Note: Descriptions are shown in the official language in which they were submitted.
Attorney Docket No. 05001H0171221CA
ANAPLEROTIC OIL PRODUCTION IN 1VHCROBIALS
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
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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.
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[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.
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[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.
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[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.
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[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.
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[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
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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
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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
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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
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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
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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
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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.
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[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
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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.
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[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,
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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
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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
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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)
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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
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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
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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
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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
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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
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[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
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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
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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
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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
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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,
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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
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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:
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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
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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
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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
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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
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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.
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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
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[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.
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[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
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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
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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.
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[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.
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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
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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
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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
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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.
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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.
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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
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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.
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[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 %.
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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
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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
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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
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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.
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[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)
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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
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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
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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
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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
C15
A = C2-C11 alkyl acid
= Ci-C12 COOMe R2 = H, C1-C10 COOMe
0 - CI
D) Stine Cross-Coupling
Pd(OAc)2, PdC12(MeCN)2 1) Cat. H2
or PdC12(PPh3)2
R2
A OR2 2) Hydrolysis
,
R1 1-
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
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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 R1
OH 0
0
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
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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
OH 0
OMe Base or Enzyme o
+ )=
0 r- OH
0 C14
OH 0y0
Glycerol
C14
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.
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[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,
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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
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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.
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[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.
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[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.
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[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).
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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
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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
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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
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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.
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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
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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.
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