Third-generation biofuels: current and future research on microalgal lipid biotechnology

One pressing issue faced by modern societies is to develop renewable energy for transportation. Microalgal biomass offers an attractive solution due to its high (annual) surface biomass productivity, efficient conversion of solar energy into chemical energy and the ability to grow on non-agricultural land. Despite these considerable advantages, microalgal biofuels are not yet commercially sustainable. Major challenges lie in improving both cultivation technologies and microalgal strains. A microalgal crop species is yet to emerge. In this review, we focus on researches aiming at understanding and harnessing lipid metabolism in microalgae in view of producing lipid-based biofuels such as biodiesel. Current biotechnological challenges and key progresses made in the development of algal models, genetic tools and lipid metabolic engineering strategies are reviewed. Possible future research directions to increase oil yields in microalgae are also highlighted.


Biofuel
With the rapid decrease in fossil fuel reserves, the increasing demand in energy, particularly for transportation and the rising concerns about global warming and other related socialeconomic issues, there is a world-wide urge to develop renewable platforms for fuel production. The solutions to meet the a Correspondence: yonghua.li@cea.fr energy demand could be many-fold, ranging from solar, water, wind, nuclear energy to bioenergy. Studies of energy production based on biomass have gained increasing attention because the knowledge generated will not only allow production of fuels but also other high-value chemical molecules essential to the oleo-chemical industries which at the moment are still heavily dependent on fossil fuel. Three generations of biofuels have emerged. The 1st generation biofuel is based on edible plant parts (oilseeds, grains, etc.); the 2nd refers to energy production from non-edible plants or non-edible parts of plants; and the 3rd is based on energy production from photosynthetic microorganisms such as microalgae. Glycerol (by-product) Biodiesel R 1,2,3 : could be any acyl chain ranging from C8 to C24 Fig. 1. Structure of a triacylglycerol and its conversion to biodiesel via acid-or based-catalyzed transmethylation reaction.

Advantages and limits of the 3rd generation biofuel
Microalgae have very high surface productivity, and can be cultivated on non-arable land (therefore not competing with food production). Because microalgae are photosynthetic (i.e. CO 2 -fixing) organisms, their use provides greenhouse gas mitigation benefits. Depending on the species, microalgae can grow on fresh, brackish, sea, or even waste water and can accumulate up to 60% oil per dry weight under stress conditions (Chisti, 2007). Therefore, microalgae have attracted increasing attention for their potential as producers of biodiesel or other lipid-based biofuels (Beer, et al., 2009;Scott, et al., 2010;Wijffels and Barbosa, 2010). Costs of cultivation in photobioreactors or open ponds, biomass harvest and oil extraction from algal biomass remain very high however (Grima, et al., 2003). In addition, many microalgal species are not suitable for industrial cultures, fatty acid composition of microalgal lipids may not be optimal for use as biofuel, and stress conditions needed to accumulate lipids result in arrest of cell growth and division, causing a strong limitation of biomass productivity (Siaut, et al., 2011). Therefore, biofuel production from microalgae is not yet sustainable, and we have not yet come up with one algal strain which can be called "an algal crop" for biofuel production. Intensive research efforts are needed in both strain development and technology innovations (Delrue, et al., 2012). Here we review key advances on the biotechnological aspects of microalgal oil research.

From microalgal lipids to biodiesel
Microalgae have been found to synthesize a large variety of fatty acids and lipids (Hu, et al., 2008;Harwood and Guschina, 2009), the composition of which often reflects adaptation to environmental conditions (Harwood and Guschina, 2009). In photosynthetic organisms, chloroplast is the central compartment of fatty acid synthesis. Fatty acids produced in the plastid are building blocks of all membrane and storage lipids. Upon stresses, almost all microalgae species can produce oil, i.e. triacylglycerols (TAGs), the major form of storage lipids in eukaryotic cells. One triacylglycerol molecule is made of three often different fatty acids which are esterified to the 3 hydroxyl groups of a glycerol backbone. Biological oils are precursors to diesel, and can be converted to biodiesel via a simple transesterification procedure (Fig. 1). This chemical process liberates fatty acid methyl esters, which are the component of biodiesel. Acyl chains determine the structural thus physical properties of the oil and the biodiesel. Modification of fatty acids (e.g. degree of unsaturation, chain length, introduction of functional groups such as hydroxyl or epoxy, etc.) has been one of the major targets of genetic engineering in plants and microalgae (Shanklin and Cahoon, 1998). Fatty acids produced by microalgae, thus, represent a potential alternative not only to fossil fuels but also to other petroleum derivatives (synthons for green chemistry).

Decoupling oil synthesis from arrest of cell division
The amount of oil produced by a microalga is dependent on species and cultivation conditions (Sheehan, 1998;Hu, et al., 2008). Substantial oil accumulation almost always requires stress conditions. One of the most potent stresses to trigger oil accumulation is nitrogen deprivation. Under such culture conditions, some microalgae such as Chlorella species can accumulate up to 60% of oils in its biomass, explaining the considerable regain for microalgae as a biofuel (biodiesel) feedstock (Chisti, 2007). However, nitrogen-starvation limits the overall productivity of the system (Hu, et al., 2008). Maximal lipid yields obtained so far in large scale cultivation systems are 10 to 20 times lower than the theoretical maximum (5000-15 000 gallons per acre per year) (Sheehan, 1998). To circumvent the dependence on stress, dissecting the cellular processes of response to nutrient status, cell division and carbon storage through the study of mutant strains is required.

Harnessing the complexity of lipid metabolism
Our current understanding of oil biosynthesis in microalgae is still rather limited, although rapid progress has been made lately (Merchant, et al., 2012;Liu and Benning, 2013). The current pathway involves three major spatially separated biochemical steps, i.e. plastidial de novo fatty acid synthesis, acylation of fatty acids to glycerol, and deposition as oil bodies, the sub-cellular compartment destined for oil storage (Fig. 2). In plants, this seemingly simple route requires the coordinated actions of several hundred proteins, and regulation across three sub-cellular compartments (Li-Beisson, 2010). For oil synthesis alone in micro algae, three distinct pathway have been proposed (Fig. 2). In plans, the best known is an acyl-CoA-dependent pathway, catalyzed by ER membrane bound enzymes (Riekhof, et al., 2005), similarly to what occurs in plants. An alternative route to oil synthesis is present in both plants and yeast, and is catalyzed by phospholipid:diacylglycerol acyltransferase (PDAT) contributing to the synthesis of triacylglycerol using phosphatidylcholine as an acyl donor and sn-1,2-diacylglycerol as an acyl acceptor. A homolog of this enzyme is present in some sequenced algal genomes including C. reinhardtii where a mutant of PDAT is found to accumulate 30% less oil than its wild-type (Yoon, et al., 2012), thus establishing the contribution of this pathway to oil synthesis in the green algal lineage. A third pathway has recently been proposed by Fan and Xu (Fan, et al has demonstrated that at least part of the triacylglycerol synthetic pathway is present in the plastid of C. reinhardtii. They have further observed under transmission electron microscope (TEM) that some of the oil droplets formed in the plastid can be secreted and re-located to the cytosol. This was later confirmed by another study (Goodson, et al., 2011). These observations point to the fact that, in C. reinhardtii at least, some triacylglycerols are made in the plastid. Thus, a deeper knowledge of the underlying biochemistry, cell biology and genetics of lipid metabolism in this group of organisms is needed.
The champions of current agriculture, for example modern maize, wheat, etc., have been obtained through millennium years of domestication (i.e. selective breeding). None of microalgae species so far have yet been subjected to domestication (selection or breeding) for oil production. The goals of microalgae lipid biologists include construction of industrially robust hyper oil-accumulators via genetic engineering, thus providing the society and industry with designer algal "crops" (Fig. 3). These goals can only be met through combining comprehensive knowledge on lipid metabolic pathways with development of novel and sophisticated genetic and genomic tools.

Current and emerging algal models for research on lipid-based biofuels
Microalgae are microscopic algae present in freshwater as well as marine systems. It encompasses the largest biodiversity with 200 000-800 000 species exist of which about 40 000 species are described (Andersen, 1992), thus constituting one of the least explored biological resources. Several high-value compounds [for example carotenoids, ω-3 polyunsaturated fatty acids (PUFAs), antioxidants, etc.] are currently commercially produced from industrially cultivated species (Wijffels and Barbosa, 2010). An algal crop destined for biofuel production is yet to emerge. Understanding of the function of biological systems in any given group of organism has largely depended on the development of models. Lately, due to the easy access to genome sequencing technologies, more than 10 microalgal species have been sequenced, and many more are in the pipeline. Intensive efforts have been put in place for the development of molecular genetic tools, thus allowing genetic manipulation of any given algal lineage. This development together with our knowledge gained through examining model systems should aid in the master design of an ideal algal cell factory for production of industrially desirable molecules (including diesel). The algae field is still lacking D606, page 3 of 8 Dossier Y. Li-Beisson et al.: OCL 2013, 20 (6) D606 essential models to accommodate its great diversity. Lately, several models for different phyla are emerging and are used for fundamental research in many laboratories. A few of these models are described briefly below.

Chlamydomonas reinhardtii
Among the diversity of microalgal species, the Chlorophyceae Chlamydomonas reinhardtii is the best studied microalga at the physiological, as well as genetic and genomic level. The success of C. reinhardtti as a model is largely due to the possibility to perform genetic analysis through sexual crosses. It has been used to study various fundamental biological processes including photosynthesis, chloroplast biogenesis, flagella function and assembly, starch accumulation, photobiological hydrogen production, and more recently on lipid accumulation (Harris, 2001;Merchant, et al., 2012).
C. reinhardtii can be grown either photoautotrophically, mixotrophically or heterotrophically (Harris, 2001). Photosynthetic function in C. reinhardtii is thus dispensable, a feature which has originally been used to isolate mutants defected in photosynthetic apparatus (Bennoun and Levine, 1967). During its vegetative phase, Chlamydomonas nuclear genome is haploid thus allowing identification of mutant phenotypes at the first generation, which facilitated forward genetic approaches. All three genomes (nuclear, plastid and mitochondrion) have been sequenced, and genetic transformation is possible for all three genomes (Rochaix, 2002). Substantial literature and infrastructure is in place, thus opens doors to new discoveries.
Many laboratories have adopted C. reinhardtii as a reference organism for studying TAG accumulation (Merchant, et al., 2012), although there has been some debates regarding if Chlamydomonas is or not an oleaginous alga. Stressed Chlamydomonas cells accumulate oils in oil bodies (Wang, et al., 2009;Moellering and Benning, 2010;Siaut, et al., 2011). Depending on the strains used and stress conditions, the oil content can reach 50% of dry biomass in starch-less mutants in response to N starvation . With the versatile molecular genetic tools available (Harris, 2001), Chlamydomonas thus serves as an excellent model organism for addressing fundamental biological questions related to oil synthesis and degradation.
If Chlamydomonas has been widely and successfully used to develop forward genetic approaches to study different processes (photosynthesis, flagellar movement, starch metabolism...) reverse genetic approaches are more limited in this model species. Major drawbacks working with Chlamydomonas are the fact that transgene expression is at low efficieng and that no efficient transformation of the nuclear genome based on homologous recombination has been demonstrated. Isolation of mutants affected in a given gene employing PCR-based techniques is feasible but tedious (Gonzalez-Ballester, et al., 2011). With the development of relatively cheap high-throughput genome sequencing (Dutcher, et al., 2012), tagged mutant collections start to emerge (personal communication S. Merchant). Another major concern over a model organism like C. reinhardtii is often the fact that it is not an industrial alga and has not historically been cultivated at large scale. But a possible solution to this drawback has lately been provided by Microphyt, a French company located in Montpellier, who has developed technologies for industrial cultivation of C. reinhardtii in tubular photobioreactors.

Chlorella sp.
Chlorella is a genus of single-cell green microalgae, belonging to the same phylum Chlorophyta as Chlamydomonas. It is spherical in shape, about 2-10 μm in diameter, and can be distinguished from Chlamydomonas because it does not have flagella. Chlorella consists of over 80 species, isolated from either freshwater or marine environment. It is an attractive food producer because it is high in protein and other essential nutrients; several species are known sources of ω-3 PU-FAs (Tokusoglu and Unal, 2003) and astaxanthin (Campenni et al. 2013). It is robust and widely cultivated commercially. One species has been sequenced (Blanc, et al., 2010), and genetic manipulation is demonstrated for three species (C. vulgaris, C. ellipsoidea, and C. kessleri) (Leon and Fernandez, 2007;Gong, et al., 2011;Niu, et al., 2011;Chia, et al., 2013).
Chlorella is an emerging model for study of photosynthetic carbon fixation in the green lineage of eukaryotic microalgae.

Ostreococcus tauri
The marine pico-eukaryote Ostreococcus belongs to Chlorophyta, and is the smallest photosynthetic unicellular eukaryote (Palenik, et al., 2007). It has an extremely small genome i.e. 12 Mb which is 10 times less than C. reinhardtii. Ostreococcus offers great advantages for functional genetic studies because it is thought to represent the minimal genes required to carry out essential biological functions. Ostreoccocus has emerged as a powerful "ready-to-use" algal model because a set of genetic tools have been developed including high efficiency gene overexpression and gene targeting by homologous recombination (personal communication F.Y Bouget), which makes it more amenable to many functional genetic studies than Chlamydomonas. Ostreococcus as a model for marine species is, thus, highly complementary to the freshwater green alga Chlamydomonas. Finally, substantial amount of literature is present which allows us minimal understanding of its physiology.

Phaeodactylum tricornutum
Diatoms are responsible for a significant proportion of primary marine biomass production. Phaeodactylum tricornutum has been developed as a model for diatom research. It is the only species in the genus Phaeodactylum. Unlike most other diatoms P. tricornutum can grow in the absence of silicon, and the biogenesis of silicified frustules is facultative. P. tricornutum can accumulate significant amount of lipids under silicon absence (Sheehan, 1998), and the fact that it can synthesis high amount of ω-3 PUFAs have brought considerable interests in research on lipid metabolism in this diatom. These features together with its high biomass productivity, high lipid content, a fully sequenced genome (Bowler, et al., 2008), and the versatile molecule toolboxes developed (Siaut, et al., 2007), made it a prominent model system for both biodiesel and ω-3 PUFA production. Nonetheless, one of the major issues associated with P. tricornutum as a model is that there is no easy way to knock out genes, knock down being mostly available (De Riso, et al., 2009).

Nannochloropsis sp.
Nannochloropsis is a heterokont microalga comprising six species, of which five are marine and one freshwater. Due to its rigorous growth, and high content of ω-3 PUFAs and astaxanthin, it is commonly used in aquaculture applications. Upon stress, it can also accumulate significant amount of neutral lipids thus attracting interests on its development for biofuel production. Draft genome sequences are available for two of the marine species (N. gaditana and N. oceanica) (Radakovits, et al., 2012;Vieler, et al., 2012). Genetic transformation is available, and more importantly homologous recombination has been demonstrated with high efficiencies for N. gaditana (Kilian, et al., 2011). Thus, Nannochloropsis represents an emerging model for lipid research on photosynthetic protists. Substantial collaborative effort at international level is required for this organism to become as the premier model for algal research.

Bio-prospecting for high performing algal strains suitable for oil production
Only a handful of the tens of thousands of microalgal species known to exist in nature are currently cultured at large scale and used for commercial applications, among which only a few hundreds have been investigated for potential biotechnological applications. Bio-prospecting for new species from local or extreme environments for oil content or other high value product has been an on-going international effort. One of the largest conducted so far has been the survey carried out by the U.S. Department of Energy (Sheehan, 1998). They studied around 300 species selected from 3000 isolated strains. More recently, several research studies for strain selection and induction of lipid biosynthesis have been carried out (de la Vega, et al., 2011;Do Nascimento et al. 2012). These studies revealed that the intrinsic ability to produce large quantity of oil and lipid is generally species/strain specific, rather than genus specific. Nonetheless, a survey of the literature by Hu et al. (Hu et al. 2008) revealed that species belonging to the group of green microalgae (Chlorophyta) generally accumulate oil to a higher level than species of other algal taxa such as cyanobacteria, brown algae, or red algae. In addition to the ability to produce biomass and accumulate oil, other criteria related to cultivation constraints (strain robustness) or to downstream processes (harvesting, oil extraction...) have to be considered together and has been proposed to impact the final productivity of the system and the economy of microalgae-based biodiesel production (Delrue, et al., 2012). Continuing efforts at bio-prospecting of strains isolated from local environment is a key to isolate strains suitable for one particular geological niche.

Genetic improvement of algal strains
Current crop plants have gone through thousands of years of selective breeding which is a common agricultural practice. However conventional breeding is not suitable for algae strains since sexual cycles are lacking or poorly defined for most species. Metabolic engineering thus represents one of the most promising strategies toward production of suitable algal strains (Rosenberg, et al., 2008). Comparing to classical breeding, genetic engineering is a time-saving technology. Indeed, metabolic engineering has revolutionized the traditional breeding programme and made high contributions to meet our societal needs. One of the most notable examples in successful genetic engineering is probably the creation of "golden rice" via genetic engineering of the rice plant to synthesize beta-carotene via introduction of two enzymes (Ye et al. 2000). Given the latest development in genomics, genetics, and molecular biology, synthetic biology is coming of age technology for addressing basic as well as applied questions in the bio-production of desirable molecules and fuels. For example, genetic engineering has already allowed the isolation of Chlamydomonas mutants with higher ability for the photobiological production of hydrogen (Tolleter, et al., 2011); and mutants with reduced antenna sizes showing improved biomass productivity in photobioreactors (Ort, et al., 2011). Heterologous gene expression has been used successfully to redirect microalgal metabolism. For example, heterologous expression of two thioesterases in Phaeodactylum tricornutum caused accumulation of novel medium chain fatty acids (Radakovits, et al., 2011). Regulatory proteins controlling oil synthesis have also been isolated in the model Chlamydomonas, and overexpression of these proteins led to altered cellular oil content (Yohn, 2011;Boyle, et al., 2012).
Despite the promising achievements, all routine work on metabolic engineering through genetic manipulation remains limited to a few model species where efficient and stable transformation is possible. Common issues associated with other transformation experiments reported in the literature are low efficiency and instability of transgenes introduced. Therefore, there is substantial need for development of molecular tools for other non-model microalgal species. One such achievement is the recent demonstration of high efficiency homologous recombination for an industrially relevant alga N. gaditana (Kilian, et al., 2011). Such genetic tools would allow the metabolic engineering of pathways or physiological processes and therefore allow the production of transgenic microalgae that are genetically tailored for the production of biodiesel.
In addition, successful genetic engineering is also dependent on the development of high-throughput screening procedures which allow the rapid and easy screening of a high number of cell lines for desirable phenotypes (high growth rate, high oil content, better fatty acid profile, etc. cytometry has been demonstrated as a powerful tool to isolate mutant with altered oil content (ongoing work in the authors' laboratory).

Advancing knowledge on lipid metabolism in microalgae
A comprehensive view of lipid metabolism in microalgae is still in its infancy. Most of the components of lipid biosynthesis are deduced from homologies to proteins of either known plant or yeast lipid synthetic pathways. To aid in genetic engineering and allow intelligent design of strains, a thorough knowledge of lipid metabolism in microalgae is urgently needed. Recent studies dedicated to advance our understanding of oil metabolism in this alga (summarized in Fig. 4) include: (i) detailed characterization of the oil accumulation kinetics in response to nitrogen depletion (Wang, et al., 2009;Siaut, et al., 2011); (ii) testing the potential impact of diverting carbon precursors from starch synthesis to oil accumulation using starchless mutants Siaut, et al., 2011); (iii) identification of proteins associated with oil droplets -the major cellular compartment for oil storage (Moellering and Benning, 2010;Nguyen, et al., 2011); (iv) comparative transcriptomic studies on cells before and after nitrogen removal (Miller, et al., 2010); (v) microscopic and biochemical analyses of the possible sub-cellular locations of oil droplets in Chlamydomonas and other algae (Fan, et al., 2011;Goodson, et al., 2011;Fan, et al., 2012); (vi) Forward genetic screening of mutants affected in oil synthesis . Please note that these are just examples, and are not meant for an exhaustive list. These pioneering works have revealed the potential complexity and divergence of oil metabolism from that of higher plants, and highlights interesting candidate genes for genetic engineering studies. Now it is the right time for verification of gene function using the latest tools developed for Chlamydomonas such artificial microRNA techniques to knock-down specific genes (Molnar, et al., 2009), or isolation of specific mutants using PCR-based screening (Gonzalez-Ballester, et al., 2011). In parallel, the development of forward genetic approaches based on efficient and high-throughput screening of mutant showing interesting properties (such as the ability to accumulate oil in optimal growth conditions), will be important to identify regulatory genes controlling oil accumulation.

Conclusion
Photosynthetic microalgae have provided us with food and feed, and now possibly also fuel. Research on microalgae lipids have come a long way starting from the 40's during the Second World War in Germany. Given the high impact of research on energy, algal fuel development is tightly related to economic, social and world development. Technologies developed today have progressed from manipulation of one or two genes to more system-based approaches. These technologies include high-throughput genome sequencing, and a suit ofomics technologies (genomics, transcriptomics, proteomics, metabolomics, and lipidomics). These powerful tools if used together with transformation and molecular genetic toolboxes developed for particular algal strains provide ample opportunities for lipid scientists to redesign algal metabolism toward production of oils or other chemical molecules useful for industrial applications.