CA3084750A1 - Anaplerotic oil production in microbials - Google Patents

Abstract

Disclosed are techniques and systems for producing microbials having anaplerotic oils that are rich in odd-chain fatty acids, and other beneficial components, at higher concentrations than those present in other natural dietary sources of OCFA, at lower cost, and higher production yield. Such compositions can comprise pentadecanoic and heptadecanoic fatty acids. The techniques described herein include methods for producing and deriving such compositions rich in odd-chain fatty acids from microbials, including microalgae and yeasts/fungi.

Description

Attorney Docket No. 05001H0171221CA

Cross-Reference to Related Applications [0001]
Background

[0002] The citric acid cycle can govern the energy metabolism in aerobic organisms. In addition, the cycle can provide precursors for biosynthesis of several amino acids, lipids, chlorophyll and other growth-related metabolites. The citric acid cycle is non-catalytic, which means that molecules used in biosynthesis need to be replenished so that the cycle can keep generating energy.
Regardless of how much acetyl CoA is fed into the citric acid cycle, the cycle is able to produce merely a limited amount of citric acid intermediates. Anaplerotic substrates can be used to produce intermediates that are used to replenish the oxidative capacity of the citric acid cycle.

[0003] Anaplerosis refers to the process of replenishing the citric acid cycle intermediates and restoring energy balance of the cell (metabolic homeostasis). Odd-chain fatty acids (OCFA) can be considered anaplerotic because, along with acetate units, they can also release propionic acid which can enter the citric acid cycle through the methylmalonate pathway (OCFA
catabolism).
Typical dietary sources of OCFA are milk and butter, but they have only trace amounts (<2 %
total fatty acids, TFA) of pentadecanoic (C15:0) and heptadecanoic (C17:0) acid. Synthetically produced concentrated sources, such as tripentanoin and triheptanoin (e.g., oils containing C5:0 and C7:0), are not considered nutritional lipids. Further, current methods that involve the use of Yarrowia lipolytica to produce odd chain fatty acids utilize genetic modification. Specifically, for example, these methods utilize the deletion of the PHD1 gene in order to improve lipid accumulation. Ref. 10.
Summary

[0004] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
factors or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0005] Disclosed are compositions rich in odd-chain fatty acids (OCFA), including pentadecanoic and heptadecanoic fatty acids, and products rich in tridecanoic, pentadecanoic and heptadecanoic fatty acids derived from microalgae, yeast or fungi; OCFA promoters that can be used to induce OCFA production; and processes that help reduce an amount of propionate used in OCFA
production. In some implementations, OCFA production in microalgae, yeast and fungi may be increased to yield useful quantities. Further, in some implementations, alternative substrates to propionic acid, such as pentanoic acid, heptanoic acid, yeast extract, proteose peptone, valine, and methionine, can be used to induce OCFA production. Additionally, in some embodiments, a method may be implemented that improves propionic acid incorporation into A.
acetophilum HS399 lipids as OCFA instead of being catabolized in the citric acid cycle.

[0006] Further, techniques and systems are disclosed for identifying propionic acid toxicity in some types of microorganisms, for example, in order to utilize an upper threshold of propionic acid during cultivation to promote OCFA production. Additionally, techniques and systems are disclosed for identifying and using promotors of OCFA production.

[0007] To the accomplishment of the foregoing and related ends, the following description and annexed drawings set forth certain illustrative aspects and implementations.
These are indicative of but a few of the various ways in which one or more aspects may be employed.
Other aspects, advantages and novel features of the disclosure will become apparent from the following detailed description when considered in conjunction with the annexed drawings.
Brief Description of the Drawings

[0008] The claimed matter may take physical form in certain parts and arrangements of parts, a preferred embodiment of which will be described in detail in the specification and illustrated in the accompanying drawings which form a part hereof, and wherein:

[0009] FIGURE 1 is a chromatogram of Aurantiochytrium acetophilum HS399 displaying the microalgal fatty acid profile.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[0010] FIGURE 2 is a line graph depicting the impact of propionate supplementation on microalgae growth under batch and fed-batch conditions.

[0011] FIGURE 3 is a flow chart of steps involved in a method according to an exemplary embodiment of the present disclosure.

[0012] FIGURE 4 is a line graph depicting Aurantiochytrium acetophilum HS399 growth in response to propionate supplementation.

[0013] FIGURE 5 is a line graph depicting Aurantiochytrium acetophilum HS399 residual glucose consumption in response to propionate supplementation.

[0014] FIGURE 6 is a line graph depicting the culture pH-drift of Aurantiochytrium acetophilum HS399 fed with varying levels of propionate.

[0015] FIGURE 7 is a line graph depicting Aurantiochytrium acetophilum HS399 growth in response to propionate supplementation.

[0016] FIGURE 8 is a line graph depicting Aurantiochytrium acetophilum HS399 residual glucose consumption in response to propionate supplementation.

[0017] FIGURE 9 is a line graph depicting the culture pH-drift of Aurantiochytrium acetophilum HS399 fed with varying levels of propionate.

[0018] FIGURE 10 is a graph depicting fatty acid distribution throughout the culture of Aurantiochytrium acetophilum HS399 fed-batch at different propionic levels.

[0019] FIGURE 11 is a graph depicting fatty acid accumulation throughout the culture of Aurantiochytrium acetophilum HS399 fed-batch at different propionic levels.

[0020] FIGURE 12 is a line graph depicting OCFA accumulation in an Aurantiochytrium acetophilum HS399 culture fed varying amounts of propionate.

[0021] FIGURE 13 is a line graph depicting cell dry weight for an Aurantiochytrium acetophilum HS399 culture fed varying amounts of propionate.

[0022] FIGURE 14 is a line graph depicting Aurantiochytrium acetophilum HS399 residual glucose consumption in response to propionate supplementation.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[0023] FIGURE 15 is a line graph depicting the culture pH-drift of Aurantiochytrium acetophilum HS399 fed with varying levels of propionate.

[0024] FIGURE 16 is a schematic diagram illustrating the active and passive transport of propionic acid inside the cell. The pH gradient across the cell controls the passive uptake of propionic acid by the cell.

[0025] FIGURE 17 is a line graph depicting the residual propionic acid as a function of pH in a pH-auxostat culture of Aurantiochytrium acetophilum.

[0026] FIGURE 18 is a line graph depicting dry cell weight for an Aurantiochytrium acetophilum HS399 culture which shows the impact of pH and an organic acid feeding regime on HS399 growth.

[0027] FIGURE 19 is a line graph depicting residual glucose as a function of the pH-set point in a pH-auxostat culture of Aurantiochytrium acetophilum HS399.

[0028] FIGURE 20 is a line graph comparing the cell dry weight for an Aurantiochytrium acetophilum HS399 culture fed propionic acid and an Aurantiochytrium acetophilum HS399 culture that is not.

[0029] FIGURE 21 is a line graph comparing the cumulative productivities for an Aurantiochytrium acetophilum HS399 culture fed propionic acid and an Aurantiochytrium acetophilum HS399 culture that is not.

[0030] FIGURE 22 is a line graph depicting the residual glucose and ammonia levels.

[0031] FIGURE 23 is a graph showing online monitoring of the fermenter HS399 cultures with other parameters such as dissolved oxygen, feedstock and titrant pumping rate, and pH control.

[0032] FIGURE 24 is a line graph comparing the total propionate consumption of an Aurantiochytrium acetophilum HS399 culture fed propionic acid and an Aurantiochytrium acetophilum HS399 culture that is not.

[0033] FIGURE 25 is a graphical representation of results of growth and substrate consumption of Yarrowia lipolytica ATCC18944 using different carbon sources.

[0034] FIGURE 26 is a micrograph illustrating the filamentous and yeast morphology of Yarrowia lipolytica while producing OCFAs.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[0035] FIGURES 27 and 28 are graphical representations of cell dry weight, residual glycerol, and pH where Y. /ipo/ytica is cultivated with increasing daily propionate concentrations.

[0036] FIGURES 29 and 30, are graphical representations of odd chain fatty acid % dry weight and total fatty acid % dry weight where Y. /4)0/pica is cultivated with increasing daily propionate concentrations.

[0037] FIGURE 31 is a graphical representation of the A. acetophilum H5399 oxygen uptake (OUR) in response to pH driven propionate toxicity.

[0038] FIGURE 32 is a 3D graphical representation of the propionic acid toxicity as cytosolic propionate is controlled by the extracellular pH and propionate concentration.

[0039] FIGURE 33 is a graphical representation of A. acetophilum H5399 in the presence of batched or fed batch propionate at different daily concentrations.

[0040] FIGURE 34 is a graphical representation illustrating results of growth of Aurantiochytrium acetophilum H5399 and residual ammonia when propionate was fed in growth or lipid phase.

[0041] FIGURE 35 is a graphical representation illustrating the growth of A.
acetophilum H5399 with different carbon sources.

[0042] FIGURE 36 is a graphical representation illustrating the residual propionate cultures of A.
acetophilum H5399 fed different carbon sources.

[0043] FIGURE 37 is a graphical representation of example results of sub-culturing A.
acetophilum H5399 in a cyanocobalamin deprived media for over 10-generations.

[0044] FIGURE 38 is a graphical representation of one implementation illustrating example results of cell dry weight and residual propionate where the culture was initially fed 3g/L
propionate.

[0045] FIGURE 39 is a graphical representation of the impact of cyanocobalamin in A.
acetophilum H5399 growth and propionic acid consumption in 10 L fermenters.

[0046] FIGURE 40 is a graphical representation showing the impact of propionic acid exposure on A. acetophilum H5399 growth and odd chain fatty acid production in 10 L
fermenters under two different growth modes.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[0047] FIGURE 41 is a graphical representation illustrating results of one implementation, where, much like the two-stage growth mode, described above, ammonia can be fed merely during the growth phase.

[0048] FIGURE 42 is a graphical representation illustrating results of one implementation, applying the impact of ammonia to sodium hydroxide ratio of the feed in the total ammonia fed and biomass yields of a double auxostat culture of Aurantiochytrium acetophilum H5399.

[0049] FIGURE 43 is a graphical representation illustrating results of one implementation, describing the impact of pH set-point control in the transition of ammonia to propionic acid pH
auxostat culture of Aurantiochytrium acetophilum H5399.

[0050] FIGURE 44 is a graphical representation illustrating results for Aurantiochytrium acetophilum H5399 double pH-auxostat cultures, showing the impact of sodium hydroxide supplementation into the glucose fed in the residual propionic acid control in a pH-auxostat.

[0051] FIGURE 45 is a graphical representation showing cell dry weight and residual glucose of A. acetophilum H5399 in a 100 L pilot fermenter.

[0052] FIGURE 46 is a graphical representation of the cumulative productivity of A. acetophilum H5399 in a 100 L pilot fermenter.

[0053] FIGURES 47A, 47B, 47C, 47D are graphical representations of online data readings exhibited by A. acetophilum 511399 in a 100 L pilot fermenter.

[0054] FIGURE 48 is a table, (Table 24) that illustrates total lipids and Fatty Acid profile from a 1000 L pilot fermenter for Aurantiochytrium acetophilum H5399 odd chain fatty acid fermentation.

[0055] FIGURES 49A, 49B, 49C, 49D are graphical representations of A.
acetophilum H5399 double pH-auxostat cultures for the production of odd chain fatty acids, growth productivity and lipid accumulation.

[0056] FIGURE 50 is a graphical representation of online data readings of A.
acetophilum H5399 double pH-auxostat cultures.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[0057] FIGURE 51 is a graphical representation of an example result illustrating growth and residual propionate in A. acetophilum HS399 cultures that are subject to propionic anaplerosis triggered by cyanocobalamin.

[0058] FIGURE 52 is a graphical representation of residual glucose in A.
acetophilum HS399 cultures subject to propionic anaplerosis triggered by cyanocobalamin.

[0059] FIGURE 53 is a graphical representation showing growth inhibition of A.
acetophilum HS399 by short chain fatty acids propionic acid (C3:0), pentanoic acid (C5:0) and heptanoic acid (C7:0).

[0060] FIGURE 54 is a table (Table 25) that illustrates a list of results for respective alternative promoters of odd chain fatty acid (0CFAs) production in Aurantiochytrium acetophilum HS399

[0061] FIGURE 55 is a table, (Table 26) that illustrates a list of fatty acid compositions of several vegetable oils.
Detailed Description

[0062] The claimed subject matter is now described with reference to the drawings, wherein like reference numerals are generally used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the claimed subject matter. It may be evident, however, that the claimed subject matter may be practiced without these specific details. In other instances, structures and devices are shown in block diagram form in order to facilitate describing the claimed subject matter.

[0063] With reference to the drawings, like reference numerals designate identical or corresponding parts throughout the several views. However, the inclusion of like elements in different views does not mean a given embodiment necessarily includes such elements or that all embodiments of the claimed subject matter include such elements. The examples and figures are illustrative only and not meant to limit the claimed subject matter, which is measured by the scope and spirit of the claims.

[0064] Anaplerosis refers to the replenishment of citric acid intermediates that have been extracted by the cell for biosynthesis. Anaplerotic substrates, such as glucose, protein and odd chain fatty Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
acids (OCFAs), could be converted into citric acid intermediates to restore an energy imbalance of the cell. Anaplerotic substrates are often referred as gluconeogenic substrates. OCFAs are different from other anaplerotic substrates because they can undergo ketosis and cross the blood-brain barrier. Therefore, OCFAs have been associated with a decrease in metabolic disease risk, and their intake has been proposed for the treatment and prevention of various gene and brain disorders. The presence of OCFAs in diet is scarce and typically limited to ruminant fat (e.g., butter), which contains only trace amounts (<2 % total fatty acid (TFA)) of pentadecanoic acid (C15:0) and heptadecanoic acid (17:0). Existing pharma OCFAs, such as tripentanoin and triheptanoin oils, are produced synthetically, and are made of fatty acids that are not typically present in a human diet. Alternatively, as described herein, a process may be devised that can result in a natural oil comprising large (e.g., >50 total fatty acid (TFA)) quantities of dietary (e.g., C15:0 and C17:0) OCFAs.

[0065] Typical anaplerotic substrates can include pyruvate (e.g., derived from carbohydrates), glutamine/glutamate (e.g., derived from protein) and precursors of propionyl-CoA, such as OCFAs. Anaplerotic substrates can be used to restore energy balance in the mitochondria; and, there is a wide range of pathologies to which OCFAs may provide benefits. As an example, in this aspect, OCFAs have been experimentally used to treat: gene metabolic disorders, such as Glutl deficiency, Fatty Acid Oxidation Disorder (FAOD), Pyruvate Carboxylase Deficiency, Carnitine Palmitoyltransferase II Deficiency, Huntington, Phenylketonuria, Adult Polyglucosan Body Disease (APBD), and Long-Chain Fat Oxidation Disorders; neural disorders, such as Epilepsy, Alzheimer's Disease, and Autism Spectrum Disorder (ASD); circulatory system disorders, and diabetes type II and other diseases associated to the metabolic syndrome epidemics.

[0066] Dietary odd chain fatty acids (OCFA), pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0), also known as margaric acid, may be derived from ruminant fat (e.g., butter), and are thought to be likely derived from bacterial anaerobic activity in the rumen of dairy producing animals. These OCFAs can be found in very small amounts (e.g., <2 % total fatty acids (TFA)) in some dairy products (e.g., milk and butter). Pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) have also been found to be produced in the human gut, which may be triggered by dietary fiber intake, presumably supporting bacterial anaerobic activity. Ref. 1.
Because only trace amounts of odd chain fatty acids (e.g., C15:0 and C17:0) are present in human diets, alternative Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
sources (i.e. nutraceuticals, medical foods or therapeutics) can be used to significantly increase the intake of this type of nutrient.

[0067] Currently, merely limited amounts of odd chain fatty acids (e.g., C15:0 and C17:0) are readily available from known natural, dietary sources, such as ruminant fat.
Techniques and systems can be devised for producing a natural anaplerotic oil that contains significant dietary OCFAs. In one aspect, compositions can be created that comprise a higher concentration, than current sources, of odd chain fatty acids, such as pentadecanoic (C15:0) and heptadecanoic (C17:0) fatty acids. Further, in one aspect, a method can be devised for efficient and affective generation of such fatty acids from a newly derived source.

[0068] Microbials can produce a variety of fatty acids, the composition of which can vary among different strains. As an example, thraustochytrid microalgae can accumulate lipids up to eighty-five (85%) of their dry weight; and, amongst the oleaginous microorganisms, they may be one of the fastest growing. Further, these organisms can be adapted to fermentation conditions (e.g., low shear sensitivity, high osmotolerance) for use in industrial production of microbe-based oils. For example, A. acetophilum HS399 is a thraustochytrid that can produce an oil containing palmitic acid (e.g., 45% total fatty acids (TFA)), n-6 docosapentaenoic acid (e.g., 8 %
TFA), and n-3 docosahexaenoic (e.g., 40% TFA) as the main fatty acids, with other fatty acids present in trace amounts.

[0069] The trace fatty acids of A. acetophilum HS399 can include pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) (e.g., at < 0.3 % TFA). The trace fatty acids, including these two identified fatty acids, are typically ignored in the lipid profile reports for these organisms. OCFA, including pentadecanoic acid and heptadecanoic acid, are fatty acids that contain an odd number of carbon atoms in the structure. OCFA are typically related to bacterial activity (e.g., propionic acid bacteria), and are less likely to be present in algae, yeast/fungi, and plants.

[0070] FIGURE 1 is a chromatogram of Aurantiochytrium acetophilum HS399 illustrating the microalgae's fatty acid profile. As shown in FIGURE 1, A. acetophilum HS399 naturally contains trace amounts of C15:0. The presence of trace amounts in A. acetophilum HS399 suggests that the pathway responsible for the synthesis of OCFA may be present in A.
acetophilum HS399.
Because of the composition of their fatty acid profile, and their ability to be grown rapidly, microbials such as A. acetophilum HS399 may provide an attractive source of odd-chain fatty Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
acids, by generating odd-chain fatty acids in a more concentrated manner than other known natural sources, such as milk fat (e.g., providing a more cost effective and efficient source of OCFA). As an example, a benefit of using microbials in place of butter and other ruminant fat is the higher concentration of OCFA found in them. In addition, as another example benefit, some microbial oil lacks residues of phytol or phytanic acid that are often present in ruminant fat. Consumption of phytol or phytanic acid can lead to health concerns in some individuals.

[0071] Techniques can be devised that provide for an increased production of naturally occurring odd-chain fatty acids from microbials than might be generated from typical microbials. The cultivated microbial and/or isolated composition may be used individually as products or as an ingredient in a variety of products. As an example, microalgae such as A.
acetophilum HS399 can be cultivated to produce a desirable fatty acid profile comprising OCFA, which may be isolated through various extraction processes. In this example, the isolated oil from A. acetophilum HS399, containing the OCFA, may comprise a composition rich in OCFA, such as pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0). As described herein, in one implementation, the algae may be cultivated using an improved method that includes the use of a complex culture media, which can promote increased production of the OCFA.

[0072] In one implementation, the microbials, such as microorganisms comprising algae, microalgae, yeast, and fungi, including species from the class Labyrinthuloycetes, such as the species Aurantiochytrium acetophilum, may be cultivated using an improved method that includes the presence of a complex media, which can promote increased production of the OCFA. In this implementation, an amount of heptadecanoic acid produced by A. acetophilum HS399 can increase from <0.3 up to 1 % TFA when a complex culture media containing yeast extract and proteose peptone is used as a replacement for previously utilized media (e.g., defined media). In this implementation, the increase of heptadecanoic acid may be proportional to the amount of proteose peptone used in the complex culture media. The increase in heptadecanoic acid in the cultured A.
acetophilum HS399, using this technique, suggests that the presence of OCFA in thraustochytrids may not be strain specific, nor stress related. Ref. 3, 5. Instead, using this technique, the increase in heptadecanoic acid is likely due to the presence of added nutrients in the media that provide for the accumulation of this OCFA. Therefore, the high levels of odd chain fatty acids reported in some thraustochytrids strains (Ref. 4) might not be a strain specific trait, nor a physiological Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
response to stress (Ref. 3,5) but rather the result of poor growth and/or high yeast extract or other OCFA precursors.

[0073] In one implementation, propionic acid (e.g., and or one or more propionates, such as the anion, salts, and/or esters of propionic acid) may be used as a precursor for production of OCFA.
Proteose peptone comprises valine, isoleucine, and methionine amino acids, respectively comprising at least a three-carbon chain, which may provide a precursor three-carbon backbone of propionic acid. In this implementation, for example, it is likely that A.
acetophilum HS399 can incorporate propionic acid in its lipid generation pathway, resulting in the production of OCFA.

[0074] Generally, fatty acid synthesis in oleaginous microbials consists of a lipid synthesis pathway involving acetyl CoA, and some metabolic cycles. As an example, acetyl-coenzyme A
(Acetyl-CoA) is a universal two carbon donor, or building block, for fatty acid biosynthesis.
Acetyl-CoA can be supplied by multiple paths, from various origins, and then subsequently activated into acetyl-acyl carrier protein (ACP) or converted to Malonyl CoA
through Acetyl-CoA
carboxylase. Later, by sequential reactions of condensation, reduction dehydration and reduction, palmitic acid will be produced.

[0075] In one aspect, analysis of the genome of A. acetophilum HS399 suggests that saturated fats are synthesized through the Fatty Acid Synthase (FAS) pathway that uses acetyl-coA as a building block for the fatty acid elongation. The production of even chain fatty acids uses a malonyl-ACP
as a substrate for elongation. As described herein, in one implementation, when propionic acid is present the acyl carrier protein (ACP) cleaves to methyl-malonyl instead of malonyl, resulting in the FAS producing of odd chain fatty acids instead of even chain fatty acids.
Palmitic acid (C16:0) is typically the primary even chain fatty acid in A. acetophilum HS399, while the primary OCFA
is typically pentadecanoic (C15:0) instead of heptadecanoic acid (17:0). In this implementation, fatty acid synthesis of palmitic acid (C16:0) undergoes through 6-consecutive elongation cycles, while the (C15:0) OCFA undergoes only 5-elongation cycles before the fatty acid is liberated from the acyl carrier protein. This suggests that FAS is governed by a chain length factor.

[0076] In one aspect, propionic acid is commonly used for its antimicrobial characteristics, among other things. For example, propionic acid can inhibit growth of mold and bacteria at low levels (e.g., < 1% by weight), and is often used as an antimicrobial agent to preserve animal and/or human food sources. Other uses include adding propionic acid to products to mitigate algae growth on Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
surfaces. In this aspect, as an illustrative example, FIGURE 2 illustrates propionic acid's growth inhibitory characteristics for A. acetophilum HS399, at concentrations as low as 3 grams/liter (g/L);
and lethality to A. acetophilum HS399 at concentrations of 10 g/L.

[0077] In this aspect, in addition to the common and traditional use of propionic acid as an antimicrobial agent that kills algae, as described herein, techniques have been devised for propionic acid to be used to facilitate in growing algae, and/or to increase OCFA production in the algae. In one implementation, in this aspect, propionic acid (e.g., and/or propionates) can be introduced into an algal bioprocess using a fed-batch approach, while reducing the potential toxic effects on the algae. FIGURE 3 is a flow diagram illustrating an exemplary method 300 for introducing propionic acid into an algal growth culture program. The exemplary method begins at 302. At reference numeral 304, a microorganism (e.g., microalgae such as A.
acetophilum HS399) can be added to the culture medium. At reference numeral 306, propionic acid may be added to the culture medium comprising the microorganism (e.g., A. acetophilum HS399) in a batch, continuous or fed-batch process, and cultured in a bioreactor having with the culture medium (e.g., organic).

[0078] In one embodiment, the propionic acid can be added at a ratio of at least 0.05 g of propionic acid per gram of A. acetophilum HS399 biomass, in order to accumulate elevated amounts of OCFA. In one embodiment, 0.15 g of propionic acid per gram of A. acetophilum HS399 biomass, in order to accumulate OCFA above 50 % TFA. In another implementation, the propionic acid can be added at a rate of above zero and up to about 3 g/L per day. In one implementation, the propionic acid can be added at a rate of above zero and up to about 3 g/L per day for three days, resulting in a total propionic acid addition of about 9 g/L. In one embodiment, adding the propionate can comprise adding the propionate in a fed-batch into the culture medium. In one embodiment, adding the propionate can comprise adjusting the propionate fed to produce OCFAs in a range of 5 and 70 % TFAs.

[0079] At reference numeral 308, anaplerotic oil containing concentrated amounts of OCFA can be extracted from the A. acetophilum HS399. In one embodiment, anaplerotic oil can be produced from the cultured microorganisms, wherein at least five percent of the total fatty acids (TFA) of the anaplerotic oil are OCFA, and OCFA make up at least one percent cell dry weight (CDW) of Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
the anaplerotic oil. Having extracted the anaplerotic oil containing concentrated amounts of OCFA
the exemplary method 300 ends at 310.

[0080] In one implementation, the propionate fed approach can cause some microorganism (e.g., A. acetophilum HS399) growth inhibition, for example, but may not result in a complete culture loss of the microorganism batch. In this implementation, the fed-batch approach can achieve similar cell densities and overall lipid accumulation as a similar control batch with no propionic acid fed, with merely a one-day difference. As one example, propionic acid can be fed into the algal culture batch on demand (e.g., automatically, using a pH-auxostat fed batch system). As another example, propionic acid can be fed into the algal batch, along with a carbon source (e.g., glucose, glycerol or acetate), at a ratio below 0.1 of weight to weight (w/w) of propionic acid to carbon source (propionic acid/carbon source). In another example, propionic acid can be fed along with the carbon source at a ratio below 0.05 w/w propionic acid/carbon source, to mitigate avoid accumulation of propionate in the culture media. In one example, propionic acid may be fed into a culture at a culture pH higher than 5. A low pH increases the toxicity of propionic acid making it more difficult to balance the window between propionate incorporation and grown inhibition.
Examples

[0081] Embodiments described herein are exemplified and additional embodiments are disclosed in further detail in the following Examples, which are not in any way intended to limit the scope of any aspects of the inventive concepts, described herein.
Example 1 - Propionic acid incorporation into A. acetophilum fatty acid synthase (FAS)

[0082] In one example implementation, the resulting impact on growth and lipid accumulation of Aurantiochytrium acetophilum HS399 when using propionic acid can be illustrated. In this implementation, four treatments can be prepared with varying concentrations of propionate (e.g., 0, 10, 20, 30 g/L), hereinafter: "PO, P10, P20 and P30" respectively. In this implementation, propionate can be batched as sodium propionate in a flask culture. Respective Erlenmeyer flasks (250 mL) can be inoculated (1 % v/v) in triplicates with a 24 hour (h) old culture of A. acetophilum HS399 and incubated in an orbital shaker at 180 rpm and 27 C.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[0083] In this implementation, the respective Erlenmeyer flasks contain 100 mL
of a medium supplemented with (g/L): dextrose (50), ammonium acetate (2.3), NaCl (12.5), MgSO4 7H20 (2.5), KH2PO4 (0.5), KC1 (0.5) and CaCl2 (0.1). This medium also contains trace element solution (5 ml/L) and vitamin solution (1 ml/L). The trace element solution contains (g/L): EDTA di-sodium salt (6), FeCl3 6H20 (0.29), H2B03 (6.84), MnC12 4H20 (0.86), ZnC12 (0.06), CoC12 6H20 (0.026), NiSat 6H20 (0.052), CuSO4 5H20 (0.002), Na2MoO4 H20 (0.005). The vitamin solution contains (mg/L): thiamine (100), biotin (0.5) and cyanocobalamin (0.5). All culture materials can be autoclaved (121 C, 15 min) and media can be filter sterilized before use. A
propionic acid stock solution (200 g/L) can be used to feed propionic acid. Daily samples can be collected to analyze the cell dry weight, residual glucose, culture pH, lipid and fatty acid composition of the cultures.
Cell dry weights are analyzed by vacuum filtration (0.2 m) and washed with a solution of ammonium bicarbonate. Residual glucose is analyzed using a colorimetric method based on glucose peroxidase activity. Biomass for lipid analysis can be centrifuged and washed using purified water. The washed biomass can be freeze dried. Total lipids are analyzed using the Folch method (AOAC 996.06) and the FAMEs are analyzed by gas chromatography using nonadecanoic (C19:0) acid as an internal standard.

[0084] FIGURE 4 illustrates the resulting cell dry weights, and the resulting residual glucose is illustrated shown in FIGURE 5. As illustrated by the results, propionic acid is lethal at concentrations of 20 g/L and 30 g/L, while concentrations of 10 g/L are strongly inhibitory to the growth of A. acetophilum HS399. FIGURE 6 illustrates the A. acetophilum HS399 growth results in the alkalization of the medium, presumably associated to the consumption of organic acids. As shown by the lipid and fatty acid data at 68 h of incubation from PO and P10 in Table 1 below, the presence of propionate decreases lipid and total fatty acid accumulation.
Further, the presence of propionate produces results in a decrease in palmitic (C16:0) and an increase in saturated odd chain fatty acids (0CFAs) tridecanoic (C13:0), pentadecanoic (C15:0) and heptadecanoic acid (C17:0), which can result in propionic incorporation/deposition (see Table 2). As illustrated, the increase of total OCFA (C13, C15, C17) from 0.2% at zero propionate to 63 % total OCFA
at 10 g/L
propionate suggests that propionate is incorporated in to the fatty acid synthase pathway (FAS).
The lack of OCFA in the polyunsaturated fraction suggests that propionate may not be incorporated/deposited in the polyketide synthase pathway (PKS).
Table 1. Impact of propionate supplementation in HS399 lipid and fatty acid profile after 68 h incubation Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Propionate (g/L) 0 10 Total Lipids (% DW) 83.0 0.0 55.3 0.6 Total Fatty Acids (% DW) 69.5 0.6 42.8 0.9 Fatty Acid Profile (% TFA) 13:0 0.0 0.0 3.0 0.1 14:0 3.1 0.0 0.9 0.0 15:0 0.2 0.0 51.7 0.6 16:0 52.7 0.1 5.1 0.3 17:0 0.0 0.0 8.3 0.0 18:0 1.6 0.0 0.2 0.1 22:5 (n-6) 7.3 0.1 3.0 0.1 22:6(n-3) 32.6 0.1 24.9 0.4 Other FA 2.2 0.0 2.2 1.2 OCFA (% TFA) 0.2 0.0 63.1 0.7 Table 2. Propionate deposition, feeding rate and productivity of OCFAs in response to propionate supplementation Propionate PA Feeding Rate Propionate deposition OCFA
Productivity (g/L) (gpA/gBio...) (%) (g/L/d) 0 0.00 0.00 0.0 0.0 0.00 0.00 1100 1.12 0.05 6.9 0.5 0.81 0.06 Example 2 - Propionic concentration in Aurantiochytrium fatty acid profile

[0085] In another implementation, fed-batching Aurantiochytrium acetophilum HS399 with propionic acid may impact its growth, and lipid accumulation. As an example, in this implementation, four treatments can be prepared with varying concentrations of propionate (0, 3, 6, 9 g/L), hereinafter "PO, P3, P6 and P9" respectively. Propionate can be fed-batch fed as sodium propionate in a flask culture with daily additions at 3 g/L. Respective Erlenmeyer flasks (250 mL) are inoculated (1 % v/v) in triplicates with a 24 h old culture of A.
acetophilum HS399. The cultures are incubated in an orbital shaker at 180 rpm and 27 C.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[0086] Respective Erlenmeyer flasks contain 100 mL of a medium supplemented with (g/L):
dextrose (100), ammonium acetate (4.6), NaCl (12.5), MgSO4 7H20 (2.5), KH2PO4 (0.5), KC1 (0.5) and CaCl2 (0.1). This medium also contains trace element solution (5 ml/L) and vitamin solution (1 ml/L). The trace element solution contains (g/L): EDTA di-sodium salt (6), FeCl3 6H20 (0.29), H2B03 (6.84), MnC12 4H20 (0.86), ZnC12 (0.06), CoC12 6H20 (0.026), NiSat 6H20 (0.052), CuSO4 5H20 (0.002), Na2MoO4 H20 (0.005). The vitamin solution contains (mg/L):
thiamine (100), biotin (0.5) and cyanocobalamin (0.5). All culture materials can be autoclaved (e.g., 121 C, 15 min) and the media can be filter sterilized before use. A
propionic acid stock solution (200 g/L) can be used as the fed propionic acid. In this example, PO
is not fed any propionate, P3 is fed 3 g/L on day 0 (inoculation day), P6 is fed 3 g/L on day 0 and 3 g/L on day 1, and P9 is fed 3 g/L on day 0, 1 and 2. Daily samples are collected to analyze the cell dry weight, residual glucose, culture pH (see FIGURE 9), and lipid and fatty acid composition of the cultures.
Cell dry weights (CDW) can be analyzed by filtration (e.g., 0.2 gm filter media) using a vacuum, and washed with a solution of ammonium bicarbonate. Residual glucose can be analyzed using colorimetric methods based on glucose peroxidase activity. Biomass for lipid analysis are centrifuged and washed using purified water. The washed biomass is freeze dried. Total lipids are analyzed using Folch method (AOAC 996.06) and the FAMEs re analyzed by gas chromatography using nonadecanoic (C19:0) acid as an internal standard.

[0087] In this example, as shown by the cell dry weights represented in FIGURE
7, and the residual glucose represented in FIGURE 8, the treatment that utilized propionic acid produced results illustrating growth inhibition at concentrations as low as 3 g/L (P3).
Further, these results illustrate that the growth inhibition effect may be dose dependent, with the strongest inhibition resulting from treatments having higher propionate concentrations (P6 and P9).
Even though growth inhibition was exhibited, the 70 h growth achieved in this example for the fed batching of 9 g/L of propionate (¨ 15 g/L) was higher than the growth achieved for the of batching 9 g/L of propionate at inoculation (see Example 1). This example result illustrates that fed-batching can be an effective strategy for mitigating propionic toxicity, while inducing the cells to produce OCFAs.

[0088] As an example of this strategy, the lipid and fatty acid data is represented in Tables 3-5, below. These results illustrate lipid accumulation observed at 68 h, 96 h, and 116 h of incubation, respectively. As shown in these Tables, the differences in lipid accumulation due to propionate toxicity decreased at 96 h and 116 h, from the 68 h observation. As illustrated, the treatments, Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
including those supplemented with propionate, accumulated lipids above 70 % DW
even with growth inhibition. Results of this example illustrate that higher, desired amount of odd chain fatty acids can be accumulated at 96 h for both P6 (62.4 % TFA) and P9 (also 62.4 %
TFA), which illustrates that the 0.18 g of propionate per gram of biomass supplied in P6 may be appropriate to improve OCFA accumulation in A. acetophilum HS399 to a desirable level.

[0089] As an example, adding more propionic acid to these treatments/cultures may not further increase the propionic acid, but may increase propionic toxicity with potentially negative impact in growth and lipid accumulation. Further, as an example, adding less propionate (e.g., 0.6 g propionate per gram of biomass), may result in low OCFAs accumulation due to palmitic acid synthesis taking over the OCFAs synthesis after 68 h, as shown in FIGURE 10, FIGURE 11 and FIGURE 12, and summarized in Table 6, below. In this example, the high (e.g., -66 %) propionic acid deposition in fatty acids suggest that propionate may be incorporated (e.g., at least once) in each fatty acid, presumably in the first condensation step of the FAS pathway (methyl malonyl acyl carrier protein condensation). In this example, the remaining propionate may be oxidized, and lost through anaplerosis into the Citric Acid Cycle.
Table 3. Lipid and fatty acid profile at 68 h Propionate (g/L) 0 3 3+3 3+3+3 Total Fatty Acids (% DW) 66.5 1.5 45.1 3.0 44.5 4.5 40.7 1.7 Fatty Acid Profile (% TFA) 13:0 0.0 0.0 3.8 0.6 5.2 0.2 5.5 0.5 14:0 4.0 0.1 2.0 0.3 1.5 0.1 1.2 0.0 15:0 0.2 0.0 44.7 6.0 49.6 0.6 48.8 0.1 16:0 47.1 0.1 10.8 5.6 5.4 0.7 5.0 0.0 17:0 0.0 0.0 5.3 0.6 5.8 0.2 5.7 0.2 18:0 1.5 0.0 0.2 0.3 0.0 0.0 0.0 0.0 22:5 (n-6) 8.6 0.0 4.5 0.5 3.6 0.1 3.5 0.1 22:6 (n-3) 36.9 0.3 26.3 0.7 25.7 0.1 26.8 0.3 Other FA 2.2 0.2 2.8 0.2 3.3 0.1 3.7 0.0 OCFA (% TFA) 0.2 0.0 52.9 7.3 60.9 0.6 60.6 0.4 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Table 4. Lipid and fatty acid profile at 96 h Propionate (g/L) 0 3 3+3 3+3+3 Total Lipids (% DW) 82.3 1.2 81.0 1.0 73.3 1.5 70.0 1.0 Total Fatty Acids (% DW) 70.6 3.7 66.8 4.8 62.5 0.3 56.3 1.2 Fatty Acid Profile (% TFA) 13:0 0.0 0.0 1.4 0.1 4.1 0.1 4.4 0.1 14:0 3.8 0.1 2.8 0.0 1.2 0.1 1.1 0.0 15:0 0.2 0.0 19.7 0.8 51.9 0.2 51.7 0.2 16:0 47.1 0.4 33.9 0.6 4.3 0.2 3.8 0.1 17:0 0.0 0.0 2.7 0.0 6.4 0.1 6.3 0.0 18:0 1.5 0.0 1.0 0.0 0.1 0.0 0.1 0.0 22:5(n-6) 8.6 0.1 6.6 0.1 3.5 0.1 3.4 0.1 22:6 (n-3) 36.5 0.4 29.4 0.2 24.6 0.1 24.9 0.1 Other FA 2.1 0.0 2.2 0.1 3.4 0.2 3.6 0.1 OCFA (% TFA) 0.2 0.0 24.0 1.0 62.8 0.2 62.9 0.2 Table 5. Lipid and fatty acid profile at 116 h Propionate (g/L) 0 3 3+3 3+3+3 Total Lipids (% DW) 82.0 1.7 74.8 0.3 77.7 0.6 74.0 1.0 Total Fatty Acids (% DW) 70.7 2.2 71.7 4.3 70.8 0.6 63.1 2.0 Fatty Acid Profile (% TFA) 13:0 0.0 0.0 1.1 0.0 2.7 0.1 3.0 0.1 14:0 3.7 0.1 2.5 0.0 1.3 0.0 1.1 0.0 15:0 0.3 0.1 18.6 0.6 46.0 1.1 49.2 0.3 16:0 46.7 0.1 33.8 0.5 8.7 1.0 5.8 0.3 17:0 0.0 0.0 3.0 0.1 7.7 0.2 8.2 0.1 18:0 1.5 0.0 1.1 0.0 0.2 0.0 0.2 0.0 22:5 (n-6) 8.7 0.1 6.8 0.0 4.4 0.2 4.0 0.0 22:6 (n-3) 36.8 0.3 30.4 0.0 25.6 0.2 24.9 0.1 Other FA 2.2 0.2 2.8 0.2 3.3 0.1 3.7 0.0 OCFA (% TFA) 0.3 0.1 22.8 0.8 57.5 1.4 60.8 0.2 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Table 6. Results of propionate feeding regime on propionate deposition and productivity of OCFAs by Aurantiochytrium acetophilum HS399 Propionate PA Feeding Rate Propionate deposition OCFA
Productivity (g/L) (gpA/gmomass) (%) (g/L/d) 0 (96 h) 0.00 0.00 0.0 0.0 0.0 0.0 3 (96 h) 0.08 0.00 57.0 0.9 1.53 0.02 3+3 (116 h) 0.18 0.00 65.0 0.6 2.89 0.06 3+3+3 (166 h) 0.28 0.00 39.5 1.7 2.56 0.04

[0090] Table 5 (above) provides results of varying use of propionic acid in an algal culture after 116 hours culturing. As an example, the result in Table 5 illustrate four different approaches to the use of propionic acid in a batch of HS399, as indicated by the four columns: 0, 3, 3+3, and 3+3+3. The first column indicates no use of propionic acid in the algal batch;
the second column indicates the use of merely one does of 3 g/L of propionic acid in the algal batch, the third column indicates the use of two separate doses of 3 g/L each of propionic acid, on separate days; and the fourth column indicates the use of three separate doses of 3 g/L each of propionic acid, at one per day. The respective rows of the Table 5 are indicative of the resulting percentage dry weight (%
DW) levels of total lipids, total fatty acids, and each fatty acid profile for the respective approaches (e.g., titled by the fatty acid name or indicator, such as C13:0 (13 chain FA), C14:0 (14 chain fatty acid), etc.).

[0091] As illustrated, the use of propionic acid (in columns 2, 3 and 4) indicates an increase in the presence of pentadecanoic acid (C15:0: fifteen-chain FA) in the resulting HS399 batch, in a dose response manner. As illustrated in column one, no use of propionic acid results in 0.3%
pentadecanoic acid of the TFA content. Column two shows that the addition of 3 g/L of propionic acid results in about 18 % (18.6%) of pentadecanoic acid of the TFA content;
column three shows the batch addition of two separate doses of 3 g/L of propionic acid results in about 40% (46%) of pentadecanoic acid of the TFA content; and column four shows the batch addition of three separate doses of 3 g/L of propionic acid (e.g., such as over a three-day period) results in greater than 40 %
(49.2%) of pentadecanoic acid of the TFA content.

[0092] Conversely, as seen in Table 5, the addition of the propionic acid indicates a reduction in the resulting palmitic acid (C16:0 or e.g., palmitate, such as the salts and esters of palmitic acid) Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
over the same four dose approaches. That is for example, in Table 5, the palmitic acid (C16:0) indicates 46.7% of the TFA profile with no propionic acid; 33.8% at one 3 g/L
dose; 8.7% at two doses of 3 g/L each; and 5.8% at three equal doses of 3 g/L. These results suggest that the number of iterative elongation cycles in fatty acid synthetase pathway may be determined by the length of the acyl chain. That is, in the presence of propionic acid, the fatty acid synthetase can prefer to eliminate one elongation cycle and produce pentadecanoic (C15:0), rather than producing heptadecanoic acid (C17:0), through full-seven-elongation cycles. This result appears to be consistent with the hypothesis that stearic acid (C18:0) is not a direct product of fatty acid synthetase, but the elongation of palmitic acid (C16:0).

[0093] Further, as illustrated in Table 5, the use of propionic acid in the algal culture batch can also result in production of other fatty acids, such as heptadecanoic acid (C17:0) and tridecanoic acid (C13:0) (e.g., both odd-chain fatty acids), with the total OCFA
indicating a result above 60%
of TFA (60.4% of TFA total for C13:0 + C15:0 + C17:0). As indicated in Table 5, as the OCFA
production increases, in the respective columns 2-4, the amount of resulting palmitic acid is reduced to 5.8% of the TFA. Additionally, results indicate that the amount of docosahexaenoic acid (DHA) also decreases to greater than about 20% (24.9%) of the TFA. This result may suggest that propionic acid enhances the synthesis of saturated fats from the fatty acid synthase (FAS) over the production of polyunsaturated fatty acids through the polyketide synthase (PKS) pathway.

[0094] In one aspect, the resulting product of an algal culture batch utilizing the multi-step propionic dose approach (e.g., three doses of 3 g/L each over three days) may be a highly concentrated anaplerotic oil from microalgae. That is, for example, the resulting product can comprise about 38% of the cell dry weight (CDW) of OCFAs (e.g., 60.4% OCFAs of the 63.1%
TFA = 38.1% OCFAs of total DW of algal product). In this aspect, no other natural source (e.g., non-synthetic) is known to produce these quantities or concentrations of odd chain fatty acids per batch product (e.g., >50% TFA; and >30% CDW).

[0095] In this implementation, as shown in the pentadecanoic (15:0) row of Table 5, the disclosed process can increase the pentadecanoic acid content from about 0.3% TFA, in the resulting product, without the use of propionate, to about 49 % in a fed-batch culture with 0.15 g propionate per gram of biomass. In one implementation, it may be desirable to control the daily propionic feed in order to control toxicity (as described herein); however, a per gram biomass fed propionate Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
may be more desirable than the g/L measurement for controlling the product OCFA composition.
The per gram biomass fed metric could be translated to different reactors, while the g/L
measurement may be reactor or process specific.

[0096] In this example, as shown in Table 5, using this same comparison, heptadecanoic acid (C17) content results are shown to increase from just a trace amount to greater than about 5% (8%) TFA. Further, in this implementation, while the concentration of DHA (C22:6) is shown to decrease from about 37% to about 25%, the presence of DHA in the resulting biomass may comprise a significant source of this oil, when compared to synthetically produced anaplerotic oils (e.g., containing odd-chain fatty acids that can improve anaplerotic conditions), which typically lack DHA entirely.

[0097] That is, for example, tripentanoin and triheptanoin (short and medium-sized odd-chain fatty acids) are currently the primary concentrated sources of odd chain fatty acids available. However, these molecules are produced synthetically and do not resemble any naturally available oil. For example, the odd-chain triheptanoin does not exist naturally, and is obtained through chemical synthesis from glycerol and heptanoic acid (C7:0). In contrast, algal anaplerotic oil can be produced naturally, as described above; and the resulting oils can contain the same odd chain fatty acids that are present in dairy products (e.g., and other natural sources), for example, which may allow for appropriate introduction into a human diet. As an example, the anaplerotic oil production, described herein, can be from algae sources, and is believed to be less costly than synthetic production because it does not utilize modified fatty acids and chemical trans esteri fic ati on.
Example 3 - Propionic acid can inhibit growth at various concentrations

[0098] As another example, a strategy can be devised for propionate feeding to Aurantiochytrium acetophilum HS399 cultures, and the results can illustrate the effect on growth and propionate deposition in the lipid fraction. In this example, five treatments (PO, P3, P0+3, P0+1+1+1, P0+2+2+2) can be used, and respective treatments can receive a different amount of propionate, which is dependent on incubation time. In this example, treatment P3 receives 3 g/L propionate on day 0 at the beginning of the protein phase, while P0+3 receives 3 g/L
propionate on day 1 at the beginning of the end of the protein phase-beginning of the lipogenic phase.
The treatment Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
P0+1+1+1 receives 1 g/L propionate on day 1, 2 and 3, and treatment P0+2+2+2 receives 2 g/L
on day 1, 2 and 3 in an attempt to reduce the high residual propionate available during lipogenesis.
Respective Erlenmeyer flasks (250 mL) are inoculated (1 % v/v) in triplicates with a 24 h old culture of A. acetophilum HS399 and incubated in an orbital shaker at 180 rpm and 27 C.

[0099] Respective Erlenmeyer flasks contain 100 mL of a medium supplemented with (g/L):
dextrose (100), ammonium acetate (4.6), NaCl (12.5), MgSO4 7H20 (2.5), KH2PO4 (0.5), KC1 (0.5) and CaCl2 (0.1). This medium also contains trace element solution (5 ml/L) and vitamin solution (1 ml/L). The trace element solution contains (g/L): EDTA di-sodium salt (6), FeCl3 6H20 (0.29), H2B03 (6.84), MnC12 4H20 (0.86), ZnC12 (0.06), C0C12 6H20 (0.026), NiSat 6H20 (0.052), CuSO4 5H20 (0.002), Na2MoO4 H20 (0.005). The vitamin solution contains (mg/L):
thiamine (100), biotin (0.5) and cyanocobalamin (0.5).

[00100] In this example, respective culture materials are autoclaved (e.g., 121 C, 15 min) and the media is filter sterilized before use. A propionic acid stock solution (200 g/L) can be used as the fed propionic acid. Daily samples are collected to analyze the cell dry weight, residual glucose, culture pH (see FIGURE 15), and lipid and fatty acid composition of the cultures. Cell dry weights are analyzed by filtration (e.g., 0.2 gm filter media) using a vacuum and washed with a solution of ammonium bicarbonate. Residual glucose is analyzed using a colorimetric method based on glucose peroxidase activity. Biomass for lipid analysis is centrifuged and washed using purified water. The washed biomass is freeze dried. Total lipids are analyzed using Folch method (AOAC
996.06) and the FAMEs are analyzed by gas chromatography and flame ionization detection using nonadecanoic (C19:0) acid as an internal standard.

[00101] As shown in FIGURES 13 and 14 respectively, the resulting cell dry weight and resulting residual glucose illustrate that feeding 3 g/L propionic acid (P3) provides a lag in growth in the first 24 h. Further, the results illustrate that this lag was mitigated by feeding 3 g/L at the end of the protein phase-beginning of lipogenesis (day 1). Additionally, the results illustrate that initial (24 h) growth for P0+3 was similar to the growth of the control treatment PO, and the growth lag observed in the P3 treatment was mitigated.

[00102] Table 7, below, provides the lipid and fatty acid analysis results, and Table 8 provides the propionate deposition. As shown in these tables, postponing the propionic feed to the lipogenic phase (P0+3) resulted in a higher production of OCFAs (27.4 % TFA) and higher propionate Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
deposition (59.3 1.1 % of total) than when feeding the propionate initially (P3), at the beginning of the protein phase (25.2 % TFA and 52.2 0.7 % of total). Further, the results suggest that propionic acid lost through its oxidation by the citric acid cycle can be higher during the protein phase than the lipid phase. As an example, this may demonstrate that waiting for lipogenesis to feed propionate can help to mitigate toxicity, and can help improve propionate incorporation into the OCFAs. Additionally, the results illustrate that OCFAs acid productivity may increase by approximately 20 % by feeding propionate merely during lipogenesis (from 1.18 0.02 at P3 to 1.18 0.02 g OCFAs/L d for P0+3).

[00103] As an example, results suggest that fractionating the 3g/L of propionate into three daily dosage of 1 g/L (P0+3 vs. P0+1+1+1) may not reduce the impact of propionic acid toxicity in A.
acetophilum HS399 growth, as illustrated in FIGURE 13. In this example, residual concentrations as low a lg/L may provide some growth inhibition. Further, the results obtained with P0+2+2+2, suggest that 6g propionate/L produces a desirable amount (e.g., higher) of OCFAs in A.
acetophilum HS399 flask cultures, which may translate into 0.18 g of propionate per g of biomass produced for other growth platforms.
Table 7. Total lipids and fatty acid profile at time of harvest (96 h) Propionate (g/L) 0 3 0+3 0+1+1+1 0+2+2+2 Harvest & Sample Day 4 6 6 6 6 Total Lipids (% DW) 79.3 0.6 78.0 0.0 79.5 0.5 79.7 0.6 76.0 1.0 Ash (% DW) Total Fatty Acids (% DW) 69.7 1.4 64.8 1.2 66.0 2.2 67.2 5.0 63.5 1.6 Fatty Acid Profile (% TFA) 13:0 0.0 0.0 1.1 0.0 1.0 0.0 1.1 0.0 1.8 0.0 14:0 4.1 0.1 2.3 0.0 2.2 0.1 2.0 0.0 1.1 0.0 15:0 0.2 0.0 20.1 0.2 22.1 0.2 23.6 0.4 42.3 0.7 16:0 47.1 0.4 31.3 0.2 29.1 0.3 27.1 0.2 7.6 0.2 17:0 0.0 0.0 4.0 0.0 4.3 0.1 4.6 0.1 8.8 0.1 18:0 1.4 0.0 1.1 0.0 1.0 0.0 0.9 0.0 0.2 0.0 22:5 (n-6) 8.1 0.0 6.1 0.0 5.8 0.1 5.8 0.0 4.2 0.2 22:6 (n-3) 36.5 0.3 30.9 0.2 31.5 0.4 31.9 0.2 30.1 0.4 Other FA 2.3 0.1 2.7 0.1 2.6 0.0 2.7 0.1 3.3 0.1 OCFA (% TFA) 0.4 0.0 25.5 0.2 27.6 0.1 29.2 0.4 53.5 0.8 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Table 8. Impact of propionate feeding regime on propionate deposition and productivity of OCFAs at 120 h incubation.
Propionate PA Feeding Rate Propionate deposition OCFA
Productivity (g/L) (gpA/gmomass) (%) (g/L/d) 0 (Day4) 0.00 0.00 0.0 0.0 0.0 0.0 3 (Day5) 0.09 0.00 52.2 0.7 1.04 0.01 0+3 (Day5) 0.09 0.00 59.3 1.1 1.18 0.02 0+1+1+1 (Day5) 0.09 0.00 59.6 1.4 1.19 0.03 0+2+2+2 (Day5) 0.20 0.00 46.7 0.9 1.86 0.04 Example 4 - Modeling propionic acid toxicity.

[00104] In one aspect, the uptake activity of propionate as an organic carbon source by microalgae may be dependent on the culture's pH. For example, when propionic acid is fed to a culture that is growing at a pH of 7, the residual organic acid can be mostly dissociated in the propionate form (propionic pKa=4.88), with only a minor amount remaining undissociated. In this aspect, while the propionate and free proton form enters the microalgae cell through a monocarboxylic symport structure, propionic acid is membrane permeable and may be diffused directly into the microalgae cell. Therefore, in this aspect, the uptake of propionate can be controlled by the cell, and the uptake of propionic acid may not be controlled by the cell. As an example, the uncontrolled uptake of propionic acid presumably lowers the internal Aurantiochytrium cell pH; in turn, the cell attempts to maintain its pH
homeostasis by pumping protons out of the cell. In this example, a build-up of propionate inside the cell can result, which is proportional to the pH gradient between the intracellular and the extracellular (see FIGURE 16).
Therefore, in one implementation in this aspect, the internal propionate concentration, which could be used to measure propionic cell toxicity, may be calculated using at least the external culture pH, the internal cell pH, and the residual acetate concentration in the culture.
In one implementation, the change in pH may be calculated with the following equation derived from the Henderson and Hasselbalch equation:
ApH = pHi - pHo = log (Pi1) [Poi Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00105] In this implementation, pHi = pH inside the cell, pHo = pH outside the cell, P =
propionate, 0 = outside cell, I = inside cell. Assumptions made in this calculation may include:
the impact of the ionic strength in the propionic acid dissociation constant as negligible, propionic acid but not propionate is membrane permeable, and there are no other protection mechanisms involved in the regulation of cell pH. The relationship may be illustrated with the non-limiting Example 1 (above), which shows that propionate may be substantially lethal at residual (extracellular) concentrations of ¨15 g/L and a pH of 6. The above-mentioned model can be used to translate this Example 1 into an intracellular propionate concentration of 176 g/L, providing a value that could be translated at different pHs and residual concentrations.
Further, the relationship may be illustrated by the non-limiting Example 2 (above), which shows that propionate may be substantially non-lethal, but growth inhibitory, at concentrations as low as 1 g/L at pH of 7. The above-mentioned model can be used to translate Example 2 into an intracellular propionate concentration of 1 g/L, providing a value that could be translated at different pHs and residual concentrations. Therefore, in this implementation, the model can help provide an understanding of the impact of medium pH in propionate toxicity, and can provide a tool to model propionic acid toxicity that could be used to improve a process to optimize OCFAs production.
Example 5 - Propionic acid/pH-auxostat led strategy to produce OCFA.

[00106] In one implementation, a propionic acid/pH-auxostat led strategy may be used to produce anaplerotic oils containing odd chain fatty acids using the microalgae A. acetophilum HS399. In this implementation, for example, three treatments can be fed propionic acid as titrant to maintain pH at a desired level (e.g., organic acids: Propionic acid-pH7, Propionic acid -pH6 and Propionic acid-pH5). Further, a control treatment can be fed acetic acid at pH
7 (e.g., Acetic acid-pH7). In this implementation, initially (24 h incubation), the culture pH may not be controlled, and the pH of the respective treatments can drift from 7.5 to 8. After 24 h incubation, the desired pH
set point of each treatment can be set using the respective organic acids.

[00107] In this implementation, for example, bubble column reactors (1.3 L) are inoculated (1 % v/v) in triplicates with a 24 h old culture of A. acetophilum HS399. The cultures are aerated at 1.4 vvm and maintained at 27 C under axenic conditions. The bubble columns contain 700 mL of a medium supplemented with (g/L): dextrose (40), ammonium acetate (1.1), NaCl (12.5), MgSat Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
7H20 (2.5), KH2PO4 (0.5), KC1 (0.5) and CaCl2 (0.1). This medium also contains a trace element solution (5 ml/L) and a vitamin solution (1 ml/L). The trace element solution can contain (g/L):
EDTA di-sodium salt (6), FeCl3 6H20 (0.29), H2B03 (6.84), MnC12 4H20 (0.86), ZnC12 (0.06), CoC12 6H20 (0.026), NiSat 6H20 (0.052), CuSO4 5H20 (0.002), Na2MoO4 H20 (0.005). The vitamin solution can contain (mg/L): thiamine (100), biotin (0.5) and cyanocobalamin (0.5).

[00108] The respective culture materials can be autoclaved (121 C, 15 min) and the media can be filter sterilized before use. In this example, propionic and acetic acid are diluted to 3 % w/w and added to the acid container, from which the cultures are fed through a peristaltic pump in response to the pH drift above their set point. Daily samples can be collected to analyze the cell dry weight, residual glucose, culture pH, and lipid and fatty acid composition of the cultures. Cell dry weights are analyzed by filtration (0.2 gm filter media) using a vacuum and washed with a solution of ammonium bicarbonate. Residual glucose is analyzed from culture supernatant (5000 g; 5 min) using a colorimetric method based on glucose peroxidase activity.
Residual acetate and propionate are analyzed by HPLC using external standard. Biomass for lipid analysis can be centrifuged and washed using purified water; and the washed biomass is freeze dried. Total lipids are analyzed using Folch method (AOAC 996.06), and the FAMEs are analyzed by gas chromatography and flame ionization detection using nonadecanoic (C19:0) acid as an internal standard.

[00109] Residual propionate results are provided in FIGURE 17, illustrating that the pH-auxostat strategy was able to successfully maintain the propionate levels at the pH 5 and 6 set points. Further, as illustrated in FIGURE 17, the treatments comprising a pH 7 set point did not appear to have sufficient residual propionate or acetate to identify whether the organic acid was present during the demonstration, and therefore they were not fed on demand, but ad libitum. The results of this example illustrate that using a propionic acid-pH7 pH-auxostat system may have interrupted the propionic acid feeding due to the displacement of residual propionate. As illustrated by the cell dry weight data represented in FIGURE 18, propionic acid was not growth inhibitory.
However, as illustrated by the data in Table 9, below, OCFA (6.3 % TFA) may not be accumulated to levels observed previously in the flask (> 50% TFA) or at lower pH
treatments, likely due to the lack of propionic acid. Therefore, in one implementation, at a higher pH, supplementation with sodium propionate, or target alkalization of the media, may be used to provide for the presence of residual propionate in the batch even at high pH-set points. These results indicate that the lower Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
pH set points can be highly growth inhibitory, as shown by the cell dry weight data (FIGURE 18) and glucose analyses (FIGURE 19). These results indicate that inhibition may be higher at lower pHs, as may be predicted by the model described in Example 4 (Table 10 below).
Table 9. Lipid and fatty acids analyses from the pH auxostat Acetic acid-pH7 Propionic acid-pH7 Propionic acid-pH6 Propionic acid-pH5 Consumed Propionate 0.0 0.0 1.0 0.1 2.4 0.1 2.3 0.2 (g/L) Propionic Acid 0.0 0.0 24.7 1.0 40.3 1.6 30.9 2.0 Deposited (%) Total Lipids 89.5 0.5 87.5 1.5 79.5 1.5 75.0 3.0 Total Fatty Acid 71.9 5.2 70.3 2.8 63.4 0.3 59.2 3.1 Fatty Acid (% TFA) (% DW) (% TFA) (% DW) (% TFA) (% DW) (%TFA) (% DW) C11:0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.1 0.1 0.0 0.0 0.0 0.0 C13:0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.2 0.3 1.4 0.2 2.1 0.1 1.2 0.0 C14:0 3.2 0.0 2.3 0.2 2.9 0.1 2.0 0.1 1.3 0.0 0.8 0.0 1.1 0.1 0.7 0.1 50.6 32.1 51.6 305 C15:0 0.0 0.0 0.0 0.0 7.3 1.7 5.1 1.0 0.3 0.0 1.7 0.6 56.4 2.9 40.6 1.7 49.3 2.6 34.7 0.0 C16:0 9.6 0.8 6.1 0.5 8.1 1.8 4.8 1.3 C17:0 0.0 0.0 0.0 0.0 1.7 0.4 1.2 0.2 7.2 0.4 4.6 0.3 7.3 0.1 4.3 0.3 C18:0 1.3 0.0 1.0 0.1 1.1 0.0 0.8 0.1 0.3 0.0 0.2 0.0 0.1 0.1 0.1 0.1 C20:3n6 &
0.2 0.0 0.2 0.0 0.1 0.1 0.1 0.1 0.2 0.2 0.1 0.1 0.3 0.0 0.2 0.0 C21:0 C20:5n3 &
0.4 0.0 0.3 0.0 0.4 0.0 0.3 0.0 0.4 0.0 0.3 0.0 0.5 0.0 0.3 0.0 C22:0 C22:5n6 DPA 6.7 0.0 4.8 0.4 6.5 0.0 4.6 0.2 4.7 0.0 3.0 0.0 4.4 0.2 2.6 0.2 31.6 22.7 30.7 21.6 23.4 14.8 24.7 14.6 C22:6n3 0.0 1.6 0.1 0.9 0.4 0.2 0.6 0.4 OCFA (%TFA) 0.0 0.0 0.0 0.0 9.0 2.1 6.3 1.2 60.0 38.0 60.9 36.0 0.2 0.1 1.7 0.9 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Table 10. The intracellular propionate, represented as an indicator of propionate toxicity for each treatment, was calculated from the propionate residual concentration and the pH set point using model described in Example 4.
Actual Values Theoretical Values Average Residual Propionic Acid (g/L) D1-D4 Average Internal Propionic Acid (g/L) Acetic acid-pH 7 0.0 0.0 0.00 Propionic acid-pH 7 0.1 0.1 0.13 Propionic acid-pH 6 0.4 0.1 4.18 Propionic acid-pH 5 0.6 0.2 56.70 Example 6¨ Single Stage Approach to producing OCFA

[00110] In another implementation, a New Brunswick 10 L Bioflo Pro 300 fermenter (Eppendorf) can be used to provide an improvement in the productivity of OCFAs accumulation, previously identified in flasks and bubble columns. In this implementation, a bioprocess can be devised to incorporate one or more portions of one or more techniques described herein. In this implementation, a propionic acid/pH-auxostat strategy can be adopted after day 1 to induce the production of OCFAs. As an example, in this implementation, the pH-auxostat is operated at a pH
set point of 6.5 to mitigate propionic toxicity. The pH-auxostat is activated through the addition of 0.5 g/L of potassium propionate to provide residual propionate availability during the culture. A
control treatment can be provided with propionate, to be used as comparison.
Fermenters containing 5 L of fresh media are inoculated (1 % v/v) in triplicates with a 24 h old culture of A.
acetophilum HS399. The fermenters are aerated at 0.5 vvm, and maintained at 27 C under axenic conditions. A stirring speed is increased in response to identified dissolved oxygen values below % saturation, from 200 rpm up to 1000 rpm.

[00111] In this implementation, for example, the 5 L of batch media can contain (g/L): corn syrup D95 (31), ammonium acetate (19.5), MgSO4 7H20 (2.5), KH2PO4 (2.5), KC1 (1) and CaCl2 (0.2). This medium can also contain a trace element solution (25 ml/L) and vitamin solution (5 ml/L). The trace element solution contains (g/L): EDTA di-sodium salt (6), FeCl3 6H20 (0.29), H2B03 (6.84), MnC12 4H20 (0.86), ZnC12 (0.06), CoC12 6H20 (0.026), NiSat 6H20 (0.052), CuSO4 5H20 (0.002), Na2MoO4 H20 (0.005); and the vitamin solution contains (mg/L): thiamine (100), biotin (0.5) and cyanocobalamin (0.5). In this example, the fermenter is fed another 5-6 L

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
of a medium containing (g/L): ammonium phosphate (2.5), ammonium hydroxide (29 % pure) (15.2), corn syrup DE95 (1143). This medium can also contain a trace element solution (25 ml/L) and a vitamin solution (5 ml/L).

[00112] In this example, this medium is fed in a DO-stat mode in response to dissolved oxygen values detected above 15 % saturation. As an example, the dissolved oxygen values can trigger a feeding pulse of 0.3 ml/L min that lasts 102 min. The pH is maintained at 5.8 using NaOH, while the batch ammonia is consumed. The pH can drift to higher values when the residual ammonia is substantially exhausted from the fermenter. At substantial residual ammonia exhaustion, the pH
can be controlled with propionic acid at a pH 6.4, in some examples, while the pH of the control treatment may drift up to 7-8 without titration. Culture materials and media can be autoclaved (e.g., 121 C, 15 min) while separating the nitrogen and the carbon source. Foam can be controlled (e.g., automatically) through the addition of (< lml/L) Hodag antifoam.

[00113] In this implementation, samples can be collected daily to analyze the cell dry weight, residual glucose, culture pH, and lipid and fatty acid composition of the cultures. Cell dry weights can be analyzed by filtration (e.g., 0.2 gm filter media) using a vacuum, and washed with a solution of ammonium bicarbonate. Residual glucose can be analyzed from culture supernatant (e.g., 5000 g; 5 min) using a colorimetric method based on glucose peroxidase activity.
Residual acetate and propionate can be analyzed by HPLC using an acceptable external standard. The biomass for lipid analysis can be centrifuged and washed using purified water, and the washed biomass can be freeze dried. Total lipids are analyzed using Folch method (AOAC 996.06) and the FAMEs are analyzed by gas chromatography and flame ionization detection using nonadecanoic (C19:0) acid as an internal standard.

[00114] Example results of the cell dry weights, in this implementation, are shown in FIGURE
20. As an example, the results illustrate that, even though the propionic acid titration slightly inhibited A. acetophilum HS399 growth compared to no propionic acid titration, the cultures presented 170 g/L cell dry weight, and accumulated lipids at 70 % DW, with 60 % TFA being OCFAs. The results illustrated in FIGURE 21 show that the average cumulative productivities are approximately 30 g/Ld, which translates into 10 g/L/day of OCFAs. In this implementation, as illustrated by these results, the example bioprocess can maintain the residual glucose and ammonia at desired levels, as illustrated by FIGURE 22, and other cultivation parameters at desired levels, Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
as shown in FIGURE 23. As illustrated in FIGURE 24, providing results of total propionate consumed during the process in this implementation, the process provided a consumption of 0.155 g of propionic per gram of biomass, which is in agreement with the values observed in flask (Example 2). In this implementation, as in previous implementations, the results of the OCFAs produced by the control treatment were negligible.
Fungi/Yeast

[00115] In another aspect, techniques and systems can be devised, as described herein, for a natural method of improving the production of OCFAs in microalgae and other microorganisms, like yeast/fungi, that do not utilize genetic modification. A process is disclosed herein for the production of an oil rich in OCFA. As one example, oil rich in OCFA may be produced at 28 g oil/L/day, and at 31 g/L/day or more. For example, such processes can produce a triacylglyceride containing OCFAs of 40 % of TFA or more, and up to approximately 67 % TFA. In one example, the cultures may achieve a final biomass yield of approximately 126 g/L.

[00116] In addition to A. acetophilum HS399 microalgae, as described above, certain microorganisms (e.g., yeast/fungus) can produce a variety of fatty acids, the composition of which can vary among different strains of microorganisms. As an example, Yarrowia lipolytica is considered to be an oleaginous yeast that can accumulate large amounts of lipids. Yeast species are typically described as "oleaginous" if the lipids they accumulate account for more than 20 %
of their biomass. For Y. /ipo/ytica, the amount of lipid accumulation is dependent on the strain and the carbon source, along with growth conditions. Under optimal growth conditions, some fed-batch cultures of Y. /ipo/ytica can store 43% lipids of their cell dry weight (CDW) in continuous fermentations using industrial glycerol, and may store up to 54% lipids of their CDW in batch cultures on a stearin-based medium. In these examples, most of the lipids accumulating in Y.
/ipo/ytica are triacylglycerols rather than free fatty acids (FFA), with C16 and C18 compounds being the most abundant, with other fatty acids present in trace amounts.

[00117] The trace fatty acids of Y. /ipo/ytica can include pentadecanoic acid (C15:0) and heptadecenoic acid (C17:1 n-8) (e.g., at < 0.3 % TFA). The trace fatty acids, including these two identified fatty acids, are typically ignored in the lipid profile reports for these organisms. Odd-chain fatty acids (OCFAs), including pentadecanoic acid and heptadecenoic acid (C17:1 n-8) are Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
fatty acids that contain an odd number of carbon atoms in the structure. OCFAs are typically related to bacterial activity (e.g., propionic acid bacteria), and are less likely to be present in other microbes, such as yeast/fungi and microalgae, or plants.

[00118] The presence of trace amounts of OCFA in yeast/fungi suggests that the pathway responsible for the synthesis of OCFA may be present in yeast/fungi. Because of the composition of their fatty acid profile, and yeast/fungi ability to be grown rapidly, yeast/fungi such as Y.
/ipo/ytica may provide an attractive source of OCFA. Such microorganisms may be able to generate OCFA in a more rapid and concentrated manner than other known natural sources, such as milk fat (e.g., providing a more cost effective and efficient source of OCFAs). Yeast/fungi may also provide alternative OCFA, such as heptadecenoic acid (17:1 n-8), which may not be found in other food sources. Further, for example, yeast can produce OCFAs without highly unsaturated fatty acids, which can help mitigate undesirable flavors often associated with this type of oil. As an example, a benefit of using yeast/fungi in place of butter and other ruminant fat is the higher concentration of OCFA found in these types of yeast/fungi. In addition, as another example, benefit, yeast/fungi oil lacks residues of phytol or phytanic acid that are often present in ruminant fat. Consumption of phytol or phytanic acid can lead to health concerns in some individuals.

[00119] In one aspect, techniques can be devised that provide for an increased production of naturally occurring odd-chain fatty acids from yeast/fungi than might be generated from typical yeast/fungi. The resulting cultivated yeast/fungi and/or resulting isolated composition may be used individually as products or as an ingredient in a variety of products. As an example, yeast/fungi such as Y. /ipo/ytica can be cultivated and produce a desirable fatty acid profile comprising OCFAs, which may be isolated through various extraction processes.
In this example, the isolated oil containing the OCFAs may comprise a composition rich in OCFAs, such as pentadecanoic acid (C15:0) and heptadecenoic acid (C17:1 n-8). In one implementation, in this aspect, the yeast/fungi may be cultivated using an improved method that includes the presence of a complex media, which can promote increased production of the OCFAs. As one example, the cultivation media may comprise propionate. Propionate is a conjugate base of propionic acid and is a short-chain fatty acid often produced by gut flora in some animals.

[00120] In one implementation, Y. /ipo/ytica can be cultivated with propionate to increase the amount of OCFA production. The following describes example implementations:

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Methods

[00121] Culture conditions. In one implementation, Yarrowia lipolytica ATCC18944 can be cultivated in triplicates (n=3) in a medium containing (g/L): glycerol (80), monosodium glutamate (5), yeast extract (1), NaCl (12.5), MgSO4 (7), H20 (2.5), KC1 (0.5), CaCl2 (0.1), KH2PO4 (0.5), trace metal solution (5 mL) and vitamin solution (1 mL). The trace element solution may contain (g/L): EDTA di-sodium salt (6), FeCl3 6H20 (0.29), H2B03 (6.84), MnC12 4H20 (0.86), ZnC12 (0.06), CoC12 6H20 (0.026), NiSat 6H20 (0.052), CuSO4 5H20 (0.002), Na2MoO4 2H20 (0.005).
The vitamin solution can be filter-sterilized (e.g., using 0.2 m pore size filter) containing (mg L-1): thiamine (100), biotin (0.5) and cyanocobalamin (0.5) unless otherwise stated. In some implementations cyanocobalamin might be added to or subtracted from the medium formulation to change or impact propionic acid deposition in OCFA. In some implementations cobalt can be reduced or eliminated from the media to avoid synthesis of cyanocobalamin that could compromise propionic acid deposition. In one implementation, a concentration of the cyanocobalamin and/or cobalt in the culture medium can be below 0.4 M.

[00122] Y. /ipo/ytica cultures can be inoculated at 1 % v/v in a 250 mL
baffled Erlenmeyer flask containing 100 mL of the above mentioned media. The flasks can be incubated in the dark in an orbital shaker at 180 rpm, 27 C. In this implementation, sodium propionate is fed batch every day according to respective treatments. As an example, treatment 0+0+2+2+2+2, is fed 2 g/L propionic acid at 48, 72, 96 and 120 hours, while treatment 0 is not fed any propionate at all.

[00123] Analyses. Cell dry weights can be obtained by drying samples that are previously vacuum filtrated (e.g., using 0.2 m pore size filters). Residual propionate is analyzed directly by high performance liquid chromatography (HPLC). Lipids and total fatty acids are analyzed using a direct extraction, transesterification followed by gas chromatography (GC) and detection by flame ionization (FID). The different fatty acids are identified and quantified using appropriate internal and external standards.

[00124] Propionic acid deposition rate. Propionate consumption can be calculated based on the residual propionate on day 0 and subtracting the final residual propionate, to find the consumption amount. The total propionate deposited in the biomass is calculated based on the final cell dry weight, multiplied by the total fatty acid (TFA) ratio in biomass, multiplied by the OCFA

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
ratio in TFA, and multiplied by the molar factor of propionate in odd chain fatty acid, averaged at 0.3. The propionate deposition rate can be calculated by dividing the propionate deposited by the propionate consumed and expressed as percent.
Results & Discussion

[00125] In this implementation, it is desirable to determine the capacity of Yarrowia lipolytica ATCC18944 to produce odd chain fatty acids (OCFAs) though the incorporation of propionate in the media. For example, propionate could be either incorporated in the lipids as OCFAs or oxidized through either the methylmalonate or methylcitrate pathways. Testing showed that the incorporation of medium propionic acid into Y. /ipo/ytica fatty acids (i.e.
OCFAs) may not be affected by the presence or absence of cyanocobalamin (see Table 11 below).
Cyanocobalamin is co-factor on the methylmalonate pathway, thus the results indicate that this pathway might not be active in Y. /ipo/ytica.
Table 11. Impact of cyanocobalamin in propionic acid deposition in Yarrowia lipolytica ATCC18944.
Cyanocobalamin (pU) 0.00037 0 Daily propionate (g/L d) 0+0+2+2+2+2 0+0+2+2+2+2 Cell dry weight (g/L) 5.3 6.0 Consumed propionate (g/L) 1.7 2.6 Odd chain fatty acids (% DW) 0.9 1.4 Odd chain fatty acids (g/L) 0.05 0.1 Propionate (MW)/OCFA (MW) 0.3 0.3 Propionate deposition (%) 0.83 0.99

[00126] In this aspect, for example, the methylcitrate pathway is the only known anaplerotic pathway involved in propionic acid catabolism. FIGURE 25 is a graphical representation 2500 of results of growth and substrate consumption of Yarrowia lipolytica ATC C18944 using different carbon sources. As illustrated in FIGURE 25, Y. /ipo/ytica may be able to grow on propionate 2502 as sole carbon source. Therefore, as an example, because anaplerosis is used to sustain the Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
growth on propionate as a sole carbon source, these results suggest that propionate is primarily oxidized through the methylcitrate pathway. Yarrowia lipolytica growth on propionate 2502 is shown to be much lower than on glucose 2504, but this trend was corrected when both substrates 2506 (propionate and glycerol) were fed simultaneously.

[00127] In some implementations, fed batch propionate in the presence of glycerol may be used to increase cell growth rates and OCFAs production. As one example, as illustrated in Tables 12 and 13, below, for Y. /ipo/ytica, propionic acid deposition rates in OCFAs may be relatively low (e.g., <3 % TFA), which indicates that methylcitrate may readily oxidize propionic acid. In one implementation, in order to mitigate the propionic acid oxidative loss, the total amount of propionate fed batch can be increased gradually from 0.3 to 8 g/L, which can result in 13.3 1.3 % TFA, 2.2 0.2 % DW and 0.33 g/L OCFAs (see Table 13 below). The main OCFAs produced by Yarrowia lipolytica were the Omega-8 heptadecenoic acid (C17:1 n-8) and pentadecanoic acid (C15:0).
Table 12 Lipid and fatty acid analyses of Yarrowia lipolytica ATCC18944 fed increasing daily propionate concentrations.
Time (hrs) Oh 168 168 168 Daily propionate (g/L) Initial 0 0.3+0.3 0+0.6 Total Lipids (% DW) 24.7 0.6 23.7 3.5 21.7 3.2 Total Fatty Acids (% DW) 10.6 24.0 1.5 21.7 2.5 21.3 2.8 Fatty Acid Profile (% TFA) 15:0 0.00 0.3 0.0 0.3 0.0 0.4 0.1 16:0 11.10 10.8 0.1 11.6 0.6 12.3 0.8 16:1 15.16 14.1 0.1 14.2 0.4 14.4 0.3 17:1 0.00 1.1 0.0 1.9 0.3 2.1 0.5 18:0 4.75 4.8 0.1 5.0 0.3 5.3 0.4 18:1(n-9) 56.59 54.9 0.3 54.2 1.4 52.0 2.4 18:2(n-6) 0.00 12.0 0.4 10.8 0.7 11.3 0.5 Other FA 12.40 1.4 0.1 1.5 0.2 1.6 0.3 OCFA (%DW) 0 0.3 0.03 0.5 0.02 0.5 0.09 OCFA (%TFA) 0 1.4 0.04 2.3 0.30 2.6 0.83 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Table 13 Lipid and fatty acid analyses of Yarrowia lipolytica ATCC18944 fed increasing daily propionate concentrations.
Time (h) Oh 168h 168h 168h Daily Propionate (g/L) Initial 0 0+0+1+1+1+1 0+0+2+2+2+2 Total Lipids (% DW) 10.00 22.7 1.2 19.0 0.0 18.3 0.6 Total Fatty Acids (% DW) 7.9 22.0 1.8 17.3 1.6 16.2 0.5 Fatty Acid Profile (% TFA) 15:0 0.0 0.3 0.0 1.3 0.0 1.4 0.1 16:0 14.0 12.4 0.5 13.2 1.0 13.8 1.6 16:1 15.7 13.8 0.4 12.2 0.1 12.0 1.1 17:1 0.0 1.1 0.0 10.0 0.7 10.0 1.2 18:0 6.4 5.2 0.3 6.3 0.7 6.9 1.4 18:1 (n-9) 49.5 52.0 0.5 40.0 0.0 38.8 0.4 18:2(n-6) 14.4 13.9 0.2 14.2 0.7 13.1 0.6 Other FA 0.0 61.3 0.8 2.7 0.5 54.0 3.0 OCFA (%FA) 0 1.4 0.1 13.1 0.6 13.3 1.3 OCFA (%DW) 0.0 0.3 0.0 2.3 0.3 2.2 0.2

[00128] As an example, unlike A. acetophilum HS399, Yarrowia lipolytica can accumulate OCFA without accumulating Docosahexaenoic acid (DHA), which might be beneficial for certain formulations when long chain polyunsaturated fatty acids (e.g., like DHA) are not desired. As illustrated in FIGURE 26, in one implementation, Y. /ipo/ytica can produce OCFAs in polymorphic cultures 2600, which are combinations of filamentous molds and yeast morphology. As an example, these results suggest that, in addition to production in algae (e.g., Aurantiochytrium), OCFAs may also be produced by fungi, regardless of their yeast or filamentous morphology.

[00129] FIGURES 27 and 28 are graphical representations of implementations where Y.
/ipo/ytica is cultivated with increasing daily propionate concentrations 2700, 2800. In these implementations, the results illustrate that growth of the Y. /ipo/ytica may not be affected by up to 2 g/L d propionic fed despite the low pH (3) present. As illustrated, under these conditions the Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
accumulation of cytoplasmic propionate equates to 328 g/L, which indicates how this strain can have a high tolerance to propionic acid toxicity.

[00130] Further, FIGURES 29 and 30, are graphical representations of implementations where Y. /ipo/ytica is cultivated with increasing daily propionate concentrations 2900, 3000. In these examples, the results illustrate that the Y. /ipo/ytica cells can accumulate lipids above 20 % DW, with OCFA concentrations above 10 % of TFA. As an example, the relatively low OCFAs levels produced by Y. /ipo/ytica may be associated with the catabolic loss of propionate through the methylcitrate pathway. In one implementation, to address this issue, and to increase the propionic acid incorporation in OCFAs, certain supplements may be added to the media during propagation to mitigate the flow of the propionate though the methylcitrate pathway, which can help mitigate propionic acid oxidative loss. As an example, itaconic acid can be used as a potential inhibitor of the methyl isocitrate enzyme. Table 14, below indicates that itaconic acid may not always provide for mitigation of the propionic acid depositions. One or more alternative inhibitor, such as 3-Nitropropionate, 3-bromo pyruvate and V-13-009920, may provide improved mitigation of the loss of propionate to the methylcitrate pathway.
Table 14. Propionic acid deposition in the presence of itaconic acid Time (h) 192 Itaconic acid (g/L)/Propionate (g/L) 0/6 0.5/6 Cell dry weight (g/L) 8.3 1.2 7.9 1.1 Consumed propionate (g/L) 5.1 1.5 5.0 1.6 Propionate (MW)/OCFA (MW) 0.3 0.3 Odd chain fatty acids (%DW) 1.3 0.5 1.1 0.6 Propionic acid deposition (%) 0.6 0.1 0.5 0.2

[00131] In summary, in this implementation, Yarrowia lipolytica could be used to produce oils rich in OCFAs. The OCFA concentration in the resulting oil, and the productivity, may be below that obtained with the microalgae A. acetophilum H5399. However, Y. /ipo/ytica provides advantages because the main OCFA is C17:1 n-8, and Y. /ipo/ytica does not produce highly unsaturated fatty acids like may be found with H5399, which might be preferred in certain applications.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Propionate Induced Growth Inhibition

[00132] In another aspect, techniques may be devised to determine intracellular propionate accumulation in microbials. In this aspect, a potentially lethal concentration of propionate may be identified for desired cultures of OCFA producing biologicals that are utilizing propionate to increase OCFA production. In this way, for example, a high threshold amount of propionate may be identified for desired biologicals that produce desired OCFA accumulation while maintaining a desired growth rate for the target biologicals.

[00133] In one implementation, the model used to determine intracellular propionate accumulation, using Henderson and Hasselbach equations, can be calibrated by establishing the lethal concentration threshold of intracellular propionate that A. acetophilum (HS399) could tolerate. The calibration can be further verified using two different approaches, propionate concentration and pH-driven propionic acid toxicity.

[00134] In a first implementation, an Erlenmeyer flask can be inoculated with 0, 10, 20, and 30 g/L initial treatments of propionate, at a substantially constant pH of 6.4.
In this implementation, observation indicates that the treatments containing 20 and 30 g/L will not survive. This model can be used to translate the extracellular pH and propionic acid concentration to intracellular propionate concentrations, a metric that could be interpreted across different pH values and propionate concentrations scenarios. Table 15 below illustrates that initial propionate treatment of propionate at 20 g/L results in lethality of the culture. Further, the cytosolic (the cytoplasmic matrix is the liquid found inside cells) propionate concentration is identified to be 97.7 g/L using the model.
Table 15. Propionic acid toxicity lethal threshold measured according to two approaches.
Toxicity Applied pH Drift Initial Propionate Toxicity Type Acute Chronic Metric Used OUR CDW
PH 5.3 6.4 Propionate Treatment (g/L) 2.3 0.1 20 Cytosolic Propionate (g/L) 135 3.5 122 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00135] In a second implementation, the evolution of the oxygen uptake rate (OUR) is measured for two cultures where the decreasing pH set point is controlled with propionic acid. FIGURE 31 is a graphical representation of an example 3100 where, in this implementation, the pH 3102 can be steadily decreased from 7 down to 4 at a rate of 0.7 pH units per hour 3104. In this example 3100, during the four hours of pH ramp, residual propionate concentration in the media is maintained relatively constant (e.g., 2.3-2.4 g/L). In this implementation, the OUR remains relatively constant until the pH reached 5.3, and the OUR 3106 measurement dropped in response to propionate toxicity. In FIGURE 31, the example 3100 shows monitoring of A.
acetophilum HS399 oxygen uptake rate (OUR) in response to pH driven propionate toxicity.
In this example, the results illustrate that cell respiration was not substantially affected until the pH reached 5.3 (vertical line I), which suggest this may be the tolerance limit for propionate by A. acetophilum HS399.

[00136] As illustrated in Table 15, above, the model illustrated in the example 3100 of FIGURE
31 can be used to translate the extracellular pH and propionic acid concentration to intracellular propionate concentrations. For example, this can be confirmed as a substantial equivalent to the concentration obtained with the "initial propionate" approach (107.5 vs 97.7 g/L). The results demonstrate that intracellular propionate concentration may be a valid metric for propionic acid toxicity and used as a metric that can be interpreted across different pH
values and propionate concentrations scenarios (see Table 15).

[00137] FIGURE 32 is a 3D graphical representation of an example expression of the propionic acid toxicity 3200. In this example, the propionic acid toxicity 3200 is expressed against a grid that defines the threshold toxicity 3208 limit for different pH 3204 and extracellular propionate combinations 3206. As an example, the graphing of the propionate toxicity model can be a very useful tool for the design of the odd chain fatty acid production process. In this example, the propionic acid toxicity 3200 represented as cytosolic propionate 3202 is controlled by the extracellular pH 3204 and propionate concentration 3206. In one implementation, the 3D graph can be built through the integration of Henderson and Hasselbalch equation.
The grid intersection line 3208 represents the lethal toxicity threshold validated experimentally according to two different approaches.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00138] In one implementation, a lower level of propionic toxicity may be determined, as described in the following example techniques:
Identify lower propionic toxicity: Batch vs. fed-batch

[00139] In one implementation, a 250 mL Erlenmeyer flask can be used to cultivate A.
acetophilum HS399. In this example, sodium propionate can either be batched or fed batch different propionate concentrations each day. FIGURE 33 is a graphical representation illustrating an example 3300 where cell dry weight is measured each day to evaluate the impact of the propionate feeding strategy in A. acetophilum HS399 growth. This example illustrates treatment of 3+3+3 (e.g., over respective days), where 3 g/L propionate is fed at 0, 24 and 48 hrs (9 g/L in total). This treatment appears to provide better growth than a treatment fed 10 g/L propionate at 0 hr. These results illustrate that fed-batching may be a better strategy to lower propionic acid toxicity than batching all the propionate at initial inoculation. Further, as illustrated in this example 3300, growth is decreased in a dose response manner with the daily amount of propionate fed. In this example, the best growth is achieved with the lowest propionate dose (1 g/L d), but this treatment still shows growth inhibition when compared against the non-propionate control treatment (0 g/L d).
Identify lower propionic toxicity: Growth vs. lipid phase.

[00140] In one implementation, a 250 mL Erlenmeyer flask can be used to cultivate A.
acetophilum HS399. In this example, 3 g/L of propionate acid can be fed during growth phase (3+0) or during lipid phase (0+3). Growth phase treatment is fed propionate at inoculation (0 hr), while lipid phase treatment can be fed propionate upon depletion of the ammonia from the media (-24 hrs). Cell dry weights may be analyzed using a filtration method. Ammonia can be measured using a Cedex bio-analyzer (Roche). Further, propionic acid can be measured using high performance liquid chromatography. The fatty acid profile of the biomass can be analyzed using gas chromatography.

[00141] In this implementation, total propionate consumed by the cell can be calculated based on the residual propionate on day 0 and subtracting the final residual propionate. The total Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
propionate deposited in the biomass can be calculated based on the final cell dry weight, multiplied by the total fatty acid (TFA) ratio in biomass, multiplied by the OCFA ratio in TFA, and multiplied by the molar factor of propionate in odd chain fatty acid, which averages at about 0.3. The propionate deposition can be calculated by dividing the propionate deposited by the propionate consumed and expressed as percent.

[00142] FIGURE 34 is a graphical representation illustrating results 3400 of growth of A.
acetophilum HS399 and residual ammonia when propionate was fed in growth or lipid phases. As illustrated, the results 3400 identify that propionic acid (0+3) is fed during the lipid phase, once the ammonia was depleted from the media. As illustrated, this treatment during the lipid phase did not appear to provide a lag phase 3402 that was present when propionate was fed during the growth phase. Additionally, the propionic acid deposition is slightly improved when propionate is fed during the lipid phase than when propionate was fed in the growth phase. These results 3400 illustrate that feeding propionate during lipid phase may be preferred over feeding during the growth phase because of the lack of lag 3402 during the lipid phase, and an increases of lipid deposition from 53 to 58.8 %, as illustrated in Table 16 below.
Table 16. Propionic acid deposition into Aurantiochytrium acetophilum HS399 according to the stage in which propionate was fed.
Propionate (g/L) Propionate Deposition (%) 0 (control) 0.0 0.0 3 (growth) 53.0 1.0 0+3 (lipid) 58.8 1.9 Pathway elucidation: A. acetophilum HS399 may not use propionic acid as sole carbon source.

[00143] In one aspect, propionic acid may be either incorporated into A.
acetophilum HS399 lipids or catabolized into the citric acid cycle. In this aspect, the propionate deposition may be controlled by both the rate of lipid synthesis and the rate of propionate catabolism. For example, there are two main catabolic pathways that are responsible for propionate oxidation into the citric acid cycle. The methyl-malonate pathway converts propionate into succinyl-CoA
which enters Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
the citric acid cycle. Alternatively, the methyl-citrate pathway, converts propionate into succinate and pyruvate, both of which enter the citric acid cycle.

[00144] As identified by one or more the techniques described herein, the methyl-citrate pathway is anaplerotic because it releases two intermediates of the citric acid cycle. Because propionate is not an anaplerotic substrate, growth on propionate (e.g., as a sole carbon source) may be sustained by an anaplerotic pathway such as the methyl-citrate. Non anaplerotic pathways, such as methyl-malonate pathway, which releases only one citric acid intermediate (succinyl-CoA
intermediate), may not sustain growth on propionate as sole carbon source.

[00145] In one implementation, the capacity of A. acetophilum HS399 to grow on propionate as sole carbon can be determined to help identify the catabolic pathway for propionate catabolism.
In this implementation, an Erlenmeyer flask (250 ml) can be inoculated (1 %
v/v) in triplicates at a pH of 7. One treatment can be fed with non-lethal concentrations of propionate (e.g., 10 g/L), and other treatments can be fed glucose as a positive control, or no carbon substrate as a negative control. FIGURE 35 is a graphical representation of one implementation that illustrates results 3500 of growth of A. acetophilum HS399 with different carbon sources. As illustrated, the results 3500 illustrate that the propionic acid treatment 3502 did not support growth above that of the starved treatment (negative control) 3504; while the glucose treatment 3506, as expected, supported high growth.

[00146] Further, as illustrated in FIGURE 36, shows example results 3600 of residual propionate cultures of A. acetophilum HS399 fed different carbon sources. The results 3600 illustrate that the residual propionate does not decrease in response to cell uptake. These results 3500, 3600, suggest that the methyl-citrate cycle may not be active in A.
acetophilum HS399.
Therefore, propionic acid is most likely catabolized via a non anaplerotic methylmalonate pathway.
Metabolic intervention: Cvanocobalamin regulating propionate catabolism in Microbials

[00147] As described above, methyl-malonate is likely the main propionate catabolic pathway in A. acetophilum HS399. The enzyme methylmalonyl-CoA mutase converts a methylmalonyl-CoA into succinyl-CoA, in a reaction that utilizes cyanocobalamin (vitamin B12) as a cofactor.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00148] In one implementation, A. acetophilum HS399 can be sub-cultured in a cyanocobalamin deprived media for at least 10-generations. FIGURE 37 is a graphical representation of example results 3700 of sub-culturing A. acetophilum HS399 in a cyanocobalamin deprived media for over 10-generations. The results 3700 illustrate that there is substantially no impact on growth of A. acetophilum HS399 between generations.
In this example, the numerous generations help dilute the possibility of potential cell reserves of cyanocobalamin and demonstrate that cyanocobalamin (Vitamin B12) is not likely essential.
These results 3700 also provide adequate evidence that A. acetophilum HS399 is not a cyanocobalamin auxotroph.

[00149] In one implementation, the impact of methionine supplementation in the growth of A, acetophilum HS399 can be illustrated. In this implementation, methionine synthetase can use cyanocobalamin as a cofactor to generate methionine from homocysteine. In this implementation, a growth media can be supplemented with 0 and 0.5 g/L methionine and the growth can be measured under the presence and absence of cyanocobalamin (0.00037 1.1M). The methionine supplementation does not appear to have an impact in growth of A. acetophilum HS399 (FIGURE
38), regardless of the presence or absence of cyanocobalamin. As an example, this may be consistent with A. acetophilum not being a cyanocobalamin auxotroph.

[00150] In one implementation, a cyanocobalamin deprived A. acetophilum HS399 can be inoculated in triplicates (n=3) in the presence of propionate (3 g/L), using three different concentration of cyanocobalamin (0.37, 0.00037, 0 1.1M). The propionic acid deposition by A.
acetophilum HS399 can be analyzed according to the method described above regarding Propionic Acid Deposition Rate. As illustrated in Table 17 below, a dose response increase in propionate deposition is identified, with decreasing concentration of cyanocobalamin in the media. Of note, a cyanocobalamin deprived media (0 1.1M) can result into 99.5 % of the propionate being incorporated into A. acetophilum HS399 lipids as OCFAs. For example, this may suggest that almost no propionate is oxidized, presumably due to the lack of methyl-malonyl-CoA mutase cofactor (e.g., cyanocobalamin) blocking the pathway. The results shown in Table 17 are consistent with the methyl citrate pathway not being active (as described above), and methyl-malonate may be the primary catabolic pathway for propionic acid oxidation. A
benefit associated with increasing propionate deposition is decreasing the propionate used for the production of OCFAs.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Table 17. Propionic acid deposition in response to cyanocobalamin (vitamin B12) concentration.
Cyanocobalamin-Vit B12 (pM) 0.37 0.00037 0 Cell dry weight (g/L) 37.0 1.4 32.0 0.7 35.0 1.2 OCFA (%DW) 2.7 0.2 14.7 0.2 25.0 0.3 Initial propionate (g/L) 3 3 3 Final Propionate (g/L) 0.06 0.1 0.36 OCFA (g/L) 1.0 0.1 4.7 0.1 8.7 0.4 Propionate (MW)/OCFA (MW) 0.3 0.3 0.3 Propionate deposition (% fed) 10.3 1.0 47.0 0.9 99.9 5.8 Metabolic intervention: Cyanocobalamin may not impact propionic acid toxicity.

[00151] In one implementation, in can be determined whether an increase of propionate deposition, through cyanocobalamin deficiency, has a negative impact in A.
acetophilum HS399 growth and productivity. For example, a testing can show if propionic acid toxicity is affected by the cyanocobalamin concentration, because, as identified above, cyanocobalamin may not be essential for A. acetophilum HS399, at least in the absence of propionate.

[00152] FIGURE 38 is a graphical representation of one implementation illustrating example results 3800 of cell dry weight and residual propionate, 3g/L propionate. In this implementation, A. acetophilum HS399 can be inoculated at different concentrations of cyanocobalamin (0.00037, 0 M) in triplicates (n=3). In this implementation, the pH can be maintained at 7 1 in respective treatments. The initial 3 g/L propionate fed to the cultures is consumed within 72 hrs, during which insignificant differences are shown in growth due to the cyanocobalamin concentrations, as illustrated by the results 3800. These results suggest that propionic acid catabolism may not be necessarily a protection mechanism for propionic acid toxicity, for example, growth can be inhibited as long as propionic acid is still present, as shown in the lower half of the graph.

[00153] In one implementation, fermenters may be used to culture A.
acetophilum HS399. In this implementation, propionic acid can be fed-batch in a pH-auxostat mode (e.g., propionic acid used as pH titrant) 24 hrs after inoculation. The fed batch system can appropriately control Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
propionic acid toxicity by maintaining residual propionate concentration at 2.0 0.1 g/L and the pH at 7 0.1. FIGURE 39 is a graphical representation showing results 3900 of the impact of cyanocobalamin in A. acetophilum HS399 growth and propionic acid consumption in 10 L
Bioflo320 fermenters. In this implementation, two treatments using different cyanocobalamin concentrations (0.00185, 0 M) are compared in duplicates. As illustrated by the results 3900, the growth of both treatments is substantially equivalent, but the treatment without cyanocobalamin consumed 30 % less propionic acid. As shown in Table 18 below, the OCFA
production may not be substantially affected by the cyanocobalamin concentration, for example, as long as propionic acid is fed on demand. However, as shown in Table 19 below, the propionate deposition is elevated in the treatment without cyanocobalamin. In other words, as an example, cyanocobalamin deficiency may produce the same OCFAs with a much smaller portion of the propionic acid.
Further, for example, Cyanocobalamin may not have any impact in propionic acid toxicity and therefore it may be freely manipulated to improve deposition rates.
Table 18. Impact of cyanocobalamin in the fatty acid profile of Aurantiochytrium acetophilum HS399 cultures grown in a 10 L Bioflo320 fermenters.
Cyanocobalamin ( M) - 0.37 0 Time (h) 0 73 73 Total Fatty Acids (% DIV) 26.7 54.7 0.5 56.9 Fatty Acid Profile (% TFA) 13:0 0.0 1.4 0.2 1.5 14:0 3.8 1.4 0.0 1.3 15:0 1.1 42.6 0.9 42.3 16:0 45.2 11.7 1.6 10.8 17:0 0.4 8.6 0.6 8.2 18:0 1.6 0.3 0.0 0.3 22:5(n-6) 7.7 3.6 0.1 3.8 22:6(n-3) 36.4 28.0 1.0 29.1 Other FA 3.8 2.5 0.3 2.7 DHA (% DW) 9.7 15.3 0.4 16.6 OCFA (% TFA) 1.5 52.6 0.5 52.0 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Table 19. Propionate deposition.
Cyanocobalamin (pM) gpropionate/gbiomass Propionate deposition 0.37 0.18 0.01 49.9%
0 0.14 65.7%
Mitigate propionic toxicity: Single vs. two-stage fermentation.

[00154] In one implementation, the impact of propionate in two different growth modes can be illustrated, using a two-stage and a single-stage growth mode, utilizing 10 L
Bioflo fermenters, for example. In this implementation, a two-stage mode can comprise a first growth stage (0-24 hrs), followed by a lipid phase (24-80 hrs), where nitrogen is not present. Thus, for example, in the two-stage mode substantially all of the nitrogen (5 g/L NH3) can be fed during growth phase, which is then depleted as it enters a lipid phase. Further, in this implementation, during the lipid phase, no additional nitrogen is fed, and the cell accumulated the lipids. In the single stage mode, half of the nitrogen (2.5 g/L NH3) can be batch fed, while the other half (2.5 g/L NH3) can be fed along with the glucose until the end of the fermentation. In this implementation, both treatments receive substantially the same nutrients; however, the single stage mode grows and accumulates lipids in a coordinated way throughout the fermentation. The single stage system can be characterized by an early lipid accumulation.

[00155] Embodiments of the single stage system are described in detail in Application No.
PCT/US2018/29602 (Ganuza et al.), entitled SINGLE-STAGE FERMENTATION METHODS
OF CULTURING MICROORGANISMS, filed on April 26, 2018 by the Applicant herein, which is incorporated herein in full by reference. The cultures can achieve higher lipid contents (-32 hrs) sooner than the two-stage system (-48 hrs) in the batch.

[00156] FIGURE 40 is a graphical representation of two results 4000, 4050 of the impact of propionic acid exposure to A. acetophilum H5399 growth and odd chain fatty acid production in L Bioflo320 fermenters under single 4000 or two-stage mode 4050. In this implementation, propionic acid can be fed fed-batch in a pH-auxostat mode (e.g., propionic acid can also be used as the pH titrant) 24 hrs after inoculation to experimental treatments in order to illustrate the impact Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
of the growth mode in propionic acid toxicity. Each of the four treatments can be cultured in duplicate (n=2) fermenters. As illustrated in Table 20 below, the growth rate can be determined, along with the amount of propionic acid fed throughout the culture. As illustrated, propionic acid slows down the growth on both systems, but the two-stage system provides a better growth rate than the single stage system in the presence of propionate. Additionally, the accumulation of OCFA in lipids (% TFA) is improves when propionic acid is fed in a two-stage process, for example, because no lipids (e.g., consisting of even chain fatty acids) may be produced in the absence of propionate (0-24 h). This implementation illustrates that the traditional two stage approach to lipid accumulation may be preferable to produce OCFAs.
Table 20. Impact of propionic acid exposure to Aurantiochytrium acetophilum HS399 biomass yield and productivity under single or two-stage mode.
Single Stage Two Stage No Acid Propionic Acid No Acid Propionic Acid Batch Time (h) 96h 96h 89h 89h Biomass Yield (g/L) 148.8 82.5 163.6 125.8 Productivity (g/L/d) 37.4 20.7 44.3 34.1 Double ammonia-propionic acid/pH-auxostat process:

[00157] Based on the results from the implementations described above, in one implementation, a fermentation process can be used to produce OCFAs under a two-stage growth mode, where ammonia is used as a nitrogen source and propionate is used as a promotor of OCFAs. Glucose can also be fed in a DO2 stat mode in response to dissolved oxygen levels rising above 15 %
saturation. Previously, as illustrated herein, fed-batch may be preferred to batch because it can reduce propionic acid toxicity (e.g., "Mitigate propionic toxicity: Batch vs.
fed-batch"). Further, as illustrated herein, propionic acid toxicity can be modulated with the propionate concentration and the pH of the media (e.g., "Establishing propionic acid toxicity limit").
Therefore, a pH-auxostat system can be used to maintain low residual concentrations of those nutrients in a fed-batch mode, while controlling their toxicity through the pH set point.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Molar NH3/NaOH ratio of the fed.

[00158] FIGURE 41 is a graphical representation illustrating results 4100 of one implementation, where, much like the two-stage growth mode, described above, ammonia can be fed merely during the growth phase. In this implementation, the results 4100 illustrate the impact of ammonia to sodium hydroxide ratio of the fed in the residual ammonia concentration of a double auxostat culture of A. acetophilum HS399. In this implementation, ammonia can be gradually displaced from the media as the sodium hydroxide from the feed interferes with the titration of the ammonia/pH-auxostat. The feeding of propionic acid can be postponed until the lipid phase (-15-25 hrs) based on the results obtained in example "Mitigate propionic toxicity:
Growth vs. lipid phase," described above.

[00159] As a result, in this implementation, a double ammonia propionic/pH-auxostat can be used, where ammonia is fed on demand during growth phase and propionate is fed on demand during the lipid phase (e.g., a two-stage process). To accommodate both auxostats, a transition can be used that would provide for the absence of ammonia during the lipid phase, which otherwise can interfere with the propionic acid auxostat titration. In this implementation, an inert base (e.g., sodium hydroxide) can be blended with the ammonia feed in a specific ratio.
The sodium hydroxide titrates the pH irreversibly, gradually displacing the residual ammonia in the medium until it is completely depleted, as shown by the results 4100. As illustrated, ammonia depletion can stop the auxostat feed, which indicates the end of the growth phase and the beginning of the lipid phase. The rate of ammonia depletion, and therefore the total ammonia fed, for example, can increase with decreasing ammonia - sodium hydroxide ratio.

[00160] FIGURE 42 is a graphical representation illustrating results 4200 of one implementation, applying the impact of ammonia to sodium hydroxide ratio of the fed in the total ammonia fed and biomass yields of a double auxostat culture of A. acetophilum HS399. In this implementation, the results 4200 illustrate the total ammonia fed to the culture and the resulting biomass yields can also be controlled by the ammonia to sodium hydroxide ratio. Further, the results 4200 indicate that the ammonia to sodium hydroxide ratio in the fed is a component for the control and operation of the double auxostat system. For example, this control may also be applied with a different inert base, such us potassium hydroxide or calcium hydroxide.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Toxicity control through a pH ramp

[00161] In one implementation, using the model described above in "Establishing propionic acid toxicity limit" a pH to provide a desired balance of the toxicity of both propionic acid and ammonia can be determined. For example, using this method, there may not be a specific pH that can accommodate the toxicity of both nutrients. FIGURE 43 is a graphical representation illustrating results 4300 of one implementation, describing the impact of pH
set-point control in the transition of ammonia to propionic acid pH auxostat culture of A.
acetophilum HS399. As illustrated, the pH ramp 4302 is used to sustain the growth of HS399. As illustrated, when A.
acetophilum HS399 is grown in a double pH-auxostat at a pH set-point of 7.0 4304, ammonia toxicity can inhibit its growth. Further, when A. acetophilum HS399 is grown at a pH of 5.5 4306 the ammonia toxicity can be greatly reduced, but the cells can be inhibited by propionate at the beginning of the lipid phase. Additionally, a ramp 4302 can be applied where pH set-point can be steadily increased from 5.5 to 7 between hour 5 to hour 17 of fermentation, which can coincide with the end of the growth phase when residual ammonia was at a low point. In this example, the pH ramp can mitigate the toxicity of both nutrients and improve the growth rate for the culture through the two-stage process 4308.
Activating and maintaining the propionic acid pH auxostat

[00162] FIGURE 44 is a graphical representation illustrating results 4400 for A. acetophilum HS399 double pH-auxostat cultures, showing the impact of sodium hydroxide supplementation into the glucose fed in the residual propionic acid control in a pH-auxostat.
In this implementation, the propionic acid pH-auxostat can be implemented throughout the lipid phase to improve the synthesis of OCFAs in A. acetophilum HS399. The lipid phase can begin when the ammonia feed naturally stops the addition. At this point, 1-3 g/L of residual propionate can be added into the culture. The propionate can be added as a salt (e.g., sodium propionate) or as an acid, in response to the simultaneous titration of sodium hydroxide that is slowly (pH 7 0.2) introduced in the reactor. In this implementation, as soon as A. acetophilum HS399 starts consuming the propionate, the pH raises, which provides for more propionate and activation of the auxostat. In one implementation, the auxostat can be maintained to keep the residual propionate levels constant, however, the residual propionate may slowly decrease, which may be due to other cell metabolites Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
interfering with the titration, as shown in FIGURE 44. In order to overcome the slow decrease, 1-2 g of sodium hydroxide can be introduced in respective liters of the glucose feed. In this implementation, the alkalization can help maintain the residual propionate constant and avoid the early interruption of propionate fed that may otherwise occur.
Propionate concentration and OCFA titers

[00163] In one implementation, the variation of propionate fed to Aurantiochytrium acetophilum HS399 cultures is reflected in the OCFA titers of the final biomass. In this example, five treatments containing different concentrations of propionate: 0, 2, 3, 4, 5 g/L respectively.
Respective Erlenmeyer flasks (250 mL) are inoculated (1 % v/v) in triplicates with a 24 h old culture of A. acetophilum HS399 and incubated in an orbital shaker at 180 rpm and 27 C.

[00164] Respective Erlenmeyer flasks contain 100 mL of a medium supplemented with (g/L):
dextrose (100), ammonium acetate (4.6), NaCl (12.5), MgSO4 7H20 (2.5), KH2PO4 (0.5), KC1 (0.5) and CaCl2 (0.1). This medium also contains trace element solution (5 ml/L) and vitamin solution (1 ml/L). The trace element solution contains (g/L): EDTA di-sodium salt (6), FeCl3 6H20 (0.29), H2B03 (6.84), MnC12 4H20 (0.86), ZnC12 (0.06), NiSat 6H20 (0.052), CuSO4 5H20 (0.002), Na2MoO4 H20 (0.005). The vitamin solution contains (mg/L): thiamine (100) and biotin (0.5).

[00165] In this example, respective culture materials are autoclaved (e.g., 121 C, 15 min) and the media is filter sterilized before use. A propionic acid stock solution (200 g/L) can be used as the fed propionic acid. Daily samples are collected to analyze the cell dry weight, residual glucose, culture pH and lipid and fatty acid composition of the cultures. Cell dry weights are analyzed by filtration (e.g., 0.2 gm filter media) using a vacuum and washed with a solution of ammonium bicarbonate. Residual glucose is analyzed using a colorimetric method based on glucose peroxidase activity. Biomass for lipid analysis is centrifuged and washed using purified water. The washed biomass is freeze dried. Total lipids are analyzed using Folch method (AOAC 996.06) and the FAMEs are analyzed by gas chromatography and flame ionization detection using nonadecanoic (C19:0) acid as an internal standard.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00166] As shown in Table 21, the results illustrate that the different propionate concentrations yield different odd chain fatty acid concentration. The results show that a wide range of OCFA
concentration in the oil can be produced by variating the propionate concentration in the culture.
Table 21. Total lipids and fatty acid profile at time of harvest (96 h).
Propionate (g/L) 0 2 3 4 5 Total Fatty Acids (% DW) 62.7 0.8 54.8 5.6 56.2 0.6 39.2 12.4 41.0 3.4 Fatty Acid Profile (% TFA) 13:0 0.0 0.0 1.6 0.1 2.6 0.1 4.2 0.4 5.1 0.7 14:0 3.7 0.0 3.0 0.2 2.4 0.1 1.8 0.3 1.9 0.2 15:0 0.7 0.0 25.2 1.5 41.6 0.9 51.6 2.6 53.0 0.4 16:0 51.5 0.4 31.7 1.3 17.0 1.1 6.4 0.5 5.7 0.5 17:0 0.3 0.0 3.9 0.4 6.7 0.3 7.2 0.3 6.6 0.7 18:0 1.9 0.3 0.9 0.0 0.5 0.0 0.1 0.1 0.1 0.1 22:5(n-6) 7.4 0.1 5.1 0.2 4.0 0.2 2.7 0.4 2.8 0.1 22:6(n-3) 33.0 0.5 26.6 0.6 23.4 0.4 22.8 1.9 21.9 0.6 Other FA 1.4 0.3 1.6 0.0 1.7 0.0 2.7 1.2 2.2 0.3 OCFA (% TFA) 0.9 0.0 30.7 1.8 50.8 1.1 63.0 2.5 64.6 0.3 Anaplerotic process in 1000 L pilot fermenter.

[00167] In one implementation, the processes described herein can be scaled up into a larger pilot facility, such as a 1000 L pilot facility, in duplicates fermenters (n=2). For example, the pilot facility may be reflective of the production in larger reactors of up to 180,000 L. In this implementation, the seed cultures can be scaled up from a 1 mL cryovial, into a 100 ml Flask, a 7 L wave bag, a 79 L fermenter, and the pilot fermenter with 800 L running volume. Thus, as an example, the cultures can be inoculated at 5 % v/v, although 1 % v/v inoculation may also be utilized. In this implementation, the fermenter cultures can be aerated at 1.3 vvm (volume of air per volume of culture per min) and agitated with Rushton impellers, for example, at 60 rpm in a dissolved oxygen (D02) cascade control. As one example, rpm may increase, but not be decreased, in response to D02, at levels below 10 % saturation. In this implementation, the temperature can be controlled at 27 1 C. Further, the pressure can be controlled at (0.7-2 kg/cm2), for example, in response to excessive foaming or rpm at its high limit. Additionally, corn syrup 95DE can be fed in a D02-stat mode at 800 g/L glucose in response to DO2 levels rising above 15 %.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00168] In this implementation, the glucose solution can be fed in a ramp from 0 to 0.2 mL/min L initially (e.g., from 8 to 20 h elapsed fermentation time), and from 20 h onwards the D02-stat triggered glucose feed can pulse 102 min at 0.3 mL/min L. The pH can be controlled with the ammonia fed at 5.5 during growth phase and propionic acid at 7.0 during lipid phase. The pH can be raised from 5.5 to 7.0 gradually between 5 and 17 h when residual ammonia is at a low point.
Upon the ammonia running out of the culture (e.g., nitrogen feeding stopped), 1 g/L of NaOH can be slowly added while correcting the pH (7.0 0.5) with propionic acid (glacial). In this example, after this point, the pH can be controlled with the propionic feed. As one example, foam can be controlled (e.g., manually or automatically) using less than 1 mL/L of Hodag K-60.

[00169] In this implementation, the 1000 L pilot fermenter can be filled with 400 L of batch media and fed with another 400 L of glucose feed media, and 40 L of ammonia fed, which may result in a final working volume of ¨850 L. The example compositions of respective media are illustrated in in Table 22, and the trace metal solution is shown in Table 23, below, along with vitamin mix contained 100 mg of thiamin and 0.5 mg/L of biotin. The medium chemicals can be dissolved in the order listed on the tables, for example, and the pH of the media can be adjusted to 5.5 using NaOH. The volume of the culture can be raised up to 345 L, accounting for another 35 L of condensation during a steam sterilization process (121 C x 30 min). In this implementation, the batch media ingredients can be filter sterilized, except for the vitamin mix, which can be filter sterilized into the reactor. Glucose fed can be pre-heated through the jackets to mitigate excessive condensation during subsequent steam sterilization (121 C x 30 min). The sodium hydroxide can be added without sterilization once the sterilized tanks have cooled down to ¨
40 C. The ammonia feed can also be prepared without sterilization.
Table 22. Medium formulation for production of OCFAs by Aurantiochytrium acetophilum HS399 in double pH-auxostat system.
H. N 3-Feed Glucose-Feed Chemicals Units Batch media Media Media Ammonium sulfate (NH4)2SO4 g/L 3.89 81.55 0 Potassium phosphate KH2PO4 g/L 5.0 0 0 Magnesium sulfate MgSO4 7.H20 g/L 2.5 0 0 Potassium chloride KC1 g/L 1.0 0 0 Calcium chloride CaCl2 g/L 0.2 0 0 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Vitamin Mix mL/L 10 0 0 Trace Metal Solution* see table below mL/L 50 0 0 Corn syrup D95 g/L 37.5 0 1143 Ammonium (29%) mL/L 0 238 0 NaOH g/L 0 64.2 2.0 Table 23: Formulation of the trace metals solution (TMS).
Required Quantity Chemicals Quantity g/L
(g for 1 L) EDTA disodium salt 6 6 FeCl3- 6H20 Iron (III) Chloride hexahydrate (cloruro 0.29 0.29 ferric o) H2B03 Boric acid 6.84 6.84 MnC12-4H20 Manganese chloride tetrahydrate 0.86 0.86 ZnC12 Zinc chloride 0.06 0.06 CoC12-6H20 Cobaltous chloride 0.026 0.026 Ni504-6H20 Nickel (II) Sulfate Hexahydrate 0.052 0.052 CuSO4-5H20 Copper (II) sulfate pentahydrate 0.002 0.002 Na2Mo04-2H20 Sodium molybdate dihydrate 0.005 0.005

[00170] In this implementation, the cultures can be monitored (e.g., periodically or continuously) for T, pH, D02, oxygen uptake rate (OUR), rpm, and running volume. In this implementation, samples of cell dry weight, lipids, fatty acids, residual glucose, ammonia and propionate can be collected twice a day, or more.

[00171] In this implementation, the pilot (1000 L) fermenter may achieve 80 g/L cell dry weight after 72 hrs of fermentation, as illustrated in the example results 4500 of FIGURE 45. These results 4500 illustrate cell dry weight 4502 and residual glucose 4504 of A.
acetophilum H5399 in a 1000 L pilot fermenter. In this implementation, productivities of over 30 g/L per day can be Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
obtained, as illustrated in the results 4600 of FIGURE 46, which illustrates the cumulative productivity of A. acetophilum H5399 in a 1000 L pilot fermenter.

[00172] In some implementations, the performance of the reactor may be lower than its 10 L
predecessor. This may be due to less than desired control of the cultivation parameters, as illustrated by the results in FIGURES 47A, 47B, 47C, 47D, which illustrate data resulting from substantially continuous monitoring (a.k.a. online data) of A. acetophilum H5399 in a 100 L pilot fermenter. Further, these results indicate that an oil containing 45 % OCFAs could be industrially produced at scale using Aurantiochytrium sp. as shown in Table 24 in FIGURE 48 using the techniques described herein. That is, for example, data generated by one or more sensors at the reactor or fermenter, such as pH sensor, dissolved oxygen sensor, off-gassing sensor, and others, can be continuously monitored (e.g., or periodically monitored, and automatically or manually recorded). In some implementations, the data may be automatically recorded, and can be communicatively transmitted to a remote location, for example. In this way, the data can be collected, as illustrated in the examples of FIGURES 47A-D and 50 (below).

[00173] FIGURES 49A, 49B, 49C, 49D are graphical representations of example results 4900 illustrating A. acetophilum H5399 growth in a 10 L bioflow-320 10 L fermenter using double pH-auxostat cultures for the production of odd chain fatty acids, growth productivity and lipid accumulation (n=2). Further, FIGURE 50 is a graphical representation of example results 5000 illustrating continuous monitoring of A. acetophilum H5399 double pH-auxostat cultures to produce odd chain fatty acids, D02, glucose fed, pH, titrant addition and agitation (n=2).
Anaplerotic oils & Type 2 Diabetes

[00174] In one aspect, epidemiological data shows that odd chain fatty acids (OCFAs) in blood plasma inversely correlate with diabetes type 2 (Forouhi et al. (2014): Lancet Diabetes Endocrinol, 2(10), 810-818.; Santaren et al. (2014). Am. J. Clin. Nutr., 100(1), 1532-1540). In one implementation, A. acetophilum H5399 can be used to evaluate if OCFAs have an impact in glucose metabolisms.

[00175] FIGURE 51 is a graphical representation of example results 5100 illustrating growth and residual propionate in A. acetophilum H5399 cultures that are subject to propionic anaplerosis Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
triggered by cyanocobalamin. In FIGURE 27, the different letters (a, b) in respective time points indicate statistically significant differences according to this implementation (p<0.05). FIGURE
52 is a graphical representation of example results 5200 illustrating residual glucose in A.
acetophilum H5399 cultures subject to propionic anaplerosis triggered by cyanocobalamin. In FIGURE 28, the different letters (e, f, g, h) in each time point indicate statistically significant differences according to a t-student test (p<0.05).

[00176] In this implementation, a culture can be fed 3 and 9 g/L of odd numbered propionic acid that is a product of the oxidation of longer chain fatty acids C15:0 and C17:0 in the presence or absence of cyanocobalamin (0 vs 0.37 M) in shake flask cultures. As described above, OCFAs anaplerosis can merely take place in the presence of cyanocobalamin. In this implementation, the cell dry weight and residual propionate can be monitored, and residual glucose in the media can be analyzed. As an example, while the growth in the first 48 hrs may not be impacted by the cyanocobalamin, as illustrated by the results 5100 of FIGURE 51, the residual glucose data indicates that glucose uptake rate may be significantly (P < 0.01 t-student) lower in the cyanocobalamin-anaplerotic treatment, as illustrated in the results 5200 of FIGURE 52. For example, these results indicate a link between OCFAs anaplerosis and glucose metabolism, in support of a protective role of odd chain fatty acids (OCFAs) against diabetes type 2.
OCFAs Promotors Alternative to Propionic Acid

[00177] In another aspect, alternative promoters for OCFA production may be utilized. Several implementations for use of alternative promoters are described below and shown in Table 25 in FIGURE 54.

[00178] In one implementation, alternative promoters to propionic acid for production of OCFA
may be implemented. According to techniques described above, A. acetophilum H5399 can accumulate OCFA in presence of propionic acid. Further, it may be beneficial to find alternative promotors to produce OCFA that are less toxic to the model organisms or that are simpler. In this implementation, pentanoate, heptanoate, yeast extract, proteose peptone, methionine, valine and isoleucine are evaluated for their capacity to induce the production of OCFAs.
Erlenmeyer flasks can be used and A. acetophilum H5399 can be cultured following the protocols described herein.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00179] In this implementation, respective flasks can be supplemented with different concentrations of the proposed promoters and the resulting biomass harvested and analyzed for total lipid and fatty acid analyses. In this implementation, valine and isoleucine are identified as a nitrogen and OCFAs source. Yeast extract, but not proteose peptone, is identified as a precursor of OCFAs, presumably because proteose peptone has a smaller proportion of the amino acids present in a free form than yeast extract. As illustrated in FIGURE 53, it pentanoate and heptanoate may be able to promote the production of OCFAs, but the toxicity of heptanoic is higher than that of propionic acid (see result 5300).
Anaplerotic oils & Health Benefits

[00180] In one aspect, epidemiological data shows that odd chain fatty acids (OCFAs) in blood plasma inversely correlate with diabetes type 2 (Forouhi et al. (2014): Lancet Diabetes Endocrinol, 2(10), 810-818.; Santaren et al. (2014). Am. J. Clin. Nutr., 100(1), 1532-1540). In one implementation, A. acetophilum HS399 can be used to evaluate if OCFAs have an impact in glucose metabolisms.

[00181] In one aspect, because anaplerotic substrates can be used to restore energy balance in mitochondria, there is a wide range of pathologies to which odd chain fatty acids have shown benefits. Namely, odd chain fatty acids have been experimentally used to treat the following conditions:
= Genetic Metabolic disorders o Glutl deficiency o Fatty acid oxidation disorder (FAOD) o Pyruvate carboxylase deficiency (Mochel et al., 2005) o Carnitine Palmitoyltransferase II deficiency (roe et al., 2008) o Rett syndrome (RTT) o Phenylketonuria (Roe & Mochel, 2006) o Adult Polyglucosan Body Disease (APBD) (Roe et al. 2010) Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
o long-chain fat oxidation disorders (Roe et al., 2002) = Neurodegenerative diseases:
o Epilepsy (Borges and Sonnewald, 2012) o Alzheimer's disease o Parkinson o Autism spectrum disorder (ASD) = Metabolic syndrome diseases o Diabetes type 2 o Obesity o Cardiovascular disease

[00182] Additionally, there is some indication that odd chain fatty acids can help in building muscle and improving athlete metabolism. For example, vigorous physical effort might result in depletion of glucose and glycogen, in which case the main anaplerotic substrate comes from the protein. Based on this process, one may hypothesize that odd chain fatty acids might spare the use of protein catabolism as anaplerotic substrate. Obesity and fat burn conditions may also benefit from use of odd chain fatty acids found in anaplerotic oils. For example, during periods of fat burn, odd chain fatty acid might restore the energy imbalance and help catalyze the energy generation from lipids. For instance, patients recovering from a surgical procedure might benefit from the OCFA anaplerosis.

[00183] Table 26, in FIGURE 55, illustrates various, commonly available natural vegetable oils.
Further, the table shows the concentrations of various fatty acids available in the respective vegetable oils. As illustrated, the OCFA, particularly C15 and C17, are not found, or are found in trace amounts in natural vegetable oils.

[00184] Table 27 below compares the features of the two types of concentrated anaplerotic oils, synthetic anaplerotic oils (e.g. tripentanoin) against naturally produced anaplerotic oils from microorganisms. Synthetic oils such as triheptanoin and tripentanoin typically have high concentrations of OCFAs. However, anaplerotic oil produced from microalgae, as described Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
herein, possesses several advantages over the synthetic triheptanoin and tripentanoin. As illustrated, the natural anaplerotic oil produced by algae includes OCFA that are naturally present in our diet (C15:0 and C17:0), while triheptanoin synthetically synthesis OCFA
of C5:0 and C7:0, which are not found in naturally occurring food sources.

[00185] Further, in this example, the anaplerotic oil produced by algae can contain a substantial amount of DHA, which is a valuable nutraceutical. For example, DHA
(docosahexaenoic acid) is a fatty acid that is commonly found in the meat of cold-water fish (e.g., tuna, salmon, cod, etc.).
DHA has been found to early brain development in infants, and may improve the vision and cognitive function development. Further, DHA has been used for treating type 2 diabetes, coronary artery disease (CAD), dementia, depression, and attention deficit-hyperactivity disorder (ADHD), as well as improving vision and cognitive function in adults.
Additionally, DHA can be converted into eicosapentaenoic acid (EPA) in the body, which is used in the prevention and reversal of heart disease, stabilizing heart rhythm, asthma, cancer, painful menstrual periods, hay fever, lung diseases, systemic lupus erythematosus (SLE), and certain kidney diseases. Both EPA
and DHA have been used in combination to treat high cholesterol, high blood pressure, psoriasis, Raynaud's syndrome, rheumatoid arthritis, bipolar disorder, certain inflammations of the digestive system (ulcerative colitis), and to prevent migraine headaches in teenagers.
Table 27. Comparison of synthetic anaplerotic oils with naturally-produced anaplerotic oils from microorganisms ANAPLEROTIC OILS
Tripentanoin Anaplerotic Dairy Fat Process Chemical Synthesis Biosynthesis Biosynthesis Type of OCFA Artificial Natural-Dietary Natural-Dietary C5:0; C 7:0 C15:0; C17:0 C15:0; C17:0 OCFA (% TFA) 100 60 1.5 DHA (% TFA) 0 25 <1 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Synthetic production of odd chain fatty acid and their triacylglycerides.

[00186] In some embodiments, odd chain fatty acids OCFAs and triacylglycerides containing OCFAs may be produced synthetically. As an example, the synthesis of saturated C15 or C17 fatty acids can be accomplished using different chemical reactions/strategies, some of which are summarized in Diagram 1 (A-I). The example methods described below can be used for synthesizing fatty acids esters as well by adopting appropriate protection/de-protection strategies.
The fatty acids or esters can then be used to synthesize triglycerides, the reaction can be catalyzed by base or preferably enzymatically to obtain OCFA enriched triglycerides. The synthetic scheme for triglyceride synthesis is described in Diagrams 1A ¨ 1! below.
A) Kumada Cross-Coupling NiCl2L2 (cat) or Pd, L 1) Cat. H2 X 2) Hydrolysis A + 0R1 _____________________________________ A
Solvent R = C13 or C15 acid X = F, Cl, Br, 1, OTf L2 = dppp, dppe, dppb A = H, C1-C11 alkyl L = PPh3 R1 = 01-C12 COOMe =MgX; X= Br, I or = Li B) Negishi cross-coupling X A 1) Cat. H2 A NiLr, or PdL, (cat.) 2) Hydrolysis or jR1 or X Solvent A A
X = Cl, Br, 1, OTf, OAc L = PPh3, P(o-toly1)3, dppe R
= C13 or C15 acid dppp,dppb, dppf, BINAP, diop, chiraphos A = H, C1-C11 alkyl R1 = 01-C12 COOMe =ZnX; X= Cl, Br, 1 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Sonogashira cross-coupling Pcl(PPh3)2C12 or Pd(PPh3)4 1) Cat. H2 X Cul or CuBr 2) Hydrolysis A
A
Solvent, Base R=
X = CI, Br, 1, OTf Base = Et2NH, Et3N, (Chx)2NH, (i-Pr)2NEt C13 or A = C2-C11 alkyl acid = Ci-C12 COOMe R2 = H, C1-C10 COOMe D) Stine Cross-Coupling Pd(OAc)2, PdC12(MeCN)2 1) Cat. H2 or PdC12(PPh3)2 A OR2 2) Hydrolysis , Solvent R = C13 or C15 acid A = Sn(alky1)3 = 01-C11 COOMe R2 = 01-C13 alkyl = Cl, Br, I, OTf, OPO(OR)2 E) Suzuki Cross-Coupling Pd (Cat.) Ligand .. R = C13 or C15 acid R1¨B(R)2 + R2-X _____________ Base or 1) Cat. H2 2) Hydrolysis R1 = C1-C15 alkyl, alkenyl, alkynyl R2R1 R2 = C1000Me-C15COOMe alkyl or alkenyl X = Cl, Br, 1, OTf, OPO(OR)2 R = C13 or C15 acid Base = Na2CO3, Ba(OH)2, K3PO4, Cs2CO3 K2CO3, TIOH, KF, CsF, Bu4F, NaOH

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
F) Alkyne Lithiation 1) Cat. H2 n-BuLi, THF R1-X, Solvent 2) Hydrolysis .õ...-----...R
_______________ > Li _____________ ,...- _____________ -R1 ,...-R = C13 or C15 acid R1 = C13-C15 COOMe alkyl, alkenyl or alkynyl X = Cl, Br, I, OTf, OMs, OTs G) Jones oxidation Cr03 or K2Cr207 acid 0 RiOH
H20, acetone Ri OH
R1 = Cualkyl or 16 alkyl Cr03 or K2Cr207 acid 0 reduction 0 R(OH ________________________________ ,... IL
H20, acetone Rr OH RI OH
R1 = C14alkenyl or c16 alkynyl R2 = C14 alkyl or C16 alkyl H) Arndt-Eistert Homologation o soci2 Ri OH ____ R o CH2N2 o Ag20, H20 CI )l' A __________ '- R1 N2 *- R1 i H OH
R1 = C13alkyl or c15 alkyl o o socI2 o CH2N2 Ag Ri"-20, H20 0 Ri)L'OH _______ .-Ri CI ._ 1.1N2 _____ Ri ..- -OH ____________________________________________________________________ .- R2----'-e OH
H
R1= C13alkenyl or ci5 alkynyl R2 =
C13 alkyl Or C15 alkyl I) Triglyceride synthesis (Chemical or enzymatic synthesis) 0 Base or Enzyme RiOR2 +
OH _______________________________________________ >
rO)Ri OH Ri y0 R1 = C14 alkyl or C16 alkyl Glycerol 0 R1 = C14 alkyl or C16 alkyl R2 = H, Me, Et, alkyl Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Diagrams 1A ¨ 1! Examples of chemical synthesis of odd chain fatty acid and their cleavage in a triacylglyceride.

[00187] In another embodiment, a representative synthesis of odd chain fatty acid triglycerides from commercially available starting materials is described in Diagram 2, below. In this embodiment, octyl bromide is first converted into corresponding boronate ester, which is then subjected to Suzuki coupling with 7-bromoheptanoic acid methyl ester to yield C15 methyl ester.
The methyl esters are then converted to triglycerides by treating with glycerol. The fatty acids/esters can be synthesized using other methods described in Diagram 2 using other commercially available starting materials.
pd2ob.)3 Nbo, p-\7 tBu2MeP. HBF4 -----'----WB-C) + '.- (ID
..>_.6B-Bb_< __ K3p04, H20, t-BuOH, heat Octyl bromide Bis(pinacolato)diboron CH3COCI, Me0H 0.y..õ..........¨Br _____________________________ .-OH OMe 7-bromoheptanoic acid 0 Br PdC12(dPID023 CH2Cl2 ,..,---,õ,...õ-- OMe t'D - __________________________ .._ OMe K2003, DME, heat 0 (:),,,C14 OMe Base or Enzyme o + )=
0 r- OH

OH 0y0 Glycerol Diagram 2 Example reactions for the synthetic production of odd chain fatty acid triglyceride from commercially available starting materials.

[00188] While this disclosure describes anaplerotic oil production using the microalgae A.
acetophilum H5399, and the yeast-fungi Yarrowia lipolytica, it should be appreciated that the method disclosed can be used with various other species of Aurantiochytrium, thraustochytrids, or other species of microorganisms including microalgae yeast, fungi and bacteria.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00189] The term "microalgae" refers to microscopic single cell organisms such as microalgae, cyanobacteria, algae, diatoms, dinoflagellates, freshwater organisms, marine organisms, or other similar single cell organisms capable of growth in phototrophic, mixotrophic, or heterotrophic culture conditions. The term fungi refers to microscopic and macroscopic single cell organisms such us yeast and filamentous fungi.

[00190] In some embodiments, microalgae biomass, excreted products, or extracts may be sourced from multiple types of microalgae, to make a composition that is beneficial when applied to plants or soil. Non-limiting examples of microalgae that can be used in the compositions and methods of the claimed subject matter comprise microalgae in the classes:
Eustigmatophyceae, Chlorophyceae, Prasinophyceae, Haptophyceae, Cyanidiophyceae, Prymnesiophyceae, P orphyri di ophyc eae, Labyrinthulomycetes, Trebouxiophyceae, Bacillariophyceae, and Cyanophyceae. The class Cyanidiophyceae includes species of Galdieria.
The class Chlorophyceae includes species of Chlorella, Haematococcus, Scenedesmus, Chlamydomonas, and Micractinium. The class Prymnesiophyceae includes species of Isochrysis and Pavlova. The class Eustigmatophyceae includes species of Nannochloropsis. The class Porphyridiophyceae includes species of Porphyridium. The class Labyrinthulomycetes includes species of Schizochytrium and Aurantiochytrium. The class Prasinophyceae includes species of Tetraselmis.
The class Trebouxiophyceae includes species of Chlorella. The class Bacillariophyceae includes species of Phaeodactylum. The class Cyanophyceae includes species of Spirulina.

[00191] Non-limiting examples of microalgae genus and species that can be used in the compositions and methods of the claimed subject matter include: Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var.
tenuis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus fakatus, Aurantiochytrium sp., Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetoceros sp., Chlamydomonas sp., Chlamydomas perigranulata, Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolate, Chlorella glucotropha, Chlorella infusionum, Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var.
aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var.
acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var.
umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgaris var.
autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var.
vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofingiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas sp., Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardawil, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Galdieria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis aff galbana, Isochrysis galbana, Lepocinclis, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenello ides, Navicula pelliculosa, Navicula saprophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrina, Nitzschia closterium, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilla, Nitzschia pusilla elliptica, Nitzschia pusilla monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilla, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlorella kessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp., Pleurochrysis camerae, Pleurochrysis dentate, Pleurochrysis sp., Porphyridium sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.

[00192] Taxonomic classification has been in flux for organisms in the genus Schizochytrium.
Some organisms previously classified as Schizochytrium have been reclassified as Aurantiochytrium, Thraustochytrium, or Oblongichytrium. See Yokoyama et al.
Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thrausochytriaceae, Labyrinthulomycetes):
emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov.
Mycoscience (2007) 48:199-211. Those of skill in the art will recognize that Schizochytrium, Aurantiochytrium, Thraustochytrium, and Oblongichytrium appear closely related in many taxonomic classification trees for microalgae, and strains and species may be re-classified from time to time. Thus, for references throughout the instant specification for Schizochytrium, it is recognized that microalgae strains in related taxonomic classifications with similar characteristics to Schizochytrium, such as Aurantiochytrium, would reasonably be expected to produce similar results.

[00193] In some embodiments, the microalgae may be cultured in phototrophic, mixotrophic, or heterotrophic culture conditions in an aqueous culture medium. The organic carbon sources suitable for growing microalgae mixotrophically or heterotrophically may comprise: acetate, acetic acid, ammonium linoleate, arabinose, arginine, aspartic acid, butyric acid, cellulose, citric acid, ethanol, fructose, fatty acids, galactose, glucose, glycerol, glycine, lactic acid, lactose, maleic acid, maltose, mannose, methanol, molasses, peptone, plant based hydrolysate, proline, propionic acid, ribose, saccharose, partial or complete hydrolysates of starch, sucrose, tartaric, TCA-cycle organic acids, thin stillage, urea, industrial waste solutions, yeast extract, and combinations thereof. The organic carbon source may comprise any single source, combination of sources, and dilutions of single sources or combinations of sources. In some embodiments, the microalgae may be cultured in axenic conditions. In some embodiments, the microalgae may be cultured in non-axenic conditions.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00194] In one non-limiting embodiment, the microalgae of the culture in an aqueous culture medium may comprise Chlorella sp. cultured in mixotrophic conditions comprising a culture medium primary comprised of water with trace nutrients (e.g., nitrates, phosphates, vitamins, metals found in BG-11 recipe [available from UTEX The Culture Collection of Algae at the University of Texas at Austin, Austin, Tex.]), light as an energy source for photosynthesis, and organic carbon (e.g., acetate, acetic acid) as both an energy source and a source of carbon. In some embodiments, the culture media may comprise BG-11 media or a media derived from BG-11 culture media (e.g., in which additional component(s) are added to the media and/or one or more elements of the media is increased by 5%, 10%, 15%, 20%, 25%, 33%, 50%, or more over unmodified BG-11 media). In some embodiments, the Chlorella may be cultured in non-axenic mixotrophic conditions in the presence of contaminating organisms, such as but not limited to bacteria. Additional detail on methods of culturing such microalgae in non-axenic mixotrophic conditions may be found in W02014/074769A2 (Ganuza, et al.), hereby incorporated by reference.

[00195] In some embodiments, by artificially controlling aspects of the microalgae culturing process such as the organic carbon feed (e.g., acetic acid, acetate), oxygen levels, pH, and light, the culturing process differs from the culturing process that microalgae experiences in nature. In addition to controlling various aspects of the culturing process, intervention by human operators or automated systems occurs during the non-axenic mixotrophic culturing of microalgae through contamination control methods to prevent the microalgae from being overrun and outcompeted by contaminating organisms (e.g., fungi, bacteria). Contamination control methods for microalgae cultures are known in the art and such suitable contamination control methods for non-axenic mixotrophic microalgae cultures are disclosed in W02014/074769A2 (Ganuza, et al.), hereby incorporated by reference. By intervening in the microalgae culturing process, the impact of the contaminating microorganisms can be mitigated by suppressing the proliferation of containing organism populations and the effect on the microalgal cells (e.g., lysing, infection, death, clumping). Thus, through artificial control of aspects of the culturing process and intervening in the culturing process with contamination control methods, the microalgae culture produced as a whole and used in the described inventive compositions differs from the culture that results from a microalgae culturing process that occurs in nature.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00196] In some embodiments, during the culturing process the microalgae culture may also comprise cell debris and compounds excreted from the microalgae cells into the culture medium.
The output of the microalgae culturing process provides the active ingredient for composition that is applied to plants for improving yield and quality without separate addition to or supplementation of the composition with other active ingredients not found in the mixotrophic microalgae whole cells and accompanying culture medium from the culturing process such as, but not limited to:
microalgae extracts, macroalgae, macroalgae extracts, liquid fertilizers, granular fertilizers, mineral complexes (e.g., calcium, sodium, zinc, manganese, cobalt, silicon), fungi, bacteria, nematodes, protozoa, digestate solids, chemicals (e.g., ethanolamine, borax, boric acid), humic acid, nitrogen and nitrogen derivatives, phosphorus rock, pesticides, herbicides, insecticides, enzymes, plant fiber (e.g., coconut fiber).

[00197] While OCFA production increase has been described herein in terms of algae, fungus yeast, and other microbial, the techniques and systems described herein may not be limited merely to these types of organisms. In one aspect, there are some plant sources that naturally contain OCFA, specifically C15 and C17, which could therefore be genetically modified to increase production of these OCFAs. That is, for example, much like with the production of OCFAs in algae, fungus and yeast, the plant genetic code may be modified to increase the already naturally occurring OCFA. As an example, the genetic code that increases OCFA production in microorganisms may be identified, and, using gene splicing techniques, such as CRISPR-type techniques, the OCFA code may be inserted into certain plants. Additionally, genetic trait selection may be undertaken to identify and breed plants that express improved OCFA production, resulting in a plant that has higher OCFA production. Also, a combination of genetic code manipulation and trait selection may be used to produce plants with improved OCFA production.

[00198] In addition to microalgae, some plants are able to serve as additional natural sources of long odd chain fatty acids. As shown in FIGURE 55, as Table 26, there are several plants that naturally contain C15:0 fatty acid, including, but not limited to: grape, silybum marianum (a thistle), wheat germ, and rapeseed. Additionally, several plants naturally contain C17:0 fatty acid, including, but not limited to: safflower, grape, silybum marianum, hemp, sunflower, wheatgerm, pumpkin seed, almond, rapeseed, and peanut. Typically, these plants merely produce trace levels of C15:0 and C17:0.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00199] Higher plants do not typically produce odd chain fatty acids (OCFAs) at commercially significant levels, however several plants (e.g., see plants in Table 16) present a metabolic pathway capable of synthetizing OCFAs. For example, the alpha oxidation pathway is a catabolic route typically associated with the degradation of 0-methyl branched fatty acids (see ref. 5 - Buchhaupt et al., 2014). The enzyme a-oxygenase (aDOX), a heme-protein, introduces an oxygen molecule to the a-C of a fatty acid, leading to a decarboxylation. An aldehyde dehydrogenase enzyme completes the oxidation of the resulting aldehyde to the corresponding fatty acid with one less carbon atom. This pathway eliminates the methyl group of the branched fatty acids, resulting in a straight chain (even) fatty acid, but it could also eliminate the methyl group of an even chain fatty acid, which would result in the production of OCFAs (see ref. 8 - Takahashi et al., 1992). This pathway has been described in pea plants (see ref. 7 - Shine and Stumpf, 1974), tobacco leaves, cucumber, potato (see ref. 6 - Hamberg et al., 1999), and could potentially be overexpressed in other plants, including oleaginous crops (e.g., soy, canola, flax, sunflower etc.), and directed to the production of OCFAs in plants. OCFAs production in oleaginous crops would benefit from the productivities and infrastructure available for such agricultural commodities.
Production of OCFA by Aurantiochvtrium limacinum SR21

[00200] Aurantiochytrium limacinum 5R21 (ATCC MYA-1381Tm), a strain previously characterized in Nakahara, et al., J Am Oil Chem Soc 73, 1421-1426 (1996); and Chi et al., Appl Microbiol Biotechnol 81,1141-1148 (2009), was grown to produce OCFA and compared to the titers achieved with A. acetophilum.

[00201] In this example, three different batch propionate concentrations (0 g/L, 3 g/L and 6 g/L) can be used. Respective Erlenmeyer flasks (250 mL) are inoculated (1 % v/v) in duplicates with a 24 h old culture of A. limacinum 5R21 and incubated in an orbital shaker at 180 rpm and 27 C.

[00202] Respective Erlenmeyer flasks contain 100 mL of a medium supplemented with (g/L):
dextrose (100), ammonium acetate (4.6), NaCl (12.5), MgSO4 7H20 (2.5), KH2PO4 (0.5), KC1 (0.5) and CaCl2 (0.1). This medium also contains trace element solution (5 ml/L) and vitamin solution (1 ml/L). The trace element solution contains (g/L): EDTA di-sodium salt (6), FeCl3 6H20 (0.29), H2B03 (6.84), MnC12 4H20 (0.86), ZnC12 (0.06), NiSat 6H20 (0.052), CuSO4 5H20 (0.002), Na2MoO4 H20 (0.005). The vitamin solution contains (mg/L): thiamine (100) and biotin (0.5).

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

[00203] In this example, respective culture materials are autoclaved (e.g., 121 C, 15 min) and the media is filter sterilized before use. Daily samples are collected to analyze the cell dry weight, residual glucose, culture pH, and lipid and fatty acid composition of the cultures. Cell dry weights are analyzed by filtration (e.g., 0.2 gm filter media) using a vacuum and washed with a solution of ammonium bicarbonate. Residual glucose is analyzed using a colorimetric method based on glucose peroxidase activity. Biomass for lipid analysis is centrifuged and washed using purified water. The washed biomass is freeze dried. Total lipids are analyzed using Folch method (AOAC
996.06) and the FAMEs are analyzed by gas chromatography and flame ionization detection using nonadecanoic (C19:0) acid as an internal standard.
As shown in Table 27, Aurantiochytrium limacinum SR21 can accumulate OCFA up to 65.7 %
TFA, and the C15:C17 ratio is similar to that of the A. acetophilum fatty acid profile.
Table 27. Fatty Acid profile at 96 h.
Propionate (g/L) 0 3 6 Total Lipids (% DW) 75.3 0.4 63.0 5.7 58.0 1.4 Total Fatty Acids (% DW) 66.4 1.1 55.3 3.0 48.1 0.0 Fatty Acid Profile (% TFA) 13:0 0.0 0.0 2.9 0.6 3.8 0.1 14:0 4.0 0.1 1.8 0.4 1.6 0.1 15:0 1.3 0.0 49.4 5.9 54.0 0.1 16:0 55.6 0.3 11.0 6.0 6.1 0.3 17:0 0.5 0.0 8.3 0.1 7.9 0.2 18:0 1.6 0.0 0.3 0.2 0.2 0.0 22:5(n-6) 6.6 0.2 3.5 0.4 3.1 0.0 22:6(n-3) 28.9 0.2 21.0 0.2 21.1 0.6 Other FA 1.6 0.0 1.9 0.2 2.2 0.1 OCFA (% TFA) 1.8 0.0 60.5 6.5 65.7 0.2 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
Fatty Acid Mixtures

[00204] In certain aspects, the disclosure provides mixtures of fatty acids including, but not limited to, the following:
Mixture A
C15:0 (%TFA) 5-70 C16:0 (%TFA) 1-30 C17:0 (%TFA) 1 -25 C22:5n6 (%TFA) 1 ¨ 10 C22:6n3 (%TFA) 5 ¨ 50 Triglycerides (% w/w) > 60 Total Saturated Fat (% w/w) 15 ¨ 90 Total Odd-Chain FA (%TFA) 5 - 95 Mixture B
C15:0 (%TFA) 10 ¨ 65 C16:0 (%TFA) 2.5 ¨25 C17:0 (%TFA) 2.5 -20 C22:5n6 (%TFA) 1 ¨ 7.5 C22:6n3 (%TFA) 10 ¨ 40 Triglycerides (% w/w) > 70 Total Saturated Fat (% w/w) 25 ¨ 85 Total Odd-Chain FA (%TFA) 12.5 - 85 Mixture C
C15:0 (%TFA) 20 ¨ 50 C16:0 (%TFA) 4-20 C17:0 (%TFA) 5-15 C22:5n6 (%TFA) 2¨ 6 Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
C22:6n3 (%TFA) 15 ¨ 35 Triglycerides (% w/w) > 80 Total Saturated Fat (% w/w) 25 ¨ 75 Total Odd-Chain FA (%TFA) 25 - 65 Mixture D
C15:0 (%TFA) 35 ¨ 50 C16:0 (%TFA) 5.0¨ 15 C17:0 (%TFA) 8.0 -13 C22:5n6 (%TFA) 2.3 ¨4.8 C22:6n3 (%TFA) 18.5 ¨29 Triglycerides (% w/w) > 88 Total Saturated Fat (% w/w) 50 ¨ 75 Total Odd-Chain FA (%TFA) 40 - 60 Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedures

[00205] The Aurantochytrium acetophilum H5399 strain was deposited on September 12, 2019 with the Bigelow National Center for Marine Algae and Microbiota, located at 60 Bigelow Drive, East Boothbay, ME 04544, USA.

[00206] The deposit was made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[00207] All references, including publications, patent applications, and patents, cited herein, are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law), regardless of any separately provided incorporation of particular documents made elsewhere herein.

[00208] Unless otherwise stated, all exact values provided herein are representative of corresponding approximate values (e.g., all exact exemplary values provided with respect to a Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
particular factor or measurement can be considered to also provide a corresponding approximate measurement, modified by "about," where appropriate). All provided ranges of values are intended to include the end points of the ranges, as well as values between the end points.

[00209] The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

[00210] The inventive concepts described herein include all modifications and equivalents of the subject matter recited in the claims and/or aspects appended hereto as permitted by applicable law.

[00211] References:
1. Weitkunat, K., Schumann, S., Nickel, D., Hornemann, S., Petzke, K. J., Schulze, M. B., ...
Klaus, S. (2017). Odd-chain fatty acids as a biomarker for dietary fiber intake: a novel pathway for endogenous production from propionate. The American Journal of Clinical Nutrition, 105(6), ajcn 152702. https://doi.org/10.3945/ajcn.117.152702.
2. kezanka, T., & Sigler, K. (2009). Odd-numbered very-long-chain fatty acids from the microbial, animal and plant kingdoms. Progress in Lipid Research, 48(3-4), 206-238.
https://doi .org/10.1016/j .plipres.2009.03 .003 .
3. Chaung, K.-C., Chu, C.-Y., Su, Y.-M., & Chen, Y.-M. (2012). Effect of culture conditions on growth, lipid content, and fatty acid composition of Aurantiochytrium mangrovei strain BL10. AMB Express, 2(1), 42. https://doi.org/10.1186/2191-0855-2-42.
4. Fan KW, Chen F, Jones EB, Vrijmoed LL. Eicosapentaenoic and docosahexaenoic acids production by and okara-utilizing potential of thraustochytrids. J Ind Microbiol Biotechnol.
2001; 27(June):199-202. doi:10.1038/sj.jim.7000169.
5. Zhu, L., Zhang, X., Ji, L., Song, X., & Kuang, C. (2007). Changes of lipid content and fatty acid composition of Schizochytrium limacinum in response to different temperatures and salinities. Process Biochemistry, 42(2), 210-214.
https://doi .org/10.1016/j .procbio.2006.08.002.
6. Buchhaupt, M., Kahne, F., Etschmann, M. M. W. & Schrader, J. Chapter 37 ¨
Biotechnological Production of Fatty Aldehydes. Flavour Science (Elsevier Inc., 2014).
doi :10.1016/B978-0-12-398549-1 .00037-4.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
7. Hamberg, M., Sanz, a & Castresana, C. a-Oxidation of fatty acids in higher plants. J.
Biol. Chem. 274, 24503 (1999).
8. Shine, W. E. & Stumpf, P. K. Fat Metabolism in Higher Plants Recent Studies on Plant a-Oxidati on Systems. Arch. Biochem. Biophys. 147-157 (1974).
9. Takahashi, T., Takahashi, H., Takeda, H. & Shichiri, M. Alpha-oxidation of fatty acids in fasted or diabetic rats. Diabetes Res. Clin. Pract. 16, 103-108 (1992).
10. Park YK, Dulermo T, Amaro RL, Nicaud JIM. Optimization of odd chain fatty acid production by Y arrowi a lipolyti c a. Biotechnol Biofuels. 2018; 11 (158):1 -12. doi :
10.1186/s13068-018-1154-4.

[00212] Although a particular feature of the disclosed techniques and systems may have been disclosed with respect to only one of several implementations, such feature may be combined with one or more other features of the other implementations as may be desired and advantageous for any given or particular application. Also, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in the detailed description and/or in the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[00213] This written description uses examples to disclose the claimed subject matter, including the best mode, and also to enable one of ordinary skill in the art to practice the claimed subject matter, including making and using any devices or systems and performing any incorporated methods. The patentable scope of the inventive concepts, described herein, are defined by the claims, and may include other examples that occur to those skilled in the art.
Such other examples are intended to be within the scope of the claims if they have structural elements that are not different from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

[00214] In the specification and claims, reference will be made to a number of terms that have the following meanings. The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Approximating language, as used herein throughout the specification and claims, may be applied to modify a quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related.
Accordingly, a value modified by a term such as "about" is not to be limited to the precise value Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA
specified. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. Moreover, unless specifically stated otherwise, a use of the terms "first," "second," etc., do not denote an order or importance, but rather the terms "first,"
"second," etc., are used to distinguish one element from another.

[00215] As used herein, the terms "may" and "may be" indicate a possibility of an occurrence within a set of circumstances; a possession of a specified property, characteristic or function;
and/or qualify another verb by expressing one or more of an ability, capability, or possibility associated with the qualified verb. Accordingly, usage of "may" and "may be"
indicates that a modified term is apparently appropriate, capable, or suitable for an indicated capacity, function, or usage, while taking into account that in some circumstances the modified term may sometimes not be appropriate, capable, or suitable. For example, in some circumstances an event or capacity can be expected, while in other circumstances the event or capacity cannot occur ¨ this distinction is captured by the terms "may" and "may be."

[00216] The best mode for carrying out the claimed subject matter has been described for purposes of illustrating the best mode known to the applicant at the time and enable one of ordinary skill in the art to practice the claimed subject matter, including making and using devices or systems and performing incorporated methods. The examples are illustrative only and not meant to limit the claimed subject matter, as measured by the scope and merit of the claims. The claimed subject matter has been described with reference to preferred and alternate embodiments.
Obviously, modifications and alterations will occur to others upon the reading and understanding of the specification. It is intended to include all such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof. The patentable scope of the inventive concepts, described herein, are defined by the claims, and may include other examples that occur to one of ordinary skill in the art. Such other examples are intended to be within the scope of the claims if they have structural elements that do not differentiate from the literal language of the claims, or if they include equivalent structural elements with insubstantial differences from the literal language of the claims.

Date Recue/Date Received 2020-06-23

Claims (52)

Attorney Docket No. 05001H0171221CA
What is claimed is:

1. A method for producing anaplerotic oil from microorganisms of the class Labyrinthuloycetes, the method comprising:
adding the microorganisms to a culture medium;
adding propionate to the culture medium resulting in cultured microorganisms;
and producing an anaplerotic oil from the cultured microorganisms, wherein at least five percent of the total fatty acids (TFA) of the anaplerotic oil are odd-chain fatty acids (OCFA), and OCFA make up at least one percent cell dry weight (CDW) of the microbial mass.

2. The method of claim 1, wherein adding propionate comprises adding the propionate to produce OCFAs in a range of 5% to 70% TFAs.

3. The method of claim 1 or 2, wherein the propionate is added with a carbon source at a weight to weight ratio below 0.1 of propionate to carbon source.

4. The method of claim 3, wherein the propionate is added with a carbon source at a weight to weight ratio below 0.05 of propionate to carbon source.

5. The method of any one of claims 1-4, wherein adding propionate comprises adding the propionate in a batch system into the culture medium

6. The method of any one of claims 1-5, wherein adding propionate comprises adding the propionate in a fed-batch system into the culture medium.

7. The method of claim 6, wherein the propionate is fed at a rate of greater than zero and up to 3 grams per liter per day.

8. The method of any one of claims 1-7, wherein the propionate is fed on demand using a pH-auxostat fed batch system to maintain a desired pH of the culture medium.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

9. The method of any one of claims 1-8, wherein the propionate is fed when the culture medium is at a pH above 5 to reduce propionic acid toxicity.

10. The method of any one of claims 1-9, wherein the propionate is added following a rise in the pH of the culture medium.

11. The method of any one of claims 1-10, wherein propionate is added at the lipid phase of the microorganisms in the culture medium to mitigate exposure to propionate toxicity of the microorganisms.

12. The method of any one of claims 1-11, wherein propionate is added at the end of the protein phase and start of lipogenesis of the microorganisms.

13. The method of any one of claims 1-12, wherein the microorganisms are oleaginous and can produce lipids to at least 20% dry cell weight.

14. The methods of any one of claims 1-13, wherein one or more of cobalt and cyanocobalamin is added to or subtracted from the culture medium to modify propionic acid deposition in OCFA.

15. The method of claim 14, wherein a concentration of the cyanocobalamin and/or cobalt in the culture medium is below 0.4 M.

16. The method of any one of claims 1-15, wherein at least 0.05 g of propionate is added per 1 gram of biomass produced.

17. The method of any one of claims 1-16, wherein the microorganisms are cultured in aerobic conditions by continuously supplying oxygen to the culture.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

18. The method of Claim 17 wherein culturing in aerobic conditions comprises continuously supplying oxygen to the culture to maintain a dissolved oxygen level of the culture between 10-15% saturation

19. The method of any one of claims 1-18, wherein the anaplerotic oil comprises saturated odd chain fatty acids (OCFAs) tridecanoic (C13:0), pentadecanoic (C15:0) and heptadecanoic acid (C17:0).

20. The method of any one of claims 1-19, wherein the microorganisms of the class Labyrinthuloycetes are thraustochytrid microalgae.

21. The method of claim 20, wherein the thraustochytrid microalgae are Aurantiochytrium sp.

22. The method of claim 21, wherein the thraustochytrid microalgae are Aurantiochytrium acetophilum HS399.

23. An anaplerotic oil obtainable from microorganisms of the class Labyrinthuloycetes grown in a culture medium comprising propionate, the anaplerotic oil comprising at least five percent of the total fatty acid (TFA) content as odd-chain fatty acids (OCFA).

24. The anaplerotic oil of claim 23, wherein pentadecanoic acid makes greater than eighteen percent of the TFA content.

25. The anaplerotic oil of claim 23 or 24, wherein heptadecanoic acid makes greater than five percent of the TFA content.

26. The anaplerotic oil of any one of claims 23-25, wherein docosahexaenoic acid (DHA) makes up greater than twenty percent of the TFA content.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

27. The anaplerotic oil of any one of claims 23-26, wherein OCFA comprise a range of 5 to 70% of the TFA content.

28. The anaplerotic oil of any one of claims 23-27, wherein the microorganisms of the class Labyrinthuloycetes are thraustochytrid microalgae.

29. The anaplerotic oil of claim 28, wherein the thraustochytrid microalgae are Aurantiochytrium sp.

30. The anaplerotic oil of claim 29, wherein the thraustochytrid microalgae are Aurantiochytrium acetophilum HS399.

31. A method for producing anaplerotic oil from microorganisms of the class Labyrinthuloycetes, the method comprising:
adding the microorganisms to a culture medium;
adding a propionate precursor to the culture medium resulting in cultured microorganisms comprising elevated levels of OCFA, at least a portion of the propionate precursor converting to propionate in the culture medium; and producing an anaplerotic oil from the cultured microorganisms, wherein at least five percent of the total fatty acids (TFA) of the anaplerotic oil are odd-chain fatty acids (OCFA), and OCFA make up at least one percent cell dry weight (CDW) of the microbial mass.

32. The method of claim 31, wherein adding a propionate precursor comprises adding one or more of: pentanoic acid; heptanoic acid; yeast extract; proteose peptone;
valine; and methionine, to induce OCFA production in the microorganism.

33. The method of claim 31 or 32, wherein adding the propionate precursor comprises adding the propionate precursor to produce OCFAs in a range of 5% to 70% TFAs.

34. The method of any one of claims 31-33, wherein the propionate precursor is added with a carbon source at a weight to weight ratio below 0.1 of propionate equivalent to carbon source.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

35. The method of claim 34, wherein the propionate precursor is added with a carbon source at a weight to weight ratio below 0.05 of propionate equivalent to carbon source.

36. The method of any one of claims 31-35, wherein adding the propionate precursor comprises adding the propionate precursor in a fed-batch into the culture medium.

37. The method of claim 36, wherein the propionate precursor is fed at a rate of greater than zero and up to 3 grams propionate equivalent per liter per day.

38. The method of any one of claims 31-37, wherein the propionate precursor is fed on demand using a pH- auxostat fed batch system to maintain a desired pH of the culture medium.

39. The method of any one of claims 31-38, wherein the propionate precursor is fed when the culture medium is at a pH above 5 to reduce propionic acid toxicity.

40. The method of any one of claims 31-39, wherein the propionate precursor is added following a rise in the pH of the culture medium.

41. The method of any one of claims 31-40, wherein propionate precursor is added at the lipid phase of the microorganisms in the culture medium to mitigate exposure to propionate toxicity of the microorganisms.

42. The method of any one of claims 31-41, wherein propionate precursor is added at the end of the protein phase and start of lipogenesis of the microorganisms.

43. The method of any one of claims 31-42, wherein the microorganisms are oleaginous and can produce lipids to at least 20% dry cell weight.

Date Recue/Date Received 2020-06-23 Attorney Docket No. 05001H0171221CA

44. The methods of any one of claims 31-43, wherein one or more of cobalt and cyanocobalamin is added to or subtracted from the culture medium to modify propionic acid deposition in OCFA.

45. The method of claim 44, wherein a concentration of the cyanocobalamin and/or cobalt in the culture medium is below 0.4 M.

46. The method of any one of claims 31-45, wherein at least 0.05 g propionate equivalent of propionate precursor is added per 1 gram of biomass produced.

47. The method of any one of claims 31-46, wherein the microorganisms are cultured in aerobic conditions by continuously supplying oxygen to the culture.

48. The method of Claim 47 wherein culturing in aerobic conditions comprises continuously supplying oxygen to the culture to maintain a dissolved oxygen level of the culture between 10-15% saturation

49. The method of any one of claims 31-48, wherein the anaplerotic oil comprises saturated odd chain fatty acids (OCFAs) tridecanoic (C13:0), pentadecanoic (C15:0) and heptadecanoic acid (C17:0).

50. The method of any one of claims 31-49, wherein the microorganisms of the class Labyrinthuloycetes are thraustochytrid microalgae.

51. The method of claim 50, wherein the thraustochytrid microalgae are Aurantiochytrium sp.

52. The method of claim 51, wherein the thraustochytrid microalgae are Aurantiochytrium acetophilum HS399.

Date Recue/Date Received 2020-06-23