WO2004081150A2

WO2004081150A2 - Methyl esters of hydroxyl-containing fatty acids as biofuels

Info

Publication number: WO2004081150A2
Application number: PCT/IB2004/000743
Authority: WO
Inventors: Jack Grushcow
Original assignee: Linnaeus Inc.
Priority date: 2003-03-14
Filing date: 2004-03-15
Publication date: 2004-09-23
Also published as: WO2004081150A3

Abstract

The invention relates to fuels, oils and fuel additives, more specifically improved biodiesels having improved oxidative stability and improved low temperature characteristics and comprising methyl esters of hydroxyl-containing fatty acids.

Description

METHYL ESTERS OF HYDROXYL-CONTAINING FATTY ACIDS AS

BIOFUELS

FIELD OF THE INVENTION The field of the invention relates to fuels and oils, more specifically biodiesels having inter alia improved oxidative stability and improved low temperature characteristics.

BACKGROUND OF INVENTION All publications and patent applications cited herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

For many years various sectors of the fuel industry have been interested in alternative energy sources based on renewable raw materials rather than being dependent on finite fossil fuel deposits. Plant oils, also referred to as fatty acid esters, are one major potential source of such renewable energy sources. Plant oils are generally biodegradable and environmentally harmless.

Under the impetus of environmental legislation, structural changes in agriculture/agribusiness and the general ecological trend, plant oils and modified plant oils are becoming increasingly important as fuels, lubricants and heating oils. For example, Marguerite Downey, the alternative-fuels manager for the U.S. Postal Service (USPS), said that biodiesel was "our most successful new fuel program, and we have more experience than any other fleet with alternative fuel." The USPS used 671,000 gallons of biodiesel blend last year; 418,000 in 2000; and 90,000 in 1999 (New York Times, May 16, 2002). Also, for information on the use of vegetable oil lubricants for internal combustion engines and total loss lubrication, see U.S. Patent No. 5,888,947.

The term biodiesel, as used herein, means a renewable fuel, typically from a plant source, comprising one or more fatty acids and or esters thereof. Typically biodiesel is combined with a petrochemical motor fuel. The ratio of petrochemical motor fuel to the renewable raw materials in the mixture, however, can vary and is not defined. An important consideration for practical use of plant oils in such applications is their physical properties at low temperatures.

As discussed above, the use of biodiesel (such as methyl esters of fatty acids) is gaining widespread acceptance as a fuel or fuel additive and, as a result, has been the subject of much innovation and experimentation. See, for example, U.S. Pat. Nos. 5,713,965, 5,578,090, 5,389,113, 5,338,471, 5,308,365, and 6,015,440. The initial driving force behind this trend has been the growing environmental awareness and the need to reduce particulate emissions. Further motivation was provided by the price subsidies for renewable energy sources instituted by governments in Europe. New regulations led to the introduction of low sulfur diesel that lacks adequate lubricity, further encouraging development of biodiesel since the incorporation of biodiesel in regular diesel helps to offset this lowered lubricity.

Biodiesel as it is currently sold is made from canola, soybean or yellow grease. The oils from these sources have a number of drawbacks, including poor low temperature properties and poor oxidative stability. The latter property results from the addition of fatty acid compositions with polyunsaturated fatty acids such as is found in the oils from canola and soybean. Similar to conventional diesel fuel, components of these fuels often crystallize out from biodiesel at low temperatures, thus impairing filterability and flowability. The use of currently available biodiesel fuels is further limited since they demonstrate a "Cloud Point" of near zero degrees centigrade (0.0° C) (i.e. thirty-two degrees Fahrenheit or 32° F), while the Cloud Point of conventional diesel is near negative sixteen degrees centigrade (-16° C).

A similar disparity exists with respect to the "Pour Point", which for biodiesel fuels is near negative two degrees centigrade (-2° C), while that for conventional diesel fuel is near negative twenty-seven degrees centigrade (-27° C). See, for example, "Low-Temperature Properties Of Triglyceride-Based Diesel Fuels: Transesterified Methyl Esters and Petroleum Middle Distillate Ester Blends", by Dunn et al, JAOCS, Vol. 72, No. 8, 1995. These adverse cold temperature flow properties of currently-used biodiesel fuel as compared to conventional diesel fuel, with accompanying reduced viscosity and low temperature flow, leads to problems such as truck fuel filter plugging below thirty-two degrees Fahrenheit (32° F).

The use of biodiesel fuels cannot become widespread unless these problems are overcome. Suggested solutions to these problems include the concept of Methyl Ester "winterization." See, for example, "Reducing The Crystallization Temperature Of Biodiesel By Winterizing Methyl Soyate", by Lee et al., JAOCS, Vol. 73, No. 5, 1996. Also see, for example, "Vegetable Oils: From Table To Gas Tank", by Chowdhury et al., Chem. Eng. February 1993, which discusses the application of biotechnology to produce biodiesel with improved specifications. The present invention solves die problems persistent in previously known formulations of biodiesel. The inventors have unexpectedly discovered that biodiesel produced by blending high oleic methyl esters and methyl esters of hydroxy fatty acids in ratios of 10:90 to 90:10 overcome many of the problems produced by using conventional biofuels and mixtures of such biofuels with regular diesel oil. These newly discovered biodiesel blends do not have the drawbacks of previous formulations due to the superior oxidative stability of oils containing hydroxyl groups" on some or all of their fatty acids when compared to polyunsaturated fatty acids. These new oil blends also have better low temperature properties and a higher oxygen content per molecule due to the presence of the hydroxyl group(s). The compositions of the instant invention can be produced by mixing the desired components, such as methyl esters of high oleic oils and methyl ricmoleate, as obtained through commercial or other sources, in the desired ratios. Further, the components of the biofuels of the instant invention may be produced by transforming plants with a high oleic background with a hydroxylase gene thereby producing high oleic oils containing hydroxyl groups on some or all of the fatty acids and subsequently esterifying the oils and blending them in the desired ratios.

SUMMARY OF THE INVENTION

The invention relates to a composition comprising a C1-C₄ ester of a Cι₂-C₂₂ fatty acid, a C1-C4 ester of a Cι₂-C₂₂ hydroxyl-containing fatty acid, and a petrochemical motor fuel.

In another aspect, the invention encompasses a composition comprising a

C ester of a C₁₂-C₂₂ fatty acid, a Cι-C₄ ester of a C₁₂-C₂₂ hydroxyl-containing fatty acid, and a petrochemical motor fuel wherein the Cι-C₄ ester of a C₁ -C₂₂ fatty acid and the Cι-C₄ ester of a Cι₂-C hydroxyl-containing fatty acid are in a ratio ranging from about 10:90 to 90:10. h another aspect, the invention encompasses a composition comprising a Cj- C₄ ester of a C₁₂-C₂₂ fatty acid, a C₁-C₄ ester of a C₁₂-C₂₂ hydroxyl-containing fatty acid, and a petrochemical motor fuel wherein the composition comprises greater than 10% petrochemical motor fuel. In another aspect, the invention encompasses a composition comprising a'Cr- C₄ ester of a Cι₂-C₂₂ fatty acid, a C₁-C₄ ester of a C₁₂-C₂₂ hydroxyl-containing fatty acid, and a petrochemical motor fuel wherein the composition comprises greater than 20% petrochemical motor fuel. In another aspect, the invention encompasses a composition comprising a C_\-

C₄ ester of a Cι₂-C₂₂ fatty acid, a -C₄ ester of a Cι₂-C₂₂ hydroxyl-containing fatty acid, and a petrochemical motor fuel wherein the composition comprises greater than 40% petrochemical motor fuel.

In another aspect, the invention encompasses a composition comprising a Ci- C₄ ester of a ₂-C₂₂ fatty acid, a -C₄ ester of a Cι₂-C₂₂ hydroxyl-containing fatty acid, and a petrochemical motor fuel wherein the composition comprises greater than 80% petrochemical motor fuel.

In another aspect, the invention encompasses a composition comprising a - C₄ ester of a Cι₂-C₂₂ fatty acid, a -C₄ ester of a Cι₂-C ₂ hydroxyl-containing fatty acid, and a petrochemical motor fuel wherein the composition comprises greater than 90% petrochemical motor fuel.

In another aspect, the invention encompasses a composition comprising a Ct- C ester of a C₁₂-C₂₂ fatty acid, a Cι-C₄ ester of a C₁₂-C₂ hydroxyl-containing fatty acid, a petrochemical motor fuel, and a pour depressant or other fuel additive standard in the industry. h another aspect, the invention encompasses a composition comprising a Ci- C₄ ester of a C₁₂-C₂₂ fatty acid, a C₁-C₄ ester of a C₁₂-C₂₂ hydroxyl-containing fatty acid, and a petrochemical motor fuel wherein the methyl esters of hydroxyl- containing fatty acids are obtained from genetically modified plants expressing a heterologous hydroxylase gene. The invention also contemplates a method of producing a biodiesel blend having at least one of improved oxidative stability, improved low temperature properties, or higher oxygen content comprising mixing one or more C esters of Ci2-C₂₂ fatty acids and one or more C₁-C₄ esters of Cι₂-C₂₂ hydroxyl-containing fatty acids in a ratio of from about 10 :90 to about 90:10.

In another aspect, the invention encompasses a method of producing a biodiesel blend having at least one of improved oxidative stability, improved low temperature properties, or higher oxygen content comprising mixing one or more Ci- C₄ esters of Cι₂-C₂₂ fatty acids and methyl ricmoleate in a ratio of from about 10:90 to about 90:10.

In another aspect, the invention encompasses a method of producing a biodiesel blend having at least one of improved oxidative stability, improved low temperature properties, or higher oxygen content comprising: transforming a plant with a gene encoding a hydroxylase, wherein the plant has a high oleic background; growing the plant under conditions under which the hydroxylase gene is expressed; isolating high oleic oils produced by the plant; and converting the high oleic oils in to C₁-C₄ esters. In another aspect the invention is a composition comprising high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in ratios from 10:90 to 90:10.

In another aspect, the present invention encompasses compositions comprising high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in ratios from 10:90 to 90:10 and further comprising greater than 10% diesel. In a further aspect the present invention encompasses compositions comprising high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in ratios from 10:90 to 90:10 and further comprising greater than 20% diesel.

In yet another aspect the present invention encompasses compositions comprising high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in ratios from 10:90 to 90:10 and further comprising greater than 40% diesel.

In a further aspect the present invention encompasses compositions comprising high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in ratios from 10:90 to 90:10 and further comprising greater than 80% diesel. hi still another aspect the present invention encompasses compositions comprising high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in ratios from 10:90 to 90:10 and further comprising at least about 90% diesel.

In still another aspect the present invention encompasses compositions comprising high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in ratios from 10:90 to 90:10 and further comprising a pour depressant and other fuel additives standard in the industry. i still a further aspect the present invention encompasses compositions comprising high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in ratios from 10:90 to 90:10 and wherein the methyl esters of hydroxyl- containing fatty acids are obtained from genetically modified plants expressing a heterologous hydroxylase gene.

The present invention further contemplates methods of producing a biodiesel blend having at least one of improved oxidative stability, improved low temperature properties, or higher oxygen content comprising mixing high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in a ratio of from about 10:90 to about 90:10.

The present invention also contemplates methods of producing a biodiesel blend having at least one of improved oxidative stability, improved low temperature properties, or higher oxygen content comprising mixing high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in a ratio of from about 10:90 to about 90:10 and wherein the methyl ester of hydroxyl-containing fatty acids is methyl ricmoleate.

The present invention is also embodied in a method of producing a biodiesel blend having at least one of improved oxidative stability, improved low temperature properties, or higher oxygen content comprising: transfoπning a plant with a gene encoding a hydroxylase, wherein the plant has a high oleic background; growing the plant under conditions under which the hydroxylase gene is expressed; isolating high oleic oils produced by the plant; and converting the high oleic oils to methyl esters.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides the basic triacylglyceride (TAG) chemical structure. Figure 2 provides the basic scheme of TAG assembly. Figure 3 provides the chemical structure of ricinoleic acid. Figure 4 provides examples of some industrial applications for high fatty acid

(HFA) oils.

Figure 5 provides a comparison of the properties of conventional plant oils and the blended oils of the instant invention..

Figure 6 provides a comparison of the fatty acid profiles of canola oil, castor oil and the oils of the present invention. Figure 7 provides a comparison of the structural and performance characteristics of castor oil, high oleic canola oil and the oils of the present invention. Figure 8 provides an explanation of how the oils of the present invention can substitute for synthetic esters. Figure 9 provides an overview of the process of agrobacterium-mediated plant transformation.

DETAILED DESCRIPTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testmg of the present invention, the preferred methods and materials are described.

The most commonly occurring fatty acids which normally or naturally occur in both membrane and storage lipids of plants are a small family of 16- and 18 -carbon fatty acids which have from zero to three methylene-interrupted cis unsaturations. All members of the family are descended from the fully saturated species as the result of a series of sequential desaturations which begin at the ω-9 carbon and progress in the direction of the ω-3 carbon. Fatty acids which cannot be described by this simple algorithm are generally considered "unusual" even though several, such as lauric (12:0), erucic (22:1), and ricinoleic (12-OH, 18:1), are of significant commercial importance. For a review of this subject, see, for example, T.S. Moore, Lipid Metabolism in Plants, CRC Press, 1993, which is specifically incorporated by reference herein in its entirety.

Glycerolipids are fatty acid esters of glycerol. Triacylglycerols (also called triglycerides or TAG) consist of a glycerol molecule that is esterified with three fatty acids. See Figure 1 for the basic TAG chemical structure and Figure 2 for the basic scheme of TAG assembly. TAGs are contained primarily in seeds but also in some fruits, such as olives or avocados. Ricinoleic acid, a rare fatty acid, which contains a hydroxyl group in the C-12 position and makes up about 90 per cent of the fatty acids in castor oil, is used in industry as a lubricant and also as a means of surface ^' protection. For example, castor oil is widely used to make polyurethanes by producing highly cross-linked products. A lower hydroxy content can provide products with interesting performance. For example, hydroxyl groups in the 1 and 3 positions can provide linear polymers when reacted with bi-functional monomers such as dissocyanatees. See Figure 3 for the chemical structure of ricinoleic acid. For an overview of plant oils, see, for example, Hans-Walter Heldt, Glycerolipids, In Plant biochemistry and molecular biology, Oxford University Press, 1997, Chapter 15, pp. 317-351, which is specifically and entirely incorporated by reference herein.

High fatty acid (HFA) oils can be used for many purposes, as shown in the following table .

The present invention solves the aforementioned problems that have limited the usefulness of biodiesel as an alternative to fossil fuels. The instant invention contemplates a bioseisel blend comprised of one or more

C1-C4 esters of a Cι₂-C₂₂ fatty acid and one or more C1-C₄ esters of a Cι₂-C₂₂ hydroxyl-containing fatty acid in ratios ranging from 10:90 to 90:10 and optionally containing up to 90% of regular diesel oil. The biodiesel blend of the instant invention further contemplates the use of pour depressants. The instant invention further encompasses biodiesel wherein the one or more C₁-C₄ esters of a Cι₂-C ₂ fatty acid and the one or more -C₄ esters of a Cι₂-C₂ hydroxyl-containing fatty acid are obtained from genetically modified plants in which a hydroxylase gene is expressed or wherein the blend is obtained by mixing one or more Cι-C₄ esters of a Cι₂-C₂₂ fatty acid and one or more C1-C₄ esters of a hydroxyl-containing C₁₂-C₂₂ fatty acid.

In one embodiment, the C₁-C₄ ester of a hydroxyl-containing C₁₂-C₂₂ fatty acid is a Cι-C₄ ester of ricinoleic acid, hi a preferred embodiment, the C₁-C₄ ester of a hydroxyl-containing C12-C22 fatty acid is a methyl ester of ricinoleic acid.

Representative C₁-C₄. esters of a C₁₂-C₂₂ fatty acid include, but are not limited to, Cι-C esters of myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, linoleic acid, or linolenic acid. Preferably, the C₁-C₄ esters of a Cι₂-C₂₂ fatty acid is a C1-C₄ esters of a Ciβ-Cis fatty acid. A more preferred Cι-C₄ ester of a C₁₂- C ₂ fatty acid is a Cι-C₄ ester of oleic acid. A most preferred C₁-C₄ ester of a C₁₂-C₂₂ fatty acid is methyl oleate.

In one embodiment, the oneOr more C₁-C₄ ester of a Cι₂-C₂₂ fatty acid are methyl esters.

In one embodiment, the one or more C1-C₄ ester of a Cι₂-C2₂ fatty acid are ethyl esters.

In one embodiment, the one or more C₁-C₄ ester of a Cι₂-C₂₂ fatty acid are n- propyl esters.

In one embodiment, the one or more C₁-C₄ ester of a Cι -C₂₂ fatty acid are iso- propyl esters. In one embodiment, the one or more -C₄ ester of a Cι₂-C₂₂ fatty acid are n- butyl esters.

In one embodiment, the one or more -C₄ ester of a Cι₂-C₂₂ fatty acid are sec-butyl esters.

In one embodiment, the one or more C₁-C₄ ester of a C₁₂-C₂₂ fatty acid are tert-butyl esters. hi one embodiment, the one or more C₁-C₄ esters of the hydroxyl-containing C₁ -C₂₂ fatty acid are methyl esters. hi one embodiment, the one or more C₁-C₄ ester of the hydroxyl-containing Ci₂-C ₂ fatty acid are ethyl esters. In one embodiment, the one or more C₁-C₄ ester of the hydroxyl-containing

C₁₂-C₂₂ fatty acid are n-propyl esters. hi one embodiment, the one or more C₁-C₄ ester of the hydroxyl-containing Cι₂-C₂₂ fatty acid are iso-propyl esters.

In one embodiment, the one or more C₁-C₄ ester of the hydroxyl-containing Cι -C₂₂ fatty acid are n-butyl esters.

In one embodiment, the one or more -C₄ ester of the hydroxyl-containing Cι₂-C₂₂ fatty acid are sec-butyl esters.

In one embodiment, the one or more -C₄ ester of the hydroxyl-containing C12-C22 fatty acid are tert-butyl esters. In one embodiment, at least one of the one or more C₁-C₄ esters of the Cι₂-C₂₂ fatty acids or the one or more -C₄ esters of the hydroxyl-containing Cι₂-C₂₂ fatty acid are methyl esters.

In one embodiment, both the one or more C₁-C₄ esters of the C₁₂-C₂₂ fatty acid and the one or more C₁-C₄ esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids are methyl esters. i one embodiment, the ratio of the one or more C₁-C₄ esters of the Cι₂-C₂₂ fatty acids and the one or more -C₄ esters of a hydroxyl-containing Cι₂-C₂₂ fatty acids is about 10:90. In one embodiment, the ratio of the one or more C1-C4 esters of the Cι₂-C₂₂ fatty acid and the one or more Cι-C esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids is about 15:85. hi one embodiment, the ratio of the one or more C1-C₄ esters of the Cι -C₂₂ fatty acid and the one or more C1-C4 esters of the hydroxyl-containing Cι₂-C ₂ fatty acids is about 20:80. i one embodiment, the ratio of the one or more C₁-C₄ ester of a Cι₂-C₂₂ fatty acid and the one or more C₁-C₄ ester of a hydroxyl-containing Cι₂-C₂₂ fatty acid is about 25:75. In one embodiment, the ratio of the one or more C₁-C₄ esters of the Cι₂-C₂₂ fatty acids and the one or more Cι-C₄ esters of the hydroxyl-containing C₁ -C₂₂ fatty acids is about 30:70.

In one embodiment, the ratio of the one or more C₁-C₄ esters of the Cι₂-C₂₂ fatty acid and the one or more C1-C4 esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids is about 35:65.

In one embodiment, the ratio of the one or more -C₄ esters of the Cι₂-C₂₂ fatty acid and the one or more C₁-C₄ esters of the hydroxyl-containing C₁₂-C₂₂ fatty acids is about 40:60.

In one embodiment, the ratio of the one or more C₁-C₄ esters of the C₁₂-C₂2 fatty acids and the one or more C C₄ esters of the hydroxyl-containing C₁₂-C ₂ fatty acids is about 45:55.

In one embodiment, the ratio of the one or more -C₄ esters of a Cι₂-C₂₂ fatty acids and the one or more C₁-C₄ esters of the hydroxyl-containing Cι -C₂₂ fatty acids is about 50:50. hi one embodiment, the ratio of the one or more C1-C₄ ester of the C₁₂-C₂₂ fatty acids and the one or more Cι-C₄ esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids is about 55:45.

In one embodiment, the ratio of the one or more C1-C₄ esters of the C₁₂-C₂₂ fatty acid and the one or more C1-C4 ester of the hydroxyl-containing Cι₂-C₂₂ fatty acid is about 60:40. hi one embodiment, the ratio of the one or more Cι-C₄ esters of the Cι₂-C₂₂ fatty acids and the one or more C1-C₄ esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids is about 65:35. In one embodiment, the ratio of the one or more C1-C₄ esters of the Cι₂-C₂₂ fatty acids and the one or more C₁-C₄ esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids is about 70:30. i one embodiment, the ratio of the one or more -C₄ esters of the Cι₂-C₂₂ fatty acids and the one or more C₁-C₄ esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids is about 75:25.

In one embodiment, the ratio of the one or more -C₄ esters of the Cι₂-C₂ fatty acids and the one or more C₁-C₄ esters of the hydroxyl-containing C₁₂-C₂₂ fatty acids is about 80:20.

In one embodiment, the ratio of the one or more Cι-C esters of the C12-G22 fatty acids and the one or more C₁-C₄ esters of the hydroxyl-containing C₁₂-C₂₂ fatty acids is about 85:15.

In one embodiment, the ratio of the one or more Cι-C esters of the Cι₂-C₂₂ fatty acids and the one or more Cι-C esters of the hydroxyl-containing Cι₂-C ₂ fatty acids is about 90:10. The present invention also encompasses any ratio that falls within these example ratios, such as, for example, 13:87 or 68:32.

Examples of some industrial applications for high fatty acid (HFA) oils is provided in Figure 4. In one embodiment the -C4 esters of a Cι₂-C22 fatty acid are a C₁-C₄ ester of oleic acid. Accordingly, the instant invention also contemplates a biodiesel blend comprised of high oleic C1-C₄ esters and one or more -G₄ esters of a ₂- 2 hydroxy fatty acid in ratios ranging from 10:90 to 90: 10 and optionally containing up to 90% of regular diesel oil. The term "high oleic C₁-C4 esters," as used herein means a mixture of one or more C₁-C₄ esters of fatty acids wherein the mixture of C₁-C4 esters of the fatty acids has a high content of C₁-C₄ esters of oleic. Preferably, the oleic acid content of the high oleic C₁-C₄ esters is at least about 50%, more preferably at least about 75%, and most preferably at least about 90% oleic acid. The high oleic C₁-C₄ esters can be obtained, for example, from high oleic triglycerides. The biodiesel blend of the instant invention further contemplates the use of pour depressants. The instant invention further encompasses biodiesel wherein the high oleic Cι-C₄ esters and the one or more Cι-C esters of a Cι₂-C₂₂ hydroxyl acid are obtained from genetically modified plants in which a hydroxylase gene is expressed or wherein the blend is obtained by mixing C C esters of high oleic oils and Cι-C₄ esters of hydroxyl-containing Cι₂-C₂₂ fatty acids.

In one embodiment, the Cι-C₄ ester of a hydroxyl-containing C₁₂-C₂₂ fatty acid is a C1-C₄ ester of ricinoleic acid. In a preferred embodiment, the -C₄ ester of a hydroxyl-containing Cι₂-C₂₂ fatty acid is a methyl ester of ricinoleic acid. In one embodiment, the high oleic C1-C4 esters are methyl esters. In one embodiment, the high oleic CrC₄ esters are ethyl esters. In one embodiment, the high oleic -C₄ esters are n-propyl esters. h one embodiment, the high oleic C₁-C₄ esters are iso-propyl esters.

In one embodiment, the high oleic -C₄ esters are n-butyl esters.

In one embodiment, the high oleic C₁-C₄ esters are sec-butyl esters. hi one embodiment, the high oleic -C₄ esters are tert-butyl esters.

In one embodiment, the one or more C₁-C₄ esters of the hydroxyl-containing Cι₂-C₂₂ fatty acid are methyl esters.

In one embodiment, the one or more C₁-C₄ ester of the hydroxyl-containing Ci₂-C₂₂ fatty acid are ethyl esters. In one embodiment, the one or more Cι-C ester of the hydroxyl-containing

Ci₂-C₂₂ fatty acid are n-propyl esters.

In one embodiment, the one or more C₁-C₄ ester of the hydroxyl-containing C12-C22 fatty acid are iso-propyl esters.

In one embodiment, the one or more Cι-C₄ ester of the hydroxyl-containing Ci₂-C₂₂ fatty acid are n-butyl esters.

In one embodiment, the one or more Cι-C₄ ester of the hydroxyl-containing C₁₂-C₂2 fatty acid are sec-butyl esters.

In one embodiment, the one or more C₁-C₄ ester of the hydroxyl-containing Ci₂-C₂₂ fatty acid are tert-butyl esters. h one embodiment, at least one of the high oleic Cι-C₄ esters or the one or more Cι-C₄ esters of the hydroxyl-containing C₁2-C₂₂ fatty acid are methyl esters. i one embodiment, both the high oleic -C esters and the one or more Ci- C₄ esters of the hydroxyl-containing C₁₂-C₂₂ fatty acid are methyl esters.

In one embodiment, the ratio of the high oleic Cι-C₄ esters to the one or more C₁-C₄ esters of the hydroxyl-containing Ci₂-C₂₂ fatty acid is about 10:90. hi one embodiment, the ratio of the high oleic Ci-C₄ esters to the one or more Cι-C esters of the hydroxyl-containing C₁₂-C₂₂ fatty acids is about 15:85. one embodiment, the ratio of the high oleic C₁-C₄ esters to the one or more Cι-C esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids is about 20:80. In one embodiment, the ratio of the high oleic C₁-C₄ esters to the one or more

C₁-C₄ esters of the hydroxyl-containing C₁₂-C₂₂ fatty acids is about 25:75.

In one embodiment, the ratio of the high oleic C₁-C₄ esters to the one or more Cι-C esters of the hydroxyl-containing Ci -C₂₂ fatty acids is about 30:70.

In one embodiment, the ratio of the high oleic C₁-C₄ esters to the one or more Cι-C esters of the hydroxyl-containing C₁₂-C₂₂ fatty acids is about 35:65. hi one embodiment, the ratio of the high oleic C₁-C₄ esters to the one or more C₁-G₄ esters of the hydroxyl-containing C₁₂-C₂₂ fatty acids is about 40:60. i one embodiment, the ratio of the high oleic -C₄ esters to the one or more -C₄ esters of the hydroxyl-containing C₁₂-C₂₂ fatty acids is about 45:55. In one embodiment, the ratio of the high oleic C₁-C₄ esters to the one or more

C₁-C₄ esters of the hydroxyl-containing Cj₂-C₂₂ fatty acids is about 50:50. hi one embodiment, the ratio of the high oleic -C₄ esters to the one or more C₁-C₄ esters of the hydroxyl-containing Cι₂-C₂ fatty acids is about 55:45.

In one embodiment, the ratio of the high oleic C₁-C₄ esters to the one or more C₁-C₄ esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids is about 60:40.

In one embodiment, the ratio of the high oleic -C₄ esters to the one or more C₁-C₄ esters of the hydroxyl-containing C₁₂-C₂₂ fatty acids is about 65:35. hi one embodiment, the ratio of the high oleic C₁-C₄ esters to the one or more C₁-C₄ esters of the hydroxyl-containing Ci₂-C₂₂ fatty acids is about 70:30. hi one embodiment, the ratio of the high oleic Cι-C₄ esters to the one or more C1-C4 esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids is about 75:25.

In one embodiment, the ratio of the high oleic C₁-C₄ esters to the one or more C₁-C4 esters of the hydroxyl-containing Cι₂-C₂₂ fatty acids is about 80:20. In one embodiment, the ratio of the high oleic C1-C4 esters to the one or more

C1-C4 esters of the hydroxyl-containing C₁₂-C₂₂ fatty acids is about 85:15. hi one embodiment, the ratio of the high oleic C₁-C esters to the one or more d-C4 esters of the hydroxyl-containing Cι₂-C ₂ fatty acids is about 90:10.

The present invention also encompasses any ratio that falls within these example ratios, such as, for example, 13:87 or 68:32.

The instant invention also contemplates a biodiesel blend comprised of high oleic methyl esters and methyl esters of one or more C₁₂-C₂₂ hydroxyl-containing fatty acids in ratios ranging from 10:90 to 90: 10 and optionally containing up to 90% of regular diesel oil. The biodiesel blend of the instant invention further contemplates the use of pour depressants. The instant invention further encompasses biodiesel wherein the high oleic methyl esters and the Cι₂-C₂₂ hydroxyl-containing fatty acid methyl esters are obtained from genetically modified plants in which a hydroxylase gene is expressed or wherein the blend is obtained by mixing methyl esters of high oleic oils and methyl esters of hydroxyl-containing fatty acids, such as methyl ricinoleate.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more C₁₂-C₂₂ hydroxyl-containing fatty acids is about 10:90.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more C₁₂-C₂2 hydroxyl-containing fatty acids is about 15:85. hi one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more Cι₂-C₂₂ hydroxyl-containing fatty acids is about 20:80.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more Cι₂-C₂₂ hydroxyl-containing fatty acids is about 25:75. hi one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more C12-C22 hydroxyl-containing fatty acids is about 30:70.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more C12-C22 hydroxyl-containing fatty acids is about 35:65.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more Cι₂-C₂2 hydroxyl-containing fatty acids is about 40:60.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more Cι₂-C₂₂ hydroxyl-containing fatty acids is about 45:55.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more Cι₂-C₂₂ hydroxyl-containing fatty acids is about 50:50. In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more Cι₂-C₂₂ hydroxyl-containing fatty acids is about 55:45.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more Cι₂-C₂₂ hydroxyl-containing fatty acids is about 60:40.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more C12-C22 hydroxyl-containing fatty acids is about 65:35.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more Ci₂-C₂₂ hydroxyl-containing fatty acids is about 70:30.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more C₁₂-C22 hydroxyl-containing fatty acids is about 75:25. In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more Ci2-C₂₂ hydroxyl-containing fatty acids is about 80:20.

In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more Cι₂-C₂₂ hydroxyl-containing fatty acids is about 85:15. In one embodiment, the ratio of the high oleic methyl esters and methyl esters of one or more C12-C22 hydroxyl-containing fatty acids is about 90:10.

The following examples serve to illustrate particular embodiments of the instant invention and are in no way intended to limit its scope.

EXAMPLES Example 1. Production of Improved Biodiesel Fuels by Mixing

The compositions of the instant invention may be produced by any of the known methods of mixing fuels components. For example, high oleic methyl esters are mixed with methyl esters of hydroxy fatty acids, such as methyl ricinoleate, in ratios from 10:90 to 90:10. For example, the ratio of high oleic methyl esters to methyl esters of hydroxy fatty acids can be 10:90 or 15:85 or 20:80 or 25:75 or 30:70 or 35:65 or 40:60 or 45:55 or 50:50 or 55:45 or 60:40 or 65:35 or 70:30 or 75:25 or 80:20 or 85:15 or 90:10, or any ratio that falls within these example ratios, such as, for example, 13:87 or 68:32.

The resulting biodiesel is then blended with conventional diesel in concentrations of up to 90% conventional diesel. Thus, for example, the ratio of high oleic fatty acids/methyl esters of hydroxy fatty acids component to the conventional diesel component can be 10:90 or 15:85 or 20:80 or 25:75 or 30:70 or 35:65 or 40:60 or 45:55 or 50:50 or 55:45 or 60:40 or 65:35 or 70:30 or 75:25 or 80:20 or 85:15 or 90:10, or any ratio that falls within these example ratios, such as, for example, 17:83 or 67:33.

A comparison of the properties of conventional plant oils and the blended oils of the instant invention is provided in Figure 5. A comparison of the fatty acid profiles of canola oil, castor oil and the oils of the present invention is provided in Figure 6.

Figure 7 provides a comparison of the structural and performance characteristics of castor oil, high oleic canola oil and the oils of the present invention. An explanation of how the oils of the present invention can substitute for synthetic esters is provided in Figure 8. i summary, the benefits of HFA-based biodiesel versus currently available biodiesel are as follows:

• Improved performance versus ASTM D6751-02 (standard biodiesel B- 100 specifications);

• Improved oxidative stability to provide cleaner burning fuel;

• Improved low temperature properties;

• Beneficial impact in emulsified fuels like Aquazole® or Purinox®;

• Improved lubricity of low sulfur diesel; • Increased cetane number;

• Lowered emissions, resulting in lower ecotoxicity; and

• Improved fuel economy.

Optionally, any of the known pour depressants, such as that taught in U.S. Patent 4,634,550 is added to the mixture in effective amounts. The instant invention contemplates the use of any high oleic methyl esters and methyl esters of hydroxy fatty acids to produce the claimed compositions. Thus, in one embodiment, the components of the instant invention are optimally produced in accordance with the methods of Example 2 below.

Example 2 Biodiesel Comprising Oil Produced by Transgenic Plants

Expression of hydroxylase in transgenic plants. A variety of methods have been developed to insert a DNA sequence of interest into the genome of a plant host to obtain the transcription or transcription and translation of the sequence to effect phenotypic changes. Recombinant DNA techniques allow plant researchers to circumvent the limitations of conventional plant breeding by enabling plant geneticists to identify and clone specific genes for desirable traits. Once the heterologous hydroxylase gene has been introduced into a plant, that plant can than be used in conventional plant breeding schemes (e.g., pedigree breeding, single-seed- descent breeding schemes, reciprocal recurrent selection) to produce progeny which also contain the heterologous hydroxylase gene.

Hydroxylase genes and the production hydroxylated fatty acids through the use of hydroxylase genes in transgenic plants have previously been described in U.S. Patent Nos: 6,433,250; 6,310,194; 6,291,742; 6,028,248; 5,801,026 and 5,668,292, herein incorporated by reference. The skilled artisan would recognize that the determination that plant fatty acyl hydroxylases are active in the in vivo production of hydroxylated fatty acids suggests several other possibilities for plant enzyme sources. Hydroxylated fatty acids are found in some natural plant species in abundance. For example, three hydroxy fatty acids related to ricinoleate occur in major amounts in seed oils from various Lesquerella species. Other natural plant sources of hydroxylated fatty acids are seeds of the Linum genus, seeds of Wrightia species, Lycopodium species, Strophanthus species, Convolvulaces species, Calendula species and many others.

Plants having significant presence of ricinoleate are preferred candidates to obtain naturally-derived oleate hydroxylases. However, it will also be recognized that other plant sources which do not have a significant presence of ricmoleate may be readily screened as other enzyme sources. For example, Lesquerella densipila contains a diunsaturated 18 carbon fatty acid with a hydroxyl group (Gunstone et al, 1986). In addition, a comparison between oleate-preferring plant fatty acyl hydroxylases and between plant fatty acyl hydroxylases which introduce hydroxyl groups at positions other than the 12-carbon or on substrates other than oleic acid may yield insights for protein modeling or other modifications to create synthetic hydroxylases using known molecular genetics methods.

Having obtained hydroxylase gene sequences, hydroxylase genes from other plant sources can be readily isolated through DNA hybridization techniques using sequences or peptide sequence information from known hydroxylase genes, by polymerase chain reaction methods based on sequence similarities between a known hydroxylase genes and other sources, and by immunological cross-reactivity using antibodies to known hydroxylase proteins as a probe. In any of these methods, cDNA or genomic libraries from the desired plants are generally necessary. Many methods of constructing cDNA or genomic libraries are provided in the scientific literature and many kits for synthesis of cDNA libraries are available commercially.

Isolation of Hydroxylase Genes through Hybridisation Techniques. The full-length cDNA clone for a hydroxylase gene is a preferred heterologous hybridization probe. However, fragments of the cDNA or a genomic clone are also useful as heterologous hybridization probes. In order to determine if the cDNA is a suitable probe for a given species, Northern analysis of RNA from various tissues of the target plant species is conducted to determine appropriate hybridization conditions. Since hydroxylated fatty acids generally accumulate preferentially in seeds but not in leaves, RNA is isolated from developing embryo tissues and leaves, electrophoresed in a formaldehyde/agarose gel and transferred to a nylon membrane filter. The labeled hydroxylase probe is added to a hybridization solution containing 50% formamide, 6.times.SSC (or ό.times.SSPE), 5.times.Denhardt's reagent, 0.5% SDS, and 100 .mu.g/ml denatured salmon sperm DNA fragments. The hybridization solution containing the labeled probe is incubated with the Northern filter at approximately 40 degrees C. for 18 hours or longer to allow hybridization of the probe to sequences which show regions of significant homology (more than about 60% identity). The filter is then washed at low stringency (room temperature to 42 degrees C. in l.times.SSC).

After exposing the filter to an X-ray film for various amounts of time, stringency conditions can be adjusted by decreasing the amount of formamide progressively to zero and duplicate filters probed until a limited number of distinct bands can be reproducibly detected. The presence of a higher degree of hybridization to the lane of RNA from tissues that accumulate hydroxylated fatty acids is taken as preliminary evidence that the probe is detecting transcripts from a hydroxylase gene which may be isolate through known methods.

Isolation of Hydroxylase Genes by PCR methods. An alternative approach to heterologous hybridization is to amplify the target gene using degenerate PCR primers. Based on the high degree of amino acid sequence identity between known hydroxylase genes probes for hydroxylases can be obtained by preparing mixed oligonucleotides of greater than 10, preferably of 15 or more, nucleotides in length representing all possible nucleotide sequences which could encode the corresponding amino acid sequences. This method is clearly documented by Gould et al. (1989). Typically, one skilled in the art would prepare a number of such primers based on the regions of conserved sequence between known hydroxylases, and would then test various combinations of these primers for their ability to produce a PCR product of the expected size.

When a PCR product of the expected size is produced, the band would be excised from an agarose gel, cloned and the nucleotide sequence determined. The cloned fragment may then be used as a hybridization probe under conditions of high stringency (i.e., 68. degree. C. in 5.times.SCC) to isolate cDNA or genomic clones from the target species. The identity of a particular clone is then verified by expression of the clone in a suitable transgenic host. The choice of a suitable host for expression of the gene is mediated by the availability in the host of the substrate for the hydroxylase enzyme and the ability to transform the particular host.

Use of immunological methods to identify hydroxylase genes. Hydroxylase genes can also be identified by immunological cross-reactivity using antibodies to the enzyme as a probe. T his experiments involves three steps: (1) isolation of large quantities of the protein from recombinant E. coli strains for the hydroxylase gene or from other biological systems for expression of recombinant proteins, (2) production of antibodies against the protein by inoculated rabbits or other antibody producing species (i.e. rats, hamsters, goats, etc.); (3) using the labeled antibodies as a probe on an expression library of MRNA sequences from the target plant. Because of the relative ease of production of large quantities of protein from a cloned gene, the use of recombinant protein is the preferred method.

It is contemplated that the foregoing methods can be used to allow the isolation of hydroxylase genes from species where hydroxylated fatty acids can be found. At least 33 structurally distinct monohydroxylated plant fatty acids have been described in the art. The approaches described above can be of utility to isolate the genes encoding the corresponding hydroxylases.

Clones identified using DNA hybridization or immunological screening techniques are then purified and the DNA is isolated and characterized, hi this manner, it is verified that the clones encode hydroxylase. The newly isolated plant hydroxylase sequences can also be used to isolate genes for hydroxylases from other plant species using the techniques described above.

Production of Transgenic Plants. Methods of producing transgenic plants are well known to those of ordinary skill in the art. Transgenic plants can now be produced by a variety of different transformation methods including, but not limited to, electroporation; microinjection; microprojectile bombardment, also known as particle acceleration or biolistic bombardment; viral-mediated transformation; and

Agrobacterium-mediated transformation (see, e.g., U.S. Patent Nos. 5,405,765,

5,472,869, 5,538,877, 5,538,880, 5,550,318, 5,641,664, 5,736,369 and 5,736369; Watson et al., Recombinant DNA, Scientific American Books (1992); Hinchee et al.,

Bio/Tech. 6:915-922 (1988); McCabe et al., Bio/Tech. 6:923-926 (1988); Toriyama et al., Bio/Tech. 6:1072-1074 (1988); Fromm et al., Bio/Tech. 8:833-839 (1990);

Mullins et al., Bio/Tech. 8:833-839 (1990); and, Raineri et al., Bio/Tech. 8:33-38

(1990)). Figure 9 provides an overview of the process of agrobacterium-mediated plant transformation. Hydroxylase genes may also be introduced in a. site directed fashion using homologous recombination. Homologous recombination permits site-specific modifications in endogenous genes and thus inherited or acquired mutations may be corrected, and/or novel alterations may be engineered into the genome. Homologous recombination and site-directed integration in plants are discussed in U.S. Patent Nos. 5,451,513, 5,501,967 and 5,527,695.

Modifications maybe made that will increase the level of accumulation of hydroxylated fatty acids in plants that expressing heterologous hydroxylase genes. Improvements in the level and tissue specificity of expression of the hydroxylase gene are contemplated. Methods to accomplish this by the use of strong, seed-specific promoters such as the B. napus napin promoter will be evident to one skilled in the art. Additional improvements resulting from increases in the amount of substrate are also envisioned. The substrate for the hydroxylase is currently believed to be oleate or other monounsaturated fatty acid esterified to phosphatidylcholine. Therefore, expression of the hydroxylase gene in plant species or particular cultivars that contain elevated levels of oleate-containing phospholipids is believed to lead to increased accumulation of hydroxylated fatty acids.

It is also contemplated that the results may be improved by modification of the enzymes which cleave hydroxylated fatty acids from phosphatidylcholine, reduction in the activities of enzymes which degrade hydroxylated fatty acids and replacement of acyltransferases which transfer hydroxylated fatty acids to the sn-1 and sn-3 positions of glycerolipids. Although genes for these enzymes are not currently available, their utility in improving the level of production of hydroxylated fatty acids will be evident based on the results of biochemical investigations of ricinoleate synthesis. Upon production of transgenic plants according to the above method one of skill in the art would then use any of the many oil extraction methods known in the art such as cold-pressing, liquid carbon dioxide extraction, screw press extraction or solvent extraction to remove the oils produced by the plant. Upon removal of the oils, the skilled artisan would then convert the oils in to methyl esters using any of the methods that are well known in the art such as those taught in US Patent Nos. 6,440,057, 6,262,285 and 5,043,485. For example, methyl esters can be prepared by a type of transesterification known as alcoholysis in which the acyl groups in the triglycerides such as soybean oil, safflower oil, com oil, lard, tallow, etc., are allowed to exchange with an excess of some methanol, so that nearly all the acyl groups are recovered as methyl esters rather than as glycerol esters.

The transesterification is accomplished by means of a transesterification catalyst such as sodium alkoxide, sodium or potassium hydroxide, and the like. Acid catalysts, such as sulfuric acid, hydrogen chloride and boron trifluoride can also be used. The acid catalysts are particularly appropriate when large amounts of free fatty acids are present in the oil. The amount of catalyst is generally in the range of from about 0.1 to about 0.5% by weight, based on the weight of the oil. The resulting methyl ester may be freed of glycerol by water washing and the excess alcohol can be removed by water washing and/or distillation. The resulting methyl esters would then be used to produce the biodiesel of the instant invention by following, for example, the methods of Example 1.

The foregoing detailed description has been given for clearness of understanding only and no unnecessary limitations should be understood therefrom as modifications will be obvious to those skilled in the art. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a C₁-C₄ ester of a Cι₂-C₂₂ fatty acid, a C₁-C₄ ester of a C₁₂-C₂₂ hydroxyl-containing fatty acid, and a petrochemical motor fuel.

2. The composition of claim 1, wherein the C₁-C₄ ester of a Cι₂-C₂₂ fatty acid and the C1-C4 ester of a C12-C22 hydroxyl-containing fatty acid are in a ratio ranging from about 10:90 to 90:10.

3. The composition of claim 1, wherein the composition further comprises greater than 10% petrochemical motor fuel.

4. The composition of claim 1, wherein the composition further comprises greater than 20% petrochemical motor fuel.

5. The composition of claim 1 , wherein the composition further comprises greater than 40% petrochemical motor fuel.

6. The composition of claim 1, wherein composition further comprises greater than 80% petrochemical motor fuel.

7. The composition of claim 1, wherein the composition further comprises at least about 90% petrochemical motor fuel.

8. The composition of claim 1, wherein the composition further comprises a pour depressant.

9. The composition of claim 1, wherein the C₁-C4 ester of a C12-C₂₂ hydroxyl- containing fatty acid is obtained from genetically modified plants expressing a heterologous hydroxylase gene.

10. A method of producing a biodiesel blend having at least one of improved oxidative stability, improved low temperature properties, or higher oxygen content comprising mixing one or more C1-C₄ esters of C₁2-C₂2 fatty acids and one or more C1-C₄ esters of Ci₂-C₂2 hydroxyl-containing fatty acids in a ratio of from about 10:90 to about 90:10.

11. The method of claim 10, wherein the -C₄ ester of a C₁₂-C22 hydroxyl- containing fatty acid is methyl ricinoleate.

12. A method of producing a biodiesel blend having at least one of improved oxidative stability, improved low temperature properties, or higher oxygen content comprising: transforming a plant with a gene encoding a hydroxylase, wherein the plant has a high oleic background; growing the plant under conditions under which the hydroxylase gene is expressed; isolating high oleic oils produced by the plant; and converting the high oleic oils in to C₁-C₄ esters.

13. A composition comprising high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in ratios from 10:90 to 90:10.

14. The composition of claim 13, wherein the composition further comprises greater than 10% conventional diesel.

15. The composition of claim 13, wherein the composition further comprises greater than 20% conventional diesel.

16. The composition of claim 13, wherein the composition further comprises greater than 40% diesel.

17. The composition of claim 13, wherein composition further comprises greater than 80% diesel.

18. The composition of claim 13, wherein the composition further comprises at least about 90% diesel.

19. The composition of claim 13, wherein the composition further comprises a pour depressant.

20. The composition of claim 13, wherein the methyl esters of hydroxyl- containing fatty acids are obtained from genetically modified plants expressing a heterologous hydroxylase gene.

21. A method of producing a biodiesel blend having at least one of improved oxidative stability, improved low temperature properties, or higher oxygen content comprising mixing high oleic methyl esters and methyl esters of hydroxyl-containing fatty acids in a ratio of from about 10:90 to about 90:10.

22. The method of claim 21, wherein the methyl esters of the hydroxyl-containing fatty acids is methyl ricinoleate.

23. A method of producing a biodiesel blend having at least one of improved oxidative stability, improved low temperature properties, or higher oxygen content comprising: transforming a plant with a gene encoding a hydroxylase, wherein the plant has a high oleic background; growing the plant under conditions under which the hydroxylase gene is expressed; isolating high oleic oils produced by the plant; and converting the high oleic oils in to methyl esters.