NZ578145A - Production of biodiesel - Google Patents

Abstract

Provided is a process for producing a biodiesel including the steps of: utilizing a PUFA-low strain of thraustochytrid to form an oil including unsaturated fatty acids; transesterifying the unsaturated fatty acids in the oil to form alkyl esters from the unsaturated fatty acids; thereby producing a biodiesel. A specific strain suitable for the purpose is also provided.

Description

New Zealand Paient Spedficaiion for Paient Number 578145 1 Production of biodiesel Field of the invention The invention relates to the production of biodiesel feedstock and biodiesel.

Background of the invention Reserves of fossil diesel and petroleum distillates are running out. Biodiesel is an alternative fuel source. Biodiesels include alkyl esters. The alkyl esters may be ) produced by the transesterification of fatty acids contained in a biodiesel feedstock.

Biodiesel feedstocks can be obtained from animal, plant or algae sources. Animal sources include fats such as tallow, lard, and yellow grease. Currently it is not 10 economically feasible to use animal sources as biodiesel feedstocks for biodiesel production on a commercial scale. In any case, the supply of animal sources cannot meet the current demand for biodiesel feedstock.

Plant sources include soybean, rapeseed and palm oil. They also include waste products such as may be obtained from the use of these oils, for example from food 15 manufacturing and processing industries and from hospitality industry, including restaurants and the like. Ultimately the infrastructure and production costs for producing ^ biodiesel from plant sources are far greater than the costs for producing diesel from fossil reserves or petroleum distillates.

Algae have also been investigated as a source of fatty acid for a biodiesel feedstock. 20 However, the "Aquatic Species Program", a research program on the use of algae for production of biodiesel which was conducted by the US National Renewable Energy Laboratory from 1978 to 1996 found that commercial production would require an algal strain having a capacity for fast growth rate and high lipid content. Such strains were found to be either very difficult to isolate or very difficult to culture on a commercial scale 25 to provide an acceptable yield of fatty acids. 2 Summary of the invention In certain embodiments there is provided a process for producing a feedstock for providing biodiesel, the process including the step of utilizing a thraustochytrid to form unsaturated fatty acids, thereby producing a feedstock for providing a biodiesel.

In other embodiments there is provided a process for producing a biodiesel, the process including the steps of utilizing a thraustochytrid to form unsaturated fatty acids and transesterifying the unsaturated fatty acids to form alkyl esters from the unsaturated fatty acids, thereby producing a biodiesel.

In other embodiments there is provided a process for selecting a thraustochytrid for 10 production of a biodiesel feedstock, the process including the step of determining that a thraustochytrid produces more monounsaturated fatty acids than polyunsaturated fatty acids.

In other embodiments there is provided a process for selecting a thraustochytrid for production of a biodiesel feedstock, the process including the step of determining that a 15 thraustochytrid can be induced to produce more monounsaturated fatty acids than polyunsaturated fatty acids.

In still further embodiments there is provided a sample of thraustochytrids for producing a biodiesel feedstock. The sample is characterised in that it contains one or more of: (i) thraustochytrids that produce more monounsaturated fatty acids than 20 polyunsaturated fatty acids; or (ii) thraustochytrids that can be induced to produce more monounsaturated fatty acids than polyunsaturated fatty acids.

In further embodiments there is provided a bioreactor for producing a feedstock for providing a biodiesel. The bioreactor includes a culture of thraustochytrids as described 25 above. 3 In other embodiments there is provided a biodiesel feedstock or a biodiesel produced according to a process described above.

In other embodiments there is provided a process for producing a feedstock for providing biodiesel, the process including the following steps: - selecting a thraustochytrid for producing an oil having unsaturated fatty acids, the thraustochytrid being characterised in that no more than 50% of the unsaturated fatty acids in the oil produced by the thraustochytrid are polyunsaturated fatty acids; and - utilizing the selected thraustochytrid to produce the oil, thereby producing a feedstock for providing a biodiesel.

In one embodiment the thraustochytrid is characterised in that no more than 25%, preferably no more than 10%, of the unsaturated fatty acids in the oil produced by the thraustochytrid are polyunsaturated fatty acids.

Detailed description of the embodiments Thraustochytrids are common marine microheterotrophs that feed as saprobes or 15 occasionally as parasites. Generally they may be characterised by the presence of a sagenogenetosome, an ectoplasmic net, a cell wall with noncellulosic scales, and a life cycle consisting of vegetative cells, zoosporangium and zoospores.

Although originally thought to be primitive fungi, thraustochytrids have more recently been assigned to the subclass Thraustochytridae (Chromista, Heterokonta), aligning 20 them more closely with the heterokont algae (e.g., brown algae with diatoms). Molecular analysis of their 18S ribosomal RNA genes shows conclusively that thraustochytrids are deeply divergent from oomycetes and close to labyrinthulids.

Examples of thraustochytrid genera include Aplanochytriu, Labyrinthuloides, Japonochytrium, Schizochytrium, Thraustochytrium and Ulkenia. Among these, a 25 number of species are recognised including A. kerguelense, A. stocchinoi, L. haliotidis, PCT/A I)2007/001881 4 L minuta, L. yorkensis, J. marinum, S. aggregatum, S. limacinum, S. mangrovei, S. minutum, T. aggregatum, T. aureum, T. kinnei, T. motivum, T. multirudimentale, T. pachydermum, T. striatum, T. visurgense, U. profunda, U. radiata, and U. visurgensis.

The primary commercial interest in thraustochytrids has been in relation to their ability to 5 produce polyunsaturated fatty acids (herein "PUFA") such as fatty acids of the omega-6 (g>6 or n-6) series (including arachidonic acid) and omega-3(o>3 or n-3) series (including eicospentaenoic acid and docosahexaenoic acid). PUFA are known to be essential for development of neural tissue and for decreasing the incidence of coronary heart ( ) disease, stroke and rheumatoid arthritis. Thus, the focus to date has been on the 10 isolation and culture of PUFA-producing thraustochytrids with the objective of providing products for improving human and animal nutrition.

The thraustochytrid strains that produce relatively high amounts of PUFA (herein "PUFAh'9h strains") produce unsaturated oil of which about 90% is PUFA. For obvious commercial reasons, PUFAh,9h strains have been much more intensely characterised 15 than strains that produce unsaturated oil of which about 10% is PUFA (herein "PUFA|0W strains"). Indeed there has been little or no characterisation of PUFAlow strains.

The inventors have now recognised that PUFA|0W strains are particularly important as these strains tend to produce unsaturated oils of which about 90% is monounsaturated 1 fatty acid (MUFA), including palmitoleic acid (C16:1), oleic acid (C18:1), eicosenoic acid acid (C20:1) and erucic acid (C22:1).

Further, it has been found that unlike other algae and like organisms, some PUFA|0W thraustochytrid strains tend to produce an abundance of a particular type of MUFA and very little other MUFA or PUFA.

The degree of saturation of fatty acid chains is highly relevant as to suitability for 25 biodiesel production because saturation at least in part determines (i) the solidification temperature of biodiesel and (ii) the oxidative and thermal stability of biodiesel.

In more detail, solidification temperature is a function of the length of alkyl chains and the number of carbon double bonds (C=C) in a biodiesel. Biodiesel having shorter alkyl chains and fewer double bonds tend to have higher solidification temperatures than those having longer chain lengths and higher saturation. This means that the latter can 5 be used at lower ambient temperatures without solidifying whereas the former are useful in a narrower range of temperatures, and not at lower temperatures.

On the other hand, biodiesel having a greater abundance of polyunsaturated oils tend to have a higher rate of oxidation and therefore less stability than those that do not. For example, the relative rate of oxidation of oleic (C18:1), linoleic (C18:2) and linolenic 10 (018:3) acid is 1:15:25.

Oxidation of biodiesel is generally undesirable as it results in the formation of products such as alcohols that reduce flash point and therefore fuel efficiency, aldehydes that cause rancidity, and short chain fatty acids that are corrosive to engine components. Anti-oxidants can be used, however this adds to production cost.

In summary of the above, it is clear that the presence and number of carbon double bonds is an important characteristic: saturated fatty acids typically have higher solidification temperatures and therefore cannot be used at lower temperatures, whereas fatty acids that are polyunsaturated are susceptible to thermal and oxidative instability. In this context, the inventors' finding that thraustochytrid strains that are 20 PUFA|0W tend to produce unsaturated oils of which about 90% is MUFA oil is particularly important to the production of biodiesel.

A further advantage of the use of thraustochytrid strains is that they can be maintained by fermentative culture and typically do not require a light source, for instance for photosynthesis. These characteristics allow the use of continuous culture, for instance 25 in a bioreactor, to produce the MUFA oil. As well, the amount of MUFA oil produced per weight of a thraustochytrid tends to be much greater than from MUFA sources presently commonly used in the art. 6 Thus, in certain embodiments there is provided a process for producing a feedstock for providing biodiesel, the process including the step of utilizing a thraustochytrid to form unsaturated fatty acids, thereby producing a feedstock for providing a biodiesel.

J The process may include a further step of harvesting the unsaturated fatty acids in the 5 form of an oil from the thraustochytrid. In a further step, the process may include fractionating the unsaturated oil to obtain a MUFA.

In other embodiments there is provided a process for producing a feedstock for providing a biodiesel. The process includes the following steps: - providing a energy source that is fermentable by a thraustochytrid; - adding a thraustochytrid to the energy source; - utilizing the thraustochytrid to ferment the energy source to form unsaturated oil; - optionally fractionating the unsaturated oil to obtain a monounsaturated oil, thereby producing the feedstock.

In a first step of the process, an energy source is provided. As known in the art, an 15 "energy source" is generally organic matter, such as plant material, vegetation, or agricultural waste that can be used as a fuel or energy source.

In one embodiment, the energy source is selected for production of MUFA. In these embodiments, PUFA may also be produced.

In other embodiments, the energy source is selected for production of MUFA only. In 20 these embodiments, the energy source may be selected to minimise PUFA production.

In certain embodiments, the energy source is selected to cause PUFAhigh strains to produce MUFA. 7 In other embodiments, the energy source is selected to maximise production of MUFA by PUFAl0W strains.

The second step of the process involves adding a thraustochytrid to the energy source. The thraustochytrid is typically one that is a PUFA|0W strain. One skilled in the art can 5 distinguish a PUFA,0W strain from a PUFAhigh strain using the techniques described in the Examples.

In some embodiments, PUFAhigh strains may be used provided that the energy source that is provided and conditions for fermentation influence a decrease in production of PUFA, so that the PUFAhigh strain essentially adopts a PUFAl0W strain phenotype.

The thraustochytrid may be naturally occurring or a genetically engineered variant. Examples of naturally occurring thraustochytrid strains are as described herein. A genetically engineered variant is typically one that has been modified to maximise MUFA production. For example, it may be derived from a PUFAhi9h strain that has been manipulated to be provided with a PUFA|0Wstrain phenotype. This could be achieved by 15 deleting the polyketide synthase system of a PUFAhigh strain. Yet another alternative is to chemically modify a PUFAh,9h strain to inhibit the polyketide synthase system. Certain antibiotics can be provided for this purpose.

In a third step the process involves utilizing the thraustochytrid to ferment the energy source to form unsaturated oil. Typically, the fermentation conditions are selected for 20 production of MUFA. In certain embodiments, only MUFA is produced. However, in most embodiments, at least a residual amount of PUFA is produced. The conditions selected for fermentation may be for minimising PUFA production, or for causing PUFAh,gh strains to produce MUFA. In other embodiments, the fermentation conditions are selected to maximise production of MUFA by PUFA,0W strains.

Typically the energy source may be fermented for a time period and at a temperature as described herein. 8 The fermentation process is typically performed in a bioreactor. One example of a suitable bioreactor is one in which the energy source is subject to continuous or periodic stirring. The bioreactor may be adapted for continuous culture.

The unsaturated oil is obtained from the fermentation culture by releasing the oil from 5 the thraustochytrids. This process may require lysing the thraustochytrids and extracting lipids from them.

The process may involve a fourth step which includes fractionating the unsaturated oil to obtain a monounsaturated oil, thereby producing the feedstock. This step may be required where a significant proportion of lipids extracted from the thraustochytrids are 10 PUFA. The fractionation of monounsaturated oils from polyunsaturated oils may be achieved by processes known in the art.

In further embodiments there is provided a feedstock produced by the above described process. The feedstock typically contains MUFA and very little, if any, PUFA. Examples of particularly preferred feedstocks are those that contain palmitoleic acid (C16:1), oleic 15 acid (C18:1), eicosenoic acid acid (C20:1) and erucic acid (C22:1). Oleic acid is particularly preferred.

In further embodiments there is provided a bioreactor for producing a feedstock for providing a biodiesel. The bioreactor includes: - a culture of thraustochytrids; and - an energy source for fermentation by the thraustochytrids to produce a feedstock in the form of an unsaturated oil.

The thraustochytrids and energy source are as described above.

In other embodiments there is provided a process for producing a biodiesel, the process including the steps of utilizing a thraustochytrid to form unsaturated fatty acids and 9 transesterifying the unsaturated fatty acids to form alkyl esters from the unsaturated fatty acids, thereby producing a biodiesel.

In other embodiments there is provided a process for producing a biodiesel. The process includes the following steps: - providing an energy source that is fermentable by a thraustochytrid; - adding a thraustochytrid to the energy source; - utilizing the thraustochytrid to ferment the energy source to form unsaturated oil; - optionally fractionating the unsaturated oil to obtain monounsaturated oil; and - forming alkyl esters from the unsaturated oils, to produce a biodiesel.

Having obtained the unsaturated oil, the biodiesel can be obtained by techniques known to the skilled addressee. The aim of these techniques is to cause a transesterification of the fatty acids to produce alkyl esters. Examples include using strong mineral acids, such as sulfuric acid or a sulfonated ion exchange resin, and methanol in counter current systems at 80 °C to 85 °C under mild pressures.

A further technique commonly used in the art is hydrogenation. Hydrogenation is also known in the art as 'hardening' of an oil. Essentially, a proportion of the unsaturated fatty acids in the oil are caused to combine with hydrogen under the influence of a catalyst to produce either less unsaturated fatty acids or saturated fatty acids. Further, the hydrogenation process can be made to be selective. This selectivity can be 20 influenced by various factors, including temperature and pressure, of the hydrogenation reaction. In the case of PUFA, the selectivity of the hydrogenation arises from the presence of the methylene interrupted double bond.

The advantage of the hydrogenation process being selective in relation to biodiesel production hydrogenation is that is can be used to convert PUFA oil to MUFA oil, rather than converting MUFA to saturated fatty acids. For example, an oil that contains a mixture of C18:2, C18:1 and C18:0 fatty acids undergoes selective hydrogenation to convert substantially all C18:2 to C18:1 before any C18:1 is converted to C18:0. Thus, in certain embodiments an oil is produced where all original PUFA is converted by 5 hydrogenation to MUFA, and the hydrogenation process does not add any further saturated fatty acid.

In certain embodiments the biodiesel produced by any of the above described embodiments should have a fatty acid methyl ester content, where the fatty acid methyl ester has 4 or more double bonds, of less than about 1 % m/m (g per 100g biodiesel). It 10 may also have a content of linolenic acid methyl ester (an esterified PUFA) of less than 12 % m/m (g per 100g biodiesel).

In other embodiments there is provided a process for producing a biodiesel, the process including the steps of utilizing a thraustochytrid to form unsaturated fatty acids and transesterifying the unsaturated fatty acids to form alkyl esters from the unsaturated 15 fatty acids, thereby producing a biodiesel. The embodiment includes the step of hydrogenating the fatty acids to form monounsaturated fatty acids from polyunsaturated fatty acids. This hydrogenation can be carried out before or after transesterification.

Thus, in other embodiments there is provided a process for producing a biodiesel. The process includes the following steps: - providing an energy source that is fermentable by a thraustochytrid; - adding a thraustochytrid to the energy source; - utilizing the thraustochytrid to ferment the energy source to form unsaturated oil; - optionally fractionating the unsaturated oil to obtain monounsaturated oil; - subjecting the unsaturated oil to a hydrogenation process to convert polyunsaturated 25 oil to monounsaturated oil; and 11 - forming alkyl esters from the unsaturated oils, to produce a biodiesel.

In further embodiments there is provided in a process for producing a biodiesel the step of utilizing a thraustochytrid to ferment an energy source to produce unsaturated oil that is useful as a feedstock for production of a biodiesel.

In further embodiments there is provided a biodiesel produced by the above described process.

In other embodiments there is provided a process for determining whether a thraustochytrid may be used to produce a feedstock for providing a biodiesel. The process includes the following steps: - obtaining a thraustochytrid; - determining whether: (i) the thraustochytrid produces more MUFA than PUFA; or (ii) the thraustochytrid can be induced to produce more MUFA than PUFA; to determine whether the thraustochytrid may be used to produce a feedstock for 15 providing a biodiesel.

The inventors have found that thraustochytrids that produce more MUFA than PUFA are generally PUFA,0W strains. One skilled in the art can distinguish a PUFA,ow strain from a PUFAh'9h strain using the techniques described in the Examples.

In still further embodiments there is provided a culture of thraustochytrids for producing 20 a biodiesel feedstock. The culture is characterised in that it contains either: (i) a PUFA|0W strain; or (ii) PUFAhigh strain that can be induced to make more MUFA than PUFA. 12 In one embodiment there is provided a thraustochytrid as deposited in accession no. V07/029468 at the National Measurement Institute (formerly AGAL) of 51-65 Clarke Street, South Melbourne, Victoria Australia 3205 on 15 November 2007 by the Applicant.

In another embodiment there is provided a use of a thraustochytrid as described herein in a process for producing or biodiesel or biodiesel feedstock as described herein.

Examples Example 1 Isolating & identifying a PUFAl0W strain.

Thraustochytrids's are degraders of plant material in marine ecosystems. They may be 10 isolated from decaying marine vegetation in estuarine waters.

Two general and one selective isolation method are used. Decaying plant material was rinsed twice in sterile 50% seawater and plated directly on selective media. Thraustochytrids were concentrated from water samples by aseptically filtering the sample through 0.2 pm filters. Filters were retained and placed onto selective media. To 15 selectively isolate thraustochytrid zoospores, samples (water or water plus plant material) were placed in a sterile Palleroni device (Palleroni, N J, 'Chamber for Bacterial Chemotaxis Experiments' (1976) 32 Appl Environ Microbiol 729-730.) Sterile 50% seawater water was added in each compartment to a level 2 mm above the bottom of the connecting channel. Sterile 5 pi capillary (Drummond "Micro-caps", Drummond 20 Scientific Co.) tubes filled with sterile chemo-attractant (e.g. glutamic acid, cellulose or pectin) in 50% seawater were immersed in the channels. After a further 2 h incubation at 20 °C, the capillaries were removed, washed externally with a jet of sterile 50% seawater and the contents spread on the surface of the agar medium. Different genera of thraustochytrid have differing response to the attractant allowing some selectivity to 25 the isolation (Fan, K W, Vrijmoed, LLP, and Jones, EBG, 'Zoospore Chemotaxis of Mangrove Thraustochytrids From Hong Kong' (2002) 94 Mycologia 569-578.). 13 Samples were plated onto glucose-yeast extract- seawater agar (0.5% glucose, 0.5% yeast extract (Oxoid), 50% artificial seawater (20 g/L sea salts; Sigma), 1.5% agar (Oxoid)) containing antibiotics to prevent bacterial growth (penicillin, streptomycin) and cultured at room temperature for up to 7 days. Characteristic colonies (nonmycelial, 5 yeast-like) were subcultured to purity on the same media.

Cultures were grown in either liquid or solid glucose-yeast extract- seawater medium for 7 days at room temperature. Biomass was recovered from liquid cultures by centrifugirig at 6000 g for 20 mins. Biomass from solid cultures was recovered by scraping the plates with cleaned glass microscope slides.

The phenotype of PUFAl0W strains is determined by analysis of fatty acid methyl esters according to Nichols, D S et al., Variation of Branched-Chain Fatty Acids Marks the Normal Physiological Range for Growth in Listeria Monocytogenes.' (2002) 68 Appl Environ Microbiol 2809-2813.

Samples were extracted by a direct transesterification procedure (Dionisi, F., P. A.

Golay, M. Elli, and L. B. Fay. 1999. Stability of cyclopropane and conjugated linoleic acids during fatty acid quantification in lactic acid bacteria. Lipids 34:1107-1115.) and demonstrated to yield fatty acid profiles comparable to those obtained by the Bligh-Dyer solvent extraction method (Bligh, E. G., and W. J. Dyer. 1959. A rapid method for total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917). Briefly, samples were added to screw-cap test tubes containing 3 mL of methanol-chloroform-hydrochloric acid (10:1:1, VA/A/). The tubes were heated at 80 °C for 1 h before they were cooled to room temperature. After addition of Milli-Q water (1 mL), the resultant fatty acid methyl esters were extracted three times with 1.5 mL of hexane-chloroform (4:1, VN).

Fatty acid methyl esters were analyzed by using a Hewlett-Packard 5890 II gas chromatograph and 5970A mass selective detector equipped with a cross-linked methyl silicone (film thickness, 0.33 pm) fused-silica capillary column (length, 50 m; interna! diameter, 0.22 mm). Fatty acid methyl esters from all samples were identified by comparing the component spectra to the spectra of known standards. The double bond 14 positions and geometry in monounsaturated isomers of selected samples were determined by producing and analyzing dimethyl disulfide adducts.

Example 2 Examples of PUFAf0W strains ACEM 0004 of Thomas E Lewis 2001 "Characterisation and application of Australia 5 thraustochytrids (PhD), Morris Miller Library, University of Tasmania and F3-1 and H1-14 of Huang et al. 2003 "Grouping newly isolated docosahexaenoic acid-producing thraustochytrids based on their polyunsaturated fatty acid profiles and comparative analysis of 18s rRNA genes" Mar. Biotechnol. 5: 450-457 are examples of PUFA|0W strains. Other thraustochytrid strains that are PUFA|0W may have about 91% nucleotide 10 sequence identity with the 18s rRNA sequences of these strains.

Example 3 Producing biodiesel feedstock Processes for culture of thraustochytrids are generally shown in US patent no. 6,451,567.

Typically, to be viable economically, the thraustochytrid strain is one that produces 15 about 30-60% of MUFA on a dry weight basis and less than 1-2% C18:2.

The strain may be grown in a medium containing glucose, cellulose and glycerol. The latter is a by-product of biodiesel production.

The strain is provided in a bio-reactor at a starting concentration of about 102-104 ce(ls/mL.

The fermentation conditions are generally aerobic as desaturase is oxygen dependant. Agitation with a propeller or turbine may be used to mix and aerate the culture. Oxygen levels may be between 30-60%. Such levels may be readily achievable with mixing and not require supplementation with gas mixtures.

Light is generally not important for culture. A temperature range of about 20-28 °C is generally provided.

The maximal concentration of the culture for a batch system generally ranges from about 0.8 to about 48 g/L.

The culture is monitored by assessing growth by measuring absorbance or by examining with a microscope. Lipid content can be estimated by staining with microscopy or spectroscopy.

Most batch cultivation is for 7-10 days. For continuous culture there is continuous harvesting. The rate of harvesting may be determined by the growth rate of the strain. 10 Typically dilution rates are set empirically based on the growth rate or in a chemistat type culture at a level to maintain an easily measurable parameter (e.g. pH, nutrient level, oxygen).

Example 4 Producing biodiesel The source of fatty acids may be an oil feedstock produced by a thraustochytrid, an 15 isolated unsaturated oil feedstock from a feedstock produced by a thraustochytrid, or a hydrogenated oil feedstock of a feedstock produced by a thraustochytrid. The oil feedstock may be mixed with methanol, hydrochloric acid and other miscible organic solvents as appropriate to form a single phase mixture. Heating of the mixture for a defined period at a defined temperature in excess of 80 °C has been determined 20 experimentally to maximise product yield of fatty acid methyl ester. The fatty acid methyl ester is then extracted from the reaction mixture with an appropriate blend of organic solvents and solvent and removed by concentration to yield the biodiesel product.

Example 5. Hydrogenation of unsaturated oil or fatty acid esters to produce biodiesel Hydrogenation may be achieved by passing a stream of hydrogen gas, generated by 25 means known in the art, through the heated oil feedstock produced by a thraustochytrid, or a transesterified oil feedstock produced by a thraustochytrid, in which is suspended a

Claims (7)

WO 2008/067605 PCT/A U2007/001881 16 finely powdered catalyst (typically a Pt or Ni related material). Modifications and variations of the process may be made, including to the catalyst, process parameters, and methods of recovery of the catalyst, to tailor selectivity and maximise yield of less polyunsaturated product. This hydrogenation process may be conducted either before or after transesterification. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention. 12946316 17 CLAIMS

1. A process for producing a biodiesel including the steps of: utilizing a PUFA|0W strain of thraustochytrid to form an oil including unsaturated fatty acids; 5 - transesterifying the unsaturated fatty acids in the oil to form alkyl esters from the unsaturated fatty acids; thereby producing a biodiesel.

2. The process according to claim 1 including the further step of: providing conditions to the oil for converting polyunsaturated fatty acids in 10 the oil to monounsaturated fatty acids.

3. The process according to claim 2 wherein said conditions are provided before the unsaturated fatty acids in the oil are transesterified.

4. The process according to claim 2 wherein said conditions are provided after the unsaturated fatty acids in the oil are transesterified. 15

5. The process according to any one of the preceding claims including the further step of: harvesting the oil formed by the thraustochytrid prior to the step of transesterifying the oil to form alkyl esters.

6. The process according to any one of the preceding claims including the further 20 step of: fractionating the oil to obtain monounsaturated fatty acids.

7. The process according to any one of the preceding claims wherein the oil produced by the thraustochytrid includes unsaturated fatty acids of which about 90% is monounsaturated fatty acids. 25 8. The process according to claim 7 wherein the oil includes unsaturated fatty acids of which about 50% is monounsaturated fatty acids. 18 The process according to claim 8 wherein the oil includes unsaturated fatty acids of which about 10% is monounsaturated fatty acids. The process according to any one of the preceding claims wherein the thraustochytrid is as deposited in accession no. V07/029468 as described herein. A process according to any one of claims 1 to 9, wherein the PUFA|0W thraustochytrid strain is derived from a PUFAh'9h thraustochytrid strain that has been manipulated to be provided with a PUFAl0W strain phenotype. A process for producing a biodiesel according to claim 1, substantially as hereinbefore described.