SpiruIina is a filamentous cyanobacterium recognized mainly by its multicellular cylindri­cal arrangement of trichomes in an open helix along the entire length (Vonshak, 1997). Under the microscope, it appears as blue-green filaments of unbranched cylindrical cells, in helical trichomes. The filaments are movable and move freely around its axis, and they are not heterocystic. They are up to 1 mm in length; the cell diameter ranges from 1-3 gm in small species and 3-12 gm in the larger species (Richmond, 1990).

This microalga inhabits various media such as soil, sand, swamps, alkaline lakes and brackish, marine, and fresh water. Through photosynthesis, it converts nutrients into cellular matter and releases oxygen. The components needed for cell growth are water, a carbon source, nitrogen, phosphorus, potassium, magnesium, iron, and other micronutrients.

In natural lakes, the limited supply of nutrients may regulate the growth cycles, and the cell density increases rapidly, reaches a maximum concentration, and retreats when nutrients are depleted. The release of nutrients from dead cells or the supply of nutrients initiates a new cycle (Henrikson, 1994).

There are many controversies in the morphology and taxonomy of cyanobacteria of the genera Spirulina and Arthospira. Many studies have described the properties of Spirulina max­ima and Spirulina platensis, and both species are considered to be of the genus Arthospira and not Spirulina. The differences between the genera have been based on the G + C content of DNA and lipid profile (Romano et al., 2000).

The helical shape is only maintained in liquid medium; in solid medium the filaments take a spiral shape, and the transition from the helical shape to the spiral shape is slow, whereas the opposite takes place instantaneously. Most species of Spirulina present a granular cyto­plasm containing gas vacuoles and septa that are easily visible. Electron microscopy reveals that the cell wall of Spirulina platensis is probably composed of four layers.

The life cycle of Spirulina begins when a trichome (filament consisting of cells) elongates, and this is followed by an increase in the number of cells as a result of repeated interspersed cell divisions. The microalga cell fragmented into several parts by the formation of special­ized, lysis-promoting necridic cells, which give rise to small chains (two or four cells) called hormogonia, which develop into new trichomes. The number of cells in the hormogonium increases by cellular fission, while the cytoplasm becomes granulated and the cells take on a bright bluish-green color. Due to this process, trichomes increase in length and take their typical helical shape (Richmond, 1990).

In both lab-scale and pilot-scale microalgae cultivation systems, the key factors that need to be considered for the design and operation of microalgae cultivation systems are as follows: (1) how to use appropriate light sources (intensity and wavelength), (2) how to enhance light conversion efficiency, and (3) how to maintain an appropriate microalgae biomass concentra­tion during prolonged operation. In addition, the stability of continuous culture of microalgae is usually poor, because the cell growth and target-product production are sensitive to changes in the environment and the medium composition.

Maintaining a sufficient cell concentration in the continuous microalgae cultivation system is also a challenge. Therefore, many large-scale outdoor microalgae cultivation systems are operated in a semibatch mode, in which a portion of microalgae culture is harvested within a specific cultivation time period and an equal amount of fresh medium is refilled into the cultivation system. In addition, most commercial-scale microalgae cultivation is carried out in open ponds, since solar light energy is directly utilized. Therefore, there are challenges such as contamination by other microorganisms or alien microalgae species, direct exposure to ultraviolet (UV) irradiation, low light intensity or uneven light energy distribution (Kim et al., 1997), day-night cycles, diurnal variation, and requirements for large areas of land (Laws et al., 1986). Moreover, since the intensity of sunlight varies greatly with the seasons, solar spectrum, and operating time, it is very difficult to maintain steady microalgae culture performance in outdoor cultivation.

The limitation of light energy is also one of the most commonly encountered problems in large-scale cultivation when the size of the microalgae cultivation system is increased. In this case, the illumination area per unit volume is often considered as a design criterion. The fac­tors mentioned here greatly limit the light conversion efficiency and productivities of outdoor microalgae cultivation systems. Other factors that may also lower the biomass productivity are consumption of biomass by respiration in the dark zones of the reactor, insufficient mixing of CO2 and nutrients, and the mechanical damage due to the shear stress on the algal cells. Variation in biomass concentration and composition (e. g., carbohydrate or lipid content) may occur when different culture media and operation modes are used.

Despite the fact that good production performances of target products can be achieved using lab-scale microalgae cultivation systems, there are still very few successful commercial-scale processes. This is mainly because of the higher operating costs, unstable light intensity, and lower mixing efficiency when the microalgae are grown outdoors on a large scale. Consequently, appropriate operating configurations with innovative design of microalgae cultivation system are required to achieve commercially viable production of microalgae biomass and target products.

Therefore, highly efficient light sources and good circulation devices are the key to pro­mote microalgae cell growth in the design of commercially feasible microalgae cultivation systems. If the light source has a narrow spectral output that overlaps the photosynthetic ab­sorption spectrum of microalgae, the emission of light at unusable wavelengths would be eliminated, thereby improving the overall energy conversion. Among the available light sources, light-emitting diode (LED) is the only one that meets the foregoing criteria. LEDs are an economic external light source that is energy-saving and small enough to fit into any microalgae cultivation system. They also have a very long life expectancy, and their elec­trical efficiency is so high that heat generation is minimized. LEDs have a half-power band­width of 20-30 nm, which can match photosynthetic needs. On the other hand, circulation is also very important in the outdoor microalgae cultivation system. The benefits include keep­ing microalgae in suspension, decreasing heat generation within the microalgae cultivation system, uniform distribution of the cells and the liquid broth, improving CO2 mass-transfer efficiency, and degassing the O2 produced during photosynthesis. Therefore, the develop­ment of economically successful production of microalgae biomass requires the improvement of both light efficiency and mixing efficiency for microalgae growth at low cost.

Sedimentation through gravity is the most usual method of harvesting microalgal biomass from wastewater treatment plants. This is because of the large volumes handled and the low commercial value of the biomass formed (Munoz and Guieysse, 2006). The density and diam­eter of the cells influence the speed of sedimentation. The collection of microalgae by sedi­mentation can be carried out in sedimentation tanks (Uduman et al., 2010). However, this method can only be applied to large-cell microalgae (>70 gm) such as Spirulina. Flocculation is usually used to increase the efficiency of sedimentation (Chen et al., 2011).

Su-Chiung Fang

Biotechnology Center in Southern Taiwan, Academia Sinica
Agricultural Biotechnology Research Center, Academia Sinica
Tainan, Taiwan R. O.C.

3.1 INTRODUCTION

Compared to other biofuel feedstocks, microalgae are the preferred option for many reasons:

1. They grow extremely fast and hence produce high biomass yield quickly.

2. Microalgae-based fuels do not compete with the food supply and hence present no food security concerns.

3. Biofuels generated from microalgae are renewable and can be carbon-reducing [generation of 100 tons of algal biomass is equivalent to removing roughly 183 tons of carbon dioxide from the atmosphere (Chisti, 2008)].

4. Microalgal farming does not require arable land and can utilize industrial flue gas as a carbon source.

5. Selected oleaginous microalgae do not require fresh water and can grow in seawater, brackish water, or waste water.

6. Biodiesel fuels derived from microalgae can be integrated into the current transportation infrastructure.

During the past few years, there have been significant advances in uncovering molecular components required for production of fuel molecules in microalgae. The availability of ge­nomic sequences in the model green alga Chlamydomonas reinhardtii has accelerated forward genetic analysis and allowed for the use of reverse genetic approaches to uncover molecular mechanisms associated with fuel production (Merchant et al., 2007). Moreover, tran — scriptomics, proteomics, and metabolomics studies have provided new insights into gene regulation networks and coordinated cellular activities governing physiological flexibility and metabolic adaptation of microalgae. Understanding the basis of microalgal biology is important in laying the foundation for innovative strategies and for ultimate development of fuel surrogates. This review summarizes recent progress in elucidating molecular and cellular mechanisms of cellular physiology that are relevant to fuel production in microalgal systems, with an emphasis on developing metabolic engineering strategies to increase fuel production.

Many algae produce antibiotics such as acrylic acid found in Phaeocystis poucht. This anti­biotic inhibits the growth of gram-positive organisms. The phenols found in macro — and microalgae have antimicrobial activity.

The microalga Scenedesmus obliquus has been used in postoperative recovery, assisting in coagulation of the skin surface. The extracts of the diatom Asterionella notata have an antifun­gal and antiviral activity. Toxic algae have been used as a depressant vessel, similar to tetro — dotoxin found in fish (Richmond, 1990).

Another drug obtained from microalgae is phycocyanin, a natural antioxidant that, when combined with caloric restriction, can contribute to mitigating the aging process. Free radicals are partly responsible for the human aging process (Finkel, 2003). The oxidative damage caused by free radicals has been linked to several diseases such as heart disease, atheroscle­rosis, lung problems, Alzheimer’s, and Parkinson’s. The DNA damage caused by free radicals plays an important role in the processes of mutagenesis and carcinogenesis.

Since outdoor sunlight cannot be controlled, carbon fixation by microalgae is usually studied indoors under artificial illumination. A good deal of scientific effort is being made to evaluate microalgae CO2 fixation potential. Most of these efforts focus the fixation into biomass (Chae et al., 2006; Jacob-Lopes et al., 2008; Kajiwara et al., 1997). However, these studies did not quantify the total carbon dioxide fixed effectively by microalgae (Jacob-Lopes et al., 2008; Fan et al., 2007), since there are other routes for carbon besides biomass generation, such as mineralization (formation of soluble bicarbonate and carbonate) and production of extracellular products such as polysaccharides, volatile organic compounds (Shaw et al., 2003), organohalogens (Scarratt and Moore, 1996), hormones, and others.

The determination of global rates of carbon dioxide sequestration through mass balances of CO2 in the liquid or gas phase of the systems (Eriksen et al., 2007) gives more complete data. One approximation for the rates may be obtained by evaluating dissolved inorganic carbon concentration in the culture media while monitoring the pH variation (see methodology at Valdes et al., 2012). This shows that carbon fixation by microalgae is a complex process whereby biomass production might be a part of the total carbon destination. In addition, little information is available with respect to the simultaneous research of both the global rates of

02 consumed (g/h) —— 02 Base Line (g/h)

FIGURE 4.3 Gas phase analysis carried by Sydney et al. (2011) showing the carbon consumption and oxygen production profiles.

carbon dioxide sequestration and the rates of incorporation of carbon into the microalgae biomass (Chiu et al., 2008).

Sydney et al. (2011) studied the global CO2 fixation rate of four microalgae through a mass balance of the gas phase. The experiments were carried out in a photobioreactor coupled with sensors to measure CO2 in the inlet and outlet gases. The net carbon dioxide mitigation during each microalgal cultivation was evaluated. Nutrient consumption, biomass production (and composition), and possible extracellular products were analyzed throughout the process. It was found that between 70% and 88% of the carbon dioxide consumed was used in biomass production. This finding indicates that, to explore the whole potential of microalgal mitiga­tion capacity (considering negotiations in the carbon market), carbon balance might be carried through (complex) carbon balance in the gas phase. The problem is that it is difficult to carry out this kind of analysis in open photobioreactors and to standardize this methodology. Figure 4.3 presents the profile of carbon dioxide consumption obtained during gas phase analysis during cultivation. It is interesting to note that CO2 consumption (in blue) has a com­plementary behavior with O2 production due to photosynthesis and respiration processes during light and dark cycles.

Chlorella spp. are simple, nonmotile, unicellular, aquatic green microalgae. They were one of the first algae to be isolated as a pure culture. The Chlorella microalga measures between 5 and 10 micrometers and, under an optical microscope one, can observe its green color and spherical shape.

Compared to higher plants, Chlorella has a high concentration of chlorophyll and photo­synthetic capacity. The microalga Chlorella is classified as a species according to the shape of the cells, characteristics of chlorophyll, and other variables. There are 20-30 species, some of which are Chlorella vulgaris, Chlorella pyrenoidosa, and Chlorella ellipsoidea. The species are differentiated within the group, known as strains (Illman et al., 2000).

The first pure culture of microalga to be scientifically proven was Chlorella vulgaris in 1890 by the microbiologist M. W. Beijerinck. In 1919, Otto Warburg published articles on the use of this microalga in culture to study its physiology. After years of research with Chlorella and other microalgae, he found that these microorganisms grow under specific conditions and can be used to produce compounds with nutritional benefits to human health.

One of the most important characteristics of Chlorella is its protein content. Depending on the culture conditions, this microalga can provide 60% of protein with essential amino acids for human consumption. Chlorella has approximately three times more protein than the same amount of red meat, which is one of the most concentrated sources of protein. Due to its high protein concentration, Chlorella is used as a food supplement. This microalga has 23% carbo­hydrates, 9% fat, and 5% minerals (Henrikson, 1994).

Chlorella is also rich in B vitamins, especially B12, which is vital in the formation and re­generation of blood cells. Because it also has a high iron content, this microalga is a product indicated for the treatment and prevention of anemia. In order for its nutrients to be fully uti­lized by the body, cells of Chlorella, which are protected by a cell wall, must be disintegrated during the drying process to enable its nutrients to be fully absorbed by the metabolism (Henrikson, 1994).

Production of sustainable biofuels from microalgae is a high-potential option for develop­ing renewable energy. Unfortunately, the production cost of microalgae-based biofuels is still too high, which prevents them from becoming commercially feasible. One of the major obsta­cles that impedes the commercialization of microalgal biofuels is the high cost of photobioreactors and the high demand of auxiliary systems or intensive energy input re­quired during the cultivation of microalgae. Basic conceptual designs for a photobioreactor for the autotrophic cultivation of microalgae are to provide efficient mixing, appropriate light intensity, and rapid gas transport (Singh and Sharma, 2012).

In light of these demands, photobioreactor designs can be generally classified as open sys­tems and closed systems (Table 2.1). Open systems can be divided into natural waters (lakes, lagoons, ponds) and artificial ponds or containers, which are presented in very different ways. Apparently, open systems are potentially subject to contamination resulting from the free gas exchange from the environment to the cultivation system. The cultivation

conditions of open systems are usually poorly controlled, and the estimated growth rate of microalgae will be mostly lower than that in closed systems.

In terms of technical complexity, open systems are more widespread than closed systems. From the aspect of operation, closed systems are more suitable for the cultivation of algae for the production of high-value products. In closed systems, the productivity of desired prod­ucts can be enhanced by controlling the microalgae cultivation under optimal operating con­ditions. The design of closed photobioreactors must be carefully optimized for each individual algal species according to its unique physiological and growth characteristics. Providing appropriate light intensity and efficient hydrodynamic mixing are key issues in the success of a productive autotrophic cultivation system (Kumar et al., 2011).

Given the advantages of closed systems over open systems, several different photo­bioreactor designs with closed systems have also been proposed, ranging from lab scale to industry scale. More detailed descriptions of microalgae cultivation in open and closed systems are presented in the following sections.

To increase the recovery of cells via sedimentation, a flocculant is added to the system. Flocculation is the first step of the harvesting; this process aims to aggregate the microalgal cells and thereby increase the particle size (Grima et al., 2003). The microalgae have a negative charge on the surface to prevent cell aggregation. The loads on the surface of algae can be altered by the addition of flocculant (Harun et al., 2010).

Flocculation may be accomplished by three methods: chemical flocculation,

bioflocculation, and electroflocculation. The most common flocculants are aluminum sul­phate, aluminum chloride, and ferric chloride. The addition of sodium hydroxide raises the pH of the culture to 8-11, coagulating the cells in just a few minutes. However, the floc — culants are toxic in high concentration. Flocculants should be inexpensive, nontoxic, and ef­fective at low concentrations. Chitosan is an organic cationic polymer, a nontoxic flocculating agent that is used in wastewater treatment and in the food industry (Pires et al., 2012).

Among the various fuel categories derived from microalgae, biodiesel receives the most attention because it shares similar chemical characteristics with petrol diesel and can be directly channeled into the current transportation infrastructure without major alterations of existing technology and fuel pipelines. Oleaginous green microalgae species that have the capacity to accumulate oil in the form of triacylglycerols, or TAGs (Chisti, 2007; Sheehan et al., 1998), have been isolated and possess great potential as a feedstock for biodiesel fuels (Converti et al., 2009; Liu et al., 2008; Xu et al., 2006). However, microalgae-based biodiesel is far from being commercially feasible, because it is not economically practical at present. From a biological point of view, one of the obvious solutions is to increase oil content. Most microalgae do not accumulate large amounts of lipid during a normal growth period. Cells begin to accumulate significant amounts of storage lipids after encountering stress conditions such as light and nutrient starvation (Hu et al., 2008; Sheehan et al., 1998). Nutrient starvation, however, slows cell proliferation and therefore limits biomass and overall lipid productivity. Despite the continuous interest in and enthusiasm about microalgal oil-to-biodiesel potential, the molecular mechanisms underlying the cellular, physiological, and metabolic networks connecting to lipid and TAG biosynthesis remain largely unknown. Recent progress in transcriptomics, proteomics, metabolomics, and lipidomics studies have started to unravel the complex molecular mechanisms and regulatory networks involved in lipid and TAG biosynthesis in microalgae.

Current efforts to isolate and characterize the repertoire of genes required for lipid and TAG biosynthesis and accumulation in microalgae have focused on a model microalga: Chlamydomonas reinhardtii (Li et al., 2008; Miller et al., 2010; Msanne et al., 2012). With com­plete genome information, many enzymes required for lipid and TAG biosynthesis and me­tabolism have been identified based on in silico predictions of orthologous genes from other organisms (Riekhof et al., 2005). Similar to oilseed crops, the most common fatty acids in microalgae are 16- and 18-carbon fatty acids (Hu et al., 2008). Genome comparison and gene prediction analyses have shown that the pathways of fatty acid and lipid biosynthesis are largely conserved between plants and green algae (Riekhof et al., 2005). In plants, de novo syn­thesis of fatty acids occurs in the plastid (Ohlrogge and Browse, 1995). The synthesized fatty acids are used as building blocks for synthesis of membrane lipids and storage lipids. Acetyl CoA serves as the basic unit for fatty acid biosynthesis. It is converted to malonyl CoA by acetyl