The present invention provides bicarbonate containing and/or bicarbonate-producing compositions and methods to induce lipid accumulation in an algae growth system, wherein the algae growth system is under light-dark cycling condition. By adding said compositions at a specific growth stage, said methods lead to much higher lipid accumulation and/or significantly reduced total time required for accumulating lipid in the algae growth system.
Goverment Interests

GOVERNMENT RIGHTS STATEMENT

[0002] This invention was made with government support under grant number DGE 0654336, awarded by National Science Foundation; grant number FA9550-09-1-0243, awarded by Air Force Office of Scientific Research; and grant number DE-FG36-08GO18161, awarded by US Department of Energy Office of Biomass Programs. The government has certain rights in the invention. Claims

1. A method of inducing lipid accumulation in an algae growth system, wherein the algae growth system is under light-dark cycling, comprising adding a composition when algae cells in the growth system are in exponential growth stage, but prior to nutrient depletion of the growth system, wherein the composition comprises bicarbonate, and/or one or more compounds that provide bicarbonate after the composition is added into the algae growth system.

2. The method of claim 1, wherein algae replication in the growth system is inhibited at the time when the composition is added.

3. The method of claim 2, wherein the inhibition happens during late phase of the exponential growth of algae in the system.

4. The method of claim 1, wherein the composition further comprises one or more agents that can increase the pH of the algae growth system.

5. The method of claim 2, wherein the inhibition comprises an aeration shift of CO.sub.2 concentration in the growth system from high to low, wherein the shift is sufficient to inhibit algae replication.

6. The method of claim 1, wherein the algae is a Scenedesmus sp., a Chlorophyta sp. or a diatom.

7. The method of claim 1, wherein the composition increases the pH of the algae growth system to at least pH 9.5.

8. The method of claim 1, wherein the composition provides the algae growth system a concentration of bicarbonate of at least 10 mM.

9. The method of claim 1, wherein the composition is added into the growth system as close to nutrient depletion of the growth system as possible.

10. The method of claim 1, wherein the lipid is triacylglycerol (TAG).

11. The method of claim 1, wherein the lipid accumulation in the algae growth system is at least 5 times the lipid accumulation in a control algae growth system.

12. A method of reducing total time required for producing lipid at a predetermined yield from an algae growth system, wherein the algae growth system is under light-dark cycling, comprising adding a composition when algae cells in the growth system are in exponential growth stage, but prior to nutrient depletion of the growth system, wherein the composition comprises bicarbonate, and/or one or more compounds that provide bicarbonate after the composition is added into the algae growth system.

13. The method of claim 12, wherein algae replication in the growth system is inhibited at the time when the composition is added.

14. The method of claim 13, wherein the inhibition happens during exponential growth of algae in the system.

15. The method of claim 12, wherein the composition comprises one or more agents that can increase the pH of the algae growth system.

16. (canceled)

17. The method of claim 12, wherein the algae is a Scenedesmus sp., a Chlorophyta sp. or a diatom.

18. The method of claim 12, wherein the composition increases the pH of the algae growth system to at least pH 9.5.

19-25. (canceled)

26. A method of inducing lipid accumulation in an algae growth system, wherein the algae growth system is under light-dark cycling, comprising adding a composition when algae cells in the growth system are in stationary growth stage after nutrient depletion of the growth system, wherein the composition comprises an inorganic nitrogen source and/or an organic nitrogen source with bicarbonate and/or one or more compounds that provide bicarbonate after the composition is added into the algae growth system.

27. The method of claim 26, wherein the inorganic nitrogen source is selected from a group comprising ammonium (NH.sub.4.sup.+) containing compounds, nitrite (NO.sub.2.sup.-) containing compounds, and nitrate (NO.sub.3.sup.-) containing compounds.

28. The method of claim 26, wherein the organic nitrogen source is selected from a group comprising urea, hypoxanthine, guanine, ornithine, glucosamine, and lysine. Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Ser. No. 61/386,260, filed Sep. 24, 2010, and U.S. Provisional Application Ser. No. 61/434,675, filed Jan. 20, 2011, which are herein incorporated by reference in their entirety.

TECHNICAL FIELD

[0003] This application relates to compositions and methods to induce lipid accumulation in algal systems.

BACKGROUND

[0004] Biodiesel, diesel fuel made from a biological source such as microalgal oil or soybean oil, is an important alternative to petroleum derived diesel. Algae are potentially a superior source of oil than plants such as soybeans because they are theoretically capable of producing more oil per acre of land/space required to grow them: for example, algae could theoretically produce 20-times the oil per acre than soybeans. Algae can be grown on non-agricultural land using little water, avoiding controversy over the diversion of food and food-production resources to fuel production when there are starving people in the world.

[0005] Triacylglycerol (TAG) is the precursor molecule of biodiesel, defined as fuel comprised of fatty acid methyl esters, and algal growth systems have the potential to produce TAG to a larger degree than land-based plant systems. Furthermore, particular fatty acid chains are desirable for altering the physical characteristics of biodiesel (e.g. gel point) or for secondary market use (e.g. nutraceutical market--omega-3 fatty acids).

SUMMARY OF THE INVENTION

[0006] The present invention provides methods of inducing lipid accumulation in an algae growth system. In some embodiments, the methods comprise adding one or more composition into the algae growth system, wherein the composition can influence the ability of an algae cell to complete a growth cycle, but is not completely toxic and does not result in cell death. In some embodiments, the composition comprises bicarbonate, and/or one or more compounds that provide bicarbonate after the composition (i.e., a bicarbonate containing and/or bicarbonate producing composition) is added into the algae growth system.

[0007] The present invention is based on the instant discovery that addition of bicarbonate (HCO.sub.3), at key growth points, is a metabolic trigger for inducing accumulation of TAG in algal growth systems. Furthermore, by using this bicarbonate trigger, TAG is produced to a much larger degree (tenfold so far) and total culture time is decreased (by several days) when compared to non-triggered systems. Therefore, the present invention discloses compositions and methods of utilizing this triggering mechanism, which has strong industrial application in biofuel production or secondary market production when algal growth systems are used.

[0008] In some embodiments, the algae growth system is under light-dark cycling conditions or under continuous dark conditions. In some embodiments, said methods comprise adding a composition when algae cells in the growth system are in exponential growth stage, but prior to nutrition depletion of the growth system and/or environmental factor changes of the growth system which induces lipid accumulation. In some embodiments, the lipid accumulation comprises TAG accumulation. In some embodiments, the nutrition is nitrate, ammonia/ammonium, urea, phosphate, sodium thiosulphate, silica, iron, or a combination thereof. In some embodiments, the environmental factor is concentration of CO.sub.2, pH, light (e.g., light intensity, light quality, light compositions etc), temperature, or a combination thereof.

[0009] In some embodiments, algae cell growth/replication in the growth system is inhibited before, at the time, or after adding the composition. In some embodiments, the inhibiting step happens during exponential growth of algae in the system.

[0010] In some embodiments, the composition comprises bicarbonate, and/or one or more compounds that provide bicarbonate after the composition (i.e., a bicarbonate containing and/or bicarbonate producing composition) is added into the algae growth system. In some embodiments, the composition further comprises one or more compounds that can increase the pH of the algae growth system. In some embodiments, the composition further comprises an inorganic nitrogen source (e.g., ammonium (NH.sub.4.sup.+) containing compounds, nitrite (NO.sub.2.sup.-) containing compounds, and nitrate (NO.sub.3.sup.-) containing compounds) and/or an organic nitrogen source (e.g., urea, hypoxanthine, guanine, ornithine, glucosamine, and lysine).

[0011] In some embodiments, the inhibiting step comprises an aeration shift of CO.sub.2 concentration in the growth system from high to low, wherein the shift is sufficient to inhibit algae cell growth/replication partially, or completely. In some embodiments, the CO.sub.2 concentration in the system after the shift from high to low is about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 5%, about 1%, about 0.1%, about 0.01%, about 0.001%, or less of the CO.sub.2 concentration before the shift. The aeration shift of CO.sub.2 concentration in the growth system from high to low is not required if the bicarbonate trigger is used for an ambient air sparged culture. Under such circumstances, the addition of bicarbonate inhibits algal replication.

[0012] In some embodiments, the algae are selected from the group consisting of Scenedesmus spp., diatoms (e.g., pennate diatoms), Botryococcus spp. (e.g., B. braunii), Chlorella, Dunaliella spp. (e.g., D. tertiolecta), Gracilaria, Pleurochrysis (e.g., P. carterae), Chlorophyta, and Sargassum. In some further embodiments, the Scenedesmus sp. is Scenedesmus WC-1 (WC-1) strain, the Chlorophyta is Chlorophyta sp. EN-2 (EN-2), and the diatom is diatom RGd-1 (RGd-1). In other embodiments, the algae may be Scenedesmus spp., diatoms (e.g., pennate diatoms), Botryococcus spp. (e.g., B. braunii), Dunaliella spp. (e.g., D. tertiolecta), Gracilaria, Pleurochrysis (e.g., P. carterae), Chlorophyta, and Sargassum but not Chlorella.

[0013] In some embodiments, the bicarbonate containing and/or bicarbonate-producing composition further comprise one or more agents that can increase the pH of the algae growth system. In some embodiments, the composition increases the pH of the algae growth system to at least 6.0, at least 6.5, at least 7.0, at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 10.5, at least 11.0, or more. The optimum pH for any one particular algal growth system may be different based upon the particular genera and/or particular species or strain of algae used in that particular growth system. In some embodiments, after the bicarbonate containing and/or bicarbonate-producing composition has been added to the algae growth system, the pH of the algae growth system may be lowered by addition of an acid. In these embodiments, addition of the bicarbonate containing and/or bicarbonate-producing composition inhibits replication of the algae cells, and after the pH is lowered, the algae cells may deplete the remaining nitrate and/or silica in the culture medium. In certain embodiments, the acid added to the algae growth system may be carbonic acid or a carbonic acid-producing composition. The algae may need CO.sub.2 to increase TAG, but bicarbonate can be used to supply the dissolved inorganic carbon (DIC) and arrest replication in the algae.

[0014] In some embodiments, the bicarbonate containing and/or bicarbonate-producing composition provides the algae growth system a concentration of bicarbonate of at least 1 mM, at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM or more. The optimum concentration of bicarbonate for any one particular algal growth system may be different based upon the particular genera and/or particular species or strain of algae used in that particular growth system.

[0015] In some embodiments, the bicarbonate containing and/or bicarbonate-producing composition is added before, at the time, or after nutrient depletion or growth condition changes that inhibit algae replication. In some embodiments, the bicarbonate containing and/or bicarbonate-producing composition is added as close to nutrient depletion as possible. In some embodiments, the bicarbonate containing and/or bicarbonate-producing composition is added into the growth system right before or right after nutrient depletion. In some embodiments, the bicarbonate containing and/or bicarbonate-producing composition is added together with an inorganic nitrogen source and/or an organic nitrogen source after nutrient depletion. In some embodiments, the bicarbonate containing and/or bicarbonate-producing and/or inorganic nitrogen source and/or organic nitrogen source composition is added into the growth system about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about half hour, about one hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, about 120 hours, or more before or after nutrient depletion of the growth system. Nutrient depletion time can be determined by running a control algae growth system without adding the composition. The optimum time point in which to add the bicarbonate and/or bicarbonate-producing composition to any one particular algal growth system may be different based upon the particular genera and/or particular species of algae used in that particular growth system.

[0016] In some embodiments, the lipid is triacylglycerol (TAG). In some embodiments, the lipid accumulation in the algae growth system is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more of the lipid accumulation in a control algae growth system.

[0017] The present invention also provides methods of reducing total time required for producing lipid at a predetermined yield from an algae growth system. In some embodiments, the algae growth system is under light-dark cycling. In some embodiments, said methods comprise adding a composition when algae cells in the growth system are in exponential growth stage, but prior to nutrient depletion of the growth system, wherein the composition comprises bicarbonate, and/or one or more compounds that provide bicarbonate after the composition is added into the algae growth system.

[0018] In some embodiments, algae cell growth/replication in the growth system is inhibited before, at the time, or after adding the bicarbonate and/or bicarbonate-producing composition.

[0019] In some embodiments, the total time required for producing lipid at a predetermined yield is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more, compared to the total time required for a control algae growth system.

[0020] The present invention also provides bicarbonate and/or bicarbonate-producing compositions for said methods. In some embodiments, the bicarbonate and/or bicarbonate-producing compositions comprise bicarbonate, and/or at least one source that can provide bicarbonate when added into the algae growth system. In some embodiments, the bicarbonate and/or bicarbonate-producing compositions are. in solid form. In some embodiments, the bicarbonate and/or bicarbonate-producing compositions are in liquid form. In some other embodiments, the bicarbonate and/or bicarbonate-producing compositions are in solid-liquid mixture form.

[0021] In some embodiments, the source for bicarbonate in the bicarbonate and/or bicarbonate-producing compositions is a bicarbonate salt. For example, the source is sodium bicarbonate, potassium bicarbonate, ammonium bicarbonate, aminoguanidine bicarbonate, choline bicarbonate, magnesium bicarbonate, or combination thereof. In some embodiments, the composition further comprises one or more agents that can increase the pH of the algae growth system.

[0022] The present invention also provides methods of increasing lipid accumulation in an algae growth system compared to a control algae growth system. A control algae growth system here is a growth system without or with little bicarbonate (i.e., a system having less than 0.001 mM of bicarbonate, or a system having less than 0.01 mM of bicarbonate) at the beginning and during algae growth. In some embodiments, the methods comprise growing algae in a medium comprising bicarbonate and/or a bicarbonate-producing composition at the starting point. In some embodiments, the concentration of bicarbonate in the medium initially is at least 1 mM, at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 150 mM, at least 200 mM, at least 250 mM, at least 300 mM, or more. In some embodiments, the algae are diatoms. In some further embodiments, the diatom is diatom Phaeodactylum tricornutum Pt-1 (Pt-1). In some embodiments, the medium is based on ASP II medium.

[0023] As discussed further herein, the inventors of the present invention have discovered that adding a solution containing bicarbonate ions to a growing algal culture prevents further growth but enhances the cell's ability to accumulate triacylglyceride. In some embodiments, to obtain maximum enhancement the bicarbonate must be added before the algal cells have utilized one, more than one, or all the available culture limiting nutrients in the medium and by increasing the pH, for example, by increasing the pH to reach a value greater than pH 10.0. Adding bicarbonate when the medium nitrogen has been utilized will not allow for maximum enhancement. This strongly implies that protein synthesis is required to obtain the desired effect. It is important to realize that this phenomenon is most likely not TAG-specific but the enhancement of product formation may be general. Not only does this invention have major ramifications for industrial production of algal material, but the finding allows "gene activity leading to desired product formation" to be monitored specifically.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 depicts average cell density, medium pH, and Nile Red Fluorescence of Scenedesmus WC-1 grown on 14:10 L:D cycle in unbuffered Bold's Basal Medium, ambient air aeration. Error is standard deviation. n=3

[0025] FIG. 2 depicts Scenedesmus WC-1 Nile Red fluorescence of three biological replicates ( , .box-solid., and .tangle-solidup.) and average medium pH (.quadrature.) monitored over 24 hours during late exponential growth (7-8 d) and prior to medium nitrate depletion in unbuffered Bold's basal medium under ambient aeration batch culturing. Bar indicates light and dark times during the 14:10 L:D cycle. n=3

[0026] FIG. 3 depicts average cell density and Nile Red Fluorescence of Scenedesmus WC-1 grown on 14:10 L:D cycle in unbuffered Bold's Basal Medium, 5% CO2 aeration. Error is standard deviation. n=3

[0027] FIG. 4 depicts average cell density (A), medium pH (B), and Nile Red Fluorescence (C) of Scenedesmus WC-1 grown on 14:10 L:D cycle in unbuffered Bold's Basal Medium, 5% CO2 aeration switched to ambient air aeration, with and without 50 mM HCO.sub.3 spike. Black circles indicate time of bicarbonate spike and error is standard deviation. n=3

[0028] FIG. 5 depicts results of bicarbonate spike treatments. Average cell density (A) and Nile Red Fluorescence (B) of Scenedesmus WC-1 grown in 14:10 L:D cycle and unbuffered Bold's Basal Medium, 5% CO2 aeration until bicarbonate was added (circles), then aeration was changed to ambient air aeration. Error is standard deviation. n=3

[0029] FIG. 6 depicts average and standard deviation of cellular growth (a), total Nile Red fluorescence intensity (b), and culture pH (c) of Scenedesmus WC-1 aerated with ambient air (.quadrature.), 5% CO.sub.2 in air (.largecircle.), and 5% CO.sub.2 in air that is changed to ambient air near nitrate depletion, both with ( ) and without (.DELTA.) a 50 mM bicarbonate addition. Arrow indicates time of bicarbonate addition. Growth was maintained in unbuffered Bold's basal medium illuminated with a 14:10 L:D cycle. n=3

[0030] FIG. 7 depicts average and standard deviation of cellular growth (a) and total Nile Red fluorescence intensity (b) of Scenedesmus WC-1 aerated with 5% CO.sub.2 in air that is switched to ambient air and 50 mM bicarbonate addition pre-nitrate depletion (.box-solid. and .quadrature.) or post-nitrate depletion ( and .largecircle.). Arrows indicate time of bicarbonate addition. Growth was maintained in unbuffered Bold's basal medium illuminated with a 14:10 L:D cycle. n=3

[0031] FIG. 8 depicts average cell density (A), medium pH (B), and Nile Red Fluorescence (C) of diatom RGd-1 grown on 14:10 L:D cycle in unbuffered Bold's Basal Medium with 2 mM Na.sub.2SiO.sub.3.9H.sub.2O, 1.5 nM cyanocobalamin (B12), and S3 vitamins from ASP II medium recipe, ambient air aeration, with and without 25 mM HCO.sub.3 spike. Culture was spiked at 7.4 d, near Si depletion, and error is standard deviation. n=3 for no spike control and n=2 for bicarbonate spiked culture.

[0032] FIG. 9 depicts major pathways for fatty acid synthesis in plants and algae. 3-PGA: 3-phosphoglycerate; Accase: acetyl CoA carboxylase; ACP: acyl carrier protein; AGPPase: ADP glucose pyrophosphorylase; ER: Endoplasmic reticulum; GDAT: putative glycolipids: DAG acyltransferase; Glc6P: glucose-6-phosphate; KAS: 3-ketoacyl-ACP; PDAT: Phospholipids: DAG acyltransferase; PDH: pyruvate dehydrogenase (putative pathways were proposed in dashed lines).

[0033] FIG. 10 depicts cell growth (A), Nile Red Fluorescence (B), and nitrate assay (C) of Phaeodactylum tricornutum Pt-1 (Pt-1) grown on 14:10 L:D cycle in ASP II medium. 5 mM, 10 mM, or 20 mM bicarbonate were provided at the starting point of algae growth.

[0034] FIG. 11 depicts average cell density (A) and Nile Red Fluorescence (B) of diatom Phaeodactylum tricornutum Pt-1 grown on 14:10 L:D cycle in ASP II Medium, with and without 5 mM HCO.sub.3 spike on day 3.

[0035] FIG. 12 depicts average cell density (A), Nile Red Fluorescence (B), and nitrate assay (C) of diatom Phaeodactylum tricornutum Pt-1 grown on 14:10 L:D cycle in ASP II Medium, with and without 10 mM HCO.sub.3 spike either before (ASP II--10 mM NaHCO.sub.3, spike at day 3) or after (ASP II+10 mM NaHCO.sub.3, spike at day 5) nitrate depletion.

[0036] FIG. 13 depicts average and standard deviation of cellular growth (a), total Nile Red fluorescence intensity (b), and culture pH (c) of Phaeodactylum tricornutum Pt-1 aerated with ambient air and 25 mM bicarbonate addition pre-nitrate depletion ( ), added post-nitrate depletion (.box-solid.), and no addition (.quadrature.). Arrow indicates time of bicarbonate addition. Growth was maintained in 50 mM Tris (pKa 7.8) buffered ASP II medium illuminated with a 14:10 L:D cycle. n=3

[0037] FIG. 14 depicts transmitted micrographs and Nile Red epifluorescent images of Scenedesmus WC-1 (top) and Phaeodactylum tricornutum Pt-1 (bottom) when bicarbonate was added pre-nitrate depletion (a and c) and when added post-nitrate depletion (b and d). Cells imaged represent average cells for each respective culture and magnification is identical between all images.

[0038] FIG. 15 depicts Scenedesmus WC-1 TAG accumulation from sodium bicarbonate and sodium carbonate both as individual, mix, or buffered additives (CAPS Buffer pKa 10.3). Growth was maintained in unbuffered Bold's Basal medium until time of addition just prior to medium nitrate depletion.

[0039] FIG. 16 depicts growth of Scenedesmus WC-1 after addition of 5, 10, 15, 20, and 25 mM NaHCO.sub.3 (A) and 0, 5, 10, 15 mM NaHCO.sub.3 and 15 mM NaCl (B). Time of addition is indicated by the circle (A) and arrow (B).

[0040] FIG. 17 depicts Nile Red fluorescence of Scenedesmus WC-1 when 50 mM NaHCO.sub.3 and 180 mM NO.sub.3 are added during stationary phase after medium nitrate had been completely consumed. Arrow indicates time of addition.

[0041] FIG. 18 depicts Nile Red fluorescence (A) and Nile Red specific fluorescence (B) of Chlorophyta EN-2 when 50 mM NaHCO.sub.3 was added prior to medium nitrate depletion. Nile Red specific fluorescence indicates TAG per cell and is calculated by Nile Red fluorescence.times.cell number.sup.-1.times.10,000 (scaling factor). Arrow indicates time of addition.

[0042] FIG. 19 depicts Nile Red fluorescence (a), pH (b), and average cell density (c) for (1) Scenedesmus sp. 131 and (2) Kirchneriella sp. 92 grown on nitrate and utilizing 50 mM NaHCO.sub.3 addition to cultures grown on 5% CO2 and switched to air (.largecircle.), no addition growing on 5% CO2 (.quadrature.), and no addition growing on air (.box-solid.). Arrow represents time of nitrate addition.

DETAILED DESCRIPTION

[0043] Lipid derived biofuel is defined as fatty acid methyl esters (FAME) derived from triacylglycerol (TAG) which can be synthesized in plant, animal, or microalgae biomass. FAMEs offer increased energy yield, over input requirements, as compared to biologically derived ethanol and can be used directly in jet or diesel engines [1, 2]. However, FAME precursor TAG availability will limit the use of this fuel to mediate global transportation needs unless non-food crop land acreage and hypersaline, brackish, high alkalinity, or wastewater can be used to produce the fuel. Due to the fast growth rate and physiological diversity microalgae exhibit [3], they are well suited to be used in intelligently designed growth systems that bypass food crop land, facilitate higher mass transfer, and potentially decrease microbial contamination [4].

[0044] Historically, a major goal of the Aquatic Species Program, which initially evaluated algae's potential for biofuel production, was to identify a "lipid trigger" [5]. This would be a set of environmental parameters or target molecule(s) that would promote algal synthesis of TAG. No trigger was identified, however nitrogen depletion has been shown to cause cellular lipid accumulation, albeit some strains can take many days to accumulate the TAG [4, 6]. Additionally, we have observed delayed cell growth/replication by inhibiting the tricarboxylic acid cycle with monofluoroacetic acid [7]), inducing cellular TAG accumulation, and similarly we have observed pH induced TAG accumulation [8]. Recently, these results have been expanded proving that pH induced TAG accumulation and nitrate depletion causing TAG accumulation are independent stress mechanisms. An advantageous interplay between pH and nitrate was identified which showed both an increase in TAG per cell and a much shortened culture time to realize nitrate depletion generated TAG accumulation [4]. However, both of these pH studies were conducted with 24 hr light conditions which will inherently limit dark cycle respiration.

[0045] The present invention discloses an extension of pH studies aimed at understanding the cellular metabolic responses during pH induced, or pH induced combined with nutrient depletion (e.g., nitrate or silica depletion), TAG accumulation and the hypothesis of the existence of a TAG accumulation inducing trigger. In some embodiments, inventors of the present invention detail a loss of pH induced TAG accumulation when cultures are shifted from 24 hr light conditions to 14:10 light-dark cycling, and by following the inventors' hypotheses that cell growth/replication inhibition leads to TAG accumulation and high pH coupled with nutrient depletion causes increased TAG accumulation, with decreased culture time to reach high TAG levels, a bicarbonate trigger was found. This trigger changes a culture from high biomass with little TAG to a high TAG accumulation state. In other words, it stops cell growth/replication but maintains photosynthesis forcing the algae to fix carbon as lipid.

[0046] Without wishing to be bound by any theory, any composition (e.g., bicarbonate) or any growth factor change (temperature, pH, light, etc) that can influence the ability of an algae cell to complete a growth cycle (wherein energy is needed for cell division) will cause TAG accumulation in an algae growth system, if adding said composition into the system or making said growth factor change to the system is not completely toxic and/or does not result in cell death. Bicarbonate is such a composition.

DEFINITION

[0047] As used herein, the verb "comprise" as is used in this description and in the claims and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements are present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0048] As used herein, the term "derived from" refers to the origin or source, and may include naturally occurring, recombinant, unpurified, or purified molecules.

[0049] The terms "algae" or "algal" are used synonymously herein and refer to the common (non-taxonomic) name for a large and diverse group of a relatively simple type of thallophytic plant which is never differentiated into distinct organs such as root, stem and leaves. They contain chlorophyll and are photosynthetic. Algae display a stunning variety of shapes, sizes, and colors and range in form from unicellular to multicellular, the latter including plants many meters in length, such as what is commonly known as seaweed. Examples of algae include but are not limited to blue green algae (cyanobacteria, Cyanophycae), red algae or Rhodophyta (e.g., Porphyra, Iridaea); dinoflagellates (e.g., Peridinium, Ceratium); diatoms (e.g., Thalassiosira); golden-brown algae (e.g., Vaucheria); brown algae or Phaeophyta (e.g., Laminaria, Postelsia, Macrocystis, Nereocystis, Fucus); and green algae or Chlorophyta (Nannochloropsis, Chlamydomonas, Volvox, Eremosphaera, Pediastrum, Hydrodictyon, Spirogyra, Zygnema, Closterium, Mougeotia, Micrasterias, Cosmarium, Desmidium, Oedogonium, Coleochaeie, Cladophora, Codium, Chara, Ulva, Bryopsis, Ulothrix). For a more comprehensive description of algae, see, for example, Lee, R. E. (2008) Phycology, Cambridge University Press 547 pages; Barsanti, et al. (2005) Algae: Anatomy, Biochemistry, and Biotechnology, First Edition, ISBN-10: 0849314674, CRC Press, 320 pages; and Brodie et al. (editors) (2007) Unraveling the algae: the past, present, and future of algal systemics, First Edition, CRC Press, 402 pages, each of which is herein incorporated by reference in their entireties.

[0050] As used herein, the term "agent", as used herein, means a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein or an oligonucleotide that modulates the function of a nucleic acid or polypeptide. A vast array of compounds can be synthesized, for example oligomers, such as oligopeptides and oligonucleotides, and synthetic organic and inorganic compounds based on various core structures, and these are also included in the term "agent". In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. Compounds can be tested singly or in combination with one another.

[0051] As used herein, the term "bicarbonate" or "hydrogencarbonate" refers to an intermediate form in the deprotonation of carbonic acid. Its chemical formula is HCO.sub.3, and as used herein is synonymous with HCO.sub.3.

[0052] As used herein, the term "transformation" refers to the transfer of nucleic acid (i.e., a nucleotide polymer) into a cell. As used herein, the term "genetic transformation" refers to the transfer and incorporation of DNA, especially recombinant DNA, into a cell.

[0053] As used herein, the term "transgenic" refers to cells, cell cultures, organisms (e.g., plants), and progeny which have received a foreign or modified gene by one of the various methods of transformation, wherein the foreign or modified gene is from the same or different species than the species of the organism receiving the foreign or modified gene.

[0054] As used herein, unless otherwise specified, the term "carbohydrate" refers to a compound of carbon, hydrogen, and oxygen that contains an aldehyde or ketone group in combination with at least two hydroxyl groups. The carbohydrates of the present invention can also be optionally substituted or deoxygenated at one or more positions. Carbohydrates thus include substituted and unsubstituted monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The saccharide can be an aldose or ketose, and may comprise 3, 4, 5, 6, or 7 carbons. In one embodiment they are monosaccharides. In another embodiment they can be pyranose and furanose sugars. They can be optionally deoxygenated at any corresponding C-position, and/or substituted with one or more moieties such as hydrogen, halo, haloalkyl, carboxyl, acyl, acyloxy, amino, amido, carboxyl derivatives, alkylamino, dialkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, thiol, imine, sulfonyl, sulfanyl, sulfinyl, sulfamonyl, ester, carboxylic acid, amide, phosphonyl, phosphinyl, phosphoryl, thioester, thioether, oxime, hydrazine, carbamate. These saccharide units can be arranged in any order and the linkage between two saccharide units can occur in any of approximately ten different ways. As a result, the number of different possible stereoisomeric oligosaccharide chains is enormous. In one embodiment, said carbohydrates are selected from the group consisting of monosaccharides, disaccharides, oligosaccharides, polysaccharides, and a combination thereof.

[0055] As used herein, the term "neutralize", "neutralizing", and "neutralization" refers to a chemical reaction in aqueous solutions, wherein an acid and a base react to form water and a salt, and wherein the pH of the solution is brought closer to 7.

[0056] As used herein, the term "aerobic conditions" refers to conditions where sufficient oxygen, is provided, and anaerobic respiration in a microorganism growing under such conditions is prohibited.

[0057] As used herein, the term "substantially aerobic conditions" refers to conditions wherein the supply of oxygen is limited, but the cellular respiration in an organism is dominantly aerobic respiration.

[0058] As used herein, the term "biofuel" (also called bioenergy) is defined as solid, liquid or gaseous fuel derived from relatively recently dead or dying biological material and is distinguished from fossil fuels, which are derived from long dead biological material. It can be produced from any biological carbon source theoretically. Biofuels can be classified into first generation biofuels (which are made from sugar, starch, vegetable oil, and animal fats, including but not limited to vegetable oil, biodiesel, bioalcohols, bioethers, biogas, syngas and solid biofuels), second generation biofuels (which are produced from biomass of non food crops, also called cellulosic biofuels, including but not limited to, biohydrogen, biomethanol, DMF, Bio-DME, Fischer-Tropsch diesel, biohydrogen diesel, mixed alcohols and wood diesel), and third generation biofuels (also called algae fuels, which are made from algae).

[0059] The term "biofuel precursor" refers to an organic molecule in which all carbon contained within is derived from biomass and is biochemically converted. It can be further converted either chemically or biochemically, into a biofuel. For example, a biofuel precursor includes, but is not limited to, e.g. isobutanol, isopropanol, propanol, 2-butanol, butanol, pentanol, 2-pentanol, 3-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 3-methyl-2-butanol, and lipid.

[0060] As used herein, the term "biodiesel" refers to a vegetable oil or animal fat-based diesel fuel consisting of long chain alkyl (e.g., methyl, propyl or ethyl) esters. It can be made by chemically reacting lipids with one or more types of alcohol in a transesterification reaction. Chemically it comprises a mix of mono-alkyl esters of long chain fatty acids. Alcohols that can be used to produce biodiesel include, but are not limited to, methanol, ethanol, propanol, isopropanol, butanol, isobutanol, and 2-ethoxyethanol. Acidic or alkaline catalyst can be applied to facilitate esterification of fatty acids. Glycerol is produced as a by-product in such reactions.

[0061] As used herein, the phrase "fatty acids" refers to long-chained molecules having a methyl group at one end and a carboxylic acid group at the other end.

[0062] As used herein, the term "fermentation" or "fermentation process" refers a process in which a biocatalyst is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, wherein the biocatalyst converts raw materials, such as a feedstock, into products.

[0063] As used herein, the term "carbon source" generally refers to a substance suitable to be used as a source of carbon for prokaryotic or eukaryotic cell growth. Carbon sources include, but are not limited to, carbon dioxide, bicarbonate, carbonate, biomass hydrolysates, carbohydrates (e.g., starch, sucrose, polysaccharides, and monosaccharides), cellulose, hemicellulose, xylose, and lignin, as well as monomeric components of these substrates. Carbon sources can comprise various organic compounds in various forms, including, but not limited to polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, etc. These include, for example, various monosaccharides such as glucose, dextrose (D-glucose), maltose, oligosaccharides, polysaccharides, saturated or unsaturated fatty acids, succinate, lactate, acetate, ethanol, etc., or mixtures thereof. Photosynthetic organisms can additionally produce a carbon source as a product of photosynthesis.

[0064] As used herein, the term "biomass" refers to biological material derived from living, or recently living organisms, e.g., stems, leaves, and starch-containing portions of green plants, or wood, waste, forest residues (dead trees, branches and tree stumps), yard clippings, wood chips, or materials derived from algae or animals, and is mainly comprised of starch, lignin, pectin, cellulose, hemicellulose, and/or pectin. Biomass may also include biodegradable wastes that can be burnt as fuel. It excludes organic material such as fossil fuel which has been transformed by geological processes into substances such as coal or petroleum. Biomass can be decomposed by either chemical or enzymatic treatment to the monomeric sugars and phenols of which it is composed (Wyman, C. E. 2003 Biotechnological Progress 19:254-62). This resulting material, called biomass hydrolysate, is neutralized and treated to remove trace amounts of organic material that may adversely affect the biocatalyst, and is then used as a feedstock for fermentations using a biocatalyst.

[0065] As used herein, the term "nutrient" is defined as a chemical compound that is used by a biocatalyst to grow and survive. Nutrients can be organic compounds such as carbohydrates and amino acids or inorganic compound such as metal or mineral salts.

[0066] As used herein, the term "recombinant microorganism" and "recombinant host cell" are used interchangeably and refer to microorganisms that have been genetically modified to express or over-express endogenous polynucleotides, or to express heterologous polynucleotides, such as those included in a vector, or which have a reduction in expression of an endogenous gene. The polynucleotide generally encodes a target enzyme involved in a metabolic pathway for producing a desired metabolite. It is understood that the terms "recombinant microorganism" and "recombinant host cell" refer not only to the particular recombinant microorganism but to the progeny or potential progeny of such a microorganism. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0067] As used herein, the term "fermentation" refers to a method to produce biofuel and other products where biomass (pretreated or unpretreated) was fermented by microorganisms (e.g., bacteria, cyanobacteria, yeast, fungi or algae).

Algae

[0068] Algae are a large and diverse group of simple, typically autotrophic organisms, ranging from unicellular to multicellular forms, such as the giant kelps that grow to 65 meters in length. The US Algal Collection is represented by almost 300,000 accessioned and inventoried herbarium specimens.

[0069] Non-limiting examples of algae include, Archaeplastida (e.g., Chlorophyta (Green algae), Rhodophyta (Red algae), Glaucophyta), Rhizaria, Excavata (e.g., Chlorarachniophytes, Euglenids), and Chromista, Alveolata (e.g., Heterokonts (e.g., Bacillariophyceae (Diatoms), Axodine, Bolidomonas, Eustigmatophyceae, Phaeophyceae (Brown algae), Chrysophyceae (Golden algae), Raphidophyceae, Synurophyceae, Xanthophyceae (Yellow-green algae)), Cryptophyta, Dinoflagellates, and Haptophyta).

[0070] All true algae have a nucleus enclosed within a membrane and plastids bound in one or more membranes. Algae constitute a paraphyletic and polyphyletic group, as they do not include all the descendants of the last universal ancestor nor do they all descend from a common algal ancestor, although their plastids seem to have a single origin.

[0071] Algae lack the various structures that characterize land plants, such as phyllids (leaves) and rhizoids in nonvascular plants, or leaves, roots, and other organs that are found in tracheophytes (vascular plants). Many are photoautotrophic, although some groups contain members that are mixotrophic, deriving energy both from photosynthesis and uptake of organic carbon either by osmotrophy, myzotrophy, or phagotrophy. Some unicellular species rely entirely on external energy sources and have limited or no photosynthetic apparatus.

[0072] Nearly all algae have photosynthetic machinery ultimately derived from the Cyanobacteria, and so produce oxygen as a by-product of photosynthesis, unlike other photosynthetic bacteria such as purple and green sulfur bacteria.

[0073] Rhodophyta, Chlorophyta and Heterokontophyta, the three main algal Phyla, have life-cycles which show tremendous variation with considerable complexity. In general there is an asexual phase where the seaweed's cells are diploid, a sexual phase where the cells are haploid followed by fusion of the male and female gametes. Asexual reproduction is advantageous in that it permits efficient population increases, but less variation is possible. Sexual reproduction allows more variation, but is more costly. Often there is no strict alternation between the sporophyte and also because there is often an asexual phase, which could include the fragmentation of the thallus.

[0074] Diatoms are eukaryotic, unicellular, algae that make a siliceous cell wall. They constitute the largest population of algae in the oceans and fix carbon for 40% of marine productivity. Most diatoms are unicellular, although they can exist as colonies in the shape of filaments or ribbons (e.g. Fragillaria), fans (e.g. Meridian), zigzags (e.g. Tabellaria), or stellate colonies (e.g. Asterionella). A characteristic feature of diatom cells is that they are encased within a unique cell wall made of silica (hydrated silicon dioxide) called a frustule. These frustules show a wide diversity in form, but usually consist of two asymmetrical sides with a split between them, hence the group name. There are more than 200 genera of living diatoms, and it is estimated that there are approximately 100,000 extant species [30-32]. Diatoms belong to a large group called the heterokonts, including both autotrophs (e.g. golden algae, kelp) and heterotrophs (e.g. water moulds). Their yellowish-brown chloroplasts are typical of heterokonts, with four membranes and containing pigments such as the carotenoid fucoxanthin. Individuals usually lack flagella, but they are present in gametes and have the usual heterokont structure, except they lack the hairs (mastigonemes) characteristic in other groups. Some species actively regulate their buoyancy with intracellular lipids to counter sinking. The entire genomes of the centric diatom, Thalassiosira pseudonana (32.4 Mb) [33] and the pennate diatom, Phaeodactylum tricornutum (27.4 Mb) [34], have been sequenced. Detailed study of diatoms can be found in Lewin et al., 1963 (Annual review of microbiology, Diatoms, Vol. 17: 373-414) and Werner, 1977 (The Biology of Diatoms, ISBN 0520034007, 9780520034006), each of which is incorporated herein by reference in its entirety.

[0075] Algae biology, strains, technology, mechanism and non-limited examples of commercialization of algal biofuel production are described in Singh et al. 2010a, 2010b, 2010c, Sims et al., 2010, Demirbas, et al., Demirbas et al., Khattar et al., Miller et al., Chen et al. (Ref. Nos 21-28), A Look Back at the U.S. Department of Energy's Aquatic Species Program: Biodiesel from Algae (National Renewable Energy Laboratory, Close-Out Report, 1998), and National Algal biofuels Technology Roadmap (U.S. Department of Energy, May 2010), each of which is incorporated herein by reference in its entirety for all purposes.

[0076] To produce biofuel, algae can be cultivated in photobioreactors, closed loop systems, or open ponds, under aerobic conditions, or substantially aerobic conditions. In a photobioreactor (PBR), nutrient-laden water is pumped through plastic tubes that are exposed to light. A PBR is a bioreactor which incorporates some type of light source. Virtually any translucent, closed container can be called a PBR. PBRs are designed to prevent the growth of unwanted species. A PBR can be operated in "batch mode", but it is also possible to introduce a continuous stream of sterilized water containing nutrients, air, and carbon dioxide. As the algae grows, excess culture overflows and is harvested. Light source can be artificial light, solar light, or mixture thereof. PBR temperature can be controlled by placing it in a constant temperature room or bath. Large scale outdoor PBRs with temperature control are also commercially available. Non-limiting exemplary PBRs include, flat-panel PBRs, tubular PBRs (horizontal or vertical), bubble column PBRs, air lift PBRs, stirred tank PBRs, immobilized PBRs, vertical-column PBRs, and those described in Lehr and Posten (Closed photo-bioreactors as tools for biofuel production, Current Opinion in Biotechnology, 2009, 20:280:285) and U.S. Pat. Nos. 5,958,761, 7,449,313, 5,981,271, 6,432,698, 7,514,247, 6,571,735, 4,743,545, 6,008,028, 6,174,720, 7,001,519, 7,229,785, 7,004,109, 5,614,097, 5,569,634, 6,986,323, and 6,923,914, each of which is hereby incorporated by reference in its entirety for all purposes. In a closed system (not exposed to open air), algae for biofuel production can be grown in large quantities, and contamination by other organisms in the air is prevented. Some algae production systems can be built next to power plants use effluent of CO.sub.2 to reduce pollution. In such systems, control over the environment is much better than in the open ponds. Open-pond systems include natural waters and artificial ponds or containers. The most commonly used systems include shallow big ponds, tanks, circular ponds and raceway ponds. One major advantage of open ponds is that they are easier and cheaper to construct and operate than most closed systems.

Lipid

[0077] Lipids are a broad group of naturally occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. Although the term lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, and monoglycerides and phospholipids), as well as other sterol-containing metabolites such as cholesterol. Although humans and other mammals use various biosynthetic pathways to both break down and synthesize lipids, some essential lipids cannot be made this way and must be obtained from the diet. Non-limiting examples of lipids include, fatty acyls, glyercolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, polyketides, and saccharolipids.

Triacylglycerol (TAG)

[0078] Triacylglycerol (a.k.a. Triglyceride, TAG or triacylglyceride) is an ester derived from glycerol and fatty acids. It is the main constituent of vegetable oil and animal fats.

[0079] Triglycerides are formed by combining glycerol with three molecules of fatty acid. The glycerol molecule has three hydroxyl (HO--) groups. Each fatty acid has a carboxyl group (HOOC--). In triglycerides, the hydroxyl groups of the glycerol join the carboxyl groups of the fatty acid to form ester bonds:

HOCH.sub.2CH(OH)CH.sub.2OH+RCO.sub.2H+R'CO.sub.2H+R'CO.sub.2H.fwdarw.RC- O.sub.2CH.sub.2CH(O.sub.2CR')CH.sub.2O.sub.2CR'+3H.sub.2O

[0080] The three fatty acids (RCO.sub.2H, R'CO.sub.2H, R'CO.sub.2H in the above equation) are usually different, but many kinds of triglycerides are known. The chain lengths of the fatty acids in naturally occurring triglycerides vary in lengths, but most contain 16-, 18-, and 20-carbon atoms. Natural fatty acids found in plants and animals are typically composed only of even numbers of carbon atoms, reflecting the pathway for their biosynthesis from the two-carbon building block acetyl CoA. Bacteria, however, possess the ability to synthesize odd- and branched-chain fatty acids. As a result, ruminant animal fat contains odd-numbered fatty acids, such as 15, due to the action of bacteria in the rumen. Many fatty acids are unsaturated, some are polyunsaturated, e.g., those derived from linoleic acid.

[0081] Algae produce triacylglycerols from fatty acids. Major pathways for fatty acids and TAG synthesis in algae are shown in FIG. 6. Under favorable growth conditions, newly photoassimilated carbon is incorporated primarily into proteins. Thus, lipid levels are generally low under log phase growth conditions. Moreover, lipids made during daylight are used as energy sources during dark periods. It has long been recognized that lipid accumulation is an adaptive response to stress for algae. Under biotic and abiotic stresses, such as low light or temperature, high light intensity or salinity, low nutrients, or fluctuations in pH, more carbon is incorporated into lipids and carbohydrates and less is incorporated into proteins. The profile of the fatty acid composition of the lipids also changes under stress. Algae must cope with fluctuations in conditions in order to survive. Lipids serve as carbon and energy storage for "hard times". It has recently been suggested that lipid accumulation may even play a protective role against excess sunlight by serving as an electron sink when excess electrons accumulate from the photosynthetic electron transport chain under photo-oxidative stress (Hu et al. (2008). Microalgal triacylglycerols as feedstocks for biofuel production: Perspectives and advances. Plant Journal. 54(4), 621-639.). The better the response to environmental stresses, the better the survival of the species, so genes in stress response pathways are often positively selected. For algae grown commercially, direct competition with conspecifics (as more and more cells are packed into a limited volume), and predation by protists are additional stresses.

[0082] Many algae are photosynthetic organisms capable of harvesting solar energy and converting CO.sub.2 and water to O.sub.2 and organic macromolecules such as carbohydrates and lipids. Under stress conditions such as high light or nutrient starvation, some microalgae accumulate lipids such as triacylglycerols (TAG) as their main carbon storage compounds. Certain microalgal species also naturally accumulate large amounts of TAG (30-60% of dry weight), and exhibit photosynthetic efficiency and lipid production at least an order of magnitude greater than terrestrial crop plants (Hu et al., 2008). Cyanobacteria and macroalgae, as a general rule, accumulate mostly carbohydrates, with lipid accumulation in macroalgae typically being less than 5% of total dry weight (McDermid et al. (2003). Nutritional composition of edible Hawaiian seaweeds. Journal of Applied Phycology, 15(6), 513-524.), although concentrations approaching 20% lipid have been reported in some species (Chu et al. (2003). Fatty Acid Composition of Some Malaysian Seaweeds. Malaysian Journal of Science. 22(2), 21-27; Mcdermid et al. 2003). Lipids and carbohydrates, along with biologically produced hydrogen and alcohols, are all potential biofuels or biofuel precursors.

[0083] Knowing how and when carbon is partitioned in a cell into lipids and/or carbohydrates could be very useful for biofuels strain development and designing cultivation strategies. Understanding carbon partitioning will require extensive knowledge of metabolic pathways. Metabolic networks have been reconstructed in various microbes from genomic and transcriptomic data, pathway analysis, and predictive modeling (Vemuri, G. N. A. A. Aristidou. (2005). Metabolic engineering in the -omics era: Elucidating and modulating regulatory networks. Microbiology and Molecular Biology Reviews. 69(2), 197-216.). Research has also been done in plant systems to understand carbon flux in biosynthetic and degradative pathways (Lytovchenko et al., 2007; Schwender et al., 2004; Allen et al., 2009; Sweetlove and Fernie, 2005; Libourel and Shachar-Hill, 2008). However, carbon partitioning in algae is less understood and research on how algal cells control the flux and partitioning of photosynthetically fixed carbon into various groups of major macromolecules (i.e., carbohydrates, proteins, and lipids) is critically needed.

[0084] Further, a link between starch and lipid metabolism has been established. Starch is a common carbon and energy storage compound in plants and algae, and shares the same precursors with the storage lipid TAG. It is, therefore, possible that TAG and starch could be inter-convertible, a potentially important implication for biofuel production. More recently, studies in higher plants showed that when starch synthesis was impaired or inhibited, plant embryos or seeds accumulated 40% less oil (Periappuram et al., 2000; Vigeolas et al., 2004). While these results provide an indication that starch synthesis is linked to lipid synthesis, the nature of the interaction is unknown.

[0085] Some algae, naturally or under stress conditions, accumulate significant quantities of neutral storage lipids such as triacylglycerols (TAG), which are important potential fuel precursors. The major pathway for the formation of TAG in plants first involves de novo fatty acid synthesis in the stroma of plastids. The synthesis of cellular and organelle membranes, as well as of neutral storage lipids such as TAG, uses 16 or 18 carbon fatty acids as precursors. TAG is formed by incorporation of the fatty acid into a glycerol backbone via three sequential acyl transfers (from acyl CoA) in the endoplasmic reticulum (ER).

[0086] TAG biosynthesis in algae has been proposed to occur via the above Kennedy pathway described in plants. Fatty acids produced in the chloroplast are sequentially transferred from CoA to positions 1 and 2 of glycerol-3-phosphate, resulting in the formation of the central metabolite phosphatidic acid (PA). Dephosphorylation of PA catalyzed by a specific phosphatase releases diacylglycerol (DAG). Since diglycerides are usually present in high amounts in rapidly growing cultures, it may be of interest to research these TAG intermediates. In the final step of TAG synthesis, a third fatty acid is transferred to the vacant position 3 of DAG by diacylglycerol acyltransferase, an enzyme that is unique to TAG biosynthesis. The acyltransferases involved in TAG synthesis may exhibit preferences for specific acyl CoA molecules, and thus may play an important role in determining the final acyl composition of TAG (Hu et al., 2008). Alternative pathways to convert membrane lipids and/or carbohydrates to TAG have recently been demonstrated in bacteria, plants and yeast in an acyl CoA-independent way (Arabolaza et al., 2008; Dahlqvist et al., 2000; Stahl et al., 2004). Such pathways have not yet been studied in algae. Moreover, PA and DAG can also be used directly as substrates for synthesis of polar lipids, such as phosphatidylcholine (PC) and galactolipids. These pathways are worth investigating when developing strains for improved lipid production.

[0087] The regulation of the synthesis of fatty acids and TAG in algae is relatively poorly understood. This lack of understanding may contribute to why the lipid yields obtained from algal mass culture efforts fall short of the high values (50 to 60%) observed in the laboratory. Understanding lipid regulation can help to maximize scenarios for lipid production and strain improvement.

[0088] Lipids in algae can be extracted using different procedures. Non-limiting examples of lipids extraction are described in King et al. (Supercritical Fluid Extraction: Present Status and Prospects, 2002, Grasa Asceites, 53, 8-21), Folch et al. (A simple method for the isolation and purification of total lipids from animal tissues, 1957, J. Biol. Chem., 226, 497-509), Bligh and Dyer (A rapid method of total lipid extraction and purification. 1959, Can. J. Biochem. Physiol., 37, 911-917), Hara et al. (Lipid extraction of tissues with a low toxicity solvent. 1978, Anal. Biochem., 90, 420-426), Lin et al. (Ethyl acetate/ethyl alcohol mixtures as an alternative to Folch reagent for extracting animal lipids. 2004, J. Agric. Food Chem., 52, 4984-4986), Whiteley et al. (Lipid peroxidation in liver tissue specimens stored at subzero temperatures. 1992, Cryo-Letters, 13, 83-86). In another example, lipid can be extracted by methods similar to the FRIOLEX.RTM. (Westfalia Separator Industry GmbH, Germany) process is used to extract the biological oils produced by the microorganisms. FRIOLEX.RTM. is a water-based physical oil extraction process, whereby raw material containing oil can be used directly for extracting oil without using any conventional solvent extraction methods. In this process, a water-soluble organic solvent can be used as a process aid and the oil is separated from the raw material broth by density separation using gravity or centrifugal forces.

[0089] After the lipids have been extracted, the lipids can be recovered or separated from non-lipid components by any suitable means known in the art. For example, low-cost physical and/or mechanical techniques are used to separate the lipid-containing compositions from non-lipid compositions. If multiple phases or fractions are created by the extraction method used to extract the lipids, where one or more phases or fractions contain lipids, a method for recovering the lipid-containing phases or fractions can involve physically removing the lipid-containing phases or fractions from the non-lipid phases or fractions, or vice versa. In some embodiments of the present invention, a FRIOLEX.RTM. type method is used to extract the lipids produced by the microorganisms and the lipid-rich light phase is then physically separated from

» Number: 20130295623

» Publication Date: 07/11/2013

» Applicant: Family ID:

» Inventor: Gardner; Robert; (Bozeman, MT) ; Peyton; Brent; (Bozeman, MT) ; Cooksey; Keith E.; (Manhattan, MT)

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