MICROALGAE AS BIOFUELS FEEDSTOCKS: AN ASSESSMENT OF THE YIELDS AND FUEL QUALITY

Transcription

1 MICROALGAE AS BIOFUELS FEEDSTOCKS: AN ASSESSMENT OF THE YIELDS AND FUEL QUALITY Lieve M.L. Laurens National Renewable Energy Laboratory National Bioenergy Center Denver West Parkway Golden, CO Robert McCormick National Renewable Energy Laboratory Center for Transportation Technologies and Systems Denver West Parkway Golden, CO Philip T. Pienkos National Renewable Energy Laboratory National Bioenergy Center Denver West Parkway Golden, CO ABSTRACT We need to move away from our dependence on fossil fuels as an energy source but there is a general reluctance to abandon liquid hydrocarbons as transportation fuels. This opens up a large market that can be addressed by renewable and sustainably produced biofuels. Algae are considered important contributors to feedstocks for future biofuels production. The high biomass productivity and associated high lipid yields make algae an attractive option. With the high oil yields, algae address the energydense fuels market, which is a niche market segment not fully addressed by other biofuels without encroaching on valuable crop land and affecting food commodity prices. We illustrate the theoretical maximum conversion efficiency of solar energy into organic products and the pathways of converting these products into fuel precursors. One of the main questions we aim to answer is how algal fuels compare to traditional biodiesel fuels, with regards to fuel quality and processing parameters. We present data on the oil yields and lipid profile of algae and extrapolate to the fuel quality of biofuels. We are also using fermentable carbohydrates present in the non-lipid portion of algae to address additional biofuels markets such as ethanol or other advanced biofuels. Our data indicate that algal fuels compare favorably to traditional biofuels, and we illustrate that algae have great potential as biofuels feedstocks, but there are a number of challenges associated with reducing the uncertainties around the overall production process. 1. BIOFUELS VERSUS FOSSIL FUELS There are two general drivers behind the development on biofuels to replace considerable quantities of liquid transportation fuels; i) reducing our dependence on fossil fuels and ii) reducing emissions of greenhouse gases. To make a substantial impact in the displacement of current fossil fuel use, production of biofuels should not interfere with valuable cropland, because of the potential for effects on the current food supply. Algae are ideal candidates to add to the future biofuels pool because algae can accumulate a lot of oil and their cultivation is not reliant on the above restrictions. The question remains about which algae feedstocks can provide the volume and quality of biofuel needed to respond to the global demand for fuels. Liquid transportation fuels are used primarily in gasoline, jet, and diesel engine applications. Each of these applications has different performance and quality requirements that are typically outlined in ASTM standard specifications in the United States or CEN standard specifications in the European Union. These standards were developed for petroleum-derived hydrocarbons, and are believed to also be adequate for bio-derived hydrocarbons. For bio-derived oxygenates such as ethanol, butanol, or biodiesel additional requirements may have to be met. For example, both ethanol and biodiesel have ASTM standard specifications for the neat blendstock. Today in the U.S. up to 15% ethanol can be blended into 1

2 conventional gasoline while still meeting the gasoline standard (D4814); and up to 5% biodiesel can be blended into conventional diesel fuel while still meeting the diesel fuel standard (D975). Up to 50% renewable hydrocarbon produced by hydrogenation of esters and fatty acids can be blended into jet fuel. Lipid feedstocks, including algal lipids, would most commonly be converted to biodiesel by transesterification, or to hydrocarbon renewable diesel or jet by hydrogenation/decarboxylation/isomerization Biofuels Quality Parameters Biodiesel is defined as the mono-alkyl esters of long-chain fatty acids and is commercially produced from triacyl glycerides (TAG) by base catalyzed transesterification or from free fatty acids (FFA) by acid catalyst esterification. The alcohol used is most commonly methanol such that the product consists predominantly of fatty acid methyl esters (FAMEs), though a number of impurities are typically present. The structure of the fatty acid chains present in the feedstock has a determining effect on many of the critical quality parameters for biodiesel. The two most important properties for biodiesel quality are i) the degree of fatty acid unsaturation, followed by ii) the chain length. The degree of unsaturation can be quantified as the iodine value (IV), which is the moles of double bonds per mass of sample (measured by, for example, ASTM method D1510). Typical terrestrial crop oils and animal fats consist almost exclusively of C16 and C18 fatty acid chains. For this narrow range of materials IV can be correlated with many important properties such as cetane number, viscosity, density, and molar H/C ratio (2, 3). A critical quality parameter for diesel fuel is cetane number, which is a measure of the ignitability of the fuel in a diesel engine. Minimum cetane number of 40 and 50 are required in the U.S. and E.U., respectively. Fully saturated FAME have high cetane number, and all saturated FAME with 10 or more carbons in the fatty acid chain will easily exceed the minimum U.S. value of 40 (1). Fully saturated FAME is also resistant to thermal and oxidative degradation. However, these saturated materials have high melting points and lower solubility in a biodiesel or hydrocarbon diesel fuel matrix at low temperatures. Thus, a biodiesel too high in saturated FAME content will not be useful, even as a blend with petroleum diesel, in cold winter climates. A blend with more highly unsaturated FAME may improve the cold-flow properties however the fuel oxidative stability may decrease (23). Therefore, a significant fraction of mono and polyunsaturated FAME (PUFA) are desirable in biodiesel. The PUFA has much lower cetane number but also much lower melting point (and much greater solubility at cold temperatures). There have been concerns that polyunsaturated FAME are not adequately stable to oxidation, however this problem can be mitigated by the use of antioxidant additives (4). As noted above, the effect of FAME makeup on biodiesel properties and performance is well understood. A much more challenging area is the impact of impurities. For biodiesel made from conventional terrestrial crop oils and animal fats these impurities are mono and diglycerides, plant sterols and steryl glucosides, free fatty acids, and residual metals. Monoglycerides and other impurities are known to have a dramatic effect on cold temperature operability which has led to the introduction of a cold soak filterability test (5) and a limit on total monoglycerides (6, 7). Additionally, there is concern that residual metals such as Na or K from transesterification catalysts, Mg from adsorbents, or Ca from hard water could poison emission control catalysts and filters. Research to date has shown some potential for problems, but much additional study is required (8). Hydrocarbon renewable diesel or green diesel (or jet) can be produced from TAG by processes involving hydrogenation, decarboxylation, and isomerization over heterogeneous catalysts. Hydrogenation is used to saturate the double bonds, and in some processes to remove the oxygen. Because of the relatively high cost of hydrogen, other process configurations remove a large fraction of the oxygen as CO 2 by decarboxylation. Using a C16/C18 feedstock, the products from these reactions are C15 to C18 normal alkanes, which are likely to be solid at room temperature. Therefore an isomerization catalyst is also needed to introduce branching, which can produce a dramatic lowering of cloud point and a moderate reduction in cetane number (9). These materials typically consist of more than 80% isoalkanes and fuels with a cloud point of C will have cetane number above 70 (10). These fuels have the advantage of being very similar to petroleum derived fuels and containing few impurities that might cause operational issues. The downside is the significantly higher capital, operating, and energy cost (11) Conversion of sunlight to algal biomass Algae are photosynthetic microorganisms that have the potential to accumulate large quantities of oils resulting from reduction of carbon dioxide to organic cell components (Figure 1). Single-celled microalgae have been the subject of many recent literature reviews in the context of biofuels, because of the high reported oil contents and the high growth rates (12, 13, 14). The cells accumulate the large quantities of oil in designated droplets in the cells, as shown in Figure 1.B and 1.D. 2

3 A" B" C" D" Oil"droplet" Oil"droplet" Fig. 1: Microscopic images of Scenedesmus sp. grown under nutrient replete (A) and deplete conditions (B), and similarly, Chlorella sp. replete (C) and deplete (D). The deplete culture condition refers to nutrient limitation, which induces the accumulation of large oil droplets in the cells (as indicated in (B) and (D)) Algae have a distinct advantage over 'traditional' biofuels such as those derived from soy and canola oils, in that certain strains can be grown in brackish and salt water and do not need to encroach on cropland. Traditional biofuels, e.g. soy and canola oil have a reported high yield and good quality oil, however, the plants are grown on crop land and the oils have a place in the food market. This means that soy and canola-derived biofuels are in direct competition with traditional agriculture and are directly contributing to the food versus fuels debate (15). For algae to produce large quantities of biomass, a culture system needs to have access to sufficient sunlight and be scalable to a size where adequate quantities of biofuels can be produced. Different growth systems exist and each have advantages and disadvantages Open ponds and photobioreactors (PBR) are two general classes of productions systems typically considered for the largescale algae cultivation. Examples of a typical open pond and a photobioreactor (PBR) are shown in Figure 2. Currently, at the fuel production scale, large-scale open ponds appear the most economically sound biomass production system. The comparison of growth systems deals with efficiency of conversion of sunlight into organic carbon and thus biomass production rates are related to surface area that exposes the algae cells to the sunlight. The major advantage of a PBR is the higher biomass productivities and the lower risk of contamination. A recent comparison of the cost of biofuel production showed that the PBR advantages do not outweigh the additional costs of installed capital (16). At the basis of biomass productivity and oil production lies photosynthesis, the process where sunlight provides the energy needed to assimilate carbon from the atmosphere into organic compounds like lipids, carbohydrates and proteins. A detailed discussion of the mechanism of photosynthesis is outside the scope of this paper, but can be found in reference (12). We just want to mention some of the limitations and constraints around the sunlight to biomass conversion. Not all of the sunlight reaching the algal culture can be converted into biomass, in fact, this conversion efficiency is on the order of ~12% due to losses associated with photosynthetic conversion efficiency (Figure 3). First of all, less than half of the sunlight s spectrum is available for photosynthesis, also referred to as photosynthetically active radiation (PAR), of which only about 80% of the energy enters the photosynthesis electron transport chain to produce metabolic energy (as ATP) and reducing power (as NAD(P)H) needed for reduction of atmospheric CO2 to organic carbon. This organic carbon will eventually be incorporated into carbohydrates, proteins and lipids and form the basis for biofuels % Solar Radiation 45% PAR 35% PS I & II (680 & 700 nm) 3 mol ATP + 2 mol NADPH [CH2O] 15% 12% Fig. 3: Illustration of stepwise losses associated with photosynthetic conversion of solar radiation to reduced carbon, which makes up the basis of algal biomass. PAR = photosynthetically active radiation, PSI &II = photosystem I & II making up the photosynthetic apparatus with their respective wavelength absorbance maxima, [CH2O] = abbreviation for reduced organic carbon (adapted from reference (12)). Fig. 2: Examples of two types of growth systems of microalgae; (l) open pond (100,000 L) and (r) photobioreactors. Facilities as presently set up at Arizona State University, Phoenix, AZ. The maximum conversion efficiency of 12% of the sunlight energy to organic products is further reduced to approximately 10% when taking the losses due to respiration into account. The theoretical yield reduces even 3

4 further due to energy losses associated with metabolic conversion of primary photosynthetic product to fuel products such as lipids and carbohydrates. However, compared with crop plants, which have a theoretical efficiency of 4.6% and 6.0% (for C3 and C4 plants respectively), microalgae are still considerably more efficient (12). A detailed study of the theoretical and practical yields alongside a description of the process is needed to provide a reality check on the algal biofuels process. A detailed discussion of how the theoretical conversion efficiencies extrapolate to oil production on a large scale is described in reference (17), where the authors conclude that an average surface area yield of between 2000 and 5000 gal. ac-1.yr-1 is achievable. This calculation assumed an average of 10% photosynthetic conversion efficiency, an average daily production rate of between 33 and 42 g/m2/day and biomass oil content of between 30 and 50%. Solar radiation and biomass production is also a function of the temperature and irradiance and thus the geographical location will play a major role in the commercialization of the technology (17, 18, 19). In addition to theoretical yields, the production rates and oil yields will vary over the course of a production cycle (discussed in more detail below). Previously, a negative relation between the algal growth rates and the lipid content for an average of 15 different strains has been demonstrated (12). This means that the highest biomass productivity is typically observed when the nutrients and sunlight are available in abundance (also referred to as nutrient replete conditions). However, this productivity is typically associated with a low lipid yield, i.e. less than 10% of the dry weight of the biomass consists of fatty acids that can be converted to biofuels. The majority of these fatty acids present in nutrient replete cells are associated with structural lipids, for example those that make up the cell membranes. When the nutrients (especially nitrogen) become limiting for algae growth, the cells start to accumulate more storage lipids, such as triglycerides, which provide the cells with an energy reserve. Nutrient limitation also causes the cells to stop dividing, which reduces the overall temporal productivity. Though the biomass that is collected can contain up to 5- fold more lipids than the biomass from an actively growing culture. A discussion of the growth and productivity rates is outside the scope of this work, however, we will discuss in detail the composition of biomass and the respective fuel precursor concentrations harvested at three different times during the growth cycle, representing a replete, and two phases of nutrient deplete stages Biofuels from Organic Carbon The organic carbon derived from photosynthetic reduction of CO 2 can enter different metabolic pathways. The first, reduced metabolites are reduced sugars, which can then enter different biochemical pathways to produce the proteins, carbohydrates and lipids that make up the cell. The distribution of organic carbon between these three different biochemicals depends on the strain of algae and, as discussed briefly above, on the nutrient status of the culture. The strain dependence of the metabolic distribution is apparent when comparing a strain like Chlamydomonas reinhardttii, which stored most of its energy as starch (20) and a Chlorella vulgaris, which can accumulate over 50% of its dry weight as lipids (21). Both have value in a biofuels process, as either diesel-type fuels from lipids or bioethanol from fermentable carbohydrates such as starch. However, when comparing the energetics, lipids have a higher calorific value (36.3 kj/g) compared with carbohydrates (17.3 kj/g). So lipids give us a bigger bang for our buck, but a biomass source that can yield both lipids and carbohydrates as dual biofuel feedstocks, may be an even better choice. 2. BIOFUELS YIELDS AND PROPERTIES 2.1. Yields from Algae Algal biomass produced changes from species to species, but also within one spices depending on the growth and nutrient conditions. Our discussion here will focus on one species (Scenedesmus sp.) and how the composition and fuel yields of biomass harvested at different times of the growth changes, early, mid and late stage. When cells are actively growing early in the growth, the protein content is high (> 30%), whereas the lipid content is relatively low (< 10% total fatty acids). Late in the growth, when nutrients are depleted, the culture slows the growth rate, which reduces the protein content (to <10%) in favor of increasing the lipid content (>40%). Interestingly, in an intermediate stage of the growth phase, the culture accumulates carbohydrates, by as much as 48% of the biomass. The respective biomass composition for one strain is shown in Figure 4. These changes in overall biomass composition can greatly affect the overall biofuels production process. The question remains whether the increase in carbohydrate at the intermediate growth phase, and bioethanol yield through fermentation, amounts to the same overall process economics as would be achieved with biomass harvested at the high lipid phase. 4

5 ate High Protein 6 29 A 33 Fatty Acids 22 High Carbohydrate B Fatty Acids High Lipid Carbohydrate Starch Protein Lipid Ash Unknown Fig. 4: Composition of Scenedesmus sp. biomass harvested at three different growth stages (early, mid and late or High Protein, High Carbohydrate and High Lipid respectively), indicating large shifts in the composition Biofuels Properties from Algal Lipids Living cells make many different types of lipids, polar and neutral lipids. Typical polar lipids are the phospholipids, which make up the structural inner membranes of the cells and help with the compartmentalization of different metabolic units in the cells. Neutral lipids include triglycerides, which make up the majority of the oil droplets shown in Figure 1. In addition to the biological differences between the lipids, there are also practical implications of conversion efficiency and yields associated with different lipids. Not all lipids are equally valuable in a biofuels process, for example a phospholipid converts to approximately 64% of its weight as FAME in a biodiesel process, whereas a triglyceride converts to 100% of its weight as FAME (21). The molecular differences between a phospholipid and a triglyceride are shown in Figure 5, indicating the large hydrophilic group attached to two fatty acid chains in a phospholipid, whereas three fatty acid chains are esterified to a glycerol backbone in a triglyceride Fig. 5: Illustration of the molecular structures of a phospholipid (A) and a triglyceride (B). Images were built in Jmol based on data from LipidMAPS.org structure database. In addition to lipid type, the fatty acid make up of the lipids also highly affects the fuel properties. In particular, the chain length is one of the major determinants of the resulting fuel properties. Traditional biodiesel fuels from seed oils consist mainly of oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and palmitic (C16:0) acid, with minor contributions from palmitoleic (C16:1) acid (12). The fatty acid profile of algae varies significantly between species and are generally much more diverse and can include major contributions from C14 through C24 chains with varying degrees of unsaturation (12). This presents an opportunity to select a species of choice based on the FAME profile to suit the needs of a biofuels process. Although lipids can be complex in algae, this complexity will affect yields in a solvent-based extraction process. The difference in polarity of the lipids will determine the solubility in different solvents. A typical solvent used in a production process model is hexane, which will preferentially extract neutral lipids. Therefore, differences in lipid composition are occurring in biomass over time and these differences can have big impacts on the overall yields. The make up of lipids will change the polarity and thus the extractability. Although practically relevant, yields determined by a solvent-based extraction may not be adequate for accurate determination of the fuel potential of algal biomass. Therefore, in the context of quantitatively determining biofuel yield and lipids in general, a better measure is the determination of total fatty acids (21, 22). Not only is this a more accurate determination of lipids, a simultaneous prediction as to the fuel properties can be made based on the fatty acid profile. 5

6 As shown in Figure 6, the fatty acid profile changes considerably as the total lipid content increases. In all three types of biomass, the major fatty acids are oleic (C18:1), linoleic (C18:2), linolenic (C18:3) and palmitic (C16:0) and hexadienoic (C16:2) acid. The most marked change is in the contribution of oleic acid, which increases from 14% to 57% of the total fatty acids in the high lipid biomass. % Biomass C16:3 C16:4 C16:2 C16:1n9 C16:1n11 C16:0 C18:2 C18:1n9 C18:3 C18:0 14% 14% 17% 20% Fig. 6: Total lipid content (as FAME) in high protein (HP), high carbohydrate (HC) and high lipid (HL) Scenedesmus sp. biomass, illustrating the individual relative contributions of fatty acids to the total lipids. The resulting fuel quality will primarily change due to differences in the level of unsaturation. The contribution of chains with no unsaturation and those with more than 2 unsaturated bonds decreases with an accompanying increase in chains with one unsaturated bond. While the overall level of unsaturation did not change significantly, the removal of the polyunsaturates will improve oxidation stability (24), while removal of fully saturated chains will improve cold weather performance (25). While the relative contribution of C16 chains decrease in favor of an increase of C18 chains, this will have only a minor effect on fuel properties Theoretical Combined Fuel Yields 6% 57% 5% 15% 10% 7% HP HC HL C18:3 C18:1n9 C18:2 C16:0 C16:2 Considering that both fatty acids and fermentable carbohydrates are biofuels precursors, the question is which of the three growth phases give the highest overall yields. As shown in Figure 4, the total carbohydrate fraction of the biomass can make up as much as 48% of the dry weight, and the measured glucose content as much as 30% of the biomass. This is promising in the development of a strategy to produce bioethanol as a fuel source in addition to extraction and conversion of the lipids. However, developing successful carbohydrate solubilization technologies and fermentation efficiencies remain a challenge. Considering the carbohydrate composition of algal biomass in theoretical fuel calculations is important and could answer questions on whether adding in the possible bioethanol yields can aid with the overall process economics (16). The total fraction of the biomass that can be converted to fuels is shown in Table 1, showing up to 75% of the biomass that is made up of biofuels precursors. Even though the fraction of fermentable carbohydrates increases in later growth phases, the highest theoretical yields will most likely be derived from biomass in the high lipid phase. TABLE 1: BIOFUEL RELEVANT BIOMASS COMPOSITION (% DRY WEIGHT) Glucose Fatty Acids Total HP HC HL FINAL REMARKS Algae have potential to make significant contributions to the future biofuels pool. Theoretical conversion efficiency of sunlight to organic carbon or biomass is around 12% and up 75% of the produced biomass could theoretically be converted to biofuels. However, the theoretical and practical yields may differ significantly due to losses inherently associated with extraction and conversion processes. Furthermore, challenges exist to render the entire process economically viable and thus all routes to increasing the biofuel yields, including fermentation of carbohydrates, should be considered. When calculating the productivity of algae as a feedstock for biofuel production one has to take the growth rate and overall composition of the biomass into account. Undoubtedly this composition and adaptation mechanisms over different growth conditions will change between species of algae. The type of approach and study of the carbohydrate and lipid yields discussed here can, and perhaps should, be applied to an algae production system to determine optimum harvest and conversion conditions for each system. 6

7 ACKNOWLEDGMENTS This work was supported by the U.S. Department of Energy under Contract No. DE-AC36-08-GO28308 with the National Renewable Energy Laboratory. The authors are grateful for the images in Figure 1 contributed by Nicholas Sweeney and the contributions from Stefanie Van Wychen and Michelle Reed for supplying the composition data shown in Figures 5 and 6. REFERENCES (1) Graboski, M.S., McCormick, R.L. Combustion of Fat and Vegetable Oil Derived Fuels in Diesel Engines Progress in Energy and Combustion Science, (1998). (2) McCormick, R.L., Alleman, T.L., Graboski, M.S., Herring, A.M., Tyson, K.S. Impact Of Biodiesel Source Material And Chemical Structure On Emissions Of Criteria Pollutants From A Heavy-Duty Engine Environ. Sci. Technol (2001). (3) Graboski, M.S., McCormick, R.L., Alleman, T.L., Herring, A.M. Effect Of Biodiesel Composition On NO x And PM Emissions From A DDC Series 60 Engine Final Report to National Renewable Energy Laboratory, NREL/SR , February (4) McCormick, R.L., Westbrook, S.R. Storage Stability of Biodiesel and Biodiesel Blends Energy&Fuels (2010). (5) Coordinating Research Council. Biodiesel Blend Low- Temperature Performance Validation, CRC Report No. 650, (6) Voegele, E. A New Standard for Quality Biodiesel Magazine, October 25, (7) Chupka, G.M., Yanowitz, J., Chiu, G., Alleman, T.A., McCormick, R.L. Effect of Saturated Monoglyceride Polymorphism on Low-Temperature Performance of Biodiesel Energy&Fuels 25 (1) (2011). (8) Williams, A., Luecke, J., McCormick, R.L., Brezny, R., Geisselmann, A., Voss, K., Hallstrom, K., Leustek, M., Parsons, J., Abi-Akar, H. Impact of Biodiesel Impurities on the Performance and Durability of DOC, DPF and SCR Technologies SAE Int. J. Fuels Lubr. 4(1) (2011). (9) Kalnes, T., Marker, T., Shonnard, D.R. Green Diesel: A second generation biofuel Int. J. Chem. React. Eng. 5 A48 (2007). (10) Smagala, T,G., Christison, K.M., Mohler, R.E., Christiansen, E., McCormick, R.L. Renewable and Synthetic Diesel Fuels: Composition and Properties to be published. (11) Huo, H., Wang, M., Bloyd, C., Putsche, V. Life- Cycle Assessment of Energy and Greenhouse Gas Effects of Soybean-Derived Biodiesel and Renewable Fuels ANL/ESD/08-2, March 12, (12) Williams, P.J. le B., Laurens L.ML. Microalgae as Biodiesel & Biomass Feedstocks: Review & Analysis of the Biochemistry, Energetics & Economics, 2010, Energy Environm. Sci, 3, (13) Greenwell, H.C., Laurens, L.ML., Shields, R.J., Lovitt, R.W., Flynn, K.J. Placing microalgae on the biofuels priority list: a review of the technological challenges, 2010, J. R. Soc. Interface, 7:46, (14) Wijffels R.H., Barbosa M.J., An outlook on microalgal bio- fuels, 2010, Science 329(5993): (15) Chisti, Y., "Biodiesel from Microalgae", 2007, Biotechnol. Adv., 25, (16) Davis R, Aden A, Pienkos PT, Techno-economic analysis of autotrophic microalgae for fuel production., 2011, Appl. Energ. 88 (10): (17) Weyer, K., Bush, D., Darzins, A., Willson, B. Theoretical maximum algal oil production. Bioenergy Res 2010;3: (18) Wigmosta, M.S., Coleman, A.M., Skaggs, R.J., Huesemann, M.H., Lane, L.J. National microalgae biofuel production potential and resource demand, 2011, Water Resources Research, 47:1-13. (19) Pate R, Klise G, Wu B. Resource demand implications for us algae biofuels production scale-up., 2011, Appl. Energ. 88: (20) Work, V.H., Radakovits, R., Jinkerson, R.E., Meuser, J.E., Elliott, L.G., Vinyard, D.J., Laurens, L.ML., Dismukes, C., Posewitz, M.C. Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless Isoamylase mutant and increased carbohydrate synthesis in complemented strains, 2010, Eukaryotic Cell, 9: (21) Laurens, L. ML., Quinn, M., Van Wychen, S., Templeton, D. T., Wolfrum, E., Accurate and reliable quantification of total microalgal fuel potential as fatty acid methyl esters by in situ transesterification, 2012, Analytical and Bioanalytical Chemistry, DOI: /s (22) Nagle N, Lemke PR., Production of methyl ester fuel from microalgae, 1990, Appl Biochem Biotechnol 24(5): (23) Knothe, G. H., A technical evaluation of biodiesel from vegetable oils vs. algae. Will algae-derived biodiesel perform?, 2011, Green Chem, 13, (24) McCormick, RL, Ratcliff, M, Moens, L, Lawrence, R, 2007 Several Factors Affecting The Stability Of Biodiesel In The United States Fuel Proc. Techn. 88, (25) Dunn, Rom 2009 Prog. Energy Comb. Sci. 35,