**3.3.2 Optical fiber technology**

The use of optical fibers as internal light sources could increase light efficiency whilst simultaneously reducing electricity consumption. For example, solar energy excited optical fiber requires only 1.0 kW-h of electricity (Chen et al., 2011). Spatial (i.e. orientation and dimensions of the photobioreactor) and temporal variations (i.e. due to weather conditions) greatly affect the availability of sunlight. Therefore, internal illumination by optical fiber is unstable. To circumvent this problem, Chen et al. (2011) have conceptualized a photobioreactor that combines optical fiber and multi-LED light sources with both solar panel and wind power generators. This has a potential to be developed into a commercially viable microalgae cultivation system with significantly reduced electricity consumption.

#### **4. Limitations and improvements of photosynthetic biomass production of microalgae**

#### **4.1 Low light intensity and distribution**

There are several key parameters which determine the microalgal productivity in a photobioreactor. These are lighting, mixing, water, CO2 pressure, O2 removal, nutrient supply, temperature, and pH (Kunjapur and Eldrige, 2010). Under nutrient-sufficient and optimal temperature conditions, the maximal culture productivity of photoautotrophic microorganisms is solely limited by the light (Richmond, 2004). The penetration of visible spectrum of light in the microalgal cultures decreases as the cell density increases (Figure 1). The appropriate intensity, duration, and wavelength of light must be provided to enhance the microalgal growth in photobioreactors. Supra-optimal light conditions lead to photoinhibition and sub-optimal light becomes a growth limiting factor. In both conditions, microalgal productivity will be lowered. The photosynthetic conversion efficiency of microalgae will generally be lower than theoretically expected under optimal conditions due to insufficient capacity to utilize the incident radiation (Zhu et al., 2008). The distribution of solar radiation over a greater photosynthetic area can spatially dilute the light in the light saturation zone, thereby reducing the mutual shading of the cells in the culture resulting in higher growth rate and lower accessory pigments content. The distribution of solar radiation can be increased by maintaining the surface to volume ratio as high as possible. The temporal and spectral distribution of irradiation and photon flux density is the main physical parameter that determines the photosynthetic productivity of microalgae. The solar conversion efficiency of microalgal mass culture grown under full sunlight is limited because of two reasons: 1) the photon absorption rate of the chlorophyll antennae of upper layers of cells far exceeds the rate of their utilization hence there is a loss of excess photons as fluorescence and heat leading to photoinhibition; 2) the deprivation of functional photons in the deeper cell layers, which is strongly attenuated due to the filtering effect of upper cells (Naus & Melis, 1991, Neidhardt et al., 1998). Gordon and Polle (2007) argued that a microalgal biomass productivity of 100 g m-2 h-1 could be obtained solely by improving the flux tolerance rather than by raising intrinsic photosynthetic efficiency.

Fig. 1. Effect of biomass concentration on the penetration of incident light into cultures of *Nannochloropsis* sp. (from Richmond 2004).

#### **4.2 Improvements of intrinsic photosynthetic efficiency**

166 Biodiesel – Feedstocks and Processing Technologies

Red LEDs were found to reduce the average cell volume of *Chlorella vulgaris* without affecting the total biomass production (Lee and Palsson, 1994). However, under the

The use of optical fibers as internal light sources could increase light efficiency whilst simultaneously reducing electricity consumption. For example, solar energy excited optical fiber requires only 1.0 kW-h of electricity (Chen et al., 2011). Spatial (i.e. orientation and dimensions of the photobioreactor) and temporal variations (i.e. due to weather conditions) greatly affect the availability of sunlight. Therefore, internal illumination by optical fiber is unstable. To circumvent this problem, Chen et al. (2011) have conceptualized a photobioreactor that combines optical fiber and multi-LED light sources with both solar panel and wind power generators. This has a potential to be developed into a commercially viable microalgae cultivation system with significantly

**4. Limitations and improvements of photosynthetic biomass production of** 

There are several key parameters which determine the microalgal productivity in a photobioreactor. These are lighting, mixing, water, CO2 pressure, O2 removal, nutrient supply, temperature, and pH (Kunjapur and Eldrige, 2010). Under nutrient-sufficient and optimal temperature conditions, the maximal culture productivity of photoautotrophic microorganisms is solely limited by the light (Richmond, 2004). The penetration of visible spectrum of light in the microalgal cultures decreases as the cell density increases (Figure 1). The appropriate intensity, duration, and wavelength of light must be provided to enhance the microalgal growth in photobioreactors. Supra-optimal light conditions lead to photoinhibition and sub-optimal light becomes a growth limiting factor. In both conditions, microalgal productivity will be lowered. The photosynthetic conversion efficiency of microalgae will generally be lower than theoretically expected under optimal conditions due to insufficient capacity to utilize the incident radiation (Zhu et al., 2008). The distribution of solar radiation over a greater photosynthetic area can spatially dilute the light in the light saturation zone, thereby reducing the mutual shading of the cells in the culture resulting in higher growth rate and lower accessory pigments content. The distribution of solar radiation can be increased by maintaining the surface to volume ratio as high as possible. The temporal and spectral distribution of irradiation and photon flux density is the main physical parameter that determines the photosynthetic productivity of microalgae. The solar conversion efficiency of microalgal mass culture grown under full sunlight is limited because of two reasons: 1) the photon absorption rate of the chlorophyll antennae of upper layers of cells far exceeds the rate of their utilization hence there is a loss of excess photons as fluorescence and heat leading to photoinhibition; 2) the deprivation of functional photons in the deeper cell layers, which is strongly attenuated due to the filtering effect of upper cells (Naus & Melis, 1991, Neidhardt et al., 1998). Gordon and Polle (2007) argued that a microalgal biomass productivity of 100 g m-2 h-1 could be obtained solely by improving the flux tolerance rather than by raising intrinsic

exposure of fluorescent light, cells regained their normal size.

**3.3.2 Optical fiber technology** 

reduced electricity consumption.

photosynthetic efficiency.

**4.1 Low light intensity and distribution** 

**microalgae** 

The photosynthetic efficiency of microalgae can potentially reach its theoretical maximum, which is calculated to be about 9-10% of total incident solar energy or 20-22% of PAR, being converted into biomass (Beilen, 2010). Such projected ultrahigh microalgal biomass yields of 100 g dry weight m-2h-1 can be realized in photobioreactors with sufficiently thin channels, ultradense cultures, and rapid light/dark cycles wherein optimal synchronization of photonic input with rate limiting dark reaction times is exploited (Gordon and Polle, 2007). However, this does not take into account the intrinsic conversion efficiency of photosynthesis, which is only likely to be improved upon through genetic engineering or synthetic biology. The integration of molecular and photobioreactor engineering is likely the only possible way of obtaining near-theoretical levels of algal biomass productivity while simultaneously augmenting lipid content. At the unicellular level, genetic modification of microalgal photophysiology could decrease light absorption, leading to enhanced availability of functional irradiance at the population level. The PSII and PSI in the light harnessing complex of green algae are associated with large numbers of chlorophyll a and chlorophyll b molecules, which are called antenna molecules. During photosynthetic biomass production in photobioreactors, high photon flux densities saturate the antenna molecules of upper cell layers with excessive photons which do not participate in the photosynthetic biomass production. These excess photons dissipate their energy as heat or fluorescence (photoinhibition) and reduce the overall solar to biomass conversion efficiency of the microalgal culture. Moreover, the lower layers do not receive appropriate amount of photons because of the

Eco-Physiological Barriers and Technological Advances for Biodiesel Production from Microalgae 169

appropriate method. Acoustic focusing, hybrid capacitive deionization or electrophoresis and use of novel materials for conventional membranes and flocculent systems are amongst the range of new innovative strategies that are currently under investigation (Cheng &

Conventional methodologies for lipid extraction involve the use of toxic organic solvents such as chloroform, methanol and hexane. While the solvent extraction process is effective it is difficult to adopt on a large scale. Novel methods for lipid extraction involve technologies such as acoustics, sonication, the use of mesoporous nanomaterials, and amphiphilic solvents (Cheng & Ogden 2011). Super critical fluid extraction has been reported to be safer and faster than the conventional solvent extractions (Andrich et al., 2005). Another technique of "milking" microalgae manipulates the hydrophobicity of the solvent system, which allows the extraction of lipids from living algal cells. A flat panel two-phase bioreactor designed by Hejazi & Wijffels (2004) was used in the milking process for *Dunaliella salina* production. In this process, microalgal cells grown under optimal growth conditions are stressed by excess light to stimulate the production of β-carotene, which is then extracted from the cells using lipophilic compounds. Important considerations for application of this "milking" process includes: a) cell wall and membrane properties of the microalgal strain; b) location and accumulation of the product inside the cell; and c) biocompatibility and chemical properties of the solvent used for the "milking" process

Lipid conversion to biodiesel can easily be achieved by chemical trans-esterification, enzymatic conversion, and catalytic cracking. Chemically, biodiesel is comprised of monoalkyl esters of fatty acids that are derived from triacylglycerols. These triacylglycerides can be produced from crop or microalgae oils, animal fats, and waste cooking oils. Such biodiesel is miscible with petroleum diesel and thus suitable blends of biodiesel-diesel can be obtained. These are denoted as BXX, where XX is the percent of biodiesel in the blend. For example, B40 is 40% biodiesel in a diesel-biodiesel blend (Tat et

There are several properties which determine the suitability of biodiesel as a biofuel including cetane number, kinematic viscosity, cold flow and oxidative stability (Ramos et al, 2009). These properties are greatly influenced by the fatty acid compositions of the feedstock oils (Figure 3, 4). Therefore, to determine the best composition of biodiesel, it is necessary to study the lipid profile of potential biomass feedstocks. The most common fatty acid methyl esters present in most biodiesel are palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2) and linolenic acid (18:3) (Knothe, 2008). Biodiesel obtained upon trans-esterification of these common fatty acids has many advantages over petroleum-derived diesel fuel. However, there are several performance problems with biodiesel, notably poor cold flow properties, lower cetane number and insufficient oxidative stability (Knothe, 2009). Ignition delay time and combustion quality of a diesel fuel is determined by the cetane number. An adequate cetane number is required for better engine performance and a high cetane number is also associated with

**6.1 Lipid extraction and biodiesel formation from microalgal biomass** 

Ogden, 2011).

(Hejazi & Wijffels 2004).

**6.2 Fuel properties of microalgal biodiesel** 

al., 2007).

**6. Biomass conversion to biodiesel** 

filtration of light by the cells of upper layer, which accounts for a further loss in the overall biomass productivities (Figure 2). Genetic modifications resulting in truncated chlorophyll antennae size could restrict the high photon absorption by the light harvesting complex. In this context, Polle et al. (2003) have cloned and functionally characterized the Chl antenna size regulatory *Tla1* gene in *Chlamydomonas reinhardtii*. The partially truncated chlorophyll antenna size of the *tla1* mutant prevents the overabsorption of irradiance by cells, thus avoiding wasteful heat losses (Polle et al., 2003). In *Dunaliella salina,* a highly truncated light-harvesting Chl antenna size resulted in aggravated photosynthetic productivity and greater oxygen production under mass microalgal culture (Melis et al., 1999). The *Stm3LR3* mutants of *C. reinhardtii* generated by RNAi technology demonstrated down-regulation of the entire LHC antenna system. The *Stm3LR3* mutant showed reduced fluorescence, increased photosynthetic quantum yield, increased resistance to photoinhibition and faster growth rate under high light levels (Mussgnug et al., 2007).

Fig. 2. A diagrammatic representation of wild-type and genetically truncated light harvesting complexes of microalgae. The incident light falling on the antenna molecules in the LHC are wasted as heat and fluorescence, while the lower layer cells are deprived of light. The modified TM has fewer antenna molecules in the TLHC that allows the absorption of light by the cells in the deeper layers. TM (thylakoid membrane), RC (reaction center), LHC (light harvesting complex), TLHC (truncated light harvesting complex).
