阅读说明：本技术 生物油及其制备与应用 (Biological oil and preparation and application thereof ) 是由 J·C·利普迈耶 J·W·普菲弗三世 J·M·汉森 K·E·阿普特 W·R·巴克利 P·W 于 2008-09-08 设计创作，主要内容包括：本发明提供了生物油及其制备与应用。生物油优选采用含纤维素作为主要的碳源的原料,由一种或多种微生物经异养发酵进行制备。本发明还提供了制备脂质基生物燃料以及使用这些生物油的食品、营养品和药品的方法。(The invention provides biological oil and preparation and application thereof. The bio-oil is preferably produced by heterotrophic fermentation of one or more microorganisms using a feedstock containing cellulose as a major carbon source. The invention also provides methods of making lipid-based biofuels and foods, nutraceuticals, and pharmaceuticals using these bio-oils.)

1. A method of producing a bio-oil, the method comprising culturing a microorganism of the genus pseudobacteriaceae via heterotrophic fermentation using a feedstock comprising cellulose as a carbon source, wherein about 11-99% of the unsaturated fatty acids in the bio-oil are polyunsaturated fatty acids.

2. The method of claim 1, wherein greater than about 50% of the unsaturated fatty acids in the bio-oil are polyunsaturated fatty acids.

3. The method of claim 1, wherein the microorganism is a thraustochytrid.

4. The method of claim 1, wherein the microorganism is selected from the group consisting of: microorganisms of the genus Schizochytrium, microorganisms of the genus Thraustochytrium, and microorganisms of the genus Uken chytrid.

5. The method of claim 1, wherein said microorganism saccharifies said cellulose.

6. The method of claim 1, wherein the microorganism is tolerant or capable of degrading a feedstock component selected from the group consisting of: lignans, hemicellulose, vegetable oils, plant extracellular polysaccharides, and combinations thereof.

7. The method of claim 1, wherein the microorganism is a genetically modified microorganism.

8. The method of claim 1, wherein the microorganism produces oil in triglyceride form in an amount of about 25 to 85% by weight of its biomass dry weight.

9. The method of claim 1, further comprising performing spontaneous lysis or induced lysis of the microorganism after the microorganism produces oil in triglyceride form in an amount of about 30 to 90% by weight of its biomass dry weight.

10. The method of claim 1, further comprising inducing lysis of said microorganism by subjecting said microorganism to conditions favorable to lysis selected from the group consisting of pH, temperature, presence of an enzyme, presence of a detergent, physical disruption, and combinations thereof.

11. The method of claim 1, wherein the cellulose-containing feedstock comprises a cellulose source selected from the group consisting of: pasture, sugar cane, agricultural waste, waste paper, sewage, wood, green plant kingdom organisms, and combinations thereof.

12. The method of claim 1, wherein the fermentation is performed in a bacterial fermentor.

13. The method of claim 1, wherein the fermentation is performed in a fermentor selected from the group consisting of: fiber-reinforced polymer fermentation tanks, metal matrix composite fermentation tanks, ceramic matrix composite fermentation tanks, thermoplastic composite fermentation tanks, metal fermentation tanks, epoxy resin-lined carbon steel fermentation tanks, plastic fermentation tanks, glass fiber fermentation tanks, and concrete fermentation tanks.

14. The method of claim 1, wherein the fermentation is performed in a fermentor submerged in water.

15. The method of claim 1, wherein the fermentation is conducted in fermenters having a cooling system in series, such that cooling water from a first fermenter or set of fermenters in series is used as cooling supply water in a second fermenter or set of fermenters in series.

16. The method of claim 1, wherein the fermentation is conducted in fermenters having a series gas system, such that off-gas discharged from a first fermenter or series of fermenters in the series is used as a gas supply in a second fermenter or series of fermenters.

17. A method of producing biodiesel, the method comprising:

(a) cultivating a microorganism of the genus Pseudomonadaceae via heterotrophic fermentation using a feedstock comprising cellulose as a carbon source to produce a bio-oil, wherein about 11-99% of the unsaturated fatty acids in the bio-oil are polyunsaturated fatty acids; and

(b) transesterifying the bio-oil to form biodiesel.

18. The method of claim 17, wherein greater than about 50% of the unsaturated fatty acids in the bio-oil are polyunsaturated fatty acids.

19. The method of claim 17, wherein the transesterification of the bio-oil is performed with an alcohol derived from an alcohol production process.

20. The method of claim 17, further comprising using glycerol produced by transesterification of the bio-oil as a carbon source for a subsequent fermentation process to produce an alcohol or bio-oil.

21. The method of claim 20, wherein the subsequent fermentation process cultures the microorganism that is capable of utilizing the glycerol as a carbon source.

22. A method of making a jet biofuel, the method comprising:

(a) cultivating a microorganism of the genus Pseudomonadaceae via heterotrophic fermentation using a feedstock comprising cellulose as a carbon source to produce a bio-oil, wherein about 11-99% of the unsaturated fatty acids in the bio-oil are polyunsaturated fatty acids; and

(b) cracking the bio-oil to form a jet biofuel.

23. The method of claim 22, wherein the bio-oil comprises about 10 wt% to about 75 wt% polyunsaturated fatty acids.

24. A lipid-based biofuel composition comprising from about 1 to about 75 wt.% of alkyl esters of long chain fatty acids having 20 or more carbon atoms.

25. The lipid-based biofuel composition of claim 24, wherein the melting temperature of the lipid-based biofuel composition is from about 30 ℃ to about-50 ℃.

26. A process for producing a bio-oil, the process comprising culturing two or more microorganisms simultaneously or sequentially by heterotrophic fermentation using a feedstock comprising cellulose as a carbon source, wherein one or more microorganisms are capable of saccharifying the cellulose.

27. A method of producing a bio-oil, the method comprising culturing a microorganism in a heterotrophic fermentation tank.

28. The method of claim 27, wherein said bio-oil is produced at a rate of about 5 to about 70 g/l/day.

29. The method of claim 28, wherein said bio-oil is produced at a rate of about 30 to about 70 g/l/day.

30. The method of claim 27, wherein culturing the microorganism achieves a cell density of about 10 to about 300 grams per liter.

31. The method of claim 30, wherein culturing the microorganism achieves a cell density of about 150 and 250 g/l.

32. The method of claim 27, wherein culturing the microorganism comprises using cellulose as a carbon source.

33. A method of producing biodiesel, the method comprising:

(a) culturing a microorganism in a bacterial fermentor to produce a bio-oil; and

(b) transesterifying the bio-oil to produce biodiesel.

34. A method of making a jet biofuel, the method comprising:

(a) culturing a microorganism in a bacterial fermentor to produce a bio-oil; and

(b) cracking the bio-oil to form a jet biofuel.

35. A method of producing biodiesel, the method comprising:

(a) culturing a microorganism with a nutrient comprising a circulating medium to produce a bio-oil; and

(b) transesterifying the bio-oil to produce biodiesel.

36. The method of claim 35, wherein the circulating medium is selected from the group consisting of: delipidated biomass, hydrolyzed biomass, partially hydrolyzed biomass, recycled metals, recycled salts, recycled amino acids, recycled extracellular carbohydrates, recycled glycerol, recycled yeast biomass, and combinations thereof.

37. The method of claim 35, wherein culturing the microorganism comprises using cellulose as a carbon source.

38. A method of making a jet biofuel, the method comprising:

(a) culturing a microorganism with a nutrient comprising a circulating medium to produce a bio-oil; and

(b) cracking the bio-oil to form a jet biofuel.

39. A method of producing biodiesel, the method comprising:

(a) culturing a microorganism with a fermentation system comprising a continuous inoculation stage and a lipid production stage to produce a bio-oil;

(b) transesterifying the bio-oil to produce biodiesel.

40. The method of claim 39, wherein the successive inoculation stages produce biomass from the microorganism such that about 10% to about 95% of the total biomass production from the microorganism is achieved in the successive inoculation stages.

41. The method of claim 39, wherein the lipid production stage is performed as a batch feed process.

42. The method of claim 39, wherein said lipid production stage produces lipids such that about 10-95% of the total lipid production by said microorganism is achieved during said lipid production stage.

43. The method of claim 39, wherein culturing the microorganism comprises using cellulose as a carbon source.

44. A method of making a jet biofuel, the method comprising:

(a) culturing the microorganism using a fermentation system having a continuous inoculation stage and a lipid production stage to produce a bio-oil;

(b) cracking the bio-oil to form jet bio-fuel.

Technical Field

The invention relates to bio-oil and preparation and application thereof. The bio-oil of the present invention is preferably prepared by microbial fermentation using a cellulose-containing raw material. The invention also relates to methods of making lipid-based biofuels and fuel additives and food, nutraceutical, and pharmaceutical products using these bio-oils.

Background

The production of bio-oil from sources such as plants (including oilseeds), microorganisms, and animals is essential in a variety of applications. For example, the production of biodiesel requires large amounts of bio-oil. Biodiesel has been proposed as a carbon-neutral liquid fuel alternative to petroleum-based diesel. Biodiesel is most commonly formed by transesterification of vegetable oil acyl groups with simple alcohols (e.g., methanol, ethanol, or isopropanol). The alkyl ester produced can then be directly combusted in most modern compression ignition internal combustion engines without any mechanical modification. It is estimated that the energy density of biodiesel will reach 95% of petroleum diesel (or "fossil diesel"). However, the higher lubricity (and hence improved fuel efficiency) of biodiesel yields about the same miles as obtained with an equal volume of fossil or biodiesel.

Because the biodiesel mainly consists of fixed CO at present₂Is obtained from the seed oil of plants, the fuel is considered "carbon-neutral" because, unlike fossil diesel, all CO escaping from the burning biodiesel₂Has recently been present in the atmosphere, while the carbon released on combustion of fossil diesel has not been present in the atmosphere for millions of years. Thus, biodiesel and other carbon-neutral fuels have a prominent contribution worldwide to reducing greenhouse gas (e.g., carbon dioxide) emissions.

Some states in the united states have regulated the blending of biodiesel with fossil diesel sold in that state, and the federal government has also established targets for the use of renewable transportation fuels. Current vegetable oil supplies for conversion to biodiesel encounter difficulties in meeting these regulations, resulting in higher prices for many oilseed plants, especially soybeans. If the current trend continues, the price of important oilseed plants will rise dramatically. The ultimate goal is to replace all fossil fuel sources with bio-based alternatives that are competitive in price. Unfortunately, this goal is not possible if the oil source of the current biodiesel is not significantly changed.

Recognizing this challenge, research has been conducted on alternative oil sources for producing biodiesel, including the feasibility of producing biodiesel from photosynthetic algae grown in open ponds. Since some algae are oily and grow very fast (less than 2 weeks from seed to harvest for some algae), the theoretical oil production per acre per year can be orders of magnitude greater than for higher plants. It should be noted that a small fraction of most oil-producing higher plants represents only a small fraction of the total population of plants, whereas photosynthetic microalgae as an oil suitable for the production of biodiesel may accumulate in a higher percentage of its population. However, the photosynthetic algae technology has serious problems that prevent its large-scale growth, and thus cannot effectively compete with the fossil diesel technology.

Photosynthetic microalgae often require CO supplementation₂To achieve high oil production. This is actually beneficial from a bioremediation point of view, since excess carbon dioxide released from coal or oil fired power plants will no longer be emitted to the atmosphere but will be used as a feedstock for the production of biodiesel. It is clear that this method does not produce a true carbon neutral fuel because the carbon dioxide released from coal-fired power plants is still ultimately released into the atmosphere (after biodiesel combustion), but the method delays the release rate of fossil-produced carbon dioxide and produces more useful energy per mass of fossil fuel. Indeed, some companies have invested in this technology, including green fuels, Inc. Green fuel company specializesA closed photobioreactor system is used that is capable of dissolving very high concentrations of carbon dioxide from fossil-fuel-fired power plants in photosynthetic algae cultures. Due to biophysical limitations of self-shading, the accumulation of biomass depends on the total illuminated surface area. Therefore, many photobioreactors are required to produce even limited amounts of biodiesel. Thus, while this technology is suitable for use as a bioremediation solution to sequester carbon (and other greenhouse gases) from fossil-fuel fired power plants, it cannot be scaled up to meet the needs of future biodiesel demand.

To address the scale issue, other agencies chose to further develop open pond technology to produce phototrophic algae-derived biodiesel. Open pond systems also rely on supplemental CO₂To achieve a hypothetical economic level of oil accumulation. Thus, these systems may be better viewed as systems for the biological discharge of fossil fuel-derived carbon waste. The annual production of useful oil per acre in these systems is orders of magnitude higher than that derived from oilseed plants. From most perspectives, these systems are the best solution to the limited supply of biodiesel. However, there are significant problems that have not yet been solved. Although the absolute theoretical yield per acre per year is very high, the actual density of biomass accumulated in open pond systems is relatively dilute. Therefore, large volumes of media need to be processed to extract the oil in the biomass, which can significantly increase the production cost of the final oil.

One approach to replacing gasoline with a renewable alternative, such as ethanol, is relatively simple. However, it should be noted that the markets for compression ignition engines (burning fossil diesel or biodiesel) and pilot-fired engines (burning gasoline or ethanol) generally meet different requirements. Compression ignition engines have excellent torque making them more suitable for industrial applications than pilot-fired engines, which can provide greater acceleration (and thus are more prevalent in general commuting). There is therefore no reason to expect that a pilot-fired engine could completely replace a compression-ignition engine even if a renewable alternative was completely used instead of gasoline.

Despite certain drawbacks, many efforts have been made to utilize ethanol as a liquid transportation fuel instead of gasoline. The brazilian model relies on sugar cane as a feedstock for ethanol fermentation, often enumerated as a pioneering example of the potential for biofuel utilization. Unfortunately, the climatic conditions in the united states do not provide the sugarcane productivity required for large-scale ethanol production. Initial efforts to scale up the U.S. ethanol fermentation have employed corn syrup and corn starch as feedstocks, but there is controversy surrounding the sustainability and scalability of this arrangement. Therefore, recent efforts have focused more on the use of "cellulosic" sources of sugars as feedstock in ethanol fermentation. The cellulosic feedstock may be any cellulose-containing feedstock.

Since most plants are composed mainly of structural polysaccharides (cellulose and hemicellulose) and lignans, the cultivation area will be more efficiently utilized if the sugar monomers of cellulose and other structural polysaccharides are used as raw materials for ethanol fermentation. This is different from the use of corn starch, which is only present in the kernel of corn plants, in a relatively low percentage of the dry weight of the plant. Furthermore, since all plants contain cellulose, faster growing and more climate tolerant plants can be used as the main source of cellulose based sugars. Examples of such plants include: switchgrass (Switchgrass), Miscanthus (Miscanthus gigantus), and Populus alba (Poplatr).

Similar to corn for ethanol, today's major biodiesel crops are inefficient in land use because only oil from the biodiesel crop seed is available for the production of biodiesel. Fiber-produced ethanol processes have been widely adopted, but fiber-produced ethanol is far from being widely accepted as a sustainable, economically competitive, possible alternative to gasoline. Cellulosic feedstocks have been considered useful for the manufacture of other petroleum-derived products (e.g., plastics).

Patent application publications WO 2005/035693, US 2005/0112735, WO 2007/027633, WO2006/127512, US2007/0099278, US 2007/0089356 and WO 2008/067605 (the entire contents of which are incorporated herein by reference) all relate to biodiesel or biofuel production systems.

Recently, microalgae Chlorella protothecoides (Chlorella protothecoides) have been studied for the production of biodiesel via fermentative heterotrophic growth. Researchers at the university of qinghua, beijing, china, used oil from heterotrophic microalgae chlorella protothecoides to conduct studies on biodiesel production. In these studies, microalgae were cultured in fermenters using glucose or corn meal hydrolysates as the carbon source. Then extracting microalgae oil, and preparing biodiesel through ester exchange. See Miao, x, and Wu, q., bioreource Technology 97: 841-846 (2006); xu, H, et al, Journal of Biotechnology 126: 499-507(2006). While these researchers suggest that hydrolysis solutions of starch and cellulose may be a low cost alternative to glucose in carbon sources in fermentation processes, they also suggest that cellulose hydrolysis is difficult and expensive. See Li, X, et al, "Large-Scale production of biodiesel from microalgae Chlorella protothecoides by heterotrophic culture in bioreactors" (Large-scale biodiesel production from microalga Chlorella serotrophic digestion in bioreactors), Biotechnology and Bioengineering, Accepted Preprint, 2007 for 20 days 4.20.

In addition to biodiesel, another oil-based fuel that requires renewable sustainable sources is jet fuel. Aircraft rely on the use of various types of jet fuel, including kerosene-type jet fuel and naphtha-type jet fuel. The severe dependence of the aviation industry on petroleum-based jet fuels has led to an urgent need to find renewable jet biofuels.

Thus, there is a need for a low cost efficient process for the preparation of lipid-based biofuels that is easy to scale up to replace fossil diesel and jet fuels. The term "lipid-based biofuel" as used herein means any fuel produced from the bio-oil of the present invention, including but not limited to biodiesel, jet biofuel and specialty fuels. To meet this need, it is necessary to develop an inexpensive and simple process for producing bio-oil that can be converted into lipid-based biofuels. In order to reduce the production cost of lipid-based biofuels, a low-cost method for producing bio-oil using abundant and inexpensive raw materials such as cellulose-containing raw materials as a main carbon source is required. In addition to the need to use inexpensive raw materials, there is also a need for improved processes aimed at reducing costs in the bio-oil production process. The improved process for preparing these bio-oils can reduce not only the cost of preparing lipid-based biofuels, but also the costs associated with the use of these bio-oils in many other applications, including food, nutrition and pharmaceuticals.

For example, it is desirable to increase the dietary intake of many beneficial nutrients present in biological oils. Particularly beneficial nutrients include fatty acids, such as omega-3 and omega-6 long chain polyunsaturated fatty acids (LC-PUFAs) and esters thereof. Omega-3 PUFA is considered to be an important compound for preventing arteriosclerosis and coronary heart disease, and is used for relieving inflammation and delaying the growth of tumor cells. Omega-6 PUFAs can be used not only as structural lipids in the human body, but also as precursors of a number of inflammatory factors such as prostaglandins, leukotrienes and hydroxylipids (oxylipins). Long chain omega-3 and omega-6 PUFAs are an important class of PUFAs.

Depending on the position of the double bond closest to the methyl end of the fatty acid, there are two main families or families of LC-PUFAs: the omega-3 series contains a double bond at the carbon at position 3, while the omega-6 series does not have a double bond until the carbon at position 6. Thus, docosahexaenoic acid ("DHA") has a chain length of 22 carbons and 6 double bonds starting from the 3 rd carbon of the methyl terminus, referred to as "22: 6 n-3". Other important omega-3 LC-PUFAs include eicosapentaenoic acid ("EPA"), referred to as "20: 5 n-3" and omega-3 docosapentaenoic acid ("DPA n-3"), referred to as "22: 5 n-3". Important omega-6 LC-PUFAs include arachidonic acid ("ARA"), referred to as "20: 4 n-6" and omega-6 docosapentaenoic acid ("DPA n-6"), referred to as "22: 5 n-6".

Because humans and many other animals cannot directly synthesize omega-3 and omega-6 essential fatty acids, they must be obtained from the diet. Traditional dietary sources of PUFAs include vegetable oils, marine animal oils, fish oils, and oilseeds. Furthermore, it was found that the oils produced by certain microorganisms are rich in LC-PUFA. To reduce the costs associated with the production of dietary sources of PUFAs, there is a need for a low cost and efficient process for producing biological oils comprising PUFAs. In order to reduce the cost of PUFA-containing bio-oils, there is a need to develop processes for producing these bio-oils using inexpensive feedstocks (e.g., cellulose-containing feedstocks) and improved processes designed to reduce production costs.

Disclosure of Invention

The present invention provides a method for producing a bio-oil comprising culturing a microorganism of the kingdom pseudobacteriaceae (Stramenopile) by heterotrophic fermentation using a feedstock comprising cellulose as a carbon source, wherein about 11-99% of the unsaturated fatty acids in the bio-oil are polyunsaturated fatty acids. In some embodiments of the invention, greater than about 50% of the unsaturated fatty acids in the biological oil are polyunsaturated fatty acids.

The microorganisms used in the present invention may include, but are not limited to, thraustochytrids (thraustochytrids), preferably selected from the group consisting of: microorganisms of the genus Schizochytrium (Schizochytrium), Thraustochytrium (Thraustochytrium) and Ulkenia (Ulkenia).

In some embodiments of the invention, the microorganism used is capable of saccharifying cellulose. Preferably, the microorganism of the invention is capable of degrading or tolerating raw material components selected from the group consisting of: lignin, hemicellulose, vegetable oils, plant extracellular polysaccharides, and combinations thereof. In some embodiments of the invention, the microorganism is a genetically modified microorganism.

The microorganisms of the present invention can produce oil in triglyceride form in yields of about 25-85% by weight of their biomass dry weight. In some embodiments of the invention, the culturing of the microbial biomass is performed at a dissolved oxygen concentration of about 10% to 100%. For example, bio-oil is produced from microorganisms under conditions of dissolved oxygen concentration of 0% to 10%. The microorganisms may be cultured at a temperature of about 15-45 ℃.

In some embodiments of the invention, the method of producing bio-oil further comprises: autolysis or induced lysis of the microorganism is performed after the microorganism produces about 30-90 wt% oil of its biomass dry weight. Induced lysis of the microorganism can be achieved by subjecting the microorganism to conditions favorable for lysis, including pH, temperature, presence of enzymes, presence of detergents, physical disruption, and combinations thereof.

In some embodiments of the invention, the cellulose-containing fermentation feedstock comprises a cellulose source selected from the group consisting of: grasses, sugar cane, agricultural wastes, waste paper, sewage, wood, organisms of the Viridiplantae kingdom (viridiplantae), and combinations thereof.

In a preferred embodiment of the invention, the fermentation is carried out in a sterile fermenter. In some embodiments of the invention, the fermentation is performed in a fermentor selected from the group consisting of: fiber-reinforced polymer fermentors, metal matrix composite fermentors, ceramic matrix composite fermentors, thermoplastic composite fermentors, metal fermentors, epoxy-lined carbon steel fermentors, plastic fermentors, glass fiber fermentors, and concrete fermentors.

In some embodiments of the invention, the fermentation is performed in a fermentor that is submerged in water. The fermentation can be carried out in fermenters with cooling systems connected in series, so that the cooling water flowing out of a first fermenter or a set of fermenters connected in series can be used as a cooling water supply for a second fermenter or set of fermenters connected in series. Similarly, fermentation may be carried out in fermenters having a gas system in series, such that sparged gas exiting a first fermentor or set of fermenters in series may be used as a gas supply in a second fermentor or series of fermenters in series.

The invention also provides a method for preparing biodiesel, which comprises the following steps: (a) culturing a Pseudomonadaceae (Stramenopile) microorganism by heterotrophic fermentation using a feedstock comprising cellulose as a carbon source to produce a bio-oil, wherein about 11-99% of unsaturated fatty acids in the bio-oil are polyunsaturated fatty acids; and (b) transesterifying the bio-oil to form biodiesel. In some embodiments of the invention, greater than about 50% of the unsaturated fatty acids in the biological oil are polyunsaturated fatty acids.

Transesterification of bio-oils can be carried out with alcohols derived from alcohol production processes. In some embodiments of the invention, glycerol produced by transesterification of a bio-oil may be used as a carbon source for subsequent fermentation processes to produce an alcohol or bio-oil. In some embodiments of the invention, the subsequent fermentation process cultures a microorganism that is capable of using glycerol as a carbon source.

The invention also provides a method for preparing jet engine biofuel, which comprises the following steps: (a) culturing a Pseudomonadaceae (Stramenopile) microorganism by heterotrophic fermentation using a feedstock comprising cellulose as a carbon source to produce a bio-oil, wherein about 11-99% of unsaturated fatty acids in the bio-oil are polyunsaturated fatty acids; and (b) cracking (cracking) the bio-oil to produce jet biofuel. In some embodiments of the invention, the bio-oil used to make jet biofuel comprises about 10 to 75 wt.% polyunsaturated fatty acids.

The present invention provides a lipid-based biofuel composition comprising from about 1 to about 75 wt.% of alkyl esters of long chain fatty acids having 20 or more carbon atoms. In some embodiments of the invention, the melting temperature of the lipid-based biofuel composition is from about 30 ℃ to about-50 ℃.

The present invention also provides a method for preparing bio-oil, comprising: (a) two or more microorganisms are cultivated simultaneously or sequentially by heterotrophic fermentation using a feedstock comprising cellulose as a carbon source, wherein one or more microorganisms are capable of saccharifying the cellulose.

The invention also provides a process for the production of biodiesel, which process comprises transesterifying bio-oils produced by two or more microorganisms cultured by heterotrophic fermentation using a feedstock comprising cellulose as a carbon source, wherein one or more microorganisms are capable of saccharifying cellulose. For the production of jet biofuel, bio-oil produced by two or more microorganisms cultured by heterotrophic fermentation using a feedstock comprising cellulose as a carbon source may be subjected to cracking, wherein one or more microorganisms are capable of saccharifying cellulose.

The present invention provides a method for producing bio-oil comprising culturing a microorganism in a sterile fermentor via heterotrophic fermentation. In some embodiments of the invention, the production rate of the bio-oil in the sterile fermentor is about 5 to 70 g/l/day, preferably about 30 to 70 g/l/day.

In some embodiments of the invention, the cell density achievable by culturing the microorganism in the sterile fermentor is about 10-300 g/L, preferably about 150-250 g/L. Preferably, the culturing of the microorganism comprises using cellulose as a carbon source.

The invention also provides a method for preparing biodiesel, which comprises the following steps: (a) culturing a microorganism in a sterile fermentor to produce a bio-oil; and (b) transesterifying the bio-oil to produce biodiesel. The invention also provides a method for preparing jet engine biofuel, which comprises the following steps: (a) culturing a microorganism in a sterile fermentor to produce a bio-oil; and (b) cracking the bio-oil to produce jet biofuel.

The invention provides a method for preparing biodiesel, which comprises the following steps: (a) culturing a microorganism with nutrients comprising a recirculating medium to produce a bio-oil; and (b) transesterifying the bio-oil to produce biodiesel. The recycled medium may be, but is not limited to, the following: delipidated biomass, hydrolyzed biomass, partially hydrolyzed biomass, recycled metals, recycled salts, recycled amino acids, recycled extracellular carbohydrates, recycled glycerol, recycled yeast biomass, and combinations thereof. Preferably, culturing the microorganism comprises using cellulose as a carbon source.

The invention also provides a method for preparing jet engine biofuel, which comprises the following steps: (a) culturing a microorganism with nutrients comprising recycled culture medium to produce a bio-oil; and (b) cracking the bio-oil to produce jet biofuel.

Some embodiments of the present invention provide a method of producing biodiesel, the method comprising: (a) culturing a microorganism with a fermentation system comprising a continuous inoculation stage (seed stage) and a lipid production stage to produce a bio-oil; and (b) transesterifying the bio-oil to produce biodiesel. Preferably, the successive inoculation stages produce microbial biomass such that about 10-95% of the total microbial biomass production is achieved in the successive inoculation stages. In some embodiments of the invention, the lipid production stage is performed as a batch feed process. Preferably, the lipids produced in the lipid production stage are such that about 10-95% of the total microbial lipid production is achieved during lipid production. In some embodiments of the invention, the culturing of the microorganism comprises using cellulose as a carbon source.

The invention also provides a method for preparing jet engine biofuel, which comprises the following steps: (a) culturing a microorganism with a fermentation system comprising a continuous inoculation stage and a lipid production stage to produce a bio-oil; and (b) cracking the bio-oil to produce jet biofuel.

Brief description of the drawings

Fig. 1 shows various embodiments of a method for producing bio-oil and bio-diesel according to the present invention.

FIG. 2 shows an example of a fermentation system design according to the present invention.

FIG. 3 shows the change over time in dry cell weight, weight percent lipid, weight percent DHA, and amount of lipid produced per liter of fermentation broth when the microorganism (ATCC20888) was cultured under aseptic and sterile conditions as described in example 4.

FIG. 4 shows the change over time in the rate of sugar consumption, the rate of oil production (g/l fermentation broth/day), the rate of biomass production (g/l/day), the amount of lipid-free biomass when the microorganism (ATCC20888) was cultured under sterile and aseptic conditions as described in example 4.

Figure 5 shows a two-stage fermentation process comprising a continuous inoculation stage and a batch-fed lipid accumulation stage.

Detailed Description

The invention provides biological oil and preparation and application thereof. Some embodiments of the present invention provide oleaginous, heterotrophic organisms and methods suitable for the conversion of carbon directly from cellulose-based or ligno-vitamin-based sugars to vegetable oils for biodiesel production by fermentation. The process of the invention is more scalable and sustainable and can produce biodiesel that is more cost competitive than processes currently used or investigated, such as seed oil biodiesel or photosynthetic algae biodiesel.

Aspects of the invention relate to high density culture of two oleaginous microorganisms using saccharified cellulosic feedstocks. For example, the protist schizochytrium and the lipomyces Yarrowia lipolytica (Yarrowia lipolytica) are suitable for this process, since both microorganisms have a transformation system for regulating the maturation development of the microorganism and are able to produce high levels of lipids by fermentation. Some aspects of the invention provide oleaginous thraustochytrids and fungi capable of growing on a variety of cellulosic and lignocellulosic substrates, as well as organisms that naturally undergo combined saccharification and fermentation and lignin degradation or resistance.

The invention also relates to improved biological strains and to a method for oil production by molecular, biological, classical genetic and physiological means using a cellulose-based substrate. Some embodiments of the invention provide for economic amplification of a fermentation process for converting cellular acylglycerols to biodiesel. The invention also provides biological and chemical reactor designs and structures for carrying out the methods of the invention, as well as commercial production protocols.

Various organisms can be used to produce the bio-oils of the present invention, including microorganisms. The microorganism may be an alga, a bacterium, a fungus or a protist. Microbial sources and methods of culturing microorganisms are known in the art (Industrial Microbiology and Biotechnology, 2 nd edition, 1999, american society for Microbiology). For example, the microorganism can be cultured with the fermentation medium in a fermentor. Microbially produced oils are useful in the methods and compositions of the present invention. In some embodiments, the microorganism comprises a microorganism selected from the group consisting of: golden algae (e.g., microorganisms of the genus Pseudocerana (Stremenopiles)), green algae, diatoms, dinoflagellates (e.g., microorganisms of the class Emmenomycetes, including members of the genus Methylocystis (Crypthecodinium), such as Crypthecodinium cohnii), yeasts (e.g., members of the genus Yarrowia (Yarrowia) (e.g., Yarrowia lipolytica), Cryptococcus (e.g., Cryptococcus albicans), Trichosporon (Trichosporon), Candida (Candida), Lipomyces (Lipomyces), Rhodosporidium (Rhodosporidium), and Rhodotorula (Rhodotorula)), Mucor (Mucor), and Mortierella (Mortierella) including, but not limited to, Mortierella alpina (Mortierella) and Mortierella (Mortierella). Microorganisms of the kingdom Planococcus (Stramenopile) include: microalgae (microalgae) and algae-like microorganisms, including the following: hammenon (Hamatories), Protomoma (Protomonads), Aphanta (Opalines), Devepita (Develpayella), Diprofecophyte (Diplophybrides), Lapril (Labringhulidoides), Thraustochytridia (Thraustochytrids), Bylera (biosciences), Oomycota (Oomycota), Depthecodinia (Hypochytrids), Commana (Commation), Reycophyta (Reticotina), Pelagomophyta (Pelagomomonas), Pelagomophycota (Pelagococcus), Ollicola (Octoglosta), Oyctolycota (Aureophycus), Microchaetophyta (Paramalles), Diatoma (Dims), Xanthophyta (Xanthophyta), Sarcophyta (Euphyridiophytidea), Schizochythora (Chlorophytridae), Schizochythora (Schizochythora) and Schizochythora (Chlorophytridae), Rhodophyceae (Chlorophytridae) (Nophyceae), Rhodophyridia (Euphyceae), Rhodophyceae (Rhodophyceae), Rhodophyceae (Euphyceae), Euphyceae (Euphyceae), Euphyceae (Euphyceae), Euphyceae) and Nophyceae (Euphyceae), Euphyceae (Euphyceae) and Nophyceae (Euphyceae) including Euphyceae (Euphyceae) and Nophyceae (Euphyceae), Euphyceae (Euphyceae) including Euphyceae (Euphyceae) and Nophyceae) including Euphyceae (Euphyceae) can, Euphyceae (Euphyceae) and Euphyceae (Euphyceae) can, Euphyceae (Euphyceae) can, Euphyceae (Euphyceae) and Euphyceae) can, Euphyceae (Euphyceae) can, Euphyceae (Euphyceae) including Euphyceae (Euphyceae ) including Euphyceae, Euphy (aggregatum), lissenesurus (limnaceum), mangifera (mangrove), mini-algae (minutum), otosporum), Thraustochytrium (Thraustochytrium) (species including aldimondo (arenmentale), oreum (aureum), bincholla (benthicola), globopom (globosum), keni (kinnei), molivum (motivum), mobadiella (mutidimentale), pekay (pacydermum), prolifra (proliferum), roscoverum (rosoum), roscovellium (roscoes), urette (striatum), stoutum (stributum), wu kensia (Ulkenia) (species including amoebeda (amateur), cokutsi (enterobacter), coelicofelis (nigrosinus), milrata (preneoides), preneocaulium (preneoides), preneophytin (preneophys), borneophytin (macrorrhiza), borealis (preneophytin (macrorrhiza), boreophytin (macrochy), borneophys) (species including echinochytium), borneophyceae (naphali), borneophytin (macrochy), borneophytin (macrothezium), borneophytin (macrochy), borneophytin (macrothezis) (species (macrotheiles) (species (macrothelen), including kayas) (species (macrotheiles) (species (macrothelen), borkayas) (species (macrochai), kayas) (species (macrothelen), kayas) (species (macrochai), kayas) (species (borneophyceae), borneophyceae (borkayas) (species (borkayas), kayas (borkayas) (species (borkayas (borgehikayas (borkayas), kayas (borkayas), including kayas (borkayas), kayas (borkayas) (species (borkayas), including kayas (borkayas) (species (borkayas) (including kayas), kayas (borkayas), including kayas), kayas (borkayas) (including kayas), kayas (borkayas), kayas (borkayas) (including kayas (borkayas), kayas (borkayas) (including kayas), kayas (borkayas) (including kayas (borkayas), kayas (borkayas) (including kayas), kayas (borkayas) (including kayas (borkayas), kayas) (including kayas (borkayas), kayas (borkayas), kayas) (including kayas, altney Chlamydia (Althornia) (species including Crowdai) and Elina (species including Marisala, Xinnaorufaca) are used. Labrintholidinota (Labrintholidis) including lappril genus (Labyrinthula) (species including Angeriensis (algeriensis), Cocinocystis (coenocystis), Chartonii (chatonii), Marcinos (macrocystis), Atlantic Marcinos (macrocystis atlantic), Dual Marcinos (macrocystis macrocystis), Maryla (marina), Mynaeta (minuta), Roscoeffenide (Roscofensis), Vancaroviet (valkanovii), Vibrio (vitellina), Vibrio paragallina (vitellina), Vibrio paragallica (vitellina), Zophyrina (zopril)), Labrinia genus Labrinthorphis (Labyrinthi) (species including Porphyrinia)), Alcarinica (Labyrinthi) (species including Porphyridia (Charcot)), Marinopril (Gray) (species including Soyrophylloridonia (Gray), Leonipril (Gray) (species including Porphyridia (Porphyra)), Leoni (Porphyra)), Phaleria (Porphyra (Pyrrosi (Porphyra)), Phaleria (Porphyra) (species including Porphyra (Porphyra)), Phaleria (Porphyra)), Phaleri) (species including Porphyra (Porphyra)), and Porphyra (Porphyra)), or (Porphyra)), or (Porphyra)) including Porphyra (Porphyra) including Porphyra (Porphyra)), or (Porphyra)), or (Porphyra) including Porphyra (Porphyra) including Porphyra)), or (Porphyra) including Porphyra (Porphyra) including Porphyra), Porphyra (Porphyra) including Porphyra (Porphyra)), or (Porphyra)), or (Porphyra) including Porphyra (Porphyra)), or (Porphyra) including Porphyra)), or (Porphyra (Porphy, species include lapinos (labyrthuloides), montana (montana). (═ accurate taxonomic localization for these genera is currently not agreed upon).

Some embodiments of the present invention provide methods of producing bio-oil comprising culturing a microorganism of the genus Pseudomycosis via heterotrophic fermentation using a feedstock comprising cellulose as a carbon source. In some embodiments of the invention, the bio-oil comprises unsaturated fatty acids, a significant portion of which are polyunsaturated fatty acids. As noted in the past, certain polyunsaturated fatty acids, such as omega-3 and omega-6 long chain polyunsaturated fatty acids, are particularly important dietary compounds. Therefore, it is desirable to produce bio-oils with significant amounts of polyunsaturated fatty acids. In some embodiments of the invention, the bio-oil is converted to a lipid-based biofuel. For these applications, it is desirable to produce hydrocarbons of various chain lengths, particularly suitable for jet biofuel applications. The presence of significant amounts of polyunsaturated fatty acids in the bio-oil used to make lipid-based biofuels will provide greater flexibility and more variety in the hydrocarbon production process, as the multiple sites of unsaturation in the polyunsaturated fatty acids provide multiple sites for cleavage to form hydrocarbons. For example, certain jet fuels require hydrocarbons having 2-8 carbons. Polyunsaturated fatty acids can be split by means known in the art (e.g., (cracking) to produce shorter hydrocarbons of various chain lengths.

In some embodiments, the biological oil produced by the methods of the present invention comprises unsaturated fatty acids, and about 11-99% of the unsaturated fatty acids in the biological oil are polyunsaturated fatty acids. The bio-oil of the present invention may comprise unsaturated fatty acids, about 20-99%, about 26-99%, about 30-99%, about 40-99%, about 51-99%, about 60-99%, about 70-99%, about 80-99%, or about 90-99% of the unsaturated fatty acids in the bio-oil being polyunsaturated fatty acids. In some embodiments of the invention, greater than about 10%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, or greater than about 90% of the unsaturated fatty acids in the biological oil are polyunsaturated fatty acids.

In some embodiments of the invention, the biological oil comprises about 10 to 75 weight percent polyunsaturated fatty acids. For certain applications, the bio-oil preferably comprises about 20-75%, about 30-75%, about 40-75%, about 50-75%, or about 60-75% by weight polyunsaturated fatty acids. In some embodiments of the invention, the biological oil comprises at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% by weight polyunsaturated fatty acids. The method of producing bio-oil according to the present invention may optionally further comprise collecting bio-oil from the microorganism.

The term "cellulose" as used herein includes unsaponified or unhydrolyzed cellulose as well as saccharified or hydrolyzed cellulose. In some embodiments of the invention, the microorganism used is a thraustochytrid. Preferably, the microorganism is a microorganism of the genus Schizochytrium, Thraustochytrium or Ukenschoenophytrium. In some embodiments of the invention, the microorganism used is Yarrowia (e.g., Yarrowia lipolytica), Cryptococcus (e.g., Cryptococcus albicans), trichosporium (trichosporine), Candida (Candida), Lipomyces (Lipomyces), Rhodosporidium (rhodosporium), or Rhodotorula (Rhodotorula). Examples of such yeasts are disclosed in patent application publication WO 2004/101757, the contents of which are incorporated herein by reference.

The present invention also contemplates the use of a combination of two or more microorganisms to produce a bio-oil or bio-oil blend. In order to reduce fermentation costs, it is preferred to culture two or more microorganisms under the same fermentation conditions. When two or more different microorganisms are combined to produce a bio-oil, one or more microorganisms can accumulate oil during fermentation. One or more microorganisms may promote the culture and accumulation of oil by another microorganism through activities that include, but are not limited to: the cleavage of a feedstock component to form unstable sugar monomers (e.g., saccharification of cellulose), the cleavage of a feedstock component that inhibits the growth of another microorganism (e.g., metabolizing or degrading a feedstock component such as lignin, hemicellulose (e.g., xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan), vegetable oil, plant extracellular polysaccharide, etc.), the synthesis of a component that promotes the growth of another microorganism (e.g., the synthesis of certain enzymes that facilitate the growth of a microorganism), and the like.

Organisms suitable for metabolizing hemicellulose include, but are not limited to: bacteroides succinogenes (Fibrobacter succinogenes), Cryptococcus yeasts (e.g.Cryptococcus albicans, Cryptococcus curvatus), Trichosporon (Trichosporon), Candida (Candida), Lipomyces (Lipomyces), Rhodosporidium (Rhodosporidium), and Rhodotorula (Rhodotorula)). Other organisms suitable for metabolizing hemicellulose include: organisms of the genera Pichia (Pichia) (e.g.Pichia stipitis), Aeromonas (Aeromonas), Aspergillus (Aspergillus), Streptomyces (Streptomyces), Rhodococcus (Rhodococcus), Bacillus (Bacillus) (e.g.Bacillus subtilis), Bacillus brevis (Bacillus brevis) and Bacillus lentus), Escherichia coli (Escherichia coli), Kluyveromyces (Kluyveromyces), Saccharomyces (Saccharomyces) and Trichoderma. Organisms suitable for metabolizing lignin include, but are not limited to: phanerochaete chrysosporium (Phanerochaete chrysosporium) or other "white rot" fungi. Patent application publication WO 91/018974, the contents of which are incorporated herein by reference, discloses examples of organisms having hemicellulosic activity.

Suitable methods for producing free sugars and oligosaccharides from lignocellulosic biomass for use in the present invention are described, for example, in patent application publication US 2004/0005674, the contents of which are incorporated herein by reference. These methods include: lignocellulosic biomass is converted to free sugars and small oligosaccharides using enzymes that break down lignocellulose (e.g., cellulases, xylanases, ligninases, amylases, proteases, lipases, and glucuronidases). These enzymes can be obtained from commercial sources or prepared recombinantly, for example by expression in microorganisms, fungi, i.e.yeasts or plants.

Oleaginous microorganisms are preferably used in the present invention. As used herein, "oleaginous microorganism" refers to a microorganism in the form of a lipid capable of accumulating more than 20% of the cell dry weight. In some embodiments of the invention, the microorganisms produce lipids that are about 30-95% by weight of their biomass dry weight. Preferably, the microorganisms of the present invention produce lipids that are about 35-93%, about 40-90%, about 45-88%, about 50-85%, about 55-83%, about 60-80%, about 65-78%, or about 70-75% by weight of their biomass dry weight. In some embodiments of the invention, the microorganism produces lipids that are at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or at least about 70% by weight of its biomass dry weight.

When two or more microorganisms are employed to produce the bio-oil of the present invention, one or more microorganisms can produce the bio-oil. In some embodiments of the invention, when two or more microorganisms are used in combination to produce a bio-oil, the weight ratio of oil produced by the first microorganism to oil produced by the second microorganism is from about 1:9 to about 1:1, from about 1:9 to about 2:3, from about 1:9 to about 3:7, or from about 1:9 to about 1: 4.

Preferably, the microorganisms of the present invention produce oil in triglyceride form at a yield of about 25-85% by weight of their dry biomass, about 30-85% by weight of their dry biomass, about 35-85% by weight of their dry biomass, about 40-85% by weight of their dry biomass, about 45-85% by weight of their dry biomass, about 50-85% by weight of their dry biomass, about 55-85% by weight of their dry biomass, about 60-80% by weight of their dry biomass, about 65-75% by weight of their dry biomass, or about 70-75% by weight of their dry biomass. In some embodiments of the invention, the microorganism produces oil in triglyceride form in an amount of at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or at least about 70% by weight of its dry biomass.

The term "triglyceride" as used herein refers to a triglyceride having the chemical formula CH₂(OOCR¹)CH(OOCR²)CH₂(OOCR³) Of three fatty acid residues and glycerol, wherein OOCR¹、OOCR²And OOCR³Each represents a fatty acid residue. In some embodiments of the invention, suitable triglycerides may comprise at least one PUFA. In some embodiments, the PUFA chain length is at least 18 carbons. These PUFAs are also referred to herein as long chain PUFAs or LC PUFAs. In some embodiments, the PUFAs may be docosahexaenoic acid C22:6n-3(DHA), omega-3 docosapentaenoic acid C22:5n-3(DPA (n-3)), omega-6 docosapentaenoic acid C22:5n-6(DPA (n-6)), arachidonic acid C20:4n-6(ARA), eicosapentaenoic acid C20:5n-3(EPA), stearidonic acid (SDA), linolenic acid (LLA), alpha Linolenic Acid (ALA), Gamma Linolenic Acid (GLA), Conjugated Linolenic Acid (CLA), eicosatetraenoic acid (C20:4n-3), homo-alpha and-gamma linolenic acid (C20:3n-6 and 20:3n-3), docosatetraenoic acid (C22:4n-6), octacosanoenoic acid (C28:8), or mixtures thereof. PUFAs can also be present in any of the common forms of natural lipids, including but not limited to triacylglycerols, diacylglycerols, monoacylglycerols, phospholipids, free fatty acids, or in the form of natural or synthetic derivatives of these fatty acids (e.g., calcium salts of fatty acids, etc.). The invention uses glycerol comprising residues with PUFAA triglyceride oil or other composition may refer to a composition comprising triglycerides with only one type of PUFA residue, such as DHA, or a composition comprising triglycerides with more than one type of PUFA residue, such as DHA, EPA, and ARA.

In a preferred embodiment of the invention, the microorganism is capable of achieving high density cell growth. In some embodiments of the invention, the microorganism is capable of achieving a cell density of at least about 10 grams per liter, at least about 15 grams per liter, at least about 20 grams per liter, at least about 25 grams per liter, at least about 30 grams per liter, at least about 50 grams per liter, at least about 75 grams per liter, at least about 100 grams per liter, at least about 125 grams per liter, at least about 135 grams per liter, at least about 140 grams per liter, at least about 145 grams per liter, or at least about 150 grams per liter. In some embodiments of the invention, the microorganism can achieve a cell density of about 10-300 g/L, about 15-300 g/L, about 20-300 g/L, about 25-300 g/L, about 30-300 g/L, about 50-300 g/L, about 75-300 g/L, about 100-300 g/L, about 125-300 g/L, about 130-290 g/L, about 135-280 g/L, about 140-270 g/L, about 145-260 g/L, or about 150-250 g/L. The high-density growth of the microorganism of the present invention can be enhanced by adjusting fermentation conditions such as temperature, pH, ion concentration and gas concentration.

The invention provides efficient preparation of bio-oil. In some embodiments of the invention, the bio-oil yield is at least about 5 g/l/day, at least about 10 g/l/day, at least about 20 g/l/day, at least about 30 g/l/day, at least about 40 g/l/day, at least about 50 g/l/day, at least about 60 g/l/day, or at least about 70 g/l/day. In some embodiments of the invention, the bio-oil yield is about 5 to about 70 g/l/day, about 10 to about 70 g/l/day, about 20 to about 70 g/l/day, or about 30 to about 70 g/l/day.

In some embodiments of the invention, the microorganism used to produce the bio-oil is a cellulolytic microorganism and is thus capable of saccharifying cellulose of a cellulosic or lignocellulosic feedstock. Cellulosic or lignocellulosic feedstocks include any source comprising cellulose. These sources include, but are not limited to: grass, sugar cane, agricultural waste, waste paper, sewage, wood, any green plant kingdom organism, or a product thereof. Preferably, the cellulose used is from a source other than a tree-based cellulose source. Grass types suitable for use as a source of cellulose include, but are not limited to: serrate, wheatgrass, oryza sativa, switchgrass (switch grasses) and Miscanthus (Miscanthus) type grasses.

In order for a microorganism to be able to utilize cellulose as a carbon source, it is necessary to break down the cellulose into its constituent sugar monomers. Cellulose is a glucose polymer linked by β -glycosidic bonds, and is a highly stable linear structure. The cleavage of cellulose to form sugar monomers (also known as saccharified cellulose) is a difficult challenge and many attempts have failed to achieve such cleavage. Enzymatic hydrolysis of cellulose is one way to degrade cellulose. Complete hydrolysis of cellulose generally requires: an endoglucanase that cleaves an interior region of the cellulose polymer; exoglucanases that cleave cellobiose units from the ends of the cellulose polymer; and a beta-glucosidase enzyme that cleaves cellobiose into glucose subunits. Cellulases can have multiple complexes that achieve endoglucanase, exoglucanase, and beta-glucosidase activities. Trichoderma reesei (Trichoderma reesei) is an important organism for the production of cellulases. Other methods of cleaving cellulose into sugar monomers include: thermochemical decomposition (with or without mechanical disruption), including but not limited to water, steam explosion, acid treatment, and/or ammonia fiber explosion.

In some embodiments of the invention, the microorganism used to produce the bio-oil is the same as the microorganism used to saccharify the cellulose. In some embodiments of the invention, two or more microorganisms may be cultured simultaneously or sequentially, using a feedstock containing cellulose as a major carbon source to produce bio-oil. According to the invention, when two or more microorganisms are fermented simultaneously or sequentially, one or more microorganisms are capable of saccharifying cellulose. In some embodiments of the invention, the microorganism can undergo heterotrophic fermentation in the presence of cellulase enzymes to enhance saccharification of the cellulose during fermentation. In some embodiments, at least one microorganism is from the kingdom pseudobacteriaceae, preferably an organism commonly referred to as a thraustochytrid.

Microorganisms suitable for use in the present invention may also be resistant to high temperatures and/or strong acid or base environments, such that their growth in high temperature and/or acidic media is not inhibited and in some cases even enhanced. In some embodiments of the invention, the microorganism is cultured using heterotrophic fermentation of a cellulose-containing feedstock at a temperature and/or pH that facilitates degradation of cellulose. In some embodiments of the invention, the fermentation is conducted at a temperature of about 15-70 ℃, about 20-40 ℃, or about 25-35 ℃. In other embodiments of the invention, the fermentation is conducted at a pH of about 3 to about 11, about 3 to about 10, about 4 to about 9.5, about 4 to about 9, about 5 to about 7, or about 6 to about 9. The pretreatment of the cellulose-containing feedstock with cellulase enzymes, chemical and/or mechanical disruption, and ammonia fiber explosion during the production of the bio-oil of the present invention may also be performed prior to use of the feedstock. Alternatively, no pretreatment is required.

Some examples of feedstock pretreatment processes are disclosed in the following patent applications: US2007/0161095, WO05/053812, WO06/086757, US2006/0182857, US2006/177551, US2007/0110862, WO06/096834, WO07/055735, US2007/0099278, WO06/119318, US2006/0172405 and US2005/0026262, the contents of which are incorporated herein by reference.

Examples of enzymes suitable for digesting cellulose are disclosed in the following patents or patent applications: US2003/0096342, WO 03/012109, US 7059993, WO 03/012095, WO 03/012090, US 2003/0108988, US 2004/0038334, US 2003/0104522, EP 1612267 and WO06/003175, the contents of which are incorporated herein by reference.

In some embodiments of the invention, the cellulosic feedstock used to culture the microorganisms comprises cellulose in an amount of about 5 to 100%, about 10 to 95%, about 20 to 90%, about 30 to 85%, about 40 to 80%, about 50 to 75%, or about 60 to 70% of the dry weight of the carbon feedstock. In some embodiments of the invention, the cellulose content of the cellulosic feedstock comprises at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the dry weight of the carbon feedstock.

Preferably, the microorganisms used in the present invention are tolerant to or capable of degrading the components of the raw materials: lignans, hemicellulose, vegetable oils, plant extracellular polysaccharides, and combinations thereof. Degradation or tolerance of these feedstock components ensures that the fermentation process of the microorganism is not inhibited by the presence of these components.

In some embodiments of the invention, the cellulosic feedstock used to culture the microorganisms comprises about 1-50%, about 5-40%, or about 10-30% by weight of a component selected from lignin, hemicellulose, or a combination thereof. In some embodiments of the invention, the cellulosic feedstock used to culture the microorganisms comprises at least about 1%, at least about 5%, at least about 10%, at least about 20%, or at least about 30% by weight of a component selected from lignin, hemicellulose, or a combination thereof.

Suitable microorganisms can be obtained from a variety of sources, including collection from the natural environment. As used herein, any particular type of any organism or microorganism includes wild-type, mutant, or recombinant types. Culture conditions for these organisms are known in the art, and suitable culture conditions for at least some of these microorganisms are described, for example, in U.S. Pat. No. 5,130,242, U.S. Pat. No. 5,407,957, U.S. Pat. No. 5,397,591, U.S. Pat. No. 5,492,938, U.S. Pat. No. 5,711,983 and U.S. Pat. No. 6,607,900, all of which are incorporated herein by reference in their entirety.

When microbial oils are used, the microorganisms are cultured in any effective medium as defined herein that is capable of promoting the production of the oil. Preferably, the effective medium also promotes rapid growth of microorganisms. The microorganisms can be cultured in conventional fermentation modes including, but not limited to, batch (batch), fed-batch (fed-batch), semi-continuous, and continuous modes. As used herein, "semi-continuous" mode refers to a fermentation mode in which a portion of the fermentation culture comprising the microorganism is not obtained from the fermentor after the fermentation process is completed. The remaining portion of the fermentation broth in the fermentor can be used to inoculate a subsequent fermentation process. In some embodiments of the invention, about 1-50 vol.%, about 1-25 vol.%, about 1-15 vol.%, about 1-10 vol.%, or about 2-8 vol.% of the fermentation broth is left in the fermentor after completion of the fermentation process to inoculate a subsequent fermentation process.

In some embodiments of the invention, the fermentation process comprises a first stage in which biomass accumulation is targeted to the microorganism and a second stage in which lipid accumulation is targeted to the microorganism. Preferably, there is no nutrient limitation during the biomass accumulation stage. The lipid accumulation phase is preferably carried out with a carbon source under nitrogen limitation.

The inventive process for producing biodiesel can comprise: (a) culturing the microorganism with a fermentation system comprising a continuous inoculation stage and a lipid production stage to produce bio-oil, and (b) converting the bio-oil to biodiesel by means known in the art, for example by transesterifying the bio-oil to produce biodiesel. The goal of the continuous inoculation stage is biomass accumulation, which is performed by providing a continuous feed of nutrients into the inoculation vessel (the vessel with the initial inoculation). The fermentation broth in the seed vessel was drained and transferred to the vessel for the lipid production phase, as in a fed-batch process, to deliver a carbon source to the batch to maintain the target sugar concentration throughout the run.

A similar two-stage fermentation process can be used to produce bio-oil for jet biofuel. In some embodiments of the invention, a method of producing jet biofuel comprises converting bio-oil produced using the fermentation system to jet biofuel by methods known in the art, utilizing a process such as cracking to facilitate conversion of the bio-oil to jet biofuel.

The two-stage fermentation process improves the efficiency of the bio-oil preparation process, thereby being beneficial to reducing the cost of preparing the lipid-based biofuel. The improved fermentation system for producing lipid-based biofuels is particularly advantageous in achieving maximum efficient large-scale production of bio-oil, and thus has a significant contribution to the more commercially viable production of lipid-based biofuels from bio-oil. The two-stage fermentation process can be used to produce bio-oils with high or low levels of polyunsaturated fatty acids, depending on the requirements of the particular application.

In some embodiments of the invention, the biomass accumulation stage (e.g., a continuous inoculation stage) produces microbial biomass that achieves biomass production of about 10-95%, about 20-95%, about 30-95%, about 40-95%, or about 50-95% of the total microorganisms at this stage. In other embodiments of the invention, the biomass production of the total microorganisms reaches about 60-95%, about 70-95%, or about 80-95% during the biomass accumulation stage. In some embodiments of the invention, the biomass accumulation stage produces a microbial biomass at which stage at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the total microbial biomass production is effected during the biomass accumulation stage. Preferably, about 50-95% of the total microbial biomass production is achieved during the biomass accumulation stage.

In some embodiments of the invention, the lipid accumulation phase produces lipids such that about 10-95%, about 20-95%, about 30-95%, about 40-95%, or about 50-95% of the total microbial lipid production is achieved during the lipid accumulation phase.

In other embodiments of the invention, about 60-95%, about 70-95%, or about 80-95% of the total microbial lipid production is achieved during the lipid accumulation phase. In some embodiments of the invention, the lipid accumulation phase produces lipids such that at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the total microbial lipid production is achieved during the lipid accumulation phase. Preferably, about 50-95% of the total microbial lipid production is achieved during the lipid accumulation phase.

Genetically modified microorganisms are also suitable for use in the present invention. The microorganism of the present invention is genetically modified to increase its ability to produce bio-oil at low cost (e.g., by increasing the ability to use cellulose-based raw materials as a primary carbon source). These genetically modified microorganisms may include, but are not limited to: microorganisms that have been genetically modified to increase the ability to saccharify cellulose or cellulosic feedstocks, microorganisms that increase oil production, microorganisms that degrade lignin or are tolerant to lignin, and microorganisms that can be cultured under culture conditions that are not optimal for the corresponding wild-type organism (e.g., high temperature or strongly acidic media). For example, the microorganism can be genetically modified to introduce or increase activity on endoglucanases, exoglucanases, and/or beta-glycosidases.

Genes from cellulase-inducing organisms can be introduced into microorganisms to increase their ability to saccharify cellulose. For example, genes encoding cellulase components from Trichoderma, Clostridium, Cellulomonas, Thermobifida, Thermoacidothermus, Schizochytrium, or Thraustochytrium organisms can be introduced into the microorganisms of the present invention by recombinant gene technology to produce microorganisms capable of directly saccharifying cellulose. Preferably, genes encoding cellulase components from Trichoderma reesei (Trichoderma reesei), Clostridium thermocellum (Clostridium thermocellum), Thermoascus cellulolyticus (Acidothermus cellulolyticus) or Schizochytrium aggregatum (Schizochytrium aggregatum) are introduced into and expressed in the microorganism of the invention. In some embodiments of the invention, cellulases from one organism are cloned into another organism.

Techniques for genetic transformation of microorganisms are well known in the art, see, e.g., Sambrook et al, 1989, molecular cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor laboratories Press). General transformation techniques for dinoflagellates suitable for Crypthecodinium cohnii are described in Lohuis and Miller, The Plant Journal (1998)13(3): 427-. General genetic transformation techniques for thraustochytriales are detailed in U.S. patent application publication 20030166207, published 9/4/2003.

In some embodiments of the invention, microbial fermentation is conducted under conditions of low concentrations of dissolved oxygen to produce bio-oil. The ability of the microorganisms to grow and produce oil at low concentrations of dissolved oxygen can reduce the energy input required for fermentation and thus can also reduce fermentation costs. In some embodiments of the invention, the culturing of the microbial biomass (biomass accumulation stage) is performed under conditions of a dissolved oxygen concentration of about 4% -100%, about 10% -80%, about 10-70%, about 10-60%, about 15-50%, or about 20-40%. The preparation of microbial oil (lipid accumulation phase) may be carried out under conditions of dissolved oxygen concentration of, for example, 0% to 10%, 0% to 8%, about 1% to 5%, or about 1% to 3%.

To reduce the energy costs associated with cooling the fermenter, the microorganisms used according to the invention are preferably resistant to a wide temperature range. In some embodiments of the invention, the microorganism can grow and produce oil at a temperature of about 15-45 ℃, about 20-45 ℃, about 25-45 ℃, about 30-45 ℃, or about 35-45 ℃.

Typically, fermentation of microorganisms is typically performed in a sterile environment to avoid contaminants that may interfere with biomass growth and/or lipid accumulation of the microorganisms. Performing fermentation under aseptic conditions increases the cost of producing bio-oil from microorganisms. To reduce fermentation costs, the present invention provides an unexpected solution to the production of bio-oil via fermentation in a germ-bearing fermentor. The use of a germ fermentor to produce oil from microorganisms is particularly useful for the production of oil for lipid-based biofuel purposes, as this approach can significantly reduce oil production costs and make lipid-based biofuel production more commercially viable. Depending on the requirements of a particular application, a germ fermenter can also be used to produce bio-oils with high or low levels of polyunsaturated fatty acids.

Preferably, low cost fermentors may be used in the fermentation process, including fiber reinforced polymer fermentors, metal matrix composite fermentors, ceramic matrix composite fermentors, thermoplastic composite fermentors, metal fermentors, epoxy lined carbon steel fermentors, plastic fermentors, glass fiber fermentors, concrete fermentors, fermentors made of polymers such as polypropylene (PP), High Density Polyethylene (HDPE), Polycarbonate (PC), Polystyrene (PS), polyvinyl chloride (PVC), kynar and nylon. Low cost fermentors may also be constructed from combinations of the above materials. Low cost tank cleaning techniques may also be employed in accordance with the present invention to further reduce fermentation costs. Low cost canister cleaning techniques include, but are not limited to: the fermenter was chemically scrubbed with methoxide or ethoxide.

In some embodiments of the invention, the production rate of bio-oil in the aerobic fermentor is about 5 to 70 g/l/day. Preferably, the amount of bio-oil produced in the fermentors is at least about 5 grams/liter/day, at least about 10 grams/liter/day, at least about 20 grams/liter/day, at least about 30 grams/liter/day, at least about 40 grams/liter/day, at least about 50 grams/liter/day, at least about 60 grams/liter/day, or at least about 70 grams/liter/day. In some embodiments of the invention, the amount of bio-oil produced in the aerobic fermentor is about 10 to about 70 grams per liter per day, about 20 to about 70 grams per liter per day, or about 30 to about 70 grams per liter per day.

The high cell density achieved by culturing the microorganism in the fermentor is preferably at least about 10 grams per liter, at least about 15 grams per liter, at least about 20 grams per liter, at least about 25 grams per liter, at least about 30 grams per liter, at least about 50 grams per liter, at least about 75 grams per liter, at least about 100 grams per liter, at least about 125 grams per liter, at least about 135 grams per liter, at least about 140 grams per liter, at least about 145 grams per liter, or at least about 150 grams per liter, or at least about 200 grams per liter. In some embodiments of the invention, the microorganism fermented in the fermentor can achieve a cell density of about 10-300 g/L, about 15-300 g/L, about 20-300 g/L, about 25-300 g/L, about 30-300 g/L, about 50-300 g/L, about 75-300 g/L, about 100-300 g/L, about 125-300 g/L, about 130-290 g/L, about 135-280 g/L, about 140-270 g/L, about 145-260 g/L, or about 150-250 g/L.

In some embodiments of the invention, depending on the intended use of the bio-oil, fermentation conditions (e.g., pH, temperature, dissolved oxygen concentration, ion ratio, etc.) of the microorganisms can be varied to alter the fatty acid profile characteristics of the resulting oil. Depending on the intended use of the biological oil of the present invention, fermentation conditions may be adjusted, for example to promote or retard microbial production of lipids in the triglyceride form, to promote or retard microbial production of a particular fatty acid or fatty acid blend (e.g., a fatty acid of a particular chain length or unsaturation), to promote or retard oil with high or low levels of energy provided per unit volume of oil, or to promote or retard the accumulation of certain by-products in the oil produced by the microbe. Various uses of the bio-oils of the present invention for lipid-based biofuel purposes include, but are not limited to: used as heating oil, biodiesel for transportation, jet engine fuel and fuel additive. In some embodiments of the invention, deuterium can be used in the fermentation medium to provide for the preparation of ultra-low capacity, extremely high value specialty fuels or lubricants. In some embodiments of the invention, the conversion of bio-oil to lipid-based biofuels involves chemical processes and refining techniques known in the art that can also be made or used to make specialty compounds (e.g., plastic components) similar to oil distillates.

Sales profits for these specialty chemicals may also offset the cost of lipid-based biofuel production. Various other uses of the bio-oil are contemplated within the scope of the present invention. For example, the bio-oil of the present invention may be used in any suitable food, nutraceutical, or pharmaceutical product.

The invention also provides a method of fermentation in a fermentor that is submerged in a cooling liquid such as water. In some embodiments of the invention, fermentation devices may be arranged in series to minimize energy usage. For example, the cooled effluent and the sparged off-gas from a series of fermentors may be used as cooling water and a gas supply (or partial supply), respectively, for a downstream or upstream fermentor in the series of pipelines. The fermentation system can be set to be a natural water system with cooling water from lakes, ponds or oceans. The fermentation system may be designed such that the cooling systems of the fermenters are connected in series such that the cooling water flowing from the first fermenter or set of fermenters in the series may be used as cooling supply water for the second fermenter or set of fermenters in the series. Similarly, a fermentation system may be designed to connect the gas supplies of fermenters in series such that the vent gas emitted from a first fermenter or set of fermenters in the series may be used as the gas supply for a second fermenter or set of fermenters in the series. The first fermentation tank or the fermentation tank group is connected with the second fermentation tank or the fermentation tank group in series in time. The fermentation according to the invention is preferably carried out in batch, fed-batch, semi-continuous or continuous mode.

While in some embodiments of the invention, the triglyceride-containing bio-oil may be crude oil (discussed in more detail below), other such oils suitable for use in the present invention may be obtained from its source by any suitable means known in the art. For example, extraction may be performed using a solvent such as chloroform, hexane, dichloromethane, methanol, or the like, or the oil may be recovered by supercritical fluid extraction or by a solvent-free extraction method. In some embodiments of the invention, the bio-oil is recovered using hexane extraction. Alternatively, the oil may be extracted using Extraction techniques, such as those described in U.S. patent 6,750,048 and PCT patent application Ser. No. US01/01806, both of which were filed as "solvent free Extraction Process" on 19.1.2001, the entire contents of which are incorporated herein by reference. Other extraction and/or purification techniques are described in: PCT patent application Ser. No. PCT/IB01/00841, entitled "Fractionation Method of Natural Raw Materials Containing oils and Polar lipids" (Method for the Fractionation of Oil and Polar Lipid-Containing Natural Raw Materials), filed on 4/12/2001; PCT patent application Ser. No. PCT/IB01/00963, filed on 12.4.2001, entitled "Method for fractionating and centrifuging a natural Raw material Containing Oil and Polar lipids Using Water-Soluble Organic solvents" (Method for the Fractionation of Oil and Polar Lipid-Containing Natural Raw Materials Using Water-Soluble Organic Solvent); U.S. provisional patent application Ser. No. 60/291,484, filed on 5/14/2001, entitled "preparation and Use of Lipid-Rich Polar fractions Containing Stearidonic Acid and Gamma-Linolenic Acid from Plant Seeds and microorganisms" (Production and Use of a Polar Lipid-Rich Fraction Containing Stearidonic Acid and Gamma-Linolenic Acid from Plant Seeds and microorganisms); filed on 14.5.2001 entitled "preparation and Use of Polar Lipid Fraction Containing Highly Unsaturated Fatty Acids Omega-3 and/or Omega-6 from microorganisms, Genetically Modified Plant Seeds and Marine Organisms (Production and Use of a Polar-Lipid Fraction Containing Omega-Fatty Acid derived Omega-3 and/or Omega-6 high unreacted Fatty Acids from Microbes, genetic Modified Plant sections and Marine Organisms U.S. provisional patent application Ser. No. 60/290,899; filed on 17.2.2000 and granted on 6.4.2002 entitled" separation method of Triglycerides Containing Docosahexaenoic Acid residual components from Triglyceride Mixture (Process for preparing Triglyceride Docosaheoic Acid from Triglyceride Mixture and method for preparing Triglyceride Mixture 6,399,803 (Process for preparing Fatty Acid Mixture for Fatty Acid rich in Triglyceride Mixture 3511) a Docosahexaenoic Acid resource from a Mixture of trigyceries) PCT patent application Ser. No. US 01/01010; the entire contents of which are incorporated herein by reference. The extracted oil was evaporated under reduced pressure to give a sample of concentrated oil. For biomass enzymatic processes for lipid recovery see: US provisional patent application 60/377,550 entitled "HIGH-QUALITY lipids released FROM Biomass enzymes and METHODS FOR their preparation (HIGH-QUALITY LIPIDS AND METHOD FOR PRODUCING BY ENZYMATIC LIBERATION FROM BIOMASS) filed on 5/3/2002; PCT/US03/14177 entitled "HIGH QUALITY lipid released FROM Biomass enzymes and method FOR preparing the same (HIGH-QUALITY LIPIDS AND METHOD FOR PRODUCING BY ENZYMATIC LIBERATION FROM BIOMASS) filed 5/2003; co-pending U.S. patent application 10/971,723, filed on 22/10/2004 FOR "HIGH QUALITY lipids FROM BIOMASS enzyme release and METHODS FOR their preparation (HIGH-QUALITY LIPIDS AND METHODS FOR PRODUCING lipids BY LIBERATION FROM bionass); european patent publication 0776356 and U.S. Pat. No. 5,928,696 entitled "Process for extracting Water-insoluble Natural products from Natural substance mixtures Using centrifugal force (Process for extracting Natural products not Water-soluble from Natural substrate fuels by centrifugal force"), the contents of all of which are incorporated herein by reference. The oil can be extracted by pressing.

In some embodiments, according to the methods of the present invention, the oil obtained from the above sources can be used as a feedstock for further modification (e.g., transesterification or cracking), even without conventional processing. Examples of such conventional processes that may be avoided include refining (e.g., physical refining, silica gel refining, or caustic refining), desolvation, deodorization, wintering, cold filtration, and/or bleaching. Thus, in certain embodiments, the triglyceride-containing oil is not subjected to one or more treatments selected from the group consisting of: refining, desolventizing, deodorizing, wintering, cold filtering, and bleaching, and in other embodiments, the oil is not subjected to any of refining, desolventizing, deodorizing, wintering, cold filtering, and bleaching.

In some embodiments, crude oil may be isolated from microorganisms using standard techniques without further refinement or purification. For example, the oil may be a microbial oil that has been treated only by solvent extraction, such as hexane extraction, isopropanol extraction, and the like. In some embodiments of the invention, crude oil may be separated from microorganisms using physical and/or mechanical extraction methods (e.g., by using a homogenizer, or by pressing), without further refining or purification.

In other embodiments, compositions comprising triglycerides with polyunsaturated fatty acid residues, such as the oils described above, may be subjected to further processing steps, such as refining, desolventizing, deodorizing, winterizing, cold filtering, and/or bleaching. Such "treated" oils include microbial oils that have undergone solvent extraction and one or more of these additional processing steps. In some embodiments, the oil is minimally treated. "minimally processed" oils include microbial oils that have been solvent extracted and filtered. In certain embodiments, the minimally processed oil is further subjected to winter storage treatment.

In some embodiments of the invention, the use is similar to(Westfalia Separator Industry GmbH, Wastfalia separation Co., Ltd., Germany)) process for the extraction of bio-oil produced by microorganisms.Is a water-based physical oil extraction process in which the oil-containing feedstock can be used directly to extract oil without the use of any conventional solvent extraction methods. In this process, oil is separated from a raw material fermentation broth by density separation using gravity or centrifugal force using a water-soluble organic solvent as a process aid. Patent application publications WO 01/76715 and WO 01/76385, the contents of which are incorporated herein by reference, disclose such extraction methods.

After extraction of the oil, the oil is recovered or separated from the non-lipid components by any suitable method known in the art. In a preferred embodiment of the invention, the lipid-containing composition is separated from the non-lipid composition using low-cost physical and/or mechanical techniques. For example, if used inThe extraction process to extract the oil produces a plurality of phases or fractions, wherein one or more of the phases or fractions comprise lipids, and the process for recovering the lipid-containing phase or fraction may include physically removing the lipid-containing phase or fraction from the non-lipid phase or fraction, or vice versa. In some embodiments of the invention, use is made ofA type of process to extract the lipids produced by the microorganisms, and then physically separate the lipid-rich light phase from the protein-rich heavy phase (e.g., by skimming the lipid-rich phase on top of the protein-rich heavy phase after density separation).

The bio-oil produced by the microorganisms of the present invention can be recovered by subjecting the microorganisms to conditions that result in spontaneous lysis or induced lysis of the microorganisms, including, but not limited to: a specific pH, a specific temperature, the presence of an enzyme, the presence of a detergent, physical disruption, or a combination thereof. In some embodiments of the invention, the microorganisms are subjected to these conditions to promote spontaneous or induced lysis after they produce oil in an amount of about 30-90 wt.%, about 40-90 wt.%, about 50-90 wt.%, about 60-90 wt.%, about 65-85 wt.%, about 70-85 wt.%, or about 75-80 wt.% of their biomass dry weight. In other embodiments of the invention, the microorganisms are subjected to these conditions to result in spontaneous or induced lysis after oil production to at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 75% by weight of their biomass dry weight. In some embodiments of the invention, lysis or spontaneous lysis of the microorganism is achieved using mechanical force. In other embodiments of the invention, the lipids are mechanically separated from the non-lipid composition after microbial lysis or spontaneous lysis.

Suitable enzymes that can be used to induce lysis of oleaginous microorganisms include, but are not limited to: commercially available enzymes or enzyme mixtures, such as proteinase K or Alcalase. It is contemplated within the scope of the invention to genetically modify a microorganism to introduce an activity of an enzyme that is capable of inducing lysis or auto-lysis of another microorganism. In some embodiments of the invention, the oleaginous microorganism undergoes induced lysis in the presence of a detergent, such as an ionic (anionic or anionic) detergent, a non-ionic detergent, a zwitterionic detergent, or a combination thereof. In other embodiments of the invention, physical disruption methods such as mechanical milling, liquid homogenization, sonication with high frequency sound waves, freeze-thaw cycling methods, pressing, extrusion, or milling may be used to induce lysis of the oleaginous microorganisms. Preferably, the extraction of oil is performed in a fermentor by in-tank lysis of oleaginous microorganisms after the end of fermentation.

Once the bio-oil is prepared according to the present invention, it can be converted to fatty acid esters using various methods known in the art for use as biodiesel, jet biofuel, or as an ingredient in food or pharmaceutical products. In some embodiments of the invention, the preparation of the fatty acid ester comprises transesterifying a bio-oil produced by the microorganism. In some embodiments of the invention, in a one-step process, the extraction of the bio-oil from the microorganism and the transesterification of the oil may be performed simultaneously. For example, a culture comprising an oleaginous microorganism may be subjected to or treated (or a combination of conditions or treatments) under conditions that promote oil extraction and transesterification of the oil. These conditions or treatments may include, but are not limited to: pH, temperature, pressure, presence of solvent, presence of water, presence of catalyst or enzyme, presence of detergent, and physical/mechanical forces. Two sets of conditions or treatments can be combined to produce a one-step process for extracting and transesterifying oil, where one set of conditions or treatments advantageously facilitates oil extraction and the other set of conditions or treatments advantageously facilitates oil transesterification, provided that the two sets of conditions or treatments can be combined without causing a significant reduction in the efficiency of oil extraction or transesterification. In some embodiments of the invention, the whole cell biomass may be directly subjected to hydrolysis and transesterification. In other embodiments of the invention, the extraction of the oil may be as a separate step from the transesterification step of the oil.

Preferably, these transesterification reactions are carried out using acid or base catalysts. In some embodiments of the invention, a method of transesterifying a bio-oil to form fatty acid esters for use as biodiesel or as a component in food or pharmaceutical products comprises reacting a triglyceride-containing bio-oil in the presence of an alcohol and a base to produce fatty acid esters having residues of triglycerides.

Alcohols suitable for use in the present invention include any lower alkyl alcohol (i.e., C) containing 1 to 6 carbon atoms_1-6Alkyl alcohols such as methanol, ethanol, isopropanol, butanol, pentanol, hexanol, and isomers thereof). Without wishing to be bound by theory, it is believed that in some embodiments of the invention, the use of a lower alkyl alcohol in the process of the invention may produce a lower alkyl ester of the fatty acid residue. For example, ethyl esters are produced using ethanol. In certain embodiments, the alcohol is methanol or ethanol. In these embodiments, the fatty acid esters produced are methyl and ethyl esters of fatty acid residues, respectively. In the process of the present invention, the amount of alcohol used in the mixture of oil composition, alcohol and base is typically from about 5 to 70 wt.%, from about 5 to 60 wt.%, from about 5 to 50 wt.%, from about 7 to 40 wt.%, from about 9 to 30 wt.%, or from about 10 to 25 wt.%. In certain embodiments, the composition and base may be added to neat ethanol or neat methanol. Generally, the amount of alcohol used will vary depending on the solubility of the triglyceride-containing oil or composition in the alcohol.

Any base known in the art may be used as a reagent in the present invention. Bases of the general formula RO-M, where M is a monovalent cation and RO is an alkoxide of a C1-6 alkyl alcohol, are particularly useful in the present invention. Examples of suitable bases include: elemental sodium, sodium methoxide, sodium ethoxide, potassium methoxide and potassium ethoxide. In some embodiments, the base is sodium ethoxide. In the process of the present invention, the amount of base used in the reaction step with the composition and alcohol is generally from about 0.05 to about 2.0, from about 0.05 to about 1.5, from about 0.1 to about 1.4, from about 0.2 to about 1.3, or from about 0.25 to about 1.2 molar equivalents of triglyceride.

The composition comprising triglycerides, alcohol and base are reacted at a temperature and for a duration such that the fatty acid residues and alcohol produce esters. Suitable reaction times and temperatures can be determined by those skilled in the art to produce esters. Without wishing to be bound by theory, it is believed that the fatty acid residues are cleaved from the glycerol backbone of the triglyceride and esters of the individual fatty acid residues are formed during the reaction step. In certain embodiments, the reacting step of the composition in the presence of the alcohol and the base is carried out at a temperature of about 20-140 ℃, about 20-120 ℃, about 20-110 ℃, about 20-100 ℃, or about 20-90 ℃. In other embodiments, the composition reacting step is carried out at a temperature of at least about 20 ℃,75 ℃,80 ℃, 85 ℃,90 ℃,95 ℃, 105 ℃, or 120 ℃ in the presence of an alcohol and a base. In some embodiments of the invention, the reacting step of the composition in the presence of an alcohol and a base is carried out at a temperature of about 20 ℃,75 ℃,80 ℃, 85 ℃,90 ℃,95 ℃, 105 ℃, or 120 ℃. In some embodiments, the step of reacting the composition is carried out in the presence of an alcohol and a base for a time period of about 2 to about 36 hours, about 3 to about 36 hours, about 4 to about 36 hours, about 5 to about 36 hours, or about 6 to about 36 hours. In certain embodiments, the reacting step of the composition is carried out in the presence of an alcohol and a base for a time period of about 0.25, 0.5, 1.0, 2.0, 4.0, 5.0, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 10, 12, 16, 20, 24, 28, 32, or 36 hours.

In one embodiment, the reaction step of the oil composition, alcohol and base is carried out by refluxing the components to produce fatty acid esters, such as PUFA esters. In other embodiments, the reacting step of the oil composition is conducted at a temperature that does not cause reflux of the reaction components. For example, conducting the reaction step of the oil composition at a pressure greater than atmospheric pressure can increase the boiling point of the solvent present in the reaction mixture. Under these conditions, the reaction is carried out at a temperature at which boiling of the solvent occurs at atmospheric pressure, but without causing reflux of the reaction components. In some embodiments, between about 5 and 20 pounds per square inch (psi); about 7-15 psi; or about 9-12 psi. In certain embodiments, the reaction is carried out at a pressure of 7, 8, 9, 10, 11, or 12 psi. The reaction under pressure may be carried out at the reaction temperature mentioned above. In some embodiments, the reaction carried out under pressure may be carried out at a temperature of at least about 70 ℃,75 ℃,80 ℃, 85 ℃, or 90 ℃. In some embodiments, the reaction under pressure can be at 70 ℃,75 ℃,80 ℃, 85 ℃ or 90 ℃.

The reaction mixture comprising the fatty acid ester may be further processed to obtain the fatty acid ester from the mixture. For example, the mixture can be cooled, diluted with water, and the aqueous solution extracted with a solvent such as hexane to prepare a composition comprising the fatty acid ester. Techniques for washing and/or extracting the crude reaction mixture are known in the art.

In some embodiments of the invention, microorganisms that produce low levels of PUFAs are used to produce bio-oils, particularly suitable for biodiesel production. The method can reduce the production cost of biodiesel. In some embodiments of the invention, less than about 50% of the unsaturated fatty acids in the biological oil are PUFAs. For certain biofuel applications, the unsaturated fatty acids in the bio-oil preferably contain less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% PUFAs. In some embodiments of the invention, the PUFA content of the biological oil is less than about 50 wt.%, less than about 40 wt.%, less than about 30 wt.%, less than about 20 wt.%, less than about 10 wt.%, or less than about 5 wt.%.

More valuable PUFA esters can be recovered by distillation to produce high potency PUFA esters, which can then be sold to reduce the overall production cost of biodiesel products.

Examples of improved lipid preparation systems are disclosed in the following patent applications: WO 06/031699, US2006/0053515, US 2006/0107348 and WO 06/039449, the contents of which are incorporated herein by reference.

In one embodiment of the invention, the fatty acid esters are separated from the reaction mixture by distilling the composition to recover a fraction comprising the fatty acid esters. In this way, a target fraction of the reaction mixture comprising the fatty acid ester of interest can be separated and recovered from the reaction mixture.

In certain embodiments, the distillation is performed under vacuum. Without wishing to be bound by theory, vacuum distillation enables distillation to be achieved at lower temperatures than when vacuum is not employed, thereby preventing degradation of the ester. Typical distillation temperatures are about 120-170 ℃. In some embodiments, the distillation step is conducted at a temperature of less than about 180 ℃, less than about 175 ℃, less than about 170 ℃, less than about 165 ℃, less than about 160 ℃, less than about 155 ℃, less than about 150 ℃, less than about 145 ℃, less than about 140 ℃, less than about 135 ℃ or less than about 130 ℃. Typical vacuum distillation pressures are about 0.1 to 10mm Hg. In some embodiments, the vacuum distillation pressure is at least about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4mm Hg. In some embodiments of the invention, the pressure of the vacuum distillation is about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, or 4mm Hg.

In some embodiments of the invention, the fatty acid esters produced by transesterification of the bio-oil are further separated by urea addition. The urea can be dissolved in the medium comprising the fatty acid ester to form a medium comprising the fatty acid ester and the dissolved urea. The medium is then cooled or concentrated to form a precipitate comprising urea and at least a portion of the saturated fatty acid esters and a liquid fraction comprising at least a major portion of the polyunsaturated fatty acid esters. The precipitate and liquid fraction are then separated to split the saturated or polyunsaturated fatty acid esters. In some embodiments of the invention, the medium comprising the fatty acid ester and the dissolved urea is cooled to about 20 ℃ to about-50 ℃, about 10 ℃ to about-40 ℃, or about 0 ℃ to about-30 ℃. U.S. patent 6,395,778, the contents of which are incorporated herein by reference, discloses a transesterification process after urea addition.

In addition to the transesterification process described above, other techniques for reducing the viscosity of the bio-oil of the present invention may also be incorporated into the process of the present invention to produce lipid-based biofuels. These techniques include, but are not limited to: using lipase, supercritical methanol catalysis, and using a whole cell system that involves cytoplasmic over-expression of lipase in a host cell, followed by permeabilization of the host to achieve transesterification catalysis of triglycerides within the cytoplasm. Patents or patent application publications US 7226771, US 2004/0005604, WO 03/089620, WO 05/086900, US 2005/0108789, WO 05/032496, WO 05/108533, US 6982155, WO 06/009676, WO 06/133698, WO 06/037334, WO 07/076163, WO 07/056786 and WO 06/124818 (the contents of which are incorporated herein by reference) disclose examples of processes for converting lipids to biodiesel.

Thraustochytrids, and in particular schizochytrium, are similar to many marine and estuary microalgae and protists in that they accumulate certain amounts of polyunsaturated fatty acids (PUFAs) in their cellular lipids. Low levels of PUFAs are useful because they can lower the gel point of fuels, making them more suitable for cold climates. Potential consumer complaints about odor generation from burning PUFA-containing biodiesel in inefficient engines (passing partially oxidized fuel into the exhaust) can be offset somewhat by microalgae biodiesel fuel being blended with fossil diesel in proportions of 1-99% to minimize this problem. To ensure that 100% microalgal oil-derived biodiesel does not present significant consumer problems during combustion, a partial or complete oil hydrogenation reaction can be used, as is conventionally used in margarine manufacture. In some embodiments of the invention, the length of the fatty acid chains can be reduced using a cracking technique (e.g., a cracking process known in the oil industry). Once the bio-oil is prepared according to the method of the present invention, the cracking of the bio-oil can be performed to produce the desired lipid-based biofuel. For certain lipid-based biofuels that require a variety of shorter hydrocarbons, such as jet biofuels, high levels of PUFAs are useful to enable the PUFAs to be cleaved at multiple sites to form a variety of hydrocarbons.

The lipid-based biofuel composition of the present invention is prepared at low cost and is an effective substitute for petroleum diesel or jet fuel. In some embodiments of the invention, the lipid-based biofuel composition comprises about 1-75 wt% alkyl esters of long chain PUFAs having 20 or more carbons. In other embodiments of the invention, the biodiesel composition comprises about 2-50%, about 4-25%, or about 5-10% by weight alkyl esters of long chain PUFAs having 20 or more carbons.

In some embodiments of the invention, the lipid-based biofuel composition (100% lipid-based biofuel, unblended petroleum diesel or jet fuel) has a melting temperature of about 30 ℃ to about-60 ℃, about 30 ℃ to about-50 ℃, about 25 ℃ to about-50 ℃, about 20 ℃ to about-30 ℃, about 20 ℃ to about-20 ℃, about 20 ℃ to about-10 ℃, about 10 ℃ to about-10 ℃, or about 0 ℃ to about-10 ℃. In other embodiments of the invention, the biodiesel composition releases about 30-45 megajoules/liter, about 35-40 megajoules/liter, or about 38-40 megajoules/liter. Various forms of biodiesel are disclosed, see for example patent or patent application publications WO 07/061903, US 7172635, EP 1227143, WO 02/38709, WO 02/38707 and US 2007/0113467, the contents of which are incorporated herein by reference.

The present invention also provides a scale-up lipid-based biofuel production facility that can be co-located with an ethanol production facility (e.g., a cellulosic ethanol facility). Examples of algal systems associated with the production of non-lipid based fuels (e.g., ethanol) are found in patents or patent application publications US 7135308 and WO 02/05932, the contents of which are incorporated herein by reference.

In some embodiments of the invention, feedstock processing is similar to or the same as cellulosic ethanol and cellulosic lipid-based biofuel fermentation. For example, after cellulosic biodiesel fermentation, the oil may be subjected to extraction and transesterification (either simultaneously or sequentially) to produce biodiesel. The alcohol used in the transesterification can be from an ethanol production process (e.g., a cellulosic ethanol production process), and the glycerol remaining from the biodiesel transesterification can be used as a supplemental carbon source for an ethanol fermentation process (or for the biodiesel process itself, as organisms such as schizochytrium tend to metabolize glycerol). In a preferred embodiment of the present invention, the microorganism used in the present invention is capable of utilizing glycerol as a carbon source. Nitrogen-containing wastes (such as yeast biomass) can also be used as a nitrogen source in biodiesel fermentation (most thraustochytrids are able to utilize yeast extract as a nitrogen source). Waste materials such as de-lipidated microbial biomass can be recycled for subsequent fermentation processes, combustion for heat or electricity generation, or used as fertilizer for crops that provide cellulosic feedstocks. The resulting biodiesel or exhaust gas can be used as a fuel for biodiesel or ethanol production facilities, making them energy independent. Furthermore, the pump in the plant may be driven by the recovered exhaust gas.

In some embodiments of the invention, a method of producing a lipid-based biofuel includes culturing a microorganism with nutrients including a circulating culture medium to produce a bio-oil. Circulating media include, but are not limited to: delipidated biomass, hydrolyzed biomass, partially hydrolyzed biomass, metals, salts, amino acids, extracellular sugars, glycerol, recycled yeast biomass, or combinations thereof, all obtained from a previous fermentation process or other process cycle. For example, residual yeast biomass and hydrolyzed pseudobacteria delipidated biomass waste may be recycled into steam pretreatment, ammonia fiber explosion, separation steps, or into enzymatic hydrolysis, separation and evaporation steps, as shown in FIG. 1. The partially hydrolyzed biomass may be recycled back to these steps with the media for further hydrolysis. Depending on the requirements of a particular application, the use of a circulating medium can be used to produce bio-oils with high or low levels of polyunsaturated fatty acids.

The cellulose-based (low cost carbon) technology of the present invention can be used to reduce the production cost of any compound produced by fermentation of a yeast or pseudomycete organism (e.g., thraustochytrium), including genetically modified organisms. Examples of compounds prepared by the process of the present invention include, but are not limited to: PUFAs, PUFA esters, proteins (including enzymes and therapeutic proteins), hydroxyllipids, carotenoids, and lipids.

In some embodiments, the present methods can be used to prepare compositions comprising a high percentage of PUFAs or PUFA esters. For example, these compositions may comprise about 50-100 wt.% PUFAs or esters of PUFAs, and in other embodiments, the compositions may comprise at least about 50 wt.%, at least about 55 wt.%, at least about 60 wt.%, at least about 65 wt.%, at least about 70 wt.%, at least about 75 wt.%, at least about 80 wt.%, at least about 85 wt.%, at least about 90 wt.%, at least about 95 wt.%, at least about 99 wt.% PUFAs or esters of PUFAs.

The compositions of the invention comprising PUFAs or PUFA esters are useful in medicine. In some embodiments, the pharmaceutical product may comprise a PUFA or PUFA ester without other pharmaceutically active ingredients. In other embodiments, the pharmaceutical product may comprise a pharmaceutically active agent. Examples of pharmaceutically active agents include: statins, antihypertensives, antidiabetics, anti-dementias, antidepressants, antiobesity agents, appetite suppressants, and agents that enhance memory and/or cognitive function. The pharmaceutical product may further comprise any pharmaceutically acceptable excipient, carrier, binder or other formulation component known in the art.

The PUFAs or PUFA esters produced by the methods of the present invention are suitable for use as therapeutic agents or as experimental reagents. Embodiments of the invention include the production of PUFAs or PUFA esters for the treatment of PUFA-deficient infants. The PUFA or PUFA esters that may be included in the parenteral formulation may be administered to the infant parenterally to boost the infant's PUFA supply. Preferred parenteral routes include, but are not limited to: subcutaneous, intradermal, intravenous, intramuscular, and intraperitoneal routes. Parenteral formulations may comprise a PUFA or PUFA ester of the invention together with a carrier suitable for parenteral delivery. The term "vector" as used herein refers to any substance suitable for use as a vehicle for delivering a molecule or composition to a suitable site of action in vivo. Examples of such vectors include, but are not limited to: water, phosphate buffered saline, ringer's solution, dextrose solution, serum-containing solution, hanks' solution, and other aqueous physiological balancing solutions. Suitable carriers also include oil-based carriers, non-aqueous solutions, suspensions, and emulsions. Examples include: propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable organic esters such as ethyl oleate, polyethoxylated castor oil (cremaphor), and others known in the art. Acceptable regimens for administering PUFAs or PUFA esters in an effective manner include single dose size, number of administrations, frequency of administration and mode of administration. Such a regimen can be determined by one skilled in the art based on various parameters such as infant weight and PUFA deficiency. Another embodiment of the invention includes the production of PUFAs or PUFA esters for use in the treatment of adults, especially pregnant women. Acceptable regimens for administering PUFAs or PUFA esters to adults include: parenteral administration techniques or encapsulation of the PUFAs or PUFA esters of the invention in a capsule, such as a gelatin (i.e., digestible) capsule, for oral administration and/or formulation in a liquid dietary formulation. Liquid meal formulations may comprise liquid compositions containing nutrients suitable for supplementing a meal or sufficient nutrients to be a complete meal.

The PUFAs or PUFA esters produced by the methods of the invention can also be used to treat subjects (e.g., humans or animals) with high levels of triglycerides, including subjects with hypertriglyceridemia. For example, subjects with triglyceride levels at or above 500mg/dL may benefit from treatment with a PUFA or PUFA ester of the invention. In some embodiments, individual PUFAs or PUFA esters can be administered to a subject to treat high triglyceride levels. In certain embodiments, the PUFA or PUFA ester may be DHA or ARA. In other embodiments, a combination of PUFAs or PUFA esters can be administered to a subject to treat high triglyceride levels. In certain embodiments, the combination of PUFAs or PUFA esters may comprise omega-3 and omega-6 PUFAs, such as DHA and DPA n-6. In some embodiments, the PUFA or PUFA ester may comprise about 90% of the composition administered to a subject. The PUFA or PUFA ester can be administered with other components and excipients, such as the carriers described above. The PUFAs or PUFA esters can also be used to treat subjects suffering from diseases associated with high levels of triglycerides, such as cardiovascular disease or hypertension.

The PUFA esters produced by the process of the invention are useful in the production of PUFA salts. In some embodiments, the PUFA esters of the present invention can be reacted in the presence of a basic metal base, such as an alkaline earth metal hydroxide (e.g., potassium hydroxide), to produce a PUFA salt. The PUFA salts formed from the PUFA esters of the invention can be used in a variety of applications, such as food, beverage, and pharmaceutical products. In some embodiments, the PUFA salts produced using the PUFA esters of the invention are water soluble and can be used directly in food, beverage, and pharmaceutical products.

The PUFA or PUFA ester prepared by the method of the invention can be used in any animal food material, especially human food material, so as to obtain food with improved PUFA concentration. The naturally occurring fatty acid content of a food varies depending on the type of food. The food products of the invention may have normal amounts of PUFAs or modified amounts of PUFAs. In the former case, a portion of the naturally occurring lipids may be replaced with a PUFA or PUFA ester of the invention. In the latter case, naturally occurring lipids may be supplemented with the PUFAs or PUFA esters of the invention.

The PUFAs or PUFA esters can be added to infant food products, such as infant formula and baby food. According to the present invention, an infant refers to an infant less than about 2 years of age, including especially premature infants. Because of the rapid growth of infants (i.e., doubling or tripling of body weight during 1 year of age), certain PUFAs are particularly important components in infant formulas and baby food. An effective amount of a PUFA or PUFA ester to supplement infant formula is an amount that approximates the PUFA concentration in human breast milk. The PUFAs or PUFA esters are preferably added to infant formula or baby food in an amount of about 0.1 to about 1.0% total fatty acids, more preferably about 0.1 to about 0.6% total fatty acids, and even more preferably about 0.4% total fatty acids.

Another aspect of the invention includes food products comprising a food material in combination with a PUFA or PUFA ester of the invention. The PUFA or PUFA ester can be added to food materials to obtain food products with increased PUFA concentration. The term "foodstuff" as used herein refers to any type of food that is administered to a human or non-human animal. Also within the scope of the present invention is a process for the preparation of a food product comprising adding a PUFA or PUFA ester produced according to the invention to a food material.

Suitable foodstuffs for the preparation of the food product of the invention include animal feeds. The term "animal" means any organism belonging to the kingdom animalia, including, but not limited to, primates (e.g., humans and monkeys), poultry, and household pets. The term "food" includes any product administered to such animals. Preferred food materials for human consumption include infant formulas and baby food. Preferred food materials for domestic pet consumption include dog food.

The PUFAs or PUFA esters prepared by the process according to the invention can be incorporated into various products, such as baked goods, vitamin supplements, dietary supplements, powdered beverages, etc., in various stages of preparation. Many finished or semi-finished powdered food products can be prepared using the compositions of the present invention.

A partial list of food products comprising the product of the invention includes: a dough; a thin batter; baked goods including, for example, cakes, cheese cakes, pies, cupcakes, cookies, loaves, breads, buns, biscuits, muffins, pastries, scones, and toasts; liquid foods, such as beverages, energy drinks, infant formula, liquid diets, fruit juices, multi-vitamin syrups, meal replacers, medicinal foods, and syrups; semi-solid food products such as baby food, yogurt, cheese, cereals, pancake mixes; food bars, including energy bars; processed meat; ice cream; freezing the dessert; freezing yogurt; a wafer blend; salad dressing; and replacing the egg mixture. Also included are baked goods such as cookies, crackers, desserts, snacks, pies, granola/snack bars, and baked pie crusts; salted snacks such as potato chips, corn chips, granola, extruded snacks, popcorn, pretzels, potato chips, and nuts; special snacks such as dips, dried fruit snacks, meat snacks, pork rolls, health food bars and rice/corn cakes; and confectionery such as candy.

Another product embodiment of the invention is a medical food. Medicinal foods include foods in formulations that are consumed and administered externally under the supervision of a physician, as well as foods that are subject to specific dietary management of a disease or condition based on unique nutritional requirements established by medical evaluation based on accepted scientific principles.

While the invention has been described in terms of particular methods, products, and organisms, these descriptions are intended to include all such methods, products, and organisms that are available and useful in accordance with the teachings herein, including all alternatives, modifications, and optimizations known to those of skill in the art. The following examples and test results are provided for illustrative purposes and are not intended to limit the scope of the invention.

Examples

Example 1

Cultures of wild-type schizochytrium or Thraustochytrium (Thraustochytrium) were grown with a source of saccharified cellulose under typical fermentation conditions using a 2-liter fermentor. Each fermentor was batch treated with medium containing carbon (saccharified cellulose), nitrogen, phosphorus, salts, trace metals and vitamins. Each fermentor can be inoculated with a typical seed culture and then incubated for 72-120 hours with a carbon (saccharified cellulose) feed and a nitrogen feed being fed during incubation. The nitrogen feed is only transported and consumed during the growth phase, while the carbon (saccharified cellulose) is transported and consumed throughout the fermentation process. After 72-120 hours, the fermenters were collected, either spontaneously lysed or hydrolyzed. The hydrolysate is separated to form an oil and a biomass fraction. The oil is then transesterified and separated from the glycerol. The monoalkyl ester is washed with water to obtain the final product.

Typical fermentation control conditions:

temperature: 20-40 deg.C

pH：3.0-10.0

Stirring: 100-400cps

Airflow: 0.25-3.0vvm

Saccharifying cellulose: 5-35g/L (in-tank concentration)

Inoculum: 1 to 50 percent

Example 2

Wild-type or transgenic schizochytrium or thraustochytrium cultures were grown with a liquefied cellulose source under typical fermentation conditions using a 10-liter fermentor. The organism will produce the necessary enzymes, simultaneously saccharifying cellulose and metabolizing glucose, xylose, hemicellulose and lignin. Each fermentor was batch treated with medium containing carbon (liquefied cellulose), nitrogen, phosphorus, salts, trace metals and vitamins. Each fermentor can be inoculated with a typical seed culture and then incubated for 72-120 hours, during which time a carbon (liquefied cellulose) feed and a nitrogen feed are given. The nitrogen feed is only transported and consumed during the growth phase, while the carbon (liquefied cellulose) is transported and consumed throughout the fermentation process. After 72-120 hours, the fermenters were collected, either spontaneously lysed or hydrolyzed. The hydrolysate is separated to form an oil and a biomass fraction. The oil is then transesterified and separated from the glycerol. The monoalkyl ester is washed with water to obtain the final product.

Typical fermentation conditions:

temperature: 20-40 deg.C

pH：3.0-10.0

Stirring: 100-400cps

Airflow: 0.25-3.0vvm

Liquefying cellulose: 5-35g/L (in-tank concentration)

Inoculum: 1 to 50 percent

Example 3

The transgenic Schizochytrium or Thraustochytrium of example 2 can be grown using existing transformation systems (e.g., as described in published patent application WO 2002/083869A 2) to express genes encoding known and suitable cellulases, hemicellulases, ligninases, sugar transporters, epimerases, and sugar isomerases. Alternatively, previously uncharacterized cellulases, hemicellulases, ligninases, sugar transporters, epimerases and sugar isomerases can be isolated from existing genomic databases or by implementing standard gene discovery strategies with uncharacterized or less characterized organisms, including PCR based on degenerate primers of conserved regions of homologous genes, or mass sequencing and mining for Expressed Sequence Tags (ESTs) or genomic sequences, or other techniques. Appropriate gene expression and gene product activity can be verified using standard techniques such as gel electrophoresis, Northern and Western blots, enzyme-linked immunosorbent assays (ELISA), and substrate conversion assays.

Example 4

Two wild-type schizochytrium cultures (ATCC20888) were grown in a 2-liter fermentor under typical fermentation conditions, comparing fatty acid profile and lipid yields under sterile and aerobic conditions. Each fermentor was batch treated with media containing carbon, nitrogen, phosphorus, salts, trace metals and vitamins. The sterile fermenter is autoclaved for 120 minutes and all the media components are sterilized in the fermenter or added after autoclaving in the form of a sterile solution. The sterile fermenter was treated batchwise with tap water, and all the ingredients were added to the fermenter without sterilization prior to inoculation. Each fermentor can be inoculated with a typical seed culture and then incubated for 50 hours, during which time carbon and nitrogen feeds are given. Nitrogen feed is only transported and consumed during the growth phase, while carbon is transported and consumed throughout the fermentation process. After 50 hours, samples were taken from each fermenter, centrifuged, lyophilized, converted to fatty acid methyl esters, and analyzed by gas chromatography.

Typical fermentation conditions:

temperature: 28-30 deg.C

pH：5.0-7.5

Stirring: 100-300cps

Airflow: 0.25-2.0vvm

Glucose: 5-55g/L (concentration)

Inoculum: 1 to 30 percent

The results are as follows:

condition	Sterile	Has bacteria
			Bacterial strains	ATCC 20888	ATCC 20888
Log hours	50	50
			Sample (I)	BN25 8.08,14	BN26 8.08,14
			％12:0	0.21	0.12
％13:0	0.16	0.16
			％14:0	9.73	6.14
％15:1	0.59	0.79
			％16:0	39.93	36.26
％16:1	0.13	0.07
			％17:0	0.17	0.28
％18:0	1.13	1.16
			％18:1n-9	0.13	0.08
％18:1n-7	0.10	0.00
			％18:3n-6	0.10	0.12
％18:3n-3	0.04	0.07
			％18:4n-3	0.12	0.13
％20:0	0.10	0.10
			％20:3n-6	0.27	0.33
％20:4ARA	0.37	0.32
			％20:5EPA	0.45	0.56
％22:5n-6	12.61	14.52
			％22:6DHA	32.67	37.43
% Fat (Fat)	40.92	35.79
			% unknown	0.98	1.10
			% saturation	51.44	44.23
% monounsaturation	0.81	0.87
			% polyunsaturated	46.64	53.48

Fig. 3 and 4 show the results of this experiment.

Example 5

Wild-type schizochytrium (ATCC20888) was cultured under low-cost fermentation conditions using a 5-liter fermentor to evaluate the efficacy of heterotrophic production of algal biomass using low-cost aerobic conditions. The fermentation tank includes low carbon steel container, polypropylene film lining, pipe sprayer, one exhaust pipeline and one other pipeline. The aerobic fermentor was batch treated with tap water and media containing carbon, nitrogen, phosphorus, salts, trace metals and vitamins. The ingredients were added to the fermentor without sterilization prior to inoculation. The fermenter was inoculated with 50 ml of culture broth from a 250 ml culture flask. The fermenter was incubated for 6 days, during which (after inoculation) no material was added to the fermenter. After 6 days, samples were taken from the fermenter, lyophilized, converted to fatty acid methyl esters and analyzed by gas chromatography.

Typical fermentation conditions:

temperature: 28-30 deg.C

pH: is not controlled

Stirring: is free of

Airflow: 2.0-4.0vvm

Glucose: 80g/L (initial concentration; no additional feed)

Inoculum: 1 percent of

Bacteria-containing batch medium:

the results are as follows:

example 6:

a two-stage fermentation system can be used in the heterotrophic fermentation process of the microorganisms. The first stage (inoculation stage) targeted accumulation of biomass, the second stage (lipid production stage) targeted accumulation of lipids. An example of a fermentation system is described below.

The fermentation system described below includes a continuous inoculation vessel and a plurality of lipid production vessels operating in a fed-batch mode. The working volume of the inoculum vessel was xx gallons, one eighth of the lipid production phase vessel (xxx gallons) based on the following assumptions:

1) the cell doubling time was 6 hours and,

2) the batch was subjected to a filling time of 24 hours for each lipid production phase

3) The initial volume of each lipid production batch (after filling) was 1/2 of the harvest volume

Continuous inoculation stage

After initial inoculation/growth to steady state, the seed container received a continuous feed of sterile nutrients at a constant rate (xx GPM, approximately 1/48 harvest volumes per hour). The fermentation broth is withdrawn from the vessel at the same rate as the nutrient feed and transferred to the lipid production stage vessel. After the desired starting volume in the production vessel is reached (about 1/2 harvest volume after about 24 hours filling), the inoculation vessel is connected to the next available lipid production vessel.

The nutrient feed will include a carbon source (cellulosic feedstock and/or simple sugars), a nitrogen source (e.g., NH3), salts, vitamins, and trace metals at concentrations that will enable proper growth (the latter supporting optimal lipid production). Recycled de-lipidated biomass and glycerol may be used as part of the nutrients to reduce raw material costs. At steady state, biomass concentrations are likely to reach at least about 50-100 grams per liter.

The gas stream is supplied to provide sufficient oxygen for cell growth. The gas flow needs to be about 0.5 to 1.0vvm to achieve an OUR (oxygen uptake rate) of about 50 to 150 mmoles/liter/hour. It is expected that this process will generate a large amount of metabolic heat, requiring sufficient heat removal to maintain the target process temperature (e.g., about 25-35 ℃). The metabolic thermogenesis of microorganisms is generally evaluated using a relationship of 0.113 kcal/millimole O2 uptake, with an estimated thermogenesis of about 6-17 kcal/l/hr. Part of the heat can be removed by the gas stream, but still requires a large heat removal capacity to maintain the target temperature. pH control with acids (e.g., sulfuric acid) and/or alkalis (caustic) may be required to maintain pH targets for optimal growth. Due to the nature of the inoculation stage, the culture medium and process conditions may be very favorable for contaminant growth; therefore, a system design with low risk of contamination is highly desirable. The two-stage process can be carried out in a sterile fermenter by selecting conditions which are not conducive to the growth of contaminants. Another option is to perform the continuous inoculation phase under sterile conditions and the lipid production phase under aseptic conditions (which may be performed under nutrient limitation such as nitrogen limitation).

Lipid production phase (fed batch)

The lipid production phase vessel is run as a fed-batch process. Most nutrients were received from the inoculation stage (during the 24 hour fill time) and a carbon source was delivered to the batch to maintain the target sugar concentration throughout the run.

The total cycle time for each lipid production batch was 120 hours, 24 hours for filling (receiving fermentation broth from the inoculation vessel), 72 hours for lipid production, and 24 hours for harvesting and turnover. Thus, each inoculation vessel should be able to provide inoculum for the 5 lipid production stage vessels. As noted above, the seed transfer rate is expected to be about xxxGMP (harvest volume of 1/48pf per hour). After a 24 hour fill time, the production vessel should have a target harvest volume of about 1/2. A carbon source such as a cellulosic feedstock (about 70% sugar concentration) is added to maintain the target sugar concentration during most of the run time. Antifoam agents are added as needed to reduce foaming. At harvest, a biomass concentration of at least about 150 g/l or at least about 300 g/l can be achieved, with 60-80% of the biomass being oil.

Lipid preparation can be carried out using continuous or semi-continuous production strategies. In a continuous process, biomass is harvested at the same rate as the lipid production vessel is filled. In a semi-continuous process, biomass is harvested at regular intervals, the amount of biomass harvested depending on the lipid production cycle. For example, in a 72 hour lipid production cycle, harvesting is half of a biomass-containing preparation tank every 36 hours; similarly, the biomass in the 25% preparation tank is harvested every 18 hours, the biomass in the 75% preparation tank is harvested every 54 hours, and so on.

Cell maintenance and lipid production require oxygen, and a gas stream is supplied to provide sufficient oxygen transfer. The gas flow requirements are about 0.5 to 1.0vvm to achieve an OUR (oxygen uptake rate) of about 40 to 150 mmoles/liter/hr. This process is expected to produce significant metabolic heat. The metabolic thermogenesis of microorganisms is generally evaluated using a relationship of 0.113 kcal/millimole O2 uptake, with an estimated thermogenesis of about 6-17 kcal/l/hr. Part of the heat is removed by the air stream, but still requires a large heat removal capacity to maintain the target temperature. pH control with acids (e.g., sulfuric acid) and/or bases (caustic) may be required to maintain the pH target for high lipid productivity.

Other information/considerations

Fermentation costs can be reduced if fermentation waste (e.g., lipid media or de-lipidated biomass) can be efficiently recycled.

The conversion of sugars to biomass is about 45-55% (on a molar basis) and the conversion of sugars to oil is about 30-40%.

To minimize potential shutdowns due to equipment failure or batch abnormalities, additional seed and lipid production vessels should be considered in the plant design.

The foregoing has described the principles, preferred embodiments and modes of operation of the present invention. However, the scope of the invention is not to be construed as limited to the particular forms described, which are to be regarded as illustrative rather than restrictive. Those skilled in the art can make changes and modifications without departing from the spirit of the present invention. Therefore, the best mode for carrying out the invention as described above is to be considered as illustrative and not restrictive on the scope and spirit of the invention, which is defined by the appended claims.