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  • 1. Algae, a New Biomass Resource 1 Algae, a New Biomass Resource needed for growth. In oxygenic photosynthesis, light-induced redox reactions occurring in the pho-CINZIA FORMIGHIERI, ROBERTO BASSI tosynthetic electron transport chain are coupled to `Dipartimento di Biotecnologie, Universita di Verona, the extraction of electrons from water.Verona, Italy Photosystem A multipigment-protein complex com- posed of a light-harvesting antenna moiety energet-Article Outline ically connected to a reaction center where the excitation energy is used for charge separation andGlossary production of a reduced product.Definition of the Subject Light saturation constant The intensity of light atIntroduction which photosynthetic oxygen evolution and specificAdvantages of Algae as Biomass Producers biomass growth rate are half the maximum level.Present Algal Productivity in the Laboratory Versus The energy absorbed in excess with respect to the Large Scale photosynthesis saturation is dissipated as fluores-Domestication cence or heat and not used for photochemistry.Light Use Efficiency Photoinhibition The light-induced inactivation ofNon-Photochemical Energy Quenching at the Molecular photosynthesis occurring when photooxidative dam- Level age of the photosynthetic machinery (particularlyInterconversion and Storage of Photosynthetic Metabolic photosystem II) overcomes the capacity for repair. Products Genetic improvement All procedures, including phe-Screening for State Transition as an Indirect Mean to notypic selection, conventional breeding, mutagen- Select Strains with Altered Redox Metabolism esis, and genetic engineering, aimed at indirectly orAccumulation of Biomass as Neutral Lipids directly influencing the genetic background ofPlanning an Algal Refinery a wild strain, which was evolved following rules ofLarge-Scale Systems natural selection. Genetic improvement is intendedFuture Perspectives and Technological Developments at improving existing characteristics or at introduc-Bibliography ing new traits to fit applications. Genetic engineering All the techniques of recombi-Glossary nant DNA to directly manipulate genotypes.Algae Oxygenic photosynthetic organisms, prokary- otic or eukaryotic, with organization ranging from Definition of the Subject unicellular to multicellular. Algae never have true stems, roots, and leaves, thus leading to their clas- Algae are oxygenic photoautotrophs, offering a very sification as “lower” plants. high level of biodiversity and thus suitable for differentBiofuel Renewable energy-rich compound derived practical applications. Today, they are mainly cultivated from living organisms or from their metabolic for human/animal food or to extract high-value by-products. chemicals and pharmaceuticals. However, their exploi-Biomass Organic raw material, stored as a result of the tation could be extended. Algae are attractive as high metabolism of a living organism, which can be used yield biomass producers, because of the short life cycle, as a resource for energy and biofuels. the ability to grow up to very high cell densities, and thePhotoautotrophy The ability of a living organism to easy large-scale cultivation that does not compete with use carbon dioxide as carbon source for biomass other demands such as those of conventional crops and light as source of energy. agriculture. Algae can be a resource of renewable, sus-Photosynthesis The overall process that converts light tainable biofuels. In addition, they can be transformed energy into chemical energy, finally used to fix into “cell factories” to produce recombinant proteins of inorganic carbon dioxide into organic compounds interest for pharmaceutical companies.M. Kaltschmitt et al. (eds.), Renewable Energy Systems, DOI 10.1007/978-1-4614-5820-3,# Springer Science+Business Media New York 2013Originally published inRobert A. Meyers (ed.) Encyclopedia of Sustainability Science and Technology, # 2012, DOI 10.1007/978-1-4419-0851-3
  • 2. 2 Algae, a New Biomass Resource Introduction a major problem for human society. Its becoming increasingly clear that finding alternative, renewable Algae are described as “lower” plants that never have energy sources to sustain our lifestyle is striking and true stems, roots, and leaves, and grow photoautotro- urgent. Such widespread and environmentally sustain- phically by performing oxygenic photosynthesis [1]. able energy source will allow single countries to be They are mostly eukaryotic, although prokaryotic more independent from the escalating prize of crude cyanobacteria are included in algae. More than oil. The public conscience that search for energy 200,000 species are estimated and form a highly het- sources alternative to fossil fuels is fundamental for erogeneous group, spread in all aquatic ecosystems future prospects is generally increasing and research is but also in other habitats such as soil. A broad spec- undertaken in many countries. Advances have been trum of phenotypes and specialized adaptation abilities made recently on the exploitation of renewable energies exists within this group while members colonize such as sun and wind, which can be considered as diverse ecological habitats, from freshwater to marine “clean” and non exhaustible. Fuels derived from living and hyper-saline, at different temperatures, pH, organisms or from their metabolic by-product are and nutrient availabilities [1, 2]. Algae are classified defined as “biofuels.” Photosynthetic organisms con- as follows: cyanobacteria (Cyanophyceae), green algae vert carbon dioxide into biomass, a form of stored (Chlorophyceae), diatoms (Bacillariophyceae), yellow- chemical energy that can be used in substitution for green algae (Xanthophyceae), golden algae fossil fuels. In some species of algae, 30% up to 80% of (Chrysophyceae), red algae (Rhodophyceae), brown the biomass is accumulated as oil that can be extracted algae (Phaeophyceae), dinoflagellates (Dinophyceae), and converted to biodiesel [2, 8–11]. Some algae, and “pico-plankton” (Prasinophyceae and Eustigma- mainly green, can photobiologically generate hydrogen tophyceae). Algae are accountable for the net primary [12–15], which is released from the culture with production of $50% of the total organic carbon pro- a purity up to 98% [16]. Spent algal biomass can be duced on earth each year [3], a reason for their massive further valorized to produce biomethane from anaero- biological importance. Long before the advent of bio- bic digestion of the biomass itself [1, 5] and high-value technology, algae were used as a human food source for ingredients for food and pharmaceuticals can be centuries by indigenous populations [4, 5]. However, produced along with biofuels in order to reach cost- aside from some attempts to cultivate algae and to effectiveness and economic competitivity over conven- extract from them valuable products back to the tional fuels. seventeenth century [6], commercial large-scale pro- duction of algae began few decades ago [4, 7]. Cur- Advantages of Algae as Biomass Producers rently, algae are mainly cultivated as human/animal food source and used in aquaculture, in agriculture, The use of algae for fuel production has notable as fertilizers, and in order to produce high-value advantages. First of all, algae are photoautotrophic chemicals, pharmaceuticals, and cosmetics, such as organisms, thus able to produce biomass from solar polyunsaturated o3 fatty acids, proteins, biopolymers, energy, water, and carbon dioxide, all renewable and and polysaccharides as agar, carrageenan, alginates, cheap components. Carbon dioxide removal, in addi- pigments, vitamins, and antioxidants [1, 4]. tion, would contribute to reduce atmospheric levels of Recently, a new interest in algal biomass as renew- this pollutant which, instead, is stoichiometrically able energy source is emerging. About 80% of world released in the atmosphere by conventional fuels. The energy demands are met by the combustion of fossil use of fossil fuels currently releases about 6 billion tons fuels: coal, oil, and natural gas. However, fossil fuel of carbon dioxide per year, only partially compensated reserves are limited and their exhaustion is estimated, by re-assimilation by photosynthetic organisms in for- assuming a stable consumption, to occur within few ests (about 1 billion ton yearÀ1) and oceans (2 billion decades while their combustion has high impact on tons yearÀ1) [17] leading to a 3-billion-ton yearÀ1 environment, due to releases of large amounts of car- increase in the global level of carbon dioxide in the bon dioxide and other pollutants. This is considered atmosphere. This has been widely suggested to cause
  • 3. Algae, a New Biomass Resource 3the so-called greenhouse effect, leading to world global 5. Algae can utilize nutrients from a variety of waste-warming [18]. To prevent undesired climate changes, water sources (agricultural runoff, and industrialcarbon dioxide emissions need to be reduced in the near and municipal wastewaters), providing additionalfuture [19]. Biofuels from algae are attractive because benefit of bioremediation.their combustion would not result in net carbon dioxide 6. The life cycle of algae is more rapid (hours to days)emissions since biofuels will only release the amount of as compared to land plants (months to years),carbon previously absorbed during growth of algae. allowing for a faster turnover and a higher biomass Advantages of algae over crop plants are many: yield. In more detail, crop plant life cycle has a long initial development phase using energy1. Photosynthesis increases with carbon dioxide con- stored in seeds for building the organism, followed centration until saturation. Algae can grow with up by a relatively short phase of full photosynthetic to 18% of carbon dioxide, being more efficient car- activity and then senescence, limiting the period bon dioxide assimilators than land plants [20] and of efficient light harvesting and utilization to a suitable for mitigation of carbon dioxide emissions fraction of the year. In contrast, algae not only when coupled to an industrial activity. Chlorella have a short life cycle but they remain productive vulgaris was shown to respond well to elevated throughout the year, allowing for a continuous bio- carbon dioxide levels (over 1,850 ppm CO2) and mass production when light is available and tem- to effectively remove up to 74% of the carbon perature allows. dioxide in the airstream to ambient levels (330 7. Many algal species are competent for using ppm CO2) with a 63.9 g mÀ3 hÀ1 bulk removal in organic carbon as energy source. This ability the experimental photobioreactor of 2 L [21]. The opens the possibility to allow for biomass pro- carbon dioxide fixation rate by algal biomass is esti- duction at night by supplying low-priced carbon mated ten times as large as that of the temperate substrates. forest [22]. 8. Algal biomass is fully photosynthetically active. In2. Cultivation of algae does not compete with other contrast, photosynthesis is localized exclusively in demands such as food production because algae can leafs of vascular plants, a fraction of the total plant grow in wastelands rather than in arable lands. body, while the rest is an energy sink, thus a disad- Using the entire US soybean crop for biodiesel vantage in terms of biomass productivity. would replace only 10% of conventional diesel con- sumed and total world plant oil production would Present Algal Productivity in the Laboratory only satisfy 80% of US demand [23]. Exploiting Versus Large Scale algae rather than crops thus represents an essential, sustainable alternative to fulfill the energetic Algae can convert solar energy into chemical energy demands without affecting food supply. through the process of photosynthesis. The whole pro-3. Algae, differently from crops, do not require pesti- cess of oxygenic photosynthesis can be summarized by cides, which avoids contamination of water and Eq. 1. soils and also fertilization is limited to the culture 6H2 O þ 6CO2 ! C6 H12 O6 þ 6O2 ð1Þ vessel without dispersion of nutrients in the envi- ronment and consequent eutrophysation of water Light energy is used to extract electrons from water bodies. (H2O), thus generating oxygen (O2). These electrons4. Algae can grow in seawater, brackish water, or are transported through a linear electron transfer wastewater making a substantial saving of fresh- chain and finally reduce NADP+ to NADPH. Photo- water that in contrast is required by conventional synthetic electron transport is coupled to the genera- crops in high amounts. Extensive cultivation of tion of a transmembrane electrochemical gradient, algae would therefore be more environmentally whose stored energy is used to synthesize ATP. sustainable than extensive cultivation of conven- NADPH and ATP are then used to produce glyceralde- tional crops. hyde-3-phosphate from CO2 in a metabolic pathway
  • 4. 4 Algae, a New Biomass Resource that is called the Calvin–Benson cycle [24]. Photosyn- would be of 77 g biomass mÀ2 dayÀ1 (280 t haÀ1 thesis enables the cell to convert inorganic CO2 into yearÀ1), corresponding to a solar-to-biomass conver- organic carbons and finally to accumulate biomass. sion efficiency of 8–10% [25]. This value is about 25 A theoretical estimation of algal biomass yield can times higher than estimations for vascular plants, pos- be assessed considering the efficiency of photosynthe- sibly due to algal biomass being fully photosyntheti- sis. A minimum of 8 photons of light energy are cally active. Such estimation assumes that all available absorbed per each oxygen molecule evolved, the actual light energy is absorbed and utilized for photosynthe- average measurement is 9.5 photons per oxygen [25]. sis. However, real algal biomass productivity achieved Per oxygen evolved, four electrons are channeled into so far in the laboratory or small-scale systems does not the linear photosynthetic electron transport chain and exceed the 73–146 t dry weight haÀ1 yearÀ1 (20–40 g in the process three molecules of ATP and two of dry weight mÀ2 dayÀ1) and 3% of solar-to-biomass NADPH are produced. In order to convert three conversion efficiency [25] in the best cases. Currently, carbon dioxide molecules into glyceraldehyde-3- commercial exploitation of algae mainly utilizes open phosphate, nine ATP and six NADPH are required. ponds. They are usually raceway cultivators driven by The ratio is 9.5 mol photons for the conversion of paddle wheels or unstirred, operating at water depths 1 mol CO2 into biomass. The average insolation, that of 15–20 cm, biomass concentration can be up to 1,000 is the solar radiation energy on a surface area in a given mg LÀ1 and productivity between 60 and 100 mg LÀ1 time, is between 3 and 5 kWh mÀ2 dayÀ1 (full solar dayÀ1 (10–25 g mÀ2 dayÀ1) [26]. Commercial rates spectrum) at temperate regions. However, only about of production of Chlorella have been reported to be 40% of solar radiation is photosynthetically active 60–75 t dry weight haÀ1 yearÀ1 (17–20 g dry weight (PAR), because only photons with wavelengths between mÀ2 dayÀ1) [27]. Another example is Dunaliella salina, 400 and 700 nm (visible spectrum) can be absorbed by which is now being cultivated on a commercial scale photosynthetic pigments, since these wavelengths carry and in pilot-scale projects to extract b-carotene and an energy equal to the change in the energetic level glycerol. Over short periods and in small-scale, pro- between the ground state and the excited one. ductivity records 60 g dry weight mÀ2 dayÀ1. However, A photon has an energy that is directly proportional commercial production requires larger culture systems to its frequency and inversely proportional to its wave- and is presently carried out in outdoor ponds (approx- length, following Eq. 2. imately 20 cm deep for 5 ha of surface, total ponds area of 50 ha), because of the still expensive constructing of E ¼ hn ¼ c =l ð2Þ closed photobioreactors. In these large-scale systems, (h, Planck constant, 6.626 Â 10À34 Js; c, light speed, the maximum productivity achievable is at present 3 Â 108 m sÀ1; n, frequency; l, wavelength) 30–40 g dry weight mÀ2 dayÀ1 but more regularly is For example, 1 mol photons (1 Einstein) at 440 nm below 30 g dry weight mÀ2 dayÀ1 [28]. Outdoor pond has energy of 272 kJ, while 1 mol photons at 670 nm has cultures of Cyclotella report a biomass productivity of energy of 178 kJ. Out of the full solar radiation of 12 g mÀ2 dayÀ1 [29]. With the present knowledge and 5 kWh mÀ2 dayÀ1, the photosynthetically active radia- with the available algal strains, real productivity is far tion (PAR) is about 35 mol photons mÀ2 dayÀ1. Con- below theoretical estimations, especially in large-scale sidering the energy requirement of 9.5 mol photons per and over long-lasting periods, and a major future goal 1 mol CO2, with such average available light radiation would be to get rid of or reduce this gap. the cell could assimilate 3.68 mol CO2 mÀ2 dayÀ1. As an alternative to open ponds, few relatively Since biomass composition can be approximated to large-scale closed photobioreactors have been devel- the formula CH2O, the previous data would translate oped (refer to “Large-Scale Systems” section for a in the synthesis of about 110 g biomass mÀ2 dayÀ1. comparison between open ponds and photobioreactors However, accumulation of organic carbons as biomass in terms of advantages), displaying a productivity of is lower, due to respiration and other metabolic activ- 2.7 g LÀ1 dayÀ1 in a small undular row tubular ities and energy losses are accounted to 30%. The photobioreactor of 11 L, 1.9 g LÀ1 dayÀ1 in a airlift resulting expected maximum biomass productivity tubular photobioreactor of 200 L and 0.05 g LÀ1 dayÀ1
  • 5. Algae, a New Biomass Resource 5in a parallel tubular photobioreactor of 25,000 L [30]. absorbed light energy in excess with respect to the satu-Constructing of photobioreactors is still at the ration level of photosynthetic electron transport doesbeginning and better productivities (up to ten not contribute to biomass accumulation, but it istimes higher g LÀ1 dayÀ1) as compared to open instead wasted as heat and/or leads to the formationponds are obtained with pilot systems. It is likely that of reactive oxygen species (ROS) that ultimately inhibitsuch interesting results would also be achieved at larger photosynthesis. Light saturation constant is defined asscale, by improving photobioreactor design and the intensity of light at which the specific biomassmechanics. growth rate is half its maximum value [9]. For example, One major reason for the real lower biomass pro- light saturation constants of microalgae Phaeodactylumductivity with respect to the theoretical calculation is tricornutum and Porphyridium cruentum are, respec-that photosynthesis light reactions have to fit down- tively, 185 mmol photons mÀ2 sÀ1 [31] and 200 mmolstream biochemical processes while excess absorbed photons mÀ2 sÀ1 [32], much lower than the maximumenergy is dissipated as heat. Photosynthesis displays outdoor sunlight level that occurs at midday ina light saturation curve [25] (Fig. 1). equatorial regions, that is about 2,000 mmol photons At low light intensities, light is the limiting factor mÀ2 sÀ1 [9]. In particular, considering a saturatingfor the photosynthesis rate, measured as photosyn- light intensity of 400 mmol photons mÀ2 sÀ1, photo-thetic oxygen evolution. At increasing light intensities, synthesis would saturate at about 7 a.m. and remainthe limiting factor becomes carbon dioxide fixation. saturated until 5 p.m. [25]. An average of 60% up toWhen light irradiance overcomes the rate of the down- more than 80% of absorbed irradiance would be wastedhill biochemical processes, excess absorbed energy is and not converted into chemical energy during thedissipated and photosynthetic oxygen evolution reaches course of a sunny day [25, 33]. These energy dissipationa plateau. If light further increases, beyond the capacity events have an important photoprotective role in theof photoprotective mechanisms, photoinhibition leads natural environment but reduce the potential growthto a decrease in photosynthesis rate. Therefore, all the rate in biomass culture conditions. P Optimal photosynthesis Heat dissipation Photoinhibition Pmax P/2 Light Light intensity saturation constantAlgae, a New Biomass Resource. Figure 1Light saturation curve. Photosynthetic oxygen evolution (P) increases with light intensity linearly until saturation. Pmax isthe maximum photosynthetic rate. P/2 is the photosynthetic oxygen evolution rate at half the maximum level. The lightintensity at P/2 is defined as the light saturation constant. Upon saturation of photosynthesis, when downstreambiochemical processes are limiting, excess absorbed energy is dissipated as heat and if light further increases beyond thecapacity of photoprotective mechanisms, photosynthetic rate decreases because of photosystem II photoinhibition
  • 6. 6 Algae, a New Biomass Resource Domestication and some of their characteristics are far from optimal for growth in mass culture conditions. A domestica- Crop species currently widely cultivated are “domesti- tion process needs to be applied to algal species pre- cated” strains that evolved from wild ancestors [34]. liminary to industrial applications are attempted. A domesticated crop has been genetically altered and In particular, selection of strains with the desired made into a resource for man through cycles of phe- properties combined with input of new alleles notypic selection, breeding, and mutagenesis. In this through mutagenesis and genetic engineering would way, man can divert evolution of plants to fit help to generate strains with improved biomass yield, agronomical, nutritional, and industrial applications. oil content, and fuel properties. For example, inser- A fully domesticated crop cannot survive without the tional mutagenesis can be used to generate random help of human mankind; meanwhile, farmers would insertional libraries to be screened for selected not work with wild species because farming with wild- phenotypes. type (ancestral) genotypes would not be economically Algal biotechnology primarily utilizes unicellular sustainable. Domestication began in Middle East species that can be propagated in the laboratory. For around 10,500 years ago when humans applied selec- this reason, a great attention is focused on unicellular tion to cereals and legumes, modifying morphological microalgae, more suitable for genetic manipulation and nutritional traits. During history, all crop plants and prospects of biofuels production [1]. Algae have were domesticated in a process that took between several advantages over higher plants in genetic decades and several centuries depending on the num- improvement: ber of genes involved. For example, modern corn was domesticated from the wild Teosinte to improve pro- 1. A short life cycle enables to genetically manipulate ductivity and the ability to grow at high density in them faster than crop plants. cornfields. High plant densities in fields are not dissim- 2. Because of the usual absence of cell differentiation ilar to the high algal cell densities that are reached up in and haploid nature of most vegetative stages, a photobioreactor. In the case of tomato, several muta- microalgae allow faster phenotypic selection. tions in genes involved, for example, in cell division, 3. Microalgae are small and can be analyzed in large carotenoid biosynthesis, anthocyanin biosynthesis, and numbers in a Petri dish. ethylene receptor altered fruit color, weight, shape, and 4. It is conceivable that substantial improvements in ripening of the wild ancestor to finally obtain the now algae cultivation could be achieved in the future. commercially cultivated tomato [35]. Indirect conven- In contrast, prospects of improvement in higher tional approaches are now being supported by recom- plants are less favorable, because current cultivated binant DNA techniques to directly manipulate crops have already been genetically improved with genotypes and speed up the process. respect to the wild ancestors and cultivation tech- The natural biodiversity of algae offers a wide spec- niques are already well optimized. Algae have been trum of phenotypes and specialized adaptation abilities less explored and exploited in the past and current that can be exploited for commercial applications. Rel- biomass productivity is likely to be improved. atively few algal strains have been examined to date, 5. Algae are suitable for growth in photobioreactors, among all the species available in nature. Strains with offering a confined environment for genetically very interesting features as accumulators of biomass, modified organisms. biofuels, or other high value products could be still In the following sections, examples of targets for unexplored and search for new strains is for domestication are described. sure a valuable strategy to pursue. However, similarly to crops, biofuel production with wild-type algal spe- Light Use Efficiency cies collected from the environment, often proposed as a “green way” to energy supply, is unlikely to be A main process affecting solar-to-biomass conversion economically sustainable, because wild-type algal efficiency is light harvesting. The photosynthetic appa- strains evolved to better adapt to their natural habitats ratus comprises two photosystems (photosystem II and
  • 7. Algae, a New Biomass Resource 7photosystem I) operating in series. Each photosystem is culture. As a consequence of the light gradient formedcomposed of an essential core complex, decorated with across the photobioreactor diameter, the real solaran antenna system of variable size with functions in radiation to biomass conversion efficiency decreaseslight harvesting and photoprotection. Chlorophyll far below the calculated value of 8–10% [25], as alsomolecules and other accessory pigments bound to pho- mentioned in the “Present Algal Productivity in thetosystems act cooperatively in the absorption of incom- Laboratory Versus Large Scale” section. The unequaling solar radiation. The ability to assemble large arrays light distribution has another negative consequence:of light-harvesting complexes has been positively algal cells are stirred into the reactor where they canselected during evolution since it represents a survival move from suboptimal illumination to strong lightstrategy and a competitive advantage in the wild, where within few seconds, without having time to adjust thelight could be limiting [36]. Meanwhile, excess light photosynthetic apparatus [47]. Intermittent light isconditions are mostly avoided by algal cells through highly stressful and contributes to the oxidative stresschanging their location in the water column. Up to 600 and photosystem II photoinhibition.chlorophyll molecules can be found in association It was first postulated 60 years ago [48] that awith photosystem II and photosystem I [25]. Light- truncated light-harvesting antenna size would optimizeharvesting complexes are however not a rigid apparatus light utilization efficiency of microalgae in a mass cul-and both short-term and long-term mechanisms to ture. Such configuration would require a higher lightadjust the light-harvesting capacity to changing light intensity to reach photosynthesis saturation (refer toconditions are present in photosynthetic organisms. “Present Algal Productivity in the Laboratory VersusThese include heat dissipation of excess absorbed energy Large Scale” section for definition of light saturation(non-photochemical quenching, NPQ) [37, 38] in the constant) and would minimize wasteful dissipation ofshort term and regulation of photosynthetic gene excess absorbed energy and photoinhibition. In partic-expression during long-term acclimation [39–41]. ular, a better transmittance of light deeper into theGrowth conditions in large-scale mass culture are culture would be allowed, enabling more cells to con-very different with respect to those found in the natural tribute to useful photosynthesis and culture productiv-environment. In photobioreactors, cellular concentra- ity. It was estimated that a reduced optical density couldtion is many orders of magnitude higher than in water improve solar radiation to biomass conversion efficiencybodies, so as to increase production per volume. In up to three to four times [33]. Figure 2 schematicallythese conditions, large-size light-harvesting antenna, compares the behavior of the wild type to a truncatedan advantage in the wild, becomes detrimental for antenna strain, with respect to light distribution andoverall biomass productivity because high optical growth inside a hypothetical tubular photobioreactor.density of antenna pigments results in self-shading While algae with a truncated light-harvesting chlo-and light attenuation in deep layers of the culture. rophyll antenna size would be useful in controlled massIncident light energy is mostly absorbed by cells at the culture conditions, they are not competitive and do notsurface, exceeding photosynthesis maximum rate and survive in the wild, so are not encountered in nature.resulting in dissipation and loss of excess photons as Although the advantages of strains with altered opticalfluorescence or heat. Up to 80% of the absorbed pho- properties were recognized long ago, only recently, withtons could thus be wasted [33]. Moreover, these cells molecular genetics coupled to biophysical phenotypewould be more subjected to photoinhibition, that is screening methods, the problem of their constructionthe light-induced inactivation of photosystem II can be addressed. Small antenna mutants can now bedue to photooxidative damage [42, 43], leading to obtained through chemical/UV mutagenesis or inser-losses in photosynthetic productivity [44]. Meanwhile, tion mutagenesis and tested for their light use effi-a suboptimal illumination would occur in the deepest ciency. Small antenna strains described earlier carriedlayers, where energy consumption by respiration would mutations in pigment biosynthesis. Pigments arereduce the overall yield [45, 46]. Only the intermediate bound to photosystem protein subunits where theylayers are in conditions for optimal photosynthetic act cooperatively in light harvesting, meanwhile pho-yield and thus determine the productivity of the mass tosystem subunits require a specific set of bound
  • 8. 8 Algae, a New Biomass Resource Wild type Truncated antenna size strain Su Heat dissipation Su Photobleaching nl Less heat dissipation nl ig ig Photoinhibition ht and photobleaching in the ht more exposed layer Photosynthesis Light intensity Growth Photosynthesis Algae, a New Biomass Resource. Figure 2 Schematic representation of a tubular photobioreactor transversal section. In wild type (left), most of light is absorbed by cells in surface layers. Excess light is transformed into heat by physiologic dissipation mechanisms. An algal strain with truncated antenna (right) is less efficient in absorbing light, thus allowing penetration of the irradiance deeper into the culture. As a consequence, a higher fraction of cells is photosynthetically active and accumulates biomass and a smaller fraction of the incident sunlight energy is dissipated into heat due to the lower number of photons intercepted by each photosystem. However, since antenna protein subunits, beside light harvesting, have important role in photoprotection preventing the formation of reactive oxygen species or scavenging them, it is essential that the reduction in the antenna system does not compromise photoprotection capacity of the strain in use pigments to be properly folded and assembled. Reduc- but no improvements in biomass productivities were tion of photosystem II antenna size was indeed observed in either laboratory cultures or outdoor observed as a consequence of mutations affecting the ponds [29]. Every isolated antenna mutant must be biosynthesis of chlorophyll b [49, 50] or the biosynthe- tested for its growth performance and oxidative stress sis of xanthophylls [51], specifically coordinated by resistance before any application. Novel genes need to light-harvesting antenna subunits, in the model unicel- be found, whose deletion or mutation leads to pheno- lular green alga Chlamydomonas reinhardtii. However, types of selective downregulation of antenna compo- photosynthetic antenna components are devoted not nents, retaining gene products with photoprotective only to light harvesting but also to photoprotection, function. Among available methods for the identifica- meant as the ability to prevent reactive oxygen species tion of these genes, random insertion mutagenesis is in the light [52]. Moreover, carotenoids of the xantho- of choice (refer to “Future Perspectives and Technolog- phyll cycle have multiple roles in photoprotection [53– ical Developments” section for further explanation). 55]. Algal mechanisms of oxidative stress resistance are Once a mutant library has been generated by transfor- particularly important for growth in photobioreactors, mation with a cassette carrying a selectable marker especially the capacity to respond to fast alterations in gene, strains with the desired characteristics must be light intensity that cells can face during stirring of the identified from the pool by high throughput methods. medium. Therefore, efforts at reducing the antenna Truncated antenna size strains can be selected for must not compromise the photoprotection ability of a lower chlorophyll fluorescence yield and/or pale the strain, otherwise the advantage obtained in terms of green appearance and/or lower accessory pigments ver- light distribution would be worthless. For example, sus chlorophyll a content with respect to wild type. The pigments mutants of the diatom Cyclotella, without fluorescence screening is performed upon application fucoxanthin, a xanthophyll synthesized in chlorophyll of a saturating light pulse to dark-acclimated cells. a and chlorophyll c containing algae, displayed Light excites chlorophylls connected to photosystem a truncated antenna size and a higher light saturation, II which undergo a transition from an “open” state
  • 9. Algae, a New Biomass Resource 9with low fluorescence to a “closed” state when all pho- Coordinated downregulation of the entire light-tosystem II primary acceptors are reduced and fluores- harvesting chlorophyll-binding lhc gene family wascence level is maximal. The maximum fluorescence achieved by RNA interference technology, at lastparameter depends on the number of chlorophylls resulting in increased efficiency of cell cultivationbound to photosystem II and its value is therefore under elevated light conditions of 1,000 mmol photonsroughly indicative of photosystem II antenna size. mÀ2 sÀ1. Peak density of RNAi cultures was detectedThus, mutants with reduced chlorophyll antenna are already after 26.5 h, at which parental strain culturesexpected to show a phenotype with a lower yield of had only been able to grow to 54% of their maximal cellchlorophyll fluorescence with respect to the wild type densities [59]. However, RNA interference-induced[56]. At room temperature, chlorophyll florescence phenotypes can become unstable after some time.derives from photosystem II, since photosystem This technology would be useful to identify antennaI fluorescence yield is negligible in this condition. Pho- components or modulators that can be depleted with-tosystem I is more efficient in quenching antenna exci- out compromising photoprotection ability. It shouldtation because of a faster turnover rate of the be noted, however, that strains for industrial usephotochemistry reactions and its fluorescence is detect- should be stable over time making deletion/insertionalable only at low temperature (77 K). For this reason, mutants preferable.room temperature fluorescence intensity is indicative Through insertional mutagenesis of C. reinhardtii,of the sole photosystem II antenna. Meanwhile, since an interesting mutant, called tla1, was isolated forantenna subunits bind different pigments with respect a reduced yield of chlorophyll fluorescence. It displayedto photosystem core complexes, namely chlorophyll b a residual chlorophyll content per cell of about 35–40%and xanthophylls, analysis of pigments by absorption the wild-type level and a functional chlorophyllspectra and/or HPLC can detect mutations in both antenna size of both photosystem II and photosystemphotosystem I and photosystem II antenna systems. I reduced to 114 and 160 chlorophyll molecules, respec-Since chlorophyll biosynthesis in the chloroplast is tively [56]. This mutation allowed to identify a novelstrictly coordinated with the expression of both nucleus gene involved in the regulation of antenna proteinsand chloroplast-encoded chlorophyll-binding proteins, accumulation in algae. The tla1 strain requireda “pale green” phenotype could arise from mutations a higher light intensity for the saturation of photosyn-affecting directly chlorophyll biosynthesis or from muta- thesis, about twofolds higher than wild type, andtions impairing expression of chlorophyll-binding pro- showed a higher photosynthetic productivity underteins, their import into the chloroplast or their assembly mass culture conditions, reaching higher cell densitiesinto photosystems. Mutations affecting these mechanisms (10 Â 106 cells mLÀ1 vs. 6.4 Â 106 cells mLÀ1) [56, 60].are expected to yield strains with constitutively reduced Simultaneous reduction of photosystem II and photo-antenna size, irrespective of the compensatory acclimative system I antenna systems is a desired advantage inmechanisms which are involved in adapting the light- terms of global optical density of the cell. In contrast,harvesting function to environmental conditions. the sole reduction in photosystem II antenna, if not Factors controlling antenna proteins expression at counterbalanced by an adjustment in photosystemthe posttranscriptional level are attractive for genetic II/photosystem I ratio, could decrease the photon useengineering manipulations [57] and a permanently efficiency possibly because a portion of the light energyactive variant of NAB1, a novel cytosolic RNA binding absorbed by photosystem I could not be efficientlyprotein, allowed to reduce photosystem II antenna size utilized in the linear photosynthetic electron transportby 10–17% via translation repression, finally improv- process [49]. The minimum number of chlorophylling light-to-biomass conversion efficiency in high light. molecules, needed for the assembly of the photosystemIn particular, cell culture densities of the antenna core complexes, has been estimated to be 37 for pho-mutant in the 200 mL bottles increased within 38 h tosystem II and 95 for photosystem I [61]. This wouldfrom 5.97 Â 105 to 1.2 Â 107 cells mLÀ1, whereas the be the minimum chlorophyll antenna size that wouldparental strain reached only 5.72Â106 cells mLÀ1 after allow for assembly of the photosystems and that couldthe same time period [58]. be theoretically achieved through mutagenesis and
  • 10. 10 Algae, a New Biomass Resource genetic engineering [25]. Reduction in the antenna that is the light-induced inactivation of photosystem II systems acquired so far is still above this estimated reaction center. More recently, qI was shown to depend minimum chlorophyll antenna size and further on the synthesis of zeaxanthin, promoted in excess improvements could be achieved in the future. light. Zeaxanthin binds to light-harvesting antenna Although truncation of the antenna size of the proteins where it becomes a quencher for chlorophyll photosystem unit can contribute to improve optical excited states. Its action relaxes when zeaxanthin properties of the algal suspension, considering the is released and back-transformed into violaxanthin important role of the antenna systems also in [63]. A third quenching component, relaxing within photoprotection, an alternative route to pursue could minutes, has been reported and called qT and proposed be to reduce the number of photosystems per cell and to depend on the phosphorylation of the outer antenna thus the pigment content per cell. In algae, acclimation proteins that controls their partition between photo- to high light could rely more on the reduction of the system II and photosystem I during the process of state photosystem density rather than on major adjustments 1–2 transition [64]. in the antenna system to prevent overexcitation [62]. Photosynthetic light harvesting in higher plants This natural light acclimation ability could be is mainly regulated through qE and requires PsbS, exploited in order to generate strains with a photosystem II subunit [65], which is sensitive to a constitutive reduced optical density through the pH through two lumen-exposed glutamate residues identification of involved regulatory factors and their whose protonation is required for qE activation [66]. genetic manipulation. Since PsbS does not bind pigments, it cannot be a quenching site but rather a pH sensitive trigger [67] transducing a conformational change to antenna pro- Non-Photochemical Energy Quenching at the teins binding chlorophylls and xanthophylls. Changes Molecular Level in the mutual distance/orientation of these chromo- Oxygenic photosynthesis involves highly reactive inter- phores reversibly promote energy-dissipation pro- mediates, such as singlet excited state of chlorophylls cesses such as transient formation of carotenoid that can decade into triplet excited state, and may lead radical cations, which decay to the ground state to the formation of reactive oxygen species (ROS). dissipating energy as heat [68, 69]. Although light- These by-products can damage the photosynthetic dependent energy quenching is a property of all apparatus and other chloroplast constituents. The photosynthetic eukaryotes, strong differences in the potential for damage is exacerbated when the amount underlying mechanisms are apparent. Although genes of absorbed light exceeds the capacity for light energy encoding PsbS are found throughout the green lineage, utilization in photosynthesis, a condition that can indicating that the protein was present before the lead to decreases in photosynthetic efficiency. Non- separation between unicellular green algae and photochemical quenching (NPQ) is the process of multicellular organisms, a PsbS protein is only heat dissipation of excess absorbed energy, which is accumulated in green macroalgae and land plants. aimed at regulating light harvesting and preventing Nevertheless, several algal species, such as Chlorella overexcitation of reaction centers. The major compo- zofingiensis and Scenedesmus communis, exhibit strong nent of NPQ, called qE, is the fastest established and light-dependent NPQ independent from light intensity more rapidly reversible quenching deriving from de- during growth [70] or upon acclimation to high light excitation of the singlet excited state of chlorophyll in as in Chlamydomonas reinhardtii [71], suggesting the light-harvesting antenna of photosystem II in other gene products might be involved. The moss response to a change in thylakoid lumen pH. Excess Physcomitrella patens and algal genomes, but not red light with respect to the capacity of CO2 fixation pro- algae and cyanobacteria, include LHCSR (stress-related motes lumen acidification since ATP synthesis is lim- members of the Lhc protein superfamily) genes that are ited by the low ADP + Pi concentration available. In essential for NPQ [72, 73]. Cyanobacteria and possibly addition to qE, a more slowly relaxing component of red algae, which have phycobilisomes for light the NPQ process is known as qI, from photoinhibition, harvesting, perform excess energy dissipation by a
  • 11. Algae, a New Biomass Resource 11mechanism distinct from qE, which depends on the activities are accounted for about 30% energy lossesOrange Carotenoid Protein (OCP) [74]. Understand- with respect to the light energy absorbed leading to aning the mechanisms of NPQ and identification of the estimated solar-to-biomass conversion efficiency ofgenes involved is valuable as a target for genetic 8–10% in algae [25]. Biomass accumulation ultimatelyimprovement of algal productivity and domestication, depends on algal photosynthetic architecture and thesince NPQ controls light stress resistance and energy intricate relationships between oxygenic photosynthe-losses in an algal mass culture. sis, mitochondrial respiration, and catabolism of endogenous substrates. In order to improve solar-to- biomass conversion efficiency, it is essential to recog-Interconversion and Storage of Photosynthetic nize bottlenecks in the processes interconnecting pho-Metabolic Products tosynthesis to other metabolic pathways to make themAside from light harvesting, solar-to-biomass conver- targets for engineering. Figure 3 is a schematic repre-sion efficiency strongly depends on downhill biochem- sentation of the main anabolic and catabolic pathwaysical reactions influencing photosynthate production influencing photosynthate production and biomassand utilization. Respiration and other metabolic accumulation. NA Lipids D(P Starch ATP )H AcetyICOA Gly col isis NA Catabolism of endogenous substrates NA Sugars D(P ½ O2 + 2H+ D(P )H, H2O ATP )H AT P CO2 Mitochondrial CO2 Proteins respiration Calvin RuBisCO NAD(P)H cycle Malate shuttle ATP ATP NADPH n tio H2 ira O2 H+ sp NADP+ ADP + Pi ore H2ase FN lor 2H + R Ch PTOX Fdx PQ Cyt Chloroplast thylakoid PSII PQH2 PSI b6f membrane ATPsynthase PC H2O ½ O2 + 2H+ 2H+Algae, a New Biomass Resource. Figure 3Metabolic overview. Components of the photosynthetic electron transport chain embedded in the chloroplast thylakoidmembrane are depicted. Black lines mark biosynthetic reactions building up the main constituents of the cell, proteins,starch, and lipids. Dark gray lines mark catabolic reactions that consume endogenous substrates. PSII photosystem II, PSIphotosystem I, Cyt b6f cytochrome b6f complex, PQ plastoquinone, PQH2 plastoquinol, PTOX plastid terminal oxidase,PC plastocyanin, Fdx ferredoxin, FNR ferredoxin-NADP+ oxidoreductase, H2ase hydrogenase, RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase
  • 12. 12 Algae, a New Biomass Resource The primary reactions of photosynthesis in algae cell. In particular, it was observed that efficient photo- and plants occur in the chloroplast thylakoid mem- synthesis depends also on mitochondrial respiration in brane and are catalyzed by the multiprotein-pigment the light [16, 78, 80, 81]. complexes photosystem II (PSII), cytochrome b6f (Cyt Carbon fixation is influenced by alternative elec- b6f), and photosystem I (PSI). The photosynthetic tron sinks as well as alternative electron sources. For apparatus itself is a very flexible system, able to modify example, reducing equivalents derived from catabo- exciton fluxes within its antenna complexes as well as lism of endogenous substrates can fuel the respiration electron fluxes among its electron transfer components chain in mitochondria but also the photosynthetic [75]. Photosystem II and photosystem I operate in electron transport chain, thanks to a respiration path- series within the photosynthetic electron transport way in the chloroplast. Respiration of the chloroplast, chain. However, alternative electron transport path- also called chlororespiration, was defined as a ways could be engaged, as well as alternative electron respiratory electron transport chain in interaction sources or sinks that ultimately would affect availability with the photosynthetic chain [82] and could have its of reducing power (NADPH) and ATP for carbon origin in the cyanobacterial endosymbiotic ancestor of dioxide fixation. As a matter of fact, the net products chloroplasts [82, 83]. This pathway was discovered of photochemistry, NADPH and ATP, that supply about 30 years ago in algae and originally proposed the Calvin–Benson cycle, are also used by other meta- to account for the changes in the redox state of bolic pathways such as nitrate assimilation, lipid, plastoquinone, a photosynthetic electron carrier, in aminoacids, and pigments synthesis. These different darkness [84]. Chlororespiration includes a NAD(P) sinks may significantly contribute to modify the ATP/ H-dehydrogenase [85, 86] and a plastid terminal oxi- NADPH ratio and, as a consequence, in vivo carbon dase PTOX [87–91]. The NAD(P)H-dehydrogenase fixation [76]. The photosynthetic process is divided enzyme of chlororespiration is possibly involved also into reactions that provide ATP and NADPH (photo- in recycling photosynthesis-generated NAD(P)H, chemical light reactions) and reactions that consume establishing a cyclic electron transport around photo- both compounds (carbon assimilation through the system I [92–94]. This alternative electron transport Calvin–Benson cycle). The rate of these two phases could be engaged to balance the ATP/NADPH stoichi- may differ in several orders of magnitude, especially ometry, since the net product is only ATP, in response in high light when carbon assimilation is limiting the to the requirements of the cell and is controlled by the overall process, and alternative electron sinks may have redox state of the plastoquinone pool. The strong inter- an important photoprotective role to consume excess play between mitochondria and chloroplast metabo- generated reductants. Respiration in mitochondria lism is also evident from the characterization of can serve as well as a sink for excess photosynthesis Chlamydomonas reinhardtii mutants with defects in generated reducing power. Complex interactions the mitochondrial electron transport chain: an between photosynthesis in the chloroplast and respira- enhanced glycolysis, to compensate for the absence tion in the mitochondria occur because both processes of mitochondrial ATP input, is thought to increase are linked by common key metabolites such as plastoquinone reduction through chlororespiration ADP/ATP, NAD(P)H, triose-P, and hexose-P [75, and favor cyclic over linear electron transport in the 77, 78]. The chloroplast continuously communicates chloroplast [80]. its metabolic state to the cell, for instance via the Cell fitness depends on the delicate equilibrium export of carbohydrates, which are synthesized during between biosynthetic and catabolic reactions, both photosynthesis. The transport of carbohydrates across essential. In order to use a photosynthetic organism the chloroplast membrane directly allows to exchange as a biofuel or biomass producer, it is essential to reducing equivalents through “redox valves,” such as control these intricate metabolic dynamics. Carbon the malate-oxaloacetate shuttle [79]. Different cellular assimilation rate may be a bottleneck, particularly in compartments are thus strictly interconnected and high light. Dunaliella salina was shown to upregulate photosynthesis is influenced by either the requirements key enzymes in the Calvin cycle at high salinity, possi- of the chloroplast or the metabolic status of the whole bly in order to enhance synthesis of glycerol as osmotic
  • 13. Algae, a New Biomass Resource 13protector [95]. It is conceivable that improvements in due to the fact that most of the photosystem II antennacarbon assimilation could be achieved in the future is displaced from photosystem II to photosystemwith the help of genetic engineering. Strategies to I during this process and chlorophyll fluorescence atachieve this aim could be altering expression and/or room temperature arises mostly from photosystem II.activity of key enzymes or acting on ATP/NADPH The difference in fluorescence between states 1 and 2 canavailability. be measured with a fluorescence video imaging system and used for screening mutants deficient in state transition. In the model plant A. thaliana, states 1 andScreening for State Transition as an Indirect 2 are induced by illuminating cells with light preferen-Mean to Select Strains with Altered Redox tially absorbed by photosystem I and photosystem II,Metabolism respectively [102]. In C. reinhardtii cells, however,The redox state of the plastoquinone pool is known to this procedure is not effective because in this algacontrol a process called “state1–2 transition.” Since the the absorption spectra of photosystem II andlight-harvesting antennae of photosystem II and pho- photosystem I antennae overlap. Thus, states 1 and 2tosystem I have distinct absorption spectra, changes in are achieved in the dark by taking advantage of thethe spectral composition of the incident light can lead ability of the chlororespiratory chain to change theto unequal excitation of the two photosystems and thus redox state of the plastoquinone pool [96, 97]. Stateto a decreased photosynthetic yield. Plants and algae transition and cyclic electron transport around photo-are able to balance the relative excitation of the photo- system I are controlled by the redox state of the plasto-systems through state transition, that is the reversible quinone pool. This diffusible electron transport elementmigration of a fraction of the light-harvesting antenna is at the crossroad between linear electron transportof photosystem II from photosystem II in state 1 from water to NADP+ and the chlororespiration path-to photosystem I in state 2, upon phosphorylation way dissipating excess redox power by reducing O2 to[96–98]. This process was discovered about 40 years H2O. Thus, plastoquinone redox state is altered eitherago in unicellular microalgae [99,100]. The state tran- by an imbalanced excitation of the photosystems or bysition process has been widely studied in the unicellular changes in the rate of catabolic degradation of storagegreen alga C. reinhardtii, where its amplitude is larger molecules and could therefore serve as a key sensorthan in plants and appears to have a major role in of both the incident photon flux and the cellular ener-balancing the ATP/NADPH stoichiometry by regulat- getic status. Mutations in state transition can affect theing the switch between linear and cyclic electron flow, non-photochemical reduction and oxidation of thein addition to the redistribution of the excitation plastoquinone, catabolism of endogenous substrates,energy between photosystems following changes in and upstream reactions involved in feeding reducinglight conditions. Indeed, recycling of electrons around equivalents into the stroma compartment of thephotosystem I was observed in state 2 [92–94]. The chloroplast. Screening for state transition mutants is ainterplay between chloroplast and mitochondria useful strategy to isolate strains with an altered redoxmetabolism is strong in algae. The mere inhibition of metabolism that could finally affect photosynthatestate transition was shown to be insufficient to modify generation/storage, growth rate, and biofuel produc-photosynthesis in the presence of active mitochondrial tion. It represents a mean of domestication alone or inrespiration, in contrast it is essential when respiration is combination with other characteristics.compromised, revealing the physiological significanceof state transition in the energetic contribution [81]. Accumulation of Biomass as Neutral LipidsUp to 80% of the excitation energy absorbed by thephotosystem II antenna can be redistributed from Assimilated carbons can be stored in high energy valuephotosystem II to photosystem I in C. reinhardtii that metabolites, such as starch, proteins, or lipids, asis therefore well suitable for screening state transition shown in Fig. 2. Some species of algae are particularlymutants [96, 97, 101]. Large chlorophyll fluorescence attractive because of their ability to produce highchanges occur during a transition from state 1 to state 2, amounts (20–50% of dry cell weight) of neutral lipids
  • 14. 14 Algae, a New Biomass Resource that can serve as a source of biodiesel. Cellular lipid than genus-specific [2]. Interesting species for biodiesel metabolism is altered toward the accumulation of production include green algae, such as Botryococcus neutral lipids, mainly triacylglycerols, under stressful braunii, Neochloris oleoabundans, Nannochloris sp., conditions, such as nutrient starvation, salinity, Chlorella sp., and Dunaliella primolecta; diatoms, nonoptimal growth medium pH, low temperature, such as Nitzschia, Phaeodactylum tricornutum, and high light, but also during aging of the culture [2]. Cylindrotheca sp.; and members of other algal taxa, These lipids do not have a structural role in membranes such as Nannochloropsis sp. and Schizochytrium sp. but serve as a storage form of carbon and energy, [9]. Relatively few algal strains have been examined to confined in lipid bodies in the cytoplasm. Lipid bodies date, among the species available in nature. The possi- also occur in the inter-thylakoid space of the chloro- bility to isolate new oleaginous strains with the ability plast in certain green algae such as Dunaliella bardawil to accumulate high levels of oils with the best proper- [103]. An attractive oleaginous green alga is ties as fuel is likely to offer substantial improvements in Botryococcus braunii that produces up to 80% of dry lipid yield of industrial cultures while mutagenesis will weight of very-long-chain (C23–C40) hydrocarbons further enhance the productivity of natural strains. similar to those found in petroleum, under adverse Fatty acid composition can vary both quantitatively environmental conditions [8, 104]. Neutral lipids can and qualitatively with the physiological status and cul- serve additional physiological roles. Fatty acids synthe- ture conditions. Since oil accumulation is enhanced sis consumes twice the NADPH required for carbohy- under stress, altering growth medium composition drate or protein synthesis and may thus provide appears as a strategy to improve oil productivity. For an electron sink under photooxidative stress [2]. More- example, high carbon dioxide concentrations (>5% v/ over, coordination with carotenoid synthesis has v) in Dunaliella salina [107] or nutrient deficiency such been observed, in particular carotenoids are seques- as nitrogen deprivation in Chlorella vulgaris [108] can tered into cytosolic lipid bodies where they function lead to a threefold increase in intracellular lipids. as a sunscreen to reduce light striking the chloroplast Lipid metabolism has been poorly studied in [2, 105, 106]. algae. Nevertheless, available data suggest that the Algae triacylglycerols can be exploited to produce basic pathways are analogous to those experimentally biodiesel, via esterification of fatty acids. Biodiesel has detailed in higher plants. However, differences are a three to four times higher energy yield than ethanol; distinguishable: however, biodiesel still represents a small percentage of 1. While in higher plants organic carbon is translocated total diesel fuel consumption (1.6% in Europe and from photosynthetically active tissues to sinks 0.21% in the USA, 2005–2007), while ethanol repre- for lipid synthesis and storage, in microalgae sents 5% of US gasoline consumption [23]. Currently, triacylglycerols accumulation takes place within a biodiesel is mainly produced from higher plants, single cell together with photosynthesis. such as palm and soybean [23]. But while extensive 2. While neutral lipids synthesis is mainly associated cultivation of crops to supply energetic demands is to seed development in higher plants, it is triggered unsustainable, competing with food industry for arable under stress conditions in algae [2]. lands, oil production per hectare from algae, based on theoretical calculations, would be 100-fold higher than A better knowledge of lipids synthesis pathways that of soybean and could meet 50% of present US and regulation mechanisms is needed in order to imple- transportation demand using less than 3% of available ment genetic engineering strategies for oil production. cropland [9]. Based on experimentally demonstrated Identification of metabolic differences between oleagi- biomass productivity, oil yield of microalgae could be nous strains and the species that do not accumulate 136 or 58 t haÀ1 yearÀ1 considering 70% or 30% oil in substantial amounts of lipids is a possible research strat- biomass, respectively [9]. However, insufficient efforts egy to be realized using comparative transcriptomic, for the establishment of algae-based biodiesel produc- proteomic, and metabolomic profiles of different strains tion plants have been made until now. The ability of or the same strain under control versus stressful algae to produce neutral lipids is species-specific rather conditions. Microarray analysis of Chlamydomonas
  • 15. Algae, a New Biomass Resource 15reinhardtii transcripts under anaerobic incubation has heterotrophic growth, concomitantly with the increasebeen performed [109], revealing fermentative pathways of C16:0 and C18:1 fatty acids in triacylglycerolsthat produce acetyl CoA, the substrate for fatty acids [118–120]. Monounsaturated fatty acids are indeedsynthesis. Genes encoding enzymes involved in these suitable for good diesel properties [23].fermentative reactions represent putative candidates Research of new genes involved in lipid metabo-for increasing triacylglycerols accumulation [2]. lism could be pursued by random mutagenesis. The limiting step in fatty acids biosynthesis is the A mutant library represents a biological resource ofreaction catalyzed by the first enzyme of the pathway: novel strains that could include improved biodieselacetyl CoA carboxylase (ACCase). The properties of producers. An easy and rapid strategy to screen forthis enzyme have been characterized in Cyclotella mutants with altered lipid content is by the use ofcryptica [110–113], but attempts to alter its expression Nile red [121]. Nile red is a hydrophobic moleculelevel did not have effects on lipid production [113]. that emits a significant fluorescence signal whenSome transformants showed two- to threefold higher dissolved in lipids at 565–585 nm, in a spectral regionACCase expression and activity than wild-type cells; where photosynthetic pigments fluorescence is negligi-however, no detectable increase in lipid levels was ble. Nile red fluorescence detection could be appliedobserved. Overexpression of the endogenous enzyme to screen and compare mutant colonies with respect tomay induce negative feedback so that increased activity the wild type in a microtiter plate. Mutants displaying anof the ACCase enzyme could be compensated for altered Nile red fluorescence yield and thus altered lipidby other pathways within the cell [113]. Indeed, feed- content with respect to the wild type can then be studiedback inhibition was reported in higher plants [114], by molecular biology techniques to indentify thewhere only a heterologous enzyme was successfully mutated gene and tested for oil productivity.overexpressed really improving oil content [115, 116].This approach could be proposed again and plants Planning an Algal Refinerygenes could be used for overexpression in algae. Although activity and expression of single enzymes Algae convert solar energy into chemical energy and pro-could be altered, a more powerful strategy might con- duce biomass through photosynthesis. The processsist in the identification of transcription factors co- requires light, carbon dioxide, water, and other essentialregulating all the genes of the pathway. nutrients and can be considered renewable. Minimal nutri- Lipids biosynthesis is strictly interconnected tional requirements can be estimated using the approxi-to other biosynthetic pathways in a unique global mate molecular formula of the microalgal biomass, that isnetwork. A Chlamydomonas reinhardtii starch-less CO0.48 H1.83 N0.11 P0.01 [9, 122]. However, inorganicmutant, defective in the ADP-glucose pyrophosphorylase, elements have to be added in excess because they formwas shown to hyper-accumulate triacylglycerol by complexes in solution that are not bioavailable.a factor of 10 [117]. A strategy to improve oil synthesis Algal biomass can be exploited in different ways,in order to produce biodiesel could be as well to act on providing several products and biofuels. A schemethe partitioning of carbon and energy between different of the possible organization of an algal refinery ispathways. reported in Fig. 4. Finally, fatty acids composition of triacylglycerols Upon harvesting of the biomass, water andinfluences diesel quality and must be considered for nutrients can be recycled, especially using closedcommercial application. Genetic engineering can addi- photobioreactors. Then, biodiesel is obtained fromtionally serve to alter the fatty acids profile thus esterification of fatty acids contained in triacylglycerolsimproving fuel properties in engines [23]. Many algae or from other neutral hydrocarbons accumulated bycan synthesize very long polyunsaturated fatty acids in oleaginous algae. A by-product from esterificationlarge amounts (arachidonic C20:4, eicosapentaenoic of fatty acids to produce biodiesel is glycerol that canC20:5, docosahexaenoic C22:6 acids), that are extracted serve as fermentative substrate. For example, algaas high added value products. Polyunsaturated fatty Schizochytrium limacinum produces docosahexaenoicacids decrease in nutrient-limited medium and during acid, a polyunsaturated o3 fatty acid with beneficial
  • 16. 16 Algae, a New Biomass Resource Glycerol as fermentative substrate Water/nutrients Extraction of Biodiesel neutral lipids H2 Purification of Light/CO2 Biomass Downstream Biomass chemicals and Water/nutrients harvesting processes pharmaceuticals CO2 Anaerobic Electric Biomethane digestion power Animal food (high protein contant) H2 Fertilizers (high nitrate contant) Algae, a New Biomass Resource. Figure 4 Scheme of the possible utilizations and products of an algal refinery effects for human health, through fermentation on nitrate. Biomethane (or biogas) has a high energetic crude glycerol [123]. Currently, algae are already com- yield and is produced by anaerobic bacteria during mercially used to extract chemicals and pharmaceuti- fermentation of the biomass. The obtained gas contains cals and this utilization can be combined with the need methane (50–75%) but also carbon dioxide (25–50%). for biofuels. High added value products derived from However, released carbon dioxide is equivalent to the algae include polyunsaturated fatty acids, proteins, bio- carbon that algae have assimilated during growth, lead- polymers, polysaccharides, pigments, vitamins, and ing to zero net carbon dioxide emissions. Carbon diox- antioxidants [1, 4]. To date, commercialized algae are ide released during the process can be immediately not transgenic, but this scenario could change in the recycled for alga growth. Electric power produced near future. For instance, algae can be transformed in from the biogas can sustain the energy demands of “cell factories” with the help of molecular biology tech- the photobioreactor plant itself [10]. Some algae have niques for manufacturing recombinant proteins with been evaluated for biomass conversion to methane, in pharmaceutical or other applications, an approach also particular the giant kelp Macrocystis pyrifera because of called “molecular farming.” For example, expression of its high growth rate and ease of harvesting [5]. Alter- human antibodies and vaccines has already proven to natively, algal biomass can serve as fermentative sub- be successful [124–128]. In general, algae are attractive strate for heterotrophic or photosynthetic anoxygenic as expression systems for the short life cycle, the cheap sulfur bacteria that use electrons coming from organic and easy large-scale cultivation, and the ability to grow carbons to produce molecular hydrogen. In particular, up to high cell densities [129]. Algae have been shown photosynthetic anoxygenic sulfur bacteria use light free of human pathogens and toxins and thus consid- energy to drive a fermentative reaction and organic ered safe. acids are electron donors for molecular hydrogen evo- After extraction of oils and other products, the lution. Generated hydrogen is a high energetic fuel and spent biomass can serve as animal food or used as clean since its combustion generates water [130]. fermentative substrate for other microorganisms In addition, algae have directly been evaluated for whose metabolic by-products include biomethane the production of biohydrogen. As a matter of fact, and biohydrogen. Effluents from the digestion can be some algae are able to evolve hydrogen by a hydroge- used as fertilizers because of their high content in nase enzyme that catalyzes reduction of protons to
  • 17. Algae, a New Biomass Resource 17molecular hydrogen. Hydrogen metabolism is primar- hydrogenase and to deplete competitive electronily the domain of bacteria and microalgae. Microbial sinks. An improved hydrogen evolution under sulfurhydrogen formation is catalyzed by either nitrogenases starvation was observed in a mutant affected in theor hydrogenases, enzymes that can only function under mitochondrial respiratory chain and simultaneouslyanaerobic conditions. Nitrogenases are used by certain impaired in the ability to activate state transitioncyanobacteria and photosynthetic bacteria, whereas [16]. In this mutant, accumulation of starch in thegreen algae use hydrogenases [12]. In Chlamydomonas chloroplast supplied the photosynthetic electronreinhardtii, the hydrogenase is expressed in anoxia transport chain through chlororespiration, whiletogether with the enzymes of the fermentative metab- downregulation of cyclic electron transport aroundolism that is active in the dark [109]. However, algae are photosystem I enabled a greater fraction of electronsattractive for hydrogen production if photosynthetic to be used by the hydrogenase (see “Interconversionelectron transport can be directly exploited, since and Storage of Photosynthetic Metabolic Products”hydrogen would be generated from the most abundant and “Screening for State Transition as an Indirectof the natural resources, sunlight and water. Interest- Mean to Select Strains with Altered Metabolism” sec-ingly, after a period of anaerobic incubation in the tions for further explanations about state transitiondark, photosynthetic hydrogen evolution is detected and cyclic electron transport). The hydrogen produc-transiently upon illumination [131]. The hydrogenase tion rates of the mutant were 5–13 times higher than inis transiently active during the dark-to-light transition, wild-type strain, yielding about 540 mL of hydrogento consume reducing power in the time needed to fully per 1 L of culture over a 10–14-day period (up to 98%activate the Calvin–Benson cycle, and receives electrons pure) [16]. However, sulfur deprivation is deleteriousfrom ferredoxin reduced by photosystem I. This activ- for the cell and this system is sustainable only for fewity lasts until oxygen evolution by photosystem II days. Upon a recovery phase of sulfur repletion, andrestores aerobiosis and inhibits the hydrogenase reconstitution of reserves as endogenous substrates,enzyme. In order to photo-produce hydrogen on hydrogen production can be resumed. Nevertheless,large scale, the major challenge to overcome is the continuous hydrogen production is not possible usinginhibition by oxygen evolved by photosystem II during this technology. An alternative two-stage approach tooxygenic photosynthesis. Up to now, encouraging overcome the oxygen sensitivity of the hydrogenase,hydrogen production rates have been obtained by with respect to sulfur deprivation, involves the controla two-stage approach: the severe oxygen sensitivity of of photosystem II expression [132]. The initial require-the hydrogenase is circumvented by temporally sepa- ment for this application is a photosystem II-lessrating photosynthetic oxygen evolution and growth mutant which would be complemented by an inducible(stage 1) from hydrogen evolution (stage 2). A transi- cassette to switch on/off photosystem II activitytion from stage 1 to stage 2 is performed upon sulfur and oxygen evolution. However, the most efficientdeprivation, which reversibly inactivates photosystem method to obtain large-scale hydrogen production isII and oxygen evolution. Under this condition, oxida- believed to be the continuous production of hydrogentive respiration by the cell in the light depletes oxygen concomitantly to photosynthesis and growth. Theand causes anaerobiosis in the culture, which is neces- limiting factor for such a technology is the finding ofsary and sufficient for the induction of the hydrogenase a hydrogenase enzyme with sufficient resistance to[13, 15]. Electrons for hydrogen production originate oxygen. Two different approaches are possible: to engi-from the residual activity of photosystem II as well as neer existing hydrogenases by introducing mutationsfrom the consumption of endogenous substrates that conferring resistance to oxygen or to find organismsgenerates extra electrons supplied to the photosyn- that can synthesize hydrogen even in the presence ofthetic chain through the chlororespiration pathway some oxygen, thus having hydrogenases less oxygen[16, 86]. Hydrogen production depends from available sensitive. Such hydrogenase could then be heterolo-electrons sources and competitive sinks of reducing gously expressed in algae [130] to generatepower and a strategy to improve its evolution would a hydrogen producing strain with increased yield ofalso be to increase electrons channeling toward the this biofuel.
  • 18. 18 Algae, a New Biomass Resource Large-Scale Systems 4. The stability of the dominating algal species is easier to be maintained in photobioreactors because con- Open ponds and closed photobioreactors have been tamination by other organisms is lower. The growth evaluated for algae mass culture [9, 26, 30]. Currently, process, thus, is more reproducible and standardized. commercial exploitation of algae mainly utilizes open Strict sterility and quality control allowed by closed ponds, easier to construct and scale up, and less expen- systems are particularly required for the production sive. Open ponds can be categorized into natural waters of pharmaceuticals and food additives [133]. (lakes, lagoons, ponds) and artificial ponds or con- 5. In closed systems, water and nutrients, such as salts, tainers. They are generally raceway cultivators driven can be recycled for algal growth and loss of water by paddle wheels or unstirred. In contrast, closed sys- due to evaporation and diffusion of CO2 to the tems like photobioreactors are more costly. Moreover, atmosphere would be minimized. while many photobioreactors are operative at laboratory 6. Efficient stirring mechanisms can be developed in scale, their scaling up is more difficult and could be photobioreactors ensuring homogeneity of the cul- a limiting step. This is counterbalanced by several advan- ture and finally influencing productivity in terms of tages over open ponds and some photobioreactors for growth. Flow is produced using either a mechanical large scale are in the process of being constructed. Closed pump or a gentler airlift pump. Mechanical pump systems are in the early stages of development but they could be more stressful and a strain with cell wall are likely to become more attractive in the future. would be more suitable, because less sensitive to 1. Although photobioreactors could be artificially shear damage. Agitation is required to prevent illuminated, to sustain growth during night, cells sedimentation and to allow proper gas the use of natural sunlight is the major option exchanges. As a consequence of the stirring and for cost-effectiveness. Photobioreactors can be depending from the light gradient inside the culture designed to improve the ratio between the illumi- due to the optical property of the strain, cells can nated surface and the total volume in order to suddenly be moved from suboptimal illumination minimize dark zones. From this point of view, to strong light at the surface without time to adjust flat-plate, horizontal and inclined tubular the photosynthetic apparatus and finally leading photobioreactors are promising [30]. A general ten- to light stress. However, it was observed that dency is to reduce the thickness, thus laminar layer light–dark cycling of above a certain frequency, plate or thin diameter tube configurations are such as 10 ms, can increase growth as compared becoming strongly favored, mimicking the with continuous illumination of the same intensity. leaves of land plants. For instance a 100-year-old, It was proposed that a short dark period could 10-m high lime tree shading an area of 100 m2 allow the photosynthetic apparatus to recover has a leaf surface area of more than 2,500 m2. from the excited state of the previous illumination Expressed as a surface/volume ratio, this amounts pulse under light saturation conditions [9]. to a value of 2.5 m2 mÀ3 [26] and the design of 7. Harvesting of biomass is expensive since it requires large-scale photobioreactors should tend to this energy. Biomass can be recovered from the value. broth by filtration or centrifugation [134]. Since 2. Large-scale cultivation of algae requires first of all concentration of biomass in photobioreactors a good understanding of the physiology and ecology is nearly 30 times higher than in open ponds of the species, for example, with respect to nutrients (2–8 g LÀ1 vs. 0.1–0.2 g LÀ1) [26], a much smaller requirements, optimum pH, and temperature. volume of the photobioreactor broth needs to Close systems allow for a better control of growth be processed to obtain a given quantity of biomass conditions, with respect to pH, salts, CO2 partial [9]. Once the biomass is harvested, processes to pressure, temperature, mixing, and light supply. disrupt the cells and to extract desired products 3. Photobioreactors allow for a shorter period are being developed, for example, the high yield until net productivity is reached, approximately and scalable recovery of high purity fatty acids 2–4 weeks over 6–8 weeks in open ponds [26]. [135].
  • 19. Algae, a New Biomass Resource 19 As a consequence of all aforementioned advantages On this basis, further improvement requires develop-of photobioreactors, productivity could be much ment of transformation protocols to introducehigher than that of open systems and could balance a foreign DNA into the cell and identification ofthe higher investment costs [9] (refer to “Present Algal markers for selection of transformants. Currently,Productivity in the Laboratory Versus Large Scale” about 25 species are accessible to genetic transforma-section for biomass productivity data). tion, mainly green and red algae [1]. However, many algal species, although of interest for commercial use, cannot be propagated in the laboratory and cannot beFuture Perspectives and Technological transformed. Overcoming this limitation will beDevelopments a major target of research in the future.Mutagenesis and genetic modification of algae for bio-fuel production has received so far little attention, Nuclear Genetic Transformationdespite molecular biology procedures appearing essen-tial in order to remove limitations in the economic Methods to introduce DNA into an algal cell exploitedsustainability of the biofuel production process. In so far include vortexing in the presence of micro- orparticular, a biotechnological approach is required to macroparticles such as glass beads, routinely used toobtain domestication of algal strains for cultivation in transform Chlamydomonas reinhardtii [136] and suc-mass culture in order to: cessful for other green algae such as Chlorella ellipsoidea [137]; microparticle bombardment, used to transform1. Improve light distribution into a photobioreactor species with a recalcitrant cell wall such as the diatoms by decreasing the optical density of the single cell Phaeodactylum tricornutum and Cyclotella criptica2. Increase the light intensity at which photosynthesis [138]; electroporation, effective in Chlamydomonas saturates, thus reducing light energy dissipation reinhardtii [139] and Dunaliella salina [124]. Cell and photoinhibition wall-less mutants or protoplasts after cell wall digestion3. Increase resistance to photooxidative stress are more easily amenable to transformation. The lim-4. Optimize light utilization and carbon assimilation, iting step is to allow survival of cells. Markers to select finally improving solar-to-biomass conversion the transformants are classified as dominant, for exam- efficiency ple, conferring resistance to paromomycin [140] or5. Channel the metabolism toward oil accumulation zeomycin [136], or recessive. The latter require auxo- for biodiesel production and to adjust the fatty acid trophic mutants and the corresponding endogenous composition to fit required fuel quality intact gene for complementation. This strategy requires6. Produce high value products and recombinant pro- no exogenous gene expression [1]. As for genome teins of interest for pharmaceutical and chemical sequence information, many genome sequencing companies projects, mainly for microalgae, are completed or in When considering the advancement that biotech- progress, including green algae Chlamydomonasnology can bring to algae cultivation, the algal system is reinhardtii, Chlorella vulgaris, Volvox carteri, Dunaliellaextremely attractive for commercial utilization if trans- salina, Ostreococcus tauri, and Ostreococcusformation and thus genetic improvement are possible. lucimarinus; red algae Cyanidioschyzon merolae,Nevertheless, the basic requirements for genetic Porphyra yezoensis, and Porphyra purpurea; and theimprovement are so far available for a low number of diatoms Thalassiosira pseudonana and Phaeodactylumspecies which are not necessarily suitable for the many tricornutum. Additionally, there has been an increase inpossible applications. The basis for domestication of expressed sequence tags (ESTs) databases.algal species is a good general knowledge of the biology Besides the methods for introduction of DNA intofor the target alga, including ecological, physiological, the cell, genetic transformation with a recombinantbiochemical, and genetic aspects. In addition, easiness cassette requires regulatory elements active in the targetof cultivation in the laboratory and availability of species to drive transgene expression and availability ofa protocol for breeding are of paramount importance. such regulatory elements is often limiting. Promoters
  • 20. 20 Algae, a New Biomass Resource can be heterologous, such as the cauliflower mosaic 5. Chlamydomonas reinhardtii has a single chloro- virus promoter CaMV35s that proved functional in plast, which contains approximately 80 copies of several algae [1]; however, heterologous sequences are its own genome. Obtaining homoplasmy, where all often not recognized by the host cell. Alternatively, copies of the plastid genome are transformed, is the endogenous promoters can be exploited. Endogenous main obstacle to the application of this technology promoters can be either constitutive, such as the pro- to many plant species. In contrast, this process is moter of the small chain of the ribulose bisphosphate easier in Chlamydomonas reinhardtii because of carboxylase, RBCS2 [136], the promoter of the PsaD its single chloroplast and is achieved by a sufficient subunit of the photosystem I core complex [141], or number of cells division under high selective pres- the promoter of the heat shock protein 70A [142] in sure, making Chlamydomonas reinhardtii chloro- Chlamydomonas reinhardtii, or inducible, such as plast a suitable factory for recombinant proteins. the promoter of the cytochrome C6 by copper depri- Nevertheless, the influence of nuclear factors vation in Chlamydomonas reinhardtii [132] or the controlling plastid gene expression at multiple levels arylsulfatase promoter inducible by sulfur deprivation may limit transgene expression level in the plastid in Volvox carteri [143]. Important regulatory elements [133, 146]. A better comprehension of these mecha- can be included within introns and their addition may nisms and of nucleus-plastid signaling is currently be useful for improving expression levels [144]. Once a major subject of research. the transgene is introduced in the host genome, trans- gene silencing often occurs due to positional effects and Mutagenesis of the Nuclear Genome action of epigenetic mechanisms [145], particularly active in the case of sequences located in the nucleus. Mutagenesis, besides its importance to investigate the Improvement of transgene expression is required for physiological role of genes by analyzing phenotypic genetic engineering and for exploiting algae as “cell effects of their inactivation, is as well a powerful tool factory.” Inducible expression systems can be useful to to generate strains with improved characteristics with partially overcome silencing of the recombinant gene. respect to the wild type. Mutagenesis of the nuclear genome is the only way to obtain mutants for the Chloroplast Transformation majority of genes. Early work was performed using chemical or UV mutagenesis, a method that is of An alternative to nuclear genome transformation is importance for species still not amenable to transfor- chloroplast transformation that has potentially many mation. Chemical mutagenesis has evolved into a novel advantages: strategy of Targeted Induced Local Lesions in Genomes 1. Transgene expression is more stable and uniform (TILLING) [147] where the chemical treatment is used between different lines, because the mechanism to generate allelic series of point mutations. The target of homologous recombination active in the chloro- gene is then amplified by PCR and analyzed to verify plast generates uniform clones, while epigenetic if mutations have occurred. TILLING is therefore control, positional effects, and gene silencing are a technique to isolate point mutations within a target not present. Thus, known variability of transgene gene that overcomes the “null phenotype” limitations of expression only derives from plastid differentiation insertional mutagenesis and generates stable mutants. stages. This technique has been so far applied to crop plants but 2. High level of expression is favored by high not yet to algae and could be used as an approach of polyploidy. reverse genetics, aimed to investigate the physiological 3. Multiple transgenes expression upon a single trans- role of a gene with a known sequence. In applied formation event is possible due to the organization research, TILLING could be pursued to obtain a stable of genes into operons. mutant for a target gene whose implications for appli- 4. The plastid genome shows uniparental inheritance cative purposes have been recognized. This approach is in many species, offering a degree of transgene of choice if transgenesis should be avoided. However, containment. limitations to this strategy are due to the high number of
  • 21. Algae, a New Biomass Resource 21induced mutations per cell in order to increase the mutation (see “Light Use Efficiency” section for furtherprobability of obtaining mutations in the target gene. informations about tla1 mutant) was identified viaSeveral backcross cycles are then needed to isolate the complementation of the mutant with a library of geno-desired mutation from all the others that decrease cell mic restriction fragments [60]. In addition, recentlyfitness. This lengthy procedure has so far discouraged developed high throughput sequencing technologiesapplication of the technique to algae and in fact makes it may prove to be effective in rapid re-sequencing ofimpossible in species where sexual reproduction is not a mutant genome and in localizing the insertion muta-available as a breeding procedure. tion. This strategy is likely to provide future potential When dealing with species in which transformation for high throughput identification of mutants andprocedures are available, insertional mutagenesis is gen- annotation of genomes in algae.erally preferred. Transforming the cell with an exoge-nous DNA vector helps to select the transformants, Concluding Remarksthanks to a selectable marker and to localize the muta-tion by identifying the genomic regions flanking the Algae currently used in industry are wild-type strains andknown insertion cassette. Integration of the exogenous mainly cultivated for human/animal food or to extractDNA in the nuclear genome of eukaryotic algae occurs high-value natural chemicals and pharmaceuticals. How-randomly, by non homologous recombination. Inser- ever, algae can be also exploited as biomass producers andtion of the cassette could take place within an encoding a source for renewable, ecologically sustainable biofuels.sequence, interrupting the gene and leading to In order to make algae suitable for cultivation in massa knockout, or it could as well perturb the promoter or culture conditions and economically competitive, domes-other regulative sequences. This strategy can thus be tication of natural strains is required. Selection of theused to generate random insertional libraries. The num- desired characteristics and introduction of new traitsber of insertions per cell can be controlled by adjusting through mutagenesis and genetic engineering can sub-the DNA/cell ratio. A haploid, mononucleated genome stantially improve algae cultivation in the future andis preferred because effects of recessive mutations on the make it practicable. Moreover, algae can serve as a “cellphenotype can be recognized without the need of homo- factory” for the production of recombinant proteins withzygosis. Multiple mutations eventually present in the pharmaceutical applications. Concerns from public opin-same cell can be sorted out if procedures for triggering ion and governments about biosafety can be satisfied andmating under laboratory conditions are available. counterbalanced by the potential advantages of transgenicBeside, breeding offers the valuable opportunity to com- algae, also considering that algae offer the advantage to bebine different mutations, contributing to the improve- grown in a confined environment. Therefore, the use ofment of the strain for industrial purposes. Since genetically modified algae for the production of renew-insertion mutagenesis is not targeted, mutants have to able and sustainable biofuels and pharmaceuticalsbe screened for the desired characteristics. While large- appears as a likely perspective. Genetic engineering isscale screening strategies have been developed, a limiting well useful to generate strains optimized for growth instep is the genetic characterization of insertion sites, that photobioreactors but uncompetitive in the wild ifis, the identification of the mutated gene responsible for released.the observed phenotype. Large genome deletions canoccur at the insertion site. The insertion cassette itself Bibliographycan be partially deleted or inserted as concatamer, so thatthe amplification of genomic regions flanking the inser- Primary Literaturetion through PCR techniques [148] can be difficult. 1. Hallmann A (2007) Algal transgenics and biotechnology.Protocol optimization and improved design of the Transgenic Plant J 1:81–98insertion cassette can decrease these limitations; how- 2. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M,ever, interesting mutants may well resist genetic char- Seibert M, Darzins A (2008) Microalgal triacylglycerols asacterization. Alternative strategies could be performed feedstocks for biofuel production: perspectives and advances.although time consuming, for instance the tla1 Plant J 54:621–639
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