ReviewMicroalgae: An alternative as sustainable source of biofuels?☆
Highlights
► Microalgal oil productivities are much higher than terrestrial plants. ► Metabolic engineering of microalgae permits sustained release of H2. ► Photosynthetic nature makes microalgae very economical alternative sources of biofuels. ► Technological bottlenecks on biocatalyst and bioreactor are reviewed.
Keywords
1. Introduction
Irreversible depletion of traditional sources of fossil fuels, coupled with accumulation of greenhouse gases derived from their combustion have been turning classical fuels for automotive engines into unsustainable pillars of energy supply. The 2009 Copenhagen Climate Conference has emphasized the urgent need for economic, CO2-neutral fuel systems, and anticipated that the global temperature rise must be limited to less than 2 °C to avoid dangerous climate changes worldwide. The Intergovernmental Panel for Climate Change has meanwhile calculated that reductions of 25–40% of CO2 emissions by 2020, and up to 80% by 2050 are required to stay within such a temperature range [1]. This calls not only for gradual replacement of carbon-based transportation fuels from fossil sources by such alternatives as biofuel and biohydrogen, but also for large-scale atmospheric CO2 sequestration [1].
Unlike other forms of renewable energy (e.g. wind, tidal and solar), biodiesel entails chemical energy that may be used in existing engines and transport infrastructures, either as such or after blending (to various degrees) with petrodiesel [2], [3]. Biodiesel and biohydrogen may be obtained from plant oils and electrolysis of water, respectively, but may as well be effectively produced via microalga-mediated routes.
Microalgae are microbial eukaryotes that populate virtually all ecosystems found on Earth. They are well-adapted to survive under a large spectrum of environmental stresses, including (but not limited to) heat, cold, drought, salinity, photo-oxidation, anaerobiosis, osmotic pressure and UV radiation [4]. They also possess a huge metabolic plasticity, which means that production of either form of biofuel can easily be triggered via medium engineering. Microalgae lay at the bottom of the aquatic food chain; as photosynthetic organisms, they take up H2O and CO2 and, with the aid of sunlight, convert them to complex organic compounds (e.g. triglycerides) or simple electron acceptors (e.g. molecular hydrogen) that are subsequently accumulated and/or secreted.
Microalgae combine indeed, in a balanced fashion, a few properties typical of higher plants, viz. efficient oxygenic photosynthesis and simple nutritional requirements, with biotechnological attributes proper of microorganisms, viz. fast growth rates and ability to accumulate or secrete metabolites. This useful combination provides the main rationale for microalgal biotechnology in the near future. Furthermore, the large number of existing species of microalgae (estimated to be above 100,000) constitutes a unique reservoir of biodiversity that supports potential commercial exploitation of many added-value products, e.g. vitamins, pigments and polyunsaturated fatty acids [5], [6], [7]. These compounds may favorably contribute to make biofuel manufacture from microalgae more competitive, based on a biorefinery approach.
A crucial factor for the eventual economic viability of microalgae is the possibility of operating large photobioreactors, able to handle biomass and metabolites to sufficiently high levels and extractable forms [8], [9]. Hence, R&D efforts focused on development of algal biofuels have grown significantly in recent years – and the Exxon-Mobil program to develop new photosynthetic algal biofuels, as a joint venture with the known biologist and entrepreneur Craig Venter, is but an example. Governments worldwide are also funding efforts towards this goal, as is the case of European Commission's call for proposals aimed at demonstrating the feasibility of microalga cultivation at industrial scale, and subsequent use for manufacture of biofuel. Furthermore, the US Department of Energy has released the National Algal Biofuels Technology Roadmap, which constitutes a participatory initiative to identify existing challenges in the production of economically viable and environmentally sound microalgal biofuels [10]. Complementarily, industrial stakholders have pursued pilot and demonstration-scale microalga cultivation, of which the European Algae Biomass Association is probably the best example - as it was created to promote the competitive development of technologies and industrial capacities encompassing microalgae. One key assumption is that current barriers to commercial implementation of microalgal-derived biofuels will likely be overcome via technological breakthroughs, thus eventually leading to biofuels from microalgae as a major energy source [11] despite the topical skepticism by some authors on their associated future commercial prospects [10].
The aforementioned trend has been consubstantiated in a number of recent reviews, either technically focused on biodiesel or biogas [11], [12], or broader in coverage but tackling only the former [13]. Hence, a more general overview that addresses synthesis of both oil and hydrogen by microalgae is in order, which is able to integrate biocatalyst and bioprocess engineering while covering the whole process, and entails discussion of both technical and economic issues. This review will accordingly attempt to add to current knowledge by briefly discussing how microalgae can help solve the present shortage of supply and environmental problems arising from extensive utilization of fossil fuels, via biodiesel and biohydrogen production thereby, and including such key issues as choice of species and processing conditions.
2. Biodiesel
Biodiesel is typically a mixture of fatty acid alkyl esters, obtained by transesterification (or ester exchange) of oils or fats. When from plant or animal origin, it is composed of 90–98% triglycerides, and much smaller amounts of mono- and diglycerides and free fatty acids, besides residual amounts of phospholipids, phosphatides, carotenes, tocopherols, sulphur compounds and water [15]. At present, the most widely available form of biodiesel is from oil crops, e.g. palm, oilseed rape and soybean. However, several concerns have been raised on the sustainability of this mode of production that can be illustrated as follows: to obtain ca. 25 billion liters of biodiesel (i.e. the current demand of petrodiesel in the whole UK) from oilseed rape, 17.5 Mha would be required for plantation, i.e. more than half the land area of UK itself [3]. A second generation of biofuels is thus in order, departing from non-food feedstocks, where microalgae probably offer the greatest opportunities on the long run.
A diagram of the microalgal biodiesel value chain is available as Fig. 1; it starts with selection of the most appropriate species, depending on local environmental conditions, and on the configuration intended for cultivation; then it goes through harvesting of biomass and extraction of oil therefrom; and ends with the biodiesel production unit. Meanwhile, byproducts generated may be recycled or used for energy generation related with each of these steps – as presented and discussed in the following subsections.
Fig. 1. Schematic representation of the various stages of manufacture of microalgal biodiesel.
2.1. Selection of microalgae
Microalgae hold clear advantages over higher plants in terms of oil productivity. Besides synthesizing storage lipids in the form of triacylglycerols, they can be induced to accumulate substantial amounts thereof via several forms of stress, e.g. N-limitation, up to yields of 60% of their dry biomass, as fully discussed elsewhere [3], [16]. Despite being grown in aqueous media, microalgae require lower rates of water renewal than terrestrial crops need as irrigation water [3], they can be cultivated in brackish water (so do not necessarily demand freshwater), they do not need application of pesticides [17], [18], and they do not require arable land as field crops do. Finally, their low environmental impacts are emphasized by the possibilities of waste upgrade, since that often contaminate effluents from agrifood processing are actual nutrients for microalgae [2], [3], [19].
Of particular interest are species able to effect wastewater tertiary treatment, coupled with biofuel production and atmospheric CO2 mitigation. This is the case of Botryococcus braunii strain LEM 14, which exhibited high rates of nitrogen and phosphorus removal (80% and 100%, respectively) and lipid accumulation (36%) when grown on domestic wastewater, both of which are almost independent of the N and P contents of that medium [20]. Furthermore, it can extensively uptake CO2 (145 mgCO2 g−1biomass day−1), in addition to its ability to accumulate lipids without need for strict control of nitrogen levels [20]. It would thus be expected to attain 3300 kglipids ha−1 year−1 if it were cultivated in 20 cm-deep lagoons of (treated) wastewater; note that this is 5-fold the productivity of soybean under identical conditions [20].
On the other hand, the biomass left after oil extraction can be fermented into ethanol or methane (both also biofuels), or else incorporated in livestock feed or simply used as organic fertilizer owing to its high N:P ratio. Finally, the spent biomass can be burned for energy cogeneration, and even electricity production [2], [19] (although this may be somehow hampered relative to other types of biomass because of its intrinsically high moisture content that would hardly match self-consumption of energy throughout processing). Furthermore, a wide range of fine chemicals for functional food formulation, e.g. polyunsaturated fatty acids, natural dyes and antioxidants, may be previously extracted from said biomass, depending on the species at stake [21]. Even though these are not bulk products as biofuels are, they hold a high added-value that contributes to the overall economic feasibility of the biofuel manufacture process. Finally, note that 1 kg of actual dry algal biomass has previously uptaken ca. 1.83 kg of CO2, which is particularly relevant if obtained from industrial flue gases via bio-fixation [3].
Many microalga species can be induced to accumulate lipids up to final contents ranging between 1 and 75% [14]. The lipid contents of selected marine and freshwater microalgae are depicted in Fig. 2; significant differences among the various species and within the same genus are apparent, as well as the greater lipid productivities of marine microalgae. The underlying high salinity also prevents extensive contamination of culture media, while allowing seawater to be directly used instead of depleting freshwater resources.
Fig. 2. Lipid content of various marine and freshwater microalga species.
A multicriterion-based strategy is, however, to be considered toward successful selection of a specific wild microalga strain: (i) growth rate; (ii) lipid quantity and quality, especially the fatty acid residue profile of acylglycerols; (iii) response to such processing conditions as temperature, nutrient input and light, and competition with other microalga and/or bacterial species; (iv) nutrient requirements and rate of uptake thereof, in particular CO2, and nitrogen and phosphorus to a lesser extent (which is especially relevant when carbon sequestration and upgrade of brackish waters and agricultural effluents are sought); (v) ease of biomass harvesting, oil extraction and further processing; and (vi) possibility of obtaining high added-value chemicals in parallel, which will call for a GRAS (Generally Recognized As Safe) status prior to general use in foods, cosmetics or pharmaceuticals [3], [19]. Said strain choice should of course be carried out interactively with medium and reactor design.
2.2. Cultivation of microalgae
Microalgae depend critically on a sufficient supply of carbon and light to carry out photosynthesis [3]. However, they entertain more than one type of metabolism, i.e. heterotrophic, mixotrophic and photoheterotrophic besides photoautothrophic, and they undergo metabolic shifts in response to changes in environmental conditions [3]. Typical examples are Chlorella vulgaris, Haematococcus pluvialis and Arthrospira (Spirulina) platensis, all of which can grow under photoautotrophic, heterotrophic and mixotrophic conditions [19]; or Selenastrum capricornutum and Scenedesmus acutus, which operate photoautotrophically, heterotrophically or photoheterotrophically [22]. Under phototrophic cultivation, there is a large variation in lipid content that ranges from 5 to 68% depending on the microalga species, with the highest lipid productivity reported to be ca. 179 mg L−1 d−1 for Chlorella spp. [3].
Phototrophic growth of microalgae can be carried out in either open ponds or enclosed photobioreactors. The latter are suitable for those cultures that are easily contaminated, whereas open systems are preferable for microalgae able to survive in extreme environments, such as high pH (e.g. Spirulina) or salinity (e.g. Dunaliella spp.), or which grow very rapidly (e.g. Chlorella spp.) [23].
Conversely, heterotrophic cultivation offers such advantages as no need for light, good control of cultivation, and low-cost harvesting owing to the associated higher cell densities [24]. In heterotrophic culture, both cell growth and metabolite biosynthesis are significantly influenced by medium nutrients and environmental factors. Microalgae can assimilate a variety of organic carbon sources during growth, e.g. glucose, acetate, glycerol, fructose, sucrose, lactose, galactose and mannose, and even corn powder hydrolysate instead of sugars, with resulting biomass productivities of up to 2 g L−1 d−1 and lipid contents of up to 932 mg L−1 d−1. However, the highest lipid productivity (3700 mg L−1 d−1) was obtained following a fed-batch culture strategy: a 20-fold better performance was indeed achieved under phototrophic cultivation [3]. The carbon source(s) is the most important requirement towards efficient production of lipids: for instance, Chlorella protothecoides can grow photoautotrophically or heterotrophically, but the latter leads to much higher biomass yields and lipid contents when using acetate or glucose as carbon source [25]. In order to decrease the production cost of microalgal oils, less expensive carbon sources should be considered (e.g. ethanol, glycerol or fructose).
Enclosed photobioreactors have the ability to scrub power plant flue gases and/or remove nutrients from wastewater, but require operation under sterile conditions, thus calling for stricter hygiene measures that add to the final cost of biodiesel [26]. On the other hand, they offer the opportunity to optimize the light path, so distinct configurations were proposed and built to improve light supply and biomass productivity: vertical reactors, flat-plate reactors, annular reactors, arrangements of plastic bags, and various forms of tubular reactors, all of them stirred mechanically or by air-lifting [27].
Because of the high cost in terms of operation and capital investment, coupled with an intrinsically smaller scale, it will hardly be feasible from an economic point of view to produce biodiesel on industrial levels using enclosed photobioreactors. Open pond systems are indeed relatively nonexpensive, and basic requirements for microalgal phototrophic growth therein reduce to atmospheric CO2 and only a few readily available micronutrients (besides sunlight). Since the oil yields relate directly to the CO2 level used as feedstock, integration with power plants is an option provided they release large amounts of waste gases rich in CO2, as happens in coal-fired power plants that may yield up to 13%(v/v) CO2 [23], [28].
In addition to promoting cellular uptake, high CO2 concentrations enhance gas transfer in open ponds. On the other hand, wastewater may contain abundant nutrients (e.g. inorganic iron, or phosphate and nitrate arising from extensive agrochemical application) that are critical for microalgal growth. One good example is Chlorella vulgaris grown on the residues from a steelmaking plant, which was able to successfully remove ammonia (0.92 g m−3 h−1) from wastewater, as well as CO2 (26.0 g m−3 h−1) from its flue gas [29].
2.3. Harvesting of microalgae
Harvesting consists of biomass recovery from the culture medium, and it may account for up to 20–30% of the total production cost [19]. This process involves large biomass volumes, so suitable harvesting usually encompasses more than one step of a physical, chemical or biological nature. Unfortunately, a universal harvesting method does not exist, so this has prompted great opportunities for research.
The classical methods of harvesting include sedimentation, centrifugation, filtration, ultra-filtration, flocculation and flotation. Flocculation has proven particularly useful in aggregating microalgal cells so as to increase their effective particle size, thus leading to faster sedimentation, centrifugal recovery or filtration [30].
The basic criterion to select harvesting steps is to apply first the processes leading to larger volume reductions, followed by those that are more selective (and also more expensive). Hence, intermediate-moisture biomass feedstocks are usually obtained, which are to be made compatible with the next processing step.
2.4. Processing of microalgal biomass
Biodiesel production requires release of lipids from their intracellular location, which should be done in the most energy-efficient and economical way possible to avoid using large amounts of organic solvents. This should maximize the pool of liquid biofuel without significant recovery of other byproducts, e.g. DNA and chlorophyll [26].
In view of the above, cell disruption should first be applied; this step is particularly important because most microalgae possess a strong cell-wall, and because the overall extraction yield depends heavily on the extent and quality of said disruption. Several methods can be followed and one's choice depends chiefly on the microalga wall and the target metabolite(s). They are based on mechanical action (e.g. cell homogenizers, bead mills, ultrasound, autoclaving and spray drying) or non-mechanical action (e.g. freezing, organic solvent extraction, osmotic shock, and acid/base- or enzyme-mediated reactions) [19].
After cell disruption, lipids are to be extracted from cell debris. This process should be lipid-specific in order to minimize co-extraction of non-lipid materials, as well as selective in order to maximize recovery of neutral lipids containing mono-, di- and triacylglycerol moieties [3]. A typical solid/liquid extraction using organic solvents is normally done directly on the biomass, and is fast and efficient enough to preclude significant degradation. Several solvents can be used, e.g. hexane, ethanol (96% v/v in water) or a mixture thereof [31]. Meanwhile, a number of alternative methods have gained their place, such as ultrasound and microwave-assisted ones, as well as supercritical carbon dioxide extraction [3].
2.5. Production of biodiesel
Triacylglycerols are typically non-volatile, so transesterification with short alkyl moieties, e.g. methyl or ethyl residues, is required to manufacture biodiesel. This is a multiple step chemical reaction that includes reversible hydrolysis, where triglycerides are converted to diglycerides, diglycerides to monoglycerides, and monoglycerides to free fatty acids and glycerol (as byproduct); followed by re-esterification with a short chain alcohol (methanol or ethanol), in the presence of a catalyst. If a lipase is used, hydrolysis and esterification may take place simultaneously, but the thermal lability of that type of enzyme makes this possibility of a lesser interest for industrial scale.
A promising alternative to the aforementioned conventional process that may reduce processing costs is in situ transesterification. This process facilitates conversion of fatty acids to their alkyl esters right inside the biomass, thereby eliminating the solvent extraction step and alleviating the need for biomass drying in harvesting. Such a form of integrated alcoholysis leads to higher biodiesel yields, up to 20% better than the conventional process; and wastes are reduced as well [32].
3. Biohydrogen
Molecular hydrogen is one of the most promising biofuels for the future; advances in hydrogen fuel cell technology, coupled with realization that combustion of H2 releases plain water, make that feedstock particularly attractive. However, its technological viability is strongly dependent on the development of cost-effective, sustainable H2 production systems at large scale that are able to replace the classical processes of steam reforming of natural gas, petroleum refining and coal gasification [33]. Furthermore, material engineering will be called upon to provide efficient containers and absorbers/adsorbers for liquid hydrogen, aimed at minimizing leakage and risk of explosion.
Photosynthetic production of H2 from water is possible via a biological process that accordingly converts sunlight into useful chemical energy, as represented in Fig. 3. The underlying phenomenon was discovered long ago [34], but little progress occurred ever since pertaining to the biotechnology thereof. Hydrogen release is indeed a feature of many phototrophic organisms [35], including several hundred species from different groups of microalgae, cyanobacteria and anaerobic photosynthetic bacteria [36]. At present, Chlamydomonas reinhardtii remains the best photosynthetic eukaryotic hydrogen producer, with Nostoc and Synechocystis cyanobacteria also holding a promising status as candidates for H2 production [1].
Fig. 3. Schematic representation of the various steps of manufacture of microalgal biohydrogen.
3.1. Production of hydrogen
Photoproduction of H2 in microalgae follows two processes, direct and indirect; both resort to reduced ferredoxin (Fd) as electron donor, coupled to (and required by) the action of hydrogenases [37]. In the direct pathway, photo-oxidation of water occurs, and both Photosystem I (PSI) and Photosystem II (PSII) play a role in supplying reductants (or electrons) to Fd via the photosynthetic electron transfer chain. The indirect pathway involves oxidative carbon metabolism (e.g. starch degradation) instead, but NADP-plastoquinone oxidoreductase and PSI activities are required to supply the reductants. Either electron source (i.e. water or starch) can be used, but the contributions of each one depend on the type of strain, culture conditions, extent of damage of PSII and specific metabolic constraints [38].
Originally, hydrogen release by microalgae was induced after anaerobic incubation in the dark; a hydrogenase (containing Fe as prosthetic group) is expressed during such an incubation, and catalyzes light-mediated production of H2 with a high specific activity [39]. This enzyme is encoded in the nucleus, but the mature protein is localized and functions in the chloroplast stroma [40]. Light absorption by the photosynthetic apparatus is essential for generation of hydrogen because it brings about oxidation of water that releases electrons and protons, and facilitates endergonic transport of said electrons to Fd. This ferredoxin thus serves as physiological electron donor to the Fe-hydrogenase, so it links that enzyme to the electron transport chain in the chloroplasts of microalgae [41]. However, the activity of hydrogenase is only transient under these conditions: it lasts from a mere several seconds to a few minutes, as a consequence of the fact that the light-dependent oxidation of water also entails release of molecular O2 that is a powerful inhibitor of Fe-hydrogenase.
Besides the aforementioned role of PSII-dependent H2 photo-evolution (which involves water as source of electrons and produces 2:1 stoichiometric amounts of H2 and O2, respectively), an alternative mechanism has been described [42]: upon dark anaerobic incubation and consequent induction of hydrogenase, electrons for the photosynthetic apparatus are derived from catabolism of the endogenous substrate and corresponding oxidative carbon metabolism. Said electrons are fed to the photosynthetic electron transport chain between PSI and PSII, and probably at the level of plastoquinone. Light absorption by PSI, and the ensuing electron transport raises the redox potential of such electrons to the redox equivalent of Fd and hydrogenase, thus permitting generation of molecular H2 [43] (Fig. 4).
Fig. 4. Schematic representation of the anaerobic phases of hydrogen production in the chloroplast of microalgae. Cytb6f = cytochrome b6f; Fd = ferredoxin; H2ase = Hydrogenase A; PC = plastocyanine; PSI = Photosystem I; PSII = Photosystem II; PQ = plastoquinone.
Since an aerobic environment is detrimental for hydrogen production, chemical and mechanical methods have been developed to remove the O2 produced by the photosynthetic activity of microalgae, which encompass addition of O2 scavengers or reductants and purging with inert gas [44]. Unfortunately, all these methods are expensive, and will realistically not withstand scale up [45].
Meanwhile, it was observed that, in the absence of sulfur but in the presence of light, C. reinhardtii decreases its PSII activity to the rate of O2 uptake by respiration. This implies that the microalga cells will consume internally all remaining O2, and sufficiently fast to generate their own anaerobic microenvironment. Therefore, cells will induce (reversible) hydrogenase and produce H2, which has been recorded for up to 4 days [39], [44]. If sulfate is subsequently re-added to the spent cultures at high concentration, further cycles of cell growth and H2 production will be observed [46]. Likewise, in the presence of the PSII inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), such a process generates 2:1 stoichiometric amounts of H2 and CO2, respectively. Hence, following sufficiently long dark anaerobic incubation, high rates of H2 production will occur upon illumination of the microalgae in the presence of DCMU [41], [47].
3.2. Design of reactors
The performance of enclosed photobioreactors, i.e. the only configuration suitable for hydrogen production, is constrained by physical parameters associated with the reactor (e.g. light penetration, area to volume ratio, temperature, transparency and durability of construction material, rate of gas exchange, and level of stirring). It is also limited by physicochemical parameters influencing the biochemical pathways involved in hydrogen production (e.g. pH, temperature, light intensity, dissolved oxygen and CO2, shear due to agitation, and nature and relative ratio of carbon and nitrogen sources.
Based on the mode of operation, enclosed photobioreactors tested for hydrogen production can be broadly classified as batch, continuous and fed-batch [36]. Continuous operation will eventually become the preferred technology, both for suspended or immobilized systems. One advantage worth mentioning in that sulphur depletion and repletion can be rapidly achieved, as reported by Laurinavichene et al. [48] for immobilized C. reinhardtii, who attained hydrogen productivities of 5.5 mL L−1 day−1. One of the most cost-effective closed bioreactors is the Biocoil system, which consists of flexible tubing wound around a cylindrical support structure, through which the microalgal culture is pumped [33], [49]; it is relatively nonexpensive, and has a modular design that permits combinations. Engineers are also developing high efficiency low-cost photobioreactors that require low energy input, while being able to produce biomass under aerobic conditions and hydrogen under anaerobic conditions [50], [51].
The highest H2 production rates reported to date have been attained under mixotrophic conditions, encompassing addition of acetate as carbon source to the growth medium. Acetate is indeed rapidly utilized as the culture switches from the aerobic into the anaerobic state, rather than acting as substrate source for the hydrogenase [1].
In terms of light capture, Fresnel lenses were used to focus light onto the bioreactor [52], and optical fibers to conduct light into the bioreactor [53], [54]. Optimization of culture mixing and cell immobilization were under scrutiny elsewhere [55], [56], as well as development of compact bioreactor designs. Optimal light capture may indeed be facilitated via light dilution, with strong incident solar radiation being spread over an increased surface area [57].
In an ideal scenario, microalgal hydrogen production would be conducted in regions characterized by a high average light intensity, so as to maximize the incident energy density, and thus minimize the area required for capture. However, microalgal photosynthesis typically saturates at light levels of ca. 100 μE, knowing that solar radiation levels of 1000–1500 μE can be obtained in subtropical regions [59]. Under such operating conditions, up to 80–90% of the incident radiation will actually be dissipated through non-photochemical quenching [59], which is a photo-protective mechanism.
3.3. Genetic engineering
Genetic transformation appears to hold a major potential in attempts to improve H2 production, especially in the case of C. reinhardtii since its genome has been fully elucidated (besides being one of the best H2 producers known to date). Several mutants have been obtained, and the mutations were performed at various levels, e.g. hydrogenase, sulfate permease and ribulose-1,5-bisphosphate carboxylase (RuBisCO) enzyme, as well as at the PSI and PSII photosystems [59].
The waste of light energy described previously can be minimized by reducing the cross-sectional area of the light harvesting antenna of Photosystem II (PSII) via genetic engineering [60], [61], [62], which prevents oversaturation of the photochemically active reaction centers. This strategy has the further advantage of reducing photodamage [60], while allowing light, which would otherwise be dissipated, to penetrate deeper into the culture thereby increasing the overall H2 yield. In vitro testing has been shown to double the biomass production efficiency [60], [61], and outdoor trials are currently underway to assess the efficiency of such strains under real-world conditions.
Technical and physiological parameters of microalga cultivation have also been optimized to increase hydrogen production efficiency: for instance, Scoma and Torzillo [62] reported on the interplay between light intensity, chlorophyll concentration and culture mixing upon H2 production in C. reinhardtii, with the best rates being achieved at 140 μE using a concentration of 24 mgChl a L−1 (ca. 2.4 × 108 cells L−1).
One illustrative example is C. reinhardtii strain Stm6, which is able to produce 5-fold more H2 than its wild type [58]: the associated strains Stm6Glc4 and Stm6Glc4T7 represent a further advance over Stm6, in terms of yield of biomass and hydrogen. A hexose symporter system from Chlorella kessleri was inserted specifically into strain Stm6glc4, thus enabling it to efficiently produce hydrogen simultaneously via water photolysis and from external sugars, with feeding of the associated H+ ions and electrons through the plastoquinone pool [63]. Stm6Glc4 was further engineered to reduce its antenna size, thus yielding Stm6Glc4T7 characterized by light capture efficiencies enhanced by 20–30%, higher light saturation threshold (800 μE) and ability to release up to 50% more H2. The overall average production rates by these strains vary between 400 and 600 mL H2 L−1 within 3–4 days, which corresponds to 8 mL H2 L−1 h−1.
4. Economic feasibility
To be a viable substitute of classical fossil fuels, any alternative fuel should entertain a lower environmental footprint, be economically competitive, and be available in sufficient amounts so as to permit a meaningful impact upon energy supply. Furthermore, it should exhibit a net energy gain over the energy consumed for its manufacture.
Production of biofuels from microalgae has proven technologically feasible, and use of microalga biomass rich in lipids may significantly reduce the use of arable land when compared to crops – although several issues relating to the quality and quantity of such land have to be addressed on a case-by-case basis. Unfortunately, microalgal biodiesel has not yet reached a clear-cut economic feasibility; the biggest challenge is the relatively high costs of production of microalgal biomass and extraction/separation of lipids for biodiesel. The cost of production of microalgal biomass with an oil content of 30% is 1.40 $/kg using an open pond, and 1.80 $/kg using a photobioreactor and assuming that CO2 is available for free; oil extraction, to an approximate yield of 1.14 L/kg, costs more than 3-fold. This compares unfavorably with crude palm oil that costs a mere 0.52 $/L, whereas petrodiesel sells at retail for 0.66–0.97 $/L [64].
An overall economic analysis indicates that microalgal biofuel feasibility hinges at present on the possibility of obtaining coproducts with a high market value, a concept known as biorefinery [65], [66] and sketched in Fig. 5. Byproducts of current interest include bulk sugars for production of bioethanol and biomethane via fermentation; intermediate-value products, e.g. proteins for animal feed; and high-value products, such as active principles exhibiting antimicrobial, antioxidant, antitumoral and anti-inflammatory features for pharmaceutical formulation. After extraction of such product(s), biomass may be pyrolyzed to biochar that holds a value as soil enhancer [1].
Fig. 5. Schematic representation of a biorefinery system for manufacture of microalgal biofuels, coupled with generation of bioenergy and added-value bioproducts.
This upgrade is particularly valid in the case of H2 because its volatile nature leaves the biomass essentially intact. Specifically, large-scale production of biogas via fermentation of microalgal biomass offers the possibility to recycle a large proportion of the original nutrients. Although not economically feasible at low production levels, it will become increasingly important as medium costs become a greater fraction of the final cost, coupled with such issues as overall phosphorous limitation [67].
Stand-alone microalgal systems in a mature biofuel market are not expected until solar energy to fuel conversion efficiencies increase substantially: the oil content should increase 2.5-fold, from the current productivity of 20 g m−2 d−1 with an oil content of 25% dry weight, to 50 g m−2 d−1 and 50%, respectively [66], [67]. Calorific values up to 29 MJ kg−1 should thus be attained, and the increase in oil yield should be accompanied by an increase in energy conversion. Doubling the oil content of microalgal biomass, from 25% w/w (23 MJ kg−1) to 50% w/w (28 MJ kg−1), would require a 20% increase in photosynthetic energy conversion over the current 2.1% level [66], [67]. Recall that the theoretical maximum photosynthetic energy conversion efficiency is 4.6% for C3 and 6.0% for C4 plants, using the whole spectrum of solar radiation; or 9.4% and 12.3%, respectively, if only photosynthetically active radiation (i.e. 400–700 nm) is used as a basis [68]. Hence, there is still room for improvement of microalga metabolism until such theoretical maxima are attained. Finally, the expensive steps of harvesting and extraction may be overridden by resorting to secretion of oil by microalgae or ‘milking’ of oil [69], [70] that simplifies oil collection via solvent interfaces.
5. Final considerations
Responses to environmental issues (including global warming) cannot be delayed much longer, otherwise Mankind may be at risk [71]. Biofuels produced from microalgae are one piece of the puzzle in what concerns energy security, since they may eventually replace traditional fossil fuels. Combined with measures on energy efficiency and savings, and with educated changes in consumer behavior, they could be helpful to meet the energy demands of the near future in a sustainable manner.
Microalgae offer in fact novel bioenergy systems, characterized by much higher oil yields and much lower water demand than terrestrial biomass, as well as much lower costs than electrolysis of water. It is estimated that the cost of hydrogen production via water electrolysis lies in the range 3-11.8 $/kg (depending on the original price of electricity), whereas hydrogen production by microalgae is estimated to be ca. 4.8 $/kg [72]. In what pertains to costs of production of microalgal biodiesel, it is estimated to range between $2.95 and $3.8/L of oil, for raceway ponds and photobioreactors, respectively [16], assuming that biomass contains 30%(w/w) oil and CO2 is available for free (e.g. from flue gas). Compared with second generation biofuels, microalgal fuels have a much higher yield: 30–100 times more energy per hectare can be produced by microalgae if compared with terrestrial oil crops [71].
However, the processes currently available are marginal in terms of net energy balance and global warming mitigation, so there is still a long way before microalgae will entail a profitable contribution as source of biofuels. Research and development efforts aimed at improving reactor designs and integrating processes are required, which address both reaction engineering and product separation schemes.
Major breakthroughs are indeed necessary towards design and development of technologies able to reduce processing costs, while increasing product yields. Integrated studies in the form of well-funded R&D programs could eventually aid in selecting microalga strains specifically adapted to regional conditions; genetic improvement and process optimization will help fulfill this endeavor as well. In particular, the biorefinery issue will be central, as it allows upgrade of spent biomass via production of alternative bulk or fine chemicals – which will contribute positively to the overall economic feasibility of microalgal biotechnology.
Careful life cycle assessments are also in order, since some biofuels may fail to provide a favorable contribution from the point of view of global sustainability. Critical analysis is thus seminal to provide an impartial picture of the scenarios available, and to eventually backup correct choices.
Acknowledgments
A PhD fellowship (ref. SFRH/BD/62121/2009, supervised by author F.X.M.) was granted to author Helena M. Amaro, under the auspices of ESF (III Quadro Comunitário de Apoio) and the Portuguese State. This work received financial support from project MICROPHYTE (PTDC/EBB-EBI/102728/2008), coordinated also by author F.X.M. and under the auspices of ESF and the Portuguese State.
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This manuscript contains material from the invited oral presentation delivered at the 6th Dubrovnik Conference on Sustainable Development of Energy, Water and Environment Systems (Croatia).