Photosynthesis is responsible for the conversion of light into chemical energy which can be used for biofuel production, including biohydrogen, biodiesel, bioethanol, and biomethane generation (Hankamer et al. 2007). Immobilized mic­roalgal cultures have also been in use for the enhancement of these secondary product formations, as explained further below.

As outlined above, using wastewater nutrients rather than synthetic nutrients improves the sustainability of microalgae biomass production. On the other hand, using microalgae rather than conventional wastewater treatment technology may also result in a more sustainable method for treating wastewaters. In conventional wastewater treatment, N and P are removed from the wastewater without being reused: N is removed primarily by denitrification and is lost to the atmosphere as N2, while P is removed from wastewater by precipitation with metal salts and disposed of in landfills. When wastewater is treated using microalgae, N and P are not only removed from the wastewater, but can also be reused to produce extra biomass. As N and P are extremely valuable resources to our society, initiatives are increasingly being taken to not only remove but also reuse N and P from wastewater (Dawson and Hilton 2011; Cordell et al. 2011; Elser 2012). Combining wastewater treatment with microalgae biomass production can achieve parts of this goal.

Eutrophication of lakes, rivers, wetlands, and coastal waters is a major envi­ronmental issue. To reduce eutrophication, regulations for discharge of effluents from wastewater treatment plants are becoming stricter. In the EU, for instance, discharge limits for wastewater have recently been decreased to 1 mg L-1 for P and to 10 mg L-1 for N (Oliveira and Machado 2013). Conventional technologies have difficulties in removing N and P from wastewater down to these levels. Residual concentrations of N and P in effluent from conventional wastewater treatment plants

are often quite high, high enough to cause eutrophication in receiving natural ecosystems. Microalgae have half-saturation constants for uptake of N and P that are well below the strictest limits. Therefore, the use of microalgae to remove nutrients from wastewater will certainly lead to lower N and P concentrations in the effluent, and less eutrophication of aquatic ecosystems.

Wastewater contains large amounts of organic matter, and it is important that it is oxidized before the effluent is discharged into the environment. In modern wastewater treatment plants, electromechanical air blowers supply oxygen that allows bacterial oxidation. This process consumes a lot of energy, and it is the major contributor to the capital and operational costs of modern wastewater treatment plants. If a proper cultivation design is developed, microalgae can produce sufficient oxygen for bac­terial oxidation of organic matter. Microalgae-based systems are equally effective as electrical air blowers for oxidation of organic matter, but have a much lower cost (Owen 1982; Craggs et al. 2013). The CO2 that is produced during degradation of organic matter can also be used as a carbon source in microalgal photosynthesis. Some microalgae are mixotrophic and can contribute to the degradation of organic matter from wastewater. This mixotrophic growth based on organic matter present in wastewater can even boost microalgal biomass production (Bhatnagar et al. 2011).

It is clear that combining microalgae production with wastewater treatment not only improves the sustainability of microalgae production but also that of waste­water treatment (Sturm and Lamer 2011). Beal et al. (2012) and Menger-Krug et al. (2012) showed that combined wastewater treatment and microalgae production has a much better energy balance than both processes operating separately. Combining microalgae biomass production with wastewater treatment would also make mic­roalgae biofuel production economically more attractive, as additional income can be generated from the treatment of wastewater (Lundquist et al. 2010; Pittman et al.

2011) . Combining microalgae production with wastewater treatment, however, is also a challenge because both processes need to be optimized simultaneously. On the one hand, the productivity and biochemical composition (e. g., lipid content) of the microalgae should be optimal. On the other hand, the quality of the wastewater effluent should comply with national water treatment standards (e. g., biological oxygen demand removal, N and P removal).

5.13 Conclusions

The high demand of microalgae for N and P poses an important environmental burden on microalgal biofuels. This environmental impact can be avoided by replacing synthetic fertilizer with N and P from wastewater. It is feasible to use wastewater as a source of N and P because microalgae have been used for many years in wastewater treatment (in facultative ponds or HRAPs). The resource base provided by wastewater nutrients is theoretically large enough to produce a similar amount of biomass as the global production of rice or wheat, yet it is not large enough to produce enough microalgal biomass to replace fossil fuels. Climatic and geographical factors limit the potential to use wastewater for microalgae produc­tion. Using wastewater as a source of nutrients rather than synthetic fertilizer poses several challenges. The N and P demand of microalgae should be matched with the variable supply of these nutrients by wastewater. Wastewater contains many types of contaminants that can interfere with the production of microalgal biomass and/or with the valorization of certain microalgal biomass fractions. The high pH that is typical of microalgal cultures may result in nutrient losses (precipitation of P and volatilization of N). Further research is needed to overcome some of these chal­lenges. Combining microalgae production with wastewater treatment not only improves the sustainability of microalgal biofuels but also increases efficiency of wastewater treatment because microalgae-based wastewater treatment has a lower energy demand, can result in a better effluent quality, and is a way to recycle valuable nutrients from the wastewater.

Genetic transformation entails introduction of foreign DNA into a cell (Gietz and Woods 2001) (Fig. 9.1). Genetic transformation has been applied to several algal strains, with C. reinhardtii obtaining the highest rates of transformation (Kindle 2004). Nuclear transformation of various microalgal species such as C. reinhardti is now routine (Walker et al. 2005).

Chloroplast transformation has plastid-specific challenges as compared to nuclear transformation. Nevertheless, chloroplast transformation has been achieved in green (C. reinhardti), red (Porphyridium sp.), and euglenoid algae (E. gracilis) (Wang et al. 2009a). Compared to nuclear transformation, chloroplast transfor­mation has some advantages: primarily, production of high protein levels; the feasibility of expressing multiple proteins from polycistronic mRNAs; and gene containment through the lack of pollen transmission (Wang et al. 2009a). On a final note, attempts in specifically targeting the chloroplast genome of C. reinhardtti and achieving a multiple loci modification in vivo have been performed (O’Neill et al. 2012). The assembly of an ex vivo chloroplast genome using cloning in yeast cells was done targeting a set of genes involved in the photosynthesis pathway (O’Neill et al. 2012). Subsequently, chloroplast transformation was done to achieve the incorporation of genes altering the photosynthesis pathway, more precisely, photosystem II (Nelson and Ben-Shem 2004; Specht et al. 2010).

C. reinhardtii remains the only algal species in which mitochondrial transfor­mation has been reported (Larosa and Remacle 2013; Remacle and Matagne 2004).

Transform into

competent E. coli cells and clone cells

Plasmids are isolated from clones and transformed as in В

Microalgae containing gene of interest

Fig. 9.1 a Transformation of microalgae starts with bacterial cloning to replicate the plasmid that is to be transformed into microalgae. The plasmids are then isolated from the cloning organism via DNA isolation techniques. b Transformation of microalgae can be performed by either vortexing glass beads in the presence of algal cells and DNA plasmids, or electroporating algal cells in a plasmid containing solution

Mitochondrial transformation is still not as common as nuclear or chloroplast transformations due to the small size of the mitochondria. This small size makes it difficult to deliver DNA into the organelles by methods that are used in other transformations. Another challenge that mitochondrial transformation faces is the absence of a relevant gene reporter. The presence of numerous mitochondria in each cell is also an obstacle for manifesting the transformed genotype at the level of the whole cell (Koulintchenko et al. 2012). Co-transformation with chloroplast or nuclear genes and initial selection for these markers is a possible work-around that facilitates the recovery of mitochondrial transformants (Remacle and Matagne 2004).

As for transformation methods, nuclear gene transfer can be achieved using var­ious methods, including electroporation, agitation with glass beads or silicon carbide whiskers, particle bombardment, and agrobacterium vector infection (Table 9.1) (Guo et al. 2013). Lack of a cell wall in the recipient cells (e. g., Dunaliella salina)

U

40

or cell wall deficiency is sometimes necessary to achieve the highest rate of trans­formation. One way to weaken the cell wall in Chlamydomonas is pretreating them with the lytic enzyme autolysin. Autolysin is produced by Chlamydomonas itself through pre-incubation of the cells in a nitrogen-free medium to induce autolysin production, followed by collection of the produced enzyme. An alternative to using autolysin is using cell-wall-deficient mutant cells, such as cw15, for transformations (Walker et al. 2005).

Nitrogen source is very important in mixotrophic and heterotrophic cultures of mic­roalgae. Adequate concentration of nitrogen is required for cell growth, while nitrogen limitation is often used to enhance lipid accumulation. Inorganic nitrogen, organic nitrogen, and various waste products have been investigated for biodiesel oil pro­duction (Becker 1994). The use of ammonium as a nitrogen source for Ellipsoidion sp. resulted in higher growth rate and lipid content than when urea and nitrate were used (Xu et al. 2001). On the other hand, Neochloris oleoabundans grew faster and accumulated higher lipid with nitrate than with urea (Li et al. 2008a), but the cell grew poorly in medium with ammonium as the nitrogen source. Complex nitrogen sources are expected to be more effective than simple nitrogen sources in the heterotrophic culture of microalgae, since most of them contain amino acids, vitamins, and growth factors. However, the effectiveness of the nitrogen source depends on the species. For example, nitrate was the best, followed by urea, for the growth of Chlorella vulgaris, while peptone and beef extract did not improve cell growth. Furthermore, ammonium sulfate and ammonium nitrate were less effective than nitrate and urea (Kong et al.

2011) . The type of nitrogen source affects not only the cell growth, but also lipid accumulation. The lipid content of Chlorella vulgaris in mixotrophic culture was highest for peptone, followed by beef extract, but the lipid productivities were low because of low biomass concentration. Ammonium sulfate and ammonium nitrate gave the least lipid content. Potassium nitrate and urea gave intermediate lipid content, yet had the highest productivity as a result of the high biomass content (Kong et al. 2011).

Among the organic nitrogen sources, urea is a promising nitrogen source for large — scale production because it is relatively cheap (Becker 1994; Danesi et al. 2002; Matsudo et al. 2009). With urea as the nitrogen source, the lipid contents of Chlorella sp. decreased with the increase in urea concentration (Hsieh and Wu 2009). The optimal concentrations differ with the nitrogen source. The optimal sodium nitrate and yeast extract concentrations for growth and lipid production by Tetraselmis sp. in mixotrophic culture were 4.70 and 0.93 g/L, respectively (Iyovo et al. 2010). Industrial wastewater rich in nitrogen, such as monosodium glutamate waste, has been reported to be a good and cheap source of nitrogen for cultivation of Rhodotorula glutinis for the production of biodiesel (Xue et al. 2006; Becker 1994). The type of nitrogen source affects the pH of the culture broth. The pH was stable when urea or potassium nitrate were used, but dropped sharply when other nitrogen sources were used.

There are a number of technical bottlenecks that need to be addressed. Some of the

basic questions yet unanswered include the following:

1. In a full-scale field operation, what are the ideal strains of algae that will yield both a high-quality effluent and a high-quality biofuel?

2. What are the most efficient methods for separating and concentrating the algae? The transition from 300 mg/L in the pond to a slurry that is 20 % solids may take two or three steps.

3. What are the most efficient and cost-effective methods for breaking open algae cells for the production of a green crude or the separation of the lipid, aqueous, and solid fractions of the lysed cells?

4. What are the most efficient and cost-effective methods for converting the green crude or purified lipid into a commercially reliable biofuel? In the past few years, several new methods have been developed on a bench and demonstration scale, but no one process has been made the leap to be an industry standard for full-scale systems.

5. The companies that favor large photobioreactor systems have yet to show how their systems could be implemented on a grand scale. There is roughly an order of magnitude difference in capital and labor costs between photobioreactors and open pond systems. For algae-based biofuel system to be commercially viable, the output would need to be on the order of thousands to millions of liters of biofuel per day. While practitioners from the fields of biotechnology and bio­chemical engineering have very reliable data from their bench and demonstra­tion-scale bioreactors, the only two fields of engineering that have a long­standing history of working on systems that reliably process liquids on a grand scale are in the chemical/petroleum industry and wastewater treatment.

6. The photobioreactors can provide the initial step of a high-quality starter culture into an open pond system, but when one takes into account the energy needs and capital cost of mega-scale bioreactors, the economic feasibility of hundreds of hectares of photobioreactors fades rapidly.

7. A number of companies are trying to outdo their competition based on the hopes of genetically altered strains of algae. Considering the fact that it takes the approval of several different local and national regulatory agencies just to restore a disturbed habitat with native plant species, the likelihood of a company being allowed to generate 50 tons/day of genetically altered algae in open ponds could face some very tough opposition that would include regulatory agencies and well-organized citizen groups. In addition, the fate of genetically introduced microbes into the environment can be precarious; for example, consider the failure of engineered Rhizobium strains introduced into soybean fields to inoc­ulate seedlings but were outcompeted by native strains (Kent and Triplett 2002)

8. What will make or break this industry is the ability to produce biofuels on a mega-scale basis with a high degree of reliability. A serious economic analysis is needed for each step (i. e, culturing, harvesting, dewatering, lysing, and bio­fuel processing) in the development of algae-based fuels.

9. Most large wastewater treatment plants that process 200-1000 ML/day are located adjacent to dense urban landscapes with little available room for large — scale algae ponds. The ideal candidates for the system proposed in this chapter would be rural wastewater treatment plants that process 2-40 ML/day. Most often, these plants are located at a good distance from populated areas, and there is ample land that could be developed into algae-culturing ponds. In parts of the world where there is a cold or monsoon season, the plant can revert back to its original treatment process and use the ponds for short-term storage of wastewater.

6.2Conclusion

The ability to culture and harvest algae has improved dramatically over the past five decades. There are numerous treatment options that can be used to make the transition from concept to demonstration to full-scale implementation of algae biofuel programs. This will require the ability to adapt preexisting technologies from several disciplines. Many of the answers are already out there but have yet to put in the proper sequence or combination. There is no one technical solution to make this process commercially viable. As demonstrated in this chapter, it will require contributions from several disciplines to go beyond their technical comfort zones. While this is an emerging field with great promise, it will be built on the fundamental principles of engineering and science.

Acknowledgment H. Ahmadzadeh thanks ATF Committee for the financial support.

Many methods can be used to validate the model’s proposed predictions; however, comparison of in silico predictions with in vivo experiments is a key method for validation. Measuring experimental growth phenotypes at specific conditions can validate the predicted growth under the same conditions. An alternative validation approach is to carry out in silico and in vivo gene deletion experiments (or to compare in silico results with available gene deletion literature) to check whether there is an agreement between model predictions and the actual deletion mutant phenotype. Moreover, omics data from transcriptomics, metabolomics, and pro- teomics experiments can be used to check the consistency of the model’s predicted results. Available simulation debugging tools may be used if the model has poor agreement with experimental data. Further refinement of the model can be done as described in the next section.

Hydrocarbons are able to be ‘milked’ (without cell death) from Botryococcus braunii using a solvent added to the growth medium (Moheimani et al. 2013a). Advances in this research further demonstrated that hydrocarbons could be repeatedly extracted (milked) from B. braunii using the solvent every 5 days for a total of 70 days with no addition of fertilisers (N and P) to the culture (Moheimani et al. 2013b). In this experiment, the cells were not dividing and therefore, nutrients were not required for the production of proteins and other cell elements. Instead, the majority of the light energy was used to convert CO2 to hydrocarbons to replace those previously milked.

Microalgae play an important role in many wastewater treatment facilities around the world. In developing economies in tropical and subtropical countries, waste­water is often treated using facultative ponds or oxidation ponds (Duncan 2004; Rahman et al. 2012). These consist of relatively deep and non-mixed ponds that are spontaneously colonized by microalgae. In these ponds, microalgae serve mainly to supply oxygen for the aerobic oxidation of organic matter present in the wastewater. Because these ponds are relatively deep and are poorly mixed, microalgal pro­ductivity is relatively low; only about 10 ton of dry biomass ha-1 year-1. The microalgal biomass is not harvested at the end of the wastewater treatment process, and either settles to the bottom of the pond or is washed out of the ponds. Because the microalgal biomass is not harvested, removal of nutrients from the wastewater by the microalgae is inefficient.

At the end of the 1950s, high-rate algal ponds (HRAPs) were proposed as an alternative to facultative ponds (Oswald and Golueke 1960). HRAPs are raceway — type ponds in which the water is mixed by a paddle wheel. Compared to facultative ponds, HRAPs are much shallower and better mixed and, as a result, have higher microalgal productivity, about 30 ton dry biomass ha-1 year-1. The productivity of HRAP’s can be further increased with CO2 addition (Craggs et al. 2013). Because microalgal productivity is higher, a larger volume of wastewater can be treated on the same land area when compared to facultative ponds. Akin to facultative ponds, microalgae in HRAPs supply oxygen for the aerobic oxidation of organic matter, and if the microalgal biomass is harvested at the end of the treatment process, the microalgae remove nutrients from the wastewater. Harvesting of the microalgal biomass, however, is costly and many HRAPs today do not harvest the biomass. Although HRAPs are used in wastewater treatment plants around the world, the technology is much less widespread than oxidation ponds or conventional elec­tromechanical wastewater treatment systems (Craggs et al. 2013).

Computational tools that help to improve synthetic biology have been developed and are currently being expanded. Improvement of algae production to increase biofuel yields through synthetic biology involves many distinct processes that can be aided by different computational tools. Genome-scale metabolic network reconstructions and models are available for a number of algal species. These can be useful for identifying and selecting gene targets for knockout and strain engi­neering. Some of the available tools and algorithms that are able to perform such tasks include (but are not restricted to) Optknock (Burgard et al. 2003) and Optstrain (Pharkya et al. 2004).

The standard approach for computing metabolic fluxes is flux balance analysis (FBA) through using toolboxes such as COBRA or Pathway Tools (these are discussed in the accompanying Chap. 10, Towards applications of Genomics and Metabolic Modeling to Improve Biomass Productivity). FBA allows prediction of optimal flux distribution throughout the network for a given cellular phenotypic state. Through the use of computational tools associated with FBA, consequences of changes introduced in a metabolic network can be predicted. For example, quantitative mapping of intracellular fluxes in relation to single or multiple gene deletions can easily be carried out by FBA. There are many strategies available to predict alterations that result in increased production of a desired metabolite. For instance, one method entails identifying key and relevant pathways that are impacted using simulated gene knockouts (Reed et al. 2010). The accuracy of this method can be enhanced by integrating experimental data; such as metabolite concentration, gene expression data, and uptake and secretion rates.

‘Pathway Tools’ (Karp et al. 2010) is an integrated reconstruction, analysis, and visualization software created by the Bioinformatics Research Group at SRI Inter­national (http://bioinformatics. ai. sri. com/ptools/). Pathway tools can automatically

generate organism-specific metabolic network databases and provides details of genes/proteins, reactions, and compound associations, as well as create pathway databases (called Pathway Genome Database, or PGDBs). To create a PGDB, one of the components of the Pathway Tools called PathoLogic is used. This tool allows users to create PGDBs using the genome annotation of an organism of interest directly from the organism’s GenBank annotation file. Users can manually adjust, edit, or add (new) content as needed. There are already many well developed and intensively curated pathway databases or PGDBs including BioCyc, EcoCyc and MetaCyc (Caspi et al. 2014), which aid metabolic analysis and network recon­structions. BioCyc alone has a collection of about 3530 PGDBs, which users can query, visualize, manage and analyze. Among these, algal PGDBs include Tha — lassiosira pseudonana, Nannochloropsis gaditana, Acaryochloris marina, Ana — baena cylindrica, Anabaena variabilis, Synechococcus elongatus and

Chlamydomonas reinhardtii. Other offered functionalities in Pathway Tools include tools that can be used in downstream analyses to identify the shortest path between two metabolites, identify dead-end metabolites, fill pathway gaps, identify choke — points (potential drug targets), and infer operons and transport reactions. Many new metabolic reactions have been added to EcoCyc using the dead-end metabolite analysis approach (Mackie et al. 2013). Pathway Tools can aid synthetic biology experiment designs by identifying potential pathways, which may be targets for alterations.

Several unicellular green algae are capable of generating hydrogen through their [FeFe]-hydrogenase enzyme by reducing water protons to molecular hydrogen. However, given the sensitivity of [FeFe]-hydrogenase to oxygen, which is gener­ated by photosystem II (PSII), new approaches have been developed for increasing the practical application of microalgae for biohydrogen production (Laurinavichene et al. 2008). For instance, higher hydrogen production efficiencies were achieved by growing the microalgal cells under sulfur-deprived conditions (Melis et al. 2000). Sulfur deprivation causes partial inactivation of PSII, which is responsible for O2 generation, resulting an enhanced synthesis of [FeFe]-hydrogenase enzyme (Laurinavichene et al. 2008).

Immobilization processes have been proposed by several researchers for enhancing hydrogen production by sulfur-deprived microalgae and also allowing an easy exchange step between “sulfur-replete” and “sulfur-depleted” stages of the experiment (Laurinavichene et al. 2006, 2008). Several challenges require addressing for scaling up the current hydrogen production systems, while immo­bilization processes offer an alternative approach to the current technology. Immobilized cells were also reported to have higher light utilization efficiencies per area and higher cell densities (Kosourov and Seibert 2009).

In a study by Kosourov and Seibert (2009), C. reinhardtii cells were immobi­lized inside alginate films for the photoproduction of hydrogen. The cells were previously deprived of sulfur and phosphorus nutrients before being entrapped inside the alginate films. They observed higher cell densities and specific hydrogen production rates after the immobilization process. An immobilization strategy also provided easy protection of the hydrogenase enzyme from oxygen inhibition, yielding higher hydrogen production rates compared to the free cells.

Laurinavichene et al. (2006, 2008) used immobilized C. reinhardtii cells on a fiber glass matrix under sulfur-deprived conditions and observed a prolonged hydrogen production phase for the immobilized cells, while the specific hydrogen production rate was similar to the free-cell counterparts. In another study, algal cells were immobilized on fumed silica particles, which had similar hydrogen production rates with the suspended cultures (Hahn et al. 2007). Song et al. (2011) recently used agar-immobilized Chlorella sp. cells for a two-stage cyclic hydrogen pro­duction involving the oxygenic photosynthesis followed by anaerobic incubation under sulfur-deprived conditions.