Some areas of interest re­lated to marine biotechnology include the understanding of genetic, nutritional, and environ­mental factors that control the production of primary and secondary metabolites, based on new or optimized products. Furthermore, there has been an emphasis on the identification of bioactive compounds and their mechanisms of action for application in the medical and chemical industry; there are also bioremediation strategies for application in damaged areas and the development of bioprocesses for sustainable industrial technologies (Zaborsky, 1999).

The cultivation of microalgae as part of biotechnology has received researcher attention. The growth conditions and the bioreactors for cultivation have been thoroughly studied (Borowitzka, 1999). The principle behind cultivation of microalgae for the production of bio­mass is the use of photosynthesis (Vonshak, 1997), which involves using solar energy and converting it into chemical energy.

Microalgae are photosynthetic prokaryotic or eukaryotic microorganisms that grow rapidly and have the ability to live in different environments due to their unicellular or simple multicellular structure. Examples of prokaryotic microalgae are the cyanobacteria; green al­gae and diatoms are examples of eukaryotics (Mata et al., 2010).

Cyanobacteria differentiate into vegetative, akinete, and heterocyst cells. The functions of vegetative, akinete, and heterocyst cells are their ability to carry oxygen in photosynthesis, resistance to climactic conditions, and potential for nitrogen fixation, respectively. Green algae have a defined nucleus, cell wall, chloroplasts containing chlorophyll and other pig­ments, pirenoide, and a dense region containing starch granules, stigma, and scourge.

Microalgae exist in various ecosystems, both aquatic and terrestrial. More than 50,000 species are known and about 30,000 are studied (Mata et al., 2010). The main advantages of microalgae cultivation as a biomass source are (Vonshak, 1997):

• They are biological systems with high capacity to capture sunlight to produce organic

compounds via photosynthesis.

• When subjected to physical and chemical stress, they are induced to produce high concentrations of specific compounds, such as proteins, lipids, carbohydrates, polymers, and pigments.

• They have a simple cellular division cycle without a sexual type stage, enabling them to complete their development cycle in a few hours. This enables more rapid development in production processes compared with other organisms.

• They develop in various environmental conditions of water, temperature, salinity, and light.

Lipid accumulation in microalgae usually occurred when microalgae are cultivated under stress conditions (e. g., nitrogen starvation, nutrient deficiency, pH variations, etc.). Among those stress conditions, nitrogen limitation is the most effective and commonly used strategy for stimulating lipid accumulation in microalgae. Recent reports demonstrated that cultivation under nitrogen starvation conditions leads to a marked increase in the oil/lipid content (Mandal and Mallick, 2009). Hu et al. (2008) collected the data of lipid contents of various microalgae and cyanobacteria species under normal growth and stress conditions in a literature
survey, indicating that under stress conditions, the lipid contents of green microalgae, diatoms, and some other microalgae species are 10-20% higher than under normal conditions. However, the lipid contents of cyanobacteria were usually very low (10%) (Hu et al., 2008).

It is thought that when microalgae are cultivated under nitrogen-starvation conditions, the proteins in microalgae will be decomposed and converted to energy-rich products, such as lipids. Siaut et al. (2011) also concluded that during microalgae growth, starch would first be synthesized to reserve energy, then lipid would be produced as a long-term storage mech­anism in case of prolonged environmental stress (such as nitrogen deficiency). Although a nitrogen-starvation strategy is very effective in increasing lipid content of microalgae, the ni­trogen deficiency conditions often lead to a significant decrease in the microalgae growth rate, thereby causing negative effects on lipid productivity. Therefore, engineering approaches should be conducted to optimize the cultivation time for the microalgae growth period (nitrogen-sufficient condition) and lipid accumulation period (nitrogen-deficient condition) to ensure high overall oil/lipid productivity.

4.1.2 Cultivation Vessels

Many different configurations of photobioreactors are possible: from simple unmixed open ponds to highly complex enclosed ones. The configuration of the bioreactor has great influence on carbon dioxide consumption during algal growth. Most of the recent research in microalgal culturing has been carried out in photobioreactors with external light supplies, large surface areas, short internal light paths, and small dark zones. Examples include open ponds (the cheapest ones), tubular reactors, flat panel reactors, and column reactors (stirred — tank reactors, bubble columns, airlift).

The applications of such systems range from the small-scale production of high-value prod­ucts to the large-scale production of biomass for feed. The choice between the different designs of photobioreactors must be specific to the intended application and local circumstances.

Open ponds can be an important and cost-effective component of large-scale cultivation technology, and optimal design parameters have been known for many years. The elongated "raceway type" of open pond, using paddlewheels for recirculation and mixing, was devel­oped in the 1950s by the Kohlenbiologische Forschungsstation in Dortmund, Germany. However, sustained open pond production proved to be feasible for only three microalgae: Spirulina platensis, Dunaliella salina, and fast-growing Chlorella, in all cases because con­tamination by other species can be avoided.

Beyond the economical difference between the types of photobioreactors feasible for algae cultivation, light incidence and CO2 availability are the two main factors influencing algae growth. Large surface areas are essential to ensure enough light diffusion to the media, but they are normally associated with very little time to mass transfer the gas to the liquid phase (short liquid column). The optimal condition of light diffusion and CO2 availability is easily achieved in a closed reactor for logical reasons: In open photobioreactors, the undissolved CO2 is lost to the atmosphere, whereas in closed ones it is possible to increase (and maintain) partial pressure.

Heterotrophic growth of algae requires organic carbons, water, and inorganic salts. The growth, lipid content, and fatty acid composition are species/strain specific and can be greatly influenced by a variety of medium nutrients and environmental factors.

Carbon is the main component of algal biomass and accounts for ca 50% of dry weight. Sugars, particularly glucose, are the commonly used organic carbon sources for heterotro­phic growth of algae (Table 6.1). Different algae may prefer diverse sugars for heterotrophic growth. Liu et al (2010) studied the effect of various monosaccharides and disaccharides on growth of C. zofingiensis and found that glucose, fructose, mannose, and sucrose were effi­ciently consumed by the cells for rapid growth, whereas lactose and galactose were poorly assimilated and hardly supported the algal growth. In contrast, C. protothecoides may be un­able to directly assimilate sucrose, and pretreatment using invertase is required to release glu­cose and fructose (Yan et al., 2011). The growth, lipid content, and fatty acid profile of heterotrophically grown C. zofingiensis were slightly affected by the sugar species, namely, glucose, fructose, mannose, and sucrose (Liu et al., 2010) but were influenced to a large extent by the initial concentration of sugars (Liu et al., 2012a). Within the tested range of sugar con­centrations (5 to 50 g L-1), higher sugar concentrations gave C. zofingiensis higher cell density but at the same time lower specific growth rate (Figure 6.3a). The slow growth at high sugar concentrations is due likely to the substrate inhibition, a common issue confronted in batch cultures. High sugar concentrations also favored the intracellular lipid accumulation of C. zofingiensis, in which the lipid content at 30 g L-1 sugar was 0.5 g g-1, 79% greater than that at 5 g L-1 sugar (Figure 6.3b). In addition, the lipid distribution was found to be associated with sugar concentrations. Neutral lipid (NL) is the major lipid class, the proportion of which increased with increased sugar concentrations and could account for up to 85.5% of total lipids. Similar to NL, TAG levels were promoted by higher sugar concentrations (Figure 6.3c). In contrast, the membrane lipids phospholipid (PL) and glycolipid (GL) decreased in re­sponse to the increased sugar concentrations (Figure 6.3c). The fatty acid profiles of hetero­trophic C. zofingiensis were investigated in response to different sugar concentrations (Liu et al., 2012a). C16:0, C16:2, C18:1, C18:2, and C18:3 are the major fatty acids and represented more than 85% of total fatty acids. The levels of C16:0, C16:2, and C18:2 remained nearly unchanged under all tested sugar concentrations. In contrast, C18:1 and C18:3 levels were significantly affected: The former was promoted by higher sugar concentrations, whereas the latter by lower sugar concentrations. In addition, the content of total fatty acids based on dry weight ascended as the sugar concentration increased and could reach as high as 42.2%. Although the mechanism underlying sugar-induced lipid accumulation remains largely unknown, preliminary data suggested the involvement of glucose in triggering the great up-regulation of fatty acid biosynthetic genes, e. g., acetyl-CoA carboxylase and stearoyl-ACP desaturase (Liu et al., 2010; Liu et al., 2012b). Glucose catabolism provides not only energy for lipid/fatty acid synthesis but also acetyl-CoA, the direct precursor of fatty acids. The high sugar levels cause the formation of excess carbon for cell generation, and the carbon flux can be directed to lipid synthesis.

It is worth noting that some algal species prefer other carbon sources over glucose in het­erotrophic mode. For example, feeding pure acetic acid enabled Crypthecodinium cohnii to yield much higher productivity of docosahexaenoic acid (DHA) of 1,152 mg L-1 d-1; the su­periority of acetic acid to glucose might be because in this alga, the conversion of glucose to acetyl-CoA needs several steps, whereas acetate only needs a single-step action to be activated to acetyl-CoA directly by acetyl-CoA synthetase (de Swaaf et al., 2003). Another alternative carbon source, glycerol, has been commonly used for those algal species naturally occurring in habitats with high osmolarity, such as seawater or saline pounds (Neilson and Lewin,

FIGURE 6.3 (A) Growth, (B) lipid content, and

(C) lipid composition of C. zofingiensis with different йГ initial sugar concentrations. (△) specific growth rate; (□) dry weight; (white column) lipid content; (light gray column) neutral lipids; (gray column) phospho­lipids; (black column) glycolipids. The horizontal line inside the neutral lipids column marks the por­tion of TAG in this fraction. Adapted from Liu et al. (2012a) and the permission for reprint requested.

ш

o.

w

1974), due possibly to that glycerol having the capability to raise the osmotic strength of the solution and consequently keep the osmotic equilibrium in cells (Perez-Garcia et al., 2011).

Nitrogen is the second main component of algal biomass. In autotrophic cultures, nitrogen is an important factor influencing intracellular lipid accumulation, and nitrogen limitation/ starvation is generally associated with the enhanced synthesis of lipids, in particular NL (Illman et al., 2000; Hsieh and Wu, 2009; Lacour et al., 2012). In heterotrophic cultures, nitro­gen availability also plays an important role in the profiles of lipids and fatty acids. A low level of nitrogen favors the accumulation of intracellular lipids (Scarsella et al., 2009; Xiong et al., 2010a). The heterotrophically grown Chlorella protothecoides produced 53.8% of lipids
(on a dry-weight basis) under nitrogen-limiting conditions—over two times of that under nitrogen-sufficient conditions (Xiong et al., 2010a). Nitrogen limitation also promoted carbo­hydrate synthesis but at the same time lowered the algal growth and protein level as well as the biomass growth yield coefficient on a glucose basis (Xiong et al., 2010a). The authors also analyzed the carbon flux by using 13C-tracer and GC-MS and indicated that C. protothecoides utilized considerably more acety-CoA for lipid synthesis under nitrogen-limiting conditions than under nitrogen-sufficient conditions (Xiong et al., 2010a). Considering that organic car­bons are used in heterotrophic cultures, the carbon/nitrogen (C/N) ratio, controlling the switch between protein and lipid syntheses, is usually employed to show the combined effect of carbon and nitrogen on lipid synthesis. Thus, it is the higher C/N ratios (corresponding to higher carbon concentrations when the initial nitrogen is fixed or lower nitrogen concentra­tions when the initial carbon is fixed) that trigger the accumulation of lipids, in particular the NLs. The NLs are likely from the excess carbon in the form of acetyl-CoA that enters the lipid synthetic pathway (Liu et al., 2012b) or from the transformation of chloroplast membrane lipids when nitrogen is depleted (Garcfa-Ferris et al., 1996). Up-regulation of enzymes in­volved in lipid biosynthesis, including acetyl-CoA carboxylase (ACCase), stearoyl-acyl car­rier protein desaturase (SAD), acyl-CoA:diacylglycerol acyltransferase (DGAT), and phospholipid:diacylglycerol acyltransferase (PDAT), was observed to be associated with lipid accumulation (Miller et al., 2010; Guarnieri et al., 2011; Boyle et al., 2012; Msanne et al 2012; Liu et al., 2012b). The enhanced lipid synthesis may be not only related to up- regulation of lipid-synthesizing enzymes under nitrogen limitation/starvation but also to the possible cessation of other enzymes associated with cell growth and proliferation (Ratledge and Wynn, 2002). For those reports that culture age affects lipid accumulation in algae (Liu et al., 2010; Liu et al., 2011b), the underlying reason maybe the nitrogen availability in that the aged cultures are accompanied by the depletion of nitrogen, which triggers the accumulation of lipids.

In addition to nitrogen availability, nitrogen sources have been demonstrated to influence the growth and biochemical composition of heterotrophic algae. Algae can utilize various forms of nitrogen, e. g., nitrate, ammonia, urea, glycine, yeast extract, and tryptone (Vogel and Todaro, 1997; Shi et al., 2000; Hsieh and Wu, 2009; Yan et al., 2011). Both nitrate-N and urea-N cannot be directly incorporated into organic compounds but have to be first re­duced to ammonia-N. Ammonia and urea are economically more favorable as nitrogen sources than nitrate in that the latter is more expensive per unit N. The uptake of ammonia results in acidification of the medium, and nitrate causes alkalinization, whereas urea leads to only minor pH changes (Goldman and Brewer, 1980). In this context, urea is the better choice of nitrogen source for avoiding large pH shifts of unbuffered medium. Shi et al (2000) reported the severe drop in culture pH (below 4) of heterotrophic C. protothecoides with am­monia, which resulted in much lower biomass yield compared to with urea or nitrate. Differ­ent algal species may favor different nitrogen sources for growth. For example, Chlorella pyrenoidosa preferred urea to nitrate or glycine for growth, whereas C. protothecoides gave a higher biomass yield when fed nitrate rather than urea (Davis et al., 1964; Shen et al.,

2010) . Those mutants deficient in nitrate/nitrite reductases have to use ammonia for growth (Dawson et al., 1997; Burhenne and Tischner, 2000).

Nitrogen limitation is not always linked to lipid accumulation in algae, e. g., the diatoms Achnanthes brevipes and Tetraselmis spp. accumulated carbohydrates rather than lipids upon nitrogen starvation (Gladue and Maxey, 1994; Guerrini et al., 2000). Diatoms need silicate for growth, and silicate metabolism in diatoms has been reviewed by Martin-Jezequel et al. (2000). In general, silicate limitation/starvation is associated with the enhanced synthesis of lipid in diatoms (Lombardi and Wangersky, 1991; Wen and Chen, 2000). In addition, the content of polyunsaturated fatty acids (e. g., EPA) increased with the depleted silicate (Wen and Chen, 2000). This may be explained by the finding that the silicate-limited diatom cells divert the energy allocated for silicate uptake when silicate is replete into energy storage lipids. Phosphorus plays an important role in the energy transfer of the algal cells as well as in the syntheses of phospholipids and nucleic acids. It was also reported that phosphorus de­ficiency promoted the accumulation of lipids in certain algae (Lombardi and Wangersky, 1991; Scarsella et al., 2009).

Aside from the medium nutrients, environmental factors play an important role in influencing the heterotrophic growth and lipid profile of algae, including but not restricted to temperature, pH, salinity, dissolved oxygen level, dilution rates, and turbulence (Chen and Johns, 1991; Jiang and Chen, 2000a, b; Chen, et al., 2008; Pahl et al., 2010; Ethier et al., 2011). When temperature shifts, the algae need to alter the thermal responses of membrane lipids to maintain the normal function of membranes (Somerville, 1995). Many studies have proved that in heterotrophic mode, a low temperature can induce the generation of unsaturated fatty acids, and vice versa (Wen and Chen, 2001a; Jiang and Chen, 2000a). There are two possible explanations: (1) a reduction in temperature leads to the decreased membrane fluidness; as a result, the algae need to speed up the desaturation of lipids as a compensation to maintain the proper cell membrane fluidity via the up-regulation of desaturase genes (Perez-Garcia et al.,

2011) ; and (2) the low temperature gives rise to more intracellular molecular oxygen and con­sequently improves the activities of desaturases and elongases that are involved in the bio­synthesis of unsaturated fatty acids (Chen and Chen, 2006). The high salinity was found to enhance the lipid accumulation in Nitzschia laevis in heterotrophic mode. Upon changing the concentration of NaCl in the medium from 10 to 20 g L-1, an increase in EPA and polar lipids was observed, accompanied by a slight decline of NLs (Chen et al., 2008). The sufficient oxygen supply is important for algal growth, especially in high cell density fermentation. Chen and Johns (1991) reported that in the heterotrophic culture of Chlorella sorokiniana, a high concentration of dissolved oxygen improved the cell growth as well as the fatty acid yield. The effect of pH on growth and lipids of Crypthecodinium cohnii was reported by Jiang and Chen (2000b), where the highest DHA content was obtained at pH 7.2.

As such, the optimization of nutritional and environmental factors is of great importance to the development of a high-yield lipid production system by heterotrophic algae. The com­monly used approaches for the production optimization are one-at-a-time and statistical methods (Kennedy and Krouse, 1999). The one-at-a-time strategy involves variation of one factor within a desired range while keeping other factors constant (Wen and Chen, 2000; Pahl et al., 2010; Liu et al., 2012a). This strategy is simple and easy to conduct and thus has been widely used for optimizing the production of biomass and desired products. However, the one-at-a-time method has its intrinsic disadvantages, e. g., failing to consider the interactions among factors and requiring a relatively large number of experiments. To overcome these problems, a good choice is the statistical approach-based optimization, which requires three steps: design, optimization, and verification (Kennedy and Krouse, 1999). The raw data obtained after experimental design can be transformed to models or

three-dimensional plots, based on which the optimal factors can be predicted. A verification experiment needs to be conducted to validate the predication. The statistical approach-based optimization has been applied to microalgae for the heterotrophic production of biomass and desired products, e. g., polyunsaturated fatty acid production by N. laevis (Wen and Chen, 2001a), biomass production by Tetraselmis suecica (Azma et al., 2011), and lipid production by Chlorella saccharophila (Isleten-Hosoglu et al., 2012).

Airlift photobioreactors comprise two interconnecting zones called the riser, where the gas mixture is sparged, and the downcomer, which does not receive the gas. Generally, an airlift photobioreactor exists in two forms: internal loop and external loop (Chisti, 1989; Miron et al., 2000). In an internal loop reactor, regions are separated either by a draft tube or a split cyl­inder; in an external loop reactor, the riser and downcomer are separated physically by two different tubes. Mixing in the system is done by bubbling the gas through a sparger in the riser tube, with no physical agitation. A riser is similar to a bubble column, where sparged gas moves upward randomly and haphazardly, which decreases the density of the riser, mak­ing the liquid move upward. Gas held up in the downcomer significantly influences the fluid dynamics of the airlift reactor. Increasing the gas hold-up, the difference between a riser and a downcomer, is an important criterion in designing airlift reactors (Chisti, 1989; Kaewpintong et al., 2007). Airlift reactors have the characteristic advantage of creating circular mixing pat­terns in which liquid culture passes continuously through dark and light phases, giving a flashing-light effect to algal cells (Barbosa et al., 2003). The biomass growth pattern of Nanochloropsis occulata and Scenedesmus quadricauda was studied inside two vertical airlift photobioreactors suitable for indoor operation, with both salt and freshwater and different lighting systems. Results depicted that the biomass productivity of the cultures was found to depend on the light regimes and the duration of operation.

Bio-oil production can be achieved along two alternative approaches: biomass pyrolysis or biomass thermochemical liquefaction, as explained in this section.

The pyrolysis process is basically an anaerobic heating process carried out at high temper­atures (between 200 °C and 750 °C). Pyrolysis may take place quickly or slowly; the former produces bio-oil (19-58% of the final product) and biochar (Miao et al., 2004; Grierson, Strezov et al., 2009). On the other hand, slow pyrolysis results in gas and biochar, with methane and CO2 accounting for most of the gaseous product.

Bio-oil produced from microalgal spent biomass is more stable than that produced from traditional crops (e. g., wood), although it is not as stable as fossil fuel (Mohan et al., 2006). Such bio-oil is composed mainly of aliphatic and aromatic hydrocarbons, phenols, long-chain fatty acids, and nitrogenous compounds (Du, Li et al., 2011). During pyrolysis 10-25% of biomass is converted into char (i. e., solid porous carbon particles), whereas 10-30% becomes a (noncondensable) gas (Grierson, Strezov et al., 2009; Ross, Biller et al., 2010).

An alternative fuel gas is synthesis gas (syngas), a gas mixture that comprises carbon mon­oxide (CO) and dioxide as well as hydrogen. It can be obtained by gasification of algal bio­mass via a process consisting of reaction of carbonaceous compounds with atmospheric air, steam, or oxygen at high temperature (ranging from 200 ° C to 700 ° C) in a gasifier (Suali and Sarbatly, 2012). As a result, one obtains clean H2 with yields from 5-56%, and CO with yields ranging from 9-52% (Abuadala, Dincer et al., 2010). Methane can be considered a coproduct since it is produced only to low levels, 2-25% (Suali and Sarbatly, 2012). The hydrocarbon products of gasification can be further processed to produce methanol: at 1000 °C, methanol production is 64% (w/w), on a biomass weight basis.

Another method for bio-oil production is thermochemical liquefaction of biomass. This requires heating the biomass at temperatures between 200 °C and 500 °C, under pressures above 20 bar in the presence of a catalyst. This process leads to bio-oil yields of 9-72%, together with a gaseous mixture (containing, for example, H2) ranging from 6-20% (Ross, Biller et al., 2010; Suali and Sarbatly, 2012). The remaining ash ranges in term from 0.2-0.5%. The product of biomass liquefaction is somewhat comparable to crude fuel: most biomass feedstock characterized a ratio of solid to water of 1:10 lead to a bio-oil yields of ca. 37% (Zou, Wu et al., 2010).

The profile of products is mainly affected by the biomass composition and the processing conditions of temperature, pressure, residence time, and catalyst. The bio-oil yield can be 5-25% higher than the lipid content of the original microalgae, depending on the composition in other compounds such as carbohydrates (Biller and Ross, 2011). For instance, Dunaliella tertiolecta is mainly composed of crude protein (63.6%) and fat (20.5%) and produces a bio-oil yield of ca. 37% on an organic basis (Minowa, Yokoyama et al., 1995); on the other hand, Spirulina sp. (a well-known food supplement, owing to its protein content) was reported to produce a bio-oil yield of up to 54% (Matsui, Nishihara et al., 1997). Microcystis viridis, which is composed of 46% carbon, 7.3% hydrogen, and 9.5% nitrogen, was able to lead to up to 33% bio-oil (Yang, Feng et al., 2004).

The aqueous coproduct of biomass liquefaction can be recycled to the microalgal culture; it is indeed rich in nitrogen, phosphorus, and potassium. The growth rate of microalgae cultured in a medium containing 0.1% aqueous coproduct was found to be one-half of that in microalgae cultured with established media, e. g., BG11 (Jena, Vaidyanathan et al., 2011).

Water is an ecologically safe substance that is widespread throughout nature. Below the critical point, the vapor pressure curve separates the liquid and vapor phases (Franck and Weingartner, 1999) and ends at the critical point (Tc = 373°C, pc = 22.1 MPa, and pc = 320 kg m-3). Beyond the critical point, the density of the supercritical water (SCW) can be varied continuously from liquid-like to gas-like values without any phase transition over a wide range of conditions.

Water plays an essential role in HTU. It is therefore critical to understand the fundamentals of water chemistry when subjected to high-temperature conditions. Water is rather benign and will not likely react with organic molecules under standard environmental conditions (20°C and 1 bar). However, when the temperature increases, two properties of water mole­cules change substantially. First, the relative permittivity (dielectric constant), er, of water
decreases quickly when the temperature increases. When the thermal energy increases, the shared electron between oxygen and hydrogen atoms tends to circulate more evenly and the electronegativity of the oxygen molecule is reduced (becomes less polar). For example, when temperature increases from 25°C to 300°C, the relative permittivity decreases from 78.85 to 19.66, resulting in water molecules from very polar to fairly nonpolar, in relative terms. This polarity change makes water more affinitive to the organic hydrocarbons, most of which are nonpolar molecules.

Second, the dissociation of water dramatically increases with the increase in temperature. Water, like any other aqueous solution, splits into H+ and OH~ ions in hydrolysis or disso­ciation. This process is reversible and the rate is sufficiently rapid that it can be considered to be in equilibrium at any instant (Zhang, 2010).

The complete miscibility of supercritical water and gases as well as many organic com­pounds makes SCW an excellent solvent for homogeneous reactions of organic compounds with gases, like the oxidation of organic compounds with oxygen and air. The absence of phase boundaries leads to a rapid and complete reaction. From the macroscopic point of view, SCW is a nonpolar solvent; from a microscopic view, water is a molecule with a strong dipole moment of 1.85 D. Water in the supercritical state is able to react with different compounds. Therefore water is both solvent and reactant in a variety of reactions.

The ionization constant of water increases with temperature and reaches a maximum near 250° C; the amount of dissociation is three times what it would be at ambient temperatures and pressures. Therefore, subcritical water in the 220-300°C region offers opportunities as both a benign solvent and a self-neutralizing catalyst. Here, water acts as both reactant and reaction medium. Water as reactant leads to hydrolysis reactions and rapidly degrades the polymeric structure of biomass to water-soluble products (Kumar, 2010).

Hot compressed water in the sub — and supercritical states exhibits exciting physical and chemical properties, which can be varied continuously from gas-like to liquid-like behavior. This opens up several promising opportunities for separation processes and chemical reactions.

The input category refers here to any product or service required at some point of the microalgae culture or transformation. It includes the materials used to build cultivation sys­tems, fertilizers and chemical reactants, production of electricity, and heat required at the facil­ity. Almost all the publications consider these inputs in exhaustive ways, except:

• Jorquera et al. (2010). Fertilizers are not taken into account.

• Clarens et al. (2010, 2011). Infrastructures are not taken into account.

• Sander et Murthy (2010). Only flows that contribute to more than 5% of the total mass, energy, and economy are taken into account.

The energy and the fertilizer are the most influencing inputs on the final environmental performance and energy balance.