**2. Microalgae biomass generation**

#### **2.1 Open cultivation**

Large scale microalgal biomass production can be achieved either through open pond cultivation under natural sunlight or under the controlled conditions of a photobioreactor. In the USA, the history of mass production of microalgae dates back at least to 1953 with the production of *Scenedesmus* species in Washington. Many systems for cultivating microalgae on a large scale have been suggested in many countries including the USA, Germany, Japan, Israel, the UK, the Czech Republic and others. Typically, microalgae are first grown in inorganic nutrients and then, in a second phase, are cultivated is done using waste water streams.

Commercial cultivation of microalgae can be done in a range of different ways including (a) open cultivation using natural sunlight, (b) closed cultivation using natural light and (c) closed cultivation using artificial light (in photobioreactors). Each of these systems has advantages and disadvantages, and the choice of system depends on the degree of parameter control needed to produce the desired product and on the value of the endproduct (Apt and Behrens, 1999). The most commonly used artificial open pond systems consist of large shallow ponds, tanks, circular ponds and raceway ponds (Ugwu et al., 2008). The construction and operation costs of such open cultivation systems are considerably less but are challenging to operate on a year round basis due to seasonal climatic variations. While open pond culture is cheaper than culture in closed photobioreactors (Borowitzka 1999), it is currently limited to a relatively small number of microalgal species. Rectangular ponds with a paddle wheel (raceway ponds) are the most widely used for the production of *Spirulina* sp*., Dunaliella salina* and *Haematococcus* sp*.* and currently represent the most efficient design for the large scale culture of most species of microalgae. Individual ponds are tyically up to 1 ha in area, with an average depth of about 20- 30 cm (Andersen 2005). The need to provide adequate light to the algal cells and maintaining an adequate water depth for mixing of the microalgae are important considerations for determining the pond depth. The diurnal natural light cycle results in the exposure of microalgae to limiting, saturating and over-saturating light conditions. High irradiances throughout the year and moderate temperatures are optimal for outdoor microalgae cultivation. For example, the geographical location of southern Spain with an average of 10-12 hours of sunlight per day, and a mean solar irradiance ranging from 400 µmol photons m-2s-1 during winter to 1800 µmol photons m-2s-1 is considered highly suitable for outdoor cultivation of microalgae. The maximal areal productivity of microalgae in outdoor conditions ranges from 20 to 30 gm-2d-1 (Cuaresma et al., 2011). To date, light-to-biomass conversion efficiencies of 1-4 % have been achieved for microalgae grown in conventional open pond cultivation systems. Because the scaling-up of microalgal biomass production in open raceway ponds is relatively easy, such systems are primarily considered for commercial applications. However, differences in weather variables such as solar irradiance, rainfall, and temperature significantly affect prospects for open cultivation of microalgae at different geographical locations. Temperature influences the rate of various reactions of photosynthesis (Raven, 1988). Therefore, microalgae exhibit an optimal growth within a narrow temperature range and die above a certain threshold temperature (Béchet et al., 2011). In addition, temperature is an important factor that affect the rate of evaporation from shallow algal ponds. In addition to changing the physical environment of open ponds, rainfall can lead to microbial contamination that inhibits microalgal growth (Hase et al., 2000).

The paddle wheels installed in open ponds are used to circulate the water, while compressed air can be introduced into the bottom of a pond to agitate the water, bringing microalgae from the lower levels upwards. Raceway channels are typically built in concrete or compacted earth, and are often lined with white plastic. During daylight, the microalgal culture is fed continuously in front of the paddlewheel where the flow begins. The biomass is harvested behind the paddlewheel, on completion of the circulation loop. The paddlewheel operates continuously to prevent sedimentation and flocculation (Chisti, 2007). The largest racewaybased biomass production facility currently occupies an area of 440,000 m2 (Spolaore et al., 2006). This facility is owned by Earthrise Nutritional (www.earthrise.com) and is used to produce cyanobacterial biomass for food. In India, Pary Nutraceuticals (part of the Chennaibased Murugappa group) has been focusing on microalgal research and development and are commercially producing *Spirulina* for nutraceuticals.

#### **2.2 Closed cultivation systems for microalgae**

162 Biodiesel – Feedstocks and Processing Technologies

biofuel involves biological processing of lignocellulosic biomass as the basis for development of second generation biofuel systems. The development of commercial-scale efficient conversion technologies for exploitation of biological wastes as an obvious source for biofuel generation is a major focus of research and development efforts associated with this generation of biofuel. One of the third generation biofuels under development aims to exploit the photosynthetic capability of microalgae for the conversion of solar energy into energy-dense biomass. A major advantage of microalgae over the use of crop biomass for biodiesel production is the lower land area requirements for production of an equivalent amount of fuel. As understanding of microalgal genomes and biochemistry increases, opportunities are emerging for development of fourth generation biofuels where metabolic engineering of microbes leads to more effective domestication of microalgae for biofuel production. However, at present the commercial production of biofuels from microalgae is limited by a lack of effective systems for biomass production, harvesting, extraction, and recovery of oils that can economically integrate all operational units from growth through to biofuel product recovery. In this chapter, we discuss the limitations of individual operational units in the context of efforts underway to establish fourth generation

microalgal biofuels that are economically and environmentally sustainable.

Large scale microalgal biomass production can be achieved either through open pond cultivation under natural sunlight or under the controlled conditions of a photobioreactor. In the USA, the history of mass production of microalgae dates back at least to 1953 with the production of *Scenedesmus* species in Washington. Many systems for cultivating microalgae on a large scale have been suggested in many countries including the USA, Germany, Japan, Israel, the UK, the Czech Republic and others. Typically, microalgae are first grown in inorganic nutrients and then, in a second phase, are cultivated is done using waste water

Commercial cultivation of microalgae can be done in a range of different ways including (a) open cultivation using natural sunlight, (b) closed cultivation using natural light and (c) closed cultivation using artificial light (in photobioreactors). Each of these systems has advantages and disadvantages, and the choice of system depends on the degree of parameter control needed to produce the desired product and on the value of the endproduct (Apt and Behrens, 1999). The most commonly used artificial open pond systems consist of large shallow ponds, tanks, circular ponds and raceway ponds (Ugwu et al., 2008). The construction and operation costs of such open cultivation systems are considerably less but are challenging to operate on a year round basis due to seasonal climatic variations. While open pond culture is cheaper than culture in closed photobioreactors (Borowitzka 1999), it is currently limited to a relatively small number of microalgal species. Rectangular ponds with a paddle wheel (raceway ponds) are the most widely used for the production of *Spirulina* sp*., Dunaliella salina* and *Haematococcus* sp*.* and currently represent the most efficient design for the large scale culture of most species of microalgae. Individual ponds are tyically up to 1 ha in area, with an average depth of about 20- 30 cm (Andersen 2005). The need to provide adequate light to the algal cells and maintaining an adequate water depth for mixing of the microalgae are important considerations for determining the pond depth. The diurnal natural light cycle results in the exposure of microalgae to limiting,

**2. Microalgae biomass generation** 

**2.1 Open cultivation** 

streams.

Several different closed systems using natural sunlight have been described for microalgae (Richmond et al. 1993, Molina Grima et al., 1995, Spektorova et al., 1997). In such systems, microalgae are grown in transparent glass or plastic vessels, and the vessels are placed under natural illumination. A higher surface to volume ratio is provided, so microalgal cell densities are often higher than in open ponds. However, these systems are also subject to variations in light intensity and temperature that make cultivation reproducibility problematic. In addition, removal of oxygen from the culture and the provision of adequate temperature control (especially if energy is required for cooling) pose a major problem with such closed systems. Large scale indoor cultivation using highly-controlled photobioreactors or fermentors have also been used successfully for microalgal biomass production. The wide range of different types of closed photobioreactors (PBRs) include vertical-column, flat-plate and tubular PBRs (Ugwu et al., 2008). These provide the ability to control and optimize culture parameters, and as a result such photobioreactors are suitable for culturing many different species of microalgae. The basic features which must be considered when designing a photobioreactor are: source of light, churning rate of algae (to avoid biomass

Eco-Physiological Barriers and Technological Advances for Biodiesel Production from Microalgae 165

illustrate how to obtain the maximum theoretical value of photosynthetic efficiency from the available PFPAR of 18,043 moles photons/m2 year. The theoretical maximum is calculated by considering both photon transmission (ηPTE) and photon conversion efficiencies (ηPUE) as

Photons utilized = PFPAR x ηPTE x ηPUE = 18,043 moles photons/m2 year x 1 x 1 In a microalgal cell, these photons power the photosynthetic production of carbohydrates (CH2O) which have an average energy content of 0.4825MJ/mole (Weyer et al., 2009). On average 10 photons are required to derive one mole of CH2O. Hence, the total energy consumed during the photosynthetic conversion reaction is obtained as follows (Cooney et

18,043 moles photons /m year x 1 x 1 . 0.4825MJ /mole E 871 MJ /m year

The estimated total photosynthetic efficiency during the conversion of PAR to microalgal biomass is calculated by dividing ECARB (871 MJ/m2year) and SEarthPAR (4065 MJ/m2year), assuming that bioconversion of carbohydrates is 100% efficient. This gives a value of 21.4%, which is stated as the overall maximum theoretical photosynthetic efficiency relative to PAR. High lipid productivity depends on both the microalgal biomass areal productivity and the lipid content that can be generated from the microalgal strain. The lipid productivity is the most important factor influencing the cost of biodiesel production. High lipid content microalgal species and strains also favor the efficiency of biomass processing

A light source with narrow spectral output that overlaps the photosynthetic absorption spectrum improves the energy conversion as the emission of light at unusable frequencies is eliminated. Light-emitting diodes (LEDs) are the only light source that currently meet this criterion. LEDs have the ability to produce high light levels with low radiant heat output and maintain useful light output for years. Thus, LEDs can have a very significantly longer life of 100,000 h as compared to 8000 h of fluorescent lights. These advantages make LEDs ideal for microalgal growth in controlled environments of growth chambers. The optimal wavelength conditions will vary from species to species of microalgae (Chen et al 2011). For example, the highest specific growth rate and biomass production from the photosynthetic cultivation of *Spirulina platensis* was obtained using red LED. The superimposed pattern of luminescence spectrum of blue LED (450-470 nm) and that of red LED (650-665 nm) corresponds to the light absorption spectrum of carotenoids and chlorophyll (Yeh & Chung, 2009). Therefore, the red LED favors microalgal growth but switching to illumination with blue LED improved the rate of astaxanthin production by *Haematococcus pluvialis* (Katsuda et al. 2004)*.* Flashing light from blue LEDs is also a promising illumination method for *H. pluvialis* growth and astaxanthin production (Katsuda et al 2006). The use of flashing LED as sources of intermittent light in indoor algal culture can yield a major gain in energy economy comparing to fluorescent light sources (Matthijs et al., 1996). The research results by Nedbal et al. (1996) also suggest that algal growth rates in intermittent light can be higher than those in equivalent continuous light.

2

<sup>2</sup>

<sup>10</sup>

100%. Thus, according to the following equation:

**3.3 Technological innovations in illumination sources** 

al 2011).

CARB

during oil extraction.

**3.3.1 Light Emitting Diodes (LEDs)** 

sedimentation and for uniform availability of nutrients and light), material for construction, CO2 supply, and removal of O2, pH and temperature control (Kaur et al., 2010). Whether, closed or open systems will be optimal for commercial cultivation of different species (or strains) of microalgae is difficult to determine. However, it is clear that photobioreactors will play a critical role to feed open ponds with a high-cell-density unialgal inoculum (Cheng et al., 2011).

#### **3. Thermodynamic efficiency of photosynthesis in microalgae**

Photosynthesis is a chemical reaction governed by the laws of thermodynamics. Assuming a microalgal cell as 'boundary' and the process of photosynthesis as 'system', then according to the law of thermodynamics, the two kinds of work associated with this chemical reaction are electrical work and work of expansion. In a biological system such as microalgae, the production of ATP derived by the transfer of charges across the biofluidic membranes can be called electrical work. The growth or increase in size of cell and cellular components (including oil bodies) is the 'work of expansion'. In the very familiar photosynthetic reaction (Albarrán-Zavala & Angulo-Brown, 2007);

> 2 2 6126 2 2 6CO 12 H O C H O 6H O 6O ∆G0 = 2880.31 kJ/molC6H12O6 at λ = 680 nm

#### **3.1 Photosynthetic conversion efficiency**

In outdoor cultivation systems, the microalgal biomass productivity derived through photosynthesis depends on the solar energy input. The estimated yearly average solar energy density, including both direct beam radiation and diffuse scattered radiation is 10,038 MJ/m2year. To account for non-sunny weather conditions, a more realistic theoretical maximum solar energy density is obtained after reducing this value by 10%, which corresponds to a value of 9034 MJ/m2year. However, the actual value will exhibit temporal and spatial variation depending on the geographical location and will generally be lower (Cooney et al., 2011). The fraction of the solar energy spectrum (SEarth ~ 9034 MJ/m2year) is further reduced by 45% to calculate the value of photosynthetically active radiation (PAR) that supports photosynthesis (SEarthPAR ~ 4065 MJ/m2year). PAR is expressed in terms of photon flux as it reaches surface of microalgal cells in the form of photons, the energy of which varies inversely with the wavelength. The upper theoretical limit for the average PAR spectrum photon flux energy (EMaxAvePAR) is 0.2253 MJ/mol that corresponds to λ531 nm (green) (Weyer et al., 2009). Hence, the available photon flux reaching the earth surface and which is available for photosynthesis is calculated by the following formula (Cooney et al., 2011):

2 EarthPAR 2 PAR MaxAvePAR S ~ 4065 MJ / m / year PF 18, 043 moles photons / m year E ~ 0.2253 MJ / mol photon

#### **3.2 Maximum theoretical photosynthetic efficiency**

The most cited values for maximum photosynthetic efficiencies in microalgae are in the range of 17-23% (Gordon & Polle 2007, Zemke et al., 2010). Cooney and coworkers (2011) illustrate how to obtain the maximum theoretical value of photosynthetic efficiency from the available PFPAR of 18,043 moles photons/m2 year. The theoretical maximum is calculated by considering both photon transmission (ηPTE) and photon conversion efficiencies (ηPUE) as 100%. Thus, according to the following equation:

#### Photons utilized = PFPAR x ηPTE x ηPUE = 18,043 moles photons/m2 year x 1 x 1

In a microalgal cell, these photons power the photosynthetic production of carbohydrates (CH2O) which have an average energy content of 0.4825MJ/mole (Weyer et al., 2009). On average 10 photons are required to derive one mole of CH2O. Hence, the total energy consumed during the photosynthetic conversion reaction is obtained as follows (Cooney et al 2011).

$$\mathbf{E}\_{\rm CAR} = \frac{\left(18,043 \text{ moles photons} / \text{m}^2 \text{year} \times 1 \times 1\right). \left(0.4825 \text{M}\right) / \text{mole}}{10} = 871 \text{ M} // \text{m}^2 \text{year}$$

The estimated total photosynthetic efficiency during the conversion of PAR to microalgal biomass is calculated by dividing ECARB (871 MJ/m2year) and SEarthPAR (4065 MJ/m2year), assuming that bioconversion of carbohydrates is 100% efficient. This gives a value of 21.4%, which is stated as the overall maximum theoretical photosynthetic efficiency relative to PAR. High lipid productivity depends on both the microalgal biomass areal productivity and the lipid content that can be generated from the microalgal strain. The lipid productivity is the most important factor influencing the cost of biodiesel production. High lipid content microalgal species and strains also favor the efficiency of biomass processing during oil extraction.

#### **3.3 Technological innovations in illumination sources 3.3.1 Light Emitting Diodes (LEDs)**

164 Biodiesel – Feedstocks and Processing Technologies

sedimentation and for uniform availability of nutrients and light), material for construction, CO2 supply, and removal of O2, pH and temperature control (Kaur et al., 2010). Whether, closed or open systems will be optimal for commercial cultivation of different species (or strains) of microalgae is difficult to determine. However, it is clear that photobioreactors will play a critical role to feed open ponds with a high-cell-density unialgal inoculum (Cheng et

Photosynthesis is a chemical reaction governed by the laws of thermodynamics. Assuming a microalgal cell as 'boundary' and the process of photosynthesis as 'system', then according to the law of thermodynamics, the two kinds of work associated with this chemical reaction are electrical work and work of expansion. In a biological system such as microalgae, the production of ATP derived by the transfer of charges across the biofluidic membranes can be called electrical work. The growth or increase in size of cell and cellular components (including oil bodies) is the 'work of expansion'. In the very familiar photosynthetic reaction

2 2 6126 2 2 6CO 12 H O C H O 6H O 6O

∆G0 = 2880.31 kJ/molC6H12O6 at λ = 680 nm

In outdoor cultivation systems, the microalgal biomass productivity derived through photosynthesis depends on the solar energy input. The estimated yearly average solar energy density, including both direct beam radiation and diffuse scattered radiation is 10,038 MJ/m2year. To account for non-sunny weather conditions, a more realistic theoretical maximum solar energy density is obtained after reducing this value by 10%, which corresponds to a value of 9034 MJ/m2year. However, the actual value will exhibit temporal and spatial variation depending on the geographical location and will generally be lower (Cooney et al., 2011). The fraction of the solar energy spectrum (SEarth ~ 9034 MJ/m2year) is further reduced by 45% to calculate the value of photosynthetically active radiation (PAR) that supports photosynthesis (SEarthPAR ~ 4065 MJ/m2year). PAR is expressed in terms of photon flux as it reaches surface of microalgal cells in the form of photons, the energy of which varies inversely with the wavelength. The upper theoretical limit for the average PAR spectrum photon flux energy (EMaxAvePAR) is 0.2253 MJ/mol that corresponds to λ531 nm (green) (Weyer et al., 2009). Hence, the available photon flux reaching the earth surface and which is available for photosynthesis is calculated by the following formula (Cooney et al.,

2

E ~ 0.2253 MJ / mol photon

**3.2 Maximum theoretical photosynthetic efficiency** 

EarthPAR 2

S ~ 4065 MJ / m / year PF 18, 043 moles photons / m year

The most cited values for maximum photosynthetic efficiencies in microalgae are in the range of 17-23% (Gordon & Polle 2007, Zemke et al., 2010). Cooney and coworkers (2011)

**3. Thermodynamic efficiency of photosynthesis in microalgae** 

(Albarrán-Zavala & Angulo-Brown, 2007);

**3.1 Photosynthetic conversion efficiency** 

al., 2011).

2011):

PAR

MaxAvePAR

A light source with narrow spectral output that overlaps the photosynthetic absorption spectrum improves the energy conversion as the emission of light at unusable frequencies is eliminated. Light-emitting diodes (LEDs) are the only light source that currently meet this criterion. LEDs have the ability to produce high light levels with low radiant heat output and maintain useful light output for years. Thus, LEDs can have a very significantly longer life of 100,000 h as compared to 8000 h of fluorescent lights. These advantages make LEDs ideal for microalgal growth in controlled environments of growth chambers. The optimal wavelength conditions will vary from species to species of microalgae (Chen et al 2011). For example, the highest specific growth rate and biomass production from the photosynthetic cultivation of *Spirulina platensis* was obtained using red LED. The superimposed pattern of luminescence spectrum of blue LED (450-470 nm) and that of red LED (650-665 nm) corresponds to the light absorption spectrum of carotenoids and chlorophyll (Yeh & Chung, 2009). Therefore, the red LED favors microalgal growth but switching to illumination with blue LED improved the rate of astaxanthin production by *Haematococcus pluvialis* (Katsuda et al. 2004)*.* Flashing light from blue LEDs is also a promising illumination method for *H. pluvialis* growth and astaxanthin production (Katsuda et al 2006). The use of flashing LED as sources of intermittent light in indoor algal culture can yield a major gain in energy economy comparing to fluorescent light sources (Matthijs et al., 1996). The research results by Nedbal et al. (1996) also suggest that algal growth rates in intermittent light can be higher than those in equivalent continuous light.

Eco-Physiological Barriers and Technological Advances for Biodiesel Production from Microalgae 167

Fig. 1. Effect of biomass concentration on the penetration of incident light into cultures of

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

*Nannochloropsis* sp. (from Richmond 2004).

**4.2 Improvements of intrinsic photosynthetic efficiency** 

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 exposure of fluorescent light, cells regained their normal size.
