IN THE UNITED STATES PATENT & TRADEMARK RECEIVING OFFICE

DERIVATIVES OF MICROBIALLY PRODUCED PALM OIL

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims the benefit of U.S. Provisional Application No. 63/340,875 filed on May 11, 2022, which is hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE DISCLOSURE

[2] The present disclosure relates to environmentally friendly and sustainable alternatives to plant-derived palm oil derivatives. The palm oil alternatives are produced by oleaginous microorganisms and share one or more features with plant-derived palm oils. These alternatives are then fractionated, treated, and/or derivatized into oleochemicals and can be used as a substitute for any palm oil derivative.

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. Derivatives of palm oil (oleochemicals) are widely used in a number of everyday products, such as home care products (for example laundry detergent), personal care products (for example shampoo), cosmetics (for example lipstick), food products (for example nut butters, baked goods, ice cream) and fuel (such as biodiesel). 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 alternatives to palm oil derivatives that do not rely upon utilization of oil palms and incur the associated negative environmental costs.

BRIEF SUMMARY

[5] The present disclosure relates to microbial oil fractions, wherein the fraction is microbial stearin, microbial olein, microbial soft mid-fraction, microbial super olein, microbial hard mid-fraction, microbial olein, and/or microbial top olein, wherein the fraction comprises at least one pigment selected from the group consisting of carotene, torulene and torulorhodin and does not comprise chlorophyll. [6] The present disclosure relates to methods of producing derivatives from a microbial oil comprising obtaining a refined microbial oil, a bleached microbial oil, and/or a deodorized microbial oil, and modifying the oil to produce a derivative.

[7] The present disclosure relates to methods of producing derivatives from whole cell or lysed microbial biomass, extracting crude microbial oil from the whole cell or lysed microbial biomass, wherein said extraction process removes toxins and produces a microbial oil safe for human use, and modifying the microbial oil to produce a derivative. In some aspects, the modifying comprises fractionation, interesterification, transesterification, hydrogenation, steam hydrolysis, distillation, saponification, amination, ethyoxylation, sulfonation, oxidation, quaternization, or combinations thereof.

[8] The present disclosure further relates to derivatives of the microbial oils disclosed herein, and derivatives produced from the methods disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[9] 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.

[10] FIG. 1 shows a representative chromatogram of a triglyceride profile of an illustrative microbial oil of the disclosure showing the main TAG region.

[11] FIG. 2 shows a representative chromatogram of a mono- and diglyceride profile of an illustrative microbial oil of the disclosure showing the mono- and diglyceride regions.

[12] FIG. 3 shows images of illustrative crude, refined, refined and bleached, and RBD microbial oil samples of the disclosure processed with an antioxidant.

[13] FIG. 4 shows images of illustrative crude, refined, refined and bleached, and RBD microbial oil samples of the disclosure processed without an antioxidant.

[14] FIGS. 5A-5D show total ion chromatograms for four different oil samples: an exemplary R. toruloides microbial oil of the disclosure (FIG. 5A); algae oil (FIG. 5B); crude palm oil (FIG. 5C); and refined, bleached, and deodorized (RBD) palm oil (FIG. 5D).

[15] FIG. 6 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.

[16] FIGS. 7A-7E show the electron-ionization spectra for five different derivatized sterols spiked into crude palm oil: ergosterol-TMS (FIG. 7A); cholesterol-TMS (FIG. 7B); campesterol-TMS (FIG. 7C); sitosterol-TMS (FIG. 7D); and stigmasterol-TMS (FIG. 7E). [17] FIGS. 8A-8B show the results of a carotenoid analysis of agricultural palm oil. FIG. 8A shows the overall UV/Vis absorbance spectrum. FIG. 8B shows the HPLC-DAD chromatogram with absorbance at 450 nm.

[18] FIGS. 9A-9B show the results of a carotenoid analysis of a strong acid-extracted exemplary R toruloides microbial oil of the present disclosure. FIG. 9A shows the overall UV/Vis absorbance spectrum. FIG. 9B shows the HPLC-DAD chromatogram with absorbance at 450 nm.

[19] FIGS. 10A-10B show the results of a carotenoid analysis of a strong acid-extracted exemplary R toruloides microbial oil of the present disclosure. FIG. 10A shows the overall UV/Vis absorbance spectrum. FIG. 10B shows the HPLC-DAD chromatogram with absorbance at 450 nm.

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

[21] FIGS. 12A-12B show the results of a carotenoid analysis of an acid-free 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.

[22] FIGS. 13A-13B show the results of a carotenoid analysis of an acid-free 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.

[23] FIG. 14 is a flow diagram of fractions produced from microbial oil.

[24] FIG. 15A is a photograph of a fractionation of crude microbial oil (left) and crude palm oil (right). FIG. 15B is a photograph of a complete fractionation of crude microbial oil. FIG. 15C is a photograph of an incomplete fractionation of crude microbial oil.

[25] FIG. 16 is a bar graph showing gas chromatography-mass spectrometry (GCMS) data highlighting how the fractionation shifts the fatty acid profile in the olein and stearin layers.

[26] FIG. 17 is a bar graph of the data shown in FIG. 16 illustrating the overall balance of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA).

[27] FIG. 18A shows the results of fatty acid methyl ester (FAME) analysis of the solid fraction resulting from solvent-based fractionation condition 1 in Example 7. FIG. 18B shows a Differential Scanning Calorimeter (DSC) chromatogram for the solid fraction resulting from solvent-based fractionation condition 1 in Example 7.

[28] FIG. 19 shows the results of FAME analysis of the liquid fraction resulting from solvent-based fractionation condition 1 in Example 7.

[29] FIG. 20A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 2 in Example 7. FIG. 20B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 2 in Example 7.

[30] FIG. 21A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 3 in Example 7. FIG. 21B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 3 in Example 7.

[31] FIG. 22A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 4 in Example 7. FIG. 22B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 4 in Example 7.

[32] FIG. 23A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 5 in Example 7. FIG. 23B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 5 in Example 7.

[33] FIG. 24A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 6 in Example 7. FIG. 24B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 6 in Example 7.

[34] FIG. 25A shows the results of FAME analysis of the liquid fraction resulting from solvent-based fractionation condition 6 in Example 7. FIG. 25B shows a DSC chromatogram for the liquid fraction resulting from solvent-based fractionation condition 6 in Example 7.

[35] FIG. 26A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 7 in Example 7. FIG. 26B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 7 in Example 7.

[36] FIG. 27A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 8 in Example 7. FIG. 27B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 8 in Example 7.

[37] FIG. 28A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 9 in Example 7. FIG. 28B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 9 in Example 7.

[38] FIG. 29A shows the results of FAME analysis of the solid fraction resulting from solvent-based fractionation condition 10 in Example 7. FIG. 29B shows a DSC chromatogram for the solid fraction resulting from solvent-based fractionation condition 10 in Example 7. [39] FIG. 30A shows the results of FAME analysis of the solid fraction resulting from dry fractionation condition 1 in Example 8. FIG. 30B shows a DSC chromatogram for the solid fraction resulting from dry fractionation condition 1 in Example 8.

[40] FIG. 31A shows the results of TAG analysis of the solid fraction resulting from dry fractionation condition 2 in Example 8. FIG. 31B shows a DSC chromatogram for the solid fraction resulting from dry fractionation condition 2 in Example 8.

[41] FIG. 32A shows the results of TAG analysis of the solid fraction resulting from dry fractionation condition 3 in Example 8. FIG. 32B shows the results of FAME analysis of the solid fraction resulting from dry fractionation condition 3 in Example 8. FIG. 32C shows a DSC chromatogram for the solid fraction resulting from dry fractionation condition 3 in Example 8.

[42] FIG. 33A shows the results of TAG analysis of the solid fraction resulting from dry fractionation condition 4 in Example 8. FIG. 33B shows the results of FAME analysis of the solid fraction resulting from dry fractionation condition 4 in Example 8. FIG. 33C shows a DSC chromatogram for the solid fraction resulting from dry fractionation condition 4 in Example 8.

[43] FIG. 34A shows the results of FAME analysis of the solid fraction resulting from dry fractionation condition 5 in Example 8. FIG. 34B shows a DSC chromatogram for the solid fraction resulting from dry fractionation condition 5 in Example 8.

[44] FIG. 35A shows a DSC chromatogram for an original microbial oil and a liquid fraction and solid fraction derived therefrom. FIG. 35B shows the same three curves as in FIG. 35A but overlaid.

[45] FIG. 36 is a flow diagram illustrating examples of various methods of processing the microbial oil and the resulting derivatives (oleochemicals).

[46] FIG. 37 is a flow diagram illustrating an example of fatty ester production from microbial oil.

[47] FIG. 38 shows a bar graph of representative fatty acid compositions of microbial oil and palm oil.

[48] FIG. 39 is a flow diagram of fatty acids produced from microbial oil.

[49] FIG. 40 is a flow diagram of fatty alcohols produced from microbial oil.

DETAILED DESCRIPTION

[50] 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

[51] 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.

[52] 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.

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

[54] 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.

[55] 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.

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

[57] “Fatty acid-ingredient” as used herein refers to an ingredient safe for human and/or animal use (i.e., in cosmetics, food, pharmaceuticals, etc.). [58] 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. The saturation levels of the fractions may be characterized by, e.g., their iodine value (IV). The IV of the fractions may differ 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.

[59] “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).

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

[61] “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.

[62] “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.

[63] “Oleochemical” as used herein refers to a chemical compound derived natural oils or fats, which could be from an animal, microbial, or plant source.

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

[65] “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.

[66] For the purposes of this disclosure, “single cell oils,” “microbial oils,” “lipid composition” and “oils” refer to microbial lipids produced by oleaginous microorganisms. [67] “ 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.

[68] “Triglyceride(s)” as used herein refers to a glycerol bound to three fatty acid molecules. They may be saturated or unsaturated. Unless the isomers are independently designated, denominations recited herein include isomers.

[69] “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.

[70] Unless otherwise noted, any terminology used herein referring to palm plant-based derivatives is intended to refer to the analogous derivative obtained from a microbial oil, e.g., the corresponding derivative derived from an oil obtained from an oleaginous yeast. For example, the term “ethyl palmate” as used herein refers to the microbial oil derivative that is analogous to the palm plant derivative known in the art by this term.

Overview

[71] The present disclosure relates to derivatives and methods of derivatization of microbial oils. These lipids may serve as palm oil alternatives and be processed, fractionated, and/or derivatized by any number of means known in the art, to produce oleochemicals such as, but not limited to, triglycerides, diglycerides, monoglycerides, free fatty acids, fatty acid salts, glycerin, fatty esters, fatty alcohols, fatty amines, fatty acid methyl esters, amide carboxylates, FOH ethoxylates, FOH sulfates, amine oxides, betaines, quats, and ether sulfates. These derivatives may be used in a variety of downstream products, such as household cleaners, personal care items, cosmetics, fuel, and feedstock, for example.

Oleaginous microorganisms

[72] 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.

[73] 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.

[74] 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 lipids 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.

[75] Suitable species of oleaginous yeast for producing the microbial lipids 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 brassicas, Trichosporon domesticum, Trichosporon laibachii, Trichosporon loubieri, Trichosporon loubieri, Trichosporon montevideense, Trichosporon pullulans, meyerae.

[76] 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.

[77] 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.

[78] In some embodiments, the oleaginous microorganisms that produce the microbial lipids 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 lipids 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 lipids of the present disclosure are a heterogeneous population comprising two or more distinct populations of microorganisms of different species.

[79] The oleaginous microorganisms that produce the microbial lipids used in the methods 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.

[80] 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 the starting microbial oil

[81] The present disclosure provides fractions, derivatives, and methods of processing to produce various derivates from microbial oils. These microbial oils may serve as palm oil alternatives and thus may be processed and/or derivatized by any number of means known in the art. In some embodiments, the fractions and/or derivatives comprise one or more fingerprints of the microbial oil.

Compositional analysis of an exemplary microbial oil

[82] Microbial oil was prepared using R. toruloides fermented on glycerol feed, lysed with acid, and extracted with heptane solvent. The composition of the oil is shown below in Table

Table 1: Oil batch analysis

Comprehensive analysis of an illustrative crude microbial oil sample

[83] Additional comprehensive analyses of exemplary crude microbial oil are shown below in Tables 2-5. 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.

[84] The three oil samples were analyzed along different physical and chemical parameters, the results of which analyses are shown in Table 2. The methods employed were those of the American Oil Chemists’ Society (AOCS) and are referenced within the Table by their AOCS identifier.

Table 2: General physical chemical characterization

[85] As shown in Table 2 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.

[86] Levels of contaminants were assessed in microbial oil, crude palm oil, and crude hybrid palm oil, with results shown in Table 3. The methods and equipment are shown in columns two and three, respectively.

Table 3: Contaminant levels

[87] 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.

Comprehensive analysis of triglyceride composition

[88] 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 4 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.

Table 4: Triglyceride composition

[89] 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-oleic-palmitic 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.

Unsaponifiable lipid content

[90] The unsaponifiable lipid content of the three microbial oil samples was analyzed, specifically measuring the amount of beta-carotene (data not shown), squalene, tocopherols, and sterols in each sample. Results are shown in Table 5. Beta-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 Johnson et al., “Sidechain 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. The other methods that were employed are indicated in Table 5. 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 5: Unsaponifiable lipid content

[91] As shown in Table 5, 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.

Characteristics unique to microbial oil

[92] In addition to the differences described above, production of microbial oil may be manipulated for the improved production of a given product, or derivative, 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 derivative. 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.

Microbial oil characteristics similar to plant-derived palm oil

[93] 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.

[94] 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 at least 10% C16 saturated fatty acid. In some embodiments, the microbial oil comprises at least 15% C16 saturated fatty acid. In some embodiments, the microbial oil comprises at least 20% C16 saturated fatty acid. In some embodiments, the microbial oil comprises at least 30% C16 saturated fatty acid. [95] In some embodiments, the microbial oil comprises 10-70% Cl 8 unsaturated fatty acid.

In some embodiments, the microbial oil comprises 35-80% Cl 8 unsaturated fatty acid. In some embodiments, the microbial oil comprises at least 20% Cl 8 unsaturated fatty acid. In some embodiments, the microbial oil comprises at least 25% Cl 8 unsaturated fatty acid. In some embodiments, the microbial oil comprises at least 30% C18 unsaturated fatty acid. In some embodiments, the microbial oil comprises at least 35% Cl 8 unsaturated fatty acid. In some embodiments, the microbial oil comprises at least 40% Cl 8 unsaturated fatty acid.

[96] 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 a saturated fatty acid composition of about 20-80% and an unsaturated fatty acid composition of about 20-80%. In some embodiments, the microbial oil has about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,

70%, 75%, 80%, or 85% saturated fatty acids. In some embodiments, the microbial oil has about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% unsaturated fatty acids.

[97] 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%, 60%, 65%, or 70% mono-unsaturated fatty acids. In some embodiments, the microbial oil comprises 30-70% mono-unsaturated fatty acids.

[98] 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. In some embodiments, the microbial oil comprises less than 25% poly-unsaturated fatty acids.

[99] 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 55-80. In some embodiments, the microbial oil has an iodine value of about 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, 75, 76, 77, 78, 79, or 80. In some embodiments, the microbial oil has an iodine value of 55-85. In some embodiments, the microbial oil has an iodine value of 62-76.

[100] Table 6 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 6. 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 6 for one or more parameters.

Table 6: Illustrative fatty acid compositions of microbial oil

[101] 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 10-50°C. In some embodiments, the microbial oil has a slip melting point of about 10, 15, 20, 25, 30, 35, 40, 45, or 50°C. In some embodiments, the microbial oil has a melting point of 10-30°C. In some embodiments, the microbial oil has a melting point of 16-25°C.

[102] 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.

[103] 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.

[104] 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.

[105] 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 a density of 0.8-1 g/cc. In some embodiments, the microbial oil has a density of 0.88-0.95 g/cc. In some embodiments, the microbial oil has a density of 0.9-0.92.

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

Triglyceride composition

[107] Tables 7 and 8 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 7, for example P-O-P, the corresponding measurements for that molecule 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 7 and Table 8. 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.

[108] 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 7: Illustrative triglyceride compositions of microbial oil

Table 8: Summary total triglyceride compositions [109] 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.

[HO] 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.

[Hl] In some embodiments, the microbial oil has a triglyceride profile wherein greater than 40% of the triglycerides have one unsaturated sidechain, and wherein greater than 30% of the triglycerides have two unsaturated sidechains.

[112] In some embodiments, between 1% and 30% of palmitic and/or stearic fatty acids comprised by the microbial oil are located at the sn-2 position of triglyceride molecules. In some embodiments, between 5% and 25% of palmitic and/or stearic fatty acids comprised by the microbial oil are located at the sn-2 position of triglyceride molecules. In some embodiments, between 10% and 15% of palmitic and/or stearic fatty acids comprised by the microbial oil are located at the sn-2 position of triglyceride molecules.

[113] In some embodiments, a microbial oil derivative herein is a microbial triglyceride. In some embodiments, a microbial oil derivative herein is the product of hydrolysis of a microbial triglyceride, e.g., a free fatty acid or glycerin.

[114] In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising less than 5% triglycerides with three saturated fatty acid chains. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising less than 2% triglycerides with three saturated fatty acid chains. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising less than 1% triglycerides with three saturated fatty acid chains. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising about 0.5% triglycerides with three saturated fatty acid chains.

[115] In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising 25%-60% triglycerides with two saturated fatty acid chains and one unsaturated fatty acid chain. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising 30%-55% triglycerides with two saturated fatty acid chains and one unsaturated fatty acid chain. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising 35%-50% triglycerides with two saturated fatty acid chains and one unsaturated fatty acid chain. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising about 40%-47% triglycerides with two saturated fatty acid chains and one unsaturated fatty acid chain.

[116] In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising 30%-65% triglycerides with one saturated fatty acid chain and two unsaturated fatty acid chains. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising 35%-55% triglycerides with one saturated fatty acid chain and two unsaturated fatty acid chains. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising 40%-50% triglycerides with one saturated fatty acid chain and two unsaturated fatty acid chains. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising about 43%-47% triglycerides with one saturated fatty acid chain and two unsaturated fatty acid chains.

[117] In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising less than 25% triglycerides with three unsaturated fatty acid chains. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising less than 20% triglycerides with three unsaturated fatty acid chains. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising less than 15% triglycerides with three unsaturated fatty acid chains. In some embodiments, a microbial oil of the disclosure has a TAG saturation profile comprising about 5%-l 5% triglycerides with three unsaturated fatty acid chains.

[118] In some embodiments, a microbial oil of the disclosure comprises 10-30% POP triglycerides. In some embodiments, a microbial oil of the disclosure comprises at least 10% POP triglycerides. In some embodiments, a microbial oil of the disclosure comprises 1-10% PLP triglycerides. In some embodiments, a microbial oil of the disclosure comprises 5-15% POS triglycerides. In some embodiments, a microbial oil of the disclosure comprises at least 5% POS triglycerides. In some embodiments, a microbial oil of the disclosure comprises 15- 40% POO triglycerides. In some embodiments, a microbial oil of the disclosure comprises 20- 35% POO triglycerides. In some embodiments, a microbial oil of the disclosure comprises at least 20% POO triglycerides. In some embodiments, a microbial oil of the disclosure comprises 3-15% PLO triglycerides. In some embodiments, a microbial oil of the disclosure comprises at least 3% PLO triglycerides. In some embodiments, a microbial oil of the disclosure comprises 5-10% PLO triglycerides. In some embodiments, a microbial oil of the disclosure comprises 2- 15% OSO triglycerides. In some embodiments, a microbial oil of the disclosure comprises at least 2% OSO triglycerides. In some embodiments, a microbial oil of the disclosure comprises 2.5-10% OSO triglycerides. In some embodiments, a microbial oil of the disclosure comprises 3-15% OOO triglycerides. In some embodiments, a microbial oil of the disclosure comprises at least 3% 000 triglycerides. In some embodiments, a microbial oil of the disclosure comprises 5-10% 000 triglycerides.

[119] In some embodiments, a microbial oil of the disclosure comprises less than 10% PPM, MOM, PPP, MOP, MLP, PPS, OMO, PLS, PLL, POLn, SOS, SLO, and/or OLO.

Oxidative stability

[120] The unsaturated lipids in vegetable oils are susceptible to oxidation over time, which can be accelerated when the oil is exposed to heat, light, or metals. Oxidation causes changes in the chemical, sensory, and nutritional properties of the oil, and can result in, among other things, an unpleasant odor.

[121] The oxidative stability of the microbial oil described herein was analyzed by detection of peroxide using methods known in the art, for example, by titration reaction of iodine and peroxide with a starch indicator. The peroxide value of the microbial oil was less than 2 mEq/kg, which is within the Malaysian Palm Oil Board (MPOB) specification.

Processing of microbial oil

[122] 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.

[123] In some embodiments, the microbial oil is refined. 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. [124] 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 deodorized.

[125] 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.

[126] 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.

[127] 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. [128] 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.

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

[130] 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.

Fractionation

[131] Fractionation is another means of processing the microbial oil described herein for use in compositions. 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.

[132] In some embodiments, the microbial oil is fractionated. In some embodiments, the disclosure teaches a method of fractioning a microbial oil. 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.

[133] In some embodiments, the disclosure relates to a microbial oil fraction, wherein the fraction is microbial stearin, microbial olein, microbial soft mid-fraction, microbial super olein, microbial hard mid-fraction, microbial olein, and/or microbial top olein, wherein the fraction comprises at least one pigment selected from the group consisting of carotene, torulene and torulorhodin and does not comprise chlorophyll.

[134] In some embodiments, the microbial fraction 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.

[135] 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.

Hydrolysis

[136] Hydrolysis is the process whereby triglycerides in fats and oils are split (“fat splitting” or “oil splitting”) into glycerol and fatty acids. It is usually carried out using great amounts of high-pressure steam (“steam hydrolysis”) but may also be performed using catalysts (for example, the tungstated zirconia and solid acid composite SAC-13 (Hydrolysis of Triglycerides Using Solid Acid Catalysts, Ngaosuwan, K, et al., Ind. Eng. Chem. Res., 2009 48 (10), 4757- 4767)). The reaction proceeds in a step-wise fashion wherein fatty acids on triglycerides are displaced one at time, generating diglycerides, then monoglycerides, and finally free fatty acids and glycerin.

[137] In some embodiments, the disclosure teaches a method of microbial oil hydrolysis. In some embodiments, the microbial oil is split into free fatty acids and glycerol. In some embodiments, the microbial oil is split by steam hydrolysis. In some embodiments, the free fatty acids are further purified and/or separated into fractions through distillation or fractionation. In some embodiments, the resulting diglycerides, monoglycerides, free fatty acids, and glycerol are used in compositions. In some embodiments, a microbial oil derivative herein is a microbial fatty acid hydrolysate. In some embodiments, a microbial oil derivative herein is the product of fractional distillation of a microbial fatty acid hydrolysate.

Distillation

[138] Distillation is a process whereby fatty acids and impurities are separated based on differences in boiling points. Fatty acids have a lower boiling point than impurities, such that the fatty acids may be vaporized, condensed, and collected, and the high-boiling impurities are left behind. In some embodiments, the disclosure teaches a method of microbial oil distillation.

Hydrogenation

[139] Hydrogenation is the process whereby liquid fats are made solid or partially solid by adding hydrogen. The extra hydrogen converts the double bonds in unsaturated fats to single bonds, generating saturated fats. Unless the process is controlled, some fats may be partially hydrogenated and this leads to “trans fats”, so named due to the trans configuration of the molecule. In the U.S., artificial trans fats have been banned from food products, however hydrogenated fats may still be used in compositions. Hydrogenated oils prevent the rancid odors caused by oxidation, thus increasing the shelf life of the product, and may also provide a thicker consistency.

[140] The FAMEs produced by transesterification may be hydrogenated to produce fatty alcohols. Fatty acids produced from hydrolysis may also be further modified via esterification to produce wax esters, which may then be hydrogenated to produce fatty alcohols. Direct hydrogenation of fatty acids is also possible and produces fatty alcohols. Thus, in some embodiments, the oil is derivatized to fatty alcohols. In some embodiments, the disclosure teaches methods of fatty acid hydrogenation, wherein the fatty acids are derived from a microbial oil. In some embodiments, the fatty alcohols are further refined and/or distilled. In some embodiments, the fatty alcohols are further derivatized by ethoxylation and/or sulfonation. In some embodiments, the fatty alcohol derivative is an ethoxylated fatty alcohol. In some embodiments, the disclosure relates to a composition of matter comprising cetearyl alcohol derived from fatty alcohols produced by an oleaginous yeast.

[141] In some embodiments, the fatty acids derived from the microbial oil are distilled. In some embodiments, the disclosure teaches methods of using free fatty acids from oleaginous microorganisms in compositions. In some embodiments, the fatty acid is stearic acid, oleic acid, palmitic acid, and myristic acid.

[142] In some embodiments, a microbial oil derivative herein is a high palmitic acid fraction of a microbial oil. In some embodiments, a microbial oil derivative herein is a high oleic acid fraction of a microbial oil. In some embodiments, a microbial oil derivative herein is a palmitic acid, or a derivatized version thereof, e.g., an ester thereof.

Amination

[143] Fatty amines are another class of oleochemicals commonly derived from C12-C18 hydrocarbons from fatty acids. They are produced through the hydrogenation of fatty nitriles, which are themselves produced from a reaction between triglycerides, fatty acids, or fatty esters with ammonia and a catalyst. Fatty amines and their derivatives may be used, for example, in antistatic products, antimicrobial products, detergents, general cleaners, and specialized cleaning products.

[144] Thus, in another embodiment, the disclosure teaches methods of producing fatty amines from fatty acids, triglycerides, and/or fatty esters derived from the microbial oil described herein. In another embodiment, the disclosure relates to compositions comprising fatty amines derived from a microbial oil. In some embodiments, the disclosure relates to compositions comprising amine surfactants derived from a microbial oil.

Saponification

[145] Saponification is the process whereby triglycerides or free fatty acids used as feedstock are converted to fatty acids salts (soaps), glycerol, and free fatty acids in the presence of a base. The base may be for example, sodium hydroxide, which for example produces hard bar soaps, or potassium hydroxide, which for example produces softer bars or liquid soaps. Saponification may be achieved via a hot or cold process. The cold process uses the heat generated from the combination of the fatty acids in the melted oils and fats with sodium hydroxide (base). This process takes longer, and an additional curing phase is needed for the soap to harder. The hot process uses heat to speed up the saponification process, and generally no additional curing step is required before use of the soap.

[146] Globally the soap bar industry is worth approximately US$186 billion. The most commonly used oil sources are vegetable oils, specifically palm and coconut oils, which contain shorter saturated fatty acids. These shorter chain saturated fatty acids increase the lathering profile, while longer saturated fatty acids contribute to the hardness of the soap. Unsaturated fatty acids provide moisture, conditioning, or skin nourishing properties. While animal fats may be used, vegetable oils generally produce higher quality soaps (Prieto Vidal N, et al., The Effects of Cold Saponification on the Unsaponified Fatty Acid Composition and Sensory Perception of Commercial Natural Herbal Soaps. Molecules. 2018;23(9):2356).

[147] In some embodiments, the disclosure teaches methods of using the microbial oil, free fatty acids, and/or triglycerides as feedstock in a saponification reaction to produce fatty acid salts, glycerol, and/or free fatty acids. In some embodiments, these fatty acid salts, glycerol, and/or free fatty acids are used in a composition.

[148] Sodium stearate is produced by saponification of stearic acid, and it one of the most commonly used commercial surfactants in soap. It is also found in solid deodorants, rubbers, latex paints, and inks. In one embodiment, the disclosure relates to a sodium stearate produced from stearic acid, wherein the stearic acid is produced by an oleaginous yeast. In another embodiment, the disclosure relates to products and compositions comprising a sodium stearate derived from an oleaginous yeast.

Surfactants

[149] Surfactants are a broad category of compounds that lower the surface tension between two liquids, for example oil and water, between a gas and a liquid, or between a liquid and a solid. Depending on the compound, they may act as an emulsifier, emollient, detergent, wetting agent, foaming agent, thickening agent, pearlescent, solubilizer, conditioning agent, cosurfactant, or dispersant. In some instances, they can act as an anti-microbial agent and/or a preservative. They can be classified by their head group as either non-ionic (neutral), anionic (negatively charged), cationic (positively charged), or amphoteric (both positive and negative charges).

[150] Nonionic surfactants: An embodiment of the present disclosure relates to nonionic surfactants derived from the microbial oils described herein. Examples of microbially derived nonionic surfactants include, but are not limited to, C10-C16 alkyl dimethyl amine oxide, laureth-6, alcohols - C12-C16 ethoxylated, soy methyl ester ethoxylate, coco methyl ester ethoxylate, D-glucopyranose, oligomeric, Cl 0-16 - alkylglycoside, D-glucopyranose, oligomeric, decyl octyl glycoside, laureth-9, decyl glucoside, lauryl glucoside, capryl/myristyl glucoside, capryl oyl/caproyl methyl glucamide, C8-C10 alkyl polyglucoside, and caprylyl/myristyl glucoside. In another embodiment, the present disclosure relates to compositions comprising a microbially derived nonionic surfactant.

[151] Anionic surfactants: An embodiment of the present disclosure relates to anionic surfactants derived from the microbial oils described herein. Examples of microbially derived anionic surfactants include, but are not limited to, sodium laureth sulfate (also known as sodium lauryl ether sulfate), sodium Cl 4- 17 alcohol sulfate, MEA C1215 alkyl ether sulfate, and sodium lauryl sulfate. In another embodiment, the present disclosure relates to compositions comprising a microbially derived anionic surfactant.

[152] Amphoteric surfactants: An embodiment of the present disclosure relates to amphoteric surfactants derived from the microbial oils described herein. Examples of microbially derived amphoteric surfactants include, but are not limited to, lauramine oxide, lauryl betaine, and lauryl di-methyl amine oxide. In another embodiment, the present disclosure relates to compositions comprising a microbially derived amphoteric surfactant.

[153] Cationic surfactants: An embodiment of the present disclosure relates to cationic surfactants derived from the microbial oils described herein. In another embodiment, the present disclosure relates to compositions comprising a microbially derived cationic surfactant.

[154] The microbially derived cationic surfactant may be a quaternary ammonium compound (“quats” or “QACs”). Quats are widely used as surface disinfectants, but can also function as antistatic agents (i.e. fabric softeners) and as the active ingredient in sanitizers. Quats can be derived from naturally occurring fats and oils, such as tallow, com oil, soybean oil, cottonseed oil, castor oil, linseed oil, safflower oil, palm oil, peanut oil and the like. In some embodiments, the disclosure teaches methods of producing a quat derived from a microbial oil. In some embodiments, the disclosure relates to compositions comprising a quat derived from a microbial oil.

[155] Esterquats: Esterquats are a type of cationic quat having two long C16-C18 fatty acid chains with two weak ester linkages. They function as fabric conditioning agents and can replace dialkyl dimethyl ammonium salts as a biodegradable alternative (Mishra S. and V.K. Tyagi, Ester Quats: The Novel Class of Cationic Fabric Softeners, J. of Oleo Sci. 2007, 56(6): 269-276). As esterquats can be derived from naturally occurring fats and oils using methods well known in the art, they can also be derived from the microbial oil described herein. Thus, in some embodiments, the disclosure teaches methods of producing esterquats from a microbial oil. In some embodiments, the disclosure relates to compositions comprising esterquats derived from a microbial oil.

[156] It is intended that the surfactants disclosed herein are to be considered illustrative rather than limiting. As will be understood by one skilled in the art, the microorganisms described herein may be tailored to produce more less of a particular lipid, for example, C12 (lauric acid). Other surfactants not listed herein but well known in the art may be combined with microbially derived surfactants in the compositions disclosed herein.

[157] Emulsifiers: In some embodiments, the microbially derived surfactant is an emulsifier. Thus, in some embodiments, the disclosure relates to methods of producing an emulsifier derived from a microbial oil, and compositions comprising microbially derived emulsifiers.

[158] Wetting agents: In some embodiments, the microbially derived surfactant is a wetting agent. Thus, in some embodiments, the disclosure relates to methods of producing a wetting agent derived from a microbial oil, and compositions comprising microbially derived wetting agents.

Esters

[159] Esterification is the general name for a reaction that generates esters, a compound derived from an acid. Esterification of fatty acids can generate nonionic surfactants (see for example, Li X., et al., Fatty acid ester surfactants derived from raffinose: Synthesis, characterization and structure-property profiles, 2019, J. of Colloid and Interface Science, Vol. 556(15); 616-627). For example, glycerol esters can be used as emulsifiers, dispersants, and solubilizing agents.

[160] Many esters have fruit-like odors and occur naturally in essential oils of plants, and may be used in fragrances to mimic those odors. In some embodiments, the microbial oil is derivatized to esters. In some embodiments, the fatty ester is isostearyl palmitate, sorbitan palmitate, ascorbyl palmitate, retinyl palmitate, sucrose palmitate, or polyglyceryl-4 oleate.

[161] In some embodiments, the microbial oil is modified via interesterification. In some embodiments, the interesterification is enzymatic. In some embodiments, the interesterification is chemical. In some embodiments, the microbial oil is modified via transesterification. In some embodiments, the oil is derivatized to fatty acid methyl esters (FAMEs). Methyl esters may be used in a number of compositions, for example, they may be a carrier for an active ingredient, an emollient, or viscosity regulator. Thus, in another embodiment, the FAMEs derived from oleaginous yeast are used in a composition of matter. In some embodiments, the methyl esters, are hydrogenated to produce fatty alcohols. Direct hydrogenation of fatty acids is also possible and produces fatty alcohols. Thus, in some embodiments, the oil is derivatized to fatty alcohols. In some embodiments, fatty alcohols derived from an oleaginous yeast are used in a composition. In some embodiments, the disclosure relates to a composition of matter comprising cetearyl alcohol derived from fatty alcohols produced by an oleaginous yeast.

[162] Fatty acid methyl ester sulfonates (MESs): MESs are traditionally derived from vegetable oils such as palm and coconut oil through transesterification (to form FAMEs) followed by sulfonation. MESs are oleochemical based anionic surfactants that are being increasingly used in detergents, soaps, hair care, and dish wash, as they provide a plant-based alternative to petroleum based surfactants, such as linear alkyl benzene sulfonates (LASs). However, plant-based does not necessarily equate to sustainable and environmentally friendly, as discussed herein with the palm oil market. The microbial oil described herein can be derivatized through the same or similar transesterification and sulfonation processes used on palm oil to produce MESs.

[163] Studies have shown that MESs are more tolerant to hard water, and have better detergency when compared to LASs (Lim Y., et al, Methyl Ester Sulfonate: A High- Performance Surfactant Capable of Reducing Builders Dosage in Detergents, J. of Surfactants and Detergents, 22(3): 549-558). It’s estimated that the global market for MESs will be worth $1,155.0 mn by 2025 (pmewswire.com/news-releases/global-methyl-ester-sulfonate-m arket- manufacturers-of-detergents-and-personal-care-products-drivi ng-demand-observes-tmr- 674822613.html, available on the world wide web Feb 2018).

[164] Thus, in some embodiments, the disclosure teaches MESs derived from microbial oil and methods of producing MESs from a microbial oil. In some embodiments, the disclosure relates to compositions comprising MESs derived from a microbial oil.

Solvents [165] Solvents dissolve solutes, resulting in a solution. Some solvents are harmful to the environment. Recent research shows that vegetable oil, such as palm oil, can be used as nontoxic bio-based solvent (Noppawan P., et al., Green Chem., 2021, 23, 5766-5774). However, for vegetable oils to truly be a renewable alternative to petroleum derived solvents, they must be sustainably sourced.

[166] Thus, in some embodiments, the disclosure relates to solvents derived from microbial oil and methods of producing thereof. In some embodiments, the disclosure relates to compositions comprising a solvent derived from a microbial oil.

Process aids

[167] Process aids are substances which are used in the production of a consumer product. They may be present in the finished product in trace amounts, but are not required to be disclosed to the consumer as an ingredient. Triglycerides, for example, soybean oil, olive oil, linseed oil, corn oil, coconut oil, hydrogenated castor oil, palm oil, and hydrogenated soybean oil, are considered safe biological substances for use as process aids (EPA’s Safer Choice Criteria for Processing Aids and Additives, available on the world wide web at epa.gov/saferchoice/safer-choice-criteria-processing-aids-an d-additives).

[168] Thus, in some embodiments, the disclosure relates process aids derived from microbial oil, and methods of producing thereof. In some embodiments, the disclosure relates to compositions comprising a process aid derived from a microbial oil.

Humectants

[169] Humectants attract water and thus are often found in personal care compositions, such as lotions and hair products, as moisturizers. They are also found in everyday compositions. An example of a humectant that may be derived from the microbial oil described herein is glycerin.

[170] Thus, in some embodiments, the disclosure relates to humectants derived from microbial oil and methods of producing thereof. In some embodiments, the disclosure relates to compositions comprising a humectant derived from a microbial oil.

Antistatic agents and softening agents

[171] Antistatic agents, also knowns as “antistats” are so named for their ability to reduce or eliminate the buildup of static electricity. Examples of antistatic agents include quaternary ammonium salts, long-chain aliphatic amines and amides, and esters, such as glycerol esters of fatty acids, and ethoxylated tertiary amines. Thus, in some embodiments, the disclosure relates to antistatic and softening agents derived from microbial oil and methods of producing thereof. In some embodiments, the disclosure relates to compositions comprising an antistatic agent derived from a microbial oil.

[172] As with antistatic agents, softening agents can reduce the buildup of static electricity, in general the term is broad and applies to any substance that increases the softness of another substance. An example of a softening agent that may be derived from the microbial oil described herein are fatty amines. Fatty amines are largely used as softening agents in the detergent industry. In some embodiments, the disclosure relates to microbial oil and derivatives thereof for use as a softening agent. In some embodiments, the disclosure teaches methods of producing a softening agent derived from a microbial oil. In some embodiments, the disclosure relates to compositions comprising a softening agent derived from a microbial oil.

Rheology modifiers/thickeners

[173] Rheology modifiers, also referred to as thickeners or viscosity modifiers, help to control the viscosity of a substance. They may also increase the suspendability of soluble ingredients and stability of a product. Naturally occurring rheology modifiers include, for example, pectin, gelatin, and collagen. Rheology modifiers that can be derived from naturally occurring fats and oils, and the microbial oil described herein, include, but are not limited to, caprylic/capric triglycerides, oleic acid, glycol stearate, and lauryl alcohol.

[174] In some embodiments, the disclosure relates to rheology modifiers and thickeners derivred from microbial oil and methods of producing thereof. In some embodiments, the disclosure relates to compositions comprising a rheology modifier derived from a microbial oil.

Foam suppressors/anti-foaming agents

[175] Foam suppressors, also known as anti-foaming agents, are substances that help prevent the formation of a foam. Common anti-foaming agents include, for example, cetostearyl alcohol, insoluble oils, stearates, ether and glycols. In some embodiments, the disclosure relates to foam suppressors or anti-foaming agents derived from microbial oil and methods of producing thereof. In some embodiments, the disclosure relates to compositions comprising a foam suppressor derived from a microbial oil.

Foam boosters/stabilizers

[176] Foam boosters, also known as stabilizers or suds enhancers are commonly used to prolong the life of the foam head generated during the washing or cleaning process. Consumers tend to associate better product performance with the presence of higher levels of foam or suds and by foam that lasts for extended periods of time. In addition to producing abundant foam and extending foam life, foam boosters typically provide other beneficial properties, for example, as mild surface-active agents, they enhance the cleaning performance of hand dish detergents, and greatly impact the aesthetic appeal of the detergent composition through viscosity modification and emolliency. Examples of common foam boosters include, but are not limited to, amine oxides, betaines, sultaines, and alkanolamides.

[177] Thus, in some embodiments, the disclosure relates to foam boosters and stabilizers derived from microbial oil. In some embodiments, the disclosure teaches methods of producing a foam booster and/or stabilizer derived from a microbial oil. In some embodiments, the disclosure relates to compositions comprising a foam booster and/or stabilizer derived from a microbial oil.

Characteristics similar to other oleochemicals

[178] The microbial oil and/or derivatives thereof described herein may also function as a replacement for other oleochemicals, such as derivatives of palm oil. As will be understood by one skilled in the art, the oleaginous microorganisms described herein may be tailored to produce more or less of a particular hydrocarbon, for example C12 (lauric acid). Additionally, many of the same derivatization processes and methods used to generate other oleochemicals can be used on the microbial oils. In some embodiments, a microbial fingerprint remains, such that the oleochemical can be distinguished from an oleochemical derived from a vegetable oil. In some embodiments, the microbial fingerprint is removed, and the oleochemical is identical or indistinguishable to an oleochemical derived from a vegetable oil.

[179] 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.

[180] 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.”

[181] 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.

[182] 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: Compositional analysis of exemplary microbial oil for use in producing derivatives

[183] Microbial oil was prepared using R. toruloides fermented on glycerol feed, lysed with acid, and extracted with heptane solvent. The composition of the oil is shown below in Table 9A.

Table 9A: Oil batch analysis

[184] Ten additional samples were analyzed in 2022 (Tables 9B-9D and FIG. 1 and 2). Samples were diluted in organic solvent and individual TAGs were separated by high resolution gas chromatography. Monoglycerides and diglycerides were determined by GC- FID using a method based on AOCS Cd 1 lb-91. The sample is dissolved in organic solvent, with added internal standard, and derivatized to trimethyl silyl derivative using N,O- Bis(trimethylsilyl)trifluoroacetamide (BSTFA) and then analyzed by GC-FID. The monoglycerides and diglycerides are quantified relative to the internal standard and calibration with monoglyceride and diglyceride standards.

Table 9B: Triglyceride profiles of the oil samples including selected positional isomers

M=Myristic C14:0; P=Palmitic C16:0; S=Stearic C18:0; A=Arachidic C20:0; B=Behenic C22:0; Po=Palmitoleic C16: l; O=Oleic C18:l; Li=Linoleic C18:2;

Ln=Linolenic C18:3; Ei=Eicosenoic C20: l

Table 9C: Mono-, di-, and triglyceride content of samples

[185] Solid fat content was determined using method T for non-stabilizing fats according to AOCS Cd 16-93 Direct, Parallel: Method I. This is a direct and parallel method meaning the NMR is calibrated using 3 samples of known solid fat content and the different temperatures are run one after the other using the same sample tubes. The samples are conditioned by melting them at 80°C for 15 minutes and then 60°C for 5 minutes to clear thermal history. The sample is then left at 0°C for 1 hour to allow the solid fat crystals to form. It is then left at the selected temperature to equilibrate for 30 minutes and following this the solid fat content percentage is measured using NMR

Table 9D: Solid fat content of the samples reported as the average of duplicate determinations

EXAMPLE 2; Microbial oil compositions following different stages of processing

[186] Microbial oil from R. toruloides was prepared and extracted as described in the foregoing examples. Crude microbial oil was then compared to the same oil following consecutive steps of refinement, bleaching, and deodorizing. The results of the analyses is provided in Tables 10A-11C below for crude (unrefined) microbial oil (“Crude Oil”), refined microbial oil (“Refined Oil”), refined and bleached microbial oil (“Refined & Bleached Oil”), and refined, bleached, and deodorized microbial oil (“RBD Oil”). Most analyses were performed with standard AOCS and ISO methodologies. After refining, 8-10% of the mass of the crude oil was recovered as soapstock, a by-product of the refining process. Spent clay was obtained as a by-product of bleaching.

[187] Tables 10A-10C include the results of processing the crude oil with an antioxidant, and are visually shown in FIG. 3, while Tables l lA-11C include the results of processing the crude oil without an antioxidant, and are shown in FIG. 4. In Table 10A and 11 A, “NR” indicates that no measurement was taken for that sample parameter. As shown in FTGs. 3 and 4, the crude oil can be processed to produce a very light-colored oil using standard RBD processes. Fatty acid profde was generally maintained among all samples, demonstrating that the microbial oil was stable throughout processing. Adding an antioxidant before processing did not appear to significantly affect the stability of the oil during the RBD process, however, the addition of an antioxidant may affect shelf-life under various conditions. As will be understood by one skilled in the art, bleaching conditions, especially dosage, can be modified based on the requirement of color of the final product.

Table 10A: Basic oil parameters of microbial oil in different stages of refinement with an antioxidant Table 10B: Fatty acid composition of microbial oil in different stages of refinement with an antioxidant

Table IOC: Diglyceride, monoglyceride, and phospholipid composition of microbial oil in different stages of refinement with an antioxidant Table 11 A: Basic oil parameters of microbial oil in different stages of refinement without an antioxidant

Table 11B: Fatty acid composition of microbial oil in different stages of refinement without an antioxidant

Table 11C: Diglyceride, monoglyceride, and phospholipid composition of microbial oil in different stages of refinement without an antioxidant

EXAMPLE 3: Sterol analysis of exemplary microbial oil of the disclosure

[188] 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. loruloides (“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.

[189] 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 (FIGS. 5A-5D) 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. 6. 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. FIGS. 7A-7E show illustrative El spectra for sterols extracted from the crude palm oil spike-in preparation.

Table 12: Mass of sterol compounds used for extraction.

[190] 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.

[191] 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 4: Carotenoid analysis of exemplary microbial oils of the disclosure

Oil samples

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

[193] - Sample 1 : agricultural palm oil.

[194] - Sample 2: exemplary microbial oil of the disclosure obtained from R. toruloides; strong acid (H2SO4) treatment with solvent extraction of lipids. [195] - Sample 3 : exemplary microbial oil of the disclosure obtained from R. toruloides,' strong acid (HC1) treatment with solvent extraction of lipids.

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

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

[198] - Sample 6: exemplary microbial oil of the disclosure obtained from R. toruloides,' acid- free extraction of lipids.

Carotenoid analysis materials and methods

[199] 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.

[200] 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)

[201] 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.

[202] 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

[203] Sample 1. The overall UV/Vis absorbance spectrum for Sample 1, agricultural palm oil, is shown in FIG. 8A 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.

[204] For Sample 1, the HPLC-DAD chromatogram reporting absorbance at 450 nm is shown in FIG. 8B 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.

[205] Sample 2. The overall UV/Vis absorbance spectrum for Sample 2, strong acid-extracted microbial oil, is shown in FIG. 9A. 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. 9B with no identifiable peaks.

[206] Sample 3. The overall UV/Vis absorbance spectrum for Sample 3, strong acid-extracted microbial oil, is shown in FIG. 10A. 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. 10B with no identifiable peaks.

[2071 Sample 4. The overall UV/Vis absorbance spectrum for Sample 4, weak acid-extracted microbial oil, is shown in FIG. 11 A. 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. 11B with individual peaks identified in Table 17. As expected for a microbial oil from R. toruloides, 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 - carotene and derivatives thereof.

Table 17: Sample 4, HPLC peak identification.

[208] Sample 5. The overall UV/Vis absorbance spectrum for Sample 5, acid-free extracted microbial oil, is shown in FIG. 12A 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.

[209] For Sample 5, the HPLC-DAD chromatogram reporting absorbance at 450 nm is shown in FIG. 12B 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.

[210] Sample 6. The overall UV/Vis absorbance spectrum for Sample 6, acid-free extracted microbial oil, is shown in FIG. 13A 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. [211] For Sample 6, the HPLC-DAD chromatogram reporting absorbance at 450 nm is shown in FIG. 13B 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.

[212] 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 u- and P-carotenes and derivatives thereof.

EXAMPLE 5: Fractionated microbial olein and stearin production

[213] Fractionation is another means of processing the microbial oil described herein. Fractionation may be used to physically separate room temperature oil into saturated and unsaturated components. As shown in FIG. 14, the primary fraction of microbial oil results in microbial stearin and microbial olein. A secondary fraction of microbial olein results in microbial soft mid-fraction and microbial super olein. A tertiary fractionation of the soft mid-fraction results in a microbial hard mid-fraction and microbial mid-olein. A tertiary fractionation of the microbial super olein results in microbial mid-olein and microbial top olein.

[214] 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. Palm oil and microbial oil were fractionated and the saturation levels of their fractions were compared. [215] 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.

[216] 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.

[217] 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. Shown in FIG. 15A is a photograph of a fractionation of crude microbial oil (left) and crude palm oil (right). The top olein layer is liquid, and the bottom stearin layer is solid. FIG. 15B is a photograph of a complete fractionation of crude microbial oil, and FIG. 15C is a photograph of an incomplete fractionation of crude microbial oil.

[218] The aqueous phase was separated by aspirating the upper olein phase into a pre-weighed 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.

[219] 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.

[220] 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 between 80.9 and 81.5 and the microbial stearin fraction had an iodine value of approximately 22.4. Crude microbial oil had an iodine value of between 62.6 and 62 9 (Table 22). The crude palm oil olein fraction had an TV of 53 and the stearin fraction had an TV 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.

Table 22: IVs for an exemplary fractionated microbial oil of the disclosure.

[221] The fatty acid profde of the fractioned oil was also analyzed. Shown in FIG. 16 is a bar graph of the fatty acid profde of crude microbial oil, microbial olein layer, microbial mid-fraction, and microbial stearin layer. Shown in FIG. 17 is a bar graph of the saturated profdes of crude microbial oil, microbial olein layer, microbial mid-fraction, and microbial stearin layer. As shown in FIG. 17, Microbial olein has a greater percentage of monounsaturated fatty acids compared to microbial stearin, which has a greater percentage of saturated fatty acids. The microbial stearin fractions shown are solid at room temperature (slip melting point > 25 °C), whereas the olein fractions are liquid at room temperature. In some instances, microbial fractionation gives rise to three layers: a stearin, olein, and mid-fraction. In some instances, the microbial oil may be re- fractioned to generate double stearin, or double olein, for example. Thus, the microbial oil of the present disclosure may be fractioned similar to other plant-derived oils, such as palm oil.

[222] Additionally, fractionated microbial olein and fractionated microbial stearin may replace rice bran oil and shea butter, respectively in compositions. Fractioned olein may also replace vegetable oils high in oleic acids, such as tea seed oil.

EXAMPLE 6: Materials and methods for fractionation and fraction analysis.

Microbial oil production

[223] Crude microbial oil was obtained from culturing an oleaginous microorganism, e g., R. toruloides. See, e.g., WO2021 T54863AT. Fractionation Method

[224] 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.

[225] 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

[226] 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 and applied chemistry 1985;57(10): 1515-22.

[227] 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 (769 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 35O°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.

[228] 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

[229] 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.

[230] 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.

[231] 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 C 18:3, 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

[232] 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.

[233] 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.

[234] 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 7: Illustrative solvent-based fractionations of microbial oils of the disclosure.

Materials & Methods

[235] For solvent-based fractionation, the following ranges were explored for each parameter, also summarized in Table 23.

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

[237] Solvent: heptane, acetone

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

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

[240] Crystallization Time: 1 hour - 4 days

[241] Fractionation, FAME analysis, and melting point determination were carried out according to Example 6.

Results

[242] The microbial oil fractions obtained from these illustrative solvent-based fractionation conditions differed significantly from the original microbial oil. Table 23 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. 18A-29B. Table 24 provides the fatty acid profiles of the solid fractions in comparison to the original crude and RBD microbial oils. Table 25 features fatty acid profiles of illustrative solid and liquid fractions from the same fractionation conditions.

Table 23: Process conditions for solvent fractionation and resulting solid fraction melting point.

Table 24: FAME profile for solid fractions resulting from different solvent conditions.

Table 25: Comparison of solid and liquid fractions from the same solvent fractionation conditions.

[243] 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 25 demonstrates significant differences in the fatty acid compositions between solid and liquid fractions, and as compared to the original samples.

EXAMPLE 8: Illustrative dry fractionations of microbial oils of the disclosure

Materials & Methods

[244] For dry fractionation, the following ranges were explored for each parameter, also summarized in Table 26.

[245] Starting material: RBD oil

[246] Temperature: 10°C to 16 °C

[247] Crystallization Time: 1 hour - 24 hours

[248] Fractionation, FAME analysis, and melting point determination were carried out according to Example 6.

Results

[249] FIG. 30A-33C show the results of TAG analysis of the solid fraction resulting from dry fractionation conditions 1-4, respectively. FIG. 34A-34B show the results of FAME analysis of the solid fraction resulting from dry fractionation condition 5. The microbial oil fractions resulting from these illustrative dry fractionation conditions differed in significant ways from the original RBD microbial oil. Table 26 provides process conditions and melting points for the solid fractions resulting from these conditions. Table 27 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 28 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 28 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 = “SSLT”, etc. Table 29 shows a more detailed breakdown of the TAG profile for the samples included in Table 28. Table 26: Process conditions and melting point for dry conditions

Table 27: FAME profile for dry conditions

Table 28: TAG saturation profile in solid fractions compared to crude samples.

Table 29: TAG Profiles of solid fractions obtained from different dry fractionation conditions.

EXAMPLE 9: Illustrative double solvent-based fractionation of a microbial oil of the disclosure.

Materials & Methods

[250] 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

[251] FIG. 35A-35B 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 30 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 30: TAG saturation in original oil and liquid and solid fractions.

EXAMPLE 10: Properties and triglyceride profiles of illustrative crude and microbial oils of the disclosure.

[252] Several batches of an illustrative crude microbial oil were produced and then refined, bleached, and deodorized. Properties of the crude and RBD oils were compared. Properties of the illustrative crude oil batches are shown in Table 31, with minimum and maximum ranges of each property established among the batches. Properties of the illustrative RBD oil batches are shown in Table 32, with minimum and maximum ranges of each property established among the batches. The crude oil was bright orange, while the RBD oil was light yellow to clear. The color of the crude oil was due to the presence of beta-carotene and torulene, which were largely removed from the RBD oil by the bleaching process. Table 31: Properties of Illustrative Crude Microbial Oil Batches

Table 32: Properties of Illustrative RBD Microbial Oil Batches

[253] Three illustrative RBD microbial oils of the disclosure were analyzed to determine triglyceride profile and TAG saturation profile. Results are shown in Tables 33 and 34.

Table 33: Illustrative Triglyceride Profiles of RBD Microbial Oils

Table 34: Illustrative TAG Saturation Profiles of RBD Microbial Oils

EXAMPLE 11: Methods of producing derivatives from microbial oil

[254] As shown in FIG. 36, the microbial oil or fraction thereof may be processed or modified by a number of means to generate derivatives for use in compositions. For example, as shown in FIG. 36, the microbial oil or fractions thereof may be modified by transesterification to produce FAMEs, or split (hydrolysis) to produce fatty acids and glycerin. These FAMEs and fatty acids may be subsequently hydrogenated to produce fatty alcohols. The fatty alcohols may undergo ethoxylation and/or sulfonation to produce fatty alcohol ethoxylates, fatty alcohol sulfates, or ether sulfates. The fatty acids may also be modified by amination, esterification (see FIG. 37), and reactions with amino acids to produce fatty amines, fatty esters, and amide carboxylates respectively. Fatty amines may further be modified by oxidation, monochloroacetic acid (MCA) reaction, and quaternization to produce amine oxides, betaines, and quats respectively. The microbial oil or fraction thereof may also be modified by saponification to produce fatty acid salts. Any of these derivatives and intermediate products may be used in compositions. Thus, in some embodiments, the microbial oil is processed and/or modified via one or more of fractionation, hydrogenation, hydrolysis, distillation, saponification, esterification, interesterification, transesterification, amination, ethoxylation, sulfonation, oxidation, quaternization, MCA reaction, and/or reaction with amino acids.

[255] In some embodiments, the disclosure relates to a derivative produced from a microbial oil. In some cases, the derivative is selected from the group consisting of sodium lauryl sulfate; sodium laureth sulfate; sodium lauryl ether sulfate; sodium oleate; MEA laureth sulfate; MEA lauryl sulfate; laureth-6; laureth-9; glycerin; D-glucopyranose, oligomeric, decyl octyl glycoside; hydrogenated castor oil; coconut fatty acid; canola-amidoethyl hydroxyethylammonium methyl sulfate; dipalmethyl hydroxyethylammonium methosulfate; dihydrogenated palmoylethyl hydroxethylmonium methyl sulfate; di (palm carboxyethyl) hydroxy ethyl methyl ammonium methyl sulfate; lauramine oxide; capryloyl methyl glucamide; caproyl methyl glucamide; soy methyl ester ethoxylate; coco methyl ester ethoxylate; lauryl betaine; lauryl di-methyl amine oxide; decyl glucoside; lauryl glucoside; capryl glucoside; caprylyl glucoside; myristyl glucoside; isostearyl palmitate; polyglyceryl-4 dipalmitate; sorbitan palmitate; polyglyceryl- 10 di oleate; polyglyceryl-4 oleate; retinyl palmitate; ascorbyl palmitate; sucrose palmitate; and ethyl palmate.

EXAMPLE 12: Ester production

[256] Esterification is the general name for a reaction that generates esters, a compound derived from an acid. In some embodiments, the disclosure relates to esters derived from fatty acids and/or microbial oils.

[257] Oil samples were converted into fatty acid methyl esters (FAMEs) and then analyzed using gas chromatography-mass spectrometry (GC-MS). 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.

[258] 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.

[259] 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.

[260] 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. [261] The results of the analysis are shown in Table 35 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. Table 35 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 35: Fatty acid composition of microbial oil samples

[262] 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. [263] 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 36. Table 36 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 36: Fatty acid composition breakdown [264] Table 37 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. 38 shows a bar graph of representative compositions of microbial oil and palm oil.

Table 37: Overall fatty acid composition

[265] As shown in the above and described herein, oleaginous microbial oil has a similar profile to that of palm oil, and thus esters derived microbial oils may be used in place of esters derived from palm oil. Methods of producing esters from fatty acids are well known in the art. See, for example, Milinsk, M. C. et al., Comparative analysis of eight esterification methods in the quantitative determination of vegetable oil fatty acid methyl esters (FAME), J. Braz. Chem. Soc., 2008, vol.19, n.8. One example use of the microbial oils described herein is esterification of a specific fatty acid produced from an oleaginous yeast.

[266] As shown in Table 36, oleaginous yeast produced approximately 9% stearic acid. Esterification of stearic acid produced by the oleaginous yeast described herein can produce stearate esters for use as emollients and thickeners. For example, a reaction with ethylhexyl alcohol can produce Ethylhexyl Stearate (also known as Octyl Stearate). Similarly, reactions with other alcohols can produce Butyl Stearate, Cetyl Stearate, Isocetyl Stearate, Isopropyl Stearate, Myristyl Stearate, and Isobutyl Stearate, and Octyldodecyl Stearoyl Stearate. Stearate esters may be used in compositions such as, for example, detergents, soaps, and smooth surface cleaners.

[267] As shown above in Table 36 above, oleaginous yeast produced approximately 28.7% palmitic acid. Esterification of palmitic acid produced by the oleaginous yeast described herein can produce palmitate esters (FIG. 37). For example, a reaction with ethylhexyl alcohol can produce ethylhexyl palmitate (also known as Octyl Palmitate), which may act as an emollient in a composition. Similarly, Isopropyl Palmitate is the ester of isopropyl alcohol and palmitic acid, and can function as an emollient, emulsifier, stabilizer, film former, spreader, and a solvent in a composition. Cetyl Palmitate is the ester of cetyl alcohol and palmitic acid, and can function as an emollient in a composition. Isostearyl Palmitate is the ester of isostearyl alcohol and palmitic acid, and can work as an emollient.

[268] Related to the above, in some embodiments, the following microbial oil derivatives are produced: isostearyl palmitate, polyglyceryl-4 dipalmitate, sorbitan palmitate, polyglyceryl- 10 dioleate, polyglyceryl-4 oleate, retinyl palmitate, ascorbyl palmitate, sucrose palmitate, ethyl palmate, methyl ester sulfonate, and esterquats.

EXAMPLE 13: Free Fatty acid and Fatty alcohol production

[269] Hydrolysis is the process whereby triglycerides in fats and oils are split (“fat splitting” or “oil splitting”) into glycerol and fatty acids. It is usually carried out using great amounts of high-pressure steam (“steam hydrolysis”) but may also be performed using catalysts (for example, the tungstated zirconia and solid acid composite SAC-13 (Hydrolysis of Triglycerides Using Solid Acid Catalysts, Ngaosuwan, K, et al., Ind. Eng. Chem. Res., 2009 48 (10), 4757- 4767)). The reaction proceeds in a step-wise fashion wherein fatty acids on triglycerides are displaced one at time, generating diglycerides, then monoglycerides, and finally free fatty acids and glycerin.

[270] Shown in FIG. 39 is a flow diagram of an example method to produce purified fatty acids from microbial oil or fractions thereof. The crude microbial oil may first be deaerated to remove un-dissolved gasses. Next, the fatty acids are produced by steam hydrolysis, wherein the temperature is raised up to 260 degrees Celsius at a pressure of 60 bar. Glycerine may be collected and further purified for various uses, and the crude fatty acids are subsequently purified by distillation. Fatty acids may be further modified to produce, for example, conjugated fatty acids, dimer acids, fatty acids ethoxylates, and fatty acid esters.

[271] Examples of fatty acids derived from microbial oils include, but are not limited to, stearic acid, oleic acid, palmitic acid, and myristic acid.

[272] Fatty alcohols may be produced via a methyl ester route or a wax ester route (FIG. 40). In the methyl ester route (also known as the Davy process), FAMEs produced by transesterification may be hydrogenated to produce crude fatty alcohols, which are then refined, polished, and purified. In the wax ester route, (also known as the Lurgi process) fatty acids produced from hydrolysis (“splitting”) are further modified via esterification to produce wax esters, which may then be hydrogenated to produce fatty alcohols. Direct hydrogenation of fatty acids is also possible and produces fatty alcohols. Fatty alcohols may be further modified to produce, for example, fatty alcohol ethoxylates, and fatty alcohol sulfates. [273] Examples of fatty alcohols derived from oleaginous microorganisms that may be used in compositions include, but are not limited to, cetearyl alcohol, cetyl alcohol, isostearyl alcohol, and myristyl alcohol.

EXAMPLE 14: Fatty acid salt production

[274] Saponification is the process whereby triglycerides or free fatty acids used as feedstock are converted to fatty acids salts (soaps), glycerol, and free fatty acids in the presence of a base. The base may be for example, sodium hydroxide, or potassium hydroxide. Saponification may be achieved via a hot or cold process. The cold process uses the heat generated from the combination of the fatty acids in the melted oils and fats with sodium hydroxide (base). This process takes longer, and an additional curing phase is needed for the soap to harder. The hot process uses heat to speed up the saponification process, and generally no additional curing step is required before use of the soap. Methods of saponification are well known in the art. See for example, Prieto Vidal N, et al., The Effects of Cold Saponification on the Unsaponified Fatty Acid Composition and Sensory Perception of Commercial Natural Herbal Soaps. Molecules. 2018;23(9):2356.

[275] The triglycerides or free fatty acids described herein (see for example, Table 36) may be used in a saponification reaction to produce salts, glycerin, and free fatty acids. For example, sodium stearate is produced by saponification of stearic acid, and it is one of the most commonly used commercial surfactants in soap. Sodium oleate is produced by the saponification of oleic acid. Saponification of palmitic acid produces sodium palmitate. Potassium stearate is the potassium salt of stearic acid. Metal salts may also be produced, for example, zinc stearate and magnesium myristate.

[276] As will be understood by one skilled in the art, saponification of the triglycerides disclosed herein may produce a number of salts and glycerin for use in compositions.

EXAMPLE 15: Derivatives of microbial oil

[277] Listed below are examples of derivatives that may be produced using the microbial oil described herein. The examples below should not be construed as limiting. Rather, these examples are provided so that this disclosure will be thorough and complete. Additional uses for the microbial oils and derivatives thereof, and various modifications to the microbial oil will be readily apparent to those skilled in the art.

• Sodium Laureth Sulfate

• Sodium Cl 2- 15 Alkyl Sulfate

• Sodium and MEA Laureth Sulfate

• Sodium and MEA Lauryl Sulfate

• C10-16 Pareth • C10-C16 alkyl dimethyl amine oxide

• Sodium and MEA Salts of Cl 2- 18 fatty acids

• Sodium Lauryl Sulfate

• Cl 0-16 Alkyl dimethyl amine oxide

• Sodium Salts of Cl 2- 18 Fatty Acids

• Laureth-6

• Glycerin

• Sodium Chloride

• Sodium Oleate

• C 10- 16 Alkyldimethylamine oxide

• Sodium Salts of Cl 2- 18 Fatty Acids

• Alcohols, C12-16, ethoxylated

• MEA Salts of C 12-18 Fatty Acids

• Capryleth-4

• Soy Methyl Ester Ethoxylate

• D-Glucopyranose, Oligomeric, Cl 0-16 - Alkylglycoside

• D-Glucopyranose, Oligomeric,

• Decyl Octyl Glycoside

• Laureth-9

• Sodium Cl 4- 17 Alcohol Sulfate

• Decyl Glucoside

• MEA C 1215 Alkyl Ether Sulfate

• Glycerin

• MEA Salts of C 12-C 18 Fatty Acids

• Hydrogenated Castor Oil

• Coconut Fatty Acid

• Sodium Lauryl Ether Sulfate

• C12-16 Pareth-7

• Canola-amidoethyl Hydroxy ethyl ammonium Methyl Sulfate

• Coco Methyl Ester Ethoxylate

• Dipalmethyl Hydroxyethylammonium Methosulfate

• Cl 6- 18 Fatty Acids

• Dihydrogenated Palmoylethyl Hydroxethylmonium Methyl Sulfate

• Cl 6- 18 Glycerides

• Di (Palm Carboxyethyl) Hydroxyethyl Methyl Ammonium Methyl Sulfates

• Lauramine Oxide

• Capryl/Myristyl Glucoside

• Sodium Lauryl Sulfate

• Lauryl Glucoside

• C 10- 16 Alkyldimethylamine Oxide

• Sodium Laureth Sulfate

• Lauramine Oxide

• Sodium Lauryl Ether Sulfate

• Capryloyl / Caproyl Methyl Glucamide

• Soy Methyl Ester Ethoxylate

• Coco Methyl Ester Ethoxylate

• Lauryl Betaine • Lauryl Di-Methyl Amine Oxide

• Sodium Cl 4- 17 Sec- Alkyl Sulfonate

• C8-C 10 Alkyl Polyglucoside

• Caprylyl/Myristyl Glucoside

• Stearyl alcohol

• Ethylhexyl Palmitate

• Ethylhexyl Stearate

• Octyldodecyl Stearate

• Sorbitan Oleate

• Sorbitan Trioleate

• Sorbitan Isostearate

• Sorbitan Stearate

• Isotridecyl Stearate

• Glyceryl Stearate

• PEG- 100 Stearate

• PEG-4 Dioleate

• PEG-4 Oleate

• PEG-4 Stearate

• PEG-6 Oleate

• PEG-6 Stearate

• PEG-8 Dioleate

• PEG-8 Oleate

• PEG- 8 Stearate

• PEG- 12 Dioleate

• PEG- 12 Di stearate

• PEG- 12 Stearate

• PEG- 150 Di stearate

• PEG-90 Glyceryl Isostearate

• Polysorbate 40

• Polysorbate 60

• Polysorbate 80

• Ceteareth-20

• Steareth-2

• Steareth-20

• Steareth-21

• Cetearyl Alcohol

• Stearic Acid

• Isostearyl PCA

Conclusion

[278] Based on the above examples and analyses, the microbial oils, fractions, and derivatives thereof described herein are a good match of palm oil/hybrid palm oil and their derivatives along a number of different parameters, demonstrating their suitability for use as an environmentally friendly alternative to plant-derived palm oil and palm oil derivatives for use in compositions. NUMBERED EMBODIMENTS OF THE INVENTION

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

1. A microbial oil fraction, wherein the fraction is microbial stearin, microbial olein, microbial soft mid-fraction, microbial super olein, microbial hard mid-fraction, microbial olein, and/or microbial top olein, wherein the fraction comprises at least one pigment selected from the group consisting of carotene, torulene and torulorhodin and does not comprise chlorophyll.

2. The microbial oil fraction of embodiment 1, wherein the fraction is microbial olein, and wherein the microbial olein comprises at least 50% (w/w) monounsaturated fatty acids.

3. The microbial oil fraction of embodiment 2, wherein the microbial olein has at least 50% (w/w) of oleic acid.

4. The microbial oil fraction of embodiment 1, wherein the fraction is microbial stearin, and wherein the microbial stearin comprises at least 50% (w/w) saturated fatty acids.

5. The microbial oil fraction of embodiment 4, wherein the microbial stearin has at least 50% (w/w) of palmitic acid.

6. The microbial oil fraction of any one of embodiments 1-5, wherein the microbial oil comprises ergosterol and does not comprise campesterol, P-sitosterol, or stigmasterol.

7. The microbial oil fraction of any one of embodiments 1-6, wherein the fraction has one or more characteristics similar to a plant-derived palm oil fraction selected from the group consisting of: apparent density, refractive index, saponification value, unsaponifiable matter, iodine value, slip melting point, and fatty acid composition.

8. The microbial oil fraction of any one of embodiments 1-7, wherein the fraction comprises less than 100 ppm of, comprises less than 50 ppm of, or does not comprise a phytosterol.

9. The microbial oil fraction of any one of embodiments 1-8, wherein the fraction comprises less than 100 ppm of, comprises less than 50 ppm of, or does not comprise cholesterol.

10. The microbial oil fraction of any one of embodiments 1-9, wherein the fraction comprises less than 100 ppm of, comprises less than 50 ppm of, or does not comprise protothecasterol.

11. The microbial oil fraction of any one of embodiments 1-10, wherein the fraction is derived from a microbial oil produced by an oleaginous yeast of the genus Yarrowia, Candida, Rhodotorula, Rhodosporidium, Metschnikowia, Cryptococcus, Trichosporon, or Lipomyces. The microbial oil fraction of any one of embodiments 1-11, wherein the fraction is derived from a microbial oil produced by Rhodosporidium toruloides. A method for producing a derivative of a microbial oil comprising: obtaining a microbial oil; and modifying the microbial oil to produce a derivative. The method of embodiment 13, wherein the microbial oil is a refined microbial oil. The method of embodiment 13 or 14, wherein the microbial oil is a bleached microbial oil. The method of any one of embodiments 13-15, wherein the microbial oil is a deodorized microbial oil. The method of any one of embodiments 13-16, wherein the microbial oil is obtained from a whole cell or lysed microbial biomass by an extraction process. The method of embodiment 17, wherein said extraction process removes toxins and produces a microbial oil safe for human use. The method of any one of embodiments 13-18, wherein the modifying comprises fractionation, interesterification, transesterification, hydrogenation, steam hydrolysis, distillation, saponification, amination, ethyoxylation, sulfonation, oxidation, quaternization, or combinations thereof. The method of any one of embodiments 13-19, wherein the microbial oil extracted from the whole cell or lysed microbial biomass has a fatty acid profile comprising: at least 10% w/w saturated fatty acids; at least 30% w/w unsaturated fatty acids; and less than 30% w/w total polyunsaturated fatty acids. The method of any one of embodiments 13-20, wherein the microbial oil has a fatty acid profile comprising: greater than 20% w/w saturated fatty acids; greater than 35% w/w mono-unsaturated fatty acids; and less than 25% w/w polyunsaturated fatty acids. The method of any one of embodiments 13-21, wherein the microbial oil comprises: less than 10% w/w palmitic-palmitic-palmitic triglycerides; greater than 10% w/w palmitic-oleic-palmitic triglycerides; and greater than 10% w/w oleic-oleic-palmitic triglycerides. The method of any one of embodiments 13-22, wherein the microbial oil comprises: less than 5% w/w palmitic-palmitic-palmitic triglycerides; greater than 15% w/w palmitic-oleic-palmitic triglycerides; and greater than 15% w/w oleic-oleic-palmitic triglycerides. The method of any one of embodiments 13-23, wherein the microbial oil comprises: a stearic-stearic-oleic triglyceride content of less than 10% w/w and a stearic-oleic-oleic triglyceride content of less than 10% w/w. The method of any one of embodiments 13-24, wherein between 10% and 15% of palmitic and/or stearic fatty acids comprised by the microbial oil are located at the sn- 2 position of triglyceride molecules. The method of any one of embodiments 13-25, wherein greater than 30% of the triglycerides comprised by the microbial oil have one unsaturated sidechain. The method of any one of embodiments 13-26, wherein greater than 30% of the triglycerides comprised by the microbial oil have two unsaturated sidechains. The method of any one of embodiments 13-27 wherein the microbial oil comprises ergosterol, at least 50 ppm ergosterol, or at least 100 ppm ergosterol. The method of any one of embodiments 13-28, wherein the microbial oil does not contain a phytosterol or chlorophyll. The method of any one of embodiments 13-29, wherein the microbial oil comprises a saponification value of 150-210. The method of any one of embodiments 13-30, wherein the microbial oil comprises an iodine value of 55-80. The method of any one of embodiments 13-31, wherein the microbial oil comprises a slip melting point of 10°C-40°C. The method of any one of embodiments 13-32, wherein the method further comprises at least one of physically refining, chemically refining, deodorizing, and bleaching the microbial oil. The method of any one of embodiments 13-33, wherein the microbial oil is produced by Rhodosporidium toruloides. The method of any one of embodiments 13-34, wherein the microbial oil derivative is a triglyceride, diglyceride, monoglyceride, free fatty acid, fatty acid salt, glycerin, fatty ester, fatty alcohol, fatty amine, fatty acid methyl ester, amide carboxylate, FOH ethoxylate, FOH sulfate, amine oxide, betaine, quat, ether sulfate, derivative thereof, or combination thereof. The method of any one of embodiments 13-35, wherein the microbial oil derivative is selected from the list consisting of: C16-18 fatty acids; sodium salts of C12-18 fatty acids; MEA salts of Cl 2- 18 fatty acids; sodium C12-C15 alkyl sulfates; MEA C12-C15 alkyl ether sulfates; C10-C16 alkyl dimethyl amine oxide; ethoxylated C12-C16 alcohols; D-glucopyranose, oligomeric, CIO-16 alkylglycosides; sodium C14-C17 alcohol sulfate; Cl 6- 18 glycerides; sodium C14-C17 sec-alkyl sulfonates; C8-C10 alkyl polyglucosides; methyl ester sulfonates; and esterquats. The method of any one of embodiments 13-36, wherein the microbial oil derivative is selected from the list consisting of: sodium lauryl sulfate; sodium laureth sulfate; sodium lauryl ether sulfate; sodium oleate; MEA laureth sulfate; MEA lauryl sulfate; laureth-6; laureth-9; glycerin; D-glucopyranose, oligomeric, decyl octyl glycoside; hydrogenated castor oil; coconut fatty acid; canola-ami doethyl hydroxyethylammonium methyl sulfate; dipalmethyl hydroxyethylammonium methosulfate; dihydrogenated palmoylethyl hydroxethylmonium methyl sulfate; di (palm carboxy ethyl) hydroxy ethyl methyl ammonium methyl sulfate; lauramine oxide; capryloyl methyl glucamide; caproyl methyl glucamide; soy methyl ester ethoxylate; coco methyl ester ethoxylate; lauryl betaine; lauryl di-methyl amine oxide; decyl glucoside; lauryl glucoside; capryl glucoside; caprylyl glucoside; myristyl glucoside; isostearyl palmitate; polyglyceryl-4 dipalmitate; sorbitan palmitate; polyglyceryl- 10 dioleate; polyglyceryl-4 oleate; retinyl palmitate; ascorbyl palmitate; sucrose palmitate; and ethyl palmate. The method of any one of embodiments 13-37, wherein the derivative is isostearyl palmitate. The method of any one of embodiments 13-37, wherein the derivative is polyglyceryl- 4 dipalmitate. The method of any one of embodiments 13-37, wherein the derivative is sorbitan palmitate. The method of any one of embodiments 13-37, wherein the derivative is polyglyceryl- 10 di oleate. The method of any one of embodiments 13-37, wherein the derivative is polyglyceryl- 4 oleate. 43. The method of any one of embodiments 13-37, wherein the derivative is retinyl palmitate.

44. The method of any one of embodiments 13-37, wherein the derivative is ascorbyl palmitate.

45. The method of any one of embodiments 13-37, wherein the derivative is sucrose palmitate.

46. The method of any one of embodiments 13-37, wherein the derivative is ethyl palmate.

47. The method of any one of embodiments 13-37, wherein the derivative is a methyl ester sulfonate.

48. The method of any one of embodiments 13-37, wherein the derivative is an esterquat.

49. An isostearyl palmitate produced by the method of any one of embodiments 13-37.

50. A polyglyceryl-4 dipalmitate produced by the method of any one of embodiments 13- 37.

51. A sorbitan palmitate produced by the method of any one of embodiments 13-37.

52. A polyglyceryl- 10 dioleate produced by the method of any one of embodiments 13-37.

53. A polyglyceryl-4 oleate produced by the method of any one of embodiments 13-37.

54. A retinyl palmitate produced by the method of any one of embodiments 13-37.

55. An ascorbyl palmitate produced by the method of any one of embodiments 13-37.

56. A sucrose palmitate produced by the method of any one of embodiments 13-37.

57. An ethyl palmate produced by the method of any one of embodiments 13-37.

58. A methyl ester sulfonate produced by the method of any one of embodiments 13-37.

59. An esterquat produced by the method of any one of embodiments 13-37.

INCORPORATION BY REFERENCE

[280] 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 applications are incorporated herein by reference in their entirety: International Patent Publication Nos. WO2021/154863 and WO2021/163194 and International Patent Application Nos. PCT/US2021/059122 and PCT/US2021/059147.