High density production of biomass and oil using crude glycerol

Abstract

A method for culturing one or more microorganisms is provided. The method includes culturing one or more microorganisms in a culture medium containing a first concentration level of crude glycerol; once the first concentration of glycerol decreases to a first threshold level, feeding an additional amount of crude glycerol into the culture medium at a concentration sufficient to achieve the first concentration level; and monitoring the crude glycerol concentration until the first concentration level of crude glycerol decreases to the first threshold level. The steps can be repeated until a desired microbial cell density is achieved.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 62 / 138,631, filed March 26, 2015, which is incorporated herein by reference in its entirety.

[0003] background

[0004] Heterotrophic microbial fermentation is a highly efficient way to produce high-value oils and biomass products. Under certain culture conditions, microorganisms synthesize intracellular oils, which can be extracted and used to produce biofuels (such as biodiesel, biojet fuel, etc.) and nutritional lipids (such as polyunsaturated fatty acids such as DHA, EPA, and DPA). The biomass of some microorganisms also possesses significant nutritional value due to its high polyunsaturated fatty acid (PUFA) and protein content, and can be used as a nutritional supplement for animal feed. However, heterotrophic fermentation is a costly and energy- and feedstock-intensive process. Carbon feedstock costs typically account for a very large portion of the total cost of producing microbial biomass and oils. Current microbial fermentation primarily uses expensive carbohydrates such as glucose as carbon sources. Cheaper carbon alternatives are being explored to make the production process economically viable. Besides glucose, several other forms of carbon, such as fructose and glycerol, are natural carbon substrates for microorganisms. However, highly purified glycerol is more expensive than glucose. Invention Overview

[0006] A method for culturing one or more microorganisms is provided. The method includes culturing one or more microorganisms in a culture medium containing a first concentration level of crude glycerol; once the first concentration of glycerol decreases to a first threshold level, feeding an additional amount of crude glycerol into the culture medium at a concentration sufficient to achieve the first concentration level; and monitoring the crude glycerol concentration until the first concentration level of crude glycerol decreases to the first threshold level. The steps can be repeated until a desired microbial cell density is achieved.

[0007] A method for producing one or more fatty acids is also provided. The method includes providing a microorganism capable of producing fatty acids, providing a culture medium containing crude glycerol, and culturing the microorganism in the culture medium under conditions sufficient to produce one or more fatty acids to provide a final concentration of at least 50% by weight of one or more unsaturated fatty acids of the microorganism. Brief description of the attached diagram

[0009] Figure 1 It is a graph showing the biomass concentration (g / L) over time during fermentation using various crude glycerol feedstocks from different biodiesel manufacturers as the sole carbon source.

[0010] Figure 2This is a graph showing the biomass concentration (g / L) over time during fermentation using mixtures of crude glycerol and glucose at different volume ratios. Glycerol refers to crude glycerol from Rothsay; glucose refers to a 750 g / L glucose solution. The ratio is the volume ratio of the two solutions.

[0011] Figure 3 This is a graph showing the biomass and lipid concentrations over time during large-scale (200,000 L) fermentation of *Ostridium lycotriensis* ONC-T18 using crude glycerol as the sole carbon source.

[0012] Figure 4 This is a schematic diagram of the methanol-based biodiesel transesterification reaction.

[0013] Detailed Explanation

[0014] The increasing global demand for and production capacity of biofuels has resulted in a significant surplus of crude glycerol as a byproduct. Refining crude glycerol requires substantial investment, while unrefined crude glycerol has very little economic value. Consequently, many biofuel manufacturers treat crude glycerol as a type of industrial wastewater. Therefore, efficient methods for converting this crude glycerol into higher-value products are of paramount importance. Although glycerol (a type of crude glycerol) has been used as a carbon source as a byproduct of biodiesel production, high-density biomass and oil production has not been achieved due to the toxicity caused by impurities in crude glycerol and the lack of appropriate fermentation carbon supply strategies to avoid such toxicity. In contrast, this paper provides a method for culturing microorganisms using crude glycerol and producing oil. The provided method results in high biomass and oil yields, achieving 172 g / L cell dry weight with 68% oil content during fermentation. This productivity is similar to that achievable through batch fermentation with a glucose-based feedstock.

[0015] Therefore, the method provided enables the use of industrial byproduct glycerol (crude) as a partial or sole carbon substrate in combination with other nutrients to grow microorganisms and achieve high cell densities (>100 g / L) and high oil content (>50%). Crude glycerol from industrial sources requires little or no pretreatment. By defining and controlling the feeding of crude glycerol substrate into the fermenter, any bioinhibitions can be minimized while achieving extremely high biomass and oil yields. This paper also demonstrates that the typical sterilization procedures required for aseptic fermentation processes can be eliminated. Furthermore, the pretreatment of crude glycerol can be reduced or eliminated, and glycerol can be fed into the microbial culture as is, upon receipt from the manufacturer. When this method is carried out on a commercial scale, this results in significant energy cost savings. As used herein, the term "pretreatment" refers to the removal of non-glycerol impurities that can affect culture growth physically or biologically. Examples of pretreatment include chemical treatments for precipitation and removal of impurities, pH adjustments to match the pH of the culture environment, and filtration or centrifugation to remove suspended solids.

[0016] This document provides a method for culturing one or more microorganisms. The method includes culturing one or more microorganisms in a culture medium containing a first concentration level of crude glycerol; once the first concentration of glycerol decreases to a first threshold level, feeding an additional amount of crude glycerol into the culture medium at a concentration sufficient to achieve the first concentration level; and monitoring the crude glycerol concentration until the first concentration level of crude glycerol decreases to the first threshold level. Optionally, the first concentration level is between 1 and 60 g / L, or any level between 1 and 60 g / L, including the extreme values. Optionally, the first concentration level is between 5 and 60 g / L, between 15 and 60 g / L, between 5 and 20 g / L, or between 15 and 20 g / L. Optionally, the first threshold level is between 0 and 5 g / L, or any level between 0 and 5 g / L, including the extreme values. Thus, the first threshold level can be, for example, 0, 1, 2, 3, 4, or 5 g / L. Optionally, the steps are repeated until a desired microbial cell density is achieved. Optionally, the desired microbial cell density is greater than 100 g / L. Optionally, the desired cell density is 50 to 200 g / L, 50 to 150 g / L, 50 to 100 g / L, 100 to 250 g / L, 100 to 150 g / L, or 80 to 100 g / L. Optionally, the cell density is any density between 50 and 250 g / L, including the extreme values. Optionally, the cell density is 80 to 100 g / L. Optionally, the cell density is 120 to 230 g / L. Optionally, the microbial cell density contains 50% to 80% by weight of total fatty acids. Optionally, the total fatty acids contain 10 to 45% DHA. Optionally, the microbial cell density contains 5 to 36% DHA by total cell weight.

[0017] Microorganisms are capable of producing one or more fatty acids. Therefore, a method for producing one or more fatty acids is provided. The method includes providing a microorganism capable of producing fatty acids, providing a culture medium containing crude glycerol, and culturing the microorganism in the culture medium under conditions sufficient to produce one or more fatty acids to provide a final concentration of at least 50% by weight of one or more unsaturated fatty acids of the microorganism. Optionally, the fatty acid is a polyunsaturated fatty acid. Polyunsaturated fatty acids may be, for example, α-linolenic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, γ-linolenic acid, linolenic acid, linolenic acid, and combinations thereof.

[0018] In the provided method, monitoring of crude glycerol concentration can be performed using a variety of methods known to those skilled in the art. Optionally, monitoring includes measuring dissolved oxygen levels. Optionally, monitoring includes obtaining a sample of the culture medium and determining the glycerol concentration in said sample. Optionally, monitoring includes analyzing the sample using calorimetry, chemical reactive calorimetry, fluorescence assay, HPLC assay, enzymatic assay, or a combination thereof.

[0019] Various microorganisms are suitable for use in the provided methods. The microorganisms described herein may be algae (e.g., microalgae), fungi (including yeast), bacteria, or protozoa. Optionally, the microorganisms include *Thraustochytrid* of the order *Thraustochytriales*, and more specifically, the genus *Thraustochytrium* of the order *Thraustochytriales*. Optionally, the population of microorganisms includes the order *Thraustochytriales* as described in U.S. Patent Nos. 5,340,594 and 5,340,742, which are incorporated herein by reference in their entirety. The microorganisms may be species of the genus *Thraustochytrium*, such as the *Thraustochytrium* species (i.e., ONC-T18) registered with ATCC Registry No. PTA-6245 as described in U.S. Patent No. 8,163,515, which is incorporated herein by reference in its entirety. Therefore, the microorganism may have the same 18S rRNA sequence as SEQ ID NO:1 by at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or greater (e.g., including 100%).

[0020] Microorganisms used in the methods described herein can produce a variety of lipid compounds. As used herein, the term lipid includes phospholipids, free fatty acids, esters of fatty acids, triglycerides, sterols and sterol esters, carotenoids, xanthophylls (e.g., oxycarotenoids), hydrocarbons, and other lipids known to those skilled in the art. Optionally, lipid compounds include unsaturated lipids. Unsaturated lipids may include polyunsaturated lipids (i.e., lipids containing at least two unsaturated carbon-carbon bonds, such as double bonds) or highly unsaturated lipids (i.e., lipids containing four or more unsaturated carbon-carbon bonds). Examples of unsaturated lipids include ω-3 and / or ω-6 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and other naturally occurring unsaturated, polyunsaturated, and highly unsaturated compounds, which significantly shorten this long lag phase when inoculated at a 10% inoculum.

[0021] The provided methods include additional steps for culturing microorganisms according to methods known in the art, or may be used in conjunction with said additional steps. For example, *Cyclochytrids*, such as a species of *Cyclochytrids*, may be cultured according to methods described in U.S. Patent Publications 2009 / 0117194 or 2012 / 0244584, which are incorporated herein by reference in their entirety for the various steps or compositions of the methods used therein.

[0022] The microorganisms are grown in a growth medium (also called a “medium”). Any of a variety of media can be used to culture the microorganisms described herein. Optionally, the medium supplies the microorganisms with a variety of nutritional components, including carbon and nitrogen sources. The medium used for culturing *Cyclochytrium* may include any of a variety of carbon sources. Examples of carbon sources include fatty acids, lipids, glycerol, triglycerides, carbohydrates, polyols, amino sugars, and any kind of biomass or waste stream. Fatty acids include, for example, oleic acid. Carbohydrates include, but are not limited to, glucose, cellulose, hemicellulose, fructose, dextrose, xylose, lactulose, galactose, maltotriose, maltose, lactose, glycogen, gelatin, starch (corn starch or wheat starch), acetate, myositol (e.g., derived from corn steep liquor), galacturonic acid (e.g., derived from pectin), L-trehalose (e.g., derived from galactose), gentiobiose, glucosamine, α-D-glucose-1-phosphate (e.g., derived from glucose), cellobiose, dextrin, α-cyclodextrin (e.g., derived from starch), and sucrose (e.g., derived from molasses). Polyols include, but are not limited to, maltitol, erythritol, and adonitol. Amino sugars include, but are not limited to, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and N-acetyl-β-D-mannosamine.

[0023] One or more culture media used in the provided methods contain crude glycerol. As used herein, the term "crude glycerol" refers to a byproduct of a manufacturing process, such as biodiesel production. The term "crude glycerol" differs from the terms "glycerol" or "glycerin" because these products contain only or substantially "glycerol" in its pure form. The terms "glycerol" and "glycerin" are used interchangeably throughout this document. Thus, crude glycerol contains reagents and / or components additional to pure glycerol. Optionally, crude glycerol is a byproduct of biodiesel production. Crude glycerol is a major byproduct of biodiesel production via triglyceride transesterification or soap production via the hydrolysis (saponification) of triglycerides. For example, a typical biodiesel process involves reacting fats or oils (triglycerides) with alcohols such as methanol to form fatty acid esters (biodiesel) and glycerol. Examples of methanol-based reactions are shown in... Figure 4Due to impurities in fats / oils and the fact that the reaction itself is often assisted by chemical catalysts such as sodium methoxide, the final glycerol fraction contains a relatively high amount of impurities. Typical crude glycerol compositions and physical properties are shown in Table 1 (Reference: REG Ralston Crude Glycerol Analysis Certificate). Ash composition of the same crude glycerol is shown in Table 2. Further examples of crude glycerol characterization can be found in Thompson and He, Applied Engineering in Agriculture, Vol. 22(2): 261-265 (2006) and Hu et al., J. Agric. Food Chem., 2012, 60(23):5915–5921 (2012), which are incorporated herein by reference in their entirety.

[0024] Table 1. REG Ralston crude glycerol byproducts.

[0025]

[0026] Table 2 Ash composition of REG's Ralston crude glycerol byproducts.

[0027]

[0028] Optionally, crude glycerol is the sole carbon source in one or more culture media. Optionally, the culture medium contains one or more additional carbon sources. Optionally, the crude glycerol is not sterilized or pretreated. Optionally, the crude glycerol comprises methanol, water, ash, non-glycerol organic matter, sodium sulfate, methyl tallow, or a combination thereof. Optionally, the ash comprises calcium, iron, magnesium, potassium, sodium, zinc, or a combination thereof.

[0029] Optionally, the microorganisms provided herein are cultured under conditions that increase the yield of biomass and / or the target compound (e.g., oil or total fatty acid (TFA) content). For example, *Cyclotridium* is typically cultured in a saline medium. Optionally, *Cyclotridium* can be cultured in a medium having a salt concentration of about 0.5 g / L to about 50.0 g / L. Optionally, *Cyclotridium* is cultured in a medium having a salt concentration of about 0.5 g / L to about 35 g / L (e.g., about 18 g / L to about 35 g / L). Optionally, the *Cyclotridium* described herein can be grown under low-salt conditions. For example, *Cyclotridium* can be cultured in a medium having a salt concentration of about 0.5 g / L to about 20 g / L (e.g., about 0.5 g / L to about 15 g / L). The medium optionally includes NaCl. Optionally, the medium comprises natural or artificial sea salt and / or artificial seawater.

[0030] The culture medium may include non-chlorinated sodium salts as a source of sodium. Examples of non-chlorinated sodium salts suitable for use in the method according to the invention include, but are not limited to, soda ash (a mixture of sodium carbonate and sodium oxide), sodium carbonate, sodium bicarbonate, sodium sulfate, and mixtures thereof. See, for example, U.S. Patent Nos. 5,340,742 and 6,607,900, the entire contents of which are incorporated herein by reference. A significant portion of the total sodium may be supplied, for example, by non-chlorinated salts such that less than about 100%, 75%, 50%, or 25% of the total sodium in the culture medium is supplied by sodium chloride.

[0031] Optionally, the culture medium has a chloride ion concentration of less than about 3 g / L, 500 mg / L, 250 mg / L, or 120 mg / L. For example, the culture medium used in the provided method may have a chloride ion concentration between about 60 mg / L and 120 mg / L, and includes both about 60 mg / L and 120 mg / L.

[0032] Culture media for *Cyclotridium perfringens* culture may include any of a variety of nitrogen sources. Exemplary nitrogen sources include ammonium solutions (e.g., H₂O containing NH₄), ammonium or amine salts (e.g., (NH₄)₂SO₄, (NH₄)₃PO₄, NH₄NO₃, NH₄OOCH₂CH₃ (NH₄Ac)), peptone, tryptone, yeast extract, malt extract, fish meal, monosodium glutamate, soybean extract, casein amino acids, and distillers' grains. Suitable concentrations of nitrogen sources in culture media are typically in the range of about 1 g / L to about 25 g / L, and include both about 1 g / L and about 25 g / L.

[0033] The culture medium may optionally include phosphates such as potassium phosphate or sodium phosphate. Inorganic salts and micronutrients in the culture medium may include ammonium sulfate, sodium bicarbonate, sodium orthovanadate, potassium chromate, sodium molybdate, selenite, nickel sulfate, copper sulfate, zinc sulfate, cobalt chloride, ferric chloride, manganese chloride, calcium chloride, and EDTA. Vitamins may be included, such as pyridoxine hydrochloride, thiamine hydrochloride, calcium pantothenate, para-aminobenzoic acid, riboflavin, niacin, biotin, folic acid, and vitamin B12.

[0034] The pH of the culture medium can be adjusted to between 3.0 and 10.0, and including 3.0 and 10.0, using an acid or base, when appropriate, and / or a nitrogen source. Optionally, the culture medium can be sterilized.

[0035] Typically, culture media used to culture microorganisms are liquid media. However, culture media used to culture microorganisms can also be solid media. In addition to carbon and nitrogen sources as discussed herein, solid media may contain one or more components that provide structural support and / or make the medium solid (e.g., agar or agarose).

[0036] Optionally, the resulting biomass is pasteurized to inactivate any unwanted substances present in the biomass. For example, the biomass may be pasteurized to inactivate degradable compounds. The biomass may be present in or separated from a fermentation medium for the pasteurization step. The pasteurization step can be carried out by heating the biomass and / or fermentation medium to a high temperature. For example, the biomass and / or fermentation medium may be heated to a temperature of approximately 50°C to approximately 95°C (e.g., approximately 55°C to approximately 90°C or approximately 65°C to approximately 80°C). Optionally, the biomass and / or fermentation medium may be heated for approximately 30 minutes to approximately 120 minutes (e.g., approximately 45 minutes to approximately 90 minutes or approximately 55 minutes to approximately 75 minutes). Suitable heating methods such as direct steam injection may be used for pasteurization.

[0037] Optionally, pasteurization may be omitted. In other words, the method taught herein may optionally omit pasteurization.

[0038] Optionally, biomass can be collected using a variety of methods, including those currently known to those skilled in the art. For example, biomass can be collected from a fermentation medium using, for instance, centrifugation (e.g., a solids discharge centrifuge) or filtration (e.g., cross-flow filtration). Optionally, the collection step includes using a precipitant to achieve accelerated collection of cellular biomass (e.g., sodium phosphate or calcium chloride).

[0039] Optionally, the biomass is washed with water. Optionally, the biomass can be concentrated up to about 20% solids. For example, the biomass can be concentrated to about 5% to about 20% solids, about 7.5% to about 15% solids, or about solids to about 20% solids, or any percentage within the stated range. Optionally, the biomass can be concentrated to about 20% solids or less, about 19% solids or less, about 18% solids or less, about 17% solids or less, about 16% solids or less, about 15% solids or less, about 14% solids or less, about 13% solids or less, about 12% solids or less, about 11% solids or less, about 10% solids or less, about 9% solids or less, about 8% solids or less, about 7% solids or less, about 6% solids or less, about 5% solids or less, about 4% solids or less, about 3% solids or less, about 2% solids or less, or about 1% solids or less.

[0040] The provided methods optionally include the isolation of polyunsaturated fatty acids from biomass or microorganisms. The isolation of polyunsaturated fatty acids can be performed using one or more of a variety of methods, including those currently known to those skilled in the art. For example, a method for isolating polyunsaturated fatty acids is described in U.S. Patent No. 8,163,515, which is incorporated herein by reference in its entirety. Optionally, the culture medium is not sterilized prior to the isolation of the polyunsaturated fatty acids. Optionally, sterilization includes increasing the temperature. Optionally, the polyunsaturated fatty acids produced by microorganisms and isolated according to the provided methods are medium-chain fatty acids. Optionally, one or more polyunsaturated fatty acids are selected from the group consisting of α-linolenic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, linoleic acid, linolenic acid, and combinations thereof.

[0041] Oils comprising polyunsaturated fatty acids (PUFAs) and other lipids produced according to the methods described herein can be used in any of a variety of applications utilizing their biological, nutritional, or chemical properties. Therefore, the provided methods optionally include separating the oil from a collection portion having a threshold volume. Optionally, the oil is used to produce fuels such as biofuels. Optionally, the oil can be used in pharmaceuticals, food supplements, animal feed additives, cosmetics, etc. Lipids produced according to the methods described herein can also be used as intermediates in the production of other compounds.

[0042] For example, oil produced by microorganisms cultured using the provided method may contain fatty acids. Optionally, the fatty acids are selected from the group consisting of α-linolenic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, gamma-linolenic acid, linoleic acid, linolenic acid, and combinations thereof. Optionally, the oil contains triglycerides. Optionally, the oil contains fatty acids selected from the group consisting of palmitic acid (C16:0), myristic acid (C14:0), palmitoleic acid (C16:1(n-7)), cis-octadecenoic acid (C18:1(n-7)), docosapentaenoic acid (C22:5(n-6)), docosahexaenoic acid (C22:6(n-3)), and combinations thereof.

[0043] Optionally, lipids generated according to the methods described herein may be incorporated into a final product (e.g., food or feed supplement, infant formula, pharmaceuticals, fuel, etc.). Suitable food or feed supplements in which lipids may be incorporated include beverages such as milk, water, sports drinks, energy drinks, tea, and fruit juices; sweets such as candy, jelly, and biscuits; fatty foods and beverages such as dairy products; processed foods such as soft rice (or porridge); infant formula; breakfast cereals; etc. Optionally, one or more lipid-generating agents may be incorporated into dietary supplements such as vitamins or multivitamins. Optionally, lipids generated according to the methods described herein may be included in dietary supplements and optionally may be directly incorporated into components of food or feed (e.g., food supplements).

[0044] Examples of feeds in which lipids produced by the methods described herein can be incorporated include pet foods such as cat food; dog food; feeds for ornamental fish, farmed fish, or crustaceans; and feeds for farm-raised animals (including livestock and fish or crustaceans raised in aquaculture). The food or feed substance into which lipids produced according to the methods described herein can be incorporated is preferably palatable to the organism intended as the recipient. This food or feed substance may have any physical properties currently known about food substances (e.g., solid, liquid, soft).

[0045] Optionally, one or more generating compounds (e.g., PUFA) may be incorporated into a nutritional food or medicine. Examples of such nutritional foods or medicines include various types of tablets, capsules, decoctions, etc. Optionally, the nutritional food or medicine is suitable for surface application. Dosage forms may include, for example, capsules, oils, granules, fine granules, powders, tablets, pills, lozenges, etc.

[0046] The oils or lipids produced according to the methods described herein can be incorporated into products as described herein in any combination of a variety of other reagents. For example, the compounds can be combined with one or more binders or fillers, chelating agents, pigments, salts, surfactants, humectants, viscosity modifiers, thickeners, lubricants, fragrances, preservatives, etc., or any combination thereof.

[0047] Disclosed are materials, compositions, and components that can be used with, in conjunction with, or as a product of the disclosed methods and compositions, the disclosed methods and compositions. These and other materials are disclosed herein, and it should be understood that when combinations, subgroups, interactions, groups, etc., of these materials are disclosed, although specific references to the various individual and collective combinations and arrangements of these compounds may not be explicitly disclosed, each is explicitly covered and described herein. For example, if a method is disclosed and discussed, and numerous modifications to the method, including to a number of molecules, are discussed, then each and every possible combination and arrangement of the method and the modifications is explicitly covered unless explicitly indicated otherwise. Similarly, any subgroups or combinations of these are explicitly covered and disclosed. This concept applies to all aspects of this disclosure, including but not limited to steps in methods using the disclosed compositions. Therefore, if multiple additional steps are possible, it should be understood that each of these additional steps can be performed with any particular method step or combination of method steps of the disclosed method, and each such combination or subgroup of combinations is explicitly covered and should be considered disclosed.

[0048] As used throughout, ranges (e.g., 1-10) and references to a given value (e.g., about 1 or about 10) include one or more of the stated values ​​(e.g., 1 and / or 10).

[0049] The publications cited in this article and the material to which they refer are hereby explicitly incorporated in their entirety by reference.

[0050] The following examples are intended to further illustrate certain aspects of the methods and compositions described herein, and are not intended to limit the scope of the claims.

[0051] Example

[0052] Example 1. Fermentation of crude glycerol

[0053] Four crude glycerol streams were obtained from three different biodiesel manufacturers (Rothsay (Winnipeg, Canada), BIOX (Hamilton, Canada), and REG Newton LLC (Newton, Iowa)). Their compositions are listed in Table 3 based on information provided by the respective manufacturers. It is clear that there is significant variability among the different crude glycerol streams.

[0054] Table 3. Crude glycerol composition of crude glycerol byproducts from biodiesel.

[0055]

[0056] MONG: Non-glycerol organic substance

[0057] For laboratory-scale fermentation experiments, the pH of Rothsay and BIOX 65 crude glycerol was adjusted to 5.5 using sodium hydroxide solution. The pH of BIOX 98 and REG Newton crude glycerol was not adjusted. All crude glycerol streams were then autoclaved and used as the sole carbon source for batch fermentation of *Ostridium lycotriensis* ONC-T18 on a single crude glycerol-based feedstock in a 2 L fermenter. All initial fermentation media contained (per liter): 60 g glycerol; 2 g soybean peptone; 1.65 g sodium chloride; 4 g magnesium sulfate heptahydrate; 2.2 g potassium dihydrogen phosphate; 2.4 g dipotassium hydrogen phosphate; 20 g ammonium sulfate; 0.1 g calcium chloride dihydrate; 0.003 g ferric chloride; 0.003 g copper sulfate pentahydrate; 0.0015 g dehydrated sodium molybdate; 0.003 g zinc sulfate heptahydrate; 0.0015 g cobalt chloride hexahydrate; 0.0015 g manganese chloride tetrahydrate; 0.0015 g nickel sulfate hexahydrate; 0.00003 g vitamin B12; 0.00003 g biotin; and 0.006 g thiamine hydrochloride. 2 L fermenters were used for Rothsay, BIOX 65, and BIOX 98 fermentations, while 5 L fermenters were used for REG Newton fermentations. The pH of all fermentations was controlled at 4.5 ± 0.2 using sodium hydroxide and phosphoric acid solutions. The temperature of all fermentations was controlled at 28°C. All fermentations were operated in a feed-batch mode, using the appropriate amount of crude glycerol as the sole carbon feedstock. At appropriate intervals, a specific amount of crude glycerol was automatically pumped into the fermenter to achieve a glycerol concentration of 60 g / L (for 2 L operations) or 20 g / L (for 5 L operations) in the culture medium.

[0058] The carbon feed is carried out throughout the fermentation process until a certain biomass concentration and intracellular oil content are reached. A final biomass concentration in the range of 140 g / L to 165 g / L is obtained. Figure 1 The crude glycerol feedstock contains 65% to 78% intracellular lipids (Table 4). Considering the quality variations between the tested crude glycerol feedstocks and the minimal pretreatment applied to them, these results demonstrate the robustness of the developed high cell / lipid density feedstock batch fermentation process for commercially viable biomass and lipid production using crude glycerol byproducts.

[0059] Table 4. Overview of fermentation results using various crude glycerol feedstocks as the sole carbon source.

[0060]

[0061] Example 2. Fermentation with mixed carbon sources

[0062] A sufficient and uninterrupted supply of carbon feedstock is critically important for the operation of fermentation-based production plants. In situations where the primary carbon feedstock is in short supply, the ability to utilize supplemental carbon feedstock is crucial for the continued operation of the plant. Therefore, a mixture of glucose and crude glycerol was used for the fermentation of *Neurochytridactylum* ONC-T18. Although glucose is a well-known carbon source for high-density fermentation of *Neurochytridactylum* to produce lipids, it remains unclear whether carbon catabolite inhibition will occur if glucose is used in the same fermentation as a second carbon source. Two 2 L fermenters were prepared using the same initial culture medium formulation as shown in Example 1. Crude glycerol from Rothway, having the specifications shown in Example 1, was obtained; and a 750 g / L glucose solution was also prepared. The crude glycerol and glucose solution were then physically mixed at an 80:20 volume ratio as the carbon feed for the first fermenter and at a 20:80 volume ratio as the feed for the second fermenter. Fermentation was carried out using the same carbon feed strategy described in Example 1. Figure 2 As demonstrated by the data shown in Table 5, the biomass and lipid results produced using crude glycerol and glucose as a mixed carbon feedstock are similar to those when crude glycerol is used as the sole carbon feedstock. No carbon catabolite repression was observed in this example, the phenomenon where the presence of a preferred carbon source inhibits the metabolism of the second carbon source when both carbon sources are present in the growth medium. During fermentation, both glycerol and glucose are consumed simultaneously throughout the culture. This example demonstrates that crude glycerol can be used as a primary or secondary carbon source during commercial cultivation of *Cyclochytrium*, and a second carbon feedstock can be used in the same fermentation. This flexibility in carbon usage ensures the stability of large-scale fermentation processes.

[0063] Table 5. Overview of fermentation results using mixtures of crude glycerol and glucose at different volume ratios.

[0064]

[0065] Example 3. Fermentation of mixed crude glycerol

[0066] Similar to Example 2, it will also be advantageous for sustained large-scale plant operation to use a mixture of crude glycerol from different biodiesel manufacturing processes. For this purpose, four crude glycerol streams were mixed in equal volume ratios to form a crude glycerol mixture. The specifications of each stream are listed in Table 6. After mixing, the crude glycerol mixture was used as a carbon feed without any further treatment (e.g., pH adjustment, filtration) or sterilization. A 30 L fermenter was prepared for this experiment using an initial culture medium with the same formulation as shown in Example 1, except that glycerol was not added in batches to the initial culture medium before the sterilization process. Immediately after inoculation, the crude glycerol mixture was fed according to the feeding strategy described in Example 1, with each feed resulting in a glycerol concentration of 20 g / L in the culture medium. By 70 hours, the biomass reached 171.54 g / L with 68.00% lipids. Despite being used without sterilization, the fermentation was contaminated.

[0067] Table 6. Composition of crude glycerol byproducts from different biodiesel manufacturing processes used to prepare crude glycerol mixtures.

[0068]

[0069] MONG: Non-glycerol organic substance

[0070] Example 4. Crude glycerol for commercial biomass and lipid production

[0071] To demonstrate the feasibility of the developed crude glycerol-based fermentation process for commercial biomass and lipid production, two large-scale (200,000 L) fermentations were conducted. REG Ralston crude glycerol was used as the sole carbon source without any pretreatment or sterilization. The culture medium formulation was the same as described in Example 1, except that only 30 g / L glucose was added batch-added to the initial medium, 1 g / L soybean peptone was used in the first batch, and 1 g / L corn soaking solids (in lieu of soybean peptone) was used in the second batch. Fermentation was carried out at pH 4.5 ± 0.3, with the temperature controlled at 29 ± 1 °C. Biomass and lipid time curves for one of the two fermentations are shown in [Figure / Table / Insert Table ... Figure 3 The full-process metrics from the two large-scale operations are listed in Table 7. On average, crude glycerol byproducts from biodiesel can be used as the sole carbon feedstock to produce 180.76 g / L of biomass with 126.53 g / L of lipids in a 200,000 L fermenter without any pretreatment or sterilization procedures. These results demonstrate the feasibility of using crude glycerol as a carbon feedstock for large-scale commercial production of biomass and lipids.

[0072] Table 7. Large-scale (200,000 L) fermentation using crude glycerol as the sole carbon source without pretreatment or sterilization.

[0073]

Claims

1. A method for culturing *Cyclochytrium* microorganisms, wherein the microorganisms produce one or more fatty acids, the method comprising: (a) Culture the *Cyclochytrium* microorganism in a medium containing a first concentration level of crude glycerol and a nitrogen source, wherein the first concentration level is between 15 and 60 g / L; (b) Once the first concentration of glycerol is reduced to a first threshold level, an additional amount of crude glycerol is fed into the culture medium at a concentration sufficient to achieve the first concentration level, the first threshold level being between 0 and 5 g / L, wherein no nitrogen source is added to the culture medium during the feeding step; (c) Monitor the crude glycerol concentration until the first concentration level of the crude glycerol decreases to the first threshold level; (d) Repeat steps (b) and (c) until the desired microbial cell density is achieved; and (e) Separate the fatty acids.

2. The method of claim 1, wherein the *Cyclochytrids* microorganism is a microorganism with ATCC accession number PTA-6245.

3. The method of claim 1, wherein the fatty acid is a polyunsaturated fatty acid.

4. The method of claim 3, wherein the polyunsaturated fatty acid is selected from the group consisting of α-linolenic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, γ-linolenic acid, linolenic acid, linolenic acid, and combinations thereof.

5. The method of any one of claims 1-4, wherein the monitoring includes measuring dissolved oxygen levels.

6. The method of any one of claims 1-4, wherein the monitoring comprises obtaining a sample of the culture medium and determining the glycerol concentration in the sample.

7. The method of claim 6, wherein the monitoring comprises analyzing the sample using calorimetry, fluorescence assay, HPLC assay, enzymatic assay, or a combination thereof.

8. The method of any one of claims 1-4, wherein the cell density is between 50 g / L and 250 g / L.

9. The method according to any one of claims 1-4, wherein the microbial cell density contains 50% to 80% by weight of total fatty acids.

10. The method of claim 9, wherein the total fatty acids comprise 10 to 45% docosahexaenoic acid (DHA).

11. The method of any one of claims 1-4, wherein the microbial cell density contains 5 to 36% DHA based on total cell weight.

12. The method of any one of claims 1-4, wherein the culture medium contains one or more additional carbon sources.

13. The method of any one of claims 1-4, wherein the crude glycerol is not sterilized.

14. The method of any one of claims 1-4, wherein the crude glycerol comprises methanol, water, ash, non-glycerol organic matter, sodium sulfate, methyl tallow, or a combination thereof.

15. The method of claim 14, wherein the ash comprises calcium, iron, magnesium, potassium, sodium, zinc, or a combination thereof.

16. The method of any one of claims 1-4, wherein the crude glycerol is a biodiesel byproduct.

17. A method for producing one or more fatty acids, comprising: (a) Provides chytrid fungi that produce fatty acids; (b) Provide a culture medium containing crude glycerol; as well as (c) Once the crude glycerol in the culture medium is between 0 and 5 g / L, the microorganisms are cultured in the culture medium by repeatedly adding fresh crude glycerol under nitrogen-free conditions to produce the one or more fatty acids and provide a final concentration of at least 50% of the total cell weight of the microorganisms of one or more unsaturated fatty acids; and (d) Separate the fatty acids.

18. The method of claim 17, wherein the *Cyclochytrids* microorganism is a microorganism with ATCC accession number PTA-6245.

19. The method of claim 17, wherein the fatty acid comprises a polyunsaturated fatty acid.

20. The method of claim 19, wherein the polyunsaturated fatty acid is selected from the group consisting of α-linolenic acid, arachidonic acid, docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, γ-linolenic acid, linolenic acid, linolenic acid, and combinations thereof.

21. The method of any one of claims 17-20, wherein the culture medium contains one or more additional carbon sources.

22. The method of any one of claims 17-20, wherein the crude glycerol is not sterilized.

23. The method of any one of claims 17-20, wherein the crude glycerol comprises methanol, water, ash, non-glycerol organic matter, sodium sulfate, methyl tallow, or a combination thereof.

24. The method of claim 23, wherein the ash comprises calcium, iron, magnesium, potassium, sodium, zinc, or a combination thereof.

25. The method of any one of claims 17-20, wherein the crude glycerol is a biodiesel byproduct.

26. The method of claim 1, wherein the crude glycerol is not pretreated.

27. The method of claim 1, wherein the required microbial cell density is from 100 g / L to 250 g / L.

28. The method of claim 1, wherein the nitrogen source is ammonium sulfate.

29. The method of claim 28, wherein the culture medium comprises 20 g / L to 25 g / L of ammonium sulfate.

30. The method of claim 12, wherein the second carbon source is glucose.

31. The method of claim 30, wherein the volume ratio of crude glycerol to glucose is 80:20 or 20:80.

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