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).

While ultimately the hard physical metrics for microalgal biocrude are essentially the energy returned on investment (EROI) and the economic viability, the maturing and scaling of the technology still require further development. During this early development phase, commercial viability requires a profitable path to technology deployment. Several approaches are possible for dealing with this problem.

High Value Products and Services (HVP&S) Algal GM is in its infancy compared to other systems. A challenge of generating GM strains for HVP&S production is to provide useful products and services that cannot be easily generated in more mature technologies. There is no rational point to replicating in algae a service that can be easily and economically performed by yeast or E. coli aside from reasons such as marketing appeal. The relative advantages of algae as GMO vehicles must therefore be carefully considered on a case by case basis. Recombinant products such as peptides larger than those able to be chemically synthesised, but small enough to be extracted with relatively harsh techniques, may be particularly suitable. Many HVP&S GM strains will be designed to operate under heterotrophic conditions which simplify reactor design.

The difference between these approaches is that modification of bulk mass and energy flows is focussed on energy production and is strictly limited by the ther­modynamics of light harvesting and carbon fixation, whereas HVP&S approaches are less thermodynamically constrained (and indeed, may not even utilise photo­autotrophic systems) but are focussed mainly on economic gains.

Enabling and Supportive Technologies Given the constraints outlined above, it is clear that GM strains for HVP&S and those for biofuels applications will have little in common and it is unlikely that a single strain (or industrial facility) will serve both purposes, which argues against the ‘biorefinery’ concept if it is confined to a single strain or process. Nonetheless, the common biology underpinning all algal systems means that most of the enabling technologies invented in this space will apply similarly to a multitude of different algal biotechnology systems, yielding substantial cross-fertilisation. It is here that the biorefinery concept may be most profitable.

Many supportive technologies will therefore need to be developed before the industry matures, and GM can make major contributions to these. Protein and lipid export systems, for example, may reduce internal product inhibition while reducing harvest costs; modified photosynthetic systems may improve the efficiency of utilisation of incident light; and fluorescent signals may be generated to monitor internal biochemical processes. None of these technologies would intrinsically compromise the ability to convert light to fuel, but might greatly simplify or reduce costs for other biotechnological aspects. Clearly, there is a vast creative space for innovative GM approaches in this area. To the extent that such technologies reduce energy wastage during production, they can improve the EROI even without an alteration of the fundamental light-harvesting efficiency.

Advantages of Algae as Heterologous Expression Systems Algae as heterologous expression systems are comparable to plant systems primarily for their ability to produce proteins with post-translational modifications. They may not replace the established and commercialised bacterial and mammalian expression systems but offer the potential for biological products which are difficult to produce in an active form in prokaryotic systems and are expensive to make in eukaryotic systems (e. g. antibodies). They also offer advantages over conventional systems to be chosen for new products which cannot be produced in other systems [e. g. anti-cancer toxin (Tran et al. 2013)] and therefore provide a valuable opportunity for the industry.

One advantage that can make transgenic microalgae systems competitive in the field of pharmaceutical proteins is that many algae lack endotoxins or human pathogens (Mayfield and Franklin 2005; Walker et al. 2005) and are therefore ‘Generally Recognized As Safe’ (GRAS). This could allow for a reduction of necessary purification steps during downstream processes as well as simplify quality control and therewith allay production costs. Another advantage of algae compared to higher plants is vegetative reproduction, leading to uniform clones with comparable production rates. This relates to product quality, e. g. demonstrated as certain beneficial post-translational modifications, product stability or biosafety. Microalgae systems display high growth rates and need only a short time from transformation to product formation so that scale up could be implemented within a few weeks within commercial processes. The cultivation can be inexpensive due to the relatively low costs of typical mineral media needed, therefore supplying a large-scale robust growing system which can yield cheaply extractable high-volume production. This provides possible cost savings during production processes, which could play a role in special fields, where large quantities of products are required at low costs such as recombinant antibodies or veterinary products.

Microalgae have already been established as biotechnological production sys­tems and approved by the US Food and Drug Administration for a number of secondary metabolites useful as food additives or cosmetics (Administration 2003, 2004, 2010a, b, 2011, 2012; Plaza et al. 2009) and for the production of carotene using Dunaliella salina (Hosseini Tafreshi and Shariati 2009) and lutein as an antioxidant and food colourant. Antiviral activities have been shown. Vaccination concepts for a large number of diseases prevalent in developing nations based on recombinant antigen expression in microalgae could result in inexpensive pro­duction and distribution as well as long-term storage at room temperature (Dreesen et al. 2010; Specht et al. 2010). Edible vaccines are a possible field of application for algal expression systems, combining biosafety issues with inexpensive pro­duction and storage and therefore opening up making products accessible for less developed countries (Gregory et al. 2013). In the context of regulatory aspects in the pharmaceutical sector, novel expression systems have to offer enormous advantages over conventional systems to be chosen for new products. The possi­bility to use a closed photobioreactor system contributes to reducing the risk of contamination and prevents transgenes dispersing into the environment.

Active research and process development are still ongoing to improve the overall cost efficiency of DAF-related systems so that they can meet the economic thresholds imposed by the biofuel marketplace. Two recent examples of this are posiDAF and ballastDAF. The term posiDAF is used to describe DAF being run with a positive surfactant added to the bubble generation system. The 2007 thesis of Henderson (2007) applied this technology to algae and is under development as a commercial product by WaterInnovate Ltd. (http://waterinnovate. co. uk/). Another technology, termed ballastDAF, uses positively charged glass beads pushed through the system instead of air bubbles to provide separation of the cells from the medium (www. rachelwhitton. co. uk/uploads/1/9/4/5/…/whitton_poster. pdf). The glass beads are collected by low-speed centrifugation (e. g., cyclones), and then, the beads can be reused for further separation. Jefferson and colleagues claimed a reduction in energy to <20 % of that required by conventional DAF.

Due to the significant potential of microalgae as a feedstock for biofuels, numerous economic viability assessments have been undertaken (Ribeiro and Silva 2013). To provide a snapshot of the current status of biofuel production from microalgae, a summary of these studies is provided in Table 17.3.

17.3.1 Techno-Economic Model Discussion

The summary in Table 17.3 demonstrates that there is a wide range of cost esti­mates for microalgae biofuel production and that none of them provide a cost estimate that will be economically competitive with current biofuels or fossil fuels (minimum selling prices of cost of production do not include fuel taxes). Further analysis of the studies is provided in the paragraphs below.

The work of Chisti (2007) was released during a time of aggressive investment and development into microalgae biofuels when oil prices were at historical highs. As a result, this work has a number of assumptions that are optimistic, and inter­estingly, the only analysis that demonstrates that PBR is better than open ponds. The major driver for this discrepancy is the extremely optimistic aerial productivity for PBR (3 times maximum recorded long-term annual averages) and the low

M

capital cost. The major concern with this work is that demonstrable operational issues (such as cleaning and contamination) were treated as trivial matters. Despite these weaknesses and overly positive assumptions, this study shows how chal­lenging the economic production is as it still demonstrates non-competitive pro­duction costs.

The work of Alabi (2009) provides a comprehensive evaluation of microalgae produced phototrophically in open ponds and PBR as well as heterotrophically in fermenters in a Canadian context. This study utilised realistic capital costs and operating data from industry and long-term research studies. The high boundary for fuel costs was due to low productivity (low solar irradiation in Canada and no production in winter), high capital costs (lined ponds) and low oil content (15 %). The low boundary for fuel costs occurred at high productivity (high solar irradia­tion), low capital costs (no pond lining) and higher oil content (30 %). Hetero­trophic solutions were deemed to be unsustainable due to the need for an organic feedstock which is typically derived from terrestrial crops and therefore not suitable for mass production.

The potential of EPA (Omega 3) to provide an economically viable solution was also investigated. This demonstrated that at optimistic conditions, microalgae could economically be grown for the purpose of EPA production; however, in these cases, biofuels were a minor by-product. Although a viable option and currently being pursued by commercial companies (Cellena and Aurora), there are concerns over market saturation at biofuel production levels (Benemann et al. 2012).

Williams and Laurens (2010) developed a model for algal biomass production from first principles that utilised solar irradiation levels and typical lipid/protein/ carbohydrate ratios to predict biomass productivity. Although this model provides indications of upper limits of aerial productivity, the economic assessment is somewhat optimistic due to the following assumptions:

• Unproven high productivities (annual average)

• Optimistic and unproven harvesting costs

• High protein prices especially considering there is no market for algae protein

• 100 % nutrient recycle from anaerobic digestion and dewatering operations

Unlike other studies, Delrue et al. (2012) used an innovative approach to address the high level of uncertainty in regard to the use of different technologies on the overall economics of microalgae biofuels. In this work, Monte Carlo simulation techniques were used to generate thousands of different scenarios based on different technologies and performance levels. Table 17.4 summarises the output of this simulation with each row representing a scenario in which one technology was kept constant and all the others changed. The minimum and maximum costs represent the lower and upper limits of the confidence level or range of the estimates, with a 50 % probability that the ultimate cost of biofuels with this technology choice will lie in between these boundaries.

The ‘all technologies’ scenario represents the overall projected price range of biofuels based on all available technologies. As an observation, these values are considered optimistic as the study assumed the flue gas could be utilised as the carbon source and wastewater would provide a source of nutrients (N and P). Unfortunately, there are very few locations that have ideal growth conditions, that is high solar irradiance and stable temperatures, large plots of low-cost non-arable land, access to waste water and close proximity to high volume and concentrated sources of CO2 (Benemann et al. 2012; Lundquist et al. 2010). This work again indicates that biofuels from microalgae are unlikely to be viable at the current levels of productivity with the current technologies.

In an attempt to develop a baseline model for techno-economic assessment, lifecycle assessment (LCA) and resource assessment of microalgae to biofuel processes, the Department of Energy (DOE) worked with the three American national laboratories to develop a harmonised model of renewable diesel production from microalgae (ANL et al. 2012). This is an extensive work that builds upon the modelling and research of multiple groups and industry representatives over a 50- year period. The fundamental data in this model are used as a reference for most other studies including those referenced in the bottom two rows of Table 17.3. The process modelled is shown in the block diagram in Fig. 17.3.

The model was built around multiple 4850 ha sites located in areas with suitable climate and water availability to achieve a total production volume of 5 billion gallons/year. The sites were chosen according to previous geographic information service (GIS)-based resource assessment modelling. The comprehensive nature of

this work provides an excellent insight into the major factors affecting the eco­nomics of large-scale biofuel production from microalgae. Key observations are as follows:

• At the baseline yield (annual average of 13.2 g/m2/day productivity and 25 % lipid content), capital costs represent over 70 % of the final diesel selling price. Due to the high capital burden, the break-even price of diesel production is $2.46/L, while the selling price to achieve a 10 % rate of return on capital investment is $4.90/L (All further costs mentioned per below are based on a 10 % rate of capital return).

• The addition of pond liners significantly increases the capital cost and therefore the unit cost of renewable diesel production, and at the baseline yield, the price is increased by $1.38/L. Alternative pond design or suitable ground would negate the need for pond liners, significantly reducing the capital cost.

• Higher yields (e. g. the baseline uses an annual average of 13.2 g/m2/day pro­ductivity and 25 % lipid content) significantly reduce cost to ($3.03/L) as the pond area, and therefore, capital investment significantly reduces. At lower yields (<20 g/m2/day), pond and liner costs dominate the capital expenditure; however, at higher yields, the other system elements have a much more significant effect.

• In a best case scenario with harvesting/extraction costs halved (due to tech­nology advancement), pond installation costs reduced by 30 % (due to opti­mised construction approach), and with the removal of liners, the selling price was approximately $2.50/L less at the low yield of 12.5 g/m2/day. If the yields were increased to 50 g/m2/day via strain improvement (selection or genetic modification), then prices would further decrease to <$1.25/L. However, at increasing yields, the capital costs of the non-growth elements and the operating costs start to dominate the cost.

• Selling the spent algal biomass as fish protein ($350/tonne) had a limited effect on the economic analysis as this was approximately equivalent to the value provided by the AD plant. To have a greater effect, the selling price of the protein would need to be in excess of $350/tonne.

As an observation, the break-even cost of production in the base case scenario of this model was $2.46/L, while under the same conditions, the minimum selling price was $4.90/L. This increase is required to deliver a rate of return on equity of 10 % (IRR). A key high-level observation from this study is that the capital cost of the growth system is the major impediment to cost competitive microalgae-based biofuels. At low productivities (<20 g/m2/day), the growth system dominates the capital expenditure, especially when liners are required. In some situations, liners can be done away with (typically resulting in a halving of pond construction cost); however, there is little opportunity to reduce the cost of pond construction due to earthmoving being a very developed field. The most effective way to reduce capital expenditure is to increase the productivity of the microalgae so that less pond area is required, from the studies considered in this work 30 g/m2/day seems to be a minimum for competitiveness. Higher yields can only come through optimisation of growth conditions, selective breeding and ultimately from genetic modification to limit photo-inhibition.

The work of Klein-Marcuschamer et al. (2013) evaluated three different paths to biomass-derived jet fuel, including microalgae, perennial oil crop and heterotrophic fermentation. Their techno-economic analysis focused on algae and processes that had sufficient data available. The major anomaly with this work is the use of centrifuges to concentrate the microalgae from harvest concentration (0.05 % ash free dry weight) to 25 %. Their motivation for this is that this has been demon­strated through the use of Evodos centrifuges. The consequence of this is that harvesting capital and operating costs (power) represent 70 % of the final cost.

This is clearly not going to be viable, with options such as settling or DAF (with or without flocculation) required as a pre-settling stage. Interestingly, however, this demonstrates another challenge associated with large-scale microalgae production— low-cost options exist; however, they are often problematic or not proven at scale.

As microalgae research and commercial R&D projects have matured, the US Department of Energy (DOE) has identified two production pathways as shown in the final row of Table 17.3[5]. In the ALU methodology, the microalgae production system is treated as a biorefinery with the microalgae constituents (lipids, carbo­hydrates and protein) being converted into green diesel, ethanol and animal feed or AD feedstock, respectively. In the latter, the whole microalgae biomass is converted to green crude via hydrothermal liquefaction. The high costs in Table 17.3 indicate the current state of the art, while the lower cost represents the proposed achievable future cost. The major savings are associated with greater yield (30 g/m2/day), no plastic liners, dewatering costs halved and high efficiencies in all processing operations.

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.

Several recent studies have investigated the potential of magnetite nanoparticles for flocculating microalgae. The nanoparticles can induce flocculation of microalgae and separation of the microalgae from the medium in a magnetic field. This tech­nology thus combines flocculation and separation of flocs in a single process step. In some studies, the magnetite nanoparticles are used as such, without func­tionalization (Xu et al. 2011; Prochazkova et al. 2012). In other studies, the nanoparticles are functionalized by coating the surface with cationic functional groups (Lim et al. 2012; Seo et al. 2014). By using functional groups that have a pH-dependent charge, it is possible to remove the nanoparticles after flocculation by adjusting pH and to reuse them in a second round of harvesting (Cerff et al. 2012; Seo et al. 2014)

In estuaries, flocculation of natural populations of microalgae is sometimes observed in the presence of high concentrations of sediment particles. This floc­culation has been ascribed to interaction between microalgae and clay minerals (Avnimelech et al. 1982). Clays have been used successfully to induce flocculation of natural blooms of microalgae in coastal waters (Sengco and Anderson 2004; Padilla et al. 2010). So far, however, only a few studies have explored the potential of clays for harvesting microalgae. One study explored the use of synthetic orga — noclays with specific surface properties for flocculating Chlorella (Lee et al. 2013b). Farooq et al. (2013) successfully demonstrated aminoclay harvesting of Chlorella vulgaris and Nannochloris oculata. Aminoclays were used recently as a template for nanoscale zerovalent iron synthesis and were shown to be efficient for harvesting Chlorella sp (Lee et al. 2014).