FIELD OF THE INVENTION

The present invention relates to the field of biofuel production, particularly to methods for efficient production of triglycerides by microalgae

BACKGROUND OF THE INVENTION

Biofuels are gaining increased public and scientific attention due to several factors, including continues increase in oil price and decrease in availability, the need for increased energy security, and concern over greenhouse gas emissions from fossil fuels. Biofuels refer to a wide range of fuels derived from biomass. Biodiesel refers to monoalkyl esters of long chain fatty acids derived from lipids present in animal, plant or microorganism, primarily triglycerides (also referred to as TG, triacylglycerol, TAG or triacylglyceride), which can be used in diesel-engine vehicles. Currently, economic oilseed crops including soybean, rapeseed and palm are the main source for biodiesel in Europe and America. However, only a small fraction of the liquid fuel demand for transportation is provided by oilseed crops, and mainly in Europe. The major limitation in extending their utilization is limited availability of agricultural lands and of water resources. Microalgae are recognized as an excellent source for biofuels for several reasons, particularly due to the ability of many species of microalgae, including green algae and diatoms, to accumulate triglycerides up to over 50% of their biomass. Combined with their fast growth rate, microalgae exceed by far the lipid productivity of the common oil crops soybean and oil palm. Microalgae can be cultured in sea water or in brackish water in areas not suitable for agriculture and can also serve as biofilters for flue gas emitted by power plants and other carbon dioxide emitting industries. As such, cultivation of microalgae does not compete with agriculture resources. In addition, byproducts of microalgae following triglyceride extraction can be utilized as a source of protein for animal and fish feed, as fertilizers in agriculture and as sources for high- value pigments and vitamins. One of the major limitations for commercial utilization of microalgae for biodiesel production is the high cost of alga biomass production, currently estimated at US$5/Kg. Lowering the cost of algae mass cultivation and processing and improvement in lipid productivity are prerequisites for economical production of biodiesel from microalgae (Rodolfi L. et al. 2009. Biotechnol Bioengineering 102: 100-112).

As mentioned above, microalgae are excellent candidates for the production of triglycerides and biodiesel as they reproduce approximately 40 times faster than higher plants; however, a major limiting element for maximizing lipid productivity from microalgae is the fact that high levels of triglycerides are accumulated only under growth limiting conditions (Guschina I. A. and Harwood J. L. 2006. Prog Lipid Res 45(2): 160-186; Roessler P. G. 1990. J Phycol 26(3):393-399). In green algae, the most common strategy to induce triglyceride accumulation is nitrogen limitation, and in diatoms, limiting the silicon concentration shows the same effect. Also sulfate and phosphate limitation, low temperature or high light intensities can induce triglycerides accumulation. Typically, nitrogen limitation increases the cellular lipid level in green microalgae by 2 to 10 fold while in parallel cell division and biomass production is inhibited. As a result, the overall lipid productivity of most oleaginous algae species per liter culture decreases at growth conditions of limited nitrogen, due to the large decrease in biomass productivity (Hu Q. et al. 2008. Plant J 54:621-639; Rodolfi et al. 2009, ibid).

Strategies proposed to optimize triglyceride accumulation by nitrogen limitation include either supplying the growth culture with a low nitrogen level that is sufficient to increase triglyceride level while having minimal inhibition of the mass production or employing a two phases growth strategy including a first phase in which the algae are grown in a medium containing sufficient nitrogen to obtain maximal biomass, followed by a second phase in which the algae are grown in nitrogen-deficient medium and massive TAG production. (Rodolfi et al. 2009, ibid).

U.S. Patent Application Publication No. 2012/0088279 discloses methods for increasing the levels by applying a stress, such as nutrient stress, after which the lipid can be harvested from the algae using a non-destructive extraction process. The stress may be provided in a periodic or "pulsed" fashion. Lipid levels in oleaginous algae can also be increased using simulated stress by treating the algae with a chemical inhibitor or by using recombinant technology to insert a sequence expressing a protein such as a nitrate reductase inhibitor that is expressed when a stressed state is desired. A method for maintaining the temperature and water levels of algae ponds using buoyant spheres is also described.

U.S. Application Publication No. 2009/0298159 discloses a method for producing biodiesel from algae using two-stage cultivation, autotrophic and heterotrophic of Chlorella. This method includes a sequence of procedures: cultivating photoautotrophic algae, concentrating the cells and then transferring the cells to a fermentor for heterotrophic cultivation. During the photoautotrophic cultivation stage, the culture is exposed to a light source, such as sunlight with carbon dioxide obtained from a carbon dioxide source or from air. Organic carbons are added during the heterotrophic cultivation stage. Fermentation conditions are optimized for maximizing lipid synthesis. After cultivation, biodiesel is made through extraction and transesterification of the algae lipids.

U.S. Application Publication No. 2010/0162620 discloses systems and processes for optimizing the conditions for each phase of biomass production and of oil/lipid production separately and independently, thereby improving overall production of oil, lipids and other useful products. However, the two-stage strategies have not reached the maximal potential triglyceride productivity, and the particular conditions to be used are typically species specific. Furthermore, transferring the algae culture from nitrogen containing to nitrogen-deprived medium requires additional equipment, space and work.

Alternatively, increased triglyceride production may be achieved by shifting assimilating carbon towards biosynthesis of storage reserves: under normal growth conditions in plants and algae, most of the photosynthetic assimilated carbon is channeled to three major destinations: biosynthesis of proteins, nucleic acids, sugars and lipids; energy production by the mitochondrial respiratory system; and biosynthesis of storage reserves such as starch and/or triglyceride. Under growth-limiting conditions, the biosynthetic channel, mostly of proteins, is greatly reduced, respiration is less affected and biosynthesis of storage reserves is greatly increased. Suppression of carbon-consuming channels other than for triglyceride production, namely starch biosynthesis or respiration may lead to accumulation of storage lipids. Li et al. (Li Y. et al. 2010. Metabol. Eng. 12:387-91) showed that inhibition of starch biosynthesis by silencing ADP-glucose pyrophosphorylase in the green algae Chlamydomonas reinhardtii, created a mutant which accumulates triglycerides rather than starch under nitrogen deprivation. However, this phenomenon could not be reproduced in a more recent report (Siaut et al, 2011 BMC Biotechnol. 11 :7).

U.S. Patent Application Publication No. 2009/0148928 describes the use of a "heterotrophic shift" for increasing lipid production, in which algae or other organisms are first grown under autotrophic conditions, under which the algae grow using photosynthesis in an energy-efficient and cost-effective manner, after which the algae are shifted to heterotrophic growth, where they feed off of supplied fixed carbon sources such as sugar without available sunlight and produce higher lipid levels.

The diverse conditions that induce triglyceride accumulation in microalgae suggest that there are several different signaling/metabolic pathways to induce triglyceride accumulation. However, the metabolic regulation of triglyceride biosynthesis and its signal transduction mechanism in microalgae are not known as of today.

Thus, there is a recognized need to, and it would be highly advantageous to have methods for optimizing oil production by microalgae as a source for biodiesel.

SUMMARY OF THE INVENTION

The present invention relates to methods for enhancing the production of triglycerides (TAG), a recognized source of biodiesel, by microalgae. Particularly, the methods of the present invention result in high algal biomass production concomitant to high production of triglycerides.

The present invention is based in part on the unexpected discovery that low concentrations of sodium azide induced accumulation of triglycerides to significantly enhanced levels. The enhancement was observed in 17 out of 19 tested species of microalgae belonging to different classes (including, for example, Chlorophyceae, Prymnesiophyceae, Eustigmatophyceae, Bacillariophyceae and Trebouxiophyceae), known to synthesize triglycerides, with little or no effect on the algae growth rate.

The present invention is advantageous over hitherto known methods for triglyceride production by algae in that the biomass production is only marginally decreased, and thus the overall accumulated amount of triglycerides per growth volume is significantly elevated. In addition the algae can be grown in the same culture medium and apparatus, thus reducing the amount of work and costs involved in the production of triglycerides and algae biomass.

Thus, according to one aspect, the present invention provides a method for triglyceride production by microalgae comprising adding to a culture of microalgae at least one inhibitor of cytochrome oxidase at a concentration sufficient to induce triglyceride accumulation, essentially without negatively affecting the growth rate of said microalgae.

According to certain embodiments, the inhibitor of cytochrome oxidase is selected from the group consisting of azide, cyanide and salts thereof.

According to certain embodiments, the inhibitor of cytochrome oxidase is an azide salt selected from the group consisting of sodium azide, potassium azide, lithium azide and cesium azide. Each possibility represents a separate embodiment of the present invention. According to certain currently typical embodiments, the inhibitor of cytochrome oxidase is sodium azide.

According to other embodiments, the inhibitor of cytochrome oxidase is cyanide salt selected from the group consisting of potassium cyanide, sodium cyanide, lithium cyanide and cesium cyanide. Each possibility represents a separate embodiment of the present invention. According to certain typical embodiments, the cyanide salt is potassium cyanide.

According to certain embodiments, the culture of the microalgae is an aqueous liquid culture. According to some embodiments, the liquid culture comprising the microalgae is diluted at least two fold before the cytochrome oxidase inhibitor is added. According to other embodiments, the microalgae are grown in the liquid culture up to mid to late logarithmic phase, before the culture is diluted and inhibitor is added. Algae known to produce triglycerides are used according to the teachings of the present invention. According to certain embodiments, the microalgae are of a species selected from the group consisting of Chlorella, Dunaliella, Nannochloropsis, Nannochloris, Scenedesmus, Tetraselmis, Crypthecodinium, Cyclotella, Navicula, Nitzschia, Phaeodactylum, Cylindrotheca, Skeletonema, Chaetoceros, Isochrysis, Pavlova, Emiliania, Rhodomonas, and Botryococcus. According to certain embodiments, the microalgae are selected from the group presented in Table 1. According to other embodiments, the microalgae are selected from the group consisting of Chlorella desiccata, Dunaliella primolecta, Phaeodactylum tricornutum, Pseudochlorococcum polymorphum, Chlorella vulgaris, Nannochloris atomus, Nanochloropsis salina, Chaetoceros muelleri, Dunaliella parva, Chlorella sorokiniana and Scenedesmus dimorphus. According to certain typical embodiments, the microalga is Chlorella desiccata. Each possibility represents separate embodiment of the present invention. The type and optimal concentration of cytochrome oxidase inhibitor depends on the microalgae species and, to a lesser extent, on the microalgae growth conditions. According to certain typical embodiments wherein the cytochrome oxidase inhibitor is azide salt, azide is added at a concentration range of from 1 μΜ to 5 mM, typically from 5 μΜ to 1 mM, more typically from 20 μΜ to 100 μΜ. Each possibility represents a separate embodiment of the present invention. According to certain typical embodiments, the azide salt is sodium azide.

Culturing apparatus, media type, growth temperature and pH range for optimal microalgae cultivation are known to a person skilled in the art, and are adapted according to the microalgae species cultivated. According to certain embodiments, the microalgae are cultivated in a liquid culture medium comprising non-limiting levels of nutrients and trace elements required for optimal increase in cell number. According to some embodiments, the medium comprises air-level or enriched level of C0 ₂ or of sodium bicarbonate. According to additional embodiments the culture medium further comprises mineral sources for nitrogen (N), phosphorous (P), sulfur (S), silicon (Si), potassium (K), calcium (Ca), magnesium (Mg) or a combination thereof. According to yet additional embodiments the culture medium further comprises microelements including, but not limited to, iron (Fe), manganese (Mn), copper (Cu), zinc (Zn) and molybdenum (Mo) as well as vitamins as required.

According to certain typical embodiments the concentration of C0 ₂ is in the range of 0.03-5%. According to other typical embodiments, the concentration of sodium bicarbonate is 5-50mM. The particular composition and component concentration is determined according to the particular species of the microalgae used, as described herein below and as is known to a person skilled in the art.

According to certain embodiments, the microalgae are grown under autotrophic conditions. According to some embodiments, the autotrophic conditions comprise light source having an intensity of from 30-500 μΕ, typically from 50-250, typically 70-130 μΕ. Each possibility represents a separate embodiment of the present invention.

According to additional embodiments, the microalgae are cultivated at an ambient temperature range of from about 10°C to about 40°C, typically at an ambient temperature of about 18°C to 25°C. Each possibility represents a separate embodiment of the present invention. According to certain embodiments, the culture obtained is diluted to reach algae concentration of 10 ⁶ -10 ⁷ cells/ml before the cytochrome oxidase inhibitor is added. It is to be explicitly understood that the optimal algal density to which the inhibitor is added varies according to the algae species. The culture can be diluted with the same or different type of medium in the same culture apparatus or with the same or different type of medium by transferring the culture to additional culture apparatus. It is to be explicitly understood that dilution can be made within a single culture growth apparatus and that no washing of the culture is required when the algae are grown in different types of media and/or apparatuses.

According to certain embodiments, the triglycerides yield ^g/L/Day) is at least 1.10 fold higher, at least 1.20, 1.25, 1.50, 2.0, 2.5, 3.0 fold or more higher compared to the yield obtained when the microalgae are grown at the same conditions without the addition of the cytochrome oxidase inhibitor. According to other embodiments, the triglycerides yield ^g/L/Day) is at least 3.5 fold, at least 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 fold or more higher compared to the yield obtained when the microalgae are grown at the same conditions without the addition of the inhibitor. Each possibility represents a separate embodiment of the present invention. Growing triglyceride-producing algae under N-deprived conditions is known to induce triglyceride production. Optimal triglyceride production is obtained when two- phase culturing system is employed, namely wherein maximal mass productivity is reached at a first growing phase under N-sufficient conditions followed by a second growing phase under N-deficient or N-deprived conditions. However, using the two phase culturing system involves cumbersome work and equipment to achieve proper media replacement.

According to certain embodiments, the triglyceride yield ^g/L/day) of algae grown according to the teaching of the present invention is at least 1.10 fold higher compared to the TAG yield of the algae grown at N-deprived conditions. According to some embodiments, the yield is at least 1.20, 1.25, 1.50, 2.0, 2.5, 3.0 fold or more higher compared TAG yield of the algae grown at N-deprived conditions. According to these embodiments, the algae are selected from the group consisting of Chlorella desiccata, Nannochloris atomus, Nanochloropsis salina, Chaetoceros muelleri Dunaliella parva, Dunaliella primolecta, Chlorella sorokiniana, Scenedesmus dimorphus, and Phaeodactylum tricornutum. Each possibility represents separate embodiment of the present invention.

According to additional aspect, the present invention provides a method for producing biodiesel from microalgae comprising utilizing lipids produced by the microalgae, wherein the microalgae are grown in a culture comprising cytochrome oxidase inhibitor at a concentration sufficient to enhance triglyceride accumulation by said microalgae essentially without negatively affecting the growth rate of said microalgae.

According to some embodiments, the microalgae are grown in a liquid culture under autotrophic conditions.

According to certain embodiments, the cytochrome oxidase inhibitor is selected from the group consisting of azide, cyanide and salts thereof. According to certain typical embodiments, the cytochrome oxidase inhibitor is azide salt, typically sodium azide. According to certain embodiments, the culture is diluted at least two fold before addition of the cytochrome oxidase inhibitor. According to certain typical embodiments, the microalgae are grown towards the mid to late logarithmic phase before the culture is diluted.

According to certain embodiments, utilizing the lipids comprises extracting the lipids from the microalgae and performing esterification of said lipids to produce biodiesel. According to some embodiments, the esterification is trans-esterification.

According to some embodiments, the algae are harvested before extraction of the lipid fraction. According to further embodiments, the step of harvesting further comprises drying the algae after harvest. According to other embodiments, the step of harvesting the microalgae is performed at an algal concentration of from 5xl0 ⁵ -5xl0 ⁸ cells/ml. It is to be explicitly understood that the optimal algal concentration at harvest varies according to the algae species. According to certain embodiments, the method further comprises collecting the culture medium after algae are harvested and neutralizing residual concentrations of the cytochrome oxidase inhibitors. According to certain typical embodiments wherein the cytochrome oxidase inhibitor is sodium azide, neutralizing the sodium azide comprises adding hypochlorite in an amount sufficient to neutralize said sodium azide.

The algal biomass left after lipid extraction can be used per se or further processed to obtain additional by products according to the algae species as is known to a person skilled in the art. According to some embodiments, the algal biomass can be used as fish, mammal or poultry feed, particularly as a source for proteins. According to certain embodiments, the algae are used as a source for at least one of starch, pigments and vitamins. Each possibility represents a separate embodiment of the present invention.

Other objects, features and advantages of the present invention will become clear from the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows azide-induced triglyceride (TAG) accumulation in Chlorella desiccata. Fig. 1A: TAG quantification by specific Nile Red fluorescence (NR Fl) in intact cells. C. desiccata was grown in vertical columns for 3 days. Emission spectra of 5xl0 ⁶ cells/ml stained with ΙμΜ Nile Red (Excitation (Ex.) 488 nm). +N, complete growth medium; -N, nitrogen deficient medium; +N+Az, complete medium with 20 μΜ NaN ₃ . Fig. IB: TAG quantification by TLC in Chlorella cells. Chlorella desiccata was grown in vertical columns for 3 days. 1 μΐ extract out of prepared total 200 μΐ lipid extract of 5xl0 ⁷ cells. T, 1 μg triolein; +N, complete growth medium; -N, nitrogen deficient medium; +N+Az, complete medium with 20 μΜ NaN ₃ . Fig. 1C: Comparison of neutral lipids (TAGs) quantification by Nile Red fluorescence and HPLC in Chlorella desiccata. Fold change of total neutral lipids (TAGs) in cells after 3 days induction, compared to cells grown in complete medium. Average fold change from 3 (for specific Nile Red fluorescence, excitation. 488; emission 580) and 2 (for HPLC) independent experiments + standard deviation (st.dev.) +N, complete growth medium; -N, nitrogen deficient medium; +N+Az, complete medium with 20 μΜ NaN ₃ . Fig.l D and E: Effect of azide on cell growth and TAG accumulation. Chlorella desiccata was grown in vertical columns in nitrogen deficient medium (-N) or in complete medium (+N) with different concentrations of NaN ₃ . Cell concentration - Fig IE; Emission at 580 nm of 5xl0 ⁶ cells/ml stained with ΙμΜ Nile Red - Fig. ID. Average of 3-4 experiments is presented.

FIG. 2 shows a comparison of TAG yields in azide-treated and nitrogen-deprived C. desiccata. Fig. 2A: TAG yields of azide-treated and nitrogen-starved cells. C. desiccata were grown in vertical columns for 2 days. TAG yield was estimated by Emission (at 580 nm) of 5xl0 ⁶ cells/ml stained with ΙμΜ Nile Red (Ex. 488 nm) multiplied by cell concentration, and normalized to control cells (+N). +N, cells grown in complete growth medium; -N, nitrogen-starved cells; +N+Az, cells grown in complete medium with 20 μΜ NaN ₃ . Average ± st. dev. of 9 independent experiments is shown. * P< 0.05. Fig. 2B: TAG yields of azide-treated and nitrogen-limited cells. In the first stage, the cells were diluted to 5xl0 ⁶ cells/ml in complete medium (+N, 10 mM KN0 ₃ ) or in nitrogen- limited medium (-N, 1.5 mM KN0 ₃ ). After two days of growth, the cells from +N were diluted 1 :5 to +N medium or to +N with 20 μΜ NaN ₃ (+N+Az), while -N cells were diluted 1 :5 again to nitrogen-limited medium (1 mM KN0 ₃ ). This was the start of the second stage of the experiment (t=o). After three days the cells were harvested to measure several parameters: TAG content by TLC, dry cell weight- by drying 50 ml cells at 110°C overnight, and cell concentration. TAG yields (average±stdev) from two independent experiments are shown. FIG. 3 shows comparison of the effects of azide and N-deprivation on photosynthesis, respiration and catalase activity in C. desiccata. Cultures were grown in vertical columns for one day, with several treatments: complete medium (+N), nitrogen starvation (-N), and complete medium with various sodium-azide concentrations: 10, 20, 50 and 100 μΜ. Oxygen evolution/uptake of the cells (2xl0 ⁷ cells/ml) was measured by the oxygen electrode connected to the recorder in three states: in light and in dark before and after the addition of 1.7 mM hydrogen peroxide (Fig. 3 A). Rates of photosynthesis, dark respiration and catalase activity of each treatment were estimated relative to +N (Fig. 3B). FIG. 4 shows the effect of light intensity on lipid accumulation in Chlorella. desiccata cells. C. desiccata nitrogen-deprived (-N, grown in nitrogen deficient medium) and azide-treated (+N+Az, complete medium with 20 μΜ NaN ₃ ) cells were grown in columns for 2 days in 5 light intensities: 15, 30, 60, 95 and 300 μΕ. On the second day, TAGs were quantified by specific NR fluorescence at emission (Em.) 580 nm (average from 2-3 independent experiments ±STDEV are shown).

FIG. 5 demonstrates the effect of C0 ₂ concentration on TAG yield of azide-treated C. desiccata (Fig. 5A, B) and N. atomus (Fig. 5C, D). The two algae species were grown either in Erlenmeyer flasks (low C0 ₂ , Fig. 5A and Fig. 5C) or in vertical columns (high C0 ₂ , 5% Fig. 5B and Fig. 5D) with different concentrations of NaN ₃ (0, 20, 50, 100, 1000 μΜ). After 2d (C. desiccata) or 3d (N. atomus) the cells were harvested for TAG estimation by TLC (performed from 5xl0 ⁷ cells of C. desiccata and from lxl 0 ⁸ cells of N. atomus).

FIG. 6 shows microscopic visualization of lipid globules. Fig.6a: Chlorella desiccata.

The algae were grown in vertical columns for 2 days. +N, complete growth medium; -N, nitrogen deficient medium; +N+Az, complete medium with 20 μΜ NaN ₃ . Panels A-

C, confocal images. Bright white (NR fluorescence): Ex. 488 nm. Em. 560-590 nm;

Gray (chlorophyll fluorescence): Ex. 635 nm. Em. 655-755 nm.; panels D-F, TEM images; panels G-I, Cryo-SEM images. C, chloroplast; N, nucleus; St, starch; LG, lipid globule; V, vacuole. Fig. 6b: D. tertiolecta. The algae were grown in closed Erlenmeyer flasks in 2M NaCl for 3 days. +N, complete growth medium; -N, nitrogen deficient medium; +N+Az, complete medium with 1 mM NaN ₃ . Panels A-C, confocal images; Panels D-F, TEM images; Panels G-I, Cryo-SEM images. C, chloroplast; N, nucleus; P, pyrenoid; St, starch; LG, lipid globule; V, vacuole; M, mitochondrion.

FIG. 7 shows fatty acid analysis and TAG level of Chlorella desiccata. Chlorella desiccata was grown in vertical columns for 3 days. Total lipid extract was prepared from 50 ml cells. % TAG of total lipids was performed by Alicia Leikin Frenkel, Tel Aviv University, Sackler School of Medicine, by TLC method (Fig.7 A). C. desiccata was grown in vertical columns for 2 days. 2xl0 ⁸ cells were taken for lipid extract, then, FA hydrolysis was performed. 10 μΐ extract out of prepared total 200 μΐ were run on HPLC. Five main fatty acids were identified by HPLC standards (Fig. 7B). +N, complete growth medium; -N, nitrogen deficient medium; +N+Az, complete medium with 20 μΜ NaN ₃ .

FIG. 8 shows Clorella desiccata TAG molecular species separated by HPLC. C. desiccata was grown in vertical columns for 3 days. 10 μΐ extract out of prepared total 200 μΐ lipid extract of lxlO ⁸ cells, run on HPLC. Seven main TAG groups are marked. The sixth group was found to contain triolein. Fig. 8 A: Fractions of 1 mL collected from TAG separation of C. desiccata (-N) by HPLC were evaporated, diluted in 5 μΐ chloroform, and run on TLC, beside triolein (T) standard. Fig. 8B: Main TAG groups of C. desiccata, separated by HPLC, were quantified by measuring the area of each TAG group peak. Percents are average of two independent experiments (Fig. 8C). FIG. 9 demonstrates the involvement of reactive oxygen species in azide-treated cells. Fig. 9A: Estimation of reactive oxygen species in Clorella desiccata, with ROS generators and scavengers. Cells at concentration of 2xl0 ⁷ cells/ml were washed in the fresh growth medium and then incubated with 5 μΜ Dihydrorhodamine 123 (DHR) in 15 mL tubes in the dark for 0.5h. After the incubation, the cells were washed again, diluted to lxl 0 ⁷ cells/ml, and exposed to different induction conditions: complete medium (+N), nitrogen deficient medium (-N) or +N with different chemicals: H ₂ 0 ₂ , NaN3, Tiron (TIR) and DABCO for 15h in the light. Then, the cells were centrifuged, resuspended in PBS(x2), and their DHR (Ex: 488 nm, Em: 529 nm) and Chlorophyll (Ex: 435 nm, Em: 685 nm) fluorescence were measured by a plate reader. DHR fluorescence normalized to Chlorophyll fluorescence is shown (average of three independent replicates + st.dev). Fig. 9B: Effect of different chemicals on cell growth and TAG accumulation in Clorella desiccata. C. desiccata was grown for two days in Erlenmeyer flasks in nitrogen deficient medium (-N), or in complete medium (+N) with different chemicals: sodium azide (NaN ₃ ), Rose bengal (RB), Benzyl viologen (BV) and potassium cyanide (KCN). Cell growth (normalized to +N, %), emission at 580 nm of 5xl0 ⁶ cells/ml stained with ΙμΜ Nile Red, and TAG quantification by TLC ^g/ 5xl0 ⁷ cells) are presented.

FIG. 10 shows the effect of DABCO on azide-induced TAG production. Clorella desiccata was grown in columns in complete medium (+N), or in nitrogen deficient medium (-N), with 20 μΜ NaN3 (Az), and/or 10 mM DABCO (D). Cell concentration (cells/mL) and specific Nile Red fluorescence (Ex. 488, Em. 580 nm) were measured after 2, 3, and 4 days. Average + standard deviation of three independent experiments is shown. TAGs from the main sixth group were quantified according to triolein standard. Average + standard deviation of two independent experiments is shown.

FIG. 11 shows the effect of D ₂ 0 on TAG induction in Clorella. desiccata. C. desiccata was grown in columns in the medium containing 30% D ₂ 0 (D ₂ 0) and in the regular medium (H ₂ 0), 2 days before the experiment. Then the cells were transferred to different induction conditions: complete medium (+N), nitrogen deficient medium (-N), or complete medium with 20 μΜ NaN ₃ (Az) and/or 10 mM DABCO (D), in H ₂ 0 or 30% D ₂ 0. After 3 days, the cells were counted and specific NR Fluorescence was measured (Ex. 488 nm, Em 580 nm). Emission at 580 nm is shown (average of 3 independent experiments + st.dev).

DETAILED DESCRIPTION OF THE INVENTION

The present invention shows for the first time that triglyceride production in microalgae can be significantly enhanced, with minor negative effect on the algae growth rate, by the addition of an inhibitor of cytochrome oxidase to the microalgae growth medium. The optimal concentration of the added inhibitor depends on the microalgae species; nevertheless, of 19 microalgae species examined, 17 have been shown to be responsive to the cytochrome oxidase inhibitor and accumulated significant amounts of triglycerides suitable for production of biodiesel while keeping close to normal growth rate. The method of the present invention is advantageous over hitherto known methods for commercial production of biodiesel in that higher productivity is achieved in terms of triglyceride amount per volume of the algae growth medium; smaller culture volumes are needed; and triglyceride production can be efficiently controlled. As used herein, the terms "microalga" or "alga" and "microalgae" or "algae" refer to microscopic photoautotrophic algae, typically found in freshwater and marine systems. Microalgae are unicellular species which exist individually, or in chains or groups. Depending on the species, their sizes can range from a few micrometers (μηι) to a few hundreds of micrometers. The terms "cytochrome oxidase inhibitor" or "inhibitor of cytochrome oxidase" are used herein interchangeably and refer to any compound that inhibits the enzyme- mediated electron transport from cytochrome c to oxygen. According to certain typical embodiments, the cytochrome oxidase inhibitor is selected from the group consisting of azide, cyanide and salts thereof. The terms "azide salt" refer to any salt of azide capable of inducing triglyceride accumulation in algae essentially without negatively affecting the growth of the algae. According to certain currently typical embodiments the azide salt is sodium azide having the formula NaN ₃ .

The terms "cyanide salt" refer to any salt of cyanide capable of inducing triglyceride accumulation in algae essentially without negatively affecting the growth of the algae. According to certain currently typical embodiments the cyanide salt is potassium cyanide having the formula KCN.

As used herein, the term "essentially without negatively affecting the growth rate" with regard to an inhibitor concentration refers to a concentration of the inhibitor that results in no more than 40% growth rate inhibition, typically not more that 35% or 30% inhibition, more typically not more than 25% or 20% inhibition, most typically not more or less than 10% growth rate inhibition.

According to one aspect, the present invention provides a method for enhancing triglyceride production by microalgae comprising adding to a microalgae culture a cytochrome oxidase inhibitor at a concentration sufficient to induce triglyceride accumulation essentially without negatively affecting the growth rate of said microalgae.

According to certain embodiments, the inhibitor of cytochrome oxidase is selected from the group consisting of azide, cyanide and salts thereof.

According to certain embodiments, the cytochrome oxidase inhibitor is an azide salt selected from the group consisting of sodium azide, potassium azide, lithium azide and cesium azide. Each possibility represents a separate embodiment of the present invention. According to certain embodiments, the azide salt is sodium azide.

According to other embodiments, the inhibitor of cytochrome oxidase is a cyanide salt selected from the group consisting of potassium cyanide, sodium cyanide, lithium cyanide or cesium cyanide. Each possibility represents a separate embodiment of the present invention. According to certain typical embodiments, the cyanide salt is potassium cyanide.

There are over 300 species of microalgae that are known to accumulate triglyceride. To be used according to the teachings of the present invention, in addition to the capability to produce triglycerides, the algae should also be suitable for large scale commercial scale in terms of availability, required growth volume and additional growth conditions.

According to certain embodiments, the microalgae are of a species selected from the group consisting of Chlorella, Dunaliella, Nannochloropsis, Nannochloris, Scenedesmus, Tetraselmis, Crypthecodinium, Cyclotella, Navicula, Nitzschia, Phaeodactylum, Cylindrotheca, Skeletonema, Chaetoceros, Isochrysis, Pavlova, Emiliania, Rhodomonas, and Botryococcus.

According to certain embodiments, the microalgea are selected from the group presented in Table 1 hereinbelow. According to other embodiments, the microalgae are selected from the group consisting of Chlorella desiccata, Dunaliella primolecta, Phaeodactylum tricornutum, Pseudochlorococcum polymorphum, Chlorella vulgaris, Nannochloris atomus, Nanochloropsis salina, Chaetoceros muelleri, Dunaliella parva, Chlorella sorokiniana and Scenedesmus dimorphus. According to additional embodiments, the microalgae are selected from the group consisting of Nannochloropsis Oceania and Nannochloropsis gadiata. According to yet further typical embodiments, the microalga is Chlorella desiccata. Each possibility represents separate embodiment of the present invention.

Table 1 : Microalgae species used according to the teachings of the present invention

The potential of the cytochrome oxidase inhibitor to increase triglyceride (TAG) yield per algal mass can be easily maximized in various algal species by the skilled artisan based on what is known in the art and the teachings of the present invention. The present invention now shows that 17 out of the 19 examined species known to accumulate TAG are responsive to cytochrome oxidase inhibitor, and further shows that in 9 species the TAG yield was higher compared to the yield obtained under N- deprivation growth conditions. The optimum conditions for TAG production disclosed herein for the microalgae Clorella desiccata and Dunaliella tertiolecta can be used as departing point for finding the optimal conditions for other TAG producing species. Particularly, the present invention now discloses that high C0 ₂ concentrations of about 5% greatly increased azide induced TAG yield in these two microalgae species. Further optimization of C0 ₂ concentration for the currently examined species as well as for other species can increase further the TAG yield. In addition, combination of azide with high bicarbonate, the latter recently reported to increase TAG productivity (Cooksey K. et al. Science daily Nov. 17 2010) can further extend the azide induced TAG accumulation. Additionally or alternatively, combinations of azide salt and a heterotrophic medium (containing carbon source) or of azide and a shift to alkaline pH can further enhance TAG accumulation (Cooksey K. 2012, 2 ^nd Int. Conf. Algal Biomass & Biofuels, San Diego).

According to certain typical embodiments, the cytochrome oxidase inhibitor is sodium azide, added to the algae culture medium at a concentration range of from 1 μΜ to 5 mM, typically from 5 μΜ to 1 mM more typically from 20 μΜ to 100 μΜ. According to certain typical embodiments, the sodium azide is added at a concentration of about 20μΜ. Each possibility represents a separate embodiment of the present invention.

It is to be noted that the cost of sodium azide is low; calculation of its cost for induction of 1,000 liter culture, at a concentration of 100 μΜ is only about US$2 based on laboratory grade product. One may be concerned about the use of sodium azide, a highly toxic respiratory inhibitor in large-scale algal cultures, that may endanger both the working staff and the environment if not properly handled. The awareness concerning toxicity of azide has surged over the last 15 years due to the extensive utilization of azide for car airbag inflatable propellant. An automobile single airbag contains between 50 to 250 grams of azide and the total amount installed in USA vehicles alone is estimated as 500,000 tons. According to the teachings of the present invention, not only that the azide can be easily monitored and neutralized in the algae growth apparatus, its initial concentration is very low. Furthermore, azide in solution can be effectively neutralized by a hypochlorite treatment that is conventional for treatment of water for human use (Betterton et al. 2010. J. Hazard. Mat. 182:716-22) or by ozonation (Betterton E. A. and Craig D. 1999. J. Air Waste Manag. Assoc. 49: 1347- 1354). The reaction of azide with hypochlorite proceeds through a chlorine-azide intermediate and results in dissociation of azide into nitrogen gas. The reaction at pH 4- 7 is fast and complete within a few minutes. Application of hypochlorite for neutralization of azide after harvesting of microalgae cultures is an attractive treatment for recycling of the culture medium and its reutilization, since the hypochlorite also destroys contaminating bacteria, fungi, predators and intruding microalgae. Alternatively, anionic N ₃ ^~ in solution can be effectively neutralized by low concentrations of aqueous ozone within a few minutes. However, ozonation is more expensive than hypochlorite treatment. Sodium azide may give rise to hydrazoic acid which may escape during algae culturing to the open air. To reduce this escape, the pH of the culture is controlled and stabilized at pH of 7.5-8.0 or above. In any case, the half life of azide in air is very short (about 2 days) due to photolysis by UV light and destruction by reactive oxygen species (ROS) (Orlando J. J. et al. 2005. Environ Sci Technol 39: 1632-40).

The microalgae according to the teachings of the present invention are typically grown in a liquid culture. The culture, typically aqueous liquid culture, comprises all nutrient required for the growth of a particular microalgae species as is known in the art. According to certain embodiments, the culture id diluted about 2 fold before the inhibitor of cytochrome oxidase is added. Dilution can be with the same or different type of medium in the same culture apparatus or with the same or different type of medium by transferring the culture to additional culture apparatus.

The effect of cytochrome oxidase inhibitor on the enhancement of triglyceride accumulation is exemplified herein with azide as the cytochrome inhibitor. As described hereinabove, hitherto known methods for enhancing triglyceride production in algae are based on deprivation of nitrogen from the algae growth medium or blocking of nitrogen or other nutrient assimilation. The present invention shows significant differences in the physiological state and the metabolism of algae grown in N-deprived medium compared to algae grown in medium comprising cytochrome oxidase inhibitor, particularly sodium azide.

As exemplified hereinbelow, azide-treated cells showed elevated metabolic activities compared to cells grown in N-deprived medium as reflected by the rate of photosynthesis and respiration, higher chlorophyll contents, more developed chloroplasts and faster growth rates. In addition, azide treated cells showed higher dependence on light intensity. Without wishing to be bound by any specific theory or mechanism of action, the effect of light intensity on TAG accumulation suggests that in azide-treated cells TAG biosynthesis is energized predominantly by photosynthesis utilizing newly photo-assimilated C0 ₂ , whereas N-deprived cells may rely partly on carbon mobilization or on alternative C0 ₂ assimilation or energization pathways, such as respiration. The higher efficiency of TAG biosynthesis in azide-treated cells may further be due to the fact that the cells enter the phase of TAG biosynthesis at a better metabolic state compared to N-deprived cells, in which large part of the chloroplast has been degraded and energy metabolism is impaired. While the present invention clearly shows that the cytochrome oxidase inhibitor sodium azide enhances the overall triglyceride production by microalgae while having a minor effect on the algae growth rate, the mechanism by which azide induces TAG biosynthesis is not clear. Without wishing to be bound by any specific theory or mechanism of action, several alternative metabolic pathways, alone or in combination, may be involved in azide induced TAG accumulation. Azide and cyanide, both shown in the present invention to significantly enhance triglyceride accumulation, are inhibitors of cytochrome oxidase (Complex IV), the latter forming part of the respiration apparatus. However, as exemplified hereinbelow, other respiratory inhibitors, such as atpenin (an inhibitor of Complex II) and antimycin A (inhibitor of Complex II), did not induce a similar level of TAG biosynthesis, indicating that respiration inhibition cannot by itself result in enhanced TAG accumulation (Table 2)·

Table 2: Effect of respiratory inhibitors on TAG accumulation in C. desiccate

C. desiccata was grown for two days in vertical columns, supplied with 5% C(¾ air mixture.

The role of reactive oxygen species (ROS) in triggering the process was also examined by replacing azide with H2O2 or other ROS generators including benzyl viologen and Rose Bengal. The examined ROS generators induced at best a small increase in TAG biosynthesis (Table 3). In addition, ROS quenchers such as 1,2- Dihydroxy-benzene-3,5-disulfonic acid (TIR), Butylated hydroxytoluene (BHT), 1,3- Dimethylurea (DMU) and a-tocopherol failed to inhibit TAG accumulation in the presence of azide. However, azide-treated cells showed differential sensitivity to DABCO (l,4-Diazabicyclo[2.2.2]octane), known as specific singlet oxygen (SO) quencher (Table 4). Table 3: ROS generators tested in C. desiccate

C. desiccata was cultured for 2 days in culture flasks at ambient C(¾

Table 4: Effect of ROS quenchers on azide induced TAG accumulation in C. desiccata

C. desiccata was grown for two days with 20 μΜ Azide in vertical columns, supplied with 5% CO ₂ air mixture (H) or with 100 μΜ Az in culture flasks at ambient C(¾ (L). DMU- 1,3-Dimethylurea, BHT- Butylated hydroxytoluene, TIR- l,2-Dihydroxy-benzene-3,5-disulfonic acid, DABCO- 1,4- Diazabicyclo[2.2.2] octane .

Without wishing to be bound by any specific theory or mechanism of action, the finding that DABCO inhibited TAG accumulation, combined with the observation that heavy water, which prolongs the lifetime of SO eliminated the effect of DABCO, suggests that SO may be involved in azide, but not in N-deprivation induction of TAG biosynthesis. This possibility seems somewhat paradoxical in view of the known fact that azide itself is an effective quencher of SO (Hall R. D. and Chignell C. F. 1987. Photochem Photobiol 45(4):459-64; Li M. Y. et al. 2001. Photochem Photobiol 74(6):760-764). In fact, attempts to measure the level of singlet oxygen with the fluorescent indicator DanePy, showed that azide treatment decreased the level of SO in the cells, consistent with its quenching effect (data not shown). However, confocal microscopy of DanePy labeled cells showed that it concentrated in the cytoplasm and was excluded from the chloroplast in the azide-treated cells. Without wishing to be bound by any specific theory or mechanism of action, the effect of azide on stimulation of SO generation within the chloroplast may be more pronounced compared to its quenching capacity, resulting in an overall increase in singlet oxygen level. Inhibition of photosynthesis either directly (by inhibiting PSII and/or ATP synthase) or indirectly through inhibition of respiration, combined with inhibition of super oxide dismutase (SOD) and catalase, known to be leading to increase in ROS species which can be converted to SO in the chloroplast, support this possibility. Because the lifetime of SO is short, its diffusion distance is limited and it is conceivable that within the chloroplast the level of SO will be significantly higher compared to its concentration in the cytoplasm.

Another clear difference observed was the higher level of reactive oxygen species (ROS) or reactive nitrogen species (RNS) production, which in azide-treated cells was higher, whereas in N-deprived cells was lower compared to their level in control cells. Without wishing to be bound by any specific theory or mechanism of action, this difference may result in part from the higher photosynthetic and respiratory activities and in part from inhibition of the ROS neutralizing enzymes catalase and SOD observed in azide-treated cells. Also partial inhibition of PS-II and of the ATP synthase could contribute to overenergization of the photosynthetic system and to an increase in ROS generation. These results are consistent also with the higher observed sensitivity of azide-treated cells to high light intensity.

The effectiveness of TAG production by azide depends on the light intensity, the culture concentration and stage of growth. Azide is effective both under continuous illumination and under light/dark growth conditions. According to certain typical embodiments, azide should be added following a small (2-fold) dilution of the culture to a starting concentration of 10 ⁶ -10 ⁷ cells/ml. Addition of azide to stationary, very dense cultures, is typically less or ineffective. As exemplified herein, highest TAG induction was obtained in high C0 ₂ culture tubes at late-log starting cell densities. These results demonstrate that azide is effective also in relatively dense cultures, which is comparable to large scale culturing conditions.

In summary, the present invention provides a novel, simple to operate and highly efficient method for enhancing triglyceride production by algae. The method of the present invention is advantageous over the hitherto commonly used method for inducing triglyceride accumulation by nutrient starvation, particularly nitrogen starvation, at least in the lower effect of the cytochrome oxidase inhibitor on the algae growth rate.

Without wishing to be bound by any specific theory or mechanism of action, the mechanism of enhancement of triglyceride production by cytochrome oxidase inhibitor is significantly different from the effect of the hitherto commonly used method for inducing triglyceride accumulation by nutrient starvation, particularly nitrogen starvation, based on the folio wings: lower inhibition of metabolic activities such as photosynthesis and respiration were observed; the size and morphology of chloroplasts and the level of chlorophyll in cells treated with cytochrome oxidase inhibitor resemble control cells whereas in N-deprived cells the chloroplasts size is reduced and chlorophyll level decreases; triglyceride accumulation in cultures treated with cytochrome oxidase inhibitor show higher dependency on light intensity compared to N-deprived cultures; triglyceride induction by cytochrome oxidase inhibitor is associated with elevated oxidative stress whereas N-deprivation involves decreased oxidative stress; and singlet oxygen species are involved in cytochrome oxidase inhibitor, but not in N-deprivation induction of TAG biosynthesis. These observations clearly indicate that triglyceride induction by cytochrome oxidase inhibitor is the result of a combined mode of activities. According to additional aspect, the present invention provides a method for producing biodiesel from microalgae comprising utilizing lipids produced by the microalgae, wherein the microalgae are grown in the presence of cytochrome oxidase inhibitor at a concentration sufficient to enhance triglyceride accumulation within said microalgae essentially without negatively affecting the growth rate of said microalgae. According to some embodiments, the microalgae are grown in a liquid culture under autotrophic conditions. According to certain embodiments, the microalgae are grown in the liquid culture towards the mid to late logarithmic phase, and the culture is diluted at least two fold before addition of the cytochrome oxidase inhibitor.

According to other embodiments, the cytochrome oxidase inhibitor is added when the culture is at mid- to late logarithmic growth phase. Once a desired algae/triglyceride concentration is achieved, the lipid fraction can be extracted from the algae. Any known method for lipid extraction from algae can be used according to the teachings of the present invention. According to certain typical embodiments, the algae are separated from the medium before lipid extraction. As is known to a person skilled in the art, various methods can be used for separating the algae cells from the growth medium. Non-limiting examples include screening, centrifugation, rotary vacuum filtration, pressure filtration, hydrocycloning, flotation and gravity settling. Other techniques, such as addition of precipitating agents, flocculating agents, or coagulating agents, etc., can also be used in conjunction with these techniques. Two or more stages of separation can also be used. When multiple stages are used, they can be based on the same or a different technique. Non-limiting examples include screening of the bulk of the algal culture contents, followed by filtration or centrifugation of the effluent from the first stage.

According to certain embodiments, residual amounts of the cytochrome oxidase inhibitor that may be left in the culture medium after the algae are harvested are neutralized by any method as is known on the art. According to some embodiments, when the cytochrome oxidase inhibitor is sodium azide, hypochlorite is added in an amount sufficient to neutralize residual sodium azide.

The harvested cell mass can optionally be dried, using any method known in the art, including, but not limited to, freeze-drying, spray-drying and heat-drying, including drying under the sunlight.

Extraction of the lipids from the algal cell biomass can be performed by solvent extraction, including high pressure solvent extraction and Soxhlet extraction. When Soxhlet extraction is used, hexane is employed as the standard extraction solvent. The lipids are separated from the algae powder by washing repeatedly with hexane. The solvent can be removed by a reduced pressure distillation. The oil obtained from the algae grown according to the methods of the present invention is rich in triglycerides. Such oils may be converted into biodiesel using well- known methods, including methanol or ethanol transesterification, pyrolysis, gasification, or thermo chemical liquefaction. Typically, biodiesel is produced by trans- esterification of the triglycerides. The conversion from fatty acids to esters of fatty acids includes, but is not limited to the process catalyzed by acid, such as concentrated sulfuric acid, or lipase. The fatty acid methyl esters resulted from this catalyzed process forms the main component of biodiesel.

In addition to the triglyceride, the algae grown according to the teachings of the present invention may be further utilized per se as a proteinaceous feed source or additional by products may be extracted. As exemplified herein, in certain algae species enhancement of triglyceride production is accompanied with enhanced accumulation of starch within the algae cells.

Thus, according to certain embodiments, the present invention further provides a method for producing starch from microalgae comprising extracting starch produced by the microalgae, wherein the microalgae are grown in the presence of cytochrome oxidase inhibitor at a concentration sufficient to enhance starch accumulation within said microalgae essentially without negatively affecting the growth rate of said microalgae. Additional by products that may be produced from the alga biomass obtained according to the teachings of the present invention are pigments and vitamins.

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention. EXAMPLES Materials and Methods Chemicals

Sodium azide (NaN ₃ ) was purchased from Merck. CM-H ₂ DCFDA (5-(and 6)- chloromethyl-2', 7 '-dichlorodihydro fluorescein diacetate) (C6827) and DHR (Dihydrorhodamine 123) were purchased from Invitrogen Molecular Probes. Atpenin A5 was purchased from Enzo Life Sciences. All other chemicals were purchased from Sigma- Aldrich Chemicals.

Algal source and cultivation conditions Dunaliella tertiolecta was obtained from the culture collection of Dr. W. H.

Thomas (La Jolla, CA). Dunaliella bardawil Ben-Amotz and Avron is a local isolated species, American Type, Culture Collection, Rockville, Md. #30861. Dunaliella parva is a local isolate, obtained from Prof. B. Ginzburg at the Hebrew University, Jerusalem. Phaeodactylum tricornutum (strain CCMP632) was a gift from Dr. A. Vardi, Dep. of Plant Sciences at the Weizmann Institute. Chlamydomonas reinhardii (strains CW15 and cc-1009) were obtained from Prof. A. Danon, Dep. of Plant Sciences at the Weizmann Institute. C. desiccata (UTEXID LB2437) was obtained from The Culture Collection of algae at the University of Texas at Austin. All other microalgae species were obtained from the Culture Collection of Algae and Protozoa, SAMS Research Services Ltd., Scottish Marine Institute, Scotland, UK.

Dunaliella tertiolecta Dunaliella salina teodoresco, Dunaliella parva and Dunaliella bardawil were cultured in medium containing 5 mM KN0 ₃ , 5 mM MgS0 ₄ , 0.2 mM CaCl ₂ , 0.2 mM KH ₂ P0 ₄ , 1.5 μΜ FeCl ₃ +6 μΜ Na ₂ EDTA, 7 μΜ MnCl ₂ , 1 μΜ CuCl ₂ , 1 μΜ ZnCl ₂ , 1 μΜ CoCl ₂ , 1 μΜ (NH ₄ )6Mo ₇ 0 ₂₄ , 50 mM NaHC0 ₃ , 50 mM Na- Tricine pH 8 and 0.6-2 M NaCl.

Pseudochlorococcum polymorphum, Scenedesmus dimorphus, Chlorella sorokiniana and Chlorella zofingiensis were cultured in medium containing 14.6 mM NaN0 ₃ , 9.2 mM KH ₂ P0 ₄ , 4 mM MgS0 ₄ , 75 μΜ CaCl ₂ , 10 μΜ FeCl ₃ +20 μΜ Na ₂ EDTA, 0.77 μΜ ZnS0 ₄ , 0.31 μΜ CuS0 ₄ , 1.61 μΜ Na ₂ Mo0 ₄ , 46.3 μΜ H ₃ B0 ₃ , 9.15 μΜ MnCl ₂ , 0.172 μΜ Co(N0 ₃ ) ₂ , 2 mM NaHC0 ₃ and 50 mM Na-tricine pH 8.0 (the two last components were not used for C. zofingiensis) .

Phaeodactylum tricornutum was cultured in F/2 medium (Guillard R.R. and Ryther J.H., 1962 Can. J. Microbiol. 8:229-239) supplemented with 2 mM NaHC0 ₃ and 50 mM Na-tricine pH 8.0. Chlamydomonas reinhardii was cultured either in Tris/ acetate/ phosphate (TAP) medium (Gorman D.S. and Levine R.P. 1965. Proc. Natl. Acad. Sci. USA 54: 1665- 1669) or in modified TAP medium, containing 0.6 M N0 ₃ ^" as a sole nitrogen source and 50 mM Na-Hepes pH 7.5 (instead of Tris base) (for photo-heterotrophic growth), or in modified high-salt (HS) minimal medium (Sueoka N. 1960. Proc. Natl. Acad. Sci. USA 46(1):83-91), containing 1.9 M N0 ₃ ^" as a sole nitrogen source (for photoautotrophic growth).

Chlorella desiccata, Chlorella vulgaris, Nannochloris atomus, Nannochloropsis salina, Dunaliella primolecta and Chaetoceros muelleri were cultured in medium containing: 461 mM NaCl, 28.5 mM MgS0 ₄ , 29 mM MgCl ₂ , 10.2 mM CaCl ₂ , 10 mM KN0 ₃ , 0.4 mM KH ₂ P0 ₄ , 0.5 mM NaHC0 ₃ , 20 mM Tris-Cl pH 7.6, 113 μΜ Na ₂ Si0 ₃ , 3 μΜ FeCl ₃ +12 μΜ Na ₂ EDTA, 39 μΜ CuS0 ₄ , 26 μΜ Na ₂ Mo0 ₄ , 72 μΜ ZnS0 ₄ , 42 μΜ CoCl ₂ , 9 μΜ MnCl ₂ , Vitamin B _i2 - lmg/1, Biotin- 1 mg/1, Thiamin-HCl - 200 mg/1.

Microalgae were cultured under two conditions: Most species were grown in culture flasks on an orbital shaker at 100 rpm, illuminated continuously with fluorescent lamps with light intensity of 70 μΕ or under 16h light/8 h dark cycles. The temperature of the culture was maintained at 24°C and pH was not controlled.

C. desiccata, N. atomus, D. primolecta and Isochrysis galbana were also grown in vertical glass columns (3.7 cm diameter, 32 cm height) submerged in transparent water bath, illuminated continuously with fluorescent lamps with light intensity of 110 μΕ, and supplied with air/C0 ₂ 5% mixture bubbled through a capillary at the bottom of the column. The temperature of the culture was maintained at 24°C and pH was not controlled.

Cell harvest and transfer to nitrogen starvation

Cells were harvested by centrifuging at 3,000 rpm for 5 min at 4°C by Multifuge 3, Heraeus. For nitrogen starvation experiments, cultures were first grown for 2-3 days in complete growth media. Next, cells were washed once and then resuspended in nitrogen deficient medium. Cell concentration was determined by Z2 Coulter Particle Analyzer (Beckman Coulter) or by Cellometer Auto M10 (Nexcelom Bioscience).

Determination of lipid content

Nile red staining and Fluorescence Spectrometry

Nile red (NR, 9-diethylamino-5H-benzo[a]phenoxazine-5-one) was prepared as a stock solution in dimethylformamide (ImM) and stored in single-use aliquots at -20°C in the dark.

Cells were washed in fresh growth medium and resuspended at final concentrations of 5xl0 ⁵ -lxl0 ⁷ cells/ml, depending on the species. NR was added 3-10 min before the measurement, to a final concentration of 1 μΜ. Emission spectra at excitation and emission wavelengths of 488 nm and 520-750 nm were measured by Cary Eclipse Spectrophotometer (Varian, Australia Pty Ltd.) Intracellular neutral lipid content was estimated from the emission intensity at 585 nm of Nile red-stained cells.

Thin-Layer Chromatography (TLC)

Lipid extraction and analysis were determined according to Pushkar (Ph.D. thesis, 2005, Ben-Gurion University, Beer-Sheva). In brief, fixed number of cells (lxl 0 ⁷ - lxlO ⁸ ) were pelleted by centrifugation, heated for 5 min to 70°C with 200 DMSO, and mixed on vortex with 3ml methanol. After 15h incubation at 4°C, the pellet was removed by centrifugation, and the extract was supplemented with 3ml diethylether, 3ml N-hexane and 3ml distilled water. The mixture was vortexed, briefly centrifuged, and the upper phase was separated. The solvent was evaporated by vacuum; the pellet was dissolved in 200 μΐ chloroform and stored in teflon-sealed vials at -20°C. Half to ten μΐ of the extract were applied to TLC silica-gel plates (5 x 7.5 cm, 60 F ₂₅₄ , Merck), and developed in a closed jar in mixture of N-hexane: Diethylether: Acetic Acid (85: 15: 1, v:v:v). Lipid spots were visualized by 5 min incubation in iodine vapor (Merck). The plate was scanned by Image Scanner III, Epson Expression™ 10000 XL using scanning software LabScan™ 6.0 (Powered by Melanie, Swiss Institute of Bio informatics). TAGs were quantified by densitometry software ImageQuant™ TL. Non-aqueous reversed phase HPLC

Waters e2695 Separations Module (supplied with Empower Pro 2 Software) was connected through a Flow Splitter (DAN Technologies Services Ltd.) to Corona Charged Aerosol Detector (CAD) (Mercury Scientific & Industrial Products Ltd.) and to Waters Fraction Collector III. The chromatographic column Halo C8 (150 x 4.6 mm, 2.7 μιη) was used at 40 C. Samples were filtered through 0.2 μιη before HPLC analysis.

The HPLC protocol for TAG separation was as follow: flow rate 1.2 mL/min; mobile phase gradient: 2 min - 100% acetonitrile, 43 min - 100% acetonitrile, 46 min - 77%) acetonitrile - 23 %> z ^' so-propanol, 50 min - 77% acetonitril e- 23% z ^' so-propanol, 60 min - 100% acetonitrile (Holcapek M et al. 2005. J Sep Sci 28(12): 1315-1333). lxlO ⁸ - 2xl0 ⁸ cells of each sample were used for lipid extract preparation (as described for TLC). The lipid pellet was dissolved in an acetonitrile- zso-propanol- hexane mixture (2:2: 1, v/v/v). 10 μΐ of this solution was injected at IO C for HPLC analysis.

The HPLC protocol for fatty acids (FAs) and polar lipid separation included (according to Plante et al, Thermo Fisher Scientific, USA): flow rate 0.8-1 mL/min, and mobile phase gradient: 40 min - 100% A, 50 min - 30% A- 70% B, 60 min - 10% A- 90% B, 65 min - 10% A- 90% B, 73 min - 100% A, where A was methanol: water: acetic acid (750:250:4, v/v/v) and B was acetonitrile: methanol: tetrahydrofuran (THF): acetic acid (500:375: 125:4, v/v/v). 2xl0 ⁸ cells of each sample were used for lipid extract preparation (as described for TLC). Then, FA hydrolysis was performed: 1 mL of 10% KOH in EtOH were added to the lipid pellet, the samples were heated for 1 hour at 80 C, and then 0.5 mL HC1 35% and 2 mL Hexane were added. After the centrifugation, the upper phase was separated, and evaporated. The FA pellet was dissolved in 200 μΐ of a methanol/ chloroform mixture (1 : 1, v/v); 10 μΐ of this solution was injected at IO C for HPLC analysis.

TAG fraction

Fifty ml of cell culture were harvested, and total lipids were extracted and dissolved in 1ml chloroform, as described above. Triglyceride percent from total lipids was determined by Dr. Alicia Leikin Frenkel, Tel Aviv University, Sackler School of Medicine. Mass spectrometry

Molecular masses of TAGs from seven main groups in nitrogen-starved C. desiccata were determined by Mass-Spectrometry and Chemical Analysis Unit, Weizmann Institute of Science, Israel. Cell dry weight

For dry cell weight determination, 50 ml of cells were harvested, dried overnight at 110°C and weighted by Sartorius CP224S.

Oxygen evolution

Samples of cell suspension (2xl0 ⁷ cells/ml) in fresh growth medium were illuminated by Edmund Industrial Optics Model 21 AC, Stocker Yale, at maximal intensity (1000 μΕ). Oxygen evolution was measured by oxygen electrode connected to Oxycorder 401, Photon Systems Instruments, at 25°C. The measurements were controlled by Oxywin software.

Measurement of Reactive Oxygen Species (ROS) Stock solution of 5 mM Dihydrorhodamine 123 (DHR) in DMSO was prepared and stored at -20°C in the dark. Washed cells (2xl0 ⁷ cells/ml) were incubated for 0.5 h in the dark with 5 μΜ DHR. Then, the cells were washed, diluted to lxl 0 ⁷ cells/ml and exposed to different induction conditions: complete medium (+N), nitrogen deficient medium (-N) or +N with different chemicals: H ₂ 0 ₂ , NaN ₃ , Tiron (1,2-Dihydroxy- benzene-3,5-disulfonic acid, TIR) and DABCO (l ,4-Diazabicyclo[2.2.2]octane) for 15 h in the light. Finally, the cells were centrifuged, resuspended in PBS (x2) and their DHR (Ex: 488 nm, Em: 529 nm) and Chlorophyll (Ex: 435, Em: 685 nm) fluorescence was measured by a plate reader Tecan infinite M200. DHR fluorescence was normalized to Chlorophyll fluorescence. Microscopy

Nile red staining of lipid globules

Cells were washed, resuspended in fresh growth medium (Ixl0 ⁶ -5xl0 ⁶ cells/ml), and incubated for 3 min with 1 μΜ NR. Ten μΐ of samples were applied to a glass slide without cover. Motile cells were supplemented with 2.5% glutaraldhyde. Stained cells were viewed and photographed by laser-scanning confocal microscope FluoView FV10-ASW Olympus, using Olympus FluoviewFV 1000 software. Laser excitation for NR was 488 nm, emission filter 560-590 nm. For chlorophyll, excitation was 635 nm, and emission 655-755 nm. Transmission electron microscopy

Cells were chemically fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer pH 7.3, 5 mM CaCl ₂ and isotonic NaCl for lh at 24°C. Then the fixative solution was replaced by fresh fixative and the cells were incubated for 24 h at 4°C. Cells were washed again with 0.1 M cacodylate buffer and isotonic NaCl, and post-fixed for 2h in 0.1 M cacodylate buffer, 1% Os0 ₄ , 5 mM CaCl ₂ , 0.5% potassium dichromate (K ₂ Cr ₂ 0 ₇ ), 0.5%> potassium hexacyonaferrate (l [Fe(CN)6] and 0.5%> potassium ferrocyanide. The staining was done by 2% uranyl acetate at room temperature for 2h. Then, the sample was dehydrated in ethanol 50-100% and embedded in Epon. After polymerization at 60°C, sections with thickness of 60-80 nm were prepared and stained with lead nitrate and lead citrate (Reynold protocol). The sample was examined by a Tecnai T12 electron microscope (FEI, Eindhoven, the Netherlands) operating at 120 kV. Images were recorded with an Eagle 2k x 2k CCD camera (FEI).

Cryo-scanning electron microscopy Cells were centrifuged and a drop of pellet was sandwiched between two aluminum platelets with depth of 25 μιη each. The sample was then high-pressure frozen in a HPM010 high-pressure freezing machine (Bal-Tec, Liechtenstein). The frozen sample was mounted on a holder and transferred to a BAF 60 freeze fracture device (Bal-Tec) using a VCT 100 Vacuum Cryo Transfer device (Bal-Tec). After fracturing at a temperature of -130°C, the sample was coated with 5nm Pt/C by double axis rotary shadowing. Samples were transferred to an Ultra 55 SEM (Zeiss, Germany) using a VCT 100 and were observed using a secondary electrons in-lens detector at 2.5 kV at a temperature of -130°C. Example 1: Accumulation of triglyceride (TAG) in microalgae grown in the presence of sodium azide

The effect of sodium azide (NaN ₃ , Az) on triglyceride (TAG) accumulation was examined in 19 different algae species, belonging to different phylogenetic groups and known to accumulate moderate to high levels of triglycerides. The presence of azide in the growth medium induced TAG accumulation in 17 out of the 19 tested species. Only Chlamydomonas reinhardtii and Chlorella zofingiensis did not respond to azide in TAG accumulation.

As is shown in Tables 5 and 6 hereinbelow, the effect of azide varied in the degree of TAG induction and in respect to the optimal azide concentration needed for maximal induction. In 9 out of the 17 species, the lipid yield was higher than in N- deprived cells. The optimal azide concentration varied from about 10-20 μΜ for most species to about 1 mM for the hypersaline Dunaliella species (D. parva, D. bardawil). Higher than optimal azide concentrations lead to growth inhibition and decrease in lipid recovery.

Table 5: Algae species showing best TAG yield

Table 6: Effect of azide on TAG accumulation in different algae species

Species Phylum Culturing Fold Fold Azide Azide

Conditions increase increase Optimal Effective

Az/ +N Az/-N Cone. Range

Chlorella desiccata Chlorophyta 2, b 83 3.1 20 μΜ 10-50 μΜ

(Green)

Dunaliella Chlorophyta 2, b 5.2 1.2 10 μΜ 10-50 μΜ primolecta (Green)

Chlorella sorokiniana Chlorophyta 3, a 3.9 1.2 100 μΜ 20-100

(Green) μΜ

Chlorella vulgaris Chlorophyta 3, a 5.2 0.3 20 μΜ

(Green)

tr cornutum atom μ

Growth Conditions:

Culturing Media

1) Dunaliella medium: 5 mM KN0 ₃ , 5 mM MgS0 ₄ , 0.2 mM CaCl ₂ , 0.2 mM KH ₂ P0 ₄ , 1.5 μΜ+6 μΜ FeCl ₃ +Na ₂ EDTA, 7 μΜ MnCl ₂ , 1 μΜ CuCl ₂ , 1 μΜ ZnCl ₂ , 1 μΜ CoCl ₂ , 1 μΜ (ΝΗ ₄ ) ₆ Μο ₇ 0 ₂₄ , 50 mM NaHCC-3, 50 mM Na-Tricine pH 8 and 0.6-2 M NaCl.

2) Artificial Sea Water (ASW) medium: 461 mM NaCl, 28.5 mM MgS0 ₄ , 29 mM MgCl ₂ , 10.2 mM CaCl ₂ , 10 mM KN0 ₃ , 0.4 mM KH ₂ P0 ₄ , 0.5 mM NaHC0 ₃ , 20 mM Tris-Cl pH 7.6, 113 μΜ Na ₂ Si0 ₃ , 3 μΜ FeCl ₃ +12 μΜ Na ₂ EDTA, 39 μΜ CuS0 ₄ , 26 μΜ Na ₂ Mo0 ₄ , 72 μΜ ZnS0 ₄ , 42 μΜ CoCl ₂ , 9 μΜ MnCl ₂ , Vitamin B ₁₂ - lmg/1, Biotin- 1 mg/1, Thiamin-HCl - 200 mg/1.

3) Fresh water medium: 14.6 mM NaN0 ₃ , 9.2 mM KH ₂ P0 ₄ , 4 mM MgS0 ₄ , 75 μΜ CaCl ₂ , 10 μΜ FeCl ₃ +20 μΜ Na ₂ EDTA, 0.77 μΜ ZnS0 ₄ , 0.31 μΜ CuS0 ₄ , 1.61 μΜ Na ₂ Mo0 ₄ , 46.3 μΜ H ₃ B0 ₃ , 9.15 μΜ MnCl ₂ , 0.172 μΜ Co(N0 ₃ ) ₂ , 2 mM NaHC0 ₃ and 50 mM Na-tricine pH 8.0.

4) F/2 medium (Guillard R.R.L. and Ryther J.H., 1962 Can. J. Microbiol. 8:229-39) supplemented with 2 mM NaHC0 ₃ and 50 mM Na-tricine pH 8.0.

Culturing setup

a) The algae were grown in culture flasks on an orbital shaker at 100 rpm, illuminated continuously with fluorescent lamps with light intensity of 70-90 μΕ. The temperature of the culture was maintained at 24°C and pH was not controlled.

b) The algae were grown in vertical glass columns (3.7 cm diameter, 32 cm height) submerged in transparent water bath, illuminated continuously with fluorescent lamps with light intensity of 110 μΕ, and supplied with 5% C0 ₂ /air mixture bubbled through a capillary at the bottom of the column. The temperature of the culture was maintained at 24°C and pH was not controlled. c) The algae were grown in culture flasks illuminated continuously with fluorescent lamps with light intensity of 80 μΕ or under 16h light/8 h dark cycles. The temperature of the culture was maintained at 20°C and pH was not controlled.

Example 2: Accumulation of TAG in Chlorella desiccata

Chlorella desiccata is marine unicellular green alga that under nitrogen deprivation accumulates high levels of triglycerides (TAG) and low levels of starch. Addition of 20 μΜ sodium azide to the growth medium of this algae induced massive accumulation of TAG, similar to the level obtained in N-deprived cells (Fig. 1 , A-D). However, in contrast to cells grown in N-deprived medium, only minor inhibition in growth rate was observed in the presence of azide (Fig. IE). The level of TAG accumulation was estimated by three different methods: increase in fluorescence of the neutral lipid indicator Nile red (NR); TLC separation and staining of TAG in lipid extracts; and resolution of lipid extracts by reverse-phase HPLC (Figs. 1A, IB and 1C, respectively). The level of TAG accumulation for the same number of cells was found to be similar in N-deprived and in azide -treated cells, amounting to 20-30 fold increase with respect to cells grown in complete growth medium (no induction) (Fig. 1C).

The level and rate of TAG accumulation was found to depend on azide concentration and was stimulated up to 20 μΜ azide, but suboptimal at higher concentrations. In comparison to N-deprived cells, the initial rate of TAG accumulation was faster in azide treated cells, but started to decline after 2-3 days. However, unlike N-deprivation, which inhibited the growth by about 70%, cells grown in the presence of 20 μΜ azide reached the same cell density as control cells within 3-4 days, after a transient growth delay (Fig. ID, E). Consequently, the overall lipid yield of azide-treated cultures was about 60%> higher compared to N-starved cells, as measured by NR quantification and cell concentration (Fig. 2A). Even after careful optimization of lipid productivity by N- limitation, obtained by dilution into low nitrate medium instead of complete N- elimination, the lipid productivity of azide-treated cells, defined as the lipid level per ml culture volume per day, was about twice as high as in N-deprived cells (Fig. 2B). As is shown in Tables 1 and 2 hereinabove, this effect of sodium azide was not restricted to Chlorella desiccata and was shown for 17 species out the 19 species examined. Example 3: Optimization of growth conditions

Photo synthetic and respiratory activities of cell cultures grown under different conditions were examined. Photosynthetic and respiratory activities were measured by the light-dependent oxygen evolution and dark oxygen uptake. As shown in Fig. 3, azide treatment inhibited in parallel photosynthesis and respiration, progressively with increasing azide concentrations. Catalase activity determined by H ₂ 0 ₂ hydrolysis was inhibited in parallel. However, in comparison to N-deprived cells, cells treated with 20 μΜ azide retained significantly higher activities (about 25% and 50%, respectively, compared to control untreated cells). Notably, cultures treated with 20 μΜ azide were visually much greener and contained higher chlorophyll levels than N-deprived cells (not shown).

Comparison of the effect of light intensity on TAG level in N-deprived and azide- treated cultures showed that the latter are more light-dependent: at light intensities lower than 60 μΕ, the lipid content of azide-treated cells was lower than in N-deprived cells, whereas high light of 300 μΕ, strongly inhibited azide-treated, but not N-deprived cultures (Fig. 4). Without wishing to be bound by any specific theory or mechanism of action, these results suggest that azide-treated cells are more dependent on photosynthesis for TAG biosynthesis than N-deprived cells.

The effectiveness of azide induction was shown to be depended on the cell density and/or growth phase. For maximal induction of TAG, azide was added to relatively dilute algae cultures, at mid to late-logarithmic growth phase.

The effect of C0 ₂ on TAG accumulation was examined by comparing the TAG yield of two azide-treated microalgae species, cultured either at ambient C0 ₂ or supplemented with 5% C0 ₂ . As shown in Fig. 5, C0 ₂ supplementation increased TAG yield by at least 5-fold in both species. However, whereas the increase in C. desiccata was mostly due to the higher cell number, in N. atomus it reflected partly an increase in cellular TAG level. Interestingly, high C0 ₂ also seems to sensitize the cells to azide: at low C0 ₂ in C. desiccata, at least 100 μΜ azide were required to increase TAG yield in comparison to 20 μΜ azide in high C0 ₂ (Fig 5 A, B; Fig. 1 D, E), and in N. atomus, 100 μΜ Az inhibited growth and TAG yield far more at high C0 ₂ than at ambient C0 ₂ (Fig. 5C and 5D). Example 4: Microscopic characterization of TAG globules in C. desiccata and D. tertiolecta

Two species were selected for microscopic analysis comparison of N-depleted and azide-treated cells: C. desiccata, which accumulates high levels of TAG and low starch, and D. tertiolecta, which accumulates high levels of starch and moderate TAG.

Control untreated cells; N-deprived cells; and azide treated cells were analyzed by three microscopic methods: Confocal fluorescence microscopy of NR-stained cells, transmission electron microscopy and scanning electron microscopy.

C. desiccata N-deprived and azide-treated cells showed similar levels of cytoplasmic NR stained lipid bodies (Fig. 6a, panels B, C) which occupy most of the cell area (Fig. 6a, panels E, F). The main observed difference between the two treatments was the higher level of chlorophyll fluorescence and larger chloroplast area in azide-treated cells (Fig. 6a, panels B, C).

In D. tertiolecta, the number and size of lipid globules is also similar in both treatments (Fig 6b, panels B, C), but they are smaller and fewer than in C. desiccata, and instead large starch bodies can be seen in the TEM preparation (Fig. 6b, panels E, F). Also in D. tertiolecta, the size of the chloroplast in azide-treated cells, indicated by chlorophyll fluorescence (Fig. 6b, panels B, C), is larger than in N-deprived cells. These results are consistent with the higher photosynthetic activity and growth rate of azide- treated cells compared to N-deprived cells.

Example 5: Fatty acid composition and TAG molecular species

Comparison of fatty acid composition and TAG contents in N-deprived and in azide-treated C. desiccata cell extracts, showed that both treatments yield similar fractional levels of TAG, amounting to about 80% of the lipids contents in the cells (Fig. 7A). Analysis of the major fatty acid composition in comparison to control cells, showed that both preparations are highly enriched in fatty acids 18:1 and 16:0 (Fig. 7B), the major fatty acids in lipid globules in many green algae species. A noticeable difference in the fatty acid composition between the two preparations is the higher content of fatty acid 18:3, the major thylakoid fatty acid, in the azide-treated cells. This finding is consistent with the higher level of chlorophyll and more developed chloroplasts in these cells compared to N-deprived cells. Example 6: Possible modes of action for azide-induced TAG accumulation

Azide is a well-known inhibitor of several haeme proteins and metalloenzymes. Azide thus has many potential targets in photosynthetic organisms, including: respiration (for example Fei M. J. et al. 2000. Acta Crystallogr D Biol Crystallogr, 56(Pt 5):529-35); photosystem II (Yu H. et al. 2005. Biochemistry 44(36): 12022-12029); ATPases (for example, Bowler M. W. et al. 2006. Proc Natl Acad Sci U.S.A 103(23):8646-8649); superoxide dismutase and catalase (Nicholls P. 1964. Biochem J 90(2):331-343).

However, its primary and best characterized targets in most living cells are the cytochrome oxidase in the respiratory system and catalase/superoxide dismutase. The effect of azide on respiration and on catalase activities was examined in vivo by following dark 0 ₂ consumption and H ₂ 0 ₂ dependent 0 ₂ production, respectively (Fig. 3). Increasing azide concentrations progressively inhibited light-dependent 0 ₂ evolution, dark respiratory 0 ₂ consumption and catalase activity to similar extents. Notably, azide concentrations which induced maximal TAG accumulation (20-50 μΜ), inhibited these activities by about 50%. For comparison, N-deprivation inhibited these activities by about 80%.

Different respiratory inhibitors were examined for their effects on cell growth and on TAG level. Specific respiratory inhibitors of Complex II (Atpenin) or of Complex III (Antimycin A) did not induce massive TAG accumulation at growth-inhibitory concentration, whereas KCN, which resembles azide in inhibiting Complex IV at similar concentrations, led to a partial TAG accumulation and to higher growth inhibition compared to azide (Fig. 9B, Table 2).

The effect of azide on the level of reactive oxygen species (ROS) in C. desiccata, was examined utilizing two fluorescent ROS (and reactive nitrogen species, RNS) detectors, CM-H ₂ DCFDA (not shown) and DHR (Fig. 9A). Both probes showed that azide induced enhanced ROS generation, and that the ROS level can be decreased by the ROS quencher TIR but not by the singlet oxygen (SO) quencher DABCO. None of the ROS or SO generators (Benzyl viologen (BV) and Rose Bengal (RB), respectively) induced significant increase in TAG level (Fig. 9B, Table 3). The effect of DABCO, a specific SO quencher, on TAG level was further examined. As shown in Fig. 10, DABCO significantly inhibited TAG accumulation in azide-treated cells but not in N- deprived cells, and the effect increased with time from about 30% to 70% inhibition after 2 and 4 day of induction, respectively (data not shown). To further test if SO species may be involved in TAG induction, C. desiccata was cultured in 30%> D ₂ 0, conditions that do not inhibit growth or photosynthesis, but are known to increase the life-time and therefore the effective concentration of SO. As shown in Fig. 11, azide- treated cells in 30% D ₂ 0 indeed had a slightly higher TAG level than cells in water, and more significantly, DABCO, which largely inhibited TAG level in azide-treated cells in water, hardly affected it in 30%> D ₂ 0. Without wishing to be bound by any specific theory or mechanism of action, these results suggest that SO might be involved in triggering TAG biosynthesis in azide-treated cells.

The method of the present invention is advantageous over hitherto known methods for commercial production of biodiesel in that higher productivity is achieved; smaller culture volumes are needed; and TAG production can be efficiently controlled. Additional advantages of this method in comparison to mineral limitations (particularly N deprivation) is a higher protein content of the product which is a valuable by-product for animal/fish feed.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the invention.