# Patent

## Title
Lauric ester compositions

## Abstract

Provided are compositions containing alkyl esters derived from triglyceride oils produced from genetically engineered microalgae. Specific embodiments relate to esters derived from oils with high C10-C12 fatty acid profile. Compositions comprising the esters include cleaning products, completion fluids, work-over fluids, drilling fluids, metal working fluids, lubricants, paints, and inks.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 61/972,026, filed Mar. 28, 2014, which is incorporated herein by reference in its entirety for all purposes.


REFERENCE TO A SEQUENCE LISTING
This application includes an electronic sequence listing in a filed named “460107-Sequence.txt”, created on Jun. 10, 2015, and containing 10,834 bytes, which is hereby incorporated by reference in its entirety for all purposes.
FIELD
Embodiments of the present invention relate to compositions containing alkyl esters derived from triglyceride oils produced from genetically engineered microalgae. Specific embodiments relate to esters derived from oils with high C10-C12 fatty acid profile. Compositions comprising the esters include cleaning products, completion fluids, work-over fluids, drilling fluids, metal working fluids, lubricants, paints, and inks.
BACKGROUND
Monoalkyl esters derived from triglyceride vegetable oils and animal fats find use as environmentally friendly compounds in variety of applications ranging from fuels to solvents. Triglycerides contain fatty acid chains that vary in length, but typically have a fatty acid profile favoring chain lengths of 12, 16, and/or 18 carbons. A fatty acid profile high in lauric acid, a 12 carbon fatty acid, is characteristic of coconut or palm kernel oils. Transesterification of these oils with an alkyl alcohol forms esters rich in laurates. However as noted in U.S. Pat. No. 8,617,317, fatty acid esters such as lauryl methyl esters have unpleasant and potent odors. These disadvantages limit acceptance of their use, particularly in applications with human exposure, for use in areas with inadequate ventilation, and/or where large quantities of the esters are required. Illustrative applications where unpleasant odor is disadvantageous include use as cleaning agents in personal care products such as hand cleaners.
Following drilling and cementing operations in an oil or gas well, a well remediation process is often necessary prior to production of the oil or gas. Drilling fluid employed in the drilling well typically contains viscosifiers and other additives that are necessary for suspending and removing the drill cuttings and for maintaining stability of the well. However these and other unwanted downhole products and deposits can prevent efficient oil/gas production such as by partially occluding the well. Completion fluids having cleaning properties capable of removing these unwanted products are thus highly desirable for maximizing flow of the production oil or gas.
In the use of a cutting or shaping tool on a workpiece, friction between the tool and the workpiece can cause wear on the tool, hinder the cutting, metal forming, or stamping process, lead to slow manufacturing cycles, and negatively affect the quality and finish of the workpiece. Lubricants are typically used to overcome these undesirable effects. In choosing the appropriate lubricants, consideration also needs to be given to the compatibility of the lubricant in the application, whether the lubricant can operate efficiently under the conditions of its use, and the ease with which the lubricant can be removed if necessary. Of further importance is the environmental impact of the lubricant in its use and disposal, and on the health of workers using the lubricant. In this regard, properties such as the biodegradability and VOC (volatile organic compound) content of the lubricant are important considerations.
SUMMARY
In one embodiment, provided is a composition comprising C1-C4 esters of fatty acids derived from oil produced by a microalgae. In some embodiments, the oil has a fatty acid profile of at least 10% C10:0 and 40% C12:0 fatty acids. The esters derived from the microalgal oils having high C10-C12 content are surprisingly found to have a reduced odor compared to the corresponding esters derived from vegetable or animal triglyceride oils with high C12 content. In some embodiments the esters provided herein and/or compositions containing the esters have one or more features of improved odor, faster drying rate, and lower VOC content in comparison to vegetable or animal based esters such as those with at least 40% C12:0 fatty acids. In some embodiments the esters provided herein are unfractionated or and/or are formed from unfractionated natural oils.
In one embodiment, provided is a composition comprising C1-C4 esters of fatty acids derived from oil produced by a microalgae, the oil having a fatty acid profile of at least 10% C10:0 and 40% C12:0 fatty acids, the composition having a reduced odor compared to a composition where all the fatty acids are derived from vegetable or animal triglyceride oil having a fatty acid profile of at least 40% C12:0 fatty acids. In some embodiments, the unpleasant odor associated with the vegetable or animal oil is characteristic of a C6 or C8 fatty acid or ester thereof.
In some embodiments, the C1-C4 ester is a methyl, ethyl, propyl, iso-propyl, butyl, or a tert-butyl ester. In some embodiments, the ester is a methyl ester. In some embodiments, the ester is an ethyl ester. In some embodiments, the ester is an iso-propyl ester.
In some embodiments, the fatty acid profile is characterized wherein C12:0>C10:0>C14:0.
In some embodiments, at least 50% of the fatty acids are C12:0 fatty acids. In some embodiments, at least 60% of the fatty acids are C:10 and C:12 fatty acids.
In some embodiments, at least 15% of the fatty acids are C10:0 fatty acids. In some embodiments, 15-25% of the fatty acids are C10:0 fatty acids.
In some embodiments, 10-15% of the fatty acids are C14:0 fatty acids.
In some embodiments, less than 8%, 7%, or 6% of the fatty acids are C16:0 fatty acids.
In some embodiments, less than 0.5% of the fatty acids are C8:0 fatty acids.
In some embodiments, less than 0.1%, 0.01%, or 0.001% of the fatty acids are C6:0 fatty acids.
In some embodiments, the compositions provided herein comprise sterols from the microalgae, wherein the amount of C28 sterols is greater than C29 sterols. In some embodiments, the C28 sterol is ergosterol. In some embodiments, β-sitosterol, camperserol, stigmaserol, or cholesterol are not the most abundant sterols.
In some embodiments, the compositions provided herein comprise one or more of: ergosterol;
ergosterol and β-sitosterol, wherein the ratio of ergosterol to β-sitosterol is greater than 6:1; ergosterol and brassicasterol;
ergosterol, brassicasterol, and poriferasterol; and
wherein the composition is optionally free from one or more of β-sitosterol, campesterol, and stigmasterol.
In some embodiments, the compositions provided herein comprise a terpene. In some embodiments, the terpene is limonene.
In some embodiments, the compositions provided herein comprise a C1-C4 ester of lactic acid. In some embodiments, the lactic acid is ethyl lactate.
In some embodiments, the compositions provided herein comprise a surfactant. In some embodiments, the surfactant is a non-ionic surfactant.
In some embodiments, the compositions provided herein comprise an emulsifier.
In some embodiments, the compositions provided herein comprise water.
In some embodiments, provided is a microalgal cell comprising an exogenous nucleic acid having at least a 85% sequence identity to SEQ ID NO: 1. In some embodiments, the exogenous nucleic acid has at least a 90% sequence identity to SEQ ID NO: 1. In other embodiments, the exogenous nucleic acid has at least a 99% sequence identity to SEQ ID NO: 1.
In some embodiments, the microalgae is Parachlorella, Chlorella, or Prototheca. In some embodiments, the microalgae is Prototheca. In some embodiments, the microalgae is Prototheca moriformis. 
In some embodiments, provided is a method for preparing a composition comprising C1-C4 esters of fatty acids derived from oil produced by a microalgae, the composition having a reduced odor compared to a composition where all the fatty acids are derived from vegetable oil, the method comprising:
a) providing oil produced by a microalgae, the oil comprising triacylglycerides having a fatty acid profile wherein the most abundant fatty acids are C12:0 fatty acids; and
b) treating the triacylglycerides with a C1-C4 alcohol under transesterification conditions to form C1-C4 esters of the microalgal derived fatty acids.
In some embodiments, provided is a method for preparing a composition comprising C1-C4 esters of fatty acids derived from oil produced by a microalgae, the composition having a reduced odor compared to a composition where all the fatty acids are derived from vegetable oil, the method comprising:
a) culturing a microalgal cell comprising an exogenous nucleic acid sequence to produce an oil comprising triacylglycerides having a fatty acid profile wherein the most abundant fatty acids are C12:0 fatty acids; and
b) treating the oil with a C1-C4 alcohol under transesterification conditions to form C1-C4 esters of microalgal derived fatty acids.
In some embodiments, the C1-C4 alcohol is methanol, ethanol, propanol, iso-propanol, butanol, or a tert-butanol. In some embodiments, the C1-C4 alcohol is methanol. In some embodiments, the C1-C4 alcohol is ethanol. In some embodiments, the C1-C4 alcohol is iso-propanol.
In some embodiments, the transesterification conditions comprise an acid or base catalyst. In some embodiments, the base catalyst is KOH.
In some embodiments, the method provided herein further comprise blending the C1-C4 esters of fatty acids with one or more of a terpene, a C1-C4 ester of lactic acid, a surfactant, an emulsifier, or water. In some embodiments, the terpene is limonene. In some embodiments, the C1-C4 ester of lactic acid is ethyl lactate.
In some embodiments, the composition is a cleaner, a corrosion inhibitor, a lubricant, a metal working fluid, an ink carrier, a mold release agent, a polystyrene recycling solvent, a drilling fluid, a drilling loss control agent, a completion fluid, a work-over fluid, or a combination thereof.
In some embodiments, the cleaner is an adhesive remover, a driveway cleaner, a degreaser, a graffiti remover, hand cleaner, a waterless hand cleaner, a paint brush cleaner, a paint stripper, a varnish remover, a rubber tire remover, a wipe, a bug remover, a tar remover, a car wash cleaner, a parts cleaner, an engine cleaner, a fuel system additive, a rig wash, a heavy residue cleaner, a down-hole cleaner, a concrete down-hole cleaner, an ink roller cleaner, or a screen printing ink wash or a combination thereof. In some embodiments, provided are cleaners for paints, inks, coatings, sealants, elastomers, and polymers.
In some embodiments, the lubricant is a hydraulic fluid, a gear lube, a bearing lube, crankcase lube, a cylinder lube, a compressor lube, a turbine lube, a chain lube, a conveyor lube, a total-loss lube, wire rope lube, a cutting lube, stamping lube, a metal forming lube, a food grade lube, a grease, or a 2-stroke engine lube or a combination thereof.
In some embodiments, the metal working fluid is a metal forming, metal cutting, or a metal finishing fluid or a combination thereof.
In some embodiments, provided is a paint or ink.
In some embodiment provided is an oil & gas down-hole fluids or mud comprising an alkyl ester provided herein.
In some embodiments, the drilling fluid is a water based drilling fluid.
In some embodiments, provided is a method for treating a wellbore, comprising administering the composition provided herein. In some embodiments, the composition removes unwanted deposits. In some embodiments the unwanted deposit is crude oil. In some embodimetns, the unwanted deposit is asphaltenes, hydrogen sulfide, paraffins, scales, fines sulfur, heavy oil by-products, water blocks, drilling fluids, cement filtrates, kill fluids, pipe, dope, hydrocarbon emulsions, oil based muds, or water based muds.
In some embodiments, the wellbore is a vertical, horizontal, or deviated wellbore. In some embodiments, the wellbore is a vertical or horizontal wellbore. In other embodiments, the wellbore comprises a cement casing. In some embodiments the compositions provided herein are for use before or after cementing of a wellbore.


DETAILED DESCRIPTION
I. Definitions
“Biomass” is material produced by growth and/or propagation of cells. Biomass may contain cells and/or intracellular contents as well as extracellular material, includes, but is not limited to, compounds secreted by a cell. Biomass isolated from fermentation broth may include nutrients and feedstock used to grow the cells.
“Exogenous gene” shall mean a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g. by transformation/transfection), and is also referred to as a “transgene”. A cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from a different species (and so heterologous), or from the same species (and so homologous), relative to the cell being transformed. Thus, an exogenous gene can include a homologous gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.
“Fatty acids” shall mean free fatty acids, fatty acid salts, or fatty acyl moieties in a glycerolipid. It will be understood that fatty acyl groups of glycerolipids can be described in terms of the carboxylic acid or anion of a carboxylic acid that is produced when the triglyceride is hydrolyzed or saponified.
“Fixed carbon source” is a molecule(s) containing carbon, typically an organic molecule that is present at ambient temperature and pressure in solid or liquid form in a culture media that can be utilized by a microorganism cultured therein. Accordingly, carbon dioxide is not a fixed carbon source.
“In operable linkage” is a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is in operable linkage with an exogenous gene if it can mediate transcription of the gene.
“Microalgae” are eukaryotic microbial organisms that contain a chloroplast or other plastid, and optionally that is capable of performing photosynthesis, or a prokaryotic microbial organism capable of performing photosynthesis. Microalgae include obligate photoautotrophs, which cannot metabolize a fixed carbon source as energy, as well as heterotrophs, which can live solely off of a fixed carbon source. Microalgae include unicellular organisms that separate from sister cells shortly after cell division, such as Chlamydomonas, as well as microbes such as, for example, Volvox, which is a simple multicellular photosynthetic microbe of two distinct cell types. Microalgae include cells such as Chlorella, Dunaliella, and Prototheca. Microalgae also include other microbial photosynthetic organisms that exhibit cell-cell adhesion, such as Agmenellum, Anabaena, and Pyrobotrys. Microalgae also include obligate heterotrophic microorganisms that have lost the ability to perform photosynthesis, such as certain dinoflagellate algae species and species of the genus Prototheca. 
An “oleaginous” cell is a cell capable of producing at least 20% lipid by dry cell weight, naturally or through recombinant or classical strain improvement. An “oleaginous microbe” or “oleaginous microorganism” is a microbe, including a microalga that is oleaginous. An oleaginous cell also encompasses a cell that has had some or all of its lipid or other content removed, and both live and dead cells.
A “natural oil” or “natural fat” shall mean a predominantly triglyceride oil obtained from an organism, where the oil has not undergone blending with another natural or synthetic oil, or fractionation so as to substantially alter the fatty acid profile of the triglyceride. In connection with an oil comprising triglycerides of a particular regiospecificity, the natural oil or natural fat has not been subjected to interesterification or other synthetic process to obtain that regiospecific triglyceride profile, rather the regiospecificity is produced naturally, by a cell or population of cells. In connection with a natural oil or natural fat, and as used generally throughout the present disclosure, the terms oil and fat are used interchangeably, except where otherwise noted. Thus, an “oil” or a “fat” can be liquid, solid, or partially solid at room temperature, depending on the makeup of the substance and other conditions. Here, the term “fractionation” means removing material from the oil in a way that changes its fatty acid profile relative to the profile produced by the organism, however accomplished. The terms “natural oil” and “natural fat” encompass such oils obtained from an organism, where the oil has undergone minimal processing, including refining, bleaching and/or degumming, that does not substantially change its triglyceride profile. A natural oil can also be a “noninteresterified natural oil”, which means that the natural oil has not undergone a process in which fatty acids have been redistributed in their acyl linkages to glycerol and remain essentially in the same configuration as when recovered from the organism.
In connection with a natural oil, a “profile” is the distribution of particular species or triglycerides or fatty acyl groups within the oil. A “fatty acid profile” is the distribution of fatty acyl groups in the triglycerides of the oil without reference to attachment to a glycerol backbone. Fatty acid profiles are typically determined by conversion to a fatty acid methyl ester (FAME), followed by gas chromatography (GC) analysis with flame ionization detection (FID). The fatty acid profile can be expressed as one or more percent of a fatty acid in the total fatty acid signal determined from the area under the curve for that fatty acid. FAME-GC-FID measurement approximate weight percentages of the fatty acids. A “sn-2 profile” is the distribution of fatty acids found at the sn-2 position of the triacylglycerides in the oil. A “regiospecific profile” is the distribution of triglycerides with reference to the positioning of acyl group attachment to the glycerol backbone without reference to stereospecificity. In other words, a regiospecific profile describes acyl group attachment at sn-1⁄3 vs. sn-2. Thus, in a regiospecific profile, POS (palmitate-oleate-stearate) and SOP (stearate-oleate-palmitate) are treated identically. A “stereospecific profile” describes the attachment of acyl groups at sn-1, sn-2 and sn-3. Unless otherwise indicated, triglycerides such as SOP and POS are to be considered equivalent. A “TAG profile” is the distribution of fatty acids found in the triglycerides with reference to connection to the glycerol backbone, but without reference to the regiospecific nature of the connections. Thus, in a TAG profile, the percent of SSO in the oil is the sum of SSO and SOS, while in a regiospecific profile, the percent of SSO is calculated without inclusion of SOS species in the oil. In contrast to the weight percentages of the FAME-GC-FID analysis, triglyceride percentages are typically given as mole percentages; that is the percent of a given TAG molecule in a TAG mixture.
The term “percent sequence identity,” in the context of two or more amino acid or nucleic acid sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. For sequence comparison to determine percent nucleotide or amino acid identity, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for comparison can be conducted using the NCBI BLAST software (ncbi.nlm.nih.gov/BLAST/) set to default parameters. For example, to compare two nucleic acid sequences, one may use blastn with the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) set at the following default parameters: Matrix: BLOSUM62; Reward for match: 1; Penalty for mismatch: −2; Open Gap: 5 and Extension Gap: 2 penalties; Gap×drop-off: 50; Expect: 10; Word Size: 11; Filter: on. For a pairwise comparison of two amino acid sequences, one may use the “BLAST 2 Sequences” tool Version 2.0.12 (Apr. 21, 2000) with blastp set, for example, at the following default parameters: Matrix: BLOSUM62; Open Gap: 11 and Extension Gap: 1 penalties; Gap×drop-off 50; Expect: 10; Word Size: 3; Filter: on.
“Recombinant” is a cell, nucleic acid, protein or vector that has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi) or dsRNA that reduce the levels of active gene product in a cell. A “recombinant nucleic acid” is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, using chemical synthesis, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention. Similarly, a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.
The terms “triglyceride”, “triacylglyceride” and “TAG” are used interchangeably as is known in the art.
“Cleaners” or “solvents” refers to substances and products used (a) to wash, de-grease, clean, disinfect, buff, polish, shine or protect (i) buildings and facilities (including, without limitation, homes, factories, offices, hotels, convention centers, hospitals and medical facilities, schools and educational facilities, shops, restaurants, places of business, government and military facilities, warehouses and storage facilities, public and private utilities, oil and gas production rigs, parks and recreation facilities), (ii) hard surfaces (including, without limitation, floors, walls, ceilings, doors, windows, counters, tables, chairs, kitchen and appliance surfaces, laboratory surfaces and toilets), (iii) human or animal skin, and (iv) automotive or other transportation interior or exterior surfaces; (b), as fuel system additives. These cleaners include those commonly characterized in the cleaning industry as Home, Industrial & Institutional Cleaners.
“Lubricants” refers to substances introduced to reduce friction between moving surfaces. Industrial applications for lubricants include but are not limited to hydraulic fluids, industrial lubricants (such as gear oils, bearing oils, crankcase oils, cylinder oils, compressor oils, turbine oils, chain & conveyor oils, “total loss” lubricants, wire rope lubricants, cutting fluids, stamping lubes, metal forming lubes, food grade lubricants, greases, and engine oils such as two-stroke engine oils
“Metalworking fluids” refers to fluids used in working with metals to create individual parts, assemblies, or large-scale structures. The fluids can be used in metal forming (e.g. sizing, casting, forging, extruding, heading, stamping, drawing, bending, hardening, rolling, shearing, blanking, fineblanking, coining), metal cutting (e.g. machining, turning, milling, drilling, grinding, sawing, threading, reaming, gun drilling, broaching, riveting, cooling, cleaning), and metal finishing (e.g. de-burring, grinding, brushing, buffing, polishing, vibratory finishing).
“Oil & Gas Down-Hole Fluids and Muds” refers to substances or formulations (both oil and water based) introduced down-hole before, during or after oil and gas exploration, drilling, cementing and completion, production, stimulation, enhanced oil recovery and clean-up and remediation.
II. General
Illustrative embodiments of the present invention feature oleaginous cells that produce altered fatty acid profiles and/or altered regiospecific distribution of fatty acids in glycerolipids, and products produced from the cells. Examples of oleaginous cells include microbial cells having a type II fatty acid biosynthetic pathway, including plastidic oleaginous cells such as those of oleaginous algae. Specific examples of cells include heterotrophic or obligate heterotrophic microalgae of the phylum Chlorophtya, the class Trebouxiophytae, the order Chlorellales, or the family Chlorellacae. Examples of oleaginous microalgae and methods of cultivation are also provided in Published PCT Patent Applications WO2008/151149, WO2010/06032, WO2011/150410, WO2011/150411, WO2012/061647, and WO2012/106560, including species of Chlorella and Prototheca, a genus comprising obligate heterotrophs. The oleaginous cells can be, for example, capable of producing 25, 30, 40, 50, 60, 70, 80, 85, or about 90% oil by cell weight, ±5%. Optionally, the oils produced can be low in highly unsaturated fatty acids such as DHA or EPA fatty acids. For example, the oils can comprise less than 5%, 2%, or 1% DHA and/or EPA. In some cases, the microalgal oils have a high oleic and low polyunsaturated fatty acid profile and derivatives of the oils, including acids, esters, epoxides, hydroxylated acids and esters, urethanes, amides, and polymers can be prepared with the same attributes for use in foodstuffs and in industrial and material applications. The above-mentioned publications also disclose methods for cultivating such cells and extracting oil, especially from microalgal cells; such methods are applicable to the cells disclosed herein and incorporated by reference for these teachings. When microalgal cells are used they can be cultivated autotrophically (unless an obligate heterotroph) or in the dark using a sugar (e.g., glucose, fructose and/or sucrose) In any of the embodiments described herein, the cells can be heterotrophic cells comprising an exogenous invertase gene so as to allow the cells to produce oil from a sucrose feedstock. Alternately, or in addition, the cells can metabolize xylose from cellulosic feedstocks. For example, the cells can be genetically engineered to express one or more xylose metabolism genes such as those encoding an active xylose transporter, a xylulose-5-phosphate transporter, a xylose isomerase, a xylulokinase, a xylitol dehydrogenase and a xylose reductase. See WO2012/154626, “GENETICALLY ENGINEERED MICROORGANISMS THAT METABOLIZE XYLOSE”, published Nov. 15, 2012.
The oleaginous cells may, optionally, be cultivated in a bioreactor/fermenter. For example, heterotrophic oleaginous microalgal cells can be cultivated on a sugar-containing nutrient broth. Optionally, cultivation can proceed in two stages: a seed stage and a lipid-production stage. In the seed stage, the number of cells is increased from s starter culture. Thus, the seeds stage typically includes a nutrient rich, nitrogen replete, media designed to encourage rapid cell division. After the seeds stage, the cells may be fed sugar under nutrient-limiting (e.g. nitrogen sparse) conditions so that the sugar will be converted into triglycerides. For example, the rate of cell division in the lipid-production stage can be decreased by 50%, 80% or more relative to the seed stage. Additionally, variation in the media between the seed stage and the lipid-production stage can induce the recombinant cell to express different lipid-synthesis genes and thereby alter the triglycerides being produced. For example, as discussed below, nitrogen and/or pH sensitive promoters can be placed in front of endogenous or exogenous genes. This is especially useful when an oil is to be produced in the lipid-production phase that does not support optimal growth of the cells in the seed stage. In an example below, a cell has a fatty acid desaturase with a pH sensitive promoter so than an oil that is low in linoleic acid is produced in the lipid production stage while an oil that has adequate linoleic acid for cell division is produced during the seed stage. The resulting low linoleic oil has exceptional oxidative stability.
The oleaginous cells express one or more exogenous genes encoding fatty acid biosynthesis enzymes. As a result, some embodiments feature natural oils that were not obtainable from a non-plant or non-seed oil, or not obtainable at all.
The oleaginous cells produce a storage oil, which is primarily triacylglyceride and may be stored in storage bodies of the cell. A raw oil may be obtained from the cells by disrupting the cells and isolating the oil. The raw oil may comprise sterols produced by the cells. WO2008/151149, WO2010/06032, WO2011/150410, WO2011/1504 WO2012/061647, and WO2012/106560 disclose heterotrophic cultivation and oil isolation techniques. For example, oil may be obtained by providing or cultivating, drying and pressing the cells. The oils produced may be refined, bleached and deodorized (RBD) as known in the art or as described in WO2010/120939. The raw or RBD oils may be used in a variety of food, chemical, and industrial products or processes. Even after such processing, the oil may retain a sterol profile characteristic of the source. Microalgal sterol profiles are disclosed below.
In some embodiments the triglycerides can be isolated from oleaginous microbes by mechanical pressing with pressure sufficient to extract oil. In various embodiments, the pressing step will involve subjecting the oleaginous microbes to at least 10,000 psi of pressure. In various embodiments, the pressing step involves the application of pressure for a first period of time and then application of a higher pressure for a second period of time. This process may be repeated one or more times (“oscillating pressure”). In various embodiments, moisture content of the oleaginous microbes is controlled during the pressing step. In various embodiments, the moisture is controlled in a range of from 0.1% to 3% by weight.
Expeller presses (screw presses) are routinely used for mechanical extraction of oil from soybeans and oil seeds. Generally, the main sections of an expeller press include an intake, a rotating feeder screw, a cage or barrel, a worm shaft and an oil pan. The expeller press is a continuous cage press, in which pressure is developed by a continuously rotating worm shaft. An extremely high pressure, approximately 10,000-20,000 pounds per square inch, is built up in the cage or barrel through the action of the worm working against an adjustable choke, which constricts the discharge of the pressed cake (spent biomass) from the end of the barrel. In various embodiments, screw presses from the following manufacturers are suitable for use: Anderson International Corp. (Cleveland, Ohio), Alloco (Santa Fe, Argentina), De Smet Rosedowns (Humberside, UK), The Dupps Co. (Germantown, Ohio), Grupo Tecnal (Sao Paulo, Brazil), Insta Pro (Des Moines, Iowa), French Oil Mill (Piqua, Ohio), Harburg Freudenberger (previously Krupp Extraktionstechnik) (Hamburg, Germany), Maschinenfabrik Reinartz (Neuss, Germany), Shann Consulting (New South Wales, Australia) and SKET (Magdeburg, Germany).
Where a fatty acid profile of a triglyceride (also referred to as a “triacylglyceride” or “TAG”) cell oil is given here, it will be understood that this refers to a nonfractionated sample of the storage oil extracted from the cell analyzed under conditions in which phospholipids have been removed or with an analysis method that is substantially insensitive to the fatty acids of the phospholipids (e.g. using chromatography and mass spectrometry). The oil may be subjected to an RBD process to remove phospholipids, free fatty acids and odors yet have only minor or negligible changes to the fatty acid profile of the triglycerides in the oil. Because the cells are oleaginous, in some cases the storage oil will constitute the bulk of all the TAGs in the cell.
III. Minor Oil Components
The oils produced according to the above methods in some cases are made using a microalgal host cell. As described above, the microalga can be, without limitation, fall in the classification of Chlorophyta, Trebouxiophyceae, Chlorellales, Chlorellaceae, or Chlorophyceae. It has been found that microalgae of Trebouxiophyceae can be distinguished from vegetable oils based on their sterol profiles. Oil produced by Chlorella protothecoides was found to produce sterols that appeared to be brassicasterol, ergosterol, campesterol, stigmasterol, and β-sitosterol, when detected by GC-MS. However, it is believed that all sterols produced by Chlorella have C24β stereochemistry. Thus, it is believed that the molecules detected as campesterol, stigmasterol, and β-sitosterol, are actually 22,23-dihydrobrassicasterol, poriferasterol and clionasterol, respectively. Thus, the oils produced by the microalgae described above can be distinguished from plant oils by the presence of sterols with C24β stereochemistry and the absence of C24α stereochemistry in the sterols present. For example, the oils produced may contain 22,23-dihydrobrassicasterol while lacking campesterol; contain clionasterol, while lacking in β-sitosterol, and/or contain poriferasterol while lacking stigmasterol. Alternately, or in addition, the oils may contain significant amounts of Δ7-poriferasterol.
In one embodiment, the oils provided herein are not vegetable oils. Vegetable oils are oils extracted from plants and plant seeds. Vegetable oils can be distinguished from the non-plant oils provided herein on the basis of their oil content. A variety of methods for analyzing the oil content can be employed to determine the source of the oil or whether adulteration of an oil provided herein with an oil of a different (e.g. plant) origin has occurred. The determination can be made on the basis of one or a combination of the analytical methods. These tests include but are not limited to analysis of one or more of free fatty acids, fatty acid profile, total triacylglycerol content, diacylglycerol content, peroxide values, spectroscopic properties (e.g. UV absorption), sterol profile, sterol degradation products, antioxidants (e.g. tocopherols), pigments (e.g. chlorophyll), d13C values and sensory analysis (e.g. taste, odor, and mouth feel). Many such tests have been standardized for commercial oils such as the Codex Alimentarius standards for edible fats and oils.
Sterol profile analysis is a particularly well-known method for determining the biological source of organic matter. Campesterol, β-sitosterol, and stigmasterol are common plant sterols, with β-sitosterol being a principle plant sterol. For example, β-sitosterol was found to be in greatest abundance in an analysis of certain seed oils, approximately 64% in corn, 29% in rapeseed, 64% in sunflower, 74% in cottonseed, 26% in soybean, and 79% in olive oil (Gul et al. J. Cell and Molecular Biology 5:71-79, 2006).
Oil isolated from Prototheca moriformis strain UTEX 1435 were separately clarified (CL), refined and bleached (RB), or refined, bleached and deodorized (RBD) and were tested for sterol content according to the procedure described in JAOCS vol. 60, no. 8, Aug. 1983. Results of the analysis are shown below (units in mg/100 g):


















Refined,





Refined &
bleached, &


Sterol
Crude
Clarified
bleached
deodorized







Ergosterol
384  
398  
293  
302  



 (56%)
 (55%)
 (50%)
 (50%)


5,22-cholestadien-24-
14.6
18.8
14  
15.2


methyl-3-ol
(2.1%)
(2.6%)
(2.4%)
(2.5%)


(Brassicasterol)


24-methylcholest-5-
10.7
11.9
10.9
10.8


en-3-ol (Campesterol
(1.6%)
(1.6%)
(1.8%)
(1.8%)


or 22,23-


dihydrobrassicasterol)


5,22-cholestadien-24-
57.7
59.2
46.8
49.9


ethyl-3-ol
(8.4%)
(8.2%)
(7.9%)
(8.3%)


(Stigmasterol or


poriferasterol)


24-ethylcholest-5-en-
 9.64
 9.92
 9.26
10.2


3-ol (β-Sitosterol or
(1.4%)
(1.4%)
(1.6%)
(1.7%)


clionasterol)


Other sterols
209  
221  
216  
213  


Total sterols
685.64
718.82
589.96
601.1 









These results show three striking features. First, ergosterol was found to be the most abundant of all the sterols, accounting for about 50% or more of the total sterols. The amount of ergosterol is greater than that of campesterol, β-sitosterol, and stigamsterol combined. Ergosterol is steroid commonly found in fungus and not commonly found in plants, and its presence particularly in significant amounts serves as a useful marker for non-plant oils. Secondly, the oil was found to contain brassicasterol. With the exception of rapeseed oil, brassicasterol is not commonly found in plant based oils. Thirdly, less than 2% β-sitosterol was found to be present. β-sitosterol is a prominent plant sterol not commonly found in microalgae, and its presence particularly in significant amounts serves as a useful marker for oils of plant origin. In summary, Prototheca moriformis strain UTEX1435 has been found to contain both significant amounts of ergosterol and only trace amounts of β-sitosterol as a percentage of total sterol content. Accordingly, the ratio of ergosterol:β-sitosterol or in combination with the presence of brassicasterol can be used to distinguish this oil from plant oils.
In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% β-sitosterol. In other embodiments the oil is free from β-sitosterol.
In some embodiments, the oil is free from one or more of β-sitosterol, campesterol, or stigmasterol. In some embodiments the oil is free from β-sitosterol, campesterol, and stigmasterol. In some embodiments the oil is free from campesterol. In some embodiments the oil is free from stigmasterol.
In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24-ethylcholest-5-en-3-ol. In some embodiments, the 24-ethylcholest-5-en-3-ol is clionasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% clionasterol.
In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 24-methylcholest-5-en-3-ol. In some embodiments, the 24-methylcholest-5-en-3-ol is 22,23-dihydrobrassicasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% 22,23-dihydrobrassicasterol.
In some embodiments, the oil content of an oil provided herein contains, as a percentage of total sterols, less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% 5,22-cholestadien-24-ethyl-3-ol. In some embodiments, the 5,22-cholestadien-24-ethyl-3-ol is poriferasterol. In some embodiments, the oil content of an oil provided herein comprises, as a percentage of total sterols, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% poriferasterol.
In some embodiments, the oil content of an oil provided herein contains ergosterol or brassicasterol or a combination of the two. In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 40% ergosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of a combination of ergosterol and brassicasterol.
In some embodiments, the oil content contains, as a percentage of total sterols, at least 1%, 2%, 3%, 4% or 5% brassicasterol. In some embodiments, the oil content contains, as a percentage of total sterols less than 10%, 9%, 8%, 7%, 6%, or 5% brassicasterol.
In some embodiments the ratio of ergosterol to brassicasterol is at least 5:1, 10:1, 15:1, or 20:1.
In some embodiments, the oil content contains, as a percentage of total sterols, at least 5%, 10%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% ergosterol and less than 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% β-sitosterol. In some embodiments, the oil content contains, as a percentage of total sterols, at least 25% ergosterol and less than 5% β-sitosterol. In some embodiments, the oil content further comprises brassicasterol.
Sterols contain from 27 to 29 carbon atoms (C27 to C29) and are found in all eukaryotes. Animals exclusively make C27 sterols as they lack the ability to further modify the C27 sterols to produce C28 and C29 sterols. Plants however are able to synthesize C28 and C29 sterols, and C28/C29 plant sterols are often referred to as phytosterols. The sterol profile of a given plant is high in C29 sterols, and the primary sterols in plants are typically the C29 sterols β-sitosterol and stigmasterol. In contrast, the sterol profile of non-plant organisms contain greater percentages of C27 and C28 sterols. For example the sterols in fungi and in many microalgae are principally C28 sterols. The sterol profile and particularly the striking predominance of C29 sterols over C28 sterols in plants has been exploited for determining the proportion of plant and marine matter in soil samples (Huang, Wen-Yen, Meinschein W. G., “Sterols as ecological indicators”; Geochimica et Cosmochimia Acta. Vol 43. pp 739-745).
In some embodiments the primary sterols in the microalgal oils provided herein are sterols other than β-sitosterol and stigmasterol. In some embodiments of the microalgal oils, C29 sterols make up less than 50%, 40%, 30%, 20%, 10%, or 5% by weight of the total sterol content.
In some embodiments the microalgal oils provided herein contain C28 sterols in excess of C29 sterols. In some embodiments of the microalgal oils, C28 sterols make up greater than 50%, 60%, 70%, 80%, 90%, or 95% by weight of the total sterol content. In some embodiments the C28 sterol is ergosterol. In some embodiments the C28 sterol is brassicasterol.
IV. Drilling, Production, and Pumping-Services Fluids
The fluids provided herein include aqueous and non-aqueous drilling fluids and other well-related fluids including those used for production of oil or natural gas, for completion operations, sand control operations, workover operations, and for pumping-services such as cementing, hydraulic fracturing, and acidification. In one embodiment, a fluid includes a fluid loss control agent that is biomass from an oleaginous microbe. In one embodiment, the biomass comprises intact, lysed or partly lysed cells with greater than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% oil. In another embodiment, the biomass is spent biomass from which oil has been removed. For example, the oil may be removed by a process of drying and pressing and optionally solvent-extracting with hexane or other suitable solvent. In a specific embodiment, the biomass is dried to less than 6% moisture by weight, followed by application of pressure to release more than 25% of the lipid. Alternately, the cells may be intact, which, when used in a drilling fluid, may impart improved fluid-loss control in certain circumstances. Generally, the drilling fluid can contain about 0.1% to about 20% by weight of said biomass, but in various embodiments, this amount may range from about 0.1% to about 10% by weight of said biomass; from about 0.1% to about 5% by weight of said biomass; from about 0.5% to about 4% by weight of said biomass; and from about 1% to about 4% by weight of said biomass.
In various embodiments, the fluid comprises a fluid loss control agent that is not derived from oleaginous microbial biomass. Suitable fluid loss control agents may include, but are not limited to, unmodified starch, hydroxypropl starch, carboxymethyl starch, unmodified cellulose, carboxymethylcellulose, hydroxyethyl cellulose, and polyanionic cellulose.
The fluid can include an aqueous or non-aqueous solvent. The fluid can also optionally include one or more additional components so that the fluid is operable as a drilling fluid, a drill-in fluid, a workover fluid, a spotting fluid, a cementing fluid, a reservoir fluid, a production fluid, a fracturing fluid, or a completion fluid.
In various embodiments, the fluid is a drilling fluid and the added biomass from the oleaginous microbe serves to help transport cuttings, lubricate and protect the drill bit, support the walls of the well bore, deliver hydraulic energy to the formation beneath the bit, and/or to suspend cuttings in the annulus when drilling is stopped.
When used in a drilling fluid, the biomass may operate to occlude pores in the formation, and to form or promote the formation of a filter cake.
In various embodiments, the fluid is a production fluid and the biomass serves to inhibit corrosion, separate hydrocarbons from water, inhibit the formation of scale, paraffin, or corrosion (e.g., metal oxides), or to enhance production of oil or natural gas from the well. In an embodiment, the biomass is used to stimulate methanogenesis of microbes in the well. The biomass may provide nutrients and/or bind inhibitors so as to increase production of natural gas in the well. In this embodiment, the well can be a coal seam having methane generating capacity. See, for example, US Patent Application Nos. 2004/0033557, 2012/0021495, 2011/0284215, US2010/0248322, 2010/0248321, 2010/0035309, and 2007/0248531.
In various embodiments, the fluid comprises a viscosifier. Suitable viscosifiers include, but are not limited to, an alginate polymer selected from the group consisting of sodium alginate, sodium calcium alginate, ammonium calcium alginate, ammonium alginate, potassium alginate, propyleneglycol alginate, and mixtures thereof. Other suitable viscosifiers include organophillic clay, polyacrylamide, xanthan gum, and mixtures of xanthan gum and a cellulose derivative, including those wherein the weight ratio of xanthan gum to cellulose derivative is in the range from about 80:20 to about 20:80, and wherein the cellulose derivative is selected from the group consisting of hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose and mixtures thereof. Other suitable viscosifiers include a biopolymer produced by the action of bacteria, fungi, or other microorganisms on a suitable substrate.
Mixtures of a bentonitic clay and additives can also be used as viscosifiers. The additives used in such mixtures can comprise, for example: (a) a nonionic, water-soluble polysaccharide selected from the group consisting of a non-ionic, water-soluble cellulosic derivative and a non-ionic water-soluble guar derivative; (b) an anionic water-soluble polysaccharide selected from the group consisting of a carboxymethyl cellulose and Xanthomonas campestris polysaccharide or a combination thereof; (c) an intermediate molecular weight polyglycol, i.e., selected from the group consisting of polyethylene glycol, polypropylene glycol, and poly-(alkanediol), having an average molecular weight of from about 600 to about 30,000; and (5) compatible mixtures thereof. Components of the mixtures may be added individually to the fluid to enhance the low shear rate viscosity thereof.
In some embodiments, the drilling fluid comprises one or more additives selected from the group consisting of an aphron, polymer particle, a thermoset polymer particle, and a nanocomposite particulate.
Aphrons can be used as additives to drilling fluids and other fluids used in creating or maintaining a borehole. Aphrons can concentrate at the fluid front and act as a fluid loss control agent and/or bridging agent to build an internal seal of the pore network along the side walls of a borehole. It is believed that aphrons deform during the process of sealing the pores and gaps encountered while drilling a borehole. Aphrons useful in the present methods are typically 50-100 μM, 25-100 μM, 25-50 μM, 5-50, 5-25 μM, 7-15 μM or about 10 μM.
In one embodiment, a drilling fluid comprises aphrons, microbial biomass in which the oil has not been extracted (unextracted microbial biomass), spent biomass or a combination of aphrons, unextracted microbial biomass, and spent biomass.
Where an aphron is used, the aphron can have an average diameter of 5 to 50 micrometers and can make up about 0.001% to 5% by mass of the fluid.
The use of drilling fluids containing polymer particle additives has several applications in construction, drilling, completion, and fracture simulation of oil and natural gas wells. These particles are generally spherical in shape, solid, and have a specific gravity of 1.06. The use of these particles provides several advantages, such as increasing mechanical lubrication, reducing equipment wear, and aiding in directional changes during sliding. These particles are generally resistant to deformation loads of up to >25,000 psi hydrostatic, and they display excellent resistance and thermal stability even at temperatures greater than 450° F. in a drilling environment. These particles can also be manufactured in fine or coarse grades, depending on the requirements of a particular drilling operation.
Polymer particles are easily added to drilling fluid through a mud-mixing hopper machine. When used to control torque and drag, these beads can be applied at concentrations of 2-8 ppb (5.71-22.87 kilograms/m3). For spotting in wire-line operations and running casing, the polymer beads may be added to concentrations of 8-12 ppb (22.87-34.31 kilograms/m3).
In some embodiments, the drilling fluid comprises a thermoset polymer particle such as those disclosed in U.S. Pat. No. 8,088,718. In some embodiments, the drilling fluid comprises a nanocomposite particulate such as those disclosed in US 2005/0272611. In some embodiments, the drilling fluid comprises a co-polymer bead such as Alpine Drill Beads commercially available from Alpine Specialty Chemicals (Houston, Tex.).
Examples of other additives that may be used in drilling applications include, but are not limited to: alkalinity agents, corrosion inhibitors, defoamers, dispersants, emulsifiers, fluid loss control agents, foaming agent for gas-based fluids, intermediates for corrosion inhibitor, lubricants, misting agents, oxygen scavengers, hydrosulfite scavengers, biocides, scale inhibitors, scale removers, shale inhibitors, solvents, specialty surfactants, thermal stabilizers, viscosifiers, and water purifiers.
The additives disclosed herein, e.g., including the polymeric and glass bead additives, can contribute to bursting and releasing oil from the microbial cells. In such instances the additives work in concert with the cells to provide delay-released lubrication to the drill bit. Though not intended to be limited by the following mechanism, in one aspect this application is directed to a pressure sensitive lubricant that allows for time-delayed release of a lubricating oil by virtue of the oil being encapsulated within a cell. In instances when the lubricant is used in a drilling fluid, the pressure that triggers the oil to be released is provided by the drill string and/or drill bit. The oil is released only when sufficient downhole pressure and/or friction is present. Such pressure and friction is provided by the drill string and/or drill bit in its interaction with the well formation, such as when it is dragged along the well-bore (particularly in the non-vertical portions of the well-bore) or during the rotational motion of the drill string/drill bit during drilling.
Additives and lubricants to be used in combination with the oleaginous cells and oils provided herein include commercially available lubricants. These lubricants can be blended with oleaginous cells and oils produced by these cells. The commercially available lubricants include those marketed by Baker Hughes (RHEO-LOGIC, MAGMA-TEQ, CARBO-DRILL, MPRESS, PERFORMAX, PERFLEX, TERRA-MAX, PYRO-DRILL, MAX-BRIDGE, CHEK-LOSS, LC-LUBE, MIL-CARB, SOLUFLAKE, FLOW-CARB, X-LINK crosslinked composition, and SOLU-SQUEEZE LCM), Haliburton (BAROID, BOREMAX, PERFORMADRIL, SHALEDRIL, SUPER-SAT, and BaraECD) and Schlumberger (DRILPLEX, DURATHERM, ENVIROTHERM NT, GLYDRIL, K-MAG, KLA-SHIELD, SAGDRIL, ULTRADRIL, ECOGREEN, MEGADRIL, NOVAPLUS, PARADRIL, PARALAND, PARATHERM, RHADIANT, VERSACLEAN, VERSADRIL, and WARP fluids).
In various embodiments, the fluid comprises a density modifier, also known as a weighting agent or a weighting additive. Suitable density modifiers include, but are not limited to, barite, hematite, manganese oxide, calcium carbonate, iron carbonate, iron oxide, lead sulfide, siderate, and ilmenite.
In various embodiments, the fluid comprises an emulsifier. Suitable emulsifiers may be nonionic, including ethoxylated alkylphenols and ethoxylated linear alcohols, or anionic, including alkylaryl sulfonates, alcohol ether sulfonates, alkyl amine sulfonates, petroleum sulfonates, and phosphate esters.
In various embodiments, the fluid comprises a lubricant. Non-limiting, suitable lubricants may include fatty acids, tall oil, sulphonated detergents, phosphate esters, alkanolamides, asphalt sulfonates, graphite, and glass beads.
The fluid can be a drilling fluid with a low shear rate viscosity as measured with a Brookfield viscometer at 0.5 rpm of at least 20,000 centipoise. In some embodiments, the low shear rate viscosity is at least about 40,000 centipoise.
Biomass added to fluid can be chemically modified prior to use. Chemical modification involves the formation or breaking of covalent bonds. For example, the biomass may be chemically modified by transesterification, saponification, crosslinking or hydrolysis. The biomass may be treated with one or more reactive species so as to attach desired moieties. The moieties may be hydrophobic, hydrophilic, amphiphilic, ionic, or zwitterionic. For example, the biomass may anionized (e.g., carboxymethylated), or acetylated. Methods for covalent modification including carboxymethylation and acetylation of biomass from oleaginous microbes are disclosed in U.S. Provisional Patent Application No. 61/615,832, filed on Mar. 26, 2012 for “Algal Plastics and Absorbants”, incorporated herein by reference in relevant part.
V. Alkyl Ester Preparation
Methods for producing alkyl esters are well known such as those described in U.S. Pat. No. 2,383,602 and U.S. Pat. No. 7,652,156. Triglycerides are contacted with excess alcohol in the presence of an acid, or more typically, a base catalyst. Suitable base catalysts include sodium hydroxide, lithium hydroxide, and potassium hydroxide. Upon completion of the transesterification reaction the heavier glycerol by-product separates from the alkyl ester as the lower of two layers and can be readily removed.
VI. Examples
Example 1
Strain A was derived from UTEX 1435 classically mutagenized for higher oil production and further transformed with plasmid pSZ1413 (SEQ ID NO:1) to disrupt a stearoyl-ACP desaturase site followed by further mutagenesis. The plasmid was constructed and transformed into Prototheca cells that were subsequently grown heterotrophically and pressed to extract oil in accordance with methods described in WO2008/151149, WO2010/063031, WO2010/063032, WO2011/150411, and WO2013/158938. The plasmid comprises a C. reinhardtii β-tubulin promoter driving the expression of a Saccharomyces cerevisiae sucrose invertase gene with a Chlorella protothecoides Ef1 3′ UTR and a Prototheca moriformis endogenous AMTS promoter driving expression of an exogenous acyl-ACP thioesterase from Cuphea. Wrightii fused to a transit peptide from Prototheca moriformis fatty acid desaturase with a Chlorella vulgaris nitrate reductase 3′ UTR. The extracted triglyceride oil was refined, bleached, and deodorized and found to have the fatty acid profile in Table 1 and the sterol content in Table 2. The fatty acid profile of strain A contain significant amounts of C12:0 fatty acids (50.72%), followed by C10:0 (19.35%) and C14:0 (13.28%) fatty acids. A comparison of the oil to coconut, palm kernel oil, and babassu oil (data from Codex Standard for Named Vegetable Oils) is also given in Table 1.







TABLE 1







FATTY ACID PROFILE (WEIGHT PERCENT) OF STRAIN A


OIL IN COMPARISON TO COCONUT AND PALM KERNEL OIL.













Coconut
Palm
Babassu


Fatty acid
Strain A
Oil
Kernel Oil
Oil














C6:0

ND-0.7 
ND-0.8 
ND


C8:0
0.28
 4.6-10.0
2.4-6.2
2.6-7.3


C10:0
19.35
5.0-8.0
2.6-5.0
1.2-7.6


C11:0
0.07


C12:0
50.72
45.1-53.2
45.0-55.0
40.0-55.0


C12:1
0.05


C13:0
0.05


C14:0
13.28
16.8-21.0
14.0-18.0
11.0-27.0


C14:1 cis-Δ9
0.02


C15:0
0.03


C16:0
5.24
 7.5-10.2
 6.5-10.0
 5.2-11.0


C16:1 cis-Δ7
0.02
ND
ND-0.2 
ND


C16:1 cis-Δ9
0.11
ND
ND
ND


C17:0
0.01
ND
ND
ND


C18:0
0.35
2.0-4.0
1.0-3.0
1.8-7.4


C18:1
6.78
 5.0-10.0
12.0-19.0
 9.0-20.0


C18:2
2.47
1.0-2.5
1.0-3.5
1.4-6.6


C18:2 trans, trans-Δ9, Δ12
0.01


C18:3 alpha
0.5
ND-0.2 
ND-0.2 
ND


(cis, cis, cis-Δ9, Δ12, Δ15)


C20:0
0.03
ND-0.2 
ND-0.2 
ND


C20:1
0.01
ND-0.2 
ND-0.2 
ND


C20:1-1
0.03


C24:0
0.01
ND
ND
ND





ND = nondetectable, defined as ≦0.05%













TABLE 2







STEROL CONTENT OF STRAIN A OIL.










Sterol
mg/100 g














Ergosterol
154



Campesterol
4.92



Stigmasterol
6.26



β-Sitosterol
23.9



Other sterols
56.1










Example 2
Microalgal Derived Alkyl Esters
The triglyceride oil from Example 1 was transesterified with methanol and ethanol according to known transesterification methods. The methyl and ethyl esters were analyzed according to the methods in Table 3 below.







TABLE 3







PROPERTIES OF ALGAL DERIVED


METHYL AND ETHYL ESTERS.














Methyl
Ethyl


Property
Method
Units
ester
ester














Kauri-butanol value
ASTM D1133
unit
  75-77.5
67


Flash Point Pensky-
ASTM D93a
° C.
113-120
116


Martens Closed Cup


Kinematic Viscosity
ASTM D445
mm{circumflex over ( )}2/s
2.7
3.2


at 40° C.


Residue on
ASTM D1353
mg/kg
62
54


Evaporation


Volatile Matter
EPA Method 24
g/g
0.93


Content












VOC Content
EPA 24/
Wt %
0.513
g/g




ASTM D2369











Vapor Pressure
ASTM D5191
psi

0.63


(DVPE)


Gardner Color
ASTM D1544
unit
3


Methanol
EN14110
mass %
0.01


Water and Sediment
ASTM D2709
volume %
0
0.0


Free Glycerin
ASTM D6584
mass %
0.001
0.012


Total Glycerin
ASTM D6584
mass %
0.034
0.143


Monoglycerides
ASTM D6584
mass %
0.09


Diglycerides
ASTM D6584
mass %
0.054


Triglycerides
ASTM D6584
mass %
0.02


Ester Content
Internal
mass %
99.8



Method












Vapor Pressure
ASTM D5191
mmHg
<1
psi
<1 psi


@ 20 C.









Example 3
Solvent Compositions
Solvents formulations with or without the methyl ester of Example 2 (Solvent M) were prepared according to Table 4.







TABLE 4







SOLVENT FORMULATIONS.












d-






limonene
C10
Solvent
Xylene


Component
solvent
Solvent
M
solvent














d-limonene (technical grade)
20
0
0
0


CE-1095 (P&G Cehmicals)
0
20
0
0


Example 2, Methyl Ester
0
0
20
0


Xylene (ACS reagent grade)
0
0
0
20


Videt ME-80
13.5
20.72
20.72
15.5


Rexonic N91-8
3
3
3
3


Water (deionized)
63.5
56.28
56.28
61.5









Example 4
Cleaning Performance Testing
The formulations in Example 3 were tested for cleaning performance in the removal of bitumen, crude oil, and drilling mud.
Equipment Used: Gardner Blue Straight-Line Washability Machine


    
    
        Nikon Camera
        Gardco Adjustable Micrometer Microm II Film Applicator, (ASTM
        D823)
        Image Analysis Software
        10.00 mL pipette
        Grieve Forced Air oven
        ASTM D4488 A5, brass template

Materials: Bitumen, LC#: 13-T0551

        Used Drilling Mud, LC#: 10-T1155
        Crude Oil, LC#: 13-T1134
        3M Cellulose Sponge
        304 brushed Stainless Steel panels, 15.0 cm×10.0 cm

Bitumen Soil Application:

Soil Application for Testing of 20% Active Solvent Emulsions

    
    


Applied Bitumen to 304 brushed SS panels using A5 brass template and thinning out to an even film via a kim wipe (length of template was along shorter edge of panel (10 cm edge), application weight was 0.35 grams. Panels sit over night at ambient conditions for a minimum of 18 hours before testing.
Soil Application for Testing of 100% Solvent
Applied the bitumen to 304 brushed SS panels, 154569, using the Gardco Adjustable Film applicator. The applicator was set to a gap of 0.500 mm. The panels were put into the forced air oven set to 800 C, (preheated). The panels were put into the oven for 24.0 hours. Cooled under ambient conditions for two hours prior to testing.
Used Drilling Mud Soil Application:
Soil Application for Testing of 20% Active Solvent Emulsions
Applied used drilling mud to 304 brushed SS panels and thinning out to an even film using the Gardco Adjustable Film applicator. The applicator was set to a gap of 0.300 mm. The panels were put into the forced air oven set to 800 C, (preheated) for 20 hours. Panels were removed at exactly 20 hours after putting into the preheated oven. The oven was preheated to 2000 C and the panels were put into the oven for 120 minutes. Cooled under ambient conditions for two hours prior to testing.
Soil Application for Testing of 100% Solvent
Applied used drilling mud to 304 brushed SS panels and thinning out to an even film using the Gardco Adjustable Film applicator. The applicator was set to a gap of 0.300 mm. The panels were put into the forced air oven set to 80° C., (preheated) for 20 hours. Panels were removed at exactly 20 hours after putting into the preheated oven. The oven was preheated to 200° C. and the panels were put into the oven for 70 minutes. Cooled under ambient conditions for two hours prior to testing.
Crude Oil Soil Application
Soil Application for Testing of 20% Active Solvent Emulsions
Applied crude oil to 304 brushed SS panels and thinning out to an even film using the Gardco Adjustable Film applicator. The applicator was set to a gap of 0.300 mm. The panels were put into the forced air oven set to 1000 C, (preheated). Panels were removed at exactly 20 hours after putting into the preheated oven. Cooled under ambient conditions for two hours prior to testing
Soil Application for Testing of 100% Solvent
Applied crude oil to 304 brushed SS panels and thinning out to an even film using the Gardco Adjustable Film applicator. The applicator was set to a gap of 0.300 mm. The panels were put into the forced air oven set to 800 C, (preheated) for 20 hours. Panels were removed at exactly 20 hours after putting into the preheated oven. The oven was preheated to 2000 C and the panels were put into the oven for 120 minutes. Cooled under ambient conditions for two hours prior to testing.
Cleaning Test
Condition the small cellulose sponge as per ASTM D4488 A5 Method.
The organic solvent product is tested at a 20% by weight concentration as per the specified base formulation or as supplied, (100% active solvent). The soiled and aged panel is placed on the Gardner Straight-line washability apparatus using a template. Apply 10.00 mL of the solvent to the preconditioned sponge and invert the sponge onto the soil. Start the Gardner machine and run the machine until approximately 70-90% of the soil has been removed by the test solvent. After the machine is stopped, rinse with a cool stream of tap water. Air-dry the panels before evaluation.
Evaluation
Using the Nikon camera and lighting booth, take a digital picture of the cleaned panel. Analyse the image using the image analysis software to quantify the percentage of grease removed from the surface. Report the average percentage removed and the average number of cycles required to achieve the percentage removed.
The results of the cleaning performance of the solvents are given in Tables 5-7 below.







TABLE 5







CLEANING EFFICIENCY OF BITUMEN GREASE REMOVAL.












Average #
Average %



Organic Solvent
Cycles
Removal















20% Xylene emulsion
30
88.5



20% d-Limonene emulsion
80
86.5



20% CE-1095 emulsion
95
84



20% Solvent M
115
76.2



100% xylene
37
94.7



100% d-Limonene
70
83.5



100% CE-1095
73
86.7



100% Solvent M
143
78.7

















TABLE 6







CLEANING EFFICIENCY OF USED


DRILLING MUD REMOVAL.












Average #
Average %



Organic Solvent
Cycles
Removal







20% Xylene emulsion
15
80.2



20% d-Limonene emulsion
19
84.1



20% CE-1095 emulsion
33
80.5



20% Solvent M
33
78.1



100% xylene
33
62.3



100% d-Limonene
48
64.6



100% CE-1095
48
73.8



100% Solvent M
47
77.6

















TABLE 7







CLEANING EFFICIENCY CRUDE OIL REMOVAL.












Average #
Average %



Organic Solvent
Cycles
Removal















20% Xylene emulsion
30
73.7



20% d-Limonene emulsion
63
82.7



20% CE-1095 emulsion
177
77.7



20% Solvent M
200
84.8



100% xylene
3
93.4



100% d-Limonene
2
92.9



100% CE-1095
4
99.1



100% Solvent M
4
89










Solvent M containing microalgal derived methyl ester was found to be efficacious in removing bitumen, used drilling mud, and crude oil.
Example 5
Cleaning Formulations
Cleaning compositions containing microalgal derived alkyl esters can be prepared as shown in Table 8.







TABLE 8







FORMULATIONS CONTAINING ALGAL ALKYL ESTER.









Use
Ingredients
Wt %












All-Purpose cleaner concentrate
Algal alkyl ester
7.95



d-limonene tech grade
1.98



NINOL 11-CM
68.41



Westvaco L-5 fatty acid
6.08



Triethanol amine, 99%
3.42



D.I. water
6.08



Polyproylene glycol
6.08


Multipurpose cleaner
NINOL 11-CM
82.14



Algal alkyl ester
17.86



Above Blend
24.19



Butyl Carbitol Solvent Butoxydiglycol
4.81



(Dow)



Versene 100 Tetrasodium EDTA (Dow)
3.21



D.I. water
q.s. to 100


Laundry pre-spotter
Algal alkyl ester
56



BIO-SOFT N25-7
19



BIO-SOFT N25-3
19



Ethanol
4



D.I. water
2


Concrete cleaner
Algal alkyl ester
70



Ethyl lactate
20



BIO-SOFT EC-690
10



Fragrance (optional)
q.s.


Concrete oil stain remover
Algal alkyl ester
50



d-limonene tech grade
25



C 12-15, 9 mole ethoxylated alcohol
25



surfactant


Parts washer solvent
Algal alkyl ester
75



Ethyl lactate
10



BIO-SOFT N1-7
10



d-limonene tech grade
5


Degreaser/cleaner
Algal alkyl ester
 5-35



Dodecylbenzensulfonic acid
 5-10



Linear alcohol ethoxylate (6-10 mole)
 5-10



Sodium xylene sulfonate
qs to pH 4 or




greater



Water
qs to 100


Parts Cleaner/Degreaser
Algal alkyl ester
 90-100



C 12-15, 9 mole ethoxylated alcohol
 0-10



surfactant


Floating Lift Station Degreaser
Algal alkyl ester
70



d-limonene tech grade
30


Heavy Duty Degreaser
Algal alkyl ester
20



NINOL 11-CM
20



MAKON 8
10



BIO-TERGE PAS-8S
10



D.I. water
qs to 100


Animal Fat Degreaser/Grease trap opener
Algal alkyl ester
25



d-limonene tech grade
75


Engine degreaser/high pressure chamber wash degreaser
Algal alkyl ester
50



Ethyl lactate
10



Dibutyl ether
5



Ethoxylated nonyl phenol
5



Defoamer
3



Mineral Oil
27


Graffiti Remover
Algal alkyl ester
62-65



Ethyl lactate
25



ViscoAbit (React)
5



NP9 or equivalent
4.95



Fragrance (optional)
0-3



BHT
0.05


Graffiti Remover
Algal alkyl ester
52-55



NMP
20



Ethyl lactate
15



ViscoAbit (React)
5



NP9 or equivalent
4.95



Fragrance (optional)
0-3



BHT
0.05


Graffiti Remover
Algal alkyl ester
85



BIO-SOFT N1-7
10



Methocel 311
5


Thick Paint Stripper
Vertec Gold 50/50
74



Algal alkyl ester
11



Nonyl phenol ethoxylate (6 mol)
6



Klucerl-H or Methocel 311 (thickener)
6



Fragrance
3


Paint Stripper
Vertec Gold 50/50
74



Algal alkyl ester
11



Tergitol 15-S-59
6



Klucerl-H or Methocel 311 (thickener)
6



Fragrance
3


Varnish remover
Algal alkyl ester
40



Ethyl lactate
10



d-limonene tech grade
46



Ethanol
4


Screen printing ink cleaner-textile inks
Algal alkyl ester
85



BIO-SOFT EC-690
15


Printing ink cleaner
Algal alkyl ester
64



Dimethyl glutarate, dimethyl adipate,
20



dimethyl succinate



BIO-SOFT N1-7
12



d-limonene tech grade (fragrance)
4


Form release agent
Algal alkyl ester
85



STEPAN GMO (glyceryl monooleate)
15


Tar asphalt remover
Algal alkyl ester
90



C 12-15, 9 mole ethoxylated alcohol
10



surfactant


Waterless Hand Cleaner
Phase 1:



Algal alkyl ester
34



NINOL 40-CO
4.5



MAKON 10
10.9



Oleic acid
1.7



Soulan 75 (Amerchol) PEG 75 Lanolin
0.5



Phase 2:



Triethanol amine, 99%
0.4



Preservative
qs



D.I. water
qs to 100


Thick gel waterless hand cleaner
Algal alkyl ester
65



Ethyl lactate
10



BIO-SOFT N1-7
15



ViscoAbit (React)
10


Hand Cleaner
Algal alkyl ester
25



E-Z-Mulse
45



Glycerin
1



Lanolin
3



Water
26


Hand cleaner
Part A:



Algal alkyl ester
30



Ethylene glycol monostearate
10



Calsuds CD-6 (Pilot Chemical)
11



Deoderized lanolin
0.5



Part B:



Propylene glycol
5



Glycerin
0.5



Water
42.5



Preservative and Fragrance
0.5

















SEQUENCE LISTING



SEQ ID MO: 1


pSZ1413; SAD2B::CrTUB2-ScSUC2-CpEF1:PmAMT3-PmFADtp_CwFATB2-CvNR:SAD2B









   1
cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag



 


  61
agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg


 


 121
ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa


 


 181
ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag


 


 241
gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact


 


 301
cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa


 


 361
attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt


 


 421
accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag


 


 481
ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca


 


 541
gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc


 


 601
agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt


 


 661
ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg


 


 721
ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca


 


 781
gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg


 


 841
ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca


 


 901
tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg


 


 961
tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct


 


1021
cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca


 


1081
tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca


 


1141
gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg


 


1201
tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac


 


1261
ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt


 


1321
attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc


 


1381
cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt atcatgacat


 


1441
taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg


 


1501
gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg taagcggatg


 


1561
ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc


 


1621
ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac


 


1681
cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc aggctgcgca


 


1741
actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg


 


1801
gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta


 


1861
aaacgacggc cagtgaattg atgcatgctc ttccgcctgg agctggtgca gagcatgggg


 


1921
cagtttgcgg aggagagggt gctccccgtg ctgcaccccg tggacaagct gtggcagccg


 


1981
caggacttcc tgcccgaccc cgagtcgccc gacttcgagg accaggtggc ggagctgcgc


 


2041
gcgcgcgcca aggacctgcc cgacgagtac tttgtggtgc tggtgggcga catgatcacg


 


2101
gaggaggcgc tgccgaccta catggccatg ctcaacacct tggacggtgt gcgcgacgac


 


2161
acgggcgcgg ctgaccaccc gtgggcgcgc tggacgcggc agtgggtggc cgaggagaac


 


2221
cggcacggcg acctgctgaa caagtactgt tggctgacgg ggcgcgtcaa catgcgggcc


 


2281
gtggaggtga ccatcaacaa cctgatcaag agcggcatga acccgcagac ggacaacaac


 


2341
ccttacttgg gcttcgtcta cacctccttc caggagcgcg ccaccaagta ggtacccttt


 


2401
cttgcgctat gacacttcca gcaaaaggta gggcgggctg cgagacggct tcccggcgct


 


2461
gcatgcaaca ccgatgatgc ttcgaccccc cgaagctcct tcggggctgc atgggcgctc


 


2521
cgatgccgct ccagggcgag cgctgtttaa atagccaggc ccccgattgc aaagacatta


 


2581
tagcgagcta ccaaagccat attcaaacac ctagatcact accacttcta cacaggccac


 


2641
tcgagcttgt gatcgcactc cgctaagggg gcgcctcttc ctcttcgttt cagtcacaac


 


2701
ccgcaaactc tagaatatca atgctgctgc aggccttcct gttcctgctg gccggcttcg


 


2761
ccgccaagat cagcgcctcc atgacgaacg agacgtccga ccgccccctg gtgcacttca


 


2821
cccccaacaa gggctggatg aacgacccca acggcctgtg gtacgacgag aaggacgcca


 


2881
agtggcacct gtacttccag tacaacccga acgacaccgt ctgggggacg cccttgttct


 


2941
ggggccacgc cacgtccgac gacctgacca actgggagga ccagcccatc gccatcgccc


 


3001
cgaagcgcaa cgactccggc gccttctccg gctccatggt ggtggactac aacaacacct


 


3061
ccggcttctt caacgacacc atcgacccgc gccagcgctg cgtggccatc tggacctaca


 


3121
acaccccgga gtccgaggag cagtacatct cctacagcct ggacggcggc tacaccttca


 


3181
ccgagtacca gaagaacccc gtgctggccg ccaactccac ccagttccgc gacccgaagg


 


3241
tcttctggta cgagccctcc cagaagtgga tcatgaccgc ggccaagtcc caggactaca


 


3301
agatcgagat ctactcctcc gacgacctga agtcctggaa gctggagtcc gcgttcgcca


 


3361
acgagggctt cctcggctac cagtacgagt gccccggcct gatcgaggtc cccaccgagc


 


3421
aggaccccag caagtcctac tgggtgatgt tcatctccat caaccccggc gccccggccg


 


3481
gcggctcctt caaccagtac ttcgtcggca gcttcaacgg cacccacttc gaggccttcg


 


3541
acaaccagtc ccgcgtggtg gacttcggca aggactacta cgccctgcag accttcttca


 


3601
acaccgaccc gacctacggg agcgccctgg gcatcgcgtg ggcctccaac tgggagtact


 


3661
ccgccttcgt gcccaccaac ccctggcgct cctccatgtc cctcgtgcgc aagttctccc


 


3721
tcaacaccga gtaccaggcc aacccggaga cggagctgat caacctgaag gccgagccga


 


3781
tcctgaacat cagcaacgcc ggcccctgga gccggttcgc caccaacacc acgttgacga


 


3841
aggccaacag ctacaacgtc gacctgtcca acagcaccgg caccctggag ttcgagctgg


 


3901
tgtacgccgt caacaccacc cagacgatct ccaagtccgt gttcgcggac ctctccctct


 


3961
ggttcaaggg cctggaggac cccgaggagt acctccgcat gggcttcgag gtgtccgcgt


 


4021
cctccttctt cctggaccgc gggaacagca aggtgaagtt cgtgaaggag aacccctact


 


4081
tcaccaaccg catgagcgtg aacaaccagc ccttcaagag cgagaacgac ctgtcctact


 


4141
acaaggtgta cggcttgctg gaccagaaca tcctggagct gtacttcaac gacggcgacg


 


4201
tcgtgtccac caacacctac ttcatgacca ccgggaacgc cctgggctcc gtgaacatga


 


4261
cgacgggggt ggacaacctg ttctacatcg acaagttcca ggtgcgcgag gtcaagtgac


 


4321
aattgacgga gcgtcgtgcg ggagggagtg tgccgagcgg ggagtcccgg tctgtgcgag


 


4381
gcccggcagc tgacgctggc gagccgtacg ccccgagggt ccccctcccc tgcaccctct


 


4441
tccccttccc tctgacggcc gcgcctgttc ttgcatgttc agcgacggat cccgcgtctc


 


4501
gaacagagcg cgcagaggaa cgctgaaggt ctcgcctctg tcgcacctca gcgcggcata


 


4561
caccacaata accacctgac gaatgcgctt ggttcttcgt ccattagcga agcgtccggt


 


4621
tcacacacgt gccacgttgg cgaggtggca ggtgacaatg atcggtggag ctgatggtcg


 


4681
aaacgttcac agcctaggga tatcgaattc ggccgacagg acgcgcgtca aaggtgctgg


 


4741
tcgtgtatgc cctggccggc aggtcgttgc tgctgctggt tagtgattcc gcaaccctga


 


4801
ttttggcgtc ttattttggc gtggcaaacg ctggcgcccg cgagccgggc cggcggcgat


 


4861
gcggtgcccc acggctgccg gaatccaagg gaggcaagag cgcccgggtc agttgaaggg


 


4921
ctttacgcgc aaggtacagc cgctcctgca aggctgcgtg gtggaattgg acgtgcaggt


 


4981
cctgctgaag ttcctccacc gcctcaccag cggacaaagc accggtgtat caggtccgtg


 


5041
tcatccactc taaagaactc gactacgacc tactgatggc cctagattct tcatcaaaaa


 


5101
cgcctgagac acttgcccag gattgaaact ccctgaaggg accaccaggg gccctgagtt


 


5161
gttccttccc cccgtggcga gctgccagcc aggctgtacc tgtgatcgag gctggcggga


 


5221
aaataggctt cgtgtgctca ggtcatggga ggtgcaggac agctcatgaa acgccaacaa


 


5281
tcgcacaatt catgtcaagc taatcagcta tttcctcttc acgagctgta attgtcccaa


 


5341
aattctggtc taccgggggt gatccttcgt gtacgggccc ttccctcaac cctaggtatg


 


5401
cgcgcatgcg gtcgccgcgc aactcgcgcg agggccgagg gtttgggacg ggccgtcccg


 


5461
aaatgcagtt gcacccggat gcgtggcacc ttttttgcga taatttatgc aatggactgc


 


5521
tctgcaaaat tctggctctg tcgccaaccc taggatcagc ggcgtaggat ttcgtaatca


 


5581
ttcgtcctga tggggagcta ccgactaccc taatatcagc ccgactgcct gacgccagcg


 


5641
tccacttttg tgcacacatt ccattcgtgc ccaagacatt tcattgtggt gcgaagcgtc


 


5701
cccagttacg ctcacctgtt tcccgacctc cttactgttc tgtcgacaga gcgggcccac


 


5761
aggccggtcg cagccactag tatggctatc aagacgaaca ggcagcctgt ggagaagcct


 


5821
ccgttcacga tcgggacgct gcgcaaggcc atccccgcgc actgtttcga gcgctcggcg


 


5881
cttcgtgggc gcgcccccaa ggccaacggc agcgccgtga gcctgaagtc cggcagcctg


 


5941
aacaccctgg aggacccccc cagcagcccc cccccccgca ccttcctgaa ccagctgccc


 


6001
gactggagcc gcctgcgcac cgccatcacc accgtgttcg tggccgccga gaagcagttc


 


6061
acccgcctgg accgcaagag caagcgcccc gacatgctgg tggactggtt cggcagcgag


 


6121
accatcgtgc aggacggcct ggtgttccgc gagcgcttca gcatccgcag ctacgagatc


 


6181
ggcgccgacc gcaccgccag catcgagacc ctgatgaacc acctgcagga caccagcctg


 


6241
aaccactgca agagcgtggg cctgctgaac gacggcttcg gccgcacccc cgagatgtgc


 


6301
acccgcgacc tgatctgggt gctgaccaag atgcagatcg tggtgaaccg ctaccccacc


 


6361
tggggcgaca ccgtggagat caacagctgg ttcagccaga gcggcaagat cggcatgggc


 


6421
cgcgagtggc tgatcagcga ctgcaacacc ggcgagatcc tggtgcgcgc caccagcgcc


 


6481
tgggccatga tgaaccagaa gacccgccgc ttcagcaagc tgccctgcga ggtgcgccag


 


6541
gagatcgccc cccacttcgt ggacgccccc cccgtgatcg aggacaacga ccgcaagctg


 


6601
cacaagttcg acgtgaagac cggcgacagc atctgcaagg gcctgacccc cggctggaac


 


6661
gacttcgacg tgaaccagca cgtgagcaac gtgaagtaca tcggctggat tctggagagc


 


6721
atgcccaccg aggtgctgga gacccaggag ctgtgcagcc tgaccctgga gtaccgccgc


 


6781
gagtgcggcc gcgagagcgt ggtggagagc gtgaccagca tgaaccccag caaggtgggc


 


6841
gaccgcagcc agtaccagca cctgctgcgc ctggaggacg gcgccgacat catgaagggc


 


6901
cgcaccgagt ggcgccccaa gaacgccggc accaaccgcg ccatcagcac ctgattaatt


 


6961
aactcgaggc agcagcagct cggatagtat cgacacactc tggacgctgg tcgtgtgatg


 


7021
gactgttgcc gccacacttg ctgccttgac ctgtgaatat ccctgccgct tttatcaaac


 


7081
agcctcagtg tgtttgatct tgtgtgtacg cgcttttgcg agttgctagc tgcttgtgct


 


7141
atttgcgaat accaccccca gcatcccctt ccctcgtttc atatcgcttg catcccaacc


 


7201
gcaacttatc tacgctgtcc tgctatccct cagcgctgct cctgctcctg ctcactgccc


 


7261
ctcgcacagc cttggtttgg gctccgcctg tattctcctg gtactgcaac ctgtaaacca


 


7321
gcactgcaat gctgatgcac gggaagtagt gggatgggaa cacaaatgga aagcttgagc


 


7381
tccagccacg gcaacaccgc gcgccttgcg gccgagcacg gcgacaagaa cctgagcaag


 


7441
atctgcgggc tgatcgccag cgacgagggc cggcacgaga tcgcctacac gcgcatcgtg


 


7501
gacgagttct tccgcctcga ccccgagggc gccgtcgccg cctacgccaa catgatgcgc


 


7561
aagcagatca ccatgcccgc gcacctcatg gacgacatgg gccacggcga ggccaacccg


 


7621
ggccgcaacc tcttcgccga cttctccgcg gtcgccgaga agatcgacgt ctacgacgcc


 


7681
gaggactact gccgcatcct ggagcacctc aacgcgcgct ggaaggtgga cgagcgccag


 


7741
gtcagcggcc aggccgccgc ggaccaggag tacgtcctgg gcctgcccca gcgcttccgg


 


7801
aaactcgccg agaagaccgc cgccaagcgc aagcgcgtcg cgcgcaggcc cgtcgccttc


 


7861
tcctggagaa gagcctctag agtcgacctg caggcatgca agcttggcgt aatcatggtc


 


7921
atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg


 


7981
aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt


 


8041
gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg


 


8101
ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga


 


8161
ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat


 


8221
acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca


 


8281
aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc


 


8341
tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata


 


8401
aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc


 


8461
gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc


 


8521
acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga


 


8581
accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc


 


8641
ggtaagacac gacttatcgc

## Claims

1. A composition comprising C1-C4 esters of fatty acids derived from triglyceride oil produced by a microalga that is a recombinant oleaginous cell of the genus Prototheca, the oil having an altered fatty acid profile relative to an oil produced by a non-recombinant microalga of the genus Prototheca, the altered fatty acid profile having at least 10% C10:0 and 40% C12:0 fatty acids, the composition having a reduced odor compared to a composition where all the fatty acids are derived from vegetable or animal triglyceride oil having a fatty acid profile of at least 40% C12:0 fatty acids.

2. The composition of claim 1, wherein the odor is an unpleasant odor characteristic of a C6 or C8 fatty acid or ester thereof.

3. The composition of claim 1, wherein the ester is a methyl, ethyl, propyl, iso-propyl, butyl, or a tert-butyl ester.

4. The composition of claim 1, having an altered fatty acid profile wherein C12:0>C10:0>C14:0.

5. The composition of claim 1, wherein at least 50% of the fatty acids are C12:0 fatty acids or the combined total amount of C:10 and C:12 fatty acids is at least 60%.

6. The composition of claim 1, wherein at least 15% of the fatty acids are C10:0 fatty acids.

7. The composition of claim 1, wherein 15-25% of the fatty acids are C10:0 fatty acids.

8. The composition of claim 1, wherein 10-15% of the fatty acids are C14:0 fatty acids.

9. The composition of claim 1, wherein less than 8%, 7%, or 6% of the fatty acids are C16:0 fatty acids.

10. The composition of claim 1, wherein less than 0.5% of the fatty acids are C8:0 fatty acids.

11. The composition of claim 1, wherein less than 0.1%, 0.01%, or 0.001% of the fatty acids are C6:0 fatty acids.

12. The composition of claim 1, comprising a terpene.

13. The composition of claim 12, wherein the terpene is limonene.

14. A method for preparing a composition comprising C1-C4 esters of fatty acids derived from triglyceride oil produced by a microalga of the genus Prototheca, the composition having a reduced odor compared to a composition where all the fatty acids are derived from vegetable oil, the method comprising:
a) providing oil produced by a microalga that is a recombinant oleaginous cell of the genus Prototheca, the oil comprising triacylglycerides having an altered fatty acid profile relative to an oil produced by a non-recombinant microalga of the genus Prototheca, wherein the most abundant fatty acids are C12:0 fatty acids; and
b) treating the triacylglycerides with a C1-C4 alcohol under transesterification conditions to form C1-C4 esters of the microalgal derived fatty acids.

15. The method of claim 14, wherein the C1-C4 alcohol is methanol, ethanol, propanol, iso-propanol, butanol, or a tert-butanol.

16. The method of claim 14 further comprising blending the C1-C4 esters of fatty acids with one or more of a terpene, a C1-C4 ester of lactic acid, a surfactant, an emulsifier, or water.