In a variation of dispersed flotation, ozone bubbles are injected in what is called “dispersed ozone flotation” that has been shown effective in the harvest S. obliquus (Cheng et al. 2011). No separation was possible with simple air flotation with this culture. The ozone air flotation separation was attributed to ozone-generated release of algal biomolecules (polysaccharides and proteins) that aided flocculation (Cheng et al. 2011). Proteins released from the cells due to the ozonation probably helped the froth formation and cell separation. Ozone dosage required to successfully separate the cells was 0.2-0.5 mg O3/mg biomass—in a range similar to that used in wastewater treatment plants. This was tested at small scale, and it is unclear how the oxidative properties of ozone would impact downstream processing and the sta­bility of oils and co-products.

There are two main types of growth systems, open ponds and closed photo-bio­reactors. As the name suggests, open ponds are growth systems that are essentially open stretches of water that are naturally occurring or artificially made, naturally circulated (by temperature differences and wind) or mechanically agitated (usually paddle wheel driven). The most common types for large-scale microalgae pro­duction are artificially designed naturally circulated ponds (e. g. Hutt Lagoon— Western Australia) or artificially constructed open raceway ponds (e. g. Sapphire energy—Mexico). The open raceway or high-rate pond utilised by most microalgae companies/researchers is considered to be the lowest capital cost and lowest energy consumption approach for microalgae growth and is typically based on the design and investigation work conducted by researchers in the aquatic species research program (Benemann et al. 2012). In this approach, shallow (15-35 cm deep)-oval — shaped ponds are gently agitated by a paddle wheel to achieve maximum productivity.

In closed Photo-bioreactors (PBR), microalgae are cultivated in a controlled environment to maximise productivity. There are numerous types of photo-biore­actors with major categories including:

• Tubular photo-bioreactors

• Flat plate bioreactors

• Bag reactors

A subclass of PBR is attached reactor systems or biofilms in which the micro­algae are grown on a substrate that is wet by the growth medium rather than being grown in solution (Ozkan et al. 2012). In the first two of these designs, construction can be either of plastic or glass, while in the latter, construction is of flexible plastics. The major stated advantages of PBR are the ability to control temperature, CO2 content and pH and provide protection from predators. Furthermore, water loss is minimised through low evaporation rates; however, evaporation loss is coun­teracted by the need to control temperature with water. The major disadvantages for closed PBR are the preventatively high capital costs (Benemann et al. 2012; Borowitzka and Moheimani 2010).

Biofilm or attached systems are fundamentally different to both open ponds and closed PBR as the microalgae grow on a substrate rather than in suspension in the liquid growth medium (Ozkan et al. 2012). The advantages of these systems are the reduction in water volume, energy consumption and the ease of harvesting; how­ever, like PBR systems, they will have a higher capital cost than open ponds.

The fundamental goal in any growth system is the low-cost production of bio­fuels from microalgae, which is achieved by maximising productivity (species specific), reducing capital cost, minimising energy consumption and maximising nutrient (N, P, trace minerals and CO2) utilisation efficiency. In general, open ponds provide the lowest capital cost and energy consumption, while PBR (closed sys­tems) offer greater protection from predators (weeds and pests), greater control and higher productivity. Numerous PBR have been trialled; however, history has demonstrated and continues to demonstrate that open ponds are the only viable methodology for large-scale microalgae production, even for high-value nutra — ceuticals (Benemann et al. 2012). As a result, the trend in research has been to identify/select or genetically modify microalgae to provide consistent high yields in the challenging environment (e. g. increasing salinity due to evaporation, predators, changing temperatures) of open ponds rather than continually refine PBR’s.

Some researchers have applied immobilization technologies to the stock culture management as an alternative to the common cryopreservation processes, since entrapment processes are cheaper and easier (Chen 2001; Faafeng et al. 1994; Hertzberg and Jensen 1989). Immobilization can also provide protection of the cells toward being consumed by any zooplankton present in the same aquatic ecosystems.

Chen (2001) observed that the immobilized Scenedesmus quadricauda cells in alginate beads can preserve their physiological activities for a long time, even after three years of storage in darkness at 4 °C. This observation was explained by the entrapped cells self-consuming their own pyrenoid reserves. Transmission electron microscopic images of immobilized S. quadricauda cells showed that they lose their pyrenoids after extended storage, which is then rebuilt when the cultures are placed back into their nutrient media under light conditions.

Lebeau et al. (1998) also established the ability to store marine diatom H. ostrearia cells for nearly 2 months after their entrapment in calcium alginate beads and later used them as a substrate source for the greening of oysters. As a follow-up study, the same group achieved a longer term storage when H. ostrearia diatom cells were entrapped in alginate beads and kept at 4 °C (Gaudin et al. 2006). Chen (2003) stored Isochrysis galbana marine microalgal cells for more than a year after immobilizing them in alginate at 4 °C in dark conditions, and the cells were then used for feeding clam cultures.

6.5.1 Cyanobacteria

Divisions of prokaryotic algae, the Cyanophyta and Prochlorophyta, include the cyanobacteria (blue-green algae) and the Prochlorophyta, which use a unique form of chlorophyll, divinyl-chlorophyll, lack red and blue phycobilin pigments, and have stacked thylakoids. These organisms are oxygen-evolving photosynthetic bacteria and have cell wall structures similar to gram-negative bacteria which include a cell membrane, a layer of peptidoglycan, and an outer membrane. Of all the microorganisms described in this chapter, the cyanobacteria pose the least difficulty in terms of cell lysis for lipid extraction. Although cyanobacteria including Spirulina and Nostoc have been used for food for centuries, they tend to have high protein concentrations but low levels of lipids (Becker 1994). However, the cyanobacteria may provide some significant advantages in biofuel production including bacterial cell walls that are easily lysed for lipid extraction and the ability to grow in extreme conditions allowing species control in outdoor ponds, and many have a filamentous morphology that enables facile harvest.

The genetics of cyanobacteria are now well developed (Park et al. 2013), and many strains are easily transformed. Another argument for the use of cyanobacteria is the production of compounds important to the chemical and nutraceutical industries whose current market value is far greater than generic algal oil. For example, Arthrospira platensis produces significant quantities of phycocyanin and gama linolenic acid (C18:3, ю6, GLA) (Colla et al. 2004).

Various groups have engineered cyanobacteria to overexpress either native alkane biosynthetic genes or regulatory genes (Rosgaard et al. 2012; Wang et al.

2013) and have determined compatibility issues between the host and high levels of alkanes synthesized through engineered pathways.

The advent of computer-aided design in biology holds the promise of greatly increasing the efficiency of biological manipulation and experimentation while easing the process of design and optimization. The hope is that CAD will allow the design and analysis of plasmids, vectors, protocols, and synthetic pathways with a minimal need for laboratory experiments.

Classically, tools used in genetic engineering perform singular specific tasks such as codon optimization, primer design, and ribosome binding site (RBS) design to optimize DNA constructs. A more inclusive set of computational tools combines multiple DNA components’ design and optimization operations via a single toolset. One example of such a tool is ApE (http://biologylabs. utah. edu/jorgensen/wayned/ ape/). ApE is a plasmid editor that, among many things, clearly highlights the different relevant features of a plasmid (restriction sites, ORF, Dam/Dcm methyl — ation sites, etc.), shows the protein translation, creates plasmid graphics and maps, performs a virtual restriction digest, selects sites matching a given criteria, and performs primer design based on an inputted criteria. Other inclusive tools include Gene Designer 2.0 (https://www. dna20.com/resources/genedesigner), which per­forms gene, operon, vector, and primer design along with codon optimization. It also performs protein translation and restriction site modification using a graphical — based interface. A similar toolkit to Gene Designer 2.0 is Gene Design. Gene Design is, however, available via a Web interface (http://54.235.254.95/gd/). Finally, Gene Composer is yet another interesting tool worth mentioning (http:// www. genecomposer. net). Gene Composer is a CAD-based software that allows alignment generation and constructs design, gene optimization, and assembly (Medema et al. 2012; Zabawinski et al. 2001).

As for molecular and synthetic biology centric computational tools, there are already a few software suits and CAD packages that include tools to aid in biological design. One such tool is Genome Compiler (http://www. genomecompiler. com), an advanced genomic design software package. Besides facilitating, viewing, and editing DNA as constructs, protein translation, and circular views, Genome Compiler allows viewing and editing a DNA construct on a functional basis. Additionally, searching and importing DNA constructs via the NCBI database is supported.

A similar CAD toolkit to Gene Compiler is GenoCAD (http://www. genocad. org). GenoCAD, however, has the added benefit of DNA construct simulation. This allows for quick performance testing of designed constructs. Other CAD tools that are focused on DNA constructs and biological parts design include TeselaGen (https:// www. teselagen. com), Clotho (http://www. clothocad. org), and SynBioSS (http:// synbioss. sourceforge. net). Both TeselaGen and Clotho offer genetic function centric DNA editing and construction tools. They also aid in the creation of DNA constructs and parts’ databases and the utilization of existing databases. Likewise, SynBioSS allows for the design and simulation of DNA constructs and biological parts, but does that through reconstructing reaction networks from a series of genetic parts that are user defined and strung together. Another CAD toolkit that allows for DNA construct simulation, in addition to DNA design and editing, is TinkerCell (http://www. tinkercell. com). TinkerCell is built with the ambition of having optimized CAD-based biological designs feed into laboratory automation tools, and thus easing the design, experimentation, and synthesis aspects of molecular and synthetic biol­ogy. Currently, TinkerCell’s analysis capability includes a plethora of deterministic and stochastic simulation and analysis options (Medema et al. 2012).

The CAD tools discussed thus far offer powerful design capabilities, but the designer must keep in mind the limitations inherent with each tool. Biological interactions are not yet fully understood, and the models and simulations of such interactions, as provided by the CAD tools, are limited by the assumptions rooted in those tools.

9.4 Conclusion

Genetic molecular techniques have allowed for tremendous progress in the fields of biology and bioengineering. The ability to manipulate, replicate, and modify DNA, RNA, proteins, and organisms via transformation techniques, cloning, and gene­editing tools has allowed for powerful biological insights and applications devel­opment. As is already apparent, the future of molecular techniques lies in devel­oping more robust editing tools, simplifying high-throughput techniques and adopting more automatable techniques. Also, the development of multiplexing techniques, techniques that are able to perform multiple manipulations at once, will allow for great progress in discovery.

Acknowledgments Major support for this work was provided by New York University Abu Dhabi Institute grant G1205, NYU Abu Dhabi Faculty Research Funds AD060, and NYU Abu Dhabi Research Enhancement Fund AD060; K. J. was supported through NYU Abu Dhabi Global Academic Fellows program. The authors thank Khalid Sam for his help in creating the figures used in this chapter.

Microalgal cultures can flocculate spontaneously when pH rises as a result of photosynthetic depletion of carbon dioxide from the medium. This spontaneous flocculation has been referred to as autoflocculation but is in fact caused by pre­cipitation of calcium and magnesium salts at high pH (Schlesinger et al. 2012; Vandamme et al. 2012a; Gonzalez-Fernandez and Ballesteros 2013).

When pH in microalgal cultures increases to pH 8.5-9, the first mineral to precipitate is often calcium phosphate. Calcium phosphate precipitation can be induced by photosynthetic depletion of carbon dioxide alone and does not require addition of a base to increase pH (Sukenik and Shelef 1984). High concentrations of calcium and phosphate are required for flocculation to occur. Therefore, this floc­culation method can only be used in relatively hard waters with a high phosphate concentration. The amount of phosphate required for autoflocculation is higher than the phosphate required for microalgal biomass production. Because phosphate is a mineral resource with a limited supply, this flocculation method is unsustainable unless perhaps when microalgae are used for treating wastewaters with excessive phosphate concentrations. Beuckels et al. (2013), however, showed that it is pos­sible to recover the phosphate from the harvested biomass by dissolving the calcium phosphate through mild acidification.

When pH is further increased above 10.5, magnesium will precipitate from the medium as magnesium hydroxide. Like aluminum or ferric hydroxide, magnesium hydroxide is a metal hydroxide and may cause flocculation through a similar mechanism as metal salts (Smith and Davis 2012). Magnesium hydroxide precip­itates are positively charged up to pH 11 and can cause flocculation through charge neutralization (Wu et al. 2012; Garcia-Perez et al. 2014). Microscopical observa­tions of flocculated microalgae, however, suggest that sweeping flocculation may be equally important as charge neutralization (Besson and Guiraud 2013). Under some conditions, magnesium hydroxide precipitation may be induced by photosynthetic depletion of carbon dioxide (Spilling et al. 2011), but more often addition of a base is needed to increase pH to a sufficiently high level to initiate precipitation. A low concentration of magnesium is sufficient to induce flocculation (about 10 mg Mg L-1; Vandamme et al. 2012a, b). Seawater contains very high magne­sium concentrations (1300 mg Mg L-1). In seawater, overdosing of base may result in massive precipitation of magnesium hydroxide and a very large sludge volume, which poses problems for further dewatering (e. g., §irin et al. 2011). Therefore, it is recommended to control addition of the base rather than target a specific pH level to maximize flocculation efficiency and minimize the sludge volume (Besson and Guiraud 2013; Garcia-Perez et al. 2014). The amount of base that needs to be added will be a function of the quantity of magnesium hydroxide required to induce flocculation, but also of the buffering capacity of the culture medium (Garzon — Sanabria et al. 2012). Repeated recycling of the culture medium after flocculation may result in depletion of magnesium from the medium. However, Vandamme et al. 2014a demonstrated that 95 % of the precipitated magnesium hydroxide could be recovered by mild acidification, allowing it to be recycled back into the culture medium. The repeated addition of base to induce flocculation and of acid to neu­tralize the medium can result in accumulation of salts (Castrillo et al. 2013). To avoid accumulation of salt, ammonia may be used as a base to induce flocculation (Chen et al. 2012). Solubility calculations that take account of aqueous speciation and the high initial solubility of freshly formed precipitates are useful means for predicting and refining autoflocculation end-points (Brady et al. 2014). Chapter 13 describes primary dewatering of microalgae using autoflocculation at an industrial scale.

At high pH, calcium may also precipitate as calcium carbonate. So far, few studies have demonstrated that calcium carbonate precipitates are effective auto­flocculation agents (Ayoub et al. 1986). Calcium carbonate precipitation is a slower process and may take more time than flocculation by magnesium hydroxide or calcium phosphate. Moreover, precipitation of calcium carbonate may be limited by carbonate concentrations in the medium. Calcite surfaces will be positively charged and able to autoflocculate microalgae only under restricted conditions, namely when Ca+2 and Mg+2 levels are relatively high and/or sulfate levels are low (Brady et al. 2014).

The surface of the Earth receives significant amounts of electromagnetic radiation from the Sun in the form of light. This irradiance has been well characterized (Neckel and Labs 1984) and varies in intensity with different wavelengths. For example, the peak irradiance from the Sun is in the visible part of the spectrum and this tails off into the infrared. The intensity and spectrum of the light that are measured on the Earth’s surface (the terrestrial spectrum) are significantly different from the spectrum of light that would be measured outside of the atmosphere (the extraterrestrial spectrum). This is due to absorbance within the Earth’s atmosphere. With its importance to many industries and sciences, the solar spectrum has been well characterized and two standard terrestrial solar spectral irradiance distributions (ASTM 2008) have been defined. These standard distributions are used in the photovoltaics industry for testing of PV modules under standard test conditions. This allows the comparison of the efficiency and performance of different solar modules.

Two different spectral distributions are described in these standards (Gueymard et al. 2002). The first of these spectral distributions is the direct normal spectrum. This is ‘“the direct component contributed to the total hemispherical (or ‘global’) radiation on a 37°-tilted surface" (Gueymard et al. 2002). The second applies specifically for photovoltaic modules and is a good approximation for modules that are tilted toward the equator at 37° (Gueymard et al. 2002). Average values for the atmospheric composition, aerosols, water vapor, and ozone content are taken into account in the standards defined in ASTM G-173-03 (ASTM 2008). Due to the absorbance of light in the atmosphere, the irradiance is dependent on the optical path length through the atmosphere. This is known as the air mass. The spectra in ASTM G-173-03 use an air mass of 1.5. For the midlatitudes, this is a reasonable average. This is a good approximation for modules in the United States of America and southern regions of Australia. As this spectrum is well defined, it is suitable for use in modeling the power absorbed by microalgae as well as the power produced by photovoltaic modules.

The extraterrestrial irradiance and the Global Tilted AM1.5 spectra described in the ASTM G-173-03 standard are shown in Fig. 15.1. This plot shows the irradiance in terms of W m-2 nm-1 which is an expression of the power in each part of the spectrum incident on a particular area. This can also be described in terms of p mole photons s-1 m-2 which is often used when discussing photosynthesis. Converting from one to the other is wavelength specific and can be done using:

Iwk

hcNA • 10-6

where Ip is the irradiance in p mole photons s-1 m-2, Iw is the irradiance in W m-2. nm-1, X is the wavelength, h is Planck’s constant, and NA is Avogadro’s number.

The daily global solar radiation exposure is defined as the total amount of solar energy falling on a horizontal surface per day. The daily solar radiation exposure typically ranges from 1 to 35 MJ m-2 and will depend on the time of year, clarity of the air, and the level of cloud cover. For example, the daily solar radiation exposure would usually be highest in clear, sunny, conditions during the summer and lowest during winter or very cloudy days. Some regions of the world have very high radiation exposures due to their location and number of cloud free days. The northern and central regions of Australia experience high levels of incident illumination.

This makes them particularly good for photovoltaic electricity production and biomass production (Clifton and Boruff 2010; Borowitzka et al. 2012). Successful conversion of solar energy into chemical energy in the way of biofuel also relies on the availability of abundant water supplies (i. e. seawater or large aquifer resource). Prime locations for algae farms and biofuel production exist where these are available and there is abundant solar radiation (Borowitzka et al. 2012).

There are two main types of microalgae cultivation systems: open ponds and closed photobioreactors (Moheimani 2012; Moheimani et al. 2011).

1.2.1.1 Closed Photobioreactors

Closed algal cultures (photobioreactors) are not exposed to the atmosphere and are covered with a transparent material or contained within transparent tubing. Pho­tobioreactors have the distinct advantage of preventing evaporation (Dodd 1986; Moheimani et al. 2011). Culturing microalgae in these kinds of systems have the added benefit of reducing the contamination risks, limiting the CO2 losses, creating reproducible cultivation conditions, and flexibility in technical design (Jeffery and Wright 1999). Closed and semi-closed photobioreactors are mainly used for pro­ducing high-value algal products (Becker 1994). In closed photobioreactors, the main challenge is being less economical than open ponds (Borowitzka 1996; Moheimani and McHenry 2013; Moheimani et al. 2013c; Pulz and Scheibenbogen 1998). A number of researchers have endeavoured to overcome a number of the limitations in closed including:

• reducing the light path (Borowitzka 1996; Janssen et al. 2002; Miron et al. 1999)

• solving shear (turbulence) complexity (Barbosa et al. 2003; Borowitzka 1996; Miron et al. 2003)

• reducing oxygen concentration (Acien Fernandez et al. 2001; Kim and Lee 2001; Rubio et al. 1999; Weissman et al. 1988), and

• temperature control system (Becker 1994; Borowitzka 1996; Carlozzi and Sacchi 2001; Morita et al. 2001; Rubio et al. 1999; Zhang et al. 1999).

Currently, the main disadvantages of closed systems are the high cost of con­struction, operation both for energy (pumping and cooling) and maintenance [such as cleaning and sterilization (Borowitzka 1996)], and scaling up difficulties (Grima et al. 2000; Janssen et al. 2002; Miron et al. 1999). However, if these difficulties can be overcome, these controlled closed systems may allow commercial mass pro­duction of an increased number of microalgal species at a wider number of locations.

Heterotrophic cultures use organic carbons as both sources of energy and carbon. There are many advantages of heterotrophic cultures over photoautotrophic cul­tures. These include the following: (i) the use conventional heterotrophic bioreac­tors that are simpler and easier to scale up, since the elimination of light requirements means that smaller reactor surface-to-volume ratios can be used; (ii) greater control of the cultivation process, since the cultures can be done indoors; and (iii) higher cell densities, which reduces the cost of harvesting the cells. The basic components of media for heterotrophic cultures are similar to those of the photoautotrophic media, with the addition of organic carbon sources. In addition, the growth rate and oil accumulation of heterotrophic cultures are affected by the C: N ratio in the medium.

Generally, the biomass concentrations obtained in heterotrophic cultures are much higher than those in photoautotrophic cultures (Ogbonna et al. 1998). Although the biomass concentration in most photoautotrophic cultures is less than 5 g/L, much higher concentrations of 15.5 g/L for Chlorella protothecoides (Xu et al. 2006), 28.8 g/L for Traselmis suecica (Azma et al. 2011), and even 53 g/L for

Chlorella zofingiensis (Sun et al. 2008) have been reported. Fed-batch cultures can be used to obtain even higher biomass concentrations. Furthermore, heterotrophi — cally grown microalgae usually accumulate more lipids than those cultivated photoautotrophically, as demonstrated for Chlorella species (Miao and Wu 2006; Xu et al. 2006; Agwa et al. 2013; Liu et al. 2008; Hsieh and Wu 2009). In the case of Chlorella vulgaris, for example, Wu et al. (2012) reported an increase from 15 % under photoautotrophic condition to more than 50 % under heterotrophic condition. Compared with photoautotrophic cultures, Jimenez et al. (2009) reported an 8 times increase in oil content of C. protothecoides under heterotrophic condition, and a 9 times increase in lipid yield was achieved in heterotrophic cultures fed with 30 g/ L of glucose (Liu et al. 2011).

The high biomass concentration and high lipid contents obtained in heterotro­phic cultures result in very high lipid productivities. A lipid productivity of 179 mg/ L/d in photoautotrophic culture is regarded as high (Chiu et al. 2008), but much higher productivities of 528.5 mg/L/d (Morales-Sanchez et al. 2013), 932 mg/L/d (Xu et al. 2006), 1.38 g/L/d (Liu et al. 2011), 2.43 g/L/d (Chen and Walker 2011),

3.0 g/L/d (Chen and Walker 2011), and 3.7 g/L/d (Xiong et al. 2008) have been reported for heterotrophic cultures. It is usually technically difficult to construct large-scale photoautotrophic photobioreactors; however, for heterotrophic cultures, conventional bioreactors can be used for large-scale processes. For example, a heterotrophic culture was scaled up from 5 to 750 L, and then 11,000 L, and the oil contents remained fairly stable at 46.1, 48.7, and 44.3 % of cell dry weight, respectively (Li et al. 2007).

It has also been reported that the quality of oil produced under heterotrophic cultures is more suitable for biodiesel production than those produced under pho­toautotrophic cultures with the same strains of microorganisms. Liu et al. (2011) reported that heterotrophic cells accumulated predominantly neutral lipids that accounted for 79.5 % of the total lipids, with 88.7 % triacylglycerol, while oleic acid accounted for 35.2 % of the total fatty acid. In contrast, photoautotrophic cells contained mainly the membrane lipids, glycolipids, and phospholipids. Further­more, C. saccharophila, C. vulgaris, N. laevis, Cylindrotheca fusiformis, Navicula incerta, and Tetraselmis suecica accumulate more lipids under heterotrophic than under photoautotrophic conditions, mainly in the form of triglycerides (Day et al. 1991; Tan and Johns 1991, 1996; Gladue and Maxey 1994). Conversely, photo­autotrophic cultures form more highly unsaturated fatty acids (polar lipids) (Tan and Johns 1991, 1996; Wen and Chen 2000a, b). Miao and Wu (2004) further noted that the oil obtained from heterotrophically grown cells possesses properties similar to those of fossil diesel in terms of oxygen content, heating value, density, and viscosity.

However, heterotrophic cultures have some limitations. Only a limited number of microalgae are capable of growing under heterotrophic conditions, and addition of an organic carbon source can significantly increase the cost of production. Furthermore, the presence of an organic carbon source further increases the risk of contamination, and depending on the species, the cell growth rate and lipid pro­ductivity in heterotrophic culture may be lower than the values obtained in mixotrophic culture. For example, Day and Tsavalos (1996) found that heterotro­phic culture of Tetraselmis with glucose yielded only about one-sixth of cellular lipid compared with the value obtained in mixotrophic culture.

Various parameters such as initial cell concentration, input CO2 concentration, aeration rate, photobioreactor design, light intensity and temperature should be taken into account for biofixation of CO2 from flue gases by microalgae. These factors are important when seeking to achieve high-productivity microalgae bio­remediation of CO2 from input gases. In this section, the effect of initial cell concentrations, input CO2 concentration, aeration rate and data analysis from these factors on microalgal CO2 bioremediation is discussed.