**5. Cultivation of microalgae**

The use of microalgae for energy generation requires large-scale, low-cost production. This demands cheap, scalable reactor design with efficient provision of the requirements for high algal productivity. Design considerations include optimum surface area to volume ratio for light provision, optimal mixing to keep cells in suspension and for distribution of nutrients, control over water balance and sterility, as well as maintenance of favorable temperature. A wide variety of reactor designs have been proposed, each with advantages and drawbacks.

#### **5.1 Reactor systems**

Microalgal production is a technology halfway between agriculture, which requires large areas for sunlight capture, and fermentation, which involves liquid culture of microorganisms (Becker, 1994). As light does not penetrate more than a few centimetres through a dense algal culture, scale-up is based on surface area rather than volume (Scott et al., 2010). Many different types of algal cultivation systems have been developed, but they can be divided into two main categories: open and closed.

TAG (Shifrin & Chisholm, 1981; Piorreck et al., 1984; Spoehr & Milner, 1949; De la Pena,

TAGs are the most suitable class of lipids for biodiesel production. Phospholipids are particularly undesirable as they increase consumption of catalyst and act as emulsifiers, impeding phase separation during transesterification (Mittelbach & Remschmidt, 2004; Van Gerpen, 2005). Phospholipids, and some sulphur-containing glycolipids, also increase the phosphorous and sulphur content of the fuel respectively, which must both be below 10 mg.L-1 to meet the European biodiesel standard EN 14214. The type of fatty acids found in the oil can have a profound effect on the biodiesel quality. The fatty acid chain length and degree of saturation (determined by the number of double bonds) affects properties such as the viscosity, cold flow plug point, iodine number and cetane number of the fuel (Ramos et al., 2009). For biodiesel production, it is therefore important to maximize not only total lipid

Lipid synthesis relies on carbon compounds generated from CO2 by photosynthesis, as well as energy and reducing power (in the form of ATP and NAD(P)H respectively). The latter are produced during the light reactions of photosynthesis, while CO2 uptake is mediated by the Calvin cycle during the dark reactions of photosynthesis. The output of the Calvin cycle is a three-carbon compound (glyceraldehyde 3-phosphate), which is converted through glycolysis into acetyl CoA. The conversion of acetyl CoA to malonyl CoA is the first committed step in lipid biosynthesis (Livne & Sukenik, 1992). Throughout metabolism there are a number of branch points at which metabolic intermediates are partitioned between the synthesis of lipids and other products such as carbohydrates and proteins (Lv et al., 2010). For example, acetyl CoA is a substrate for lipid synthesis as well as entry into the TCA cycle, which generates energy and biosynthetic precursors for proteins and nucleic acids. Both external and internal constraints, such as the availability of nutrients and the enzymatic reaction rates, limit the supply of metabolic intermediates. The production of storage lipids is particularly energy and resource intensive (Dennis et al., 1998; Roessler, 1990) and therefore usually occurs at

The use of microalgae for energy generation requires large-scale, low-cost production. This demands cheap, scalable reactor design with efficient provision of the requirements for high algal productivity. Design considerations include optimum surface area to volume ratio for light provision, optimal mixing to keep cells in suspension and for distribution of nutrients, control over water balance and sterility, as well as maintenance of favorable temperature. A wide variety of reactor designs have been proposed, each

Microalgal production is a technology halfway between agriculture, which requires large areas for sunlight capture, and fermentation, which involves liquid culture of microorganisms (Becker, 1994). As light does not penetrate more than a few centimetres through a dense algal culture, scale-up is based on surface area rather than volume (Scott et al., 2010). Many different types of algal cultivation systems have been developed, but they

production, but also TAG content and appropriate fatty acid profile.

2007; Becker, 1994).

conditions of reduced growth.

**5. Cultivation of microalgae** 

with advantages and drawbacks.

can be divided into two main categories: open and closed.

**5.1 Reactor systems** 

Open systems consist of natural waters such as lakes, ponds and lagoons, or artificial ponds and containers that are open to the atmosphere. Most commercial production to date has taken place in open ponds as these systems are easy and cheap to construct (Pulz, 2001). The most common technical design is the raceway pond: an oblong, looped pond mixed by a paddlewheel, with water depths of 15 to 20 cm (Becker, 1994). Biomass concentrations of between 0.1 and 1 g.L-1 and biomass productivities of between 50 and 100 mg.L-1.day-1 are possible (Chisti, 2007; Pulz, 2001). The main advantages of open systems are their low cost and ease of construction and operation. They also offer the potential for integration with wastewater treatment processes or aquaculture systems (Chen, 1996).

Disadvantages of open systems include contamination with unwanted species such as foreign algae, yeast, bacteria and predators, evaporation of water, diffusion of CO2 to the atmosphere and low control over environmental conditions, particularly temperature and solar irradiation (Becker, 1994; Pulz, 2001). In addition, the relatively low cell densities achieved can lead to higher cost of cell recovery (Chen, 1996). Only a few microalgal species have been successfully mass cultivated in open ponds. These tend to be either fast-growers that naturally outcompete contaminating algae (e.g. *Chlorella* and *Scenedesmus*), or species that grow in a specialised environment such as high salt (e.g. *Dunaliella salina*) or high pH (*Spirulina platensis*), which limits growth of competitors and predators (Chen, 1996). Due to the lack of control over cultivation conditions resulting in low productivity, and the fact that many desirable species cannot be effectively maintained in open systems, attempts have been made to overcome some of these limitations through the use of enclosed reactor systems.

Closed systems, or photobioreactors, consist of containers, tubes or clear plastic bags of various sizes, lengths and orientations (Pulz, 2001). Commonly used designs include vertical flat-plate reactors and tubular reactors, either pumped mechanically or by airlift (Scott et al., 2010). Closed reactors offer a much higher degree of control over process parameters, leading to improved heat and mass transfer, and thus higher biomass yields. They can also offer a much higher surface area to volume ratio for light provision, better control of gas transfer, reduction of evaporation and easier installation in any open space (Chen, 1996). Additionally, the risk of contamination is reduced, CO2 can be contained, production conditions can be reproduced and temperature can be controlled.

Productivity in closed systems can be much higher than open systems, with biomass concentrations of up to 8 g.L-1 and productivities of between 800 and 1300 mg.L-1.day-1 (Pulz, 2001). However, they are generally much more costly to build and more energy demanding to operate than open systems (Table 2). Closed systems can also have problems with fouling and oxygen build-up. Large systems can be difficult to clean and sterilize and long sections of enclosed tubing may require oxygen purging. High oxygen concentrations cause the key enzyme Rubisco to bind oxygen instead of carbon dioxide, leading to photorespiration instead of photosynthesis (Dennis et al., 1998). Although closed bioreactors offer a much higher degree of control over process parameters and can have higher yields, it is uncertain whether the increased productivity can offset the higher cost and energy requirements. For a commodity product such as vegetable oil for biodiesel, low cost, high volume production is demanded, while quality is less critical (Pulz, 2001). In this case, the more favourable economics and energy requirements of open ponds may well outweigh the advantages of closed reactors.

A hybrid system combining the cost effectiveness of open ponds with the controlled environment of closed systems is appealing and has been tested in a few cases. Generally

Advantages and Challenges of Microalgae as a Source of Oil for Biodiesel 183

Above the saturation point, damage to photosynthetic machinery can occur in a process known as photoinhibition (Scott et al., 2010). In a dense culture exposed to direct sunlight, cells at the surface are likely to be photoinhibited, while those at the centre of the reactor are in the dark. Mixing is therefore important not only in preventing cell settling and improving mass transfer, but also exposing cells from within a dense culture to light at

The frequency of light-dark cycling has been reported to affect algal productivity (Grobbelaar, 1994; Grobbelaar, 2000). Algae are less likely to become photoinhibited when the light is supplied in short bursts because the photosystems have time to recover during the dark period (Nedbal et al., 1996). While high rates of mixing facilitate rapid circulation of cells between light and dark zones in the reactor, high liquid velocities can damage algal cells due to increased shear stress (Mata et al., 2010). High rates of mechanical mixing or gas sparging also have large energy requirements, jeopardizing the process energy balance and

In order to maintain a high photosynthetic rate, the influx of carbon and energy must be non-limiting. In photoautotrophic growth, energy is provided by light and carbon in the form of CO2. In order to be taken up by cells, the CO2 must dissolve in the water. The rate of dissolution is determined by the CO2 concentration gradient as well as by the temperature, rate of gas sparging and surface area of contact between the liquid and gas (a function of agitation and bubble size). Reactor geometry, methods of gas introduction and reactor mixing can all influence the rate of CO2 delivery (Bailey & Ollis, 1977). Certain strains of microalgae can tolerate up to 12% CO2 (Pulz, 2001). The 0.03% CO2 content of ambient air is suboptimal for photosynthesis (Pulz, 2001), hence for optimal microalgal growth, additional CO2 must be provided. This is usually done by direct injection of a CO2 enriched air stream. As the addition of CO2 acidifies the medium, care must be taken not to adversely decrease the pH (Anderson, 2005). It is debatable whether direct gas injection is the optimal method of CO2 delivery. Efficiencies of carbon uptake are very low at high CO2 concentrations, as most CO2 exits the top of the reactor. Novel strategies of CO2 provision include microporous hollow fibre membranes and separate gas exchanger

The major nutrient requirements for microalgal growth are nitrogen and phosphorous, with certain diatoms, silicoflagellates and chrysophytes also requiring silicon (Anderson, 2005). Requirements of nutrients, pH and osmolarity are species dependent. Deviation from optimal levels may cause a decrease in biomass productivity, but can have other advantages, for example, high salinity may limit contamination. Sufficient supply of all essential nutrients is a prerequisite for efficient photosynthesis and growth, but limitation of key nutrients (e.g. nitrate, phosphate or silica) may cause accumulation of desired products

Most microalgae are photoautotrophs (utilizing sunlight as their source of energy and CO2 as a carbon source). This is the most common growth mode employed in algal cultivation

the surface.

increasing costs (Richardson, 2011).

systems (Carvalho et al., 2006).

**5.2.4 Salinity, nutrients and pH** 

such as lipid.

**5.2.5 Nutritional mode** 

**5.2.3 Gas exchange** 


production is divided into an initial growth or inoculum production stage in closed reactors, followed by a stress or scaling up stage in open ponds (Huntley & Redalje, 2006).

Table 2. Comparison of open ponds and closed photobioreactors. Adapted from Pulz (2001).
