MICROBIAL OILS

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Application No. 63/399,566 filed on August 19, 2022, which is hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE DISCLOSURE

[2] The present disclosure relates to microbially produced oils and fats. The microbial oils and fats are produced by oleaginous microorganisms. The present disclosure also relates to methods of fractionating microbial oils. The disclosed microbial oil fractions comprise saturation levels, fatty acid profiles, melting temperatures, and differential scanning calorimetry curves that differ from those of the microbial oil from which they are derived.

BACKGROUND

[3] Palm oil is currently the most widely produced vegetable oil on the planet, as it finds uses in the manufacture of a large variety of products. It is widely used in food, as a biofuel precursor, and in soaps and cosmetics. The global demand for palm oil is approximately 57 million tons and is steadily increasing. However, the high demand for palm oil has resulted in environmentally detrimental practices related to the expansion of plantations devoted to palm oil-producing plants. Palm oil production is a leading contributor to tropical deforestation, resulting in habitat destruction, increased carbon dioxide emissions, and local smog clouds across South East Asia.

[4] Thus, there is an urgent need for palm oil alternatives that do not rely upon utilization of oil palms and incur the associated negative environmental costs.

BRIEF SUMMARY

[5] The present disclosure provides novel microbial fats obtained from oleaginous yeast.

[6] In one aspect, the disclosure provides a microbial fat obtained from an oleaginous yeast, wherein the microbial fat comprises the following amounts of fatty acids relative to the total fatty acids: at least about 35% w/w saturated fatty acids; and less than about 15% w/w total polyunsaturated fatty acids.

[7] In one aspect, the disclosure provides a microbial fat obtained from an oleaginous yeast, wherein the microbial fat comprises an average desaturation level of less than about 0.72.

[8] In one aspect, the disclosure provides a microbial oil obtained from an oleaginous yeast, wherein the microbial oil comprises the following amounts of fatty acids relative to the total fatty acids: less than about 40% w/w total saturated fatty acids; and at least about 10% w/w total polyunsaturated fatty acids.

[9] In one aspect, the disclosure provides a method of fractionating a first microbial oil to obtain a microbial fat and a second microbial oil, wherein the method comprises: (a) melting the first microbial oil; (b) crystallizing the melted first microbial oil; and (c) separating solid and liquid phases of the crystallized first microbial oil, wherein the solid phase of step (c) is the microbial fat and the liquid phase of step (c) is the second microbial oil.

BRIEF DESCRIPTION OF THE DRAWINGS

[10] The accompanying figures, which are incorporated herein and form a part of the specification, illustrate some, but not the only or exclusive, example embodiments and/or features. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

[11] FIG. 1A shows a chromatogram of the fatty acid composition analysis of exemplary crude microbial oil; FIG. IB shows a chromatogram of the fatty acid composition analysis of exemplary crude palm oil; FIG. 1C shows a chromatogram of the fatty acid composition analysis of exemplary crude hybrid palm oil; and FIG. ID shows a bar graph of representative fatty acid compositions of microbial oil and palm oil.

[12] FIG. 2A shows a chromatogram of the triglyceride composition analysis of exemplary crude microbial oil; FIG. 2B shows a chromatogram of the triglyceride composition analysis of exemplary crude palm oil; and FIG. 2C shows a chromatogram of the triglyceride composition analysis of exemplary crude hybrid palm oil.

[13] FIG. 3 shows a chromatogram of the tocopherols analysis of exemplary crude microbial oil, crude palm oil, and crude hybrid palm oil. Notable peaks are annotated, with “External ISTD” illustrating the location of the standard.

[14] FIG. 4A-4B show the results of a fatty acid analysis of exemplary microbial oils of the disclosure produced by three illustrative strains of the oleaginous yeast/?, toruloides. FIG. 4A shows the overall fatty acid composition broken down by percentage of poly-unsaturated fatty acid (PUFA), mono-unsaturated fatty acid (MUFA), and saturated fatty acid (SFA). FIG. 4B shows the breakdown of the fatty acid composition for the microbial oils in terms of specific fatty acids.

[15] FIG. 5A-5B show the results of fractionation of hydrolyzed free fatty acids on fatty acid composition for an exemplary microbial oil. FIG. 5A shows the results of fractionation of hydrolyzed free fatty acids on overall fatty acid composition in terms of PUFA, MUFA, and SFA. FIG. 5B shows the breakdown in terms of specific fatty acids for the crude microbial oil and each of the fractions.

[16] FIG. 6A-6B show a visual comparison of fractionated hydrolyzed microbial oils, nonfractionating microbial oil, and fractionated palm oil. FIG. 6A, left shows the visual results of fractionation on a hydrolyzed microbial oil from R. toruloides on the right is a fractionated palm oil. FIG. 6B shows the visual results of fractionation on a fractionable microbial oil (left) and a non-fractionating microbial oil (right).

[17] FIG. 7A-7D show total ion chromatograms for four different oil samples: an exemplary R. toruloides microbial oil of the disclosure (FIG. 7A); algae oil (FIG. 7B); crude palm oil (FIG. 7C); and refined, bleached, and deodorized (RBD) palm oil (FIG. 7D).

[18] FIG. 8 shows a representative extracted peak for a compound of interest (ergosterol- TMS) from the total ion chromatogram of an exemplary microbial oil of the present disclosure.

[19] FIG. 9A-9E show the electron-ionization spectra for five different derivatized sterols spiked into crude palm oil: ergosterol-TMS (FIG. 9A); cholesterol-TMS (FIG. 9B); campesterol-TMS (FIG. 9C); sitosterol-TMS (FIG. 9D); and stigmasterol-TMS (FIG. 9E).

[20] FIG. 10A-10B show the results of a carotenoid analysis of agricultural palm oil. FIG. 10A shows the overall UV/Vis absorbance spectrum. FIG. 10B shows the HPLC-DAD chromatogram with absorbance at 450 nm.

[21] FIG. 11A-11B show the results of a carotenoid analysis of a strong acid-extracted exemplary R. toruloides microbial oil of the present disclosure. FIG. 11A shows the overall UV/Vis absorbance spectrum. FIG. 11B shows the HPLC-DAD chromatogram with absorbance at 450 nm.

[22] FIG. 12A-12B show the results of a carotenoid analysis of a strong acid-extracted exemplary R. toruloides microbial oil of the present disclosure. FIG. 12A shows the overall UV/Vis absorbance spectrum. FIG. 12B shows the HPLC-DAD chromatogram with absorbance at 450 nm.

[23] FIG. 13A-13B show the results of a carotenoid analysis of a weak acid-extracted exemplary R. toruloides microbial oil of the present disclosure. FIG. 13A shows the overall UV/Vis absorbance spectrum. FIG. 13B shows the HPLC-DAD chromatogram with absorbance at 450 nm.

[24] FIG. 14A-14B show the results of a carotenoid analysis of an acid-free extracted exemplary R. toruloides microbial oil of the present disclosure. FIG. 14A shows the overall UV/Vis absorbance spectrum. FIG. 14B shows the HPLC-DAD chromatogram with absorbance at 450 nm. [25] FIG. 15A-15B show the results of a carotenoid analysis of an acid-free extracted exemplary R. toruloides microbial oil of the present disclosure. FIG. 15A shows the overall UV/Vis absorbance spectrum. FIG. 15B shows the HPLC-DAD chromatogram with absorbance at 450 nm.

[26] FIG. 16A shows the results of fatty acid methyl ester (FAME) analysis of the solid fraction resulting from solvent-based fractionation condition 1 in Example 9. FIG. 16B shows a Differential Scanning Calorimeter (DSC) chromatogram for the solid fraction resulting from solvent-based fractionation condition 1 in Example 9.

[27] FIG. 17 shows the results of FAME analysis of the liquid fraction resulting from solvent-based fractionation condition 1 in Example 9.

[28] FIG. 18A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 2 in Example 9. FIG. 18B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 2 in Example 9.

[29] FIG. 19A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 3 in Example 9. FIG. 19B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 3 in Example 9.

[30] FIG. 20A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 4 in Example 9. FIG. 20B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 4 in Example 9.

[31] FIG. 21A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 5 in Example 9. FIG. 21B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 5 in Example 9.

[32] FIG. 22A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 6 in Example 9. FIG. 22B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 6 in Example 9.

[33] FIG. 23A shows the results of FAME analysis of the liquid fraction resulting from solvent-based fractionation condition 6 in Example 9. FIG. 23B shows a DSC chromatogram for the liquid fraction resulting from solvent-based fractionation condition 6 in Example 9.

[34] FIG. 24A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 7 in Example 9. FIG. 24B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 7 in Example 9.

[35] FIG. 25A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 8 in Example 9. FIG. 25B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 8 in Example 9. [36] FIG. 26A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 9 in Example 9. FIG. 26B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 9 in Example 9.

[37] FIG. 27A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 10 in Example 9. FIG. 27B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 10 in Example 9.

[38] FIG. 28A shows the results of FAME analysis of the solid fraction resulting from dry fractionation condition 1 in Example 10. FIG. 28B shows a DSC chromatogram for the solid fraction resulting from dry fractionation condition 1 in Example 10.

[39] FIG. 29A shows the results of TAG analysis of the solid fraction resulting from dry fractionation condition 2 in Example 10. FIG. 29B shows a DSC chromatogram for the solid fraction resulting from dry fractionation condition 2 in Example 10.

[40] FIG. 30A shows the results of TAG analysis of the solid fraction resulting from dry fractionation condition 3 in Example 10. FIG. 30B shows the results of FAME analysis of the solid fraction resulting from dry fractionation condition 3 in Example 10. FIG. 30C shows a DSC chromatogram for the solid fraction resulting from dry fractionation condition 3 in Example 10.

[41] FIG. 31A shows the results of TAG analysis of the solid fraction resulting from dry fractionation condition 4 in Example 10. FIG. 31B shows the results of FAME analysis of the solid fraction resulting from dry fractionation condition 4 in Example 10. FIG. 31C shows a DSC chromatogram for the solid fraction resulting from dry fractionation condition 4 in Example 10.

[42] FIG. 32A shows the results of FAME analysis of the solid fraction resulting from dry fractionation condition 5 in Example 10. FIG. 32B shows a DSC chromatogram for the solid fraction resulting from dry fractionation condition 5 in Example 10.

[43] FIG. 33A shows a DSC chromatogram for an original microbial oil and a liquid fraction and solid fraction derived therefrom. FIG. 33B shows the same three curves as in FIG. 33A, but overlaid.

DETAILED DESCRIPTION

[44] The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed disclosures, or that any publication specifically or implicitly referenced is prior art. Definitions

[45] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[46] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques and/or substitutions of equivalent techniques that would be apparent to one of skill in the art.

[47] As used herein, the singular forms “a,” "an,” and “the: include plural referents unless the content clearly dictates otherwise.

[48] The term “about” or “approximately” when immediately preceding a numerical value means a range (e.g., plus or minus 10% of that value). For example, “about 50” can mean 45 to 55, “about 25,000” can mean 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example in a list of numerical values such as “about 49, about 50, about 55, ...”, “about 50” means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g., more than 49.5 to less than 52.5. Furthermore, the phrases “less than about” a value or “greater than about” a value should be understood in view of the definition of the term “about” provided herein. Similarly, the term “about” when preceding a series of numerical values or a range of values (e.g., “about 10, 20, 30” or “about 10-30”) refers, respectively to all values in the series, or the endpoints of the range.

[49] A “fatty acid” is a carboxylic acid with a long aliphatic chain, which is either saturated or unsaturated. Most naturally occurring fatty acids have an unbranched chain of an even number of carbon atoms, from 4 to 28. Fatty acids are usually not found free in organisms, but instead within three main classes of esters: triglycerides, phospholipids, and cholesteryl esters. Within the context of this disclosure, a reference to a fatty acid may refer to either its free or ester form.

[50] “Fatty acid profile” as used herein refers to how specific fatty acids contribute to the chemical composition of an oil.

[51] As used herein, the term “fractionable” is used to refer to a microbial oil or lipid composition which can be separated into at least two fractions that differ in saturation levels and wherein the at least two fractions each make up at least 10% w/w (or mass/mass) of the original microbial oil or lipid composition. In some embodiments, the saturation levels of the fractions are characterized by their iodine value (IV). In some embodiments, the IV of the fractions differs by at least 10. Accordingly, a “fraction” as used herein refers to a separable component of a microbial oil that differs in saturation level from at least one other separable component of the microbial oil.

[52] “Lipid” means any of a class of molecules that are soluble in nonpolar solvents (such as ether and hexane) and relatively or completely insoluble in water. Lipid molecules have these properties, because they are largely composed of long hydrocarbon tails that are hydrophobic in nature. Examples of lipids include fatty acids (saturated and unsaturated); glycerides or glycerolipids (such as monoglycerides, diglycerides, triglycerides or neutral fats, and phosphoglycerides or glycerophospholipids); and nonglycerides (sphingolipids, tocopherols, tocotrienols, sterol lipids including cholesterol and steroid hormones, prenol lipids including terpenoids, fatty alcohols, waxes, and polyketides).

[53] “Microorganism” and “microbe” mean any microscopic unicellular organism and can include bacteria, algae, yeast, or fungi.

[54] “Oleaginous” as used herein refers to material, e.g., a microorganism, which contains a significant component of oils, or which is itself substantial composed of oil. An oleaginous microorganism can be one that is naturally occurring or synthetically engineered to generate a significant proportion of oil.

[55] “Oleaginous yeast” as used herein refers to a collection of yeast species that can accumulate a high proportion of their biomass as lipids (namely greater than 20% of dry cell mass). An oleaginous yeast can be one that is naturally occurring or synthetically engineered to generate a significant proportion of oil.

[56] As used herein, “RBD” refers to refinement, bleaching, and deodorizing or refers to an oil that has undergone these processes.

[57] “Rhodosporidium toruloides” refers to a particular species of oleaginous yeast. Previously called Rhodotorula glutinis or Rhodotorula gracilis. Also abbreviated as R. toruloides. This species includes multiple strains with minor genetic variation.

[58] For the purposes of this disclosure, “single cell oils,” “microbial oils,” “lipid composition” and “oils” refer to microbial lipids produced by oleaginous microorganisms.

[59] “ Tailored fatty acid profile” as used herein refers to a fatty acid profile in a microbial oil which has been manipulated towards target properties, either by changing culture conditions, the species of oleaginous microorganism producing the microbial oil, or by genetically modifying the oleaginous microorganism. [60] “Triglyceride(s)” as used herein refers to a glycerol bound to three fatty acid molecules. They may be saturated or unsaturated, and various denominations may include other isomers. For example, reference to palmitic-oleic-palmitic (P-O-P) would also include the isomers P- P-0 and O-P-P.

[61] “W/W” or “w/w”, in reference to proportions by weight, refers to the ratio of the weight of one substance in a composition to the weight of the composition. For example, reference to a composition that comprises 5% w/w oleaginous yeast biomass means that 5% of the composition's weight is composed of oleaginous yeast biomass (e.g., such a composition having a weight of 100 mg would contain 5 mg of oleaginous yeast biomass) and the remainder of the weight of the composition (e.g., 95 mg in the example) is composed of other ingredients.

[62] Within the context of this disclosure, as it pertains to microbial oil fractions, the term “original microbial oil” is used to refer to the microbial oil that was fractionated to produce the microbial oil fractions. In some embodiments, the original microbial oil is at various stages of refinement. In some embodiments, the original microbial oil is a crude, i.e., unrefined, microbial oil. In some embodiments, the original microbial oil is one or more of refined, bleached, and deodorized. In some embodiments, the original microbial oil is a refined, bleached, and deodorized (RBD) oil. In some embodiments, the original microbial oil is itself a fraction of another microbial oil.

[63] As used herein, the term “microbial oil fraction” or “fraction” refers to a component of an original microbial oil that is separated out via a fractionation technique. In some embodiments, the fractionation is dry fractionation. In some embodiments, the fractionation is wet fractionation. In some embodiments, the fractionation is solvent-based fractionation. In some embodiments, the fraction is characterized based on melting temperature, DSC curve, saturation level, fatty acid profile, and/or weight percent of the original microbial oil. Throughout the disclosure, any discussion of techniques that may be performed on microbial oils should be understood to be equally applicable to any fractions thereof. As used herein, the term “microbial oils” should be understood to encompass microbial fats and microbial oil fractions unless the context indicates otherwise.

[64] A “first microbial oil” as used herein refers to a microbial oil that is subject to fractionation, while the “second microbial oil” refers to the liquid fraction resulting therefrom.

[65] As used herein, in the context of fractionation, the term “yield” is used to refer to the amount of a fraction obtained from a fractionation of an original microbial oil, as compared to the amount of the original microbial oil. In some embodiments, the yield is given as a weight or volume percentage. In some embodiments, the yield of a given fraction is at least about 5% w/w. In some embodiments, the yield is at least about 10% w/w. In some embodiments, the yield of the liquid fraction is at least about 50% w/w. In some embodiments, the yield of the solid fraction is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% w/w.

[66] As used herein, the term “fat” refers to an oil with a melting point above room temperature, wherein room temperature is 20-25°C. In some embodiments, a microbial oil herein is a fat, referred to herein as a “microbial fat.” In some embodiments, a fraction of a microbial oil herein is a microbial fat. In some embodiments, the solid fraction of a microbial oil, also referred to as microbial stearin, is a microbial fat.

[67] As used herein, the term “average fatty acid desaturation level” or “Avg. Desat” as applied to a microbial oil and/or microbial fat refers to the average number of double bonds in the aliphatic chains of its fatty acids by weight. E.g., a pure C 12:0 oil would have an average fatty acid desaturation level of 0, while a 50% C16:0 and 50% C16: 1 oil would have an average fatty acid desaturation level of 0.5. In some embodiments, a microbial fat of the disclosure has an average fatty acid desaturation level of less than about 0.7.

Overview

[68] The present disclosure relates to novel microbial oils. In some embodiments, the microbial oils are microbial fats. In some embodiments, the microbial oils have been refined, bleached, deodorized, and/or fractionated. These microbial oils and fractions thereof may serve as alternatives to palm oil and fractions thereof in a variety of downstream products of interest. Oleaginous microorganisms

[69] The present disclosure provides microbial oils produced by oleaginous microorganisms. In some embodiments, the oleaginous microorganism is a microalgae, yeast, mold, or bacterium.

[70] The use of oleaginous microorganisms for lipid production has many advantages over traditional oil harvesting methods, e.g., palm oil harvesting from palm plants. For example, microbial fermentation (1) does not compete with food production in terms of land utilization; (2) can be carried out in conventional microbial bioreactors; (3) has rapid growth rates; (4) is unaffected or minimally affected by space, light, or climate variations; (5) can utilize waste products as feedstock; (6) is readily scalable; and (7) is amenable to bioengineering for the enrichment of desired fatty acids or oil compositions. In some embodiments, the present methods have one or more of the aforementioned advantages over plant-based oil harvesting methods.

[71] In some embodiments, the oleaginous microorganism is an oleaginous microalgae. In some embodiments, the microalgae is of the genus Botryococcus, Cylindrotheca, Nitzschia, or Schizochytrium. In some embodiments, the oleaginous microorganism is an oleaginous bacterium. In some embodiments, the bacterium is of the genus Arthrobacter, Acinetobacter, Rhodococcus, o Bacillus. In some embodiments, the bacterium is of the species Acinetobacter calcoaceticus, Rhodococcus opacus, or Bacillus alcalophilus . In some embodiments, the oleaginous microorganism is an oleaginous fungus. In some embodiments, the fungus is of the genus Aspergillus, Mortierella, or Humicola. In some embodiments, the fungus is of the species Aspergillus oryzae, Mortierella isabellina, Humicola lanuginosa, ox Mortierella vinacea.

[72] Oleaginous yeast in particular are robust, viable over multiple generations, and versatile in nutrient utilization. They also have the potential to accumulate intracellular lipid content up to greater than 70% of their dry biomass. In some embodiments, the oleaginous microorganism is an oleaginous yeast. In some embodiments, the yeast may be in haploid or diploid forms. The yeasts may be capable of undergoing fermentation under anaerobic conditions, aerobic conditions, or both anaerobic and aerobic conditions. A variety of species of oleaginous yeast that produce suitable oils and/or lipids can be used to produce microbial oils in accordance with the present disclosure. In some embodiments, the oleaginous yeast naturally produces high (20%, 25%, 50% or 75% of dry cell weight or higher) levels of suitable oils and/or lipids. Considerations affecting the selection of yeast for use in the invention include, in addition to production of suitable oils or lipids for production of food products: (1) high lipid content as a percentage of cell weight; (2) ease of growth; (3) ease of propagation; (4) ease of biomass processing; and (5) glycerolipid profile. In some embodiments, the oleaginous yeast comprise cells that are capable of producing at least 20%, 25%, 50% or 75% or more lipid by dry weight. In other embodiments, the oleaginous yeast contains at least 25-35% or more lipid by dry weight.

[73] Suitable species of oleaginous yeast for producing the microbial oils of the present disclosure include, but are not limited to Candida apicola, Candida sp., Cryptococcus albidus. Cryptococcus curvatus, Cryptococcus terricolus, Cutaneotrichosporon oleaginosus, Debaromyces hansenii, Endomycopsis vernalis, Geotrichum carabidarum, Geotrichum cucujoidarum, Geotrichum histeridarum, Geotrichum silvicola, Geotrichum vulgare, Hyphopichia burtonii, Lipomyces lipofer, Lypomyces orentalis, Lipomyces starkeyi, Lipomyces tetrasporous, Pichia mexicana, Rodosporidium sphaerocarpum, Rhodosporidium toruloides Rhodotorula aurantiaca, Rhodotorula dairenensis, Rhodotorula diffluens, Rhodotorula glutinus, Rhodotorula glutinis var. glutinis, Rhodotorula gracilis, Rhodotorula graminis Rhodotorula minuta, Rhodotorula mucilaginosa, Rhodotorula mucilaginosa, Rhodotorula terpenoidalis, Rhodotorula toruloides, Sporobolomyces alborubescens, Starmerella bombicola, Torulaspora delbruekii, Torulaspora pretoriensis, Trichosporon behrend, Trichosporon brassicae, Trichosporon domesticum, Trichosporon laibachii, Trichosporon loubieri, Trichosporon loubieri, Trichosporon montevideense, Trichosporon pullulans, Trichosporon sp., Wickerhamomyces canadensis, Yarrow ia lipolytica, and Zygoascus meyerae.

[74] In some embodiments, the yeast is of the genera Yarrowia, Candida, Rhodotorula, Rhodosporidium, Metschnikowia, Cryptococcus, Trichosporon, or Lipomyces. In some embodiments, the yeast is of the genus Yarrowia. In some embodiments, the yeast is of the species Yarrowia lipolytica. In some embodiments, the yeast is of the genus Candida. In some embodiments, the yeast is of the species Candida curvata. In some embodiments, the yeast is of the genus Cryptococcus. In some embodiments, the yeast is of the species Cryptococcus albidus. In some embodiments, the yeast is of the genus Lipomyces. In some embodiments, the yeast is of the species Lipomyces starkeyi. In some embodiments, the yeast is of the genus Rhodotorula. In some embodiments, the yeast is of the species Rhodotorula glutinis. In some embodiments, the yeast is of the genus Metschnikowia. In some embodiments, the yeast is of the species Metschnikowia pulcherrima.

[75] In some embodiments, the oleaginous yeast is of the genus Rhodosporidium. In some embodiments, the yeast is of the species Rhodosporidium toruloides. In some embodiments, the oleaginous yeast is of the genus Lipomyces. In some embodiments, the oleaginous yeast is of the species Lipomyces Starkeyi.

[76] In some embodiments, the oleaginous microorganisms that produce the microbial oils of the present disclosure are a homogeneous population comprising microorganisms of the same species and strain. In some embodiments, the oleaginous microorganisms that produce the microbial oils of the present disclosure are a heterogeneous population comprising microorganisms from more than one strain. In some embodiments, the oleaginous microorganisms that produce the microbial oils of the present disclosure are a heterogeneous population comprising two or more distinct populations of microorganisms of different species.

[77] The oleaginous microorganisms that produce the microbial oils of the present disclosure may have been improved in terms of one or more aspects of lipid production. These aspects may include lipid yield, lipid titer, dry cell weight titer, lipid content, and lipid composition. In some embodiments, lipid production may have been improved by genetic or metabolic engineering to adapt the microorganism for optimal growth on the feedstock. In some embodiments, lipid production may have been improved by varying one or more parameters of the growing conditions, such as temperature, shaking speed, growth time, etc. The oleaginous microorganisms of the present disclosure, in some embodiments, are grown from isolates obtained from nature (e.g., wild-types). In some embodiments, wild-type strains are subjected to natural selection to enhance desired traits (e.g., tolerance of certain environmental conditions such as temperature, inhibitor concentration, pH, oxygen concentration, nitrogen concentration, etc.). For example, a wild-type strain (e.g., yeast) may be selected for its ability to grow and/or ferment in a feedstock of the present disclosure, e.g., a feedstock comprising one or more microorganism inhibitors. In other embodiments, wildtype strains are subjected to directed evolution to enhance desired traits (e.g., lipid production, inhibitor tolerance, growth rate, etc.). In some embodiments, the cultures of microorganisms are obtained from culture collections exhibiting desired traits. In some embodiments, strains selected from culture collections are further subjected to directed evolution and/or natural selection in the laboratory. In some embodiments, oleaginous microorganisms are subjected to directed evolution and selection for a specific property (e.g., lipid production and/or inhibitor tolerance). In some embodiments, the oleaginous microorganism is selected for its ability to thrive on a feedstock of the present disclosure.

[78] In some embodiments, directed evolution of the oleaginous microorganisms generally involves three steps. The first step is diversification, wherein the population of organisms is diversified by increasing the rate of random mutation creating a large library of gene variants. Mutagenesis can be accomplished by methods known in the art (e.g., chemical, ultraviolet light, etc.). The second step is selection, wherein the library is tested for the presence of mutants (variants) possessing the desired property using a screening method. Screens enable identification and isolation of high-performing mutants. The third step is amplification, wherein the variants identified in the screen are replicated. These three steps constitute a "round" of directed evolution. In some embodiments, the microorganisms of the present disclosure are subjected to a single round of directed evolution. In other embodiments, the microorganisms of the present disclosure are subjected to multiple rounds of directed evolution. In various embodiments, the microorganisms of the present disclosure are subjected to 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more rounds of directed evolution. In each round, the organisms expressing the highest level of the desired trait of the previous round are diversified in the next round to create a new library. This process may be repeated until the desired trait is expressed at the desired level.

Properties of microbial oil

[79] The present disclosure provides microbial oils produced by oleaginous microorganisms. In some embodiments, the microbial oils of the present disclosure are characterized by fatty acid composition, triglyceride composition, sterol composition, pigment composition, ability to be fractionated, slip melting point, iodine value, saponification value, and the like.

Sterol composition

[80] In some embodiments, the microbial oil comprises one or more sterols. In some embodiments, the microbial oil comprises ergosterol. In some embodiments, the microbial oil comprises at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500, or 2000 ppm, or any ranges or subranges therebetween, of ergosterol. In some embodiments, the microbial oil comprises at least 50 ppm ergosterol. In some embodiments, the microbial oil comprises at least 100 ppm ergosterol. In some embodiments, at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%, or any ranges or subranges therebetween, of the sterols in the microbial oil are ergosterol. In some embodiments at least 60% of the overall sterol composition is ergosterol.

[81] In some embodiments, the microbial oil comprises less than 100 ppm of a phytosterol, cholesterol, or a protothecasterol. In some embodiments, the microbial oil comprises less than 50 ppm of of a phytosterol, cholesterol, or a protothecasterol. In some embodiments, the microbial oil does not comprise a sterol selected from a phytosterol, cholesterol, or a protothecasterol.

[82] In some embodiments, the microbial oil does not comprise plant sterols. In some embodiments, the microbial oil does not comprise one or more phytosterols. In some embodiments, the microbial oil does not comprise campesterol, P-sitosterol, or stigmasterol. In some embodiments, the microbial oil does not comprise cholesterol. In some embodiments, the microbial oil does not comprise protothecasterol.

[83] In some embodiments, the microbial oil comprises one or more sterols or stanols in addition to ergosterol. In some embodiments, the microbial oil comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 ppm, or any ranges or subranges therebetween, of one or more of 3,5- Cycloergosta-6,8(14),22-triene, anthraergostatetraenol p-chlorobenzoate, ergosta- 5, 7, 9(1 l),22-tetraen-3P-ol, ergosta-7,22-dien-3-ol, l'-Methyl-l'H-5a-cholest-3-eno[3,4- b]indole, 5%-ergost-7-en-3P-ol, anthraergostatetraenol hexahydrobenzoate, 4,4- dimethylcholesta-8,24-dien-3-ol, and 9,19-cyclolanost-24-en-3-ol. Pigments

[84] In some embodiments, the microbial oil comprises a pigment. In some embodiments, the microbial oil comprises at least one pigment selected from the group consisting of carotene, torulene and torulorhodin.

[85] In some embodiments, the microbial oil comprises carotene. In some embodiments, the microbial oil comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 ppm, or any ranges or subranges therebetween, of carotene. In some embodiments, the microbial oil comprises at least 25 ppm of carotene. In some embodiments, the microbial oil comprises at least 50 ppm of carotene. In some embodiments, the microbial oil comprises at least 100 ppm of carotene. In some embodiments, the carotene is P-carotene and/or a derivative thereof. In some embodiments, the carotene is (13Z)-P-Carotene. In some embodiments, the carotene is (9Z)-P-Carotene.

[86] In some embodiments, the microbial oil comprises torulene. In some embodiments, the microbial oil comprises torulorhodin. In some embodiments, the microbial oil comprises a derivative of torulene and/or torulorhodin. In some embodiments, the microbial oil comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 ppm, or any ranges or subranges therebetween, of torulene, torulorhodin, and/or derivatives thereof. In some embodiments, the microbial oil comprises at least 25 ppm of torulene, torulorhodin, and/or derivatives thereof. In some embodiments, the microbial oil comprises at least 50 ppm of torulene, torulorhodin, and/or derivatives thereof. In some embodiments, the microbial oil comprises at least 100 ppm of torulene, torulorhodin, and/or derivatives thereof. In some embodiments, the microbial oil comprises at least 300 ppm of torulene, torulorhodin, and/or derivatives thereof.

[87] In some embodiments, the microbial oil comprises each of carotene, torulene and torulorhodin. In some embodiments, the microbial oil does not comprise chlorophyll.

Fractionable

[88] In some embodiments, the microbial oil is fractionable. In some embodiments, the microbial oil is fractionable into two or more fractions. In some embodiments, the microbial oil is fractionable into more than two fractions. In some embodiments, the microbial oil is fractionable into two fractions, which may then be further fractionated. [89] In some embodiments, the microbial oil is fractionable into two fractions. In some embodiments, the two fractions are microbial olein and microbial stearin. In some embodiments, each fraction comprises at least 10% of the microbial oil’s original mass. In some embodiments, the iodine value (IV) of the fractions differs by at least 10. In some embodiments, the iodine value of the fractions differs by at least 20. In some embodiments, the iodine value of the fractions differs by at least 30.

Fatty acid composition

[90] The composition of the microbial oil may vary depending on the strain of microorganism, feedstock composition, and growing conditions. In some embodiments, the microbial oil produced by the oleaginous microorganisms of the present disclosure comprise about 90% w/w triacylglycerol with a percentage of saturated fatty acids (% SFA) of about 44%. In some embodiments, the most common fatty acids in microbial oils of the disclosure are oleic acid (C18: l), palmitic acid (C16:0), linoleic acid (C18:2), stearic acid (C18:0), myristic acid (C14:0), and palmitoleic acid (C16: l).

[91] In some embodiments, the microbial oil comprises C10:0 (capric acid). In some embodiments, the microbial oil comprises at least 0.01%, at least 0.05%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, or at least 1% capric acid, or any ranges or subranges therebetween.

[92] In some embodiments, the microbial oil comprises myristic acid (C14:0). In some embodiments, the microbial oil comprises at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, or at least 5% myristic acid, or any ranges or subranges therebetween.

[93] In some embodiments, the microbial oil comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60% w/w palmitic acid (C16:0), or any ranges or subranges therebetween. In some embodiments, the microbial oil comprises at least 5% w/w palmitic acid. In some embodiments, the microbial oil comprises at least 10% w/w palmitic acid. In some embodiments the microbial oil comprises about 10-50% w/w palmitic acid. In some embodiments the microbial oil comprises about 20-40% w/w palmitic acid.

[94] In some embodiments, the microbial oil comprises at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10% w/w palmitoleic acid (Cl 6: 1), or any ranges or subranges therebetween. In some embodiments, the microbial oil comprises at least 0.1% w/w palmitoleic acid. In some embodiments, the microbial oil comprises at least 0.5% w/w palmitoleic acid. In some embodiments, the microbial oil comprises about 0.5-10% w/w palmitoleic acid. In some embodiments, the microbial oil comprises about 0.5-5% w/w palmitoleic acid.

[95] In some embodiments, the microbial oil comprises at least 1%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, or at least 25% w/w stearic acid (C18:0), or any ranges or subranges therebetween. In some embodiments, the microbial oil comprises at least 1% w/w stearic acid. In some embodiments, the microbial oil comprises at least 5% w/w stearic acid. In some embodiments, the microbial oil comprises about 1-25% w/w stearic acid. In some embodiments, the microbial oil comprises about 2-10% w/w stearic acid.

[96] In some embodiments, the microbial oil comprises at least 5%, at least 10%, at least

15%, at least 20%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least

30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least

37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least

44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least

51%, at least 52%, at least 53%, at least 54% at least 55%, at least 56%, at least 57%, at least

58%, at least 59%, or at least 60% w/w oleic acid (C18: l), or any ranges or subranges therebetween. In some embodiments, the microbial oil comprises at least 25% w/w oleic acid. In some embodiments, the microbial oil comprises at least 30% w/w oleic acid. In some embodiments, the microbial oil comprises about 30-60% w/w oleic acid. In some embodiments, the microbial oil comprises about 35-55% w/w oleic acid.

[97] In some embodiments, the microbial oil comprises Cl 8:2 (linoleic acid). In some embodiments, the microbial oil comprises at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15% linoleic acid, or any ranges or subranges therebetween. In some embodiments, the microbial oil comprises about 1-25% linoleic acid. In some embodiments, the microbial oil comprises about 5-15% linoleic acid.

[98] In some embodiments, the microbial oil comprises C18:3 (linolenic acid). In some embodiments, the microbial oil comprises at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, or at least 5% linolenic acid, or any ranges or subranges therebetween. In some embodiments, the microbial oil comprises about 0.5-5% linolenic acid. [99] In some embodiments, the microbial oil comprises C20:0 (arachidic acid). In some embodiments, the microbial oil comprises at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, or at least 5% arachidic acid, or any ranges or subranges therebetween.

[100] In some embodiments, the microbial oil comprises C12:0. In some embodiments, the microbial oil comprises C15: l. In some embodiments, the microbial oil comprises C16: l. In some embodiments, the microbial oil comprises C17:0. In some embodiments, the microbial oil comprises C17: l. In some embodiments, the microbial oil comprises C20: l. In some embodiments, the microbial oil comprises C22:0. In some embodiments, the microbial oil comprises C22:l. In some embodiments, the microbial oil comprises C22:2. In some embodiments, the microbial oil comprises C24:0. In some embodiments, the microbial oil comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, or about 5% of any one of these fatty acids, or any ranges or subranges therebetween. In some embodiments, the microbial oil comprises about 0-5% of any one of these fatty acids. In some embodiments, the microbial oil comprises about 0.1-2% of any one of these fatty acids.

Characteristics similar to plant-derived palm oil

[101] In some embodiments, the microbial oils of the present disclosure have differences from plant-derived palm oil. In some embodiments, these differences are useful and allow for manipulation of the microbial oil for the improved production of a given product compared to plant-derived palm oil. For example, in some embodiments, the fatty acid profile of a microbial oil is tailored so as to produce a higher fraction of one or more fatty acids of interest for use in production of a product. In some embodiments, other parameters of the microbial oil are also able to be manipulated for increased production of a component of interest or decreased production of an undesired component relative to plant-derived palm oil.

[102] However, in some embodiments, the present compositions are also useful as environmentally friendly alternatives to plant-derived palm oil. Therefore, in some embodiments, the microbial oil has one or more properties similar to those of plant-derived palm oil. Exemplary properties include apparent density, refractive index, saponification value, unsaponifiable matter, iodine value, slip melting point, and fatty acid composition.

[103] In some embodiments, the microbial oil has a fatty acid profile similar to that of plant- derived palm oil. In some embodiments, the microbial oil has a significant fraction of Cl 6:0 fatty acid. In some embodiments, the microbial oil has a significant fraction of Cl 8: 1 fatty acid. In some embodiments, the microbial oil comprises 10-45% C16 saturated fatty acid. In some embodiments, the microbial oil comprises 10-70% Cl 8 unsaturated fatty acid.

[104] In some embodiments, the microbial oil has a similar ratio of saturated to unsaturated fatty acids as plant-derived palm oil. Some plant-derived palm oils have approximately 50% of each. In some embodiments, the microbial oil has a saturated fatty acid composition of about 50% and an unsaturated fatty acid composition of about 50%. In some embodiments, the microbial oil has a saturated fatty acid composition of about 40-60% and an unsaturated fatty acid composition of about 40-60%. In some embodiments, the microbial oil has a saturated fatty acid composition of about 30-70% and an unsaturated fatty acid composition of about 30- 70%. In some embodiments, the microbial oil has about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% saturated fatty acids.

[105] In some embodiments, the microbial oil has a similar level of mono-unsaturated fatty acids as plant-derived palm oil. Some plant-derived palm oils contain approximately 40% mono-unsaturated fatty acids. In some embodiments, the microbial oil contains about 40% mono-unsaturated fatty acids. In some embodiments, the microbial oil contains about 30-50% mono-unsaturated fatty acids. In some embodiments, the microbial oil contains about 5-60% mono-unsaturated fatty acids. In some embodiments, the microbial oil has about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% mono-unsaturated fatty acids.

[106] In some embodiments, the microbial oil has a similar level of poly-unsaturated fatty acids as plant-derived palm oil. Some plant-derived palm oils contain approximately 10% polyunsaturated fatty acids. In some embodiments, the microbial oil contains about 10% polyunsaturated fatty acids. In some embodiments, the microbial oil contains about 5-25% polyunsaturated fatty acids. In some embodiments, the microbial oil has about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% poly-unsaturated fatty acids.

[107] In some embodiments, the microbial oil has a similar iodine value as plant-derived palm oil. Some plant-derived palm oils have an iodine value of about 50.4-53.7. In some embodiments, the microbial oil has an iodine value of about 49-65. In some embodiments, the microbial oil has an iodine value of about 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65.

[108] Table 1 shows ranges for the fatty acid composition of an illustrative plant-derived palm oil and ranges of values for the fatty acid composition of illustrative microbial oil. In some embodiments, the microbial oil has one or more fatty acid composition parameters similar to those of Table 1. For example, in some embodiments, the microbial oil has a value within the plant-derived palm oil range for a given fatty acid composition parameter. In some embodiments, the microbial oil has a value within the microbial oil ranges provided in Table 1 for one or more parameters.

Table 1: Illustrative fatty acid compositions of microbial oil

[109] Tables 2A and 2B show ranges for the triglyceride composition of an illustrative plant- derived palm oil and ranges of values for the triglyceride composition of illustrative microbial oil. The abbreviations used are as follows: S: Stearic fatty acid; P: Palmitic fatty acid; O: Oleic fatty acid. For each component shown below in Table 2A, for example P-O-P, the corresponding measurements for that molecule may also include other isomers, for example P- P-0 and O-P-P. In some embodiments, the microbial oil has one or more triglyceride composition parameters similar to those of Table 2A and Table 2B. For example, in some embodiments, the microbial oil has a value similar to or within the plant-derived palm oil range for a given triglyceride composition parameter. For example, plant-derived palm oil has an O- O-P of approximately 23.24% and microbial-derived oil has an O-O-P of approximately 20.78.

In some embodiments, the microbial oil has a similar triglyceride content to that of plant- derived palm oil. For example, the total triglyceride content of sat-unsat-sat in plant-derived palm oil is approximately 49.53 and microbial-derived oil has approximately 49.42. In some embodiments, the microbial oil has a value different than plant-derived palm oil. For example, plant-derived palm oil has approximately 9.04% sat-sat-sat chains, whereas microbial-derived oil has approximately 3.36%. Some plant-derived palm oils have a triglyceride content of over 95%. In some embodiments, the microbial oil has a triglyceride content of 90-98%. In some embodiments, the microbial oil has a triglyceride content of about 90, 91, 92, 93, 94, 95, 96, 97, or 98%.

Table 2A: Illustrative triglyceride compositions of microbial oil

Table 2B: Summary total triglyceride compositions

[HO] In some embodiments, the microbial oil has a similar diacylglycerol content as a plant- derived palm oil. Percentage of diacylglycerol varies between about 4-11% for some plant- derived palm oils. In some embodiments, the microbial oil comprises 0-15% diacylglycerol content.

[Hl] In some embodiments, the microbial oil has a similar triacylglycerol profile to plant- derived palm oil. Some plant-derived palm oils have over 80% C50 and C52 triacylgylcerols. In some embodiments, the microbial oil has a triacylglycerol profile comprising at least 40% C50 and C52 triacylglycerols.

[112] In some embodiments, the microbial oil has a similar slip melting point to plant-derived palm oil. Some plant-derived palm oils have a slip melting point of about 33.8-39.2°C. In some embodiments, the microbial oil has a slip melting point of about 30-40°C. In some embodiments, the microbial oil has a slip melting point of about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C.

[113] In some embodiments, the microbial oil has a saponification value similar to that of plant-derived palm oil. Some plant-derived palm oils have a saponification value of about 190- 209. In some embodiments, the microbial oil has a saponification value of about 150-210. In some embodiments, the microbial oil has a saponification value of about 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, or 210.

[114] In some embodiments, the microbial oil has a similar unsaponifiable matter content to that of plant-derived palm oil. Some plant-derived palm oils have an unsaponifiable matter content of about 0.19-0.44% by weight. In some embodiments, the microbial oil has an unsaponifiable matter content of less than 5% by weight.

[115] In some embodiments, the microbial oil has a similar refractive index to that of plant- derived palm oil. Some plant-derived palm oils have a refractive index of about 1.4521-1.4541. In some embodiments, the microbial oil has a refractive index of about 1.3-1.6.

[116] In some embodiments, the microbial oil has a similar apparent density to that of plant- derived palm oil. Some plant-derived palm oils have an apparent density of about 0.8889- 0.8896. In some embodiments, the microbial oil has an apparent density of about 0.88-0.9.

[117] In some embodiments, the microbial oil has one or more parameters similar to those of hybrid palm oil.

[118] In some embodiments, the microbial oil may be used as a palm oil substitute or alternative. In some embodiments, the microbial oil may be used in the manufacture of any product for which palm oil can be employed. For example, in some embodiments, the microbial oil may be used in the production of soap bases, detergents, and oleochemicals. In some embodiments, the microbial oil may be used in the production of food products.

Processing of microbial oil

[119] In some embodiments, once the microbial oil is obtained from the oleaginous microorganism, it is subjected to some form of processing. In some embodiments, the microbial oil is refined, bleached, deodorized, fractionated, treated, and/or derivatized.

[120] In some embodiments, the microbial oil is refined. In some embodiments, prior to refinement, the microbial oil is referred to as crude microbial oil. In some embodiments, the refinement process comprises the removal of one or more non-triacylglycerol components. Typical non-triacylglycerol components removed or reduced via oil refinement include free fatty acids, partial acylglycerols, phosphatides, metallic compounds, pigments, oxidation products, glycolipids, hydrocarbons, sterols, tocopherols, waxes, and phosphorous. In some embodiments, refinement removes certain minor components of the crude microbial oil with the least possible damage to the oil fraction (e.g., trans fatty acids, polymeric and oxidized triacylglycerols, etc.) and minimal losses of desirable constituents (e.g., tocopherols, tocotrienols, sterols, etc.). In some embodiments, processing parameters are adapted for retention of desirable minor components like tocopherols and tocotrienols and minimal production of unwanted trans fatty acids. See Gibon (2012) “Palm Oil and Palm Kernel Oil Refining and Fractionation Technology,” incorporated by reference herein in its entirety, for additional details of oil processing that are useful for the present microbial oils.

[121] Common processing methods include physical refining, chemical refining, or a combination. In some embodiments, chemical refining comprises one or more of the following steps: degumming, neutralization, bleaching and deodorization. In some embodiments, physical refining comprises one or more of the following steps: degumming, bleaching, and steam-refining deodorization. While “physical refining” and “chemical refining,” as used herein and in the art, may refer to a general process of oil purification comprising multiple steps, possibly including bleaching and/or deodorizing, in the context of the present disclosure, the term “refined” as it relates to a microbial oil, e.g., a refined microbial oil, refers to a microbial oil from which one or more impurities or constituents have been removed other than odor and pigment. As such, stating that a microbial oil is refined does not indicate that the microbial oil has been deodorized and/or bleached. The term “RBD,” as used herein and as applied to a microbial oil, indicates that the microbial oil has been each of refined, bleached, and/or deodorized.

[122] In some embodiments, in chemical refining, the free fatty acids and most of the phosphatides are removed during alkali neutralization. In some embodiments, the nonhydratable phosphatides are first activated with acid and further washed out together with the free fatty acids during alkali neutralization with caustic soda. In some embodiments, chemical refining comprises one or more steps of acid treatment, centrifugation, bleaching, deodorizing, and the like.

[123] In some embodiments, during physical refining, phosphatides are removed by a specific degumming process and the free fatty acids are distilled during the steam refining/deodorization process. In some embodiments, the degumming process is dry degumming or wet acid degumming. In some embodiments, physical refining is employed when the acidity of the crude microbial oil is sufficiently high. In some embodiments, physical refining is employed for crude microbial oil with high initial free fatty acid (FFA) content and relatively low phosphatides. [124] In some embodiments, the microbial oil is deodorized. In some embodiments, the deodorization process comprises steam refining. In some embodiments, deodorization comprises vacuum steam stripping at elevated temperature during which free fatty acids and volatile odoriferous components are removed to obtain bland and odorless oil. Optimal deodorization parameters (temperature, vacuum, and amount of stripping gas) are determined by the type of oil and the selected refining process (chemical or physical refining) but also by the deodorizer design.

[125] In some embodiments, the microbial oil is bleached. In some embodiments, the bleaching is performed through the use of bleaching earth, e.g., bleaching clays. In some embodiments, the bleaching method employed is the two stage co-current process, the countercurrent process, or the Oehmi process. In some embodiments, the bleaching method is dry bleaching or wet bleaching. In some embodiments, bleaching is accomplished through heat bleaching. In some embodiments, bleaching and deodorizing occur concurrently.

[126] In some embodiments, the microbial oil is refined, bleached, and/or deodorized.

[127] In some embodiments, the microbial oil is not bleached or is only partially bleached. For example, in some embodiments, the microbial oil still retains pigments after processing. In some embodiments, the microbial oil comprises any one or more of the pigments referenced herein. Therefore, in some embodiments, the microbial oil is refined and deodorized, but not bleached or not fully bleached.

[128] In some embodiments, the microbial oil is processed and/or modified via one or more of fractionation, interesterification, trans-esterification, hydrogenation, steam hydrolysis, distillation, and saponification.

[129] In some embodiments, the microbial oil is fractionated. In some embodiments, fractionation is carried out in multiple stages, resulting in fractions appropriate for different downstream indications. In some embodiments, the microbial oil is fractionated via dry fractionation. In some embodiments, the microbial oil is fractionated via wet fractionation. In some embodiments, the microbial oil is fractionated via solvent/detergent fractionation.

[130] In some embodiments, the microbial oil is modified via interesterification. In some embodiments, the interesterification is enzymatic. In some embodiments, the interesterification is chemical.

[131] In some embodiments, the microbial oil is derivatized. In some embodiments, the oil is derivatized to free fatty acids and glycerol. In some embodiments, the oil is derivatized to fatty alcohols. In some embodiments, the oil is derivatized to esters. In some embodiments, the oil is derivatized to fatty acid methyl esters (FAMEs). Fractionation of microbial oils

[132] Oil fractionation can be accomplished through a variety of means. Fractionation of microbial oils disclosed herein can be accomplished according to any fractionation methods known in the art and/or familiar to the skilled person. Generally, fractionation is the process wherein an oil is cooled to a temperature such that a portion of the oil crystallizes, after which the solid oil crystals are separated from the liquid portion of the oil.

Altered properties of microbial oil fractions

[133] Fractionation generates oil fractions with divergent properties. In some embodiments, fractionation results in a fraction having a property that differs from that of the original microbial oil. In some embodiments, the property is melting point, saturation level, fatty acid profile, TAG profile, DSC curve, appearance, texture, specific fatty acid content, emulsifying ability, hardness, spreadability, viscosity, brittleness, plasticity, or stickiness.

Starting material for fractionation

[134] In some embodiments, the starting material is a crude microbial oil. In some embodiments, the starting material is a refined, bleached, and/or deodorized microbial oil. In some embodiments, the starting material is a refined, bleached, and deodorized (RBD) microbial oil.

Dry fractionation

[135] In some embodiments, the fractionation is carried out in the absence of solvent, i.e., via “dry” fractionation. In some embodiments, dry fractionation is used to produce microbial oil fractions without detectable solvent from the fractionation method. In some embodiments, dry fractionation is used to produce microbial oil fractions with an improved safety profile, making them well suited for use in, e.g., foods and cosmetics.

Solvent-based fractionation

[136] In some embodiments, the original oil can be dissolved in a solvent which may help with the separation by reducing viscosity. In some embodiments, solvents alter the profile of TAGs that are present in the solid fraction. In some embodiments, the solvent employed is acetone, hexane, and/or heptane. In some embodiments, solvent-based fractionation is used to produce microbial oil fractions with more divergent properties compared to the original microbial oil, e.g., saturation level, melting point, and the like.

Exemplary fractionation protocol

[137] The following exemplary protocol is based on the fractionation of illustrative microbial palm oil alternatives having relatively comparable saturated and unsaturated fatty acid content. As will be appreciated by one of skill in the art, the particular temperatures, ratios, and times employed in the fractionation method are adjusted based on the characteristics of the starting material.

[138] 1. Melting: In some embodiments, the original microbial oil is raised to a temperature at which the oil can fully melt, e.g., via water bath. In some embodiments, the temperature is raised to about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C. In some embodiments, the original microbial oil is raised to a temperature of about 45-55°C. In some embodiments, the original microbial oil is maintained at this temperature until fully melted.

[139] 2. Solvent addition: Once melted, in some embodiments, the sample is agitated as solvent is added. In some embodiments, for solvent fractionation, the desired solvent is added in sufficient quantity to obtain the desired solvent to oil ratio. In some embodiments, sufficient solvent is added to achieve a solvent to oil ratio of about 20:1, 19: 1, 18: 1, 17: 1, 16: 1, 15: 1, 14: 1, 13: 1, 12: 1, 11 : 1, 10:1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4, or 1 :5. In some embodiments, sufficient solvent is added to achieve a solvent to oil ratio of about 10: 1 to about 1 :2. In dry fractionation, no solvent is added.

[140] 3. Temperature decrease: In some embodiments, following melting and optional solvent addition, the temperature of the microbial oil is lowered to a temperature between the melting temperature and the crystallization temperature. In some embodiments, this temperature is close to room temperature, e.g., 25°C. In some embodiments, the sample is agitated while the temperature is lowered. In some embodiments, the sample is held at this temperature for about 5 minutes to about 24 hours.

[141] In some embodiments, the temperature is ramped down to crystallization temperature over a period of minutes, hours, or days. In some embodiments, the temperature is ramped down from melting temperature to a target temperature, with continual crystallization and removal of high-melting point solid fractions.

[142] 4. Crystallization: In some embodiments, after fully melting, the temperature of the oil is lowered to induce crystallization. In some embodiments, the crystallization temperature is between about -20°C to about 35°C. In the case of solvent-based fractionation, in some embodiments, lower temperatures are used in order to obtain crystals because of the effects of the solvent, which vary based on the type and amount of solvent employed. In some embodiments, solvent-based fractionation employs a crystallization temperature of between about -15°C to about 25°C. In some embodiments, dry fractionation employs a higher crystallization temperature than solvent-based fractionation. In some embodiments, the crystallization temperature for dry fractionation is chosen based on the DSC curve for the original microbial oil to occur approximately below the temperature of the lowest peak on the chromatogram. In some embodiments, the crystallization temperature for dry fractionation is between about -5°C to about 35°C. In some embodiments, the crystallization temperature for dry fractionation is between about 5°C to about 30°C.

[143] In some embodiments, the crystallization temperature is about -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35°C.

[144] In some embodiments, the temperature of the microbial oil is maintained at the crystallization temperature for 30 minutes to 7 days. In some embodiments, the crystallization time is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, the crystallization time is 1, 2, 3, 4, 5, 6, or 7 days.

[145] 5. Filtration: Following crystallization, the solid crystals are separated from the liquid portion of the oil. In some embodiments, a temperature-controlled jacketed filtration system is used to do the solid-liquid separation. In some embodiments, the filtration system temperature is set to the crystallization temperature. In some embodiments, the filter on the filtration system is in the size range of 0.1-50 pm, e.g., 1 pm. In some embodiments, a gentle vacuum, e.g., of less than 50 torr, is applied to the filtration system to facilitate separation. At the end of crystallization, the mixture of crystals and liquid is delivered to the filtration system, and the liquid is allowed through the filter, while the solids are retained. At the end of separation, the solid fraction is collected from the filter.

[146] 6. Analysis of solid and liquid fractions: In some embodiments, following filtration, there is a liquid fraction and a solid fraction. In some embodiments, the yield of a fraction is quantified, e.g., in comparison to the volume or mass of the original microbial oil. In some embodiments, the fractions are assessed visually based on color and opacity. In some embodiments, a TAG analysis is performed on a fraction. In some embodiments, a FAME profile is obtained for a fraction. In some embodiments, a DSC curve is generated for a fraction. In some embodiments, the melting point of a fraction is determined.

Illustrative microbial fats of the disclosure

[147] The present disclosure provides a microbial fat obtained from an oleaginous yeast.

[148] In some embodiments, the microbial fat is characterized based on content of saturated mono-unsaturated, and poly-unsaturated fatty acid. In some embodiments, the microbial fat comprises the following amounts of fatty acids relative to the total fatty acids: at least about 35% w/w saturated fatty acids; and less than about 15% w/w total polyunsaturated fatty acids. In some embodiments, the microbial fat comprises between about 35% and about 85% w/w saturated fatty acids. In some embodiments, the microbial fat has a high proportion of saturated fatty acids with chain lengths between 16 and 18 carbons long. In some embodiments, the microbial fat comprises at least about 35% w/w saturated fatty acids with chain lengths between 16 and 18 carbons long. In some embodiments, the microbial fat comprises between about 35% and about 85% w/w saturated fatty acids with chain lengths between 16 and 18 carbons long. In some embodiments, the microbial fat comprises between about 5% and about 20% w/w saturated Cl 8 fatty acid. In some embodiments, the microbial fat comprises between about 3% and about 15% w/w total polyunsaturated fatty acids.

[149] In some embodiments, the microbial fat is characterized based on the average fatty acid desaturation level. In some embodiments, the microbial fat has an average fatty acid desaturation level less of less than about 0.8. In some embodiments, the microbial fat has an average fatty acid desaturation level less of less than about 0.72. In some embodiments, the microbial fat has an average fatty acid desaturation level less of less than about 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1. In some embodiments, the microbial fat comprises an average fatty acid desaturation level of between about 0.1 and 0.72.

[150] In some embodiments, the microbial fat has a melting point of at least about 23°C. In some embodiments, the microbial fat has a melting point above room temperature. In some embodiments, the microbial fat has a melting point of between about 23°C and about 75°C. In some embodiments, the microbial fat has a melting point of about 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75°C.

[151] In some embodiments, the microbial fat is characterized based on triglyceride profile. In some embodiments, the microbial fat comprises, as a percentage of overall triacylglycerols (TAGs), less than about 10% w/w TAGs with three unsaturated fatty acids. In some embodiments, the microbial fat comprises, as a percentage of overall TAGs, about 1% to about 10% w/w TAGs with three unsaturated fatty acids.

[152] In some embodiments, the microbial fat comprises, as a percentage of overall TAGs, less than about 40% w/w TAGs with one saturated fatty acid and two unsaturated fatty acids. In some embodiments, the microbial fat comprises, as a percentage of overall TAGs, between about 20% and about 40% w/w TAGs with one saturated fatty acid and two unsaturated fatty acids.

[153] In some embodiments, the microbial fat comprises, as a percentage of overall TAGs, at least about 40% w/w TAGs with two saturated fatty acids and one unsaturated fatty acids. In some embodiments, the microbial fat comprises, as a percentage of overall TAGs, between about 40% and about 80% w/w TAGs with two saturated fatty acids and one unsaturated fatty acid. In some embodiments, the microbial fat comprises, as a percentage of overall TAGs, at least about 25% w/w palmitic-oleic-palmitic TAGs.

[154] In some embodiments, the microbial fat comprises, as a percentage of overall TAGs, at least about 0.3% w/w TAGs with three saturated fatty acids. In some embodiments, the microbial fat comprises, as a percentage of overall TAGs, at least about 1% w/w TAGs with three saturated fatty acids.

[155] In some embodiments, the microbial fat is a fraction of an original microbial oil. In some embodiments, the microbial fat is harder, more viscous, thicker, stickier, and/or more opaque than the microbial oil. In some embodiments, the microbial fat has a higher melting point and/or fatty acid saturation level than the original microbial oil. In some embodiments, the microbial fat has a lower fatty acid desaturation level than the original microbial oil. In some embodiments, the microbial fat differs from the original microbial oil in terms of fatty acid profile, TAG profile, DSC curve, appearance, texture, specific fatty acid content, emulsifying ability, hardness, spreadability, viscosity, brittleness, plasticity, or stickiness.

[156] In some embodiments, the yield of microbial fat from a fractionation of an original microbial oil is at least about 1% w/w. In some embodiments, the yield of microbial fat from a fractionation of an original microbial oil is at least about 5% w/w. In some embodiments, the yield of microbial fat from a fractionation of an original microbial oil is at least about 10% w/w. In some embodiments, the yield of microbial fat from a fractionation of an original microbial oil is at least about 20% w/w. In some embodiments, the yield of microbial fat from a fractionation of an original microbial oil is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% w/w.

[157] The present description is made with reference to the accompanying drawings and Examples, in which various example embodiments are shown. However, many different example embodiments may be used, and thus the description should not be construed as limited to the example embodiments set forth herein. Rather, these example embodiments are provided so that this disclosure will be thorough and complete. Various modifications to the exemplary embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the disclosure. Thus, this disclosure is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein. [158] Although the disclosure may not expressly disclose that some embodiments or features described herein may be combined with other embodiments or features described herein, this disclosure should be read to describe any such combinations that would be practicable by one of ordinary skill in the art. Unless otherwise indicated herein, the term “include” shall mean “include, without limitation,” and the term “or” shall mean non-exclusive “or” in the manner of “and/or.”

[159] Those skilled in the art will recognize that, in some embodiments, some of the operations described herein may be performed by human implementation, or through a combination of automated and manual means. When an operation is not fully automated, appropriate components of embodiments of the disclosure may, for example, receive the results of human performance of the operations rather than generate results through its own operational capabilities.

[160] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world, or that they disclose essential matter.

EXAMPLES

EXAMPLE 1: Fatty acid composition of exemplary microbial oil.

[161] To compare the fatty acid composition of an exemplary microbial oil to that of a plant- derived palm oil, the oil samples were converted into fatty acid methyl esters and then analyzed using gas chromatography-mass spectrometry (GC-MS).

FAME preparation

[162] A method of using commercial aqueous concentrated HC1 (cone. HC1; 35%, w/w) as an acid catalyst was employed for preparation of fatty acid methyl esters (FAMEs) from microbial oil and palm oil for GC-MS. FAME preparation was conducted according to the following exemplary protocol.

[163] Commercial concentrated HC1 (35%, w/w; 9.7 ml) was diluted with 41.5 ml of methanol to make 50 ml of 8.0% (w/v) HC1. This HC1 reagent contained 85% (v/v) methanol and 15% (v/v) water that was derived from cone. HC1 and was stored in a refrigerator.

[164] A lipid sample was placed in a screw-capped glass test tube (16.5 x 105 mm) and dissolved in 0.20 ml of toluene. To the lipid solution, 1.50 ml of methanol and 0.30 ml of the 8.0% HC1 solution were added in this order. The final HC1 concentration was 1.2% (w/v) or 0.39 M, which corresponded to 0.06 ml of concentrated HC1 in a total volume of 2 ml. The tube was vortexed and then incubated at 45°C overnight (14 h or longer) for mild methanolysis/methylation or heated at 100°C for 1 h for rapid reaction. After cooling to room temperature, 1 ml of hexane and 1 ml of water were added for extraction of FAMEs. The tube was vortexed, and then the hexane layer was analyzed by GC-MS directly or after purification through a silica gel column.

GC-MS

[165] For the analysis of fatty acid composition, a Shimadzu GCMS-TQ8040/GC-2010 Plus instrument was employed. The FAME samples were concentrated at 5 g/L in hexane/chloroform/heptane prior to analysis.

[166] The results of the analysis are shown in Table 3 comparing the fatty acid composition of three exemplary microbial oil samples produced by Rhodosporidium toruloides to the measurements expected for crude palm oil, as set forth by guidelines from the Malaysian government. For Microbial oil sample 3, the fatty acid compositions were determined via fatty acid methyl ester analysis with a GC-SSL/FID (7890A, Agilent) instrument. The methods employed were using AOCS Ce la- 13 and AOCS C2 2-66. (see also FIG. 1A-1D). Table 3 shows the breakdown of the individual fatty acid constituents by w/w percent, with the percentages for each sample adding up to 100%. Fatty acids that were assayed but not detected in any sample include C4, C6, C13, C15, C15:l, C18:2 tt, C18:2 5,9, C18:2 tc, C18:3, C18:3 etc, C18:3 fit, C18:3 ttc+tct, C20:4 n6ARA, C22, and C24.

Table 3: Fatty acid composition of microbial oil samples

[167] These results show that exemplary microbial oil samples of the present disclosure have a similar breakdown of saturated vs. unsaturated fatty acids compared to plant-derived palm oil, though the specific identities of the predominant fatty acids differs between the microbial samples and typical palm oil. Similar to palm oil, though, Cl 6:0 was a significant source of saturated fatty acid in the microbial samples and Cl 8 unsaturated fatty acids made up the majority of the unsaturated fatty acids in the sample.

[168] The fatty acid composition breakdown of the samples were determined via fatty acid methyl ester analysis with a GC-SSL/FID (7890A, Agilent) instrument. The methods employed were using AOCS Ce la-13 and AOCS C22-66. The results these analyses are shown in Table

4 and FIG. 1A-1C. Table 4 below shows the breakdown of the individual fatty acid constituents by w/w percent, with the percentages for each sample adding up to 100%. Fatty acids that were assayed but not detected in any sample include C4, C6, C13, C15, C15:l, C18:2 tt, C18:2 5,9, C18:2 tc, C18:3, C18:3 etc, C18:3 ttt, C18:3 ttc+tct, C20:4 n6ARA, C22, and C24.

Table 4: Fatty acid composition breakdown

[169] Table 5 shows the w/w percentage of saturate, trans, mono-unsaturated, polyunsaturated, and unknown fatty acids in each sample. The fatty acid compositions were determined via fatty acid methyl ester analysis with a GC-SSL/FID (7890A, Agilent) instrument. The methods employed were using AOCS Ce la-13 and AOCS C2 2-66. FIG.

1A-1C show the chromatograms for the crude microbial oil (FIG. 1A), palm oil (FIG. IB), and hybrid palm oil (FIG. 1C), respectively. FIG. ID shows a bar graph of representative compositions of microbial oil and palm oil.

Table 5: Overall fatty acid composition

EXAMPLE 2: Fractionation and saturation analysis of exemplary microbial oil composition

[170] Fats and oils are mixtures of hydrocarbons of various chain lengths and saturation levels. Fractionation may be used to physically separate room temperature oil into saturated and unsaturated components. The melting points of full oil mixtures and their saturated/unsaturated components differ. Hydrophilization makes use of surface active agents (surfactants) that dissolve solidified fatty crystals and emulsify liquid oils. By centrifuging this hydrophilized suspension, fats can be separated into different fractions based on saturation. A palm oil and a microbial oil were fractionated and the saturation levels of their fractions were compared.

Fractionation

[171] Crude palm oil and an R. toruloides microbial oil were fractionated using a method as set out in, e.g., Stein, W., "The Hydrophilization Process for the Separation of Fatty Materials," Henkel and Cie, GmbH, Presented at AOCS Meeting, New Orleans, May 1967.

[172] The oil sample was weighed and then incompletely melted to 50°C. The temperature was then brought down to 32°C over the course of 10 min. The temperature was then slowly lowered to 20°C with periods of time held at select temperatures between 32°C-20°C as follows: 32°C - 30 min; 26°C - 15 min; 24°C - 15 min; 22°C - 15 min; 21°C - 15 min; 20°C - 15 min. The oil sample was then maintained at 20°C for an additional 1 hr.

[173] After this temperature manipulation, the oil sample was emulsified in a wetting agent solution at a ratio of 1 : 1.5 w/w fat to wetting agent. The wetting agent was comprised of a salt and a detergent in DI water: 0.3% (w/w) sodium lauryl sulfate; 4% (w/w) magnesium sulfate. The oil/wetting agent mixtures were vortexed until thoroughly mixed. The samples were centrifuged at 4700 rpm for 5 min in a benchtop centrifuge. The lighter oil phase migrated to the top, while the heavier aqueous phase (containing solid, saturated fatty particles) migrated to the bottom. The aqueous phase was separated by aspirating the upper olein phase into a preweighed scintillation vial. The aqueous phase was heated - with its solidified stearin layer interspersed atop - until all fatty materials melted. This heated aqueous phase was centrifuged (4700 rpm, 1 min, 40°C) and the stearin fraction was also aspirated into a pre-weighed scintillation vial.

[174] The separated olein and stearin fractions were weighed and their masses compared to the original mass of oil pre-fractionation. By mass, an exemplary microbial oil produced by R. toruloides was 68.4% w/w olein and 31.6% w/w stearin. By comparison, a crude plant-derived palm oil sample was analyzed as comprising 72% w/w olein and 28% w/w stearin using this fractionation method.

Saturation level measurement

[175] Next, the iodine value (IV) for each fraction was calculated, which is expressed as the number of grams of iodine absorbed by 100 g of the oil sample. The microbial olein fraction had an iodine value of 81 and the microbial stearin fraction had an iodine value of 22. The crude palm oil olein fraction had an IV of 53 and the stearin fraction had an IV of 40. These results indicate an even more distinct fractionation of saturated and unsaturated fatty acids between the microbial fractions, a distinction that could be useful for the manufacture of downstream products, as plant-derived palm oil may require multiple fractionation steps to achieve this level of differentiation between fractions.

EXAMPLE 3: Comprehensive analysis of an illustrative crude microbial oil sample.

[176] A 100g sample of crude microbial oil produced by the oleaginous microorganism R. toruloides was analyzed for general physical chemical characterization; fatty acid content; triglyceride composition; unsaponifiable lipid content; oxidative stability; FAs at Sn-2 position; and contaminant (3-MCPD, GEs) levels. These analyses were carried out in comparison to standard Colombian palm oil and hybrid palm oil samples over the course of 70 days. Samples were stored in the dark, at cold temperatures, and at atmospheric nitrogen conditions.

General physical chemical characterization

[177] The three oil samples were analyzed along different physical and chemical parameters, the results of which analyses are shown in Table 6. The methods employed were those of the American Oil Chemists’ Society (AOCS) and are referenced within the Table by their AOCS identifier.

Table 6: General physical chemical characterization

[178] As shown in Table 6 above, crude microbial oil has similar amounts of free fatty acids, triglycerides, and monoglyceride as those found in crude palm oil and crude hybrid oil. Specific triglycerides were also measured and shown below. Triglyceride composition

[179] The triglyceride compositions of the three samples were analyzed on a GC-COC/FID (7890A, Agilent) instrument according to the AOCS Ce 5-86 method. Table 7 shows the results of the triglyceride analysis, with values as w/w percentages. The abbreviations used are as follows. M: Myristic fatty acid; S: Stearic fatty acid; P: Palmitic fatty acid; O: Oleic fatty acid;

L: Linoleic fatty acid; La: Lauric fatty acid; Ln: linoleic fatty acid. The chromatogram for crude microbial oil is shown in FIG. 2A, the chromatogram for crude palm oil is shown in FIG. 2B, and the chromatogram for crude hybrid palm oil is shown in FIG. 2C.

Table 7: Triglyceride composition [180] The microbial oil sample showed similarity to both palm oil and hybrid palm oil along different parameters of fatty acid and triglyceride content. For example, microbial oil comprised approximately 1.2% w/w palmitic-palmitic-palmitic triglycerides, approximately 22.53% w/w palmitic-palmitic-oleic triglycerides, approximately 20.78% w/w oleic-oleic- palmitic triglycerides, approximately 1.53% w/w stearic-stearic-oleic triglycerides, and approximately 4.29% w/w stearic-oleic-oleic triglycerides.

Fatty acids at Sn-2 position

[181] The three samples were analyzed for the amount of palmitic and stearic fatty acids located at the sn-2 position of triglyceride molecules, with results shown in Table 8. Methods used were adapted from Luddy et al., “Pancreatic lipase hydrolysis of triglycerides by a semimicro technique,” Journal of the American Oil Chemists' Society 1964;41(10):693-6, and Pina-Rodriguez et al., “Enrichment of amaranth oil with ethyl palmitate at the sn-2 position by chemical and enzymatic synthesis,” Journal of Agricultural and Food Chemistry 2009;57(l l):4657-62, each incorporated herein by reference in its entirety.

Table 8: Fatty acids at sn-2 position of triglycerides

[182] The microbial oil sample contained an acceptable amount of palmitic and stearic fatty acids located at the sn-2 position of the triglyceride molecules, suggesting the oil has suitability for use in various food products.

Unsaponifiable lipid content

[183] The unsaponifiable lipid content of the three samples was analyzed, specifically measuring the amount of P-carotene (data not shown), squalene, tocopherols, and sterols in each sample. Results are shown in Table 8. P-carotene was analyzed using the method of Luterotti et al., “New simple spectrophotometric assay of total carotenes in margarines,” Analytica Chimica Acta 2006;573:466-473, incorporated by reference herein in its entirety. The sterol composition was analyzed using the method of Johnsson et al., “Side-chain autoxidation of stigmasterol and analysis of a mixture of phytosterol oxidation products by chromatographic and spectroscopic methods,” Journal of the American Oil Chemists' Society 2003;80(8):777-83, incorporated by reference herein in its entirety, with the HPLC-DAD chromatogram results shown in FIG. 3. The other methods that were employed are indicated in Table 9. The sterol composition of the microbial oil sample showed an atypical sterols chromatographic profile differentiating it from the palm oil and hybrid palm oil samples and warranting further investigation. In this illustrative sample, the unexpected sterol composition acts as a unique fingerprint for the microbial oil sample.

Table 9: Unsaponifiable lipid content

[184] As shown in Table 9, the microbial oil sample does not contain significant levels of unsaponifiable lipids, or tocopherols. Specifically, microbial oil has approximately 122 ppm of squalene, compared to 389 ppm and 260 ppm in palm oil and hybrid palm oil respectively. Microbial oil also contained less than 10 ppm of tocopherols, whereas palm oil and hybrid palm oil contained 869 ppm and 761 ppm respectively.

Oxidative stability

[185] The oxidative stability of the samples was analyzed (data not shown) via The Ferric Reducing Ability of Plasma (FRAP) using the method of Szydlowska-Czerniak et al., “Effect of refining processes on antioxidant capacity, total contents of phenolics and carotenoids in palm oils,” Food Chemistry 2011 ; 129(3): 1187-92, herein incorporated by reference in its entirety.

Contaminant (3-MCPD, GEs, and phosphorus) levels

[186] Levels of contaminants were assessed in each sample, with results shown in Table 10. The methods and equipment are shown in columns two and three, respectively. Table 10: Contaminant levels

[187] All three samples had contaminant levels below the limit of quantitation (LOQ). However, the samples differed greatly in the amount of phosphorous detected. Unlike crude palm oil and crude hybrid palm oil, which had 25 ppm and 20 ppm respectively, crude microbial oil had less than 1 ppm of phosphorous.

Conclusion

[188] Based on the above analyses, the crude microbial oil was a good match of palm oil/hybrid palm oil along a number of different parameters, demonstrating its suitability for use as an environmentally friendly alternative to plant-derived palm oil.

EXAMPLE 4: Exemplary microbial oils from three different strains of R. toruloides Fatty acid profile of microbial oil produced by three exemplary strains of oleaginous yeast

[189] Using the FAME and GC-MS protocols of Example 1, exemplary microbial oils according to the present disclosure were analyzed from three illustrative strains of oleaginous yeast of the species Rhodosporidium toruloides'. strain A, strain B, and strain C.

[190] FIG. 4A shows the overall fatty acid composition broken down by percentage of polyunsaturated fatty acid (PUFA), mono-unsaturated fatty acid (MUFA), and saturated fatty acid for exemplary microbial oils produced by these three strains. This breakdown shows a comparable ratio of saturated to unsaturated fatty acids within each sample, especially for strain A, which produced approximately equal amounts of saturated and unsaturated fatty acids. FIG. 4B shows the breakdown of the fatty acid composition for the microbial oils in terms of specific fatty acids. For all three microbial oils, C18: 1 was most prevalent, comprising between 40-50% of each sample. The next most prevalent was C16:0, comprising 15-35% of each sample, followed by C18:0 and C18:2, which each made up about 10-20% of the samples. C14:0, C16: 1, and C18:3 (not shown) each comprised less than 3% of the samples. The remaining less than 1% was made up of other fatty acids. EXAMPLE 5: Fractionation of additional exemplary microbial oils

Fractionation protocol

[191] A 5 g sample of an exemplary R. toruloides microbial oil of the disclosure was melted to 50°C over a hot plate. Temperature was brought down to 32°C over 10 min and then slowly down to 20°C, allowing the sample to remain held at temperature every two degrees for 15 min. The sample was then held at 20°C for Ihr.

[192] Wetting agent comprised of 0.3% (w/w) sodium lauryl sulfate and 4% (w/w) magnesium sulfate was added to the oil sample (1 : 1.5 w/w oil to wetting agent). The oil sample was vortexed thoroughly and then centrifuged at 4100g for 5 min.

[193] The liquid, upper lipid phase comprising a higher percentage of unsaturated fatty acids (olein) was transferred to a pre-weighed vial. The lower lipid phase (stearin), along with the remaining aqueous material, was heated until the stearin was fully melted. Then the sample was centrifuged for 1 min before the stearin layer was transferred to a separate pre-weighed vial. This process was repeated with a 10g sample of crude palm oil.

Effect of fractionation on fatty acid profile of exemplary microbial oil

[194] An exemplary R. toruloides microbial oil of the disclosure was fractionated. FIG. 5A shows the results of fractionation on overall fatty acid composition for a representative microbial oil. This figure demonstrates a higher percentage of unsaturated fatty acids in the olein fraction and a higher percentage of saturated fatty acids in the stearin fraction compared to the crude microbial oil. The microbial mid-fraction has a profile in between the olein and stearin profiles. FIG. 5B shows the breakdown in terms of specific fatty acids for the crude microbial oil and each of the fractions.

Iodine value calculation

[195] Iodine value was determined based on the Malaysian Palm Oil Board’s test method. Briefly, approximately 0.5 g of oil was dissolved in 20mL 1 : 1 cyclohexane/glacial acetic acid. 25mL of Wijs reagent (iodine mono chloride dissolved in acetic acid) was added, and the solution was well stirred before being placed in the dark for 1 hr. A blank sample was prepared identically, without the addition of any oil sample.

[196] At the end of the incubation time, 20mL of 100 g/L potassium iodide and 150mL of DI water were added. A standard volumetric solution of 0.1M sodium thiosulfate was added in a dropwise fashion until the solution’s yellow color began to fade. 5g/L starch solution was added until the solution turned a deep blue color. Additional thiosulfate titrant is added until the solution became clear upon mixing. The blank solution was titrated in parallel. For some samples, Metrohm’s 892 professional rancimat was also used to confirm iodine values, in which case the starch solution was no longer needed as an indicator.

[197] Iodine value was calculated as IV = 12.69 x C x (V 1-V2)/M, where C is the concentration of sodium thiosulfate, VI is the volume in mL of sodium thiosulfate used for the blank test, V2 is the volume in mL of sodium thiosulfate used for the determination, and is the mass in g of the test oil sample.

Effect of fractionation on iodine value (IV) for an exemplary microbial oil

[198] The effect of fractionation on iodine value was evaluated using the protocol above for an illustrative crude R. toruloides microbial oil of the disclosure, along with its stearin and olein fractions. The results are summarized in Table 11 below.

Table 11: IVs for an exemplary fractionated microbial oil of the disclosure.

Visual effects of fractionation on exemplary microbial oils of the disclosure

[199] Exemplary crude microbial oils from R toruloides were fractionated. FIG. 6A-6B exhibit the visual effects of fractionation on various samples. FIG. 6A shows a fractionated microbial oil (left) compared to a fractionated crude palm oil (right). Both fractionated samples contain a top olein layer that is liquid at room temperature and a bottom stearin layer that is solid at room temperature. FIG. 6B shows another fractionated microbial oil (left) and a microbial oil that did not fractionate (right). These images demonstrate a characteristic of exemplary microbial oils of the disclosure which demonstrate the ability to fractionate similar to plant-derived palm oil, a characteristic which does not hold for all microbial oils.

EXAMPLE 6: Sterol analysis of exemplary microbial oil of the disclosure

Materials and Methods

[200] The following procedure was followed in order to measure the content of sterols present in each of these samples: an exemplary microbial oil of the disclosure obtained from R toruloides (“yeast microbial oil”), Crude Palm Oil (CPO), RBD Palm Oil (RBDPO) and Algae oil. First, each oil was weighed to obtain 40 mg. All oil samples were dissolved in 200 pL of hexane containing 200 pg/mL of a tridecanoic acid methyl ester internal standard (ISTD). The oil samples were then set at 60°C for 2 h in the vacuum oven to remove the organic solvent by evaporation. Then, one half of each sample was resuspended in 100 pL of pyridine (“plain” preparation). The other half of each sample was resuspended in 100 pL pyridine solution comprising 0.4 mg/mL of each of 5 purified sterol standards corresponding to targets of interest (“spike-in” preparations). Finally, both plain and spike-in preparations were further derivatized by addition of 100 pL of BSTFA + 10% TCMS (Thermo Scientific, USA) and incubated at 92°C for 2 h.

[201] Derivatized oil samples were analyzed using an Agilent® 7890B GC System coupled to an Agilent® 5975 mass selective detector. The GC was operated in splitless mode with constant helium gas flow at 1 mL/min. 1 pL of derivatized oil was injected with the PAL3 Sampler (Model Pal RSI 120 from CTC Analytics, Switzerland) onto an HP-5ms Ultra Inert column. The total ion chromatograms for each oil (FIG. 7A-7D) were obtained by using a GC oven program as follows: the initial oven temperature was first held at 70°C for one minute, and then ramped from 70°C to 255°C at a rate of 20°C/min; the oven temperature was then further increased at a rate of 1.5°C/min to reach 283°C; finally, the ramp rate was increased to 15°C/min until the oven temperature reached 300°C, where it was held for 9 min. The total run time was 39 minutes. Peaks representing compounds of interest were extracted and integrated using MassHunter software (Agilent Technologies®, USA), e.g., as visually represented in FIG. 8. Each extracted, integrated peak was then normalized to both the ISTD and their corresponding spike-in sterol peak area. The masses of molecular ions used for extraction are shown in Table 12. All peaks were manually inspected and their electron ionization (El) spectra were verified relative to known spectra for each sterol. FIG. 9A-9E show illustrative El spectra for sterols extracted from the crude palm oil spike-in preparation.

Table 12: Mass of sterol compounds used for extraction.

[202] Extracted peaks were first normalized to the ISTD peak for the corresponding runs. For each spike-in run, residual peaks for each sterol standard were calibrated by subtracting normalized peak areas of the plain runs from the spike-in runs. Residual peaks for each sterol were averaged across the 4 oil sample runs, and then used to re-normalize plain peak areas for differences in detector signal across targets. These final, re-normalized peak areas were used to calculate total sterol content (Table 13) and sterol profiles (Table 14) for each of the oil samples.

Table 13: Total sterol content.

Table 14: Sterol profiles.

[203] The results demonstrate that an exemplary yeast microbial oil of the disclosure only comprised ergosterol and did not comprise cholesterol, campesterol, stigmasterol, or sitosterol, in contrast to the other three samples derived from agricultural palm plants or algae.

EXAMPLE 7: Carotenoid analysis of exemplary microbial oils of the disclosure

Oil samples

[204] Six oil samples were analyzed to identify the carotenoids present within each one.

[205] - Sample 1 : agricultural palm oil.

[206] - Sample 2: exemplary microbial oil of the disclosure obtained from R. loruloides: strong acid (H2SO4) treatment with solvent extraction of lipids.

[207] - Sample 3: exemplary microbial oil of the disclosure obtained from R. loruloides strong acid (HC1) treatment with solvent extraction of lipids.

[208] - Sample 4: exemplary microbial oil of the disclosure obtained from R. loruloides weak acid (H3PO4) treatment with solvent extraction of lipids.

[209] - Sample 5: exemplary microbial oil of the disclosure obtained from R. loruloides,' acid- free extraction of lipids.

[210] - Sample 6: exemplary microbial oil of the disclosure obtained from R. loruloides,' acid- free extraction of lipids. Carotenoid analysis materials and methods

[211] Sample Preparation. Oil samples were diluted in diethyl ether. Each solution was saponified in homogeneous phase for 1 hr. After acidification and washing, UV/Vis and HPLC analysis were performed.

[212] UV/Vis analysis. For each sample, an initial overall UV/Vis absorbance spectrum was collected between 200 and 600 nm wavelengths. This overall spectrum shows the total overlapping absorbance of all of the sample’s carotenoids, which allows for estimation of the total carotenoid content within the sample. UV/Vis spectra were recorded with a Jasco V-530 spectrophotometer in benzene. (E ^1% icm= 2500)

[213] High performance liquid chromatography (HPLC) diode array detector (DAD) analysis. The HPLC-DAD assay was conducted using a Dionex Ultimate 3000 HPLC system detecting absorbance at X = 450 nm. Temperature was maintained at 22°C. Data acquisition was performed by Chromeleon 7.2 software. The column employed was a YMC Carotenoid C30 column, with 3 pM bead size and dimensions of 250 x 4.6 mm i.d. Buffer A had the following composition: 81% MeOH, 15% TBME, 4% H2O. Buffer B had this composition: 6% MeOH, 90% TBME, 4% H2O. The chromatograms were performed in linear gradient: 0 min 100% Buffer A to 70 min 70% Buffer B. The flow rate was maintained at 1.00 cm ³ /min.

[214] Carotenoid identification. An absorbance spectrum was collected for each analyte with a corresponding peak in the HPLC-DAD chromatogram. Identities of individual carotenoids were confirmed based on comparing the retention time and UV/Vis spectrum for that analyte to known standards.

Results

[215] Sample 1. The overall UV/Vis absorbance spectrum for Sample 1, agricultural palm oil, is shown in FIG. 10A with the absorbance at individual wavelengths identified in Table 15. The overall UV/Vis spectrum shows the expected distribution centered around 450 nm. The total carotenoid content, roughly estimated using the absorbance at 459 nm, was determined to be approximately 478 ppm.

Table 15: Sample 1, UV/Vis Abs at specific wavelengths. [216] For Sample 1, the HPLC-DAD chromatogram reporting absorbance at 450 nm is shown in FIG. 10B with individual peaks identified in Table 16. As expected, this sample contained the known agricultural palm oil-associated carotenoids a- and P-carotene, and derivatives thereof.

Table 16: Sample 1, HPLC peak identification.

[217] Sample 2. The overall UV/Vis absorbance spectrum for Sample 2, strong acid- extracted microbial oil, is shown in FIG. 11 A. The overall UV/Vis spectrum shows essentially no absorbance in the 300-500 nm range, likely because of carotenoid degradation due to the strong acid treatment. For Sample 2, the HPLC-DAD chromatogram reporting absorbance at 450 nm is shown in FIG. 11B with no identifiable peaks.

[218] Sample 3. The overall UV/Vis absorbance spectrum for Sample 3, strong acid- extracted microbial oil, is shown in FIG. 12A. The overall UV/Vis spectrum shows essentially no absorbance in the 300-500 nm range, likely because of carotenoid degradation due to the strong acid treatment. For Sample 3, the HPLC-DAD chromatogram reporting absorbance at 450 nm is shown in FIG. 12B with no identifiable peaks.

[219] Sample 4. The overall UV/Vis absorbance spectrum for Sample 4, weak acid-extracted microbial oil, is shown in FIG. 13A. The total carotenoid content, roughly estimated using the absorption at 496 nm, was determined to be approximately 169 ppm. For Sample 4, the HPLC- DAD chromatogram reporting absorbance at 450 nm is shown in FIG. 13B with individual peaks identified in Table 17. As expected for a microbial oil from R. loruloides. the microbial oil was identified as comprising both torularhodin and torulene, as well as other unidentified carotenoids some of which may correspond to derivatives of these carotenoids. The sample also contained P-carotene and derivatives thereof. Table 17: Sample 4, HPLC peak identification.

[220] Sample s. The overall UV/Vis absorbance spectrum for Sample 5, acid-free extracted microbial oil, is shown in FIG. 14A with the absorbance at individual wavelengths identified in Table 18. The overall UV/Vis spectrum shows a peak around 475 nm. The total carotenoid content, roughly estimated using the absorbance at 496 nm, was determined to be approximately 471 ppm.

Table 18: Sample 5, UV/Vis Abs at specific wavelengths. [221] For Sample 5, the HPLC-DAD chromatogram reporting absorbance at 450 nm is shown in FIG. 14B with individual peaks identified in Table 19. As with sample 4, this sample contained torulene, possible derivatives of torulene, P-carotene and P-carotene derivatives.

Table 19: Sample 5, HPLC peak identification.

[222] Sample 6. The overall UV/Vis absorbance spectrum for Sample 6, acid-free extracted microbial oil, is shown in FIG. 15A with the absorbance at individual wavelengths identified in Table 20. The overall UV/Vis spectrum shows a peak around 475 nm. The total carotenoid content, roughly estimated using the absorbance at 496 nm, was determined to be approximately 802 ppm.

Table 20: Sample 6, UV/Vis Abs at specific wavelengths.

[223] For Sample 6, the HPLC-DAD chromatogram reporting absorbance at 450 nm is shown in FIG. 15B with individual peaks identified in Table 21. As with samples 4 and 5, this sample contained torulene, possible derivatives of torulene, P-carotene and P-carotene derivatives.

Table 21: Sample 6, HPLC peak identification.

[224] Overall, these results demonstrate that exemplary microbial oils of the disclosure comprise torulenes and/or torulorhodins, as well as P-carotene and derivatives thereof. This is in contrast to agricultural palm oil, which contains predominantly a- and P-carotenes and derivatives thereof. EXAMPLE 8: Materials and methods for fractionation and fraction analysis.

Microbial oil production

[225] Crude microbial oil was obtained from culturing an oleaginous microorganism, e.g., R. toruloides. See, e.g., WO2021154863A1.

Fractionation Method

[226] Crude or RBD microbial oil was melted by placing the storage container in a water bath at 50°C. Once the sample was fully melted, it was place in a jacketed vessel with agitation. For solvent fractionation, the desired solvent was then added in sufficient quantity to obtain the desired solvent to oil ratio. The agitation was set to the desired mixing rate and the chiller was set to 25°C. The temperature was controlled by a heater/chiller (USA Lab Recirculating Heater Chiller RHC-7L) that flowed a solution of glycol into the jacket of the vessel. Internal temperature in the liquid was measured using a type J stainless steel thermocouple. Once the system was at 25 °C, it was held at that temperature for 30 min. Then the chiller was set to the desired temperature for crystallization. Once that temperature was reached, the system was held at that temperature for the desired duration.

[227] A jacketed filter was used for solid-liquid separation. The filter temperature was also controlled by a heater/chiller (USA Lab Recirculating Heater Chiller RHC-7L) that flowed a solution of glycol into the jacket of the filter. The filter temperature was set to the desired crystallization temperature and the filter was allowed to stabilize at that temperature. A 1 pm filter was placed in the filter. A vacuum of less than 50 torr was applied to the filtration system which facilitated the separation. At the end of crystallization, the mixture of crystals and liquid was poured into the filter, and the liquid was allowed through the filter paper, while the solids were retained. At the end of the separation, the solid fraction was collected from the filter paper and placed in a separate container.

Triglyceride quantification method

[228] The method to measure the content of triglycerides (TAGs) in oil was developed using the AOCS Official Method Ce 5-86 and the first international collaborative study to standardize the method for different type of oils as reference with few modifications. See “Triglycerides by Gas Chromatography,” AOCS Official Method Ce 5-86, revised 2017; and Pocklington and Hautfenne, “Determination of triglycerides in fats and oils: results of a collaborative study and the standardised method,” Pure andapplied chemistry 1985;57(10): 1515-22.

[229] Briefly, TAG profiles in different samples of oil were estimated by first weighing out 10 mg of each oil to be analyzed. All the oil samples were dissolved in 1 mL of chloroform and vortexed to guarantee a completely homogenized solution. Oil samples were then set at room temperature on an Agilent autosampler (7693 A model) to be injected in a GC 8890 model coupled to an Agilent® 5975 mass selective detector, equipped with a split/splitless injector (split ratio 1 : 100) and set at a working temperature of 350°C. The system used a CP-TAP CB capillary column 25 m long with an internal diameter (i.d) =0.25 mm (Agilent technologies, Santa Clara, CA) to efficiently separate TAGs base on their total carbon number (CN) and unsaturation levels. The experimental chromatography conditions were as follow: the initial oven temperature of 300°C was held for 1 minute and raised to 355°C at a rate of l°C/min and then held at this temperature for 14 minutes for a total run time of 70 minutes. Helium was used as a carrier gas and the system was delivering a pressure at the top of the column of 23.234 psi and a flow of 1.3 mL/min.

[230] TAG analysis was performed by calculating the correction factor (F) that corrects for losses of TAGs during injection and column separation. F was estimated following the indications described in the AOCS Official Method Ce 5-86. Peaks representing individual TAGs of interest were manually integrated using MassHunter Qualitative Analysis software (Agilent Technologies, Santa Clara, CA). Each integrated peak was then corrected applying the corresponding correction factor and the individual TAG abundance was expressed as percentage of the total area of all TAGs present in the chromatogram.

Fatty Acid Methyl Ester (FAME) Quantification Method

[231] The method employed herein was modified from the procedure described by Van Wychen et al, used to measure free fatty acids in oil extracted from algae. See Van Wychen et al., “Determination of total lipids as fatty acid methyl esters (FAME) by in situ transesterification: laboratory analytical procedure (LAP),” (2016) National Renewable Energy Lab (NREL), Golden, CO (United States). Briefly, oil extracted from yeast was dissolved in hexane at 20 mg/mL. The dissolved oil was then diluted further twenty times in hexane containing Tridecanoic acid (C13:0) as internal standard (ISTD) at 200 pg/mL. The oil samples (100 pL) were esterified in 2 mL glass vials at 85°C for 1 h. At the end of the esterification reaction, samples were extracted with 500 pL of hexane containing ISTD to collect all the products of the FAME reaction. The samples were set at room temperature for 20 minutes to promote separation of the phases and 200 pL of the top phase were transferred to the GC vial for injection into an Agilent GC 7890B model equipped with a Flame Ionization Detector (FID). The FAME samples were injected with the PAL 3 Sampler Robot (Model Pal RSI 120 from CTC Analytics, Switzerland) into a split/splitless injector at 250°C connected to a DB-FAST FAME capillary column 20 m long with an internal diameter (i.d) =0.2 pm (Agilent Technologies, Santa Clara, CA). The chromatographic conditions were as follow: the initial oven temperature of 50°C was held for 0.5 minute and raised to 194°C at a rate of 30°C/min and then held at this temperature for 3.5 minutes. This was followed by a further increase to 240°C at a rate of 5°C/min and held for 1 minute for a total run time of 19 minutes. The system used helium as carrier gas and delivered a pressure at the top of the column of 20 psi and a flow of 0.72476 mL/min.

[232] FAME quantification analysis was performed using MassHunter Quantitative Analysis Software (Agilent Technologies®, USA) that allowed automatic peak integration, accelerating the quantification process of the FAME data collected for each sample.

[233] To calculate the average desaturation level by weight, the percent weight of each fatty acid species was multiplied by the number of double bonds in its aliphatic chain. E.g., given a microbial oil with 90% w/w Cl 6:0 and 10% w/w Cl 8:3, the average desaturation level would be calculated as follows: (0.9 percent weight x 0 double bonds) + (0.1 percent weight x 3 double bonds) = 0.3.

Melting Point Determination by Differential Scanning Calorimetry

[234] The method to measure the melting point of an oil on the Differential Scanning Calorimeter (DSC) was developed based on the capillary melting point method (AOCS Cc 1- 25) used by accredited labs. See also Nassu and Goncalves, “Determination of melting point of vegetable oils and fats by differential scanning calorimetry (DSC) technique,” Grasas y Aceites, 1999; 50: 16-22. The method was optimized such that the melting point as determined via DSC came within 2°C of a capillary melting point measured independently.

[235] Briefly, oil samples were heated in a water bath at 40-50°C until fully liquid before obtaining a sample. Using a spatula, 2-10 mg of oil were placed in a 40 pL aluminum pan and hermetically sealed and run against air (empty pan) as reference on a Mettler Toledo DSC 3 STARe System. Nitrogen was used as both the purge gas held at 150 mbar and method gas of 50 mL/min. The instrument calibration was performed with indium and zinc. For melting point calculation, the oil sample was initially held at 80°C for 3 min to remove any previous crystalline structure, cooled at 5°C/min to -80°C, held at this temperature for 5 min to fully crystallize, and finally heated from -80°C to 80°C at a heating rate of 10°C/min.

[236] The resulting DSC data was collected and processed by the STARe Software (V16.30). The final plateau on the DSC curve corresponds to the sample being completely liquified. The Evaluation Window within the STARe Software was used to determine the end set of the last occurring melting peak via computer-generated tangent lines. This value is what is reported as the melting point of the oil sample. EXAMPLE 9: Illustrative solvent-based fractionations of microbial oils of the disclosure.

Materials & Methods

[237] For solvent-based fractionation, the following ranges were explored for each parameter, also summarized in Table 22.

[238] Starting material: crude microbial oil and RBD microbial oil

[239] Solvent: heptane, acetone

[240] Solvent to oil ratio: 1 :1 - 6: 1

[241] Crystallization Temperature: Heptane, -15°C to 5°C; Acetone, 5°C to 12°C

[242] Crystallization Time: 1 hour - 4 days

[243] Fractionation, FAME analysis, and melting point determination were carried out according to Example 8.

Results

[244] The microbial oil fractions obtained from these illustrative solvent-based fractionation conditions differed significantly from the original microbial oil. Table 22 provides process conditions and melting points for the solid fractions resulting from these conditions. FAME and DSC chromatograms for representative solid and liquid fractions are shown in FIG. 16A- 27B. Table 23 provides the fatty acid profiles of the solid fractions in comparison to the original crude and RBD microbial oils. Table 24 features fatty acid profiles of illustrative solid and liquid fractions from the same fractionation conditions.

Table 22: Process conditions for solvent fractionation and resulting solid fraction melting point

Table 23: FAME profile for solid fractions resulting from different solvent conditions.

Table 24: Comparison of solid and liquid fractions from the same solvent fractionation conditions.

[245] Solid fractions obtained via the solvent-based fractionation conditions described above exhibited melting points that were double or more than the starting material. Solid fractions also exhibited a FAME profile with a significant increase in saturation levels. Finally, Table 24 demonstrates significant differences in the fatty acid compositions between solid and liquid fractions, and as compared to the original samples.

EXAMPLE 10: Illustrative dry fractionations of microbial oils of the disclosure

Materials & Methods

[246] For dry fractionation, the following ranges were explored for each parameter, also summarized in Table 25.

[247] Starting material: RBD oil

[248] Temperature: 10°C to 16 °C

[249] Crystallization Time: 1 hour - 24 hours

[250] Fractionation, FAME analysis, and melting point determination were carried out according to Example 8.

Results

[251] The microbial oil fractions resulting from these illustrative dry fractionation conditions differed in significant ways from the original RBD microbial oil. Table 25 provides process conditions and melting points for the solid fractions resulting from these conditions. Table 26 provides fatty acid profiles for the resulting solid fractions in comparison to the original RBD microbial oil, and the crude oil from which it was derived. Table 27 provides an overall summary of the TAG profiles for the solid fractions from three of these conditions in comparison to two representative crude microbial oils. Table 27 characterizes the TAGs based on the saturation state of their three fatty acids; e.g., three saturated fatty acids = “SSS”, two saturated and one unsaturated = “SSU”, etc. Table 28 shows a more detailed breakdown of the TAG profile for the samples included in Table 27.

Table 25: Process conditions and melting point for dry conditions

Table 26: FAME profile for dry conditions

Table 27: TAG saturation profile in solid fractions compared to crude samples.

Table 28: TAG Profiles of solid fractions obtained from different dry fractionation conditions.

EXAMPLE 11: Illustrative double solvent-based fractionation of a microbial oil of the disclosure.

Materials & Methods

[252] A microbial oil was fractionated using solvent-based fractionation, e.g., as disclosed in the Examples supra. The solvent was acetone. For the first round of fractionation, the solvent to oil ratio was 4:1. In the second round of fractionation, the solid fraction from the first round was fractionated again, with a 6: 1 solvent to oil ratio. In both rounds, the crystallization temperature was 6°C, and the crystallization time was 1 hr.

Results

[253] FIG. 33A-33B show the DSC curves from the solid and liquid fractions from the second round of fractionation in comparison to the curve for the original microbial oil. These figures demonstrate a significant upward shift in melting point for the solid fraction compared to the original oil. In addition, these chromatograms demonstrate a simplification of the peak structure in the DSC curve for the solid fraction, signifying the distillation of a more purified saturated/mono-unsaturated fatty acid composition within the solid fraction compared to the original oil sample. Table 29 shows the TAG saturation profile of the original oil compared to the liquid and solid fractions from the second fractionation, as well as the percent change in composition between the solid fraction compared to the original oil.

Table 29: TAG saturation in original oil and liquid and solid fractions.

INCORPORATION BY REFERENCE

[254] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not, be taken as an acknowledgement or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. The following international PCT publications are incorporated herein by reference in their entireties for all purposes: International Patent Application Nos. WO2018227184A1, WO2021154863A1, WO2021163194A1, W02022104046A1, and WO2022104063 A2.

NUMBERED EMBODIMENTS OF THE INVENTION

[255] Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments:

1. A microbial fat obtained from an oleaginous yeast, wherein the microbial fat comprises the following amounts of fatty acids relative to the total fatty acids: a) at least about 35% w/w saturated fatty acids; and b) less than about 15% w/w total polyunsaturated fatty acids.

2. A microbial fat obtained from an oleaginous yeast, wherein the microbial fat comprises an average desaturation level of less than about 0.72.

3. The microbial fat of any one of embodiments 1-2, wherein the microbial fat comprises an average fatty acid desaturation level of less than about 0.7.

4. The microbial fat of any one of embodiments 1-3, wherein the microbial fat comprises an average fatty acid desaturation level of less than about 0.5.

5. The microbial fat of any one of embodiments 1-4, wherein the microbial fat comprises an average fatty acid desaturation level of between about 0.1 and 0.72.

6. The microbial fat of any one of embodiments 1-5, wherein the microbial fat comprises between about 35% and about 85% w/w saturated fatty acids.

7. The microbial fat of any one of embodiments 1 -6, wherein the microbial fat comprises at least about 35% w/w saturated fatty acids with chain lengths between 16 and 18 carbons long.

8. The microbial fat of any one of embodiments 1-7, wherein the microbial fat comprises between about 35% and about 85% w/w saturated fatty acids with chain lengths between 16 and 18 carbons long.

9. The microbial fat of any one of embodiments 1-8, wherein the microbial fat comprises between about 5% and about 20% w/w saturated Cl 8 fatty acid.

10. The microbial fat of any one of embodiments 1-9, wherein the microbial fat comprises between about 3% and about 15% w/w total polyunsaturated fatty acids.

11. The microbial fat of any one of embodiments 1-10, wherein the microbial fat has a melting point of at least about 23 °C. The microbial fat of any one of embodiments 1-11, wherein the microbial fat has a melting point of between about 23°C and about 75°C. The microbial fat of any one of embodiments 1-12, wherein the microbial fat comprises, as a percentage of overall triacylglycerols (TAGs), less than about 10% w/w TAGs with three unsaturated fatty acids. The microbial fat of any one of embodiments 1-13, wherein the microbial fat comprises, as a percentage of overall TAGs, about 1% to about 10% w/w TAGs with three unsaturated fatty acids. The microbial fat of any one of embodiments 1-14, wherein the microbial fat comprises, as a percentage of overall TAGs, less than about 40% w/w TAGs with one saturated fatty acid and two unsaturated fatty acids. The microbial fat of any one of embodiments 1-15, wherein the microbial fat comprises, as a percentage of overall TAGs, between about 20% and about 40% w/w TAGs with one saturated fatty acid and two unsaturated fatty acids. The microbial fat of any one of embodiments 1-16, wherein the microbial fat comprises, as a percentage of overall TAGs, at least about 40% w/w TAGs with two saturated fatty acids and one unsaturated fatty acids. The microbial fat of any one of embodiments 1-17, wherein the microbial fat comprises, as a percentage of overall TAGs, between about 40% and about 80% w/w TAGs with two saturated fatty acids and one unsaturated fatty acid. The microbial fat of any one of embodiments 1-18, wherein the microbial fat comprises, as a percentage of overall TAGs, at least about 25% w/w palmitic-oleic-palmitic TAGs. The microbial fat of any one of embodiments 1-19, wherein the microbial fat comprises, as a percentage of overall TAGs, at least about 0.3% w/w TAGs with three saturated fatty acids. The microbial fat of any one of embodiments 1-20, wherein the microbial fat comprises, as a percentage of overall TAGs, at least about 1% w/w TAGs with three saturated fatty acids. A microbial oil obtained from an oleaginous yeast, wherein the microbial oil comprises the following amounts of fatty acids relative to the total fatty acids: a) less than about 40% w/w total saturated fatty acids; and b) at least about 10% w/w total polyunsaturated fatty acids.

23. The microbial oil of embodiment 22, wherein the microbial oil comprises between about 15% and about 40% w/w total saturated fatty acids.

24. The microbial oil of embodiment 22 or 23, wherein the microbial oil comprises less than about 10% w/w saturated Cl 8 fatty acid.

25. The microbial oil of any one of embodiments 22-24, wherein the microbial oil comprises less than about 30% w/w saturated Cl 6 fatty acid.

26. The microbial oil of any one of embodiments 22-25, wherein the microbial oil comprises between about 10% and about 20% w/w total polyunsaturated fatty acids.

27. The microbial oil of any one of embodiments 22-26, wherein the microbial oil has a melting point of less than about 25°C.

28. The microbial oil of any one of embodiments 22-27, wherein the microbial oil has a melting point below room temperature.

29. The microbial oil of any one of embodiments 22-28, wherein the microbial oil has a melting point of between about 25°C and about 10°C.

30. The microbial fat or the microbial oil of any one of embodiments 1-29, which comprises ergosterol and does not comprise campesterol, P-sitosterol, or stigmasterol.

31. The microbial fat or the microbial oil of any one of embodiments 1-30, which does not comprise, comprises less than 50 ppm, or comprises less than 100 ppm of a sterol selected from a phytosterol, cholesterol, and a protothecasterol.

32. The microbial fat or the microbial oil of any one of embodiments 1-31, which does not comprise chlorophyll.

33. The microbial fat or the microbial oil of any one of embodiments 1-32, which comprises a pigment selected from the group consisting of carotene, torulene and torulorhodin.

34. The microbial fat or the microbial oil of any one of embodiments 1-33, which comprises each of carotene, torulene and torulorhodin.

35. The microbial fat or the microbial oil of any one of embodiments 1-34, wherein the oleaginous yeast is a recombinant yeast.

36. The microbial fat or the microbial oil of any one of embodiments 1-35, wherein the oleaginous yeast is of the genus Yarrowia, Candida, Rhodotorula, Rhodosporidium, Metschnikowia, Cryptococcus, Trichosporon, or Lipomyces. 37. The microbial fat or the microbial oil of any one of embodiments 1-36, wherein the oleaginous yeast is of the genus Rhodosporidium.

38. The microbial fat or the microbial oil of any one of embodiments 1-37, wherein the oleaginous yeast is of the species Rhodosporidium toruloides.

39. The microbial fat or the microbial oil of any one of embodiments 1-38, which is obtained by a fractionation method.

40. The microbial fat or the microbial oil of any one of embodiments 1-39, which is obtained by a fractionation method performed on a refined, bleached, and/or deodorized microbial oil.

41. The microbial fat or the microbial oil of any one of embodiments 1-40, which is obtained by a fractionation method performed on a crude microbial oil.

42. The microbial fat or the microbial oil of any one of embodiments 1-41, which is obtained by a fractionation method performed on a microbial palm oil alternative, wherein the microbial palm oil alternative has one or more characteristics similar to plant-derived palm oil selected from the group consisting of: apparent density, refractive index, saponification value, unsaponifiable matter, iodine value, slip melting point, fatty acid composition, triglyceride content, overall saturation level, and level of mono- and poly-unsaturated fatty acids.

43. The microbial fat or the microbial oil of any one of embodiments 1-42, which is obtained by a fractionation method performed on a microbial palm oil alternative, wherein the microbial palm oil alternative has one or more characteristics similar to plant-derived palm oil selected from the group consisting of: a saponification value of 150-210, an iodine value of 50-65, a slip melting point of 30°C-40°C, a saturated fatty acid composition of 30-70%, an unsaturated fatty acid composition of 30-70%, 30-50% mono-unsaturated fatty acids as a percentage of overall fatty acids, 5-25% poly-unsaturated fatty acids as a percentage of overall fatty acids, and a triglyceride content of 90-98% as a percentage of overall glycerides.

44. The microbial fat or the microbial oil of any one of embodiments 1-43, which is obtained by a fractionation method performed on a balanced microbial oil, wherein the balanced microbial oil comprises the following amounts of fatty acids relative to the total fatty acids: a) at least about 30% w/w saturated fatty acids with chain lengths between 16 and 18 carbons long; b) at least about 30% w/w unsaturated fatty acids with 18 carbon chain lengths; and c) less than about 30% w/w total polyunsaturated fatty acids.

45. The microbial fat or the microbial oil of any one of embodiments 1-44, which is obtained by serial fractionation.

46. The microbial fat or the microbial oil of any one of embodiments 1-45, which is obtained by a dry fractionation method.

47. The microbial fat or the microbial oil of any one of embodiments 1 -46, which is obtained by a solvent-based fractionation method.

48. The microbial fat or the microbial oil of any one of embodiments 1-47, which is obtained by a solvent-based fractionation method, wherein the solvent is acetone, hexane, or heptane.

49. The microbial fat of any one of embodiments 1-21 and 30-48, wherein the microbial fat is obtained by a fractionation method performed on a microbial oil, and wherein the method comprises the steps of: a) melting the microbial oil, b) crystallizing the melted microbial oil, and c) separating the liquid and solid phases of the crystallized microbial oil, wherein the microbial fat is the solid phase of step (c).

50. The microbial oil of any one of embodiments 22-48, wherein the microbial oil is a second microbial oil obtained by a fractionation method performed on a first microbial oil, and wherein the method comprises the steps of: a) melting the first microbial oil, b) crystallizing the melted first microbial oil, and c) separating the liquid and solid phases of the crystallized first microbial oil, wherein the second microbial oil is the liquid phase of step (c).

51. A method of fractionating a first microbial oil to obtain a microbial fat and a second microbial oil, wherein the method comprises: a) melting the first microbial oil; b) crystallizing the melted first microbial oil; and c) separating solid and liquid phases of the crystallized first microbial oil wherein the solid phase of step (c) is the microbial fat and the liquid phase of step (c) is the second microbial oil.

52. The method of embodiment 51 , wherein the first microbial oil is a refined, bleached, and/or deodorized microbial oil.

53. The method of any one of embodiments 51-52, wherein the first microbial oil is a crude microbial oil.

54. The method of any one of embodiments 51-53, wherein the first microbial oil is the product of a fractionation process.

55. The method of any one of embodiments 51 -54, wherein the first microbial oil is a microbial palm oil alternative, wherein the microbial palm oil alternative has one or more characteristics similar to plant-derived palm oil selected from the group consisting of: apparent density, refractive index, saponification value, unsaponifiable matter, iodine value, slip melting point, fatty acid composition, triglyceride content, overall saturation level, and level of mono- and poly-unsaturated fatty acids.

56. The method of any one of embodiments 51-55, wherein the first microbial oil is a microbial palm oil alternative, wherein the microbial palm oil alternative has one or more characteristics similar to plant-derived palm oil selected from the group consisting of: a saponification value of 150-210, an iodine value of 50-65, a slip melting point of 30°C- 40°C, a saturated fatty acid composition of 30-70%, an unsaturated fatty acid composition of 30-70%, 30-50% mono-unsaturated fatty acids as a percentage of overall fatty acids, 5- 25% poly-unsaturated fatty acids as a percentage of overall fatty acids, and a triglyceride content of 90-98% as a percentage of overall glycerides.

57. The method of any one of embodiments 51-56, wherein the first microbial oil is a balanced microbial oil, wherein the balanced microbial oil comprises the following amounts of fatty acids relative to the total fatty acids: a) at least about 30% w/w saturated fatty acids; b) at least about 30% w/w unsaturated fatty acids; and c) less than about 30% w/w total polyunsaturated fatty acids. 58. The method of any one of embodiments 51-57, wherein the microbial fat and/or the second microbial oil differ in one or more parameters from the first microbial oil.

59. The method of any one of embodiments 51-58, wherein the microbial fat and/or the second microbial oil differ in a parameter from the first microbial oil, and wherein the parameter is selected from the list consisting of: appearance, opacity, texture, consistency, melting point, saturation, fatty acid composition, TAG composition, emulsifying ability, hardness, spreadability, viscosity, brittleness, plasticity, and stickiness.

60. The method of any one of embodiments 51-59, wherein the method does not comprise the use of solvent.

61. The method of any one of embodiments 51-60, wherein the method does not comprise the use of solvent, and wherein the resulting microbial fat and second microbial oil are solvent- free or contain an undetectable level of solvent.

62. The method of any one of embodiments 51-61, wherein the method comprises the use of a solvent.

63. The method of any one of embodiments 51-62, wherein the method comprises the use of a solvent, and wherein the solvent is acetone, hexane, or heptane.

64. The method of any one of embodiments 51-63, wherein the method comprises the use of a solvent, and wherein the solvent is added to the melted original microbial oil prior to crystallization.

65. The method of any one of embodiments 51 -64, wherein step (a) comprises melting the first microbial oil at a temperature of between about 30°C and about 70°C until the first microbial oil is fully melted.

66. The method of any one of embodiments 51-65, wherein step (b) comprises lowering the temperature of the first microbial oil to a crystallization temperature of between about - 20°C and about 15°C.

67. The method of any one of embodiments 51-66, wherein step (b) comprises lowering the temperature of the first microbial oil to a crystallization temperature and then maintaining the first microbial oil at the crystallization temperature for between about 30 minutes and 7 days.

68. The method of any one of embodiments 51-67, wherein the microbial fat and the second microbial oil each comprise at least 10% of the first microbial oil’s original mass 69. The method of any one of embodiments 51-68, wherein the iodine value (IV) of the microbial fat and the IV of the second microbial oil differ by at least 10.

70. The method of any one of embodiments 51-69, wherein the IV of the microbial fat and the IV of the second microbial oil differ by at least 20.

71. The method of any one of embodiments 51-70, wherein the IV of the microbial fat and the IV of the second microbial oil differ by at least 30.

72. The method of any one of embodiments 51-71, wherein the microbial fat comprises an average desaturation level of less than about 0.72.

73. The method of any one of embodiments 51-72, wherein the microbial fat comprises an average fatty acid desaturation level of less than about 0.7.

74. The method of any one of embodiments 51-73, wherein the microbial fat comprises an average fatty acid desaturation level of less than about 0.5.

75. The method of any one of embodiments 51-74, wherein the microbial fat comprises an average fatty acid desaturation level of between about 0.1 and 0.72.

76. The method of any one of embodiments 51-75, wherein the microbial fat comprises the following amounts of fatty acids relative to the total fatty acids: a) at least about 35% w/w saturated fatty acids; and b) less than about 15% w/w total polyunsaturated fatty acids.

77. The method of any one of embodiments 51-76, wherein the microbial fat comprises between about 35% and about 85% w/w saturated fatty acids.

78. The method of any one of embodiments 51 -77, wherein the microbial fat comprises at least about 35% w/w saturated fatty acids with chain lengths between 16 and 18 carbons long.

79. The method of any one of embodiments 51-78, wherein the microbial fat comprises between about 35% and about 85% w/w saturated fatty acids with chain lengths between 16 and 18 carbons long.

80. The method of any one of embodiments 51-79, wherein the microbial fat comprises at least about 5% w/w saturated Cl 8 fatty acid.

81. The method of any one of embodiments 51-80, wherein the microbial fat comprises between about 5% and about 20% w/w saturated Cl 8 fatty acid.

82. The method of any one of embodiments 51-81, wherein the microbial fat comprises between about 3% and about 15% w/w total polyunsaturated fatty acids. 83. The method of any one of embodiments 51-82, wherein the microbial fat has a melting point of at least about 23 °C.

84. The method of any one of embodiments 51-83, wherein the microbial fat has a melting point of between about 23°C and about 75°C.

85. The method of any one of embodiments 51-84, wherein the microbial fat comprises, as a percentage of overall triacylglycerols (TAGs), less than about 10% w/w TAGs with three unsaturated fatty acids.

86. The method of any one of embodiments 51-85, wherein the microbial fat comprises, as a percentage of overall TAGs, about 1% to about 10% w/w TAGs with three unsaturated fatty acids.

87. The method of any one of embodiments 51-86, wherein the microbial fat comprises, as a percentage of overall TAGs, less than about 40% w/w TAGs with one saturated fatty acid and two unsaturated fatty acids.

88. The method of any one of embodiments 51-87, wherein the microbial fat comprises, as a percentage of overall TAGs, between about 20% and about 40% w/w TAGs with one saturated fatty acid and two unsaturated fatty acids.

89. The method of any one of embodiments 51-88, wherein the microbial fat comprises, as a percentage of overall TAGs, at least about 40% w/w TAGs with two saturated fatty acids and one unsaturated fatty acids.

90. The method of any one of embodiments 51-89, wherein the microbial fat comprises, as a percentage of overall TAGs, between about 40% and about 80% w/w TAGs with two saturated fatty acids and one unsaturated fatty acid.

91. The method of any one of embodiments 51-90, wherein the microbial fat comprises, as a percentage of overall TAGs, at least about 25% w/w palmitic-oleic-palmitic TAGs.

92. The method of any one of embodiments 51-91, wherein the microbial fat comprises, as a percentage of overall TAGs, at least about 0.3% w/w TAGs with three saturated fatty acids.

93. The method of any one of embodiments 51-92, wherein the microbial fat comprises, as a percentage of overall TAGs, at least about 1% w/w TAGs with three saturated fatty acids.

94. The method of any one of embodiments 51 -93, wherein the second microbial oil comprises the following amounts of fatty acids relative to the total fatty acids: a) less than about 40% w/w total saturated fatty acids; and b) at least about 10% w/w total polyunsaturated fatty acids.

95. The method of any one of embodiments 51 -94, wherein the second microbial oil comprises between about 15% and about 40% w/w total saturated fatty acids.

96. The method of any one of embodiments 51 -95, wherein the second microbial oil comprises less than about 10% w/w saturated Cl 8 fatty acid.

97. The method of any one of embodiments 51 -96, wherein the second microbial oil comprises less than about 30% w/w saturated Cl 6 fatty acid.

98. The method of any one of embodiments 51 -97, wherein the second microbial oil comprises between about 10% and about 20% w/w total polyunsaturated fatty acids.

99. The method of any one of embodiments 51-98, wherein the second microbial oil has a melting point of less than about 25°C.

100. The method of any one of embodiments 51-99, wherein the second microbial oil has a melting point below room temperature.

101. The method of any one of embodiments 51-100, wherein the second microbial oil has a melting point of between about 25°C and about 10°C.

102. The method of any one of embodiments 51-101, wherein the first microbial oil, second microbial oil, and/or microbial fat comprises ergosterol and does not comprise campesterol, P-sitosterol, or stigmasterol.

103. The method of any one of embodiments 51-102, wherein the first microbial oil, second microbial oil, and/or microbial fat does not comprise, comprises less than 50 ppm, or comprises less than 100 ppm of a sterol selected from a phytosterol, cholesterol, and a protothecasterol.

104. The method of any one of embodiments 51-103, wherein the first microbial oil, second microbial oil, and/or microbial fat does not comprise chlorophyll.

105. The method of any one of embodiments 51-104, wherein the first microbial oil, second microbial oil, and/or microbial fat comprises a pigment selected from the group consisting of carotene, torulene and torulorhodin.

106. The method of any one of embodiments 51-105, wherein the first microbial oil, second microbial oil, and/or microbial fat comprises each of carotene, torulene and torulorhodin.

107. The method of any one of embodiments 51-106, wherein the oleaginous yeast is a recombinant yeast. 108. The method of any one of embodiments 51-107, wherein the oleaginous yeast is of the genus Yarrowia, Candida, Rhodotorula, Rhodosporidium, Metschnikowia, Cryptococcus, Trichosporon, or Lipomyces.

109. The method of any one of embodiments 51-108, wherein the oleaginous yeast is of the genus Rhodosporidium.

110. The method of any one of embodiments 51-109, wherein the oleaginous yeast is of the species Rhodosporidium toruloides.

111. The method of any one of embodiments 51-110, wherein the yield of the microbial fat is at least about 1% w/w. 112. The method of any one of embodiments 51-111, wherein the yield of the microbial fat is at least about 5% w/w.

113. The method of any one of embodiments 51-112, wherein the yield of the microbial fat is at least about 10% w/w.