Open ponds are the most usual setting for large-scale outdoor microalgae cultiva­tion (Fon Sing et al. 2013; Jeffery and Wright 1999). The major commercial pro­duction of algae is today based on open channels (raceway) which are less expensive, and easier to build and operate compared with closed photobioreactors (Borowitzka 2013b; Tredici and Materassi 1992). In addition, the growth of mic­roalgae meets is less challenging in open than closed cultivation systems; however, just a few species of microalgae (e. g. Chlorella, D. salina, Spirulina. sp., Chlorella sp. and P. carterae) have been successfully grown in open ponds (Moheimani and Borowitzka 2006; Tredici and Materassi 1992). Large-scale outdoor commercial microalgal culture has been methodically developed over the last sixty years (Borowitzka and Moheimani 2013a). Profitable production of microalgae, at present, are limited to a comparatively few small-scale (<10 ha) plants producing high-value health foods, most located in south-east Asia, Australia and the USA (Benemann 1992; Borowitzka and Borowitzka 1990; Richmond 1992). Two major types of large-scale open cultivation systems have been developed and have been used on a commercial basis. These are (a) unstirred ponds and (b) stirred ponds (circular and raceway) (Borowitzka 1993a, b; Borowitzka and Moheimani 2013b). The most common commercial microalgal culture system in use today is the paddlewheel-driven raceway pond (Richmond et al. 1993). The advantages and disadvantages of growing microalgae in open ponds and closed photobioreactors are summarised in Table 1.2. Relatively low cost of construction and operation are the main reasons for culturing algae in open ponds (Tredici and Materassi 1992). However, the high contamination risks and low productivity, induced mainly by poor mixing regime and light penetration, are the main disadvantages of open systems.

Mixotrophic cultures are culture systems where light and organic carbons are used as the energy source, while inorganic and organic carbons are used as the carbon source. Although it is sometimes used interchangeably with photoheterotrophic culture, in a strict sense, photoheterotrophic culture involves the use of light as the energy source, while organic carbon is used as the carbon source. In other words, light is required to metabolize the organic carbon source in photoheterotrophic culture. Photoheterotrophic cultivation requires both organic carbons and light at the same time, whereas in mixotrophic culture, both are present, but either can be used without the other. From practical point of view, both mixotrophic and pho­toheterotrophic cultures can be regarded as culture systems where light, organic carbon, and inorganic carbon are present at the same time.

As already discussed, heterotrophic culture has many advantages over photo­autotrophic cultures. However, there are many metabolites whose syntheses are promoted by light, and thus are not efficiently produced in heterotrophic cultures (Chen and Zhang 1997; Lee and Zhang 1999; Cohen 1999; Sukenik et al. 1991). This disadvantage can be overcome by mixotrophic culture which involves simultaneous use of light and organic carbon sources. Mixotrophic cultures have many advantages over other culture systems. For example, inhibition of photo­synthesis by high dissolved oxygen concentration is a major problem in photoau­totrophic cultures, while oxygen limitation is a major problem in heterotrophic cultures. In mixotrophic culture, dissolved oxygen concentration does not increase to inhibitory levels since it is simultaneously used for heterotrophic metabolism of the organic carbon. On the other hand, organic carbon assimilation is hardly limited by dissolved oxygen concentration since oxygen is constantly produced by pho­tosynthetic activities. Furthermore, heterotrophic growth generates carbon dioxide which is used for photoautotrophic growth (photosynthesis).

In mixotrophic cultures, the presence of an organic substrate means that cell growth is not strictly dependent on photosynthesis, and hence, light is not an indispensable growth factor. Read et al. (1989) and Fernandez Sevilla et al. (2004) have reported that mixotrophic growth requires relatively low light intensities and, consequently, can reduce energy costs. In some strains, it has been found that mixotrophic cultures reduced photoinhibition and that the growth rates are higher than those observed in both photoautotrophic and heterotrophic cultures. Further­more, mixotrophic cultivation reduces biomass loss at night and decreases the amount of organic substances utilized during growth (Chojnacka and Noworyta 2004).

In mixotrophic cultures of many strains of microalgae, there are additive or synergistic effects of photoautotrophic and heterotrophic metabolic activities, leading to increases in productivity. Park et al. (2012) reported higher biomass and fatty acid productivities of 14 species of microalgae in mixotrophic culture over photoautotrophic culture. Bhatnagar et al. (2011) found that the mixotrophic growth of Chlamydomonas globosa, Chlorella minutissima, and Scenedesmus bijuga resulted in 3-10 times more biomass production compared to those obtained under photoautotrophic growth conditions. It has also been shown that the addition of glycerol as the carbon source resulted in increased biomass productivity of Phae — odactylum tricornutum (Ceron Garcia et al. 2005, 2006; Moraisa et al. 2009). One of the possible reasons for better growth in mixotrophic cultures is the stability of pH, since carbon dioxide is simultaneously assimilated and released during pho­tosynthesis and respiration. In photoautotrophic cultures, the pH increased to more than 10, but remained stable around 7 in mixotrophic culture (Kong et al. 2011). It is important to note, however, that biomass productivity in mixotrophic cultures depends on many factors such as the strain, type, and concentration of the carbon source, and other medium components, as well as the light intensity. In some strains, for example, the addition of some carbon sources to photoautotrophic cultures inhibits growth, while others stimulate growth (Heredia-Arroyo et al.

2011) . This is because photosynthesis and oxidative phosphorylation of organic carbon substrates seem to function independently in some algae, and growth rates in mixotrophic cultures are the sum of those in photoautotrophic and heterotrophic cultures. This has been reported for Chlorella sp., Spirulina sp., and Haemato — coccus (Ogawa and Aiba 1981; Marquez et al. 1993; Martinez and Orus 1991; Hata et al. 2001). Under certain culture conditions, the presence of organic carbon in some microalgae depresses photosynthetic O2 evolution and inhibits respiration and enzymes of Calvin cycle (Liu et al. 2009). In mixotrophic cultures, photosynthetic fixation of inorganic carbon is influenced by light intensity, while the heterotrophic assimilation of carbon is influenced by the availability of organic carbon. Thus, the ratio of photoautotrophic growth to heterotrophic growth depends on the light intensity, type, and concentration of organic carbon and carbon dioxide concen­tration (Ogbonna et al. 2002a, b). These factors must be controlled to ensure high rates of growth and lipid accumulation.

Aside from increased biomass concentration and productivities (Lodi et al. 2005), mixotrophic cultures can lead to increases in lipid accumulation over the values obtained in photoautotrophic cultures. This has been reported for several species of microalgae such as Chlorella sp. (glucose), P. tricornutum (glycerol) (Fernandez Sevilla et al. 2004), Nannochloropsis sp. (glycerol) (Wood et al. 1999; Liang et al. 2009), and C. vulgaris (Kong et al. 2011). However, the oil contents of the cells in mixotrophic cultures are dependent on the nature of the carbon source. In some cases, the lipid contents of the cells are even lower or the same as those in the photoautotrophic cultures (Park et al. 2012). Nevertheless, because of the higher growth rate, the lipid productivities are, generally, much higher than those in photoautotrophic cultures. Ratha et al. (2013) reported that lipid production by twenty different strains of cyanobacteria and green algae was highest under mixotrophic condition, compared to heterotrophic and photoautotrophic cultures. With either glucose, starch, or acetate, the maximum lipid productivities of Phaeodactylumtricornutum in mixotrophic cultures were several times higher than those obtained in the corresponding photoautotrophic control cultures (Wang et al.

2012) .

Lipid productivity in mixotrophic culture is also dependent on the strain used. For example, the lipid content and lipid productivity were higher under mixotrophic conditions as compared to both photoautotrophic and heterotrophic cultures in all the members of Chlorococcales tested. Yet, the filamentous alga Ulothrix and all the cyanobacterial strains had slightly higher lipid content and lipid productivity in photoautotrophic cultures (Ratha et al. 2013). The increases in fatty acid produc­tivity under mixotrophic conditions can result from the combined increases in biomass productivity and fatty acid content, or from increased biomass productivity at relatively constant fatty acid content. In some strains and under certain culture conditions, there is no positive effect of mixotrophic culture on cell lipid content; thus, the increase in lipid productivity is mainly due to increases in biomass pro­ductivity, shown for C. vulgaris with various carbon sources (Kong et al. 2011). In contrast to photoautotrophic cultures, where conditions that favor lipid accumula­tion often suppress cell growth (Chisti 2007; Hu et al. 2008), in mixotrophic cultures, there can be a linear relationship between biomass and fatty acid pro­ductivities (Griffiths and Harrison 2009; Park et al. 2012).

Mixotrophic cultivation affects the fatty acid profile of microalgae. In 10 out of 14 isolates grown under mixotrophic condition with acetate as the organic carbon source, the percentage of oleic acid content increased significantly (Park et al.

2012) . However, the fatty acid profile was not affected when glycerol was used (Fernandez Sevilla et al. 2004), indicating that high oleic acid content is not a general feature of fatty acids in mixotrophically grown cells and that the carbon source is likely to be an important determinant of the fatty acid profile.

Other advantages of mixotrophic cultures include the feasibility of using open ponds for large-scale cultivation (Perez-Garcia et al. 2011), and the use of waste­waters as sources of organic carbon and other nutrients for reduced production costs (Zhao et al. 2012). When open ponds or non-sterilized bioreactors are used, the addition of the organic carbon sources must be controlled to avoid contamination by fast-growing heterotrophs. In some cases, the organic carbon substrate is only introduced during daylight hours, or alternatively is added only once toward the end of the culture to avoid bacterial contaminants from accumulating to unacceptable levels (Abeliovich and Weisman 1978; Lee 2001).

The main disadvantages of mixotrophic culture, as with heterotrophic culture, are that the cost of carbon source can be high and an excess/uncontrolled addition of organic substrates in an open system is likely to stimulate growth of invasive heterotrophic bacteria, resulting in a low microalgae biomass yield. There is also the problem of photoinhibition of organic carbon metabolism, in some cases, while maintaining an optimum balance of photoautotrophic to heterotrophic metabolic activities can be challenging.

Initial cell concentration covers the range of 1-6.76 g L-1 which is summarized in Table 7.5. For example, two mutants of Chlorella sp. (MT-7 and MT-15) was investigated at different initial cell concentrations on CO2 fixation rates from 1 to 3 g L 1 (Ong et al. 2010). The CO2 fixation rate increased from 0.0124 to 0.0168 g L-1 d-1 and from 0.0109 to 0.0177 g L-1 d-1 for Chlorella sp. MT-7 and MT-15, respectively (Table 7.5), indicating a significantly higher CO2 fixation rate at higher biomass concentrations (Ong et al. 2010). Furthermore, Table 7.5 shows the effect of initial cell concentration on CO2 removal and biomass concentration of cyanobacterium Synechococcus sp. (Takano et al. 1992). When the initial micro­algal cell concentration increased from 1.4 to 6.8 g L-1, the CO2 fixation rate increased from 1.06 to 2.22 g L-1 and biomass concentration increased from 1.92 to 7.76 g L-1 (Takano et al. 1992). Additionally, CO2 retention times will be increased in higher biomass concentration as a result of higher culture medium viscosity, resulting in an enhanced CO2 removal rate and fixation efficiency (Ong et al. 2010).

Genome-scale metabolic networks are often computationally explored to charac­terize functional relationships between their reactions. For instance, identification of correlated reaction sets (cosets), i. e., reactions that are always “on” or “off” con­currently (Papin et al. 2004) can define functional relationships between reactions that are not necessarily in the same pathway or obvious. The significance of these reactions is evident from the observation that mutations in correlated reactions may lead to a manifestation of the same aberrant (or disease) phenotype (Jamshidi and Palsson 2006). Because, the solution space of genome-scale networks can be

enormous, uniform sampling of the space is often carried out using the Monte Carlo method to identify the cosets and the overall shape and size of the steady state flux space (Becker et al. 2007). This sampling method, which is implemented in COBRA, identifies a set of randomly distributed solutions to serve as a proxy for the entire space. In Monte Carlo sampling, points are picked randomly from the space and the fraction inside the defined constraints is counted. This sampling method allows a uniform exploration of the metabolic network space while reducing the computational power demand required for the analysis.

Entrapment is one of the most common immobilization methods which consists of capturing the cells in a three-dimensional gel lattice, made of either natural (agar, cellulose, alginate, carrageenan) or synthetic (polyacrylamide, polyurethane, polyvinyl, polypropylene) polymers (de-Bashan and Bashan 2010; Hameed and Ebrahim 2007; Liu et al. 2009). Synthetic polymers are reported to be more stable in wastewater samples than the natural polymers, whereas natural polymers have higher nutrient/product diffusion rates and are more environmentally friendly (de-Bashan and Bashan 2010; Leenen et al. 1996).

Polysaccharide gel-immobilized algal cells have often been used for the removal of nitrate, phosphate, and heavy metal ions from their aqueous environment, in providing an alternative to the current physicochemical wastewater treatment technologies (Bayramoglu et al. 2006). Microalgae cells entrapped within either alginate or carrageenan beads were shown to have sufficient immobilization and significant nutrient removal efficiencies from aqueous environments (Chevalier et al. 2000). Aguilar-May et al. (2007) reported that the immobilization of Syn — echococcus sp. cells in chitosan gels had a positive effect on protecting the cell walls from the toxic effect of high NaOH concentration, with immobilized cells displaying higher growth than their free-cell counterparts.

Alginate beads are one of the most common encapsulation matrices, being an anionic polysaccharide found mostly in the cell walls of brown algae (Andrade et al. 2004). Major advantages of alginate gel are it being nontoxic, easy to process, cost-effective, and transparent and permeable (de-Bashan and Bashan 2010). Despite these advantages, alginate beads have some drawbacks such as not retaining their polymeric structure in the presence of high phosphate concentrations or high content of some cations such as K+ or Mg2+ (Kuu and Polack 1983). Faafeng et al. (1994) observed the degradation of sodium alginate beads, used for the immobilization of Selenastrum capricornutum, after keeping them in polluted wastewater with high phosphorous (P) and nitrogen (N) content for longer than two weeks. This degradation problem can be minimized if the stability of the target gel is enhanced. In this context, Serp et al. (2000) found that the mechanical resistance of alginate beads was doubled after mixing them with chitosan. Japanese konjac flour was also used to increase the stability of chitosan gels during tertiary treatment of wastewaters with high phosphate concentrations (Kaya and Picard 1996). Kuu and Polack (1983) suggested that increasing the gel strength of carrageenan and agar gels by integrating them with polyacrylamide results in a more rigid support for microorganisms.

Most of the entrapment processes have a similar protocol, namely mixing the microalgal suspension with the monomers of the selected polymer, followed by solidification of the resulting algae/polymer mixture by some physical or chemical process such as cross-linking of the monomers of the polymer with di- or multi­valent cations (Cohen 2001; de-Bashan and Bashan 2010). As an illustration, a general procedure for the entrapment of microalgae within alginate beads includes the following steps: (1) mixing of algal suspension with sodium alginate solution, (2) placing the homogenously distributed algae/alginate mixture in a vessel with a small orifice, such as a syringe, (3) gently dripping the mixture from the syringe as small droplets/beads into a cross-linking solution such as calcium chloride, (4) optimizing the time for algae/alginate beads inside the cross-linking solution to form cross-linked/hardened beads, (5) collecting the final algae/alginate beads, and rinsing them with deionized water several times (Smidsrad and Skjak-Brsk 1990). Since a manual dripping process for bead production is not practical for larger scale processes, automated prototypes were also proposed for the mass production of gel beads (de-Bashan and Bashan 2010; Hunik and Tramper 1993).

There are some drawbacks of cellular entrapment due to limitations of the oxygen and/or carbon dioxide transfer from the liquid environment through the immobilization matrix, which would cause difficulties mainly for aerobic micro­organisms (Toda and Sato 1985). Co-immobilization of the target microorganism with microalgal cells has been proposed as an interesting alternative to overcome any oxygen transfer limitations. Since microalgae are capable of generating oxygen from the photolysis of water, they function as ideal oxygen generators for their surrounding microenvironments (Adlercreutz et al. 1982; Chevalier and de la Node 1988). Selected microalgae-bacteria pairs have already been shown to benefit from each other, with microalgal cells generating oxygen and some organic compounds that are assimilated by bacteria. On the other hand, bacteria release some vitamins and phytohormones or provide an additional CO2 source that can enhance the algal growth (de-Bashan et al. 2005; Gonzalez and Bashan 2000; Mouget et al. 1995). Mouget et al. (1995) also found that Pseudomonas diminuta and Pseudomonas vesicularis bacterial cells isolated from the algal cultures of Chlorella sp. and Scenedesmus bicellularis stimulate the growth of those microalgal cells.

Previous attempts to immobilize viable algal cells inside gels faced other limi­tations, as the volume-to-surface ratios of spherical encapsulating materials are usually orders of magnitude larger than that of thin films. As a consequence, algal viability is a concern since the nutrients or reactants have to diffuse far into these materials to reach the algal cells. In order to overcome these problems, several other immobilization matrices have been proposed in the recent literature. Three different

immobilization matrices with different geometries and chemical properties are given in Fig. 2.1.

Algal biofilms are one of the alternatives to overcome the harvesting problems of algae in larger scale processes, where microalgal cells stick to each other on external surfaces (Chevalier et al. 2000; Wuertz et al. 2003). Microorganisms form a biofilm as a response to several factors, such as the cellular recognition of the specific functional groups on the targeted surfaces (Karatan and Watnick 2009). Microorganisms forming a biofilm on a surface secrete extracellular polymeric substance, which is mainly composed of phospholipids, proteins, polysaccharides, and extracellular DNA (Hall-Stoodley et al. 2004; Qureshi et al. 2005). Polystyrene disks (Przytocka-Jusiak et al. 1984), textured steel surfaces (Cao et al. 2009), aluminum disks (Torpey et al. 1971), and polystyrene surfaces (Johnson and Wen

2010) are some examples of biofilm surfaces used for algal growth for the primary application of nutrient removal from wastewaters.

The shape of algal cell composite material has two components, a global geometrical form and the surface detail which determines the texture of the surface, with nanomaterial processing techniques being the useful approaches for creating different shapes, from fibers to spheres and flat membranes (Crandall 1996). Var­ious nanofabrication processes have featured in recent research from the authors’ laboratories, albeit in using more unconventional types of immobilization matrices for the immobilization of Chlorella vulgaris cells, such as electrospun nanofibers (Eroglu et al. 2012), laminar nanomaterials such as graphene and graphene oxide nanosheets (Wahid et al. 2013a, b), microfibers of ionic liquid-treated human hair (Boulos et al. 2013), and magnetic polymer matrix composed of magnetite nano­particles embedded in polyvinylpyrrolidone (Eroglu et al. 2013). Electrospinning processes can create nanofiber mats with high porosities and surface-to-volume ratios and are generated by forcing a charged polymer solution through a very

small-sized nozzle while applying an electrical field (Kelleher and Vacanti 2010). On the other hand, a recently developed vortex fluidic device has been successfully used for the exfoliation of laminar materials within the dynamic thin films formed on the walls of this microfluidic platform (Wahid et al. 2013a, b). Scanning electron microscopic images of different nanomaterial matrices, used for the immobilization of C. vulgaris microalgal cells, are given in Fig. 2.2.

The unique environmental conditions in microalgal cultures may result in signifi­cant losses of nutrients from wastewater. In microalgal cultures, pH is high due to photosynthetic depletion of carbon dioxide (CO2) from the culture medium, and this may result in volatilization of ammonia or precipitation of P. In concentrated wastewaters such as animal manure, N is often present as ammonium. When pH is high, ammonium is converted to free ammonia and escaped as a gas from the culture medium through volatilization. Volatilization of ammonia can be significant in open algal ponds used for wastewater treatment, particularly when water tem­peratures are high (Garcia et al. 2000); this not only results in losses of N, but also causes eutrophication in the surrounding landscape through N deposition. However, maintaining the pH of the culture medium at 8 by addition of CO2 is effective to prevent ammonia volatilization (Park et al. 2011b). At a high pH, phosphate can also precipitate as calcium phosphates (when Ca concentrations are high; Beuckels et al. (2013) or as struvite when ammonium and magnesium (Mg) concentrations are high. Phosphate precipitation can result in significant losses of P from the wastewater (e. g., Lodi et al. 2003), causing additional turbidity in medium and reducing microalgal production (Belay 1997).

In contrast to conventional recombinant DNA techniques, synthetic DNA synthesis can generate nucleotide sequences de novo. In this way, DNA synthesis technology allows for custom design of novel nucleotide sequences. The increased throughput of DNA synthesis now allows entire genetic regions and even small genomes to be derived synthetically (Carr and Church 2009; Gibson et al. 2008). This break­through has given rise to the field of ‘synthetic genomics’. DNA synthesis tech­niques hold promising applications in algae biofuel research.

DNA synthesis, coupled with recombinant techniques, can generate over 1 Mb of synthetically derived nucleotide sequence. DNA synthesis technology alone can produce customized sequences of up to 10 kb (known as a cassette) (Gibson et al. 2010a). Assembly of multiple cassettes using in vitro recombination techniques can create large synthetic DNA constructs (up to 150 kb). Larger constructs (>500 kb) can be achieved when in vivo recombination techniques are used (Gibson et al.

2008) . Synthesizing small genomes synthetically is now possible. Bacteriophage and viral genomes (5-8 kb) can be created by synthetic oligonucleotides alone (Cello et al. 2002; Liu et al. 2012; Smith et al. 2003). Mitochondrial and chloroplast genomes have also been synthesized and assembled (16 and 242 kb respectively) (Gibson et al. 2010b; O’Neill et al. 2012). The Mycoplasma mycoides bacterial genome (1.08 Mb) is currently the largest published assembly. Importantly, the M. mycoides synthetic genome was shown to be biologically viable. This synthetic genome was able to ‘boot-up’ and co-ordinate normal cell function when it replaced genomic DNA of a M. capricolum recipient cell (Gibson et al. 2010a).

Genome synthesis promises unrestricted editing of whole genome sequence. Presently, small autonomous or semi autonomous genetic circuits have been introduced into cells to perform a desired role (Havens et al. 2012; Tigges et al.

2009) . Synthetic genomics strives to widen the scale and complexity of circuitry, ultimately delivering new novel phenotypes. Crucially, synthetic genomics must be partnered with CAD tools (such as SynBioSS) to ensure ease of hypothesis testing in silico before embarking on lengthy wet-lab experiments.

Synthetic genomics also enables global editing of cis-regulatory elements. This approach was recently trialed in a biologically active synthetic yeast chromosome. No gross changes to gene circuitry were attempted; rather 98 small elements (loxPsym sites) were introduced throughout the chromosome. When ectopically activated, these sites could initiate chromosomal deletion events (Annaluru et al.

2014) . This demonstrated that incorporating small sequence additions by synthetic DNA synthesis can provide unprecedented control of chromosome architecture.

Synthetic genomics is still in its infancy. Technical problems of synthetic assembly exist. The error rate of DNA synthesis, even at 1 x 10-5 bp, is still problematic when synthesizing large nucleotide stretches (Carr et al. 2004). Such base misincorporations have shown to render entire genomic assemblies biologi­cally inactive (Katsnelson 2010). Stability of constructs in host cells during in vivo recombination (e. g. E. coli or Saccharomyces cerevisiae) significantly limits assembly size. The Prochlorococcus marinus genome assembly (1.7 Mb) is cur­rently the largest stably maintained synthetic construct (however it has not been proven biologically active) (Tagwerker et al. 2012). Furthermore small synthetic genomes that have shown to be biologically active were replicates of known genomes. DNA synthesis may support the introduction of novel genetic regions, however building functional gene circuits from the bottom up has been shown problematic (Katsnelson 2010).

immobilization of cells brings several advantages over current suspension biopro­cessing, such as (1) providing flexibility to the photobioreactor designs; (2) increasing reaction rates arising from higher cell density; (3) enhancing oper­ational stability; (4) avoiding cell washouts; (5) facilitating cultivation and easy harvesting of microorganisms; (6) minimizing the volume of growth medium as the immobilized cellular matter occupies less space; (7) easier handling of the products;

(8) permitting the easy replacement of the algae at any stage of the experiment;

(9) protecting the cell cultures from the harsh environmental conditions such as salinity, metal toxicity, variations in pH, and any product inhibition; and

(10) allowing continuous utilization of algae in a non-destructive way. Enhanced survival rates of immobilized cells in toxic environments provide a significant alternative to achieve sufficient bioremediation of chemically contaminated envi­ronments. It is also important to stress that continuous biomass production, opportunity for product recycling, and nearly spontaneous biomass harvesting will have the potential to outweigh the difficulties and added costs associated with applying the technology on a larger scale.

Conventional wastewater treatment methods are mostly focused on the separa­tion of pollutants from the liquid effluents with a requirement for a further stage to eliminate them. Developing integrated wastewater treatment processes that elimi­nate the undesired portion of the wastewater while converting it into valuable products is important in developing sustainable processes for the future. immobi­lization of algal cells is important in the development of an integrated process while simplifying the harvesting of biomass and providing the retention of the high-value algal biomass for further processing.

There are, however, technical issues to address, such as the hybridization of different polymers for creating more efficient and stronger immobilization matrix for algal cells. Immobilization of viable algae inside three-dimensional gel lattices also faces several limitations given that the encapsulating materials can have high volume-to-surface ratios. As a consequence, algal viability decreases since the light, nutrients, or reactants have to diffuse far into these materials to reach the algal cells. One of the other restrictions for the gel-entrapped cultures is their lower growth rates compared to their free-living counterparts. Such drawbacks can be addressed by optimizing the immobilization processes, that is, by choosing different encap­sulating materials with lower volume-to-surface ratios such as thin films. Over­coming the difficulties of the current technology will increase the applicability of immobilized algae systems for various industrial applications.

Current immobilization projects have been often confined to the laboratory in providing an effective proof-of-concept rather than quick-install industrial proto­types. For larger scale wastewater treatment and biofuel production bioprocesses, the cost of immobilization matrix becomes a significant parameter that needs to be improved by further innovative designs and additional profits through generating valuable by-products.

Discovering the optimal microalgae-bacteria combinations for co-immobilization processes can also be a good alternative for large-scale wastewater treatment prac­tices, since algal cultures in nature are usually associated with bacteria.

Application of innovative composite materials for use as the algal immobiliza­tion matrices can have a significant contribution to the economic and environmental development by sustainable utilization and recovery of the local resources, while bringing valuable strategies for solving important environmental issues.

Potentials of Exploiting Heterotrophic Metabolism for Biodiesel Oil Production by Microalgae

James Chukwuma Ogbonna and Navid R. Moheimani

Abstract The current prices of microalgae oils are much higher than oils from higher plants (vegetable oils) mainly due to the high cost of photoautotrophic cultivation of microalgae. However, many strains of microalgae can also grow and produce oil using organic carbons, as the carbon source under dark (heterotrophy) or light conditions (mixotrophy). Lipid productivities of most strains of microalgae are higher in culture systems that incorporate heterotrophic metabolisms (presence of organic carbon source) than under photoautotrophic conditions. This is because for many strains, cell growth rates and final cell concentrations are higher in het­erotrophic cultures than in photoautotrophic cultures. Furthermore, in some cases, the oil contents of the cells are also higher in cultures incorporating heterotrophic metabolisms. It has also been reported for some strains that the quality of oil produced in the presence of organic carbon sources are more suitable for biodiesel oil production than those produced under photoautotrophic conditions. Thus, het­erotrophy can be used to reduce the cost of biodiesel oil production, but the effectiveness of the various organic carbons in supporting cell growth and oil accumulation depends on the strain and other culture conditions. Use of waste­waters for cultivation of microalgae can further substantially reduce the cost of production (since they contain carbon, nitrogen, and other nutrients) and also reduce the requirement for freshwater. Generally, many factors such as nitrogen limitation, phosphate limitation, silicon limitation, control of pH, and low tem­perature can be used to increase oil accumulation, although their effectiveness depends on the strain and other culture conditions.

J. C. Ogbonna (H)

Department of Microbiology, University of Nigeria, Nsukka, Nigeria e-mail: james. ogbonna@unn. edu. ng

N. R. Moheimani

Algae R&D Center, School of Veterinary and Life Sciences, Murdoch University, Murdoch, WA 6150 Australia

© Springer International Publishing Switzerland 2015

N. R. Moheimani et al. (eds.), Biomass and Biofuels from Microalgae,

Biofuel and Biorefinery Technologies 2, DOI 10.1007/978-3-319-16640-7_3

1.1 Introduction

Interest in production of biodiesel continues to be sustained because, unlike fossil diesel which is non-renewable and associated with various environmental problems, biodiesel is biodegradable, renewable, non-toxic, and emits less gaseous pollutants. The cost of biodiesel will determine to what extent it will be able to replace or complement fossil diesel production. Vegetable oil remains a major source of oil for large-scale industrial biodiesel production. However, the cost of vegetable oil is high, and waste oils often contain large amounts of free fatty acids which are difficult to convert to biodiesel through transesterification. Microalgae oil has a high potential for biodiesel production as it contains large proportions of fatty acid triglycerides, and the composition of the oil can be controlled by varying the culture conditions (Jiang and Chen 2000; Widjaja et al. 2009; Wen and Chen 2001a, b; Zhila et al. 2005). Microalgae oil is characterized by lower oxygen content, higher calorific value, and higher H/C ratio which make it more suitable for biodiesel, as compared to terrestrial plant oils (Miao and Wu 2004, 2006).

However, the cost of microalgae biodiesel is still too high to compete with the fossil diesel. The cost of microalgae cultivation accounts for 60-75 % of the total cost of the microalgae biodiesel fuel (Krawczyk 1996). It has been estimated that the cost of production of a liter of oil ranges from $1.40 to $1.81, depending on the type of photobioreactor used, and assuming that the biomass contains 30 % oil by weight (Azimatun-Nur and Hadiyanto 2013). Reduction in the cost of microalgae oil requires improvement in growth rate, oil content of the cells, and reduced cost of construction and operation of bioreactors. Reports from various studies have shown that it is already very difficult to increase cell growth rates and productivities in photoautotrophic cultures. However, many strains of microalgae can grow het — erotrophically, using various organic carbons in dark. Heterotrophic cultures can be used to overcome most of the problems associated with photoautotrophic cultures. Generally, in comparison with photoautotrophic cultures, higher cell densities are achieved in heterotrophic cultures, with consequent reduction in the cost of downstream processing. Thus, heterotrophic cultures can be used to significantly reduce the cost of microalgae biodiesel production. The feasibility of exploiting heterotrophy for efficient biodiesel oil production is discussed in this chapter.

The haptophytes, predominately marine phytoplankton, are recognized as a division divided into two classes, Pavlovophyceae and Prymnesiophyceae (Cavalier-Smith

2007) . The chloroplast pigments are similar to those of the heterokonts, and in both divisions, chloroplasts are derived from red algal symbionts (Anderson 2004). Several members of the class Prymnesiophyceae including the Prymnesium (Becker 1994), Isochrysis spp. (Chisti 2007), and the Coccolithophores are oleaginous algae. Pavlova and Isochrysis spp. are widely used in the aquaculture industry because of their favorable lipid content (Walker et al. 2005). While the Haptophyta and Heterokonts are predominantly marine organisms, there are a number of species in both groups that are found in freshwater systems. In addition, certain types of wastewater such as the effluent from food processing plants have a much higher salinity than domestic wastewater.

The nuclear genome of C. reinhardtii was published in 2007 (Merchant et al. 2007) after the first wave of next-generation sequencing (NGS) became commercially available in 2005 (Margulies et al. 2005; Shendure et al. 2005). Nevertheless, the Chlamydomonas genome was sequenced through a conventional shotgun sequencing and assembly pipeline with 13X coverage (Merchant et al. 2007). Following the completion of Chlamydomonas genome sequencing, Thalassiosira pseudonana, a diatom, was the first eukaryotic marine alga that was sequenced (Bowler et al. 2008). A draft genome sequence of Nannochloropis gaditana was also made available in 2012 (Radakovits et al. 2012).

The continued development of NGS platforms, among them Illumina, and Ion Torrent semiconductor sequencing in the main stream, have brought down the time, effort, and cost of genome sequencing well beyond the exponential drop predicted by Moore’s law (Moore 1998). This enabled the sequencing of many new algal genomes (Fig. 10.2). The main limitation of NGS has been that the relatively short read length (50-500 bp) introduces inaccuracy in the assembly of sequences (Zhang et al. 2011). Furthermore, the high demand on bioinformatics analysis due to the increased data volume by several orders of magnitudes (Morey et al. 2013) intro­duces challenges in the use of NGS, particularly when the investigators do not have access to high performance computing infrastructure and appropriate bioinformatics support. Third-generation sequencing (TGS) technologies are being developed to address these problems. For instance, single molecule real-time (SMRT) sequenc­ing makes the whole genome sequencing of single cells from uncultivable organ­isms possible (Schadt et al. 2010). Many more TGS technologies are expected to be on the way. Moreover, user-friendly software such as the CLC Genomics Work­bench (CLC bio, a QIAGEN Company, Denmark) is enabling investigators to carry out genome assembly without the need of high performance computers or dedicated informatics specialists.

While advances in technology will ultimately lead to the generation and in-depth analysis of sequenced genomes, this would still be an initial step to be

complemented by transcriptomic, proteomic, and metabolic analysis in order to reach a better understanding of the system per se. The integration of all of these levels of analyses, compiling them into a predictive model, and describing the interactions between their respective components, is in fact the main feat of systems biology. In such endeavors, metabolic network models occupy a central and key position in advancing bioproduct optimization.