In comparison to crystalline solar cells, thin-film solar cells use significantly less material. The development of thin-film solar cells was in part driven by high material costs of crystalline silicon. Thin-film solar cells were much more efficient in terms of material usage and had potentially lower fabrication costs. Thin-film solar cells are fabricated from layers of doped semiconducting materials. These layers produce a charge separating junction often in the form of a p-i-n junction (McEvoy et al. 2003). As they use significantly less material and are inherently thin, thin-film solar cells can be semitransparent to visible light. When a transparent substrate (such as glass) is used, this can allow some irradiance to pass through the device (Shah et al. 2004). Although a single-junction thin-film solar cells can be relatively inefficient in comparison with their crystalline counterparts, several junctions can be stacked so as to produce a more efficient device (Shah et al. 2004). These multi-junction devices can be made from identical junctions or the junctions can be tuned to different wavelengths and parts of the solar spectrum so as to absorb as much of the spectrum as possible. There are a range of examples of thin-film solar cells, and one of the most well-known examples is amorphous silicon.

Hydrogenated amorphous silicon solar cells have been in development since the late 1970s (Wilson 1980). Although thought to be a good alternative to crystalline silicon, amorphous silicon solar cells had one significant drawback in the form of a light-induced degradation, photodegradation, known as the Staebler-Wronski Effect. This is the process whereby the performance and efficiency of the amor­phous silicon solar cell degrade upon extended exposure to light (Staebler and Wronski 1977). The degradation occurs over a period of time as the solar cell is exposed to light. The efficiency and performance of the cell degrade asymptotically to a stabilized minimum, upon which point the stabilized cell does not degrade any further. Any additional exposure to light after this point has minimal effect on the solar cell’s performance. This light-induced degradation can be revered by annealing the cell above 150 °C for a period of time (Staebler and Wronski 1977). Despite these drawbacks to amorphous silicon, a thin-film amorphous silicon solar cell has been produced on a antireflection-coated glass substrate with a reported stabilized efficiency of 9.47 % (Meier et al. 2004). The spectral response from this solar cell is shown in Fig. 15.3. As can be seen, the device had lower quantum efficiency in the blue and the infrared portions of the spectrum compared to the crystalline silicon PERL solar cell. However, it does have a comparable peak in efficiency in other parts of the solar spectrum.

The economic viability of the current microalgae to fuel (or chemical) processes (summarised in Fig. 1.1) is limited by the high cost and energy burdens for growth inputs, capital and operating costs for dewatering, and the operating and capital costs of the growth system (Clarens et al. 2010; Lardon et al. 2009; Stephenson et al. 2010). Advances in growth, harvesting and extraction systems provide incremental improvement to these systems. The persistence of companies/research institutions and governments in continuing to pursue these systems indicates that many believe that such incremental improvement over time will ultimately result in an economically viable process. Others believe that a step change is required and are pursuing an entirely different biofuel production model in which the product of interest is continually secreted by the microalgae. This novel method is generally referred to as ‘milking’, as the product of interest is ‘milked’ from the algae without the need to destroy it and subsequently regrow it.

Koenraad Muylaert, Annelies Beuckels, Orily Depraetere, Imogen Foubert, Giorgos Markou and Dries Vandamme

Abstract Production of microalgal biomass requires large amounts of nitrogen (N) and phosphorus (P). The sustainability and economic viability of microalgae pro­duction could be significantly improved if N and P are not supplied by synthetic fertilizers but with wastewater. Microalgae already play an important role in wastewater treatment, yet several challenges remain to optimally convert waste­water nutrients into microalgal biomass. This book chapter aims to give an over­view of the potential of using wastewater for microalgae production, as well some challenges that should be taken into account. We also review the benefits of combining microalgal biomass production with wastewater treatment.

5.1 Introduction

Microalgae have a high areal productivity, do not require fertile land, and are seen as a promising new source of biomass that could complement production by conventional agricultural crops. Microalgae have attracted a lot of interest in recent [1] [2]

years as a novel feedstock for biofuels (Schenk et al. 2008). But microalgae also hold a lot of potential for production of food (Draaisma et al. 2013), animal feed (Benemann 2013), or feedstock for the chemical industry (Wijffels et al. 2010). When compared to conventional agricultural crops, microalgae have a high content of proteins and lipids, and a low content of structural carbohydrates such as cel­lulose (Lam and Lee 2011). This is an attractive property of microalgae, because it implies that most of the biomass can be valorized. On the contrary, with conven­tional crops, only a small fraction of the biomass is used (e. g., seeds or tubers) and a large proportion of the biomass is left on the field (i. e., the fraction that contains mostly cellulose and lignin). Because of the low content of structural carbohydrates such as lignin or cellulose, microalgal biomass has a high content of nitrogen (N) and phosphorus (P): about 10 % N and 1 % P per unit dry weight. This is almost three times higher than the N and P content of terrestrial plants (Elser et al. 2000). Because of the high content of N and P in microalgal biomass, production of microalgae requires vast quantities of inorganic fertilizer, much more than the production of terrestrial crops (Sialve et al. 2009). This high fertilizer demand is a challenge to the sustainability of microalgae biomass production, and several life cycle analyses studies have shown that the energy required for synthetic fertilizer production contributes significantly the total energy demand for microalgal biofuels (Lardon et al. 2009; Clarens et al. 2010; Benemann et al. 2012). Production of N fertilizers through the Haber-Bosch process is highly energy-intensive and is reliant on fossil fuels (Smil 2002; Pfromm et al. 2011). Extraction and processing of mineral phosphates for production of P fertilizer is also energy-intensive (Johnson et al. 2013). Moreover, mineral phosphates reserves are limited and are rapidly being depleted (Cordell et al. 2011). If microalgae are to be produced on a large scale, e. g., large enough to contribute significantly to fuel demand, consumption of synthetic fertilizers is expected to increase strongly above current levels (Venteris et al. 2014). Microalgae are often promoted as a biomass source that does not compete with agricultural biomass production, and thus avoids the food versus fuel discussion. However, as both microalgae and agricultural crops require mineral fertilizers, microalgae may indirectly compete with agricultural crops and thus indirectly impact food production through increases in fertilizer prices (Pate et al.

2011) . Many studies and opinion papers have in recent years suggested to use wastewater rather than synthetic fertilizers as a source of nutrients for microalgae production and showed that this could significantly improve the sustainability and economic feasibility of microalgae production (Lundquist et al. 2010; Clarens et al. 2010; Christenson and Sims 2011; Pittman et al. 2011; Park et al. 2011b; Olguin 2012; Prajapati et al. 2013). Because microalgae are already being used for wastewater treatment, replacing synthetic fertilizer with nutrients from wastewater is feasible. The goal of this book chapter is to discuss both the potential as well as limitations of using wastewater as a nutrient source for microalgae production.

Part registries are lists and collections of standardized biological parts that can be strung together to build advanced and more complicated biological devices. Parts in this context mean DNA constructs that encode a given function. On the other hand, biological devices perform more complicated functions and are made from combining different parts. An example would be that a part encodes a specific enzyme in a pathway, while a device performs a complicated function like biological arsenic detection or bioplastics production. These registries are a list of DNA constructs (parts) and synthetic circuits (biological devices) that are ready for genetic insertion. The idea behind creating such registries is to list reliable and well tested biological parts that can be manufactured to a given standard and that serve as the building blocks of sophisticatedly engineered biological tools and devices (Canton et al. 2008). This standardization liberates the process of designing and fabricating complex biological devices from the daunting task of designing and fabricating each individual and necessary component. In addition, the fabricators of those devices can rely on specialized manufacturers of those standard parts to provide the components needed for fabricating the advanced biological devices. All in all, this has the effect of opening up the field and providing the impetus for more advanced applications (Baker 2006).

In biological part registries, standardization is of essence. Standardization in the context of biological parts refers to the ability of a part to assimilate into a larger structure without any complications. In other words, the part does not have any restriction sites that interfere with the process of assembling into larger devices. Another important aspect of standardization is with respect to function. Biological parts should serve a given function that is both well-defined and consistent. Perhaps the most widely known and used registry is the Registry for Standard Biological Parts (http://parts. igem. org/Main_Page) (Kahl and Endy 2013). The registry, like most other registries, relies on community contributions to expand its biological part offerings. The community accessible approach of the registry has expanded the registry’s offering to include thousands of parts serving numerous functions. The registry’s part types include promoters, ribosome binding sites, protein domains, protein coding sequences, translational units, terminators, plasmid backbones, primers and composite parts which are a composition of two or more simpler parts. To ensure openness, efficiency and consistency, parts offered by the registry comply with the BioBricks assembly standard (http://parts. igem. org/Help:Standards/Assembly). The current standard relies on defining a DNA prefix and suffix on a standard plasmid backbone. The part is then inserted into the plasmid backbone specifically between the prefix and suffix. The prefix and suffix also contain specific restriction sites, and this precise definition that is included in the standard also forbids the introduction of restriction sites that interfere with the part assembly and usage process. In general, those features allow the smooth and immediate use of the biological parts.

Algae-specific registries with the ambition of the registry for standard biological parts are yet to be fully developed. However many repositories are available that provide cell lines, several thousand algal strains, DNA constructs, and specific genetic engineering tools. One prominent example is the Chlamydomonas Resource Center, a repository of Chlamydomonas reinhardtii strains, plasmids, kits, and cDNA libraries among other things (http://chlamycollection. org/). While most of these registries are not registries of synthetic biological parts specifically, they still offer many tools and products of value to synthetic biology endeavors in algae. Also, the availability of standard and customized algal optimized plasmids prepared and sold by private companies, such as Life Technologies, (a brand of Thermo Fisher Scientific, Carlsbad, CA, USA), is another step forward into easing up synthetic biology applications with microalgae.

While the breadth of registries and repositories up and running is a call for optimism in the field of synthetic biology, certain challenges still permeate the part registry model. For example, characterization of many biological parts and the precise definition of their functions are still lacking. Furthermore, many parts display different behavior in different cells or organisms and in different laboratory conditions; this introduces a major challenge to the field with respect to repro­ducibility of function. Stability and reliability become even more daunting chal­lenges as the organism’s complexity increases. This means that extending biological parts and the registry model to algae, or organisms of higher complexity than simple microbes, becomes additively challenging quite quickly. Still, another challenge is in the long-term behavior of parts and their behavior as components of increasingly complex devices. Cell functions are prone to seemingly random behavior and noise which can complicate the ability of biological parts and complex devices to behave consistently for a significantly long period (Kwok 2010).

Nuclear Transformation There are a variety of established transformation meth­ods to integrate heterologous DNA into the nuclear genome including particle bombardment (Debuchy et al. 1989; Kindle et al. 1989; Mayfield and Kindle 1990), agitation of cell-wall-deficient strains with glass beads (Kindle 1990), electropor­ation (Shimogawara et al. 1998), agitation with silicon carbide whiskers (Dunahay 1993) and biologically mediated gene transfer by Agrobacterium tumefaciens (Kumar et al. 2004). In C. reinhardtii, transformation of the nuclear genome occurs by random insertion through non-homologous end joining (Tam and Lefebvre 1993) or by using linear DNA that promotes the insertion of multiple copies in one locus (Cerutti et al. 1997b). Phenotypic and genetic screening of transformants can minimise undesirable non-target effects of the random insertion of a transgene that can lead to disrupted genes or regulatory elements. In return, this effect is used to study genes of unknown function using high-throughput insertional mutagenesis (Dent et al. 2005). In C. reinhardtii, targeted gene integration through homologous recombination (HR) using single-stranded transforming DNA (Zorin et al. 2005,

2009) is possible, but only at low frequencies to date. However, high rates of homologous recombination have been reported in another green algal species Nannochloropsis (Kilian et al. 2011), showing promise for reverse genetics and targeted gene knockouts.

Random and Insertional Mutagenesis The creation of mutants of an organism by the use of irradiation or chemical mutagenesis is the classic approach pioneered by Morgan in Drosophila and used for a century to study the effect of significant alteration in the behaviour of a gene, typically by partial or total deletion. These methods produce base pair changes leading to a range of disturbances including altered amino acid sequence, small deletions, truncations, frameshifts and splicing defects; the resultant mutants include temperature-sensitive and dominant mutants as well as functional knockouts sometimes giving rise to complex phenotypes. In the last 3 decades, the insertion of foreign transgenes (usually carrying a marker to allow selection and identification) has also been well established, as described above. Insertion mutants usually have knocked out or disabled genes, though insertion into promoters can lead to alterations in expression levels and splicing. Gene inactivation is largely on a random basis (Zhang et al. 2014) across the genome (at least where the chromatin structure is sufficiently open) which allows the unbiased identification of genes relevant to biological processes and has been very useful for research into biological mechanisms and physiology in Chla — mydomonas and other algae. The difficulty is usually that a specific phenotypic screen is needed to identify relevant genes. Lethal mutations will not be identified, nor will mutants in genes which are redundant or which show no overt phenotype under the conditions of the screen. Of the estimated * 15,000 genes in the Chla- mydomonas genome, only a few hundred have been reported in the literature, and many genes known to be important are not represented in collections of mutants. A good example is the multigene family of LHC genes. An insertion mutant in a single LHC gene will usually not produce any easily measurable phenotype, due to compensation by other LHCs, while the highly specific physiological or genetic tests needed to demonstrate the loss of a specific LHC gene are not suitable for screening assays. Finally, each transformation produces only a few hundred colo­nies, with very likely a biased set of genes being affected. This makes it a major project to uniquely identify mutants of each gene in an alga. Fortunately, this is being performed at Stanford University, and the resulting collection of mutants will be an invaluable research resource for the Chlamydomonas community (Zhang et al. 2014). However, it is unlikely that this resource will be duplicated for every species of algae of research or commercial interest.

Homologous Recombination (HR) in the Nucleus Homologous recombination, the recombination between homologous DNA sequences, is essential for eukaryotes to repair DNA double-strand breaks and introduce genetic diversity during cell division, and two main pathways (‘double-strand break repair’ and ‘synthesis — dependent strand annealing’) have been proposed (Sung and Klein 2006). In plants and algae, nuclear located RecA homologues show high similarity to the prokary­otic RecA genes which suggests an endosymbiotic transfer from mitochondria and chloroplasts to the nucleus of ancestral eukaryotes (Lin et al. 2006). Although the introduction of foreign DNA into the nucleus occurs predominantly via random insertional mutagenesis, successful targeted homologous recombination has been reported in several algal species such as C. reinhardtii (Gumpel et al. 1994; Sodeinde and Kindle 1993; Zorin et al. 2009), Nannochloropsis sp. (Kilian et al.

2011) and Cyanidioschyzon merolae 10D (Minoda et al. 2004) with as little as 230- bp DNA sequence homology in the haploid cell (Gumpel et al. 1994). It has been demonstrated that the introduction of single-stranded repair DNA leads to a more than 100-fold reduction of non-homologous DNA integration in comparison with double-stranded DNA (Zorin et al. 2005). Attempts to increase the low frequency of homologous recombination in plants by the over-expression of well-characterised enzymes involved in homologous recombination such as the recA and ruvC pro­teins have been reported to increase homologous recombination and double-strand break repairs; however, these reports suggest that foreign gene targeting is not improved (Reiss et al. 1996, 2000; Shalev et al. 1999).

RNA Interference (RNAi) In the absence of tools for precise genome manipula­tion, RNAi-mediated knock-down of gene expression enables the creation of highly specific research mutants without the need for phenotypic screening or selection. RNAi techniques also enable the study of reduced levels of gene expression where total ablation would be lethal. The phenomenon of RNA interference is produced by the action of specific cellular machinery [Dicer and Argonaute (AGO) proteins (Casas-Mollano et al. 2008; Cerutti and Casas-Mollano 2006)] on mRNAs, guided by microRNAs which are naturally occurring small double-stranded RNAs (dsR — NAs) in a process called mRNA cleavage (Bartel 2004). RNAi represents the use of this natural process for experimental ends by the artificial provision of small RNA molecules designed to interfere with the expression of a target gene. MicroRNAs (miRNAs) have also been discovered in C. reinhardtii (Molnar et al. 2009; Zhao et al. 2007) enabling a highly specific genetic tool (Moellering and Benning 2010; Molnar et al. 2009; Schmollinger et al. 2010; Zhao et al. 2007). Difficulties in achieving direct nuclear gene knockout via homologous recombination for C. reinhardtii (Nelson and Lefebvre 1995) led to RNAi becoming a widely used method to accomplish post-transcriptional gene silencing for gene function dis­covery (knock-down approaches via reverse genetics) and metabolic engineering (Fuhrmann et al. 2001; Molnar et al. 2009; Rohr et al. 2004; Schroda et al. 1999; Zhao et al. 2009). Artificial microRNAs have also been engineered to create functional knock-downs of several nuclear genes in Dunaliella salina (Sun et al. 2008) and in the diatom Phaeodactylum tricornutum (De Riso et al. 2009). This method could potentially be used for algal genetic engineering for biofuel pro­duction (Cerutti et al. 2011; Grossman 2000; Wilson and Lefebvre 2004). The weakness of RNAi is the need to maintain the expression of the RNAi construct, typically requiring ongoing selective pressure, for example with antibiotics.

Genome Editing The ability to precisely edit the genome enables the specific deletion or mutation of genes and regulatory regions, with the resultant ability to create targeted mutations for study or industrial applications. As the resultant mutations are permanent, the stability problems inherent in RNAi knock-down constructs are eliminated. Genome editing also offers the prospect of precise mutants lacking foreign DNA, which consequently are technically non-GMO organisms. Several systems of genome editing have been developed in recent years, including zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), meganuclease and the CRISPR/Cas system. Both ZFN (Sizova et al. 2013) and TALENS (Gao et al. 2014) have been used for genome editing in Chlamydomonas. The drawback of these systems is the substantial investment of time and effort required. In contrast, the CRISPR/Cas system promises a simpler, more facile approach. CRISPR (clustered regularly interspaced short palindromic repeats) sequences are found in prokaryotes and are short repetitive DNA sequences, corresponding to part of the DNA of bacteriophage genomes. In con­junction with a specific nuclease (Cas), they form the basis of a prokaryotic ‘immune system’ to recognise and eliminate viral genomes. The CRISPR RNA acts as a guide for Cas-mediated cleavage of the viral DNA or provirus. For genome editing, first described in 2013 (Cho et al. 2013; Mali et al. 2013), the addition of a targeted ‘guide RNA’ with co-expression of the Cas9 nuclease enables precise and specific genome editing and has been rapidly adopted in many organisms including both animals (Hsu et al. 2014) and plants (Feng et al. 2013). Although first attempts to use CRISPR/Cas in algae have encountered difficulties, it is anticipated that these will be overcome in the near future, enabling rapid and flexible genome editing in algae.

Chloroplast Transformation The chloroplast is the site of photosynthesis and storage of the resultant starch and is also important for the production of fatty acids and photosynthesis-related pigments especially carotenoids. Transformation of the chloroplast requires transfer of the transforming DNA to the interior of the chlo — roplast, and consequently, particle bombardment (biolistics) using DNA-coated gold or tungsten particles is the most commonly used method for chloroplast transformation. Some particles penetrate the cell wall, plasma and chloroplast membrane and deposit the transforming DNA in the plastid, which can then inte­grate into the local genome through homologous recombination (Boynton et al. 1988). Flanking endogenous sequences that are homologous to the targeted inser­tion site can make chloroplast transformation events highly targeted to any region in the genome (Rasala et al. 2013) which in C. reinhardtii is a great advantage over nuclear transformation where homologous recombination occurs only at low effi­ciency (Sodeinde and Kindle 1993).

Homologous Recombination in the Chloroplast In the chloroplast, homologous recombination is mediated by an efficient RecA-type system which, due to its homology to the Eschericia coli RecA system, is suggested to be related to the cyanobacterial ancestors of chloroplasts (Cerutti et al. 1992, 1995; Inouye et al. 2008; Nakazato et al. 2003). In cyanobacteria, the RecA system is essential for cell viability especially under DNA damaging conditions (Jones 2014; Matsuoka et al. 2001). DNA repair is also believed to be the main function for homologous recombination in the chloroplast (Cerutti et al. 1995; Rowan et al. 2010). Several different models exist for the precise mechanism of homologous recombination and are not presented in detail here; however, the main steps of prokaryotic homologous recombination include initial strand breakage, formation of an enzyme complex to unwind the double-stranded DNA to form a single strand, followed by a mechanism called ‘strand invasion’ which searches for similar sequences on a homologous DNA fragment for pairing. This is then followed by DNA synthesis in accordance with the new template strand and resolution of the structure (Amundsen et al. 2007; Smith 2012).

Genome Stability in the Chloroplast With 50-100 genome copies, the chloroplast is highly polyploid, and apart from a few exceptions, each plastome shows a tet — rapartite organisation containing two inverted repeat sequences that are mirror images of one another, separated by a large and a small single-copy unit. Though absent in present-day cyanobacteria and not essential for the general chloroplast genome function, the inverted repeat sequences show properties which suggest their involvement in gene maintenance and increased genome stability (Goulding et al.

1996) . Chloroplast genomes can undergo homologous recombination between the inverted repeat sequences as several studies have shown two populations of plast — omes in the same organism differing only in the single-copy sequence orientation (Aldrich et al. 1985; Palmer 1983; Stein et al. 1986). Furthermore, it has been observed that the inverted repeat regions accumulate nucleotide substitution muta­tions 2.3 times more slowly than the single-copy regions (Perry and Wolfe 2002; Ravi et al. 2008; Shaw et al. 2007). It was also demonstrated that DNA rearrange­ments occur more frequently when a large inverted repeat sequence is lost (Palmer and Thompson 1982). This suggests that homologous recombination between plastomes and inverted repeats contributes to this increased genome stability.

Manipulation of the chloroplast genome via homologous recombination offers the potential for exact gene deletions and insertions. Generally, chloroplast trans­formation vectors are E. coli plasmid derivatives carrying the foreign DNA flanked by DNA sequences (>400 bp each) which are homologous to the target region of the plastid DNA (Bock 2001; Hager and Bock 2000). Due to the high ploidy of the plastid genome and the fact that initially only a single plastome copy is transformed, the resulting phenotype may be weak in primary transformants and it is important to establish an efficient and suitable selection system or strategy to identify and enrich cells containing transformed ptDNA copies, which may easily revert to wild type (Hager and Bock 2000; Rasala et al. 2013). Once the genome is homoplastomic and thus lacking template for further undesired homologous recombination events, the selectable marker can be removed (Day and Goldschmidt-Clermont 2011), though selective pressure against the transgene can still exist.

Mitochondrial Transformation Respiration and photosynthesis are coupled pro­cesses, and mitochondrial mutations are known to affect photosynthesis (Schonfeld et al. 2004) as well as many other aspects of cellular metabolism. Although in Chlamydomonas homologous recombination in mitochondria DNA is only detected after crosses between different mating types in mitotic zygotes (Remacle et al.

2012) , it demonstrates that the cellular machinery is available for recombination — based foreign gene integration. Although the first transformation of the mito­chondrion was published in 1993 (Randolph-Anderson et al. 1993) using biolistic bombardment, reports of mitochondrial transformation are rare and initially limited to the restoration of the wild-type mitochondria genome in mutant strains. In 2006, Remacle et al. reported the first and, to date, the only modification of the mito­chondrial genome in vivo in a photosynthetically active organism. The work pre­sented the introduction of a nucleotide substitution in the cob gene, conferring resistance to myxothiazol, and an internal deletion in nd4 (Remacle et al. 2006). Homologous recombination was facilitated with as little as 28-bp homology between the introduced and endogenous DNA (Remacle et al. 2006).

Selection Techniques Following Transformation The identification of chloroplast and nuclear transformants can be achieved through the rescue of mutants impaired in endogenous cellular functions (non-photosynthetic, flagellar or auxotrophic mutants) or the introduction of antibiotic and herbicide resistances by point mutation of endogenous genes or the expression of heterologous resistance genes. A range of auxotrophic mutants have been developed (including mutations in arginine biosynthesis, nitrate reductase, nicotinamide biosynthesis and thiamine biosynthesis (Debuchy et al. 1989; Ferris 1995; Kindle et al. 1989; Rochaix and Vandillewijn 1982)) as well as mutants in photosynthetic capability or flagellar motility (Diener et al. 1990; Mayfield and Kindle 1990; Mitchell and Kang 1991; Smart and Selman 1993).

Classical antibiotic resistance markers include kanamycin [chloroplast: (Bateman and Purton 2000); nuclear: (Hasnain et al. 1985; Sizova et al. 2001)], spectinomycin/ streptomycin [chloroplast: (Goldschmidt-Clermont 1991); nuclear: (Cerutti et al. 1997a)], neamine/kanamycin and erythromycin [chloroplast: (Harris et al. 1989)] paromomycin and neomycin [nuclear: (Sizova et al. 2001)], cryptopleurine and emetine [nuclear: (Nelson et al. 1994)], zeocin and phleomycin [nuclear: (Stevens et al. 1996)] and hygromycin B [nuclear: (Berthold et al. 2002)]. Screening for resistance to herbicides, such as atrazine, which inhibits the function of photosystem II [chloroplast: (Erickson et al. 1984)], or Basta, leading to disruption of the chloroplast structure [chloroplast: (Cui et al. 2014)], is also possible. The use of antibiotic resistance markers is not problematic for research purposes, but is less desirable for industrial-scale production of vaccines or therapeutics, and for large — scale outdoor or agricultural production, antibiotic-free media is usually required for both sociopolitical and socio-economic reasons.

Recombinant Protein Expression Regardless of the energy return on investment (EROI) of microalgal biocrude, the low net value poses a significant problem for economic biocrude production from microalgae. One potential strategy to offset this is to exploit the fact that high-value products such as recombinant proteins are typically only a small fraction by weight of the biomass and could potentially be extracted prior to thermochemical processing of remaining biomass to biocrude. Consequently, an additional and lucrative revenue stream can arguably be obtained to subsidise the process without sacrificing overall biofuel productivity. The demand for recombinant proteins is growing with increasing population and bio­technological applications, and ultimately, the expression systems with the greatest cost-benefit are likely to dominate these markets. In this context, microalgal sys­tems offer significant advantages over traditional microbial fermentation or mam­malian cell culture systems. So-called molecular ‘pharming’ is the production of pharmaceutical proteins, therapy peptides, vaccine subunits, industrial enzymes and secondary metabolites or other compounds of interest in plants, and algae also offer commodity-scale opportunities. Proteins with biopharmaceutical or biotechnologi­cal relevance can be, e. g. monoclonal antibodies, vaccines, blood factors, hor­mones, growth factors and cytokines.

Nucleus Although the chloroplast is the expression system of choice for high protein expression levels, the nucleus as expression location has its advantages which have to be carefully considered in the context of each product. Despite the comparatively low expression rate of 1 % of TSP claimed by commercial vendors and the risk of gene silencing, nuclear expression tools enable the fusion of the desired product to a selection marker (to avoid silencing) with subsequent self­cleavage, as well as secretion of the expressed compound into the media (Lauersen et al. 2013; Rasala et al. 2012). So far, most proteins expressed from the nucleus have been reporter genes used to quantitatively measure protein expression levels as well as the localisation and accumulation (e. g. within the C. reinhardtii cell) (Rasala et al. 2013). Six fluorescent proteins (blue mTagBFP, cyan mCerulean, green CrGFP, yellow Venus, orange Tomato and red mCherry) were expressed from the nuclear genome to allow protein detection in whole cells by fluorescence microplate reader analysis, live-cell fluorescence microscopy and flow cytometry.

Chloroplast The production of recombinant proteins including reporters (e. g. GUS, luciferase or GFP) (Franklin et al. 2002b; Mayfield and Schultz 2004; Minko et al. 1999; Sakamoto et al. 1993), protein therapeutics (antibodies, hormones, growth factors or vaccines) (Franklin and Mayfield 2005; Manuell et al. 2007; Mayfield and Franklin 2005; Mayfield et al. 2003; Rasala et al. 2010; Surzycki et al. 2009) and industrial enzymes has established the chloroplast as a suitable production platform for a broad range for protein candidates. The potential for high expression levels [over 40 % of TSP (Surzycki et al. 2009)] and the ability to form complex proteins including disulphide bonds (Tran et al. 2009) allow for limited post­translational modification without interference caused by ‘gene silencing’ making the chloroplast the site of choice for protein expression. Furthermore, the pro­karyotic characteristics of the chloroplast genome such as gene organisation into operon-like structures (Holloway and Herrin 1998) potentially allow the expression of multiple transgenes from a single operon (transgene stacking) and with this the introduction of complete biochemical pathways. The additional development of a Gateway-compatible transformation system (Oey et al. 2014) allows the rapid production of multiple transformants. Success rates and costs for successfully establishing expression of new proteins in C. reinhardtii are estimated to be similar to those of yeast and mammalian cells (Rasala et al. 2013), but with potentially much lower production and purification costs.

Microfiltration employs the use of filtration media with <1 pm pores and a pressure differential created by flow to dewater biomass slurry. Various conformations of filtration media that are made from various materials are available at industrial scale, including hollow fibers, plates, and spiral wound. The membranes can be made of polymers (e. g., PVDF, PES, PS), ceramics, or metals. There is an established history of using microfiltration technology for the purpose of cell harvesting, as in the fermentation industry and wastewater treatment. Advantages of microfiltration are that the algal cells retain their structure, properties, and motility (Chen et al. 2011).

Microfiltration methods have been advancing rapidly since the early studies in algal harvesting. Stacked filters that are subject to blocking have been replaced by hollow fiber filters and tangential flow filters that are now being applied to dewa­tering of extremely dirty solutions, such as those in wastewater treatment plants (e. g., Koch Membrane Systems PURONPlus MBR (http://www. kochmembrane. com/Engineered-Systems/Standard/PURON-MBR. aspx). Blocking of the filter pores (membrane fouling) by algal and bacterially derived materials, due to their small cell sizes, is a chief problem of any of these microfiltration methods and is a concern for microfiltration (Bosma et al. 2003). The use of hollow fiber filters and tangential flow filters provides some scouring of the membrane surface to help reduce blockage and provide high flux rates.

Not much data are publically available on the performance of membrane fil­tration at an industrial scale for algal biofuel systems. Important performance values include flux rate (LMH or L m-2 h-1), recirculation rate (L min-1), and filter pressures (kPa). However, engineers at Phycal, Inc. performed studies at a subpilot scale (hundreds of liters) using polymeric, hollow fiber filtration membranes with

0. 2-pm pore sizes. A typical performance over an 8-h trial for the alga Chlorella vulgaris (Fig. 14.1.) demonstrated steady state flux rates around 150 LMH, and a final biomass concentration of 22 g/L. Other trials with similar performance metrics were able to reach 80 g/L, showing this membrane filtration system could provide primary and secondary harvesting for an algal biofuel production system that could tolerate significant water content, such as aqueous extraction and hydrothermal liquefaction. However, because flux rates are lower when biomass concentrations

are higher, scaled systems would have two or more filtration stages with different operating parameters, membrane pore sizes, and operating concentrations at each stage in order to optimize throughput and lower OpEx (e. g., pumping and mem­brane replacement).

Work on improved microfiltration in conjunction with algal biofuels continues with novel metallic membranes being developed, which are proposed to lower the membrane replacement costs (lower OpEx) and lower overall fouling to increase throughput (NAABB 2014).

Lipids contained within microalgae are attractive for the production of methane biogas. Lipids have a higher theoretical methane potential when compared to proteins and carbohydrates (Zamalloa 2012). However, due to the chemistry of lipids and the low alkalinity and buffering capacity associated with them, high lipid concentrations can be inhibitory to anaerobic digestion (Park and Li 2012; Ward et al. 2014). Inhibition by lipids is caused by the intermediate products produced during their breakdown, such as long chain fatty acids and VFAs (Park and Li 2012). When the lipid content of the microalgae is below 40 %, it has been sug­gested that the direct conversion of microalgae to methane by anaerobic digestion is more energetically favourable when compared to lipid removal from microalgae biomass (Sialve et al. 2009). However, lipid concentrations have been reported to be inhibitory to aerobic digestion at concentrations of 31 % or higher (Cirne et al. 2007). When considering many of the microalgae species used commercially for biofuel production, many have been purposely selected for a lipid concentration of 30 % or higher. Therefore, the removal of the lipid fraction for lipid-based biofuel production is highly beneficial and crucial for the anaerobic digestion process. (Cirne et al. 2007; Sialve et al. 2009). The lipid extraction methods utilised to extract microalgae lipid can also have an effect on the digestibility of the residual biomass (Ehimen et al. 2009). The solvents butanol, hexane and methanol have been shown to have no detrimental effect or inhibition to the anaerobic microbial community if the residual biomass is heated sufficiently to remove any entrained solvents (Ehimen et al. 2009). However, chloroform from the Bligh and Dyer extraction process has been shown to be detrimental to the anaerobic digester microbial community (Bligh and Dyer 1959; Thiel 1969).

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).