Method of producing plant suspension cells in a growth medium enriched with carbonic acid

Abstract

The present invention provides a method of producing a photosynthetic product, the method comprising maintaining a photosynthetic plant or algal cell suspension culture, in the presence of water, light and a carbonic acid-enriched growth medium. The carbonic acid may, for example be provided by feeding the photosynthetic plant cell suspension culture with a carbonic acid solution, a solid or liquid precursor thereof, or a gaseous mixture of carbon dioxide and one or more other gases. The invention also provides a method for producing a photosynthetic product, the method comprising maintaining a photosynthetic plant or algal cell suspension culture, in the presence of water, light and a carbon source selected from carbon dioxide and carbonic acid, wherein the culture is maintained at a pH of less than 7.0, preferably 4.5 to 5.5.

Claims

1. A method of producing a sugar-comprising photosynthetic product, wherein the method comprises maintaining a photosynthetic non-algal plant cell suspension culture, in the presence of water, light and in a carbonic acid-enriched growth medium, wherein the concentration of carbonic acid in the carbonic acid-enriched growth medium is at least about 0.1% w/v.

2. The method of claim 1, wherein the concentration of carbonic acid in the carbonic acid-enriched growth medium is of from about 0.1% w/v to about 10% w/v.

3. The method of claim 1, wherein the level of carbonic acid in the carbonic acid-enriched growth medium is maintained at a steady level.

4. The method of claim 1, wherein the culture medium is maintained at a pH of less than 7.0.

5. The method of claim 1, wherein the culture is maintained in the presence of constant light.

6. The method of claim 1, wherein the photosynthetic non-algal plant cell suspension culture is a suspension culture of differentiated photosynthetic non-algal plant cells.

7. The method of claim 6, wherein the photosynthetic plant cell suspension culture is a suspension culture of plant palisade cells, leaf mesoderm cells or petiole cells.

8. The method of claim 1, wherein the sugar-comprising photosynthetic product is selected from the group consisting of sugar, glyceraldehyde, glycerose, one or more starches, and a combination of any of the preceding.

9. The method of claim 8, wherein the photosynthetic product is a sugar selected from the group consisting of a mono-saccharide, a di-saccharide, glucose, sucrose, fructose, and a combination of any of the preceding.

10. The method of claim 1, further comprising the step of extracting or recovering the sugar-comprising photosynthetic product from the carbonic acid-enriched growth medium.

11. The method of claim 10, wherein the step further of extracting or recovering the photosynthetic product is a continuous process.

12. The method of claim 10, wherein the extracted or recovered photosynthetic product is provided in the form of a syrup, crystals, or solution.

13. The method of claim 1, wherein the photosynthetic non-algal plant cell suspension culture has a volume that is at least 10,000 L.

14. The method of claim 1, further comprising the production of a biological product of a second culture, the method comprising (i) maintaining a first cell suspension culture of photosynthetic non-algal plant cells in accordance with the method as defined by claim 1 such that cells of the photosynthetic non-algal plant cell suspension culture photosynthesize and thereby generate and release sugar-comprising photosynthetic product into the surrounding culture medium; and (ii) maintaining a second cell culture in the presence of the photosynthetic product generated by the first cell suspension culture to allow growth of the second culture and the production of a biological product by the second culture.

15. The method of claim 14, wherein the biological product is biomass.

16. The method of claim 14, wherein the biological product is selected from the group consisting of a fatty acid, oil, a combination of a fatty acid and oil, a proteinaceous product and a metabolite.

17. The method of claim 14, wherein the second cell culture is a culture of prokaryotic cells or eukaryotic cells.

18. The method of claim 14, wherein the second cell culture is a culture of microorganisms.

19. The method of claim 14, wherein the second cell culture is a cell suspension culture of oil-producing plant cells, the method comprising maintaining the second cell suspension culture of oil-producing plant cells in the presence of the photosynthetic product generated by the first cell suspension culture and under conditions such that the cultured oil-producing plant cells produce fatty acid, oil, or a combination of fatty acid and oil.

20. The method of claim 14, further comprising the step of extracting the biological product from the second cell culture.

21. The method of claim 19, further comprising the step of extracting the fatty acid, oil, or combination of fatty acid and oil, from the plant cell culture of oil-producing plant cells.

22. The method of claim 20, further comprising purifying, or processing, or purifying and processing, the extracted biological product.

23. The method of claim 21, wherein the fatty acid, oil, or combination of fatty acid and oil, that is extracted is then further processed to convert it to a biofuel.

24. The method of claim 19, wherein the oil-producing plant cells present in the second cell suspension culture are differentiated plant cells.

25. The method of claim 24, wherein the differentiated plant cells are cells that are specialized in the production and storage of oils.

26. A two-culture system for producing a biological product, comprising a first cell suspension culture and a second cell culture, each as defined by claim 14 wherein the first cell suspension culture is a cell suspension culture of photosynthetic non-algal plant cells that is maintained in the presence of water, light, and in a carbonic acid-enriched growth medium, wherein the concentration of carbonic acid in the carbonic acid-enriched growth medium is at least about 0.1% w/v.

27. A carbon dioxide capture system comprising at least the first plant cell suspension culture as defined by claim 14 wherein the first cell suspension culture is a cell suspension culture of photosynthetic non-algal plant cells that is maintained in the presence of water, light, and in a carbonic acid-enriched growth medium, wherein the concentration of carbonic acid in the carbonic acid-enriched growth medium is at least about 0.1% w/v.

28. A photosynthetic non-algal plant cell suspension culture that is capable of producing a sugar-comprising photosynthetic product when maintained in the presence of water, light and in a carbonic acid-enriched growth medium, wherein the concentration of carbonic acid in the carbonic acid-enriched growth medium is at least about 0.1% w/v.

29. A carbonic acid-enriched growth medium as defined by, or suitable for use in, the method of claim 1, wherein the concentration of carbonic acid in the medium is at least about 0.1% w/v.

30. The method of claim 4, wherein the culture medium is maintained at a pH of about 4.5 to about 6.5, or about 4.5 to about 5.5, or up to about 6.4.

31. The method of claim 6, wherein the plant cell specialized for photosynthesis is a cell from the leaf or green tissue of a plant.

32. The method of claim 13, wherein the photosynthetic non-algal plant cell suspension culture has a volume that of at least 20,000 L, at least 30,000 L, at least 40,000 L, or at least 50,000 L.

33. The method of claim 16, wherein the proteinaceous product is a recombinantly-encoded proteinaceous product and the metabolite is ethanol.

34. The method of claim 17, wherein the cells of the second culture are bacterial cells, fungal cells, plant cells, animal cells or human cells.

35. The method of claim 18, wherein the microorganisms are yeast cells.

36. The method of claim 18, wherein the biological product is an alcohol.

37. The method of claim 19, wherein the photosynthetic product is a sugar.

38. The method of claim 21, wherein the fatty acid, oil, or combination of fatty acid and oil, that is extracted is purified to produce a purified extract.

39. The method of claim 21, wherein the fatty acid, oil, or combination of fatty acid and oil, that is extracted is used in a downstream process.

40. The method of claim 39, wherein the downstream process involves incorporation of the fatty acid, oil, or combination of fatty acid and oil, that is extracted into a product selected from the group consisting of a food product, a cosmetic and a lubricant.

41. The method of claim 21, wherein the fatty acid, oil, or combination of fatty acid and oil that is extracted is further purified to produce a purified extract, and the purified extract is used in a downstream process.

42. The method of claim 41, wherein the downstream process involves incorporation of the purified extract into a product selected from the group consisting of a food product, a cosmetic and a lubricant.

43. The method of claim 25, wherein the cells specialized in the production and storage of oils are mesoderm cells.

44. The method of claim 6, wherein the plant cell specialized for photosynthesis is a palisade, leaf mesoderm or petiole cell.

45. The method of claim 35, wherein the yeast cells are Saccharomyces species.

46. The method of claim 36, wherein the alcohol is ethanol.

Description

(1) The invention will be further understood with reference to the following non-limiting figures and experimental examples.

(2) FIG. 1 shows level of sugar in the culture medium of a subculture of photosynthetic cell suspension cultures, as described in section 2.2 and 6.0 of Example 2, grown in light conditions in a growth medium with no detectable levels of carbonic acid (the sole carbon source for photosynthesis was a gaseous mixture of 10% carbon dioxide and 90% air), allowing the culture to grow for 14 weeks, before modifying the conditions to generate carbonic acid levels of 35-40 g/L (i.e. about 3.5 to 4% w/v) in the culture medium (by increasing the concentration of carbon dioxide relative to air to 40% carbon dioxide by volume) and continuing to grow. For both the 10% and 40% CO.sub.2 feeds, the mean average diameter bubble size was 0.2 mm, the path length was 1.8 m, and the culture pressure was 3.2 atm.

EXAMPLES

Example 1

(3) A plant cell suspension culture was produced in which an unusually highly concentrated level of carbon dioxide, at 40% by volume, was bubbled, using liquid carbon dioxide from a tank which is piped directly to the reactor for conversion to gas and mixing with air to produce the bubbles. The mean average diameter bubble size was 0.2 mm, the path length was 1.8 m, the culture was maintained under a pressure is 3.2 atm, and the culture pH was 3.75, resulting in a level of carbonic acid of 35-40 g/L (i.e. about 3.5 to 4% w/v).

(4) As discussed above, to the Applicant's knowledge there is no other known or published plant culture which operates at this unusually high carbon dioxide concentration in a buffered media, and no previous reports that would have lead the skilled person to focus on providing carbonic acid in an adequate amount for a plant or algal cell suspension culture to use it as the substrate for photosynthesis, instead of using gaseous carbon dioxide.

(5) As shown below, the use of 40% carbon dioxide bubbles, under conditions suitable to form a carbonic acid-enriched growth medium, drives the reaction kinetics of photosynthesis forward to produce sugars and starches. Important to repeat, this is not at all classical photosynthesis. This is a unique liquid culture reaction system with very unusually high carbon dioxide concentrations under conditions that lead to enrichment of the medium with carbonic acid, wherein we must account for the Gibbs free energy and entropy values.

(6) As a result of this, we are able to reduce the amount of light required to drive photosynthesis, and thereby markedly reduce the energy consumption of the process. The system does not use a white light broad spectrum light process like classical photosynthesis.

(7) A production tank uses 12 LED arrays (+4 arrays of 1,500 W each) which are each rated for 500 watts. Thus, each tank has available electrical power of 12,000 total watts for sugar production, plus the energy derived from the high concentration of carbon dioxide as described below. The LED arrays were chosen at a very select frequency. Sunlight or white light in general contains a broad spectrum of frequencies. Another innovation in the technology package is the select use of 652 nm wavelengths, optimized for the particular plant cell component. In field crops, most of the energy incoming is wasted in the form of heat striking the plant leaves and other energy hits the ground and is absorbed and radiated back into the air. The LED arrays used are mounted both internally and externally to the tanks. In the past, some variations were attempted to utilize natural sunlight by conducting it through mirrors or other optics to enter the first step tank. However, very little increase in overall efficiency was observed in part because the wavelengths of natural sunlight are broad and only enter the tank during daylight hours. The arrays do contribute to the temperature of the tank fluids. The total temperature differential is 9 F. This indicates the overall efficiency of the LEDs is still not 100% and some energy is given up at the LED to glass external interface. In general, for optical energy going across a barrier with a differential in refractive index, in this case air and glass, the energy loss is about 0.4 dB, of which some is reflected back and some heats the glass on the outside of the tank. In any case, the temperature rise is anticipated and reasonable.

(8) This process achieves an overall energy efficiency of about 50%, wherein some goes to waste heat requiring cooling in the building in the summer months. If we were to have used broad spectrum lights and generate excessive amounts of heat, that overall efficiency would plummet. Thus it is not simply that the current process takes advantage of the thermodynamics of concentrated species begin added to the liquid culture but also the fact that the energy input is highly targeted to a wavelength optimized for this particular component of the plant cell culture.

(9) In a 24 hour period, once the tank has achieved optimal cell densities, the rate of sugar production is 1,000 kg/day in a solution that achieves a concentration of 50% sugars and starches. In addition, electrical power is used to move fluids inside the reactor. The duty cycle for the pumps is 100% meaning they are on a total of 24 hours during the day. The hp of these pumps are 0.81 hp. This translates to 1.1 kWatts. The total electrical power required for oil manufacture using the two step process is approximately 2 kWatt-hr per 1 liter of oil. That oil has an energy content of 34,000 BTU/liter. Overall, the net energy efficiency of electrical power and chemical energy from the carbon dioxide concentration to oil is about 50%. The balance is in wasted heat, released oxygen, and cell biomass growth and maintenance.

(10) There are several interesting things to note here. One is that part of the process is taking compressed carbon dioxide or gas under pressure and introducing it into the tank. The gas pressure exceeds the tank head pressure which is roughly 6 feet of head plus the gas backpressure. The tank gauge gas pressure is measured as 10 psi. Thus, some of the energy used in the process must consider the gas blowers or compressors used for the carbon dioxide. It may be for free in terms of gas transfer into the reactor but energy is required to move that gas from the flue stack or carbon dioxide tank into the liquid reactor. All energy required to concentrate and liquefy the carbon dioxide must be accounted for in the energy balance because this energy is returned in step one in the photosynthesis reactions.

(11) At a pH of 7.0, the partial pressure of carbon dioxide in the liquid is only 10.sup.8 whereas at a pH of 6.4 the partial pressure goes up to 10.sup.5. By the time the pH hits 5.92, the partial pressure of carbon dioxide has hit 10.sup.4 or 4 orders of magnitude higher concentration. It is quite evident that gas concentration changes significantly with pH. This brings us to several conclusions as demonstrated in the lab and demonstration scale reactors. That is, the reaction rate kinetics are a function of pH and energy is input into the system from the entering 40% carbon dioxide vapor.

(12) Conclusions:

(13) The rate and direction of a chemical reaction depends on the free energy, entropy, and concentration of the reactants and products as well as the temperature and pH of the system. Chemical reactions progress in the direction of high to low energy. We can estimate the direction of the chemical reaction, as well as the equilibrium concentrations of reactant and product, by examining the energy of the reactants and products.

(14) In nature, the concentration of the CO.sub.2 reactant (i.e. maximum CO.sub.2 concentration in air) is 0.04% v/v (i.e. 1 liter air contains 0.4 ml carbon dioxide), and so provides 0.0007904 g of CO.sub.2 per liter of air (since CO.sub.2 has a mass of 1.976 g per liter).

(15) The present example uses the CO.sub.2 at 40% volume with air so each liter of air contains 400 ml CO.sub.2 and so provides 0.7904 g of CO.sub.2 per liter of air.

(16) Accordingly, the present example uses 1,000 times the concentration of carbon dioxide compared to the use of atmospheric air. As all other concentrations can be considered to be constant it is this increase in reactant concentration that lowers the energy required by a calculated 1,000 times.

(17) Reported energy of formation for glucose is +2,826 KJ/mol. We have measured Energy of formation for glyceraldehyde as 65.98 KJ/mol. Reported energy of formation for glyceraldehydes is 59.8 KJ/mol.

(18) The sugar concentration in a leaf is usually 10 mg/g or 1% w/w. In contrast, the sugar concentration obtained by the in the current process is 500 mg/g or 50%.

(19) Therefore, if the energy is proportional to the product concentration/reactant concentration, and we consider carbon dioxide and glucose as the only variables (due to excess water and oxygen in both halves of the reaction) then: Plant ratio is 1/(molarity of carbon dioxide)=1/1.79636E-05=55,668.02 The ratio achieved by the process of the present example is 50/(molarity of carbon dioxide)=50/0.017964=2,783.40

(20) Therefore, we can calculate the photosynthetic efficiency ratio of a plant versus process of the present example as 55,668.016/2,783.4008=20. In other words, the current process is calculated to require 20 times less energy to produce photosynthetic sugars than the plant.

(21) Two molecules of glyceraldehyde form one molecule of glucose. Therefore in the current process, energy=65.98220=2,639.2 KJ/mol

(22) The difference in the energies in the two systems is due to the fact that energy is released as a compound moves from high concentration to a low concentration. This complies with the laws of thermodynamics.

(23) We can calculate this energy for the sugar production from the information above.

(24) As we know the difference in the energy per mole required to be inputted to make sugars in the plant and in the current process we can calculate the potential chemical energy available from the concentration of the carbon dioxide. This is because free energy also depends on the concentration of reactants and products. This is because the movement of molecules from a more to less concentrated state can perform work.

(25) If we take the difference in energy per mole glucose in plant and current process we get 2,507.24 KJ/mol.

(26) This equates to 13.93 KJ/g glucose.

(27) As we know that 1,000 kg glucose converts to 650 liters of oil (at 100% efficiency) then we can work out the energy provided by the carbon dioxide concentration.

(28) 1,000 kg glucose contains 13,929,111 KJ of this energy. This equates to 21,429.4 KJ/liter of oil produced.

(29) As demonstrated by measuring electrical power inputs and oil produced in the experimental system, that rather than traditional photosynthesis, a pseudo photosynthesis process more properly named Photo Mediated Enzymatic Glycogenesis takes place.

(30) This is due to the higher concentrations of carbon dioxide and its presence as carbonic acid in the media. Carbonic acid will react with water to form HCOO.sup. and H.sup.+ ions. Formation of these ions releases energy in the form of heat that can be used to enhance the rate of subsequent reactions. The Rubisco enzymes will be able to attach to the HCOO.sup. ion and convert it to C.sub.3H.sub.6O.sub.3 and oxygen.

(31) ##STR00003##

(32) As can be seen above, the C.sub.3H.sub.6O.sub.3 can exist as two isomers, glyceraldehyde and dihydroxyacetone. It is well reported in literature that these two isomers can combine with the release of energy to form glycerose (a simple aldose sugar) that is the basis of glycerol and fatty acid production.

(33) Total energy provided in the current process: Light energy=6,000 J/s=518,400,000 J/Day Moles of sugar produced=1,000,000 gm/(180 gm/mole)=5,555.56 per day Light energy per mole=93,312 J Energy from reactants per mole=2,639.2 KJ/mol(65.98*2)=2,507.24 KJ/mol.

(34) Extra energy from temperature increase above 298 K is not significant as the 6 degree difference in temperatures is only 2% and so gives a contribution of 55.8 KJ/mol glucose.

(35) If the current process is changed so that the final concentration of products is kept to 250 gm/liter of sugars, the energy required is lowered considerably as the contribution from the reactants stays the same but the required energy for the final concentration of products is reduced by half.

Example 2

(36) This example relates to an analysis of the energy balance for rapeseed oil production, by making a photosynthetic product in a first culture tank according to an exemplary method of the present invention and using the photosynthetic product as a carbon source for the growth of a suspension culture of plant cells that produce rapeseed oil in a second culture tank.

(37) Summary

(38) The purpose of this example is to outline the energy balance in an exemplary process. The known energy inputs are compared to the energies that can be potentially released from the process. This example does not seek to provide a full biochemical model for the process; rather it describes the reactions that are known in the process.

(39) Materials and Methods:

(40) 1. Induction and Maintenance of Photosynthetic Cell Suspension Culture

(41) 1.1 Initiation of Callus Cultures: Preparation of Callus Induction Media

(42) Materials: Callus induction media solution; Distilled H.sub.2O to 100%; 3.0% sucrose; 1.0% NAA (naphthalene acetic acid) 0.004% stock solution; 0.44% Murashige and Skoog Basal powdered medium.

(43) Equipment. Glass bottle with cap; Magnetic stirrer; Sterile plastic plant culture dishes; Glass pipettes; pH meter; Autoclave; Laminar flow cabinet; Balance; Nescofilm; Phytagel; 1M NaOH solution; 0.1M NaOH solution.

(44) Callus induction media was prepared using Murashige and Skoog (MS) media obtained from Sigma, with 3% sucrose and 1% naphthalene acetic acid (from a concentrated stock solution of 0.004% w/v.

(45) The prepared media was pH was adjusted to pH 5.75 and solidified with 0.2% phytagel.

(46) The media was autoclaved for 20 mins at 121 C. and then poured out into sterile plastic plant tissue culture dishes.

(47) 1.2 Initiation of Callus Cultures: Sterilisation of Plant Tissue

(48) Reagents: Media prepared previously (section 1.1); Agrostis tenuis plant tissue.

(49) Equipment: Sterile glass beakers; Sterile distilled water; Sterile scalpel; Sterile tweezers; 10% bleach solution; 70% ethanol solution; 1M NaOH solution; 0.1M NaOH solution.

(50) Plant tissue of Agrostis tenuis was sterilised by immersion in 70% ethanol for 2 minutes, followed by immersion in 10% bleach solution for 10 minutes; then washed three times with sterile (autoclaved) distilled water. The sterile plant tissue was aseptically cut into disk shapes in a sterile laminar flow cabinet. Slices were placed onto the prepared plates containing callus induction media, and plates were sealed with Nescofilm. The plates were placed in the dark at 27 C. and callus formation began to appear after about 1 month.

(51) 1.3 Media Preparation for Established Cultures

(52) Reagents: Distilled H.sub.2O to 100%; 3% sucrose; 0.44% Murashige and Skoog Basal powdered medium; 1% NAA (naphthalene acetic acid) 0.004% stock solution; 0.01% Vitamin solution (0.05% pyridoxalhydrochlorid, 0.10% thiamine dichloride and 0.05% g nicotinic acid); 1M NaOH solution; 0.1M NaOH solution.

(53) Equipment: 1 L glass bottle; Magnetic stirrer; 20 250 m conical flasks; 20 sheets of foil approximately 2020 cm; Glass pipettes; pH meter; Autoclave; Laminar flow cabinet; Balance.

(54) Method: Mix 3% sucrose, 0.44% MS powder, 1% NAA stock and 0.01% vitamin stock and prepare to 100% with distilled H.sub.2O. Mix using a magnetic stirrer until all dry components dissolved, then pH adjust with 1M and 0.1M NaOH, to 5.75. Take 20 250 ml conical flasks. To each add 50 ml media and seal neck of flask with foil. Sterilize in autoclave, at 121 C., 103 kPa, for 25 minutes. Immediately following sterilization, place flasks in laminar flow cabinet and allow to cool to ambient temperature.

(55) 1.4 Inoculation and Subculture of Established Cultures

(56) Reagents: Friable callus; 70% Ethanol.

(57) Equipment: Laminar flow cabinet; Bunsen burner; Prepared media; 20 sterile sheets of foil approximately 2020 cm; Several pairs of tweezers or small forceps; Wide spatulas with holes.

(58) Method: Sterilize inside of laminar flow cabinet with 70% ethanol. Sterilize all tweezers and spatulas by dipping in 70% ethanol, then flaming till red hot. Allow to cool inside laminar flow cabinet.

(59) Initial inoculation: Remove foil from prepared media flask. Take sterilized tweezers and remove thumbnail sized pieces of friable callus from the plant tissue. Break up into finely dispersed cells and add to flask. Aim to add approximately 5 g tissue to 50 ml media (10% w/v). Flame the neck of the flask, and cover with a sterile sheet of foil. Place the flask on a shaker at 120 rpm, in a light room heated to 27 C. Leave until a thick, dispersed cell suspension culture can be observed (approximately 2 weeks).

(60) Subculture: Remove foil from prepared media flask. Remove foil from flask containing dispersed cell suspension cultures (produced by initial inoculation, as above). Take wide spatula with holes, sterilize, allow to cool and scoop out the cells. Add these cells to the fresh media. Aim to add approximately 5 g tissue to 50 ml media. Flame the neck of the flask, and cover with a sterile sheet of foil. Place the flask on a shaker at 120 rpm, in a dark room heated to 27 C. After 14 days, use the cell suspension culture for further subcultures.

(61) 2.0 Photosynthetic Cell Suspension Culture

(62) 2.1 Media Preparation for Cell Suspension Cultures

(63) Reagents: Distilled H.sub.2O to 100%; 3% sucrose; 0.44% Murashige and Skoog Basal powdered medium; 1% NAA (naphthalene acetic acid) 0.004% stock solution; 0.01% Vitamin solution (0.05% pyridoxalhydrochlorid, 0.10% thiamine dichloride and 0.05% nicotinic acid); 1M NaOH solution; 0.1M NaOH solution; Compressed Air; Compressed Carbon Dioxide (vapour release).

(64) Method: Mix 3% sucrose, 0.44% MS powder, 1% NAA stock and 0.01% vitamin stock and prepare to 100% with distilled H.sub.2O. Mix until all dry components have dissolved, then pH adjust with 1M and 0.1M NaOH, to 5.75. Sterilize media and allow to cool to ambient temperature before use.

(65) 2.2 Subculture of Cell Suspension Cultures

(66) Reagents: Friable cells; Media prepared previously (section 1.1).

(67) Method: Take cell suspension culture in the exponential phase of growth. Filter cells from media, and use these cells to inoculate fresh media. Aim to add cells to media at approximately 10% w/v. Agitate the culture vessel at 120 rpm, at 27 C., and in light conditions. In light conditions pass through carbon dioxide and air mixture at a concentration of 10% carbon dioxide by volume, allowing the culture to grow under these conditions for 14 weeks, before increasing the concentration of carbon dioxide relative to air to 40% carbon dioxide by volume and continue to grow. For both the 10% and 40% CO.sub.2 feeds, the mean average diameter bubble size was 0.2 mm, the path length was 1.8 m, and the culture pressure was 3.2 atm. There were no detectable levels of carbonic acid in the culture fed with 10% level of CO.sub.2, and the CO.sub.2 feed caused no significant change in the culture medium pH. The 40% CO.sub.2 feed resulted in a carbonic acid level of 35-40 g/L (i.e. about 3.5 to 4% w/v) but higher levels can be achieved and used in the practice of this method, and (as a result of the formation of carbonic acid) the pH of the medium dropped from about 5.5 to 3.7.

(68) For further subcultures, the cells should be used when the culture has reached the logarithmic growth phase. For harvesting of the photosynthetic product, glycerose, the cells should be used when the culture has reached the stationary phase. Glycerose was harvested from the culture fed with 40% CO.sub.2 by removal of the glycerose-enriched media from the cells in the cell culture, using chromatography separation.

(69) 3.0 Induction and Maintenance of Rapeseed Oil-Producing Cell Suspension Culture

(70) 3.1 Initiation of Callus Cultures from Brassica napus; Preparation of Callus Induction Media

(71) Materials: Callus induction media solution; Distilled H.sub.2O to 100%; 3.0% sucrose; 1.0% NAA (naphthalene acetic acid) 0.004% stock solution; 0.44% Murashige and Skoog Basal powdered medium.

(72) Equipment: Glass bottle with cap; Magnetic stirrer; Sterile plastic plant culture dishes; Glass pipettes; pH meter; Autoclave; Laminar flow cabinet; Balance; Nescofilm; Phytagel; 1M NaOH solution; 0.1M NaOH solution.

(73) Callus induction media was prepared using Murashige and Skoog (MS) media obtained from Sigma, with 3% sucrose and 1% naphthalene acetic acid (from a concentrated stock solution of 0.004% w/v. The prepared media was pH was adjusted to pH 5.75 and solidified with 0.2% phytagel. The media was autoclaved for 20 mins at 121 C. and then poured out into sterile plastic plant tissue culture dishes.

(74) 3.2 Initiation of Callus Cultures from Brassica napus: Sterilisation of Plant Tissue

(75) Reagents: Media prepared previously (section 1.1); Brassica napus plant tissue.

(76) Equipment: Sterile glass beakers; Sterile distilled water; Sterile scalpel; Sterile tweezers; 10% bleach solution; 70% ethanol solution; 1M NaOH solution; 0.1M NaOH solution.

(77) Plant tissue of Brassica napus was sterilised by immersion in 70% ethanol for 2 minutes, followed by immersion in 10% bleach solution for 10 minutes; then washed three times with sterile (autoclaved) distilled water. The sterile plant tissue was aseptically cut into disk shapes in a sterile laminar flow cabinet. Slices were placed onto the prepared plates containing callus induction media, and plates were sealed with Nescofilm. The plates were placed in the dark at 27 C. and callus formation began to appear after about 1 month.

(78) 3.3 Media Preparation for Established Cultures

(79) Reagents: Distilled H.sub.2O to 100%; 3% sucrose; 0.44% Murashige and Skoog Basal powdered medium; 1% NAA (naphthalene acetic acid) 0.004% stock solution; 0.01% Vitamin solution (0.05% pyridoxalhydrochlorid, 0.10% thiamine dichloride and 0.05% g nicotinic acid); 1M NaOH solution; 0.1M NaOH solution.

(80) Equipment: 1 L glass bottle; Magnetic stirrer; 20 250 m conical flasks; 20 sheets of foil approximately 2020 cm; Glass pipettes; pH meter; Autoclave; Laminar flow cabinet; Balance.

(81) Method: Mix 3% sucrose, 0.44% MS powder, 1% NAA stock and 0.01% vitamin stock and prepare to 100% with distilled H.sub.2O. Mix using a magnetic stirrer until all dry components dissolved, then pH adjust with 1M and 0.1M NaOH, to 5.75. Take 20 250 ml conical flasks. To each add 50 ml media and seal neck of flask with foil. Sterilize in autoclave, at 121 C., 103 kPa, for 25 minutes. Immediately following sterilization, place flasks in laminar flow cabinet and allow to cool to ambient temperature.

(82) 3.4 Inoculation and Subculture of Established Cultures

(83) Reagents: Friable callus; 70% Ethanol.

(84) Equipment: Laminar flow cabinet; Bunsen burner; Prepared media; 20 sterile sheets of foil approximately 2020 cm; Several pairs of tweezers or small forceps; Wide spatulas with holes.

(85) Method: Sterilize inside of laminar flow cabinet with 70% ethanol. Sterilize all tweezers and spatulas by dipping in 70% ethanol, then flaming till red hot. Allow to cool inside laminar flow cabinet.

(86) Initial inoculation: Remove foil from prepared media flask. Take sterilized tweezers and remove thumbnail sized pieces of friable callus from the plant tissue produced in section 3.2. Break up into finely dispersed cells and add to flask. Aim to add approximately 5 g tissue to 50 ml media (10% w/v). Flame the neck of the flask, and cover with a sterile sheet of foil. Place the flask on a shaker at 120 rpm, in a dark room heated to 27 C. Leave until a thick, dispersed cell suspension culture can be observed (approximately 2 weeks).

(87) Subculture: Remove foil from prepared media flask. Remove foil from flask containing dispersed cell suspension cultures (produced by initial inoculation, as above). Take wide spatula with holes, sterilize, allow to cool and scoop out the cells. Add these cells to the fresh media. Aim to add approximately 5 g tissue to 50 ml media. Flame the neck of the flask, and cover with a sterile sheet of foil. Place the flask on a shaker at 120 rpm, in a dark room heated to 27 C. After 14 days, use the cell suspension culture for further subcultures.

(88) 4.0 Oil-Producing Cell Suspension Culture

(89) 4.1 Media Preparation for Cell Suspension Cultures

(90) Reagents: Distilled H.sub.2O to 100%; 3% sucrose; 0.44% Murashige and Skoog Basal powdered medium; 1% NAA (naphthalene acetic acid) 0.004% stock solution; 0.01% Vitamin solution (0.05% pyridoxalhydrochloride, 0.10% thiamine dichloride and 0.05% nicotinic acid); 1M NaOH solution; 0.1M NaOH solution; Compressed Air.

(91) Method: Mix 3% sucrose, 0.44% MS powder, 1% NAA stock and 0.01% vitamin stock and prepare to 100% with distilled H.sub.2O. Mix until all dry components have dissolved, then pH adjust with 1M and 0.1M NaOH, to 5.75. Sterilize media and allow to cool to ambient temperature before use.

(92) 4.2 Subculture of Cell Suspension Cultures

(93) Reagents: Friable cells; Media prepared previously (section 1.1).

(94) Method: Take cell suspension culture from section 3.4 in the exponential phase of growth. Filter cells from media, and use these cells to inoculate fresh media. Aim to add cells to media at approximately 10% w/v. Agitate the culture vessel at 120 rpm, at 27 C., and in dark conditions, with aeration using the compressed air. For further subcultures, the cells should be used when the culture has reached the logarithmic growth phase. Due to the pH of 4.0-5.5 the oil is secreted from the cells and rises to the top of the media where it may be floated off.

(95) 5.0 Two-Culture Oil Production System

(96) The sugar produced by the cell culture of photosynthetic cells (section 2.2) secretes naturally into the surrounding media. As the air and carbon dioxide mix is fed into the vessel via diffuser plates located at the bottom of the vessel, this gas flow also provide lift to the cells and so performs a constant mixing function.

(97) Conversely, the culture medium of the oil-producing cell culture (section 4.2) becomes sugar depleted during growth as the sugar in the medium is used by the cells for the production of oil.

(98) Once per day sugar-enriched media is removed from the established culture of photosynthetic cells in carbonic-acid enriched medium (fed with 40% CO.sub.2), and the sugar-enriched media is used to feed the cell culture of oil-producing cells, using the following steps Step 1. Remove 10% by volume of the sugar-depleted medium from the culture of oil-producing cells, and store the removed sugar-depleted medium for later addition to the cell culture of photosynthetic cells in step 3, below. Step 2. Turn off the gas (air and carbon dioxide) feed into the cell culture of photosynthetic cells, to allow the cells in culture to settle to the bottom of the culture tank. Extract 10% by volume of the sugar enriched media from the top of the vessel, and add it to the culture of oil-producing cells to enhance the level of sugars available to the cells in the oil-producing cell culture. Step 3. Feed the sugar-depleted media that is stored in step 1, above, into the sugar producing vessel, so that the photosynthetic cells in the culture replenish it with sugar.

(99) It will be appreciated that other volumes of sugar-enriched culture medium and sugar-depleted medium can be transferred between the cultures of photosynthetic cells and the culture of oil-producing cells, and that the transfer may occur at a greater or lesser frequency than once per day. However, we have found that a transfer of 10% volume every 24 hours provides suitable results.

(100) The rapeseed oil produced by the cells in the oil-producing cell culture is excreted from the cells due to the pH of the culture being maintained in the range of 4.0-5.5. Since the excreted oil has a lower specific gravity than the surrounding medium, and is also immiscible with the medium, it floats to the surface where it forms a layer which is then removed via a pipe located above the level of the interface between the medium and that oil layer.

(101) 6.0 Results

(102) The subculture of photosynthetic cell suspension cultures, as described in section 2.2 above was grown in light conditions in a growth medium with no detectable levels of carbonic acid (the sole carbon source for photosynthesis was a mixture of 10% carbon dioxide and 90% air), allowing the culture to grow for 14 weeks, before modifying the conditions to generate carbonic acid levels of 35-40 g/L (i.e. about 3.5 to 4% w/v) in the culture medium (increasing the concentration of carbon dioxide relative to air to 40% carbon dioxide by volume) and continuing to grow.

(103) The level of sugar in the culture medium was determined at the start of each week of culture. The results are shown below in Table 1 and in FIG. 1.

(104) TABLE-US-00001 TABLE 1 CO.sub.2 level/ Measured carbonic sugar level Week acid level (g/L) 0 10%/ND 0 1 10%/ND 0.19 2 10%/ND 0.27 3 10%/ND 0.41 4 10%/ND 0.47 5 10%/ND 0.47 6 10%/ND 2.56 7 10%/ND 3.14 8 10%/ND 3.15 9 10%/ND 6.57 10 10%/ND 9.54 11 10%/ND 10.11 12 10%/ND 10.17 13 10%/ND 13.94 14 10%/ND 16.57 15 40%/ 53.83 3.5-4% w/v 16 40%/ 81.71 3.5-4% w/v 17 40%/ 69.89 3.5-4% w/v [. . .] [. . .] [. . .] 21 40%/ 67.57 3.5-4% w/v 22 40%/ 67.57 3.5-4% w/v 23 40%/ 66.97 3.5-4% w/v 24 40%/ 67.14 3.5-4% w/v 25 40%/ 67.15 3.5-4% w/v 26 40%/ 67.05 3.5-4% w/v 27 40%/ 67.05 3.5-4% w/v 28 40%/ 71.02 3.5-4% w/v 29 40%/ 70.75 3.5-4% w/v 30 40%/ 73.98 3.5-4% w/v 31 40%/ 73.26 3.5-4% w/v ND = Not detectable

(105) The data indicate that, after about 10 weeks, the photosynthetic culture fed on 10% gaseous CO.sub.2 as the carbon source is well established and, despite having carbonic acid below detectable levels, thereafter shows relatively stable levels of sugar production during the continued use of the 10% CO.sub.2 feed, albeit that there is a gradual increase observable as the culture grows between weeks 10-14.

(106) However, the effect of modifying the conditions to produce a medium with enhanced levels of carbonic acid, at about 3.5-4% w/v, produces an immediate, dramatic and stable increase in sugar production without any increase in the light energy input to the system. This shows that, compared to the use of 10% gaseous CO.sub.2 as the sole carbon source for photosynthesis, the energy efficiency with which photosynthesis is able to proceed is approximately or greater than 4-fold higher when the conditions used provide a culture medium with enhanced levels of carbonic acid.

(107) Common Misconceptions:

(108) In farming practice, rapeseed is grown in air and soil. The air contains low amounts of carbon dioxide. Growth rates are governed by a classical photosynthesis reaction which is carbon dioxide, gas, and water, combining with light to form solid (soluble) glucose.

(109) In an exemplary process according to the present invention, we do not grow in air or soil. The formation of sugars and starches is catalytic, in an aqueous media that enables carbon dioxide levels 1,000 times that used in traditional farming. The catalysts used are naturally occurring enzymes, used at unusually high concentrations compared to whole plants with roots and leaves.

(110) The exemplified process does not use any GMO (Genetically Modified Organisms). The oil thus produced can be considered food grade.

(111) Additionally, the exemplified process does not use any solvents for oil extraction. Thus, the mass and energy balance does not include oil recovery costs. An advantageous feature of the process is the ability to harvest oil by floatation without cell destruction, a dramatic difference compared to oil seeds or algae.

(112) In some operations, which further involve conversion of the oil to biodiesel using sodium methoxide, will include the step of drying the oil to remove 1% moisture (water), and this can have a slight impact to the overall energy balance to end-product form, although that is not calculated in this example. Rather, this example assesses the amount of energy required to produce the oil, via an exemplary method according to the present invention, and its potential energy in the form of heat of combustion.

(113) General Principles:

(114) Energy is only ever displaced or changed. The amount of input energy is usually greater than the amount of output energy as there are always slight inefficiencies in any process. Furthermore, the energy of a substance will vary depending upon the state it is in, (i.e. a solid, a gas, a liquid) and when energy is transferred to a different state, there is an energy reaction, e.g. heat.

(115) Input Energy:

(116) We have three energy inputs: light, carbon dioxide and activation energy. We can measure the energy of each of the inputs into the exemplified system.

(117) We measure the energy input for light by the amount of electricity consumed. In this example, the light used for CO.sub.2 conversion to sugars and starches in the chloroplast tank is not a white light broad spectrum light like the sun. Rather the light is from LED arrays which are chosen to be at a select frequency between 600-700 nm (for example, 652 nm may be used), a wavelength optimized for the particular plant cell component in the example, as this is the wavelength that is most efficiently used by chloroplasts. The LED arrays contribute to a temperature rise of the tank fluids by 1-2 C., and can be as high as 6 C., but this is anticipated and reasonable.

(118) Energy is also consumed by preparing concentrated carbon dioxide for use in the exemplified process. We know from published information (e.g. Leskovac et al, 2008, Indian Journal of Biochemistry & Biophysics, 45, 157-165) that the energy of the concentration of carbon dioxide is 62 kJ per mole. From our experiments, we have shown that 3.117 Kg of carbon dioxide is required for 1.0 kg of oil. The molecular weight of CO.sub.2 is 44 so there are (311744) moles added per kg of oil, which equates to 70.84 moles of CO.sub.2 per Kg of oil. The energy input required to supply concentrated carbon dioxide for the product of 1 kg of oil is therefore: 62 kJ70.84=4,392.13 kJ/kg of oil.

(119) Activation and Transition Energy: A Three Step Process

(120) Reaction 1: Chemical Reaction

(121) Much of the chemical energy is a function of the manner in which the carbon dioxide is added to the media.

(122) In this example, this is performed by passing a stream of carbon dioxide gas into a stream of air which then mixes to form a stream of input gas with a level of carbon dioxide of 40% by volume.

(123) This gas stream is passed into the liquid media via diffusion plates which provide micro bubbles of gas which are quickly absorbed into the liquid media. Note that growing seed crops in soil, the carbon dioxide level in air is only 380 ppm. In contrast, the exemplified process operates in liquid (not air) at 1,000 times the concentration of carbon dioxide in air, and with an enzyme concentration many times that of whole plants in soil which expend energy on roots, seeds and vascular tissues that are not required in the photosynthetic cell suspension culture used in the present invention.

(124) The absorption of the gas into the media means that the carbon dioxide reacts with the water in the media which leads to the production of carbonic acid. We have determined the optimum reaction rate kinetics (i.e. the speed at which the reaction takes place and how complete the reaction will be) for the absorption of the CO.sub.2 into the media. Conclusions demonstrated in the lab, and in demonstration scale reactors, also show that the reaction rate kinetics is a function of the pH and the concentration level of CO.sub.2.

(125) The equation that describes the first step is:
CO.sub.2+H.sub.2O.fwdarw.H.sub.2CO.sub.3(carbonic acid)
Reaction 2: Activation

(126) As the carbon dioxide is absorbed into the media, and turns into carbonic acid, energy (activation energy) is given off, in line with the laws of thermodynamics (i.e. energy is released as a compound moves from high concentration to a low concentration).

(127) Carbonic acid is not stable at the temperatures in the tanks and will also react with the enzymes within the cells that are present in the media. As a result the carbonic acid will break down into hydrogen carbonate (HCO.sub.3.sup.) and hydrogen (H.sup.+) ions. This reaction can be described as follows:
H.sub.2CO.sub.3.fwdarw.H.sup.++HCO.sub.3.sup.(bi-carbonate)
Reaction 3: Transition

(128) The enzymes of the Rubisco pathway can use the carbonate ions (HCO.sub.3 or CO.sub.3) to produce hydrocarbon units and these hydrocarbon units will be joined together. In humans and plants, the highest activity enzyme, with the highest turnover of any known enzyme, is carbonic anhydrase. It allows carbon dioxide exchange in the lungs at an incredible rate. The plant cells in cell suspension culture used in the present example contain these enzymes and the process is able to take exceptional advantage of the high turnover rate in the catalytic reaction of carbon dioxide to form complex carbohydrates.

(129) The reaction can be described as follows:
H.sup.++HCO.sub.3.sup..fwdarw.H.sub.2CO(carbohydrate)+2OH.sup.(hydroxide ion)

(130) Energy is needed for these units to be joined together. This energy is provided by NADPH being broken down to NADP+H.sup.+. As the amount of NADPH available in the reaction system is limited to the amount present in the cultures cells, then in order to keep the reaction going forward, NADP must be converted back to NADPH. This happens by using the spare hydrogen ions from the breakdown of carbonic acid as described in Reaction 3. It is cyclical.

(131) Overall, the production of glyceraldehyde from CO.sub.2, via Reactions 1-3 as defined above, can be described as follows:
3H.sub.2CO.fwdarw.H.sub.6C.sub.3O.sub.3

(132) The process uses up 4 NADPH to NADP for each molecule of glyceraldehyde produced.

(133) As the hydrogen ions are used up the pH would be expected to rise. But we have observed that, in practice, it does not, which must mean that hydrogen ions are continually formed. This is due to the continual supply of carbonic acid (in this example, by the continued supply of CO.sub.2), which in turn continues to be broken down into the carbon and hydrogen ions.

(134) Our results show that the amount of hydrogen ions is proportional to the amount of CO.sub.2. From that understanding, and knowing the pH measurements, the volume of liquid and the amount of CO.sub.2 added to the system, the amount of H ions present in the system can be calculated. Furthermore, knowing the amount of energy associated with a single hydrogen ion, the amount of energy at any given time in the system can be calculated. Note that this liquid enzymatic catalytic system is a dramatic departure from classical farming.

(135) Calculating the Amount of System Energy:

(136) We have experimentally determined that 3.117 Kg of carbon dioxide is required for the production of 1.0 Kg of oil in the exemplified Two-Culture oil production system as defined above in Section 5.0. We have determined that the amount of oil that is made per minute is 0.415 Kg.

(137) Therefore (3.1170.415)=1.2935 Kg of CO.sub.2 is used per minute by the exemplified system. Since the molecular weight of CO.sub.2 is 44, the exemplified system is therefore using (1293.5 g44=) 29.39 moles of CO.sub.2 per minute.

(138) The amount of energy associated with one H.sup.+ is 13.6 electron volts which equals=2.1810.sup.18 joules. In one mole of H.sup.+ there therefore are 6.02210.sup.23 molecules (Avagadro's constant). Therefore, one mole of a hydrogen ions has an energy of 2.1810.sup.186.02210.sup.23 which=1.3110.sup.3 kJ.

(139) Therefore, knowing that the exemplified system uses 29.39 moles of CO.sub.2 per minute, and knowing the CO.sub.2 forms carbonic acid which then breaks down to form at least one hydrogen ion, there will be (29.391.3110.sup.3 kJ) or 38,501 kJ energy associated with the hydrogen ions per minute.

(140) 38,501 kJ per minute energy is created which produces 0.415 Kg of oil per minute. The amount of energy therefore within the system to create 1.0 kg of oil is (38,5010.415)=1.5510.sup.5 i.e. 155,000 kJ per kg of oil.

(141) Total Input Energy is therefore:

(142) TABLE-US-00002 Energy Description Measurement Gauge Measurement Input Energy The energy associated with the 1. Consumption of electricity. 1. 1,990 kJ/kg different inputs i.e. light, CO.sub.2, 2. Accepted published criteria. 2. 4392.136 kJ/kg and the media: 3. Measured as a function of pH. 3. Included in activation energy. 1. Light Measured as a function of pH. 155,000 KJ/Kg 2. CO.sub.2 3. Media Activation The diffusion of the CO.sub.2 into the media causes the CO.sub.2 to breakdown which releases energy. TOTAL 161,382 KJ/Kg
Release of Oil:

(143) The Rubisco enzymes in the cultured plant cells will be able to attach to the HCO.sub.3.sup. and convert it to C.sub.3H.sub.6O.sub.3 and oxygen. The glyceraldehyde is removed from the first tank and passed into the second tank to act as a carbon course for a cell suspension culture of plant cells that produce and release rapeseed oil.

(144) C.sub.3H.sub.6O.sub.3 can exist as two isomers, which are glyceraldehyde and dihydroxyacetone. Literature reports that these two isomers can combine, with the release of energy, to form glycerose (a simple aldose sugar) which is the basis of glycerol and fatty acid production to create oil, which is represented in the following formula:

(145) ##STR00004##
Energy Outputs:

(146) The measurable energy outputs of the exemplified system are the potential energy (combustion) of the rapeseed oil, and the heat produced. Similarly to field grown crops, the process also releases oxygen.

(147) Combustion of rapeseed oil is known to be 39.59 MJ/kg or 39,590 kJ/kg.

(148) As a result of the combination of the inputs, there is a temperature rise proportional to the amount and rate of CO.sub.2 and air mix. The more CO.sub.2 that is added, the higher the temperature rises. Our data shows that there is about a 5 C. rise in the exemplified system. Formation of the hydrogen ions also releases energy in the form of heat. The heat generated is a form of energy that can be used to enhance the rate of subsequent reactions, as the hotter the temperature, the more a molecule vibrates and therefore combines more easily.

(149) The heat evolved from the tank is 13,196 kJ, which is 22.0675 kJ/kg. This is calculated based on the heat capacity of the stainless steel tanks and the temperature rise of 5 C.

(150) Total Energy output is therefore:

(151) TABLE-US-00003 Energy Description Measurement Gauge Measurement Oil The energy Combustion of Oil 39,590 kJ/kg in the oil. Heat Heat Temperature 22.06 kJ/kg Cellular The energy Very difficult Negligible % Metabolism used up to to measure maintain the cells TOTAL 39,612 kJ/kg
Energy Balance:

(152) The total energy input is a combination of three factors, light, CO.sub.2, and the media. The combination of those elements creates additional significant energy within the system. The output energy is the combustion of oil and heat.

(153) TABLE-US-00004 Input Energy: (KJ/Kg) Output Energy: (KJ/Kg) LEDs 1990 39,590 Combustion of oil Concentration of CO.sub.2 4392 22 Heat Activation Energy 155,000 Total 161,382 .fwdarw. 39,612

(154) All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.