Hydrothermal liquefaction is a process in which biomass is converted in hot compressed water to a liquid biocrude (Brown et al., 2010; Biller et al., 2012). Processing temperatures range from 200-350 °C with pressures of around 15-20 MPa, depending on the temperature, because the water has to remain in the subcritical region to avoid the latent heat of vaporiza­tion (Biller et al., 2012). At these conditions, complex molecules are broken down and repolymerized to oily compounds (Peterson et al., 2008). This procedure is ideal for the con­version of high-moisture-content biomass such as microalgae because the drying step of the feedstock is not necessary.

Depending on the primary biofuel target, a broad group of naturally occurring lipids remain in algal spent biomass; these include fats; waxes; sterols; fat-soluble vitamins (e. g., A, D, E and K); mono-, di-, and triacylglycerols; diglycerides, and phospholipids (Williams and Laurens, 2010). Of particular importance are polyunsaturated fatty acids (PUFA); interest has emerged in recent years owing to their potential therapeutic uses in addition to nutritional applications derived from physiological roles in actual cells. PUFAs have been thoroughly studied, especially o3 long-chain PUFA (LC-PUFA), in regard to docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and a-linolenic acid (ALA). Their importance to human health has backed up market demand for them (Guedes et al., 2011a).

The predominant PUFA in various marine algae is EPA at concentrations as high as 50% of its total fatty acid content (Murata and Nakazoe, 2001; Dawczynski, Schubert et al., 2007). Marine algae also contain 18:4 n-3, which is hardly found in other organisms; notably, red alga species contain significant quantities of EPA and arachidonic acid (20:4), whereas green algae are unique in their content of 16:4, varying from 4.9% to 23.1% of the total fatty acids, besides 16:0, 18:1, and 18:3 acids. Unsaturated fatty acids predominate in all brown algae and saturated fatty acids in red algae, both groups being balanced sources of n-3 and n-6 acids (Mabeau and Fleurence, 1993; Sanchez-Machado, Lopez-Hermndez et al., 2004).

The main effects of n-3 fatty acids on human health can be classified into three categories: (1) structural components of cell and organelle membranes, (2) significant role in lowering blood lipids, and (3) precursors in mediating biochemical and physiological responses. Human beings have to include ALA, EPA, and/or DHA in their daily diet, especially via inclusion of marine products. However, algae exhibit competitive advantages as sources of PUFAs: Fish (the most common source) have typically lower contents (on a mass basis) and are subjected to seasonal variations in fatty acid profile, besides their being proven to be contaminated by heavy metals (Guil-Guerrero, Navarro-Juoirez et al., 2004). Fur­thermore, they have a limited capacity to synthesize PUFA, so most of them are simply accumulated from their microalgal diet (Guedes et al., 2011a). Algae are indeed a good source of EPA (Plaza, Cifuentes et al., 2008) and an important source of n-3 PUFAs (Murata and Nakazoe, 2001). Besides the well-accepted effect of 18:4 n-3 on the immune system in humans (Ishihara, Murata et al., 1998 ), several other bioactivities have been reported, as tabulated in Table 10.4.

The relative composition of algal lipids depends greatly on the species as well as available nutrients and prevailing environmental conditions during cell culture and harvest. For instance, it has been shown that the composition of algal lipids varies considerably with the growth cycle, under nutrient limitation and a diurnal light/dark cycle (Ekman A, Bulow L et al., 2007; Greenwell, Laurens et al., 2010). Many algal species can be induced to accumulate substantial contents of lipids; although average lipid contents vary between 1% and 70%, some species may reach 90% (w/wDW) (Guedes et al., 2011a). Concerning their extraction, several methods can be applied, but the most common are expeller/oil pressing, liquid-liquid extraction (solvent extraction), supercritical fluid extraction (SFE), and ultrasound techniques, all of which bear advantages and limitations, as discussed elsewhere (Singh and Gu, 2010).

To make algal biofuel economically viable, extraction of value-added coproducts, along with oil, appears absolutely necessary. The major barrier in algal coproduct development is the lack of an efficient separation technology. To address this issue, a unique two-step

sequential hydrothermal liquefaction (SEQHTL) technology for the simultaneous production of value-added polysaccharides and bio-oil from algal-biomass was developed. The first step involves the subcritical water extraction of valuable algal (Chlorella sorokiniana) polysaccha­rides at 160°C. The polysaccharide-rich water extract was removed and precipitated with ethanol. In the next step, the extracted biomass was liquefied to bio-oil at 300°C. The yield of bio-oil by SEQHTL was 24% of the dry wt. In addition, this method also extracted 26% of the polysaccharides present, whereas direct hydrothermal liquefaction (DIRHTL) generated only 28% bio-oil. In the SEQHTL method, biochar formation was remarkably low, and as such, SEQHTL produced 63% less biochar than DIRHTL. The yield of biochar production is negligible correlated to polysaccharide content (p > 0.98), suggesting a majority of carbohy­drates present in algal biomass were converted into biochar. This conversion did not signif­icantly influence the bio-oil production. Comparative GC-FID, GC-MS, NMR, FT-IR analysis and ESI-MS of the bio-oil extracted by SEQHTL with DIRHTL showed no significant differences. Elemental analysis of the SEQHTL bio-oil demonstrated that it contained 70% carbon, 0.8% nitrogen, and 11% oxygen (Chakraborty et al., 2012).

Very scarce data are available to build up an inventory of microalgal oil extraction. Char­acterization of the lipid content of microalgae is based on techniques and solvents that cannot be extrapolated to industrial-scale techniques, and often the characterization is done on ly — ophilized algae, which of course is not an option for bioenergy production. Hence inventories for oil extraction and methylester production are usually based on inventories of vegetable oil production and transesterification (e. g., rapeseed or soybean). Some studies specify a phase of pretreatment based on homogenizers. The rapid compression and decompression of the algal slurry is supposed to disrupt cell walls and hence increase extraction efficiency and digest­ibility of extraction residues (Stephenson et al., 2010; Clarens et al., 2011). Triglycerides are extracted with an organic solvent; hexane, lipids, and aqueous phases are then separated and the oil/hexane mixture is finally purified by distillation. During distillation, most of the hexane is recovered, so only a small quantity is lost by volatilization.

In all the concerned studies, triglyceride esterification is performed by reaction with meth­anol and with alkaline catalysis. This step requires heating, mixing, and the addition of a base, usually potassium hydroxide. The reaction yield can be significantly reduced by a concurrent saponification reaction, which is enhanced by water. Consequently there is a trade-off be­tween the energy to invest for dewatering and drying the biomass and the energy for extracting and down-processing the lipid fraction, with reaction yields drastically affected by the water content. Other approaches have been proposed, such as supercritical CO2 extrac­tion of lipids or in situ esterification. Both approaches could suffer from too high water con­tent. More recently, in situ esterification with supercritical methanol has been proposed as a way to overcome this issue. This last option was selected in the LCA-based optimization pro­posed Brentner et al. (2011).

One of the decisions to be taken in the cultivation of microalgae is regarding the use of open or closed photobioreactors. Closed photobioreactors of the vertical tubular, helical tubular, and flat panel type are considered to have high photosynthetic efficiency and degree of control. Closed reactors have some advantages and disadvantages over open ones.

1.1.1 Closed Photobioreactors

Due to the high productivity achieved in cultures carried out in closed photobioreactors, much attention has been paid to these systems. The configurations tested on a laboratory or pilot scale include vertical reactors, flat plate, annular, plastic bags, green wall panel (GWP), and various forms of tubular reactors, stirred mechanically or by airlifting.

Closed photobioreactors are highly efficient at biofixation of CO2, mainly due to better homogeneity of the medium and mass transfer. However, these reactors are limited by the excess O2 produced (Ho et al., 2011). The costs of these reactors are generally high (Table 1.1). Contamination can be controlled in sterile systems; however, this causes an in­crease in production costs (Amaro et al., 2011). The scale-up of open photobioreactors gener­ally occurs by increasing the diameter of the tube, but the cells do not receive sufficient light for growth (Ugwu et al., 2008).

Microalgae are a very heterogeneous group of microorganisms. The term microalgae includes prokaryotes and eukaryotes. Cyanobacteria (blue-green algae) are frequently unicel­lular, with some species forming filaments or aggregates. The internal organization of a cyanobacterial cell is prokaryotic, where a central region (nucleoplasm) is rich in DNA and a peripheral region (chromoplast) contains photosynthetic membranes. The sheets of the photosynthetic membranes are usually arranged in parallel, close to the cell surface. Eukary­otic autotrophic microorganisms are usually divided according to their light-harvesting pho­tosynthetic pigments: Rhodophyta (red algae), Chrysophyceae (golden algae), Phaeophyceae (brown algae), and Chlorophyta (green algae). Their photosynthetic apparatus are organized in special organelles, the chloroplasts, which contain alternating layers of lipoprotein mem­branes (thylakoids) and aqueous phases (Staehelin, 1986).

All photosynthetic organisms contain organic pigments for harvesting light energy. There are three major classes of pigments: chlorophylls (Chl), carotenoids, and phycobilins. The chlorophylls (green pigments) and carotenoids (yellow or orange pigments) are lipophilic and associated in ChI-protein complexes, while phycobilins are hydrophilic. Chlorophyll molecules consist of a tetrapyrrole ring (polar head, chromophore) containing a central magnesium atom and a long-chain terpenoid alcohol. Structurally, the various types of Chl molecules, designated a, b, c, and d, differ in their side-group substituent on the tetrapyr­role ring. All Chl have two major absorption bands: blue or blue-green (450-475 nm) and red (630-675 nm) (Niklas Engstrom, 2012). Chl a is present in all oxygenic photoautotrophs.

Photoautotrophic cultures seldom reach very high cell densities; they are more than an order of magnitude less productive than many heterotrophic microbial cultures, the reason that microalgal cultures are carried in very large volumes. However, the microalgal photosyn­thetic mechanism is simpler than that of higher plants, providing more efficient solar energy conversion. This makes microalgae the most important carbon-fixative group and oxygen producer on the planet. Microalgae cultures have some advantages over vascular plants (Benemann and Oswald, 1996): All physiological functions are carried out in a single cell, they do not differentiate into specialized cells, and they multiply much faster.

An injected air stream containing ozone gas was used in separating microalgae from high rate oxidation pond effluent by ozone flotation. Use of ozone-induced flotation for algae recovery and effluent treatment was studied (Betzer et al., 1980). The ozone gas promotes cell flotation by modification of algae cell wall surface and by releasing some surface active agents from algae cells. The ozone-flotation process has been studied in numerous applications (Jin et al., 2006; Benoufella et al., 1994).

Elimination of a Microcystis strain of cyanobacteria (blue-green algae) by the use of ozone flotation was investigated in a pilot study (Benoufella et al., 1994). The oxidizing properties of ozone and the physical aspects of flotation were exploited in the flotation process. A specific ozone utilization rate of Microcystis was calculated, and ozone concentration and contact time curves were plotted versus algal removal. The study found that use of ozone in pretreatment leads to an inactivation of the algal cells. A prior coagulation stage was necessary for satis­factory cyanobacteria removal, and use of ferric chloride as a coagulant produced the best performance. Preozonation was also of influence on enhancement of the coagulation efficiency. Coupling ozone flotation with filtration can improve water quality, with treated effluent indicating low turbidity and low organic content.

Palmitate is the precursor of stearate and longer-chain saturated fatty acids as well as palmitoleate and oleate (Pollard and Stumpf, 1980). The palmitic acid gets modified further and lengthened to form stearate (18:0) or even to longer saturated fatty acids (oleiceate, linealate, etc.) by further additions of acetyl groups through the action of fatty acid elongation systems present in the smooth endoplasmic reticulum (ER) and in mitochondria (Thelen and Ohlrogge, 2002). The mechanism of elongation in the ER is identical to palmitate synthesis, which involves donation of two carbons by malonyl-CoA, followed by reduction, dehydra­tion, and reduction to the saturated 18-carbon product, stearoyl-CoA. Figure 8.4 shows the formation of higher fatty acids from the palmitic acid through different steps of chain elonga­tion. In algae, oleate (from stearoyl-CoA) gets converted to the a and g linolenates (Thelen and Ohlrogge, 2002). a-linolenate further getsconverted to other polyunsaturated fatty acids, while g-linolenate converts to the eicosatrienoate and further arachidonate. Mammals cannot

convert oleate to linoleate or linolenate because of the lack of enzymes to introduce double bonds at carbon atoms beyond C9 (Nelson and Cox, 2009). All fatty acids containing a double bond at positions beyond C9 have to be supplied in the diet and are called essential fatty acids.

Direct photolysis involves water oxidation and a light-dependent transfer of electrons to the [Fe]-hydrogenase, leading to the photosynthetic hydrogen production. Electrons are derived from water upon the photochemical oxidation by photosystem II (PSII or water — plastoquinone oxidoreductase), which is an enzyme located in the thylakoid membrane of algae and cyanobacteria. PSII uses photons from sunlight to energize electrons that are then transferred through the thylakoid membrane electron-transport chain and, via photosystem I (PSI or ferredoxin oxidoreductase) and ferredoxin (Fd), to the hydrocarbon cluster of [Fe]-hydrogenase (Florin et al., 2001). Plastoquinone is reduced to plastoquinol from the transferred electrons, which are used to reduce NADP+ to NADPH or are used in cyclic photophosphorylation. The energized electrons are replaced by oxidizing water to form hydrogen ions and molecular oxygen, as shown in Figure 9.1. By obtaining these electrons from water, PSII provides the electrons needed for the photosynthesis. The hydrogen ions (protons) generated by the oxidation of water help create a proton gradient that is used by ATP synthase to generate ATP. Protons are the terminal acceptors of these photosynthetically generated electrons in the algal chloroplast. The process results in the simultaneous produc­tion of oxygen and hydrogen gases (Spruit, 1958; Greenbaum et al., 1983).

Direct photolysis capitalizes on the photosynthetic capability of microalgae and cyanobacteria to split water directly into oxygen and hydrogen. Cyanobacteria, also known as blue-green algae, belong to a phylum of bacteria that obtain their energy through photo­synthesis. Microalgae have evolved the ability to harness solar energy by extracting protons

and electrons from water via water-splitting reactions. The biohydrogen production takes place via direct absorption of light and transfer of electrons to two groups of enzymes: hydrogenases and nitrogenases (Manis and Banerjee, 2008). Under anaerobic conditions or when too much energy is captured in the process, some microorganisms vent the excess electrons by using a hydrogenase enzyme that converts the hydrogen ions to hydrogen gas (Sorensen, 2005; Turner et al., 2008). It has been reported that the protons and electrons extracted via the water-splitting process are recombined by a chloroplast hydrogenase to form molecular hydrogen gas with a purity of up to 98% (Hankamer et al., 2007).

In addition to producing hydrogen, the microorganisms also produce oxygen, which in turn suppresses hydrogen production (Kovacs et al., 2006; Kapdan and Kargi, 2006). Research work has been carried out to engineer algae and bacteria so that the majority of the solar energy is diverted to hydrogen production, with bare energy diverted to carbohydrate production to solely maintain cells. Researchers are attempting to either identify or engineer less oxygen-sensitive microorganisms, isolate the hydrogen and oxygen cycles, or change the ratio of photosynthesis to respiration to prevent oxygen buildup (U. S. DOE, 2007). Addition of sulfate has been found to suppress oxygen production. However, the hydrogen production mechanisms are also inhibited (Sorensen, 2005; Turner et al., 2008).

The merit of direct photolysis is that the principal feed is water and the driver energy is derived from sunlight, both of which are readily available. Although this technology has significant promise, it is also facing tremendous challenges. A major challenge is the incom­patibility in the simultaneous molecular hydrogen and oxygen production. Photosynthetic hydrogen can only be produced transiently, since oxygen is a strong suppressor of hydroge­nate reactions and a powerful inhibitor of the [Fe]-hydrogenase. In addition, the photolysis process requires a significant algae cultivation area to collect sufficient light. Another challenge is achieving continuous hydrogen production under aerobic conditions (U. S. DOE, 2007).

Because algae require a variety of organic nutrients, it is possible to use them in wastewater treatment. In fact, wastewater has a significantly higher content of individual amino acids that support growth of algae, and they have been shown to reduce the chemical oxygen demand (COD) and biochemical oxygen demand (BOD) in wastewater (Christenson and Sims, 2011).

10.4 FEED

A final important use of spent biomass is a feed—for plants as fertilizers but also for such aquatic animals as fish or even zooplankton. These two applications are discussed in further detail in this section.