Solar cells are devices for producing electricity which use incident illumination to supplying electrons to an external circuit. The use of these has even been described as the “art of converting sunlight directly into electricity” (Wenham et al. 1994). There are a range of technologies and materials used to produce solar cells, each with their own benefits and drawbacks. By far, the most common and familiar example of a solar cell is that of crystalline silicon. Crystalline silicon solar cells currently dominate the world market and held over 93.5 % market share in 2005 (Singh and Jennings 2007), decreasing to 83 % in 2010 (Tyagi et al. 2013) and 86 % in 2011 (Fraunhofer 2012). Crystalline silicon solar cells have a long history and have undergone major improvements in efficiency over the years. The first crystalline silicon solar cell had a limited efficiency of 6 % (Chapin et al. 1954); however, new solar cells have been developed with efficiencies greater than 25 % in the laboratory and 22 % in full modules (Green et al. 2012; Beardall et al. 2009). Although solar cells are generally optimized to absorb strongly across the whole solar spectrum, each individual technology will have variations in performance. These variations are due to a number of factors including the properties of the semiconductor, such as the bandgap.

Energy-efficient and cost-effective microalgae dewatering, nutrient recycling and effluent water quality control are some of the major challenges facing industrial — scale microalgae production for commodity feeds and fuels (Benemann 2013; Borowitzka and Moheimani 2013b; Wyman and Goodman 1993a). Irrespective of the cultivation system, the biomass concentration of the algae culture is generally low (a few mg L 1 in open ponds to a few g L 1 in intensive closed photobiore­actors). Dewatering is therefore critical for producing any materials from micro­algae. The objective of harvesting and dewatering is to raise the concentration of the microalgal biomass by more than two orders of magnitude to over 10 % solids, sufficiently concentrated for subsequent processing or drying. It is widely believed that this is best achieved using a combination of technologies in a two-stage process (Benemann et al. 1982; Shelef et al. 1984; Vandamme et al. 2013), such as floc­culation followed by centrifugation. This necessitates that large volumes of water need to be processed to harvest the biomass. This concentration process is typically energy intensive and results in high harvesting, thickening and dewatering costs (Mohn 1988). Available harvesting and dewatering process selection often interacts with both up — and downstream process steps in microalgae production, such as strain selection and medium composition, biomass fractionation (e. g. in a biore­finery) and water or nutrient recycling (de Boer et al. 2012; Wijffels et al. 2010).

Depending on the strain and organic carbon source, a major disadvantage of het­erotrophic culture is that light is required for increased productivity. In some strains, for example, lipid productivity is higher under photoautotrophic cultures when compared with heterotrophic cultures. Furthermore, the cost of organic carbon source can be very high, making heterotrophic production of biodiesel oil uneconomical. Solar light energy is abundant and freely available in outdoor photoautotrophic cultures. Thus, it is desirable to use light from solar energy to reduce production costs. Depending on the location and season, however, only a few hours of the day have the high light intensity needed to support photoautotrophic growth. During the night, cells not only cease to grow, but they metabolize the already intracellularly stored energy for cell maintenance, thereby leading to a decrease in biomass con­centration (Ogbonna and Tanaka 1996). Cyclic photoautotrophic-heterotrophic culture seeks to overcome this problem by cultivating cells photoautotrophically during the day, while adding the required amount of organic carbon source to grow the cells heterotrophically at night. By taking advantage of both alternating photo­autotrophic and heterotrophic cultures, the cells grow continuously during both day and night, leading to increased productivity (Ogbonna and Tanaka 1998; Ogbonna et al. 2001). This is especially useful for the cultivation of some microalgae that are not truly mixotrophs, yet can switch between phototrophic and heterotrophic metabolisms, depending on environmental conditions (Kaplan et al. 1986). However, the technical challenge of minimizing contamination risk through the selection and control of the organic carbon source added during the night remains.

Data in Table 7.1 present the effect of CO2 concentration of the input gas on biomass concentration and CO2 removal of cyanobacterium Synechococcus sp. (Takano et al. 1992). When the CO2 concentration of the input gas increased from 0.03 to 0.55 %, the biomass concentration rose by 1.5-fold and the CO2 removal rate more than doubled. However, when the input gas concentration further increased from 0.55 to 1.10 %, only a slight increase in CO2 removal occurred (Takano et al. 1992). Furthermore, Fig. 7.4 shows the influence of the input CO2 content on S. obliquus WUST4 (Li et al. 2011) in the range of 6-18 %. The highest CO2 fixation efficiency (67 %) was achieved at 12-14 % CO2, indicating higher CO2 concentration is an inhibitory factor to CO2 fixation, and is an species — dependent variable tolerance.

Evan Stephens, Juliane Wolf, Melanie Oey, Eugene Zhang, Ben Hankamer and Ian L. Ross

Abstract An advanced understanding of the genetics of microalgae and the availability of molecular biology tools are both critical to the development of advanced strains, which offer efficiency advantages for primary production, and more specifically in the context of production for biocrude and renewable energy. Consequently, we outline the current state of the art in microalgal molecular biology including the available genome sequences, molecular techniques and toolkits, amenable strains for transformation of nuclear and plastid genomes, and the control of transgenes at both transcriptional and translational levels. We also examine some strategies for improvement of expression and regulation. We suggest the primary strategies in strain improvement that are most relevant to biocrude applications; briefly illustrate the process of photosynthesis to enable identification of targets for improvement of net photosynthetic conversion efficiency in mass cultivation; and further discuss how improvement of metabolic systems may also be achieved and benefit production models. Finally, we acknowledge the aspects of prudent risk assessment and consequent regulation that are developing and how our knowledge of natural algae in existing ecosystems, and GM work in conventional agriculture both contribute lessons to these discussions. We conclude that if properly managed, these developments provide significant potential for increasing global capacity for renewable fuel production from microalgae and that these developments could also have benefits for other applications.

E. Stephens • J. Wolf • M. Oey • E. Zhang • B. Hankamer • I. L. Ross (H)

Institute for Molecular Bioscience, The University of Queensland, Queensland, Australia e-mail: i. ross@imb. uq. edu. au

E. Stephens • J. Wolf • B. Hankamer • I. L. Ross

Solar Biofuels Research Centre, The University of Queensland, Queensland, Australia

© Springer International Publishing Switzerland 2015 191

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

Biofuel and Biorefinery Technologies 2, DOI 10.1007/978-3-319-16640-7_11

11.1 Introduction

The Need for Strain Improvement While microalgae are a proven and promising platform for the production of high-value products, their greatest potential arguably lies in their ability to capture solar energy and convert it to chemical energy in the form of high energy density fuel feedstocks with low net carbon emissions. The importance of this is highlighted by the fact that * 80 % of global energy demand is supplied in the form of fuels, while only *20 % is utilised as electricity (BP 2014; Stephens et al. 2013b). Consequently, there is a great need for renewable fuel production systems that have an economic and energetically positive return on investment (ROI), and microalgae are one of the very few options for making this a reality at scale.

Thermochemical processing of whole biomass to biocrude is a promising area of research and current commercialisation. At first glance this processing strategy does appear to promise increased yields, since in addition to lipids, other organic mol­ecules such as proteins, starch and cellulose can be converted. It may also address some of the conventional cost/energy bottlenecks, particularly as complete dewa­tering and cell disruption are not needed. But it must also be considered that, in contrast to the extraction of a relatively homogenous product such as TAGs and neutral lipids, the resultant output product from hydrothermal liquefaction (HTL) can vary in quality from a type I kerogen (a complex carbonaceous organic com­pound) (Speight 2006) to a higher grade biocrude, equivalent to the best petroleum crudes. The quality of the output depends upon the efficiency of the HTL process as well as the composition of the initial biomass. While the upgrading of kerogen to biocrude can be a much simpler process than lipid extraction from microalgae biomass, it remains an economic and energetic cost in the process. Thus, the technology can be streamlined partly by the development of microalgal production strains that have a more desirable composition for HTL processing and conse­quently improve the quality of biocrude output. The marketability of the biocrude product to fuel producers depends upon specific quality criteria including high carbon and hydrogen content and low oxygen, nitrogen and sulphur levels. Other qualitative considerations include acyl chain lengths and saturation, as well as finer points related to fuel standards. To achieve such standards and ultimately obtain a biocrude product that is comparable to conventional petroleum crude, HTL kero — gens and oils can require fractionation to a higher grade product. This additional process step results in material losses and increased energy costs which offset some of the anticipated benefit of this production strategy. This poses a significant operational loss unless the residual fraction can be efficiently recycled back to production (e. g. through strategies such as anaerobic digestion or gasification) or otherwise contributes to cost recovery and energy balance.

Strain development through the use of molecular biology has greater flexibility than conventional breeding and strain development techniques. This may translate to increases in overall productivity (greater volumes to process) and greater carbon density (higher grade biocrude output) and so is of importance for advancing this production strategy. Knowledge of algal genetics is not yet as sophisticated as other model systems. The ability to engineer algal biology is correspondingly limited at present, but is growing rapidly. Here, we discuss the ongoing development of molecular research for greater understanding of microalgae systems. In particular, we summarise the increasing set of available molecular techniques (Sect. 11.2), their application to microalgae technologies (Sect. 11.3) and the establishment of prudent regulatory systems to ensure these systems become environmentally responsible and socially accepted (Sect. 11.4).

Many of the available harvesting methods are not suitable for complete dewatering of the algal biomass and must be used in combination with other methods to achieve the cell concentrations necessary for the production of a feedstock for algal biofuel production. This has been referred to as either primary and secondary concentration or bulk harvesting and thickening. While the terminology is different, the processes are the same. Primary or bulk harvesting takes low concentration cultures typical in photoautotrophic mass algal culturing and brings them to roughly 2-7 % solids. For most downstream processing, a secondary or thickening step is required to increase the solid concentration. The final solid content needed varies for this secondary concentration step and ranges from 50 % solids for downstream processes that can tolerate significant moisture content (e. g., hydrothermal lique­faction and aqueous extraction) to >90 % solids for dry feedstocks needed for traditional solvent extraction.

While primary harvesting remains a huge challenge for photoautotrophically produced biomass (due to large volumes at low density), processes that bypass the higher energy secondary concentration step could provide an advantage for com­mercial production of biofuels. It is beyond the scope of this review to discuss methods for drying algae (for delivery of >90 % solids) and will focus on methods to deliver concentrated algal feedstocks at <90 % solids as well technologies to avoid harvesting altogether. In Table 14.1, the different primary harvesting methods are listed with a comparison of their properties and the current scale of deployment in the algal industry (not limited to biofuels).

If the technique has an advantage in a particular property, it is marked by a plus (+) sign under the applicable Properties column. For example, in settling/sedi- mentation, very little energy is required, so it has a (+) in that column, but since product stability is not maintained, it has a (—) in that column. If the process could have advantages for some biofuels applications and not work for another, a (±) is provided in that column. If there is not enough information to make a decision, a question mark is inserted in the column in Table 14.1. The current scale section of Table 14.1 provides an estimate of the applicability of the method to rapid com­mercial deployment.

Many species of microalgae are encapsulated by a rigid cell wall and can be prob­lematic in anaerobic digesters (Chen and Oswald 1998; Golueke and Oswald 1959; Gonzalez-Fernandez et al. 2012a, b; Mussgnug et al. 2010; Samson and Leduy 1983; Sialve et al. 2009; Zamalloa 2012). Anaerobic digester associated bacteria need to be able to access the contents of the microalgae cells to allow the digestible components to be converted to methane biogas (Ward et al. 2014). The microalgae cell wall material and chemistry have a major influence on the biogas potential (Ras et al.

2010) , and some microalgae species have been shown to survive intact after 6 months within an anaerobic digester, changing to heterotrophic growth rather than photo­trophic growth (Mussgnug et al. 2010). Several studies conclude that a pre-treatment step is required to disrupt the cell wall to increase bacterial hydrolysis before addition to the anaerobic digester (Chen and Oswald 1998; Golueke and Oswald 1959; Mussgnug et al. 2010; Sialve et al. 2009; Zamalloa 2012). Within an integrated system, cell lysis or disruption is also essential for solvent extraction of the lipid fraction of microalgae biomass, allowing solvents to react with internal cell lipids (Lee et al. 2013). Therefore, microalgae cell wall disruption processes are essential for both lipid-based biofuel applications and for optimal anaerobic digestion. The multiple methods of disruption and cell lysis include mechanical, physical, thermal, chemical and enzymatic methods and have the twin applicability of allowing solvent to react with lipids increasing lipid yield in the extraction process and also microbial hydrolysis of the cell contents during anaerobic digestion (Ward et al. 2014). However, the cost of the extra pre-treatments must be considered within the anaer­obic digester and biofuel production systems as the energy consumption for the pre­treatment of microalgae biomass can be equal or higher than the energy gained from the microalgae cell (Lakaniemi et al. 2011; Lee et al. 2012, 2013; Lu et al. 2013; Sialve et al. 2009; Yen and Brune 2007).

Wastewater treatment consists of the removal of unwanted chemicals, or biological contaminants from impure water sources, such as from the liquid wastes released by houses, industrial operations, or agricultural processes. Conventional wastewater treatment methods include physical processes such as filtration and sedimentation; chemical processes such as flocculation and chlorination; and biological processes such as generation of activated sludge (Metcalf and Eddy 2003). However, these methods are mainly based on the separation of pollutants from the wastewater with a requirement for a further stage to eliminate these pollutants. This brings a need for an integrated wastewater treatment process that eliminates the undesired portion of the wastewater while converting them into valuable products, which can be successfully achieved by applying a selected immobilization process. Immobilized algal systems are particularly effective for the removal of nutrients (i. e., phosphate and nitrate) and various metals from wastewaters, which will be discussed in the following sections.

Wastewater not only contains nutrients such as N and P, but also a range of other contaminants that may interfere with microalgal growth. The presence of growth — inhibiting substances probably explains why microalgal growth rates in real wastewaters are often slightly lower than in synthetic wastewaters. Contaminants not only pose a problem because they inhibit microalgal growth, but they can also accumulate in the microalgal biomass and limit the valorization of the microalgal biomass, or fractions thereof. Wastewater can contain a wide range of toxic chemicals such as heavy metals, persistent organic pollutants, and surfactants.

This is particularly the case in industrial wastewaters, although domestic waste­water or animal manure may also contain substantial quantities of pollutants such as heavy metals (Nicholson et al. 1999). Even chemicals with a low toxicity (such as those used in personal care products), may have an inhibitory effect on microalgal growth rates (e. g., Wilson et al. 2003).

Animal manure wastewaters often contain very high concentrations of N present in reduced form as ammonium. Ammonium is converted to free ammonia at high pH and is toxic to many microalgae, with some microalgae inhibited by concen­trations as low as 20 mg L 1 (Azov and Goldman 1982). High concentrations of ammonium may result in ammonia toxicity even when pH is low (Peccia et al. 2013). Some substances do not inhibit microalgal growth, but accumulate in the microalgal biomass and limit valorization. The cell wall of microalgae is often rich in carboxyl, amino, hydroxyl, or sulfate groups. These groups are anionic and can bind metals through ion exchange (Wang and Chen 2009). Microalgae are efficient absorbers of heavy metals and even low concentrations of heavy metals present in wastewater can be absorbed into microalgal biomass. This is not necessarily a problem when the biomass is converted into biofuels using chemical or physical methods. It may be a problem when biological methods are used to convert the biomass into fuel (e. g., anaerobic digestion, fermentation). It may also be a problem when the protein-rich residue of the biomass remaining after extraction of lipids for biodiesel production is to be used as animal feed, as is proposed in the microalgal biorefinery context (Wijffels et al. 2010). Rwehumbiza et al. (2012) showed that metals used for flocculating microalgae remain in the protein-rich residue after extraction of lipids. However, microalgal absorbtion of heavy metals from waste­water can also be an advantage, and numerous studies have demonstrated that microalgae can be applied to remove heavy metals from a variety of wastewaters (see for instance Wilde and Benemann 1993; Gadd 2009). Wastewater may also contain microbial contaminants such as cysts of parasites, infectious bacteria, or viruses. These may also interfere with the use of microalgal biomass fractions as animal feed. However, due to the high pH, high oxygen concentrations, and exposure to light, many harmful microorganisms tend to be inactivated in micro­algal cultures (Davies-Colley et al. 1999).

Many wastewaters of agricultural origin such as piggery waste or anaerobic digestion wastewater often have a dark color. This dark color is predominantly due to the presence of humic substances derived from incomplete breakdown of lignin in plant material (Brezonik and Arnold 2011). This dark color limits the light pene­tration in the water and reduces microalgal productivity (Martin et al. 1985); potentially a 20-30 % lower productivity when compared to growth rates in a non­colored culture medium (De Pauw et al. 1980). In many laboratory studies on production of microalgae in animal manure wastewater, the wastewater is diluted prior to the experiments and inhibition of microalgal productivity by the dark col­oring is barely noticed. However, in large-scale systems, dilution of wastewater with pure water will be unsustainable due to the high water demand. To prevent inhibition of growth by colored substances, the wastewater can be pre-treated with oxidizing agents such as sodium hypochlorite, ozone or hydrogen peroxide, or by coagulants, flocculants, or adsorbents (Markou et al. 2012b; Depraetere et al. 2013). Alterna­tively, nutrients can be separated from the wastewater containing colored substances and then added to the microalgae culture medium. This can be carried out, for instance, using dialysis membranes (Blais et al. 1984). Also nutrients can be sorbed onto zeolites and released from the zeolites in fresh medium (Markou et al. 2014).

The ongoing progress in sequencing of algal genomes will permit annotation, comprehensive cloning and manipulation of genes, which altogether allow omics approaches to generate large-scale experimental datasets. This advancement will aid in identification of key regulators of metabolism and enables the eventual manip­ulation of cellular pathways. Synthetic biology combines the use of molecular tools with knowledge gained from systems level analysis of organisms to generate innovative experimental designs. For example, advances in long DNA synthesis make it possible to construct complex genetic circuits designed and informed by metabolic modeling and pathway analyses. With these advances, synthetic biolo­gists have made tremendous progress on the construction of genetic circuits and even entire chromosomes.

The majority of synthetic biology efforts are focused on microbes as many of the most pressing problems, such as sustainability in food and energy production ultimately rely on modification of microorganisms. As such, synthetic modifica­tions of algal strains to enhance desired physiological properties is likely needed to improve their productivity. There has been increasing efforts by synthetic biologists to push for the creation of accessible tools that would improve the potential of algal technology. With synthetic biology, still a young field, the future of this auspicious approach is clearly apparent. While much remains to be achieved to exploit the full potential of algae through various approaches, synthetic biology is likely to play a central role in this process in the coming years.

Acknowledgments Support for this work was provided by New York University Abu Dhabi (NYUAD) Institute grant G1205, NYUAD Research Enhancement Fund AD060, and NYUAD Faculty Research Funds; K. J. was supported through NYUAD Global Affiliate Fellow program; L. Y. was supported in part by NIH R01EB013584, DOD W81XWH-10-10327, and OCRF PPD/ BCM/01.12.