**5.2 Cultivation parameters**

Several factors need to be considered in the cultivation of algal biomass. These include the provision of light, carbon and nutrients such as nitrate, phosphate and trace metals, the mixing regime, maintenance of optimal temperature, removal of O2 and control of pH and salinity (Becker, 1994; Grobbelaar, 2000; Mata et al., 2010). The optimal and tolerated ranges tend to be species specific, and may vary according to the desired product.

#### **5.2.1 Temperature**

Light and temperature are among the most difficult parameters to optimise in large-scale outdoor culture systems. Daily and annual fluctuations in temperature can lead to significant decreases in productivity. Optimal growth temperatures are generally between 20 and 30C (Chisti, 2008). Many algal species can tolerate temperatures of up to 15C lower than their optimum, with reduced growth rates, but a temperature of only a few degrees higher than optimal can lead to cell death (Mata et al., 2010). Closed systems in particular often suffer from overheating during hot days, when temperatures inside the reactor can reach in excess of 50C. Heat exchangers or evaporative water-cooling systems may be employed to counteract this (Mata et al., 2010). Low seasonal and evening temperatures can also lead to significant losses in productivity.

## **5.2.2 Light and mixing**

The efficient production of algal biomass relies on the optimal provision of light energy to all cells within the culture. Most algal growth systems become light limited at high cell densities. Due to absorption and shading by the cells, light only penetrates a few centimetres into a dense algal culture (Richmond, 2004). The average provision of light is linked to reactor depth or diameter, cell concentration and mixing. A larger surface area to volume ratio, usually achieved through areas of thin panelling or narrow tubing, results in higher light provision.

Photosynthetic efficiency is highest at low light intensities. At high light levels, although photosynthetic rate may be faster, there is less efficient use of absorbed light energy. 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 surface.

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 increasing costs (Richardson, 2011).

#### **5.2.3 Gas exchange**

182 Biodiesel – Feedstocks and Processing Technologies

production is divided into an initial growth or inoculum production stage in closed reactors,

**Parameter Open Closed**  Control over process parameters Low High Contamination risk High Low Water loss due to evaporation High Low CO2 loss High Low O2 build-up Low High Area required High Low Productivity Low High Consistency and reproducibility Low High Weather dependence High Low Cost Low High Energy required Low High

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

Several factors need to be considered in the cultivation of algal biomass. These include the provision of light, carbon and nutrients such as nitrate, phosphate and trace metals, the mixing regime, maintenance of optimal temperature, removal of O2 and control of pH and salinity (Becker, 1994; Grobbelaar, 2000; Mata et al., 2010). The optimal and tolerated ranges

Light and temperature are among the most difficult parameters to optimise in large-scale outdoor culture systems. Daily and annual fluctuations in temperature can lead to significant decreases in productivity. Optimal growth temperatures are generally between 20 and 30C (Chisti, 2008). Many algal species can tolerate temperatures of up to 15C lower than their optimum, with reduced growth rates, but a temperature of only a few degrees higher than optimal can lead to cell death (Mata et al., 2010). Closed systems in particular often suffer from overheating during hot days, when temperatures inside the reactor can reach in excess of 50C. Heat exchangers or evaporative water-cooling systems may be employed to counteract this (Mata et al., 2010). Low seasonal and evening temperatures can

The efficient production of algal biomass relies on the optimal provision of light energy to all cells within the culture. Most algal growth systems become light limited at high cell densities. Due to absorption and shading by the cells, light only penetrates a few centimetres into a dense algal culture (Richmond, 2004). The average provision of light is linked to reactor depth or diameter, cell concentration and mixing. A larger surface area to volume ratio, usually achieved through areas of thin panelling or narrow tubing, results in higher

Photosynthetic efficiency is highest at low light intensities. At high light levels, although photosynthetic rate may be faster, there is less efficient use of absorbed light energy.

tend to be species specific, and may vary according to the desired product.

**5.2 Cultivation parameters** 

also lead to significant losses in productivity.

**5.2.1 Temperature** 

**5.2.2 Light and mixing** 

light provision.

followed by a stress or scaling up stage in open ponds (Huntley & Redalje, 2006).

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 systems (Carvalho et al., 2006).

#### **5.2.4 Salinity, nutrients and pH**

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 such as lipid.

#### **5.2.5 Nutritional mode**

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

Advantages and Challenges of Microalgae as a Source of Oil for Biodiesel 185

Fig. 1. Conceptual overview of microalgae process options (adapted from Chisti, 2008)

**Zeta potential** 

*vulgaris* 1070 -17.4 7 Single cell

*Microcystis* 1200 -7.5 to -26 5.6 - 9.5 Globular sphere 3-7

*Cyclotella* sp. 1140 -19.8 to -22.3 4 - 10 Chains of spheres 6.1

*Syendra acus* 1100 -30 to -40 7.6 Needles 4.5-6, 100-300 Table 3. Characteristics relevant to harvesting of some microalgae (Henderson et al., 2008). Zeta potential is a measure of the degree of repulsion between adjacent particles due to

**(mV)**

Several natural properties of microalgal cells affect the choice and efficiency of harvesting methods. Factors relevant to separation include density, surface charge, size, shape, hydrophobicity, salinity of the medium, adhesion and cohesion properties and settling or floating velocities. Table 3 highlights the variability in some of these parameters between species, indicating that species-specific solutions may be required. Algal cell characteristics can vary with culture age and growth conditions. For example, changes in biochemical composition such as lipid content could affect the buoyancy of algal cells. Surface charge, the chemical structure of the cell wall and the amount and composition of the extracellular organic matrix (EOM) can vary with growth phase and greatly influence the degree to which cells repel or stick to one another (Bernhardt & Clasen, 1994; Henderson et al., 2008). Danquah et al. (2009) found a strong correlation between growth phase and settling efficiency, with improved filtration, flocculation and sedimentation rates during the

**Culturing** 

**pH Morphology Diameter,** 

spherical 3.5

**length (µm)** 

**6.1 Factors affecting separation** 

stationary phase.

*Chlorella* 

surface charge.

**Species Density** 

**(kg.m-3)**

(Chen, 1996). However, several species (e.g. *Chlorella*, *Chlamydomonas, Phaeodactylum, Nitzschia, Tetraselmis* and *Crypthecodinium*) are also capable of heterotrophic growth (utilizing organic carbon such as glucose, acetate or glycerol as the sole source or carbon and energy) or mixotrophic growth (photoautotrophic growth supplemented by an organic carbon source).

The advantages of using an organic carbon substrate are that it decreases dependence on light provision, allowing growth in conventional fermenters in the dark. Optimal growth conditions can be maintained, allowing higher cell concentrations and hence increased volumetric productivities to be reached (Chen, 1996). Higher productivities of both biomass and lipid have been reported under heterotrophic growth compared to autotrophic (Ceron Garcia, 2000; Miao & Wu, 2006). Disadvantages of feeding an organic carbon source include the fact that there are a limited number of algal species that can utilize organic carbon sources, the risk of bacterial contamination is greatly increased and the carbon substrate adds an additional cost, along with the environmental burden of its production. The use of a substrate such as glucose, commonly sourced from crop plants, adds a trophic level to the process, thereby removing the simplicity of the concept of microalgae as cellular factories producing liquid fuel from pure sunlight and CO2.

#### **5.2.6 Cultivation strategy**

The optimal cultivation strategy (e.g. batch, fed-batch or continuous cultivation mode) is determined by the kinetics of growth, product accumulation and substrate uptake (Shuler & Kargi, 2005). For production of a primary product such as protein or biomass for food or feed, optimisation of biomass productivity is the main objective. In this case, batch or continuous systems are generally used. For production of a secondary product such as carotenoids or storage lipids, the use of two or more production stages to enhance yield has been proposed (Ben-Amotz, 1995; Huntley & Redalje, 2006; Richmond, 2004). The first stage is designed to optimize growth, while the second stage provides conditions that retard growth and encourage product synthesis, usually by applying some form of stress, e.g. nutrient deprivation in the case of lipid accumulation. Another potential two-stage strategy that could enhance lipid productivity is an initial photosynthetic stage, followed by a second heterotrophic phase, where feeding with an organic carbon source such as glucose may boost lipid content.
