FIELD OF THE INVENTION
The present invention is directed to a novel process for
growing microorganisms and recovering microbial lipids. In particular, the present
invention is directed to producing microbial polyunsaturated lipids.
BACKGROUND OF THE INVENTION
Production of polyenoic fatty acids (fatty acids containing
2 or more unsaturated carbon-carbon bonds) in eukaryotic microorganisms has been
generally believed to require the presence of molecular oxygen (i.e., aerobic
conditions). This is because it is believed that the cis double bond formed in the
fatty acids of all non-parasitic eukaryotic microorganisms involves a direct oxygen-dependent
desaturation reaction (oxidative microbial desaturase systems). Other eukaryotic
microbial lipids that are known to require molecular oxygen include fungal and plant
sterols, oxycarotenoids (i.e., xanthophylls), ubiquinones, and compounds made from
any of these lipids (i.e., secondary metabolites).
Certain eukaryotic microbes (such as algae; fungi, including
yeast; and protists) have been demonstrated to be good producers ofpolyenoic fatty
acids in fermentors. However, very high density cultivation (greater than about
100 g/L microbial biomass, especially at commercial scale) can lead to decreased
polyenoic fatty acid contents and hence decreased polyenoic fatty acid productivity.
This may be due in part to several factors including the difficulty of maintaining
high dissolved oxygen levels due to the high oxygen demand developed by the high
concentration of microbes in the fermentation broth. Methods to maintain higher
dissolved oxygen level include increasing the aeration rate and/or using pure oxygen
instead of air for aeration and/or increasing the agitation rate in the fermentor.
These solutions generally increase the cost of lipid production and capital cost
of fermentation equipment, and can cause additional problems. For example, increased
aeration can easily lead to severe foaming problems in the fermentor at high cell
densities and increased mixing can lead to microbial cell breakage due to increased
shear forces in the fermentation broth (this causes the lipids to be released in
the fermentation broth where they can become oxidized and/or degraded by enzymes).
Microbial cell breakage is an increased problem in cells that have undergone nitrogen
limitation or depletion to induce lipid formation, resulting in weaker cell walls.
As a result, when lipid-producing eukaryotic microbes are
grown at very high cell concentrations, their lipids generally contain only very
small amounts of polyenoic fatty acids. For example, the yeast Lipomyces starkeyi
has been grown to a density of 153 g/L with resulting lipid concentration of 83
g/L in 140 hours using alcohol as a carbon source. Yet the polyenoic fatty acid
content of the yeast at concentration greater than 100 g/L averaged only 4.2% of
total fatty acids (dropping from a high of 11.5% of total fatty acid at a cell density
of 20-30 g/L).
Yamauchi et al., J. Ferment. Technol., 1983, 61, 275-280
. This results in a polyenoic fatty acid concentration of only about 3.5
g/L and an average polyenoic fatty acid productivity of only about 0.025 g/L/hr.
Additionally, the only polyenoic fatty acid reported in the yeast lipids was C18:2.
Another yeast, Rhodotorula glutinus, has been demonstrated
to have an average lipid productivity of about 0.49 g/L/hr, but also a low overall
polyenoic fatty acid content in its lipids (15.8% of total fatty acids, 14.7% C18:2
and 1.2% C18:3) resulting in a polyenoic fatty acid productivity in fed-batch culture
of only about 0.047 g/L/hr and 0.077 g/L/hr in continuous culture.
One of the present inventors has previously demonstrated
that certain marine microalgae in the order Thraustochytriales can be excellent
producers of polyenoic fatty acids in fermentors, especially when grown at low salinity
levels and especially at very low chloride levels. Others have described Thraustochytrids
that exhibit an average polyenoic fatty acid (DHA, C22:6n-3; and DPA, C22:5n-6)
productivity of about 0.158 g/L/hr, when grown to cell density of 59 g/L in 120
hours. However, this productivity was only achieved at a salinity of about 50% seawater,
a concentration that would cause serious corrosion in conventional stainless steel
Costs of producing microbial lipids containing polyenoic
fatty acids, and especially the highly unsaturated fatty acids, such as C18:4n-3,
C20:4n-6, C20:5n3, C22:5n-3, C22:5n-6 and C22:6n-3, have remained high in part due
to the limited densities to which the high polyenoic fatty acid containing eukaryotic
microbes have been grown and the limited oxygen availability both at these high
cell concentrations and the higher temperatures needed to achieve high productivity.
Therefore, there is a need for a process for growing microorganisms
at high concentration which still facilitates increased production of lipids containing
polyenoic fatty acids.
SUMMARY OF THE INVENTION
The present invention provides a process for growing eukaryotic
microorganisms that are capable of producing at least about 20% of their biomass
as lipids and a method for producing the lipids. Preferably the lipids contain one
or more polyenoic fatty acids. The process comprises adding to a fermentation medium
comprising eukaryotic microorganisms a carbon source, preferably a non-alcohol carbon
source, and a limiting nutrient source. Preferably, the carbon source and the limiting
nutrient source are added at a rate sufficient to increase the biomass density of
the fermentation medium to at least about 100 g/L.
In one aspect of the present invention, the fermentation
condition comprises a biomass density increasing stage and a lipid production stage,
wherein the biomass density increasing stage comprises adding the carbon source
and the limiting nutrient source, and the lipid production stage comprises adding
the carbon source without adding the limiting nutrient source to create conditions
which induce lipid production.
In another aspect of the present invention, the amount
of dissolved oxygen present in the fermentation medium during the lipid production
stage is lower than the amount of dissolved oxygen present in the fermentation medium
during the biomass density increasing stage.
In yet another aspect of the present invention, microorganisms
are selected from the group consisting of algae, fungi (including yeasts), protists,
bacteria, and mixtures thereof, wherein the microorganisms are capable of producing
polyenoic fatty acids or other lipids that had been generally believed to require
molecular oxygen for their synthesis. Particularly useful microorganisms of the
present invention are eukaryotic microorganisms that are capable of producing lipids
at a fermentation medium oxygen level of about less than 3% of saturation.
In still another aspect of the present invention, microorganisms
are grown in a fed-batch process.
Yet still another aspect of the present invention provides
maintaining an oxygen level of less than about 3% of saturation in the fermentation
medium during the second half of the fermentation process.
Another embodiment of the present invention provides a
process for producing eukaryotic microbial lipids comprising:
wherein greater than about 15% of said lipids are polyunsaturated lipids.
- (a) growing eukaryotic microorganisms in a fermentation medium to increase the
biomass density of said fermentation medium to at least about 100 g/L;
- (b) providing fermentation conditions sufficient to allow said microorganisms
to produce said lipids; and
- (c) recovering said lipids,
Another aspect of the present invention provides a lipid
recovery process that comprises:
- (d) removing water from said fermentation medium to provide dry microorganisms;
- (e) isolating said lipids from said dry microorganisms.
Preferably, the water removal step comprises contacting
the fermentation medium directly on a drum-dryer without prior centrifugation.
Another aspect of the present invention provides a lipid
recovery process that comprises:
- (d) treating the fermentation broth to permeabilize, lyse or rupture the microbial
- (e) recovering the lipids from the fermentation broth by gravity separation,
and preferably centrifugation, with or without the aid of a water-soluble solvent
to aid in breaking the lipid/water emulsion.
Preferably, the microbial cells are treated in step (c)
in a fermentor or a similar vessel.
In a further aspect of the present invention, a method
for enriching the polyenoic fatty acid content of a microorganism is provided. The
method includes fermenting the microorganisms in a growth medium having a level
of dissolved oxygen of less than 10%.
A further aspect of the invention is a heterotrophic process
for producing products and microorganisms. The process includes culturing the microorganisms
containing polyketide synthase genes in a growth medium and maintaining the level
of dissolved oxygen in the culture at less than about 10 percent.
BRIEF DESCRIPTION OF THE DRAWINGS
DETAILED DESCRIPTION OF THE INVENTION
- Figure 1 is a table and a plot of various lipid production parameters of a microorganism
versus the amount of dissolved oxygen in a fermentation medium.
The present invention provides a process for growing microorganisms,
such as, for example, algae, fungi (including yeast), protists, and bacteria. Preferably,
microorganisms are selected from the group consisting of algae, protists and mixtures
thereof. More preferably, microorganisms are algae. Moreover, the process of the
present invention can be used to produce a variety of lipid compounds, in particular
unsaturated lipids, preferably polyunsaturated lipids (i.e., lipids containing
at least 2 unsaturated carbon-carbon bonds, e.g., double bonds), and
more preferably highly unsaturated lipids (i.e., lipids containing 4 or more
unsaturated carbon-carbon bonds) such as omega-3 and/or omega-6 polyunsaturated
fatty acids, including docosahexaenoic acid (i.e., DHA); and other naturally
occurring unsaturated, polyunsaturated and highly unsaturated compounds. As used
herein, the term "lipid" includes phospholipids; free fatty acids; esters of fatty
acids; triacylglycerols; sterols and sterol esters; carotenoids; xanthophylls (e.g.,
oxycarotenoids); hydrocarbons; isoprenoid-derived compounds and other lipids known
to one of ordinary skill in the art.
More particularly, processes of the present invention are
useful in producing eukaryotic microbial polyenoic fatty acids, carotenoids, fungal
sterols, phytosterols, xanthophylls, ubiquinones, and other isoprenoid-derived compounds
which had been generally believed to require oxygen for producing unsaturated carbon-carbon
bonds (i.e., aerobic conditions), and secondary metabolites thereof. Specifically,
processes of the present invention are useful in growing microorganisms that produce
polyenoic fatty acid(s), and for producing microbial polyenoic fatty acid(s).
While processes of the present invention can be used to
grow a wide variety of microorganisms and to obtain polyunsaturated lipid containing
compounds produced by the same, for the sake of brevity, convenience and illustration,
this detailed description of the invention will discuss processes for growing microorganisms
which are capable of producing lipids comprising omega-3 and/or omega-6 polyunsaturated
fatty acids, in particular microorganisms that are capable of producing DHA (or
closely related compounds such as DPA, EPA or ARA). Preferred microorganisms include
microalgae, fungi (including yeast), protists and bacteria. One group of preferred
microorganisms is the members of the microbial group called Stramenopiles which
includes microalgae and algae-like microorganisms. The Stramenopiles include the
following groups of microorganisms: Hamatores, Proteromonads, Opalines, Developayella,
Diplophrys, Labrinthulids, Thraustochytrids, Biosecids, Oomycetes, Hypochytridiomycetes,
Commation, Reticulosphaera, Pelagomonas, Pelagococcus, Ollicola, Aureococcus, Parmales,
Diatoms, Xanthophytes, Phaeophytes (brown algae), Eustigmatophytes, Raphidophytes,
Synurids, Axodines (including Rhizochromulinaales, Pedinellales, Dictyochales),
Chrysomeridales, Sarcinochrysidales, Hydrurales, Hibberdiales, and Chromulinales.
Other preferred groups of microalgae include the members of the green algae and
dinoflagellates, including members of the genus Crypthecodium. More particularly,
preferred embodiments of the present invention will be discussed with reference
to a process for growing marine microorganisms, in particular algae, such as Thraustochytrids
of the order Thraustochytriales, more specifically Thraustochytriales of the genus
Thraustochytrium and Schizochytrium, including Thraustochytriales
which are disclosed in commonly assigned
U.S. Patent Nos. 5,340,594
5,340,742, both issued to Barclay
, all of which are incorporated herein by reference in their entirety.
It should be noted that many experts agree that Ulkenia is not a separate
genus, but is in fact part of the genus Schizochytrium. As used herein, the
genus Schizochytrium will include Ulkenia.
Preferred microorganisms are those that produce the compounds
of interest via polyketide synthase systems. Such microorganisms include microorganisms
having an endogenous polyketide synthase system and microorganisms into which a
polyketide synthase system has been genetically engineered. Polyketides are structurally
diverse natural products that have a wide range of biological activities, including
antibiotic and pharmacological properties. Biosynthesis of the carbon chain backbone
of polyketides is catalyzed by polyketide synthases. Like the structurally and mechanistically
related fatty acid synthases, polyketide synthases catalyze the repeated decarboxylative
condensations between acyl thioesters that extend the carbon chain two carbons at
a time. However, unlike fatty acid synthases, polyketide synthases can generate
great structural variability in the end product. Individual polyketide synthase
systems can do this by using starting units other than acetate, by utilizing methyl-
or ethyl-malonate as the extending unit and by varying the reductive cycle of ketoreduction,
dehydration and enoyl reduction on the beta-keto group formed after each condensation.
Of particular interest here is that the carbon-carbon double bonds that are introduced
by the dehydration step can be retained in the end product. Further, although these
double bonds are initially in the trans configuration, they can be converted
to the cis configuration found in DHA (and other polyenoic fatty acids of
interest) by enzymatic isomerization. Both the dehydrase and isomerization reactions
can occur in the absence of molecular oxygen.
Preferably, in accordance with the present invention a
heterotrophic process is provided for producing products and microorganisms. The
process preferably comprises culturing the microorganisms in a growth medium wherein
the microorganisms contain a polyketide synthase system. Preferably, the level of
dissolved oxygen is maintained at less than about 8 percent, more preferably at
less than about 4 percent, more preferably at less than about 3 percent, and more
preferably at less than about 1 percent.
It is to be understood, however, that the invention as
a whole is not intended to be so limited, and that one skilled in the art will recognize
that the concept of the present invention will be applicable to other microorganisms
producing a variety of other compounds, including other lipid compositions, in accordance
with the techniques discussed herein.
Assuming a relatively constant production rate of lipids
by an algae, it is readily apparent that the higher biomass density will lead to
a higher total amount of lipids being produced per volume. Current conventional
fermentation processes for growing algae yield a biomass density of from about 50
to about 80 g/L or less. The present inventors have found that by using processes
of the present invention, a significantly higher biomass density than currently
known biomass density can be achieved. Preferably, processes of the present invention
produces biomass density of at least about 100 g/L, more preferably at least about
130 g/L, still more preferably at least about 150 g/L, yet still more preferably
at least about 170 g/L, and most preferably greater than 200 g/L. Thus, with such
a high biomass density, even if the lipids production rate of algae is decreased
slightly, the overall lipids production rate per volume is significantly higher
than currently known processes.
Processes of the present invention for growing microorganisms
of the order Thraustochytriales include adding a source of carbon and a source of
a limiting nutrient to a fermentation medium comprising the microorganisms at a
rate sufficient to increase the biomass density of the fermentation medium to those
described above. As used herein, the term "limiting nutrient source" refers to a
source of a nutrient (including the nutrient itself) essential for the growth of
a microorganism in that, when the limiting nutrient is depleted from the growth
medium, its absence substantially limits the microorganism from growing or replicating
further. However, since the other nutrients are still in abundance, the organism
can continue to make and accumulate intracellular and/or extracellular products.
By choosing s specific limiting nutrient, one can control the type of products that
are accumulated. Therefore, providing a limiting nutrient source at a certain rate
allows one to control both the rate of growth of the microorganism and the production
or accumulation of desired products (e.g., lipids). This fermentation process, where
one or more substrates (e.g., a carbon source and a limiting nutrient
source) are added in increments, is generally referred to as a fed-batch fermentation
process. It has been found that when the substrate is added to a batch fermentation
process the large amount of carbon source present (e.g., about 200 g/L or
more per 60 g/L of biomass density) had a detrimental effect on the microorganisms.
Without being bound by any theory, it is believed that such a high amount of carbon
source causes detrimental effects, including osmotic stress, for microorganisms
and inhibits initial productivity of microorganisms. Processes of the present invention
avoid this undesired detrimental effect while providing a sufficient amount of the
substrate to achieve the above-described biomass density of the microorganisms.
Processes of the present invention for growing microorganisms
can include a biomass density increasing stage. In the biomass density increasing
stage, the primary objective of the fermentation process is to increase the biomass
density in the fermentation medium to obtain the biomass density described above.
The rate of carbon source addition is typically maintained at a particular level
or range that does not cause a significant detrimental effect on productivity of
microorganisms, or the viability of the microorganisms resulting from insufficient
capabilities of the fermentation equipment to remove heat from and transfer gases
to and from the liquid broth An appropriate range of the amount of carbon source
needed for a particular microorganism during a fermentation process is well known
to one of ordinary skill in the art. Preferably, a carbon source of the present
invention is a non-alcohol carbon source, i.e., a carbon source that does
not contain alcohol. As used herein, an "alcohol" refers to a compound having 4
or less carbon atoms with one hydroxy group, e.g., methanol, ethanol and
isopropanolbut for the purpose of this invention does not include hydroxy organic
acids such as lactic acid and similar compounds. More preferably, a carbon source
of the present invention is a carbohydrate, including, but not limited to, fructose,
glucose, sucrose, molasses, and starch. Other suitable simple and complex carbon
sources and nitrogen sources are disclosed in the above-referenced patents. Typically,
however, a carbohydrate, preferably corn syrup, is used as the primary carbon source.
Fatty acids, in the form of hydroxy fatty acids, triglycerides, and di- and monoglycerides
can also serve as the carbon source
Particularly preferred nitrogen sources are urea, nitrate,
nitrite, soy protein, amino acids, protein, corn steep liquor, yeast extract, animal
by-products, inorganic ammonium salt, more preferably ammonium salts of sulfate,
hydroxide, and most preferably ammonium hydroxide. Other limiting nutrient sources
include carbon sources (as defined above), phosphate sources, vitamin sources (such
as vitamin B12 sources, pantothenate sources, thiamine sources), and
trace metal sources (such as zinc sources, copper sources, cobalt sources, nickel
sources, iron sources, manganese sources, molybdenum sources), and major metal sources
(such as magnesium sources, calcium sources,, sodium sources, potassium sources,
and silica sources, etc.). Trace metal sources and major metal sources can include
sulfate and chloride salts of these metals (for example but not limited to MgSO4•7H2O;
FeSO4•7H2O; CaCl2; K2SO4;
KCl; and Na2SO4).
When ammonium is used as a nitrogen source, the fermentation
medium becomes acidic if it is not controlled by base addition or buffers. When
ammonium hydroxide is used as the primary nitrogen source, it can also be used to
provide a pH control. The microorganisms of the order Thraustochytriales, in particular
Thraustochytriales of the genus Thraustochytrium and Schizochytrium,
will grow over a wide pH range, e.g., from about pH 5 to about pH
11. A proper pH range for fermentation of a particular microorganism is within the
knowledge of one skilled in the art.
Processes of the present invention for growing microorganisms
can also include a production stage. In this stage, the primary use of the substrate
by the microorganisms is not increasing the biomass density but rather using the
substrate to produce lipids. It should be appreciated that lipids are also produced
by the microorganisms during the biomass density increasing stage; however, as stated
above, the primary goal in the biomass density increasing stage is to increase the
biomass density. Typically, during the production stage the addition of the limiting
nutrient substrate is reduced or preferably stopped.
It was previously generally believed that the presence
of dissolved oxygen in the fermentation medium is crucial in the production of polyunsaturated
compounds, including omega-3 and/or omega-6 polyunsaturated fatty acids, by eukaryotic
microorganisms. Thus, a relatively large amount of dissolved oxygen in the fermentation
medium was generally believed to be preferred. Surprisingly and unexpectedly, however,
the present inventors have found that the production rate of lipids is increased
dramatically when the dissolved oxygen level during the production stage is reduced.
Thus, while the dissolved oxygen level in the fermentation medium during the biomass
density increasing stage is preferably at least about 8% of saturation, and preferably
at least about 4% of saturation, during the production stage the dissolved oxygen
in the fermentation medium is reduced to about 3% of saturation or less, preferably
about 1% of saturation or less, and more preferably about 0% of saturation. At the
beginning of the fermentation the DO can be at or near saturation and as the microbes
grow it is allowed to drift down to these low DO setpoints. In one particular embodiment
of the present invention, the amount of dissolved oxygen level in the fermentation
medium is varied during the fermentation process. For example, for a fermentation
process with total fermentation time of from about 90 hours to about 100 hours,
the dissolved oxygen level in the fermentation medium is maintained at about 8%
during the first 24 hours, about 4% from about 24th hour to about 40th
hour, and about 0.5% or less from about 40th hour to the end of the fermentation
The amount of dissolved oxygen present in the fermentation
medium can be controlled by controlling the amount of oxygen in the head-space of
the fermentor, or preferably by controlling the speed at which the fermentation
medium is agitated (or stirred). For example, a high agitation (or stirring) rate
results in a relatively higher amount of dissolved oxygen in the fermentation medium
than a low agitation rate. For example, in a fermentor of about 14,000 gallon capacity
the agitation rate is set at from about 50 rpm to about 70 rpm during the first
12 hours, from about 55 rpm to about 80 rpm during about 12th hour to
about 18th hour and from about 70 rpm to about 90 rpm from about 18th
hour to the end of the fermentation process to achieve the dissolved oxygen level
discussed above for a total fermentation process time of from about 90 hours to
about 100 hours. A particular range of agitation speeds needed to achieve a particular
amount of dissolved oxygen in the fermentation medium can be readily determined
by one of ordinary skill in the art.
A preferred temperature for processes of the present invention
is at least about 20°C, more preferably at least about 25°C, and most
preferably at least about 30°C. It should be appreciated that cold water can
retain a higher amount of dissolved oxygen than warm water. Thus, a higher fermentation
medium temperature has the additional benefit of reducing the amount of dissolved
oxygen, which is particularly desired as described above.
Certain microorganisms may require a certain amount of
saline minerals in the fermentation medium. These saline minerals, especially chloride
ions, can cause corrosion of the fermentor and other downstream processing equipment.
To prevent or reduce these undesired effects due to a relatively large amount of
chloride ions present in the fermentation medium, processes of the present invention
can also include using non-chloride containing sodium salts, preferably sodium sulfate,
in the fermentation medium as a source of sodium. More particularly, a significant
portion of the sodium requirements of the fermentation is supplied as non-chloride
containing sodium salts. For example, less than about 75% of the sodium in the fermentation
medium is supplied as sodium chloride, more preferably less than about 50% and more
preferably less than about 25%. The microorganisms of the present invention can
be grown at chloride concentrations of less than about 3 g/L, more preferably less
than about 500 mg/L, more preferably less than about 250 mg/L and more preferably
between about 60 mg/L and about 120 mg/L.
Non-chloride containing sodium salts can include soda ash
(a mixture of sodium carbonate and sodium oxide), sodium carbonate, sodium bicarbonate,
sodium sulfate and mixtures thereof, and preferably include sodium sulfate. Soda
ash, sodium carbonate and sodium bicarbonate tend to increase the pH of the fermentation
medium, thus requiring control steps to maintain the proper pH of the medium. The
concentration of sodium sulfate is effective to meet the salinity requirements of
the microorganisms, preferably the sodium concentration is (expressed as g/L of
Na) at least about 1 g/L, more preferably in the range of from about 1 g/L to about
50 g/L and more preferably in the range of from about 2 g/L to about 25 g/L.
Various fermentation parameters for inoculating, growing
and recovering microorganisms are discussed in detail in
U.S. Patent No. 5,130,242
, which is incorporated herein by reference in its entirety. Any currently
known isolation methods can be used to isolate microorganisms from the fermentation
medium, including centrifugation, filtration, ultrafiltration, decantation, and
solvent evaporation. It has been found by the present inventors that because of
such a high biomass density resulting from processes of the present invention, when
a centrifuge is used to recover the microorganisms it is preferred to dilute the
fermentation medium by adding water, which reduces the biomass density, thereby
allowing more effective separation of microorganisms from the fermentation medium.
The very high biomass densities achieved in the present
invention also facilitate "solventless" processes for recovery of microbial lipids.
Preferred processes for lysing the cells in the fermentor are described in
U.S. Provisional Patent Application Serial No. 60/177,125
entitled "SOLVENTLESS EXTRACTION PROCESS" filed January 19, 2000, U.S.
Patent Application Serial No.
entitled "SOLVENTLESS EXTRACTION PROCESS" filed January 19, 2001, and PCT Patent
Application Serial No.
entitled "SOLVENTLESS EXTRACTION PROCESS" filed January 19,2001, which are incorporated
herein by reference in their entirety. Preferred processes for recovering the lipids
once the cells are permeabilized, broken or lysed in the fermentor (which enables
the lipid emulsion to be broken, and the lipid-rich fraction to be recovered) include
the deoiling process outlined in
which is incorporated herein by reference in its entirety. In this process
a water soluble compound, e.g., alcohol or acetone, is added to the oil/water emulsion
to break the emulsion and the resulting mixture is separated by gravity separation,
e.g., centrifugation. This process can also be modified to use other agents (water
and/or lipid soluble) to break the emulsion.
Alternatively, the microorganisms are recovered in a dry
form from the fermentation medium by evaporating water from the fermentation medium,
for example, by contacting the fermentation medium directly (i.e.,
without pre-concentration, for example, by centrifugation) with a dryer such as
a drum-dryer apparatus, i.e., a direct drum-dryer recovery process. When
using the direct drum-dryer recovery process to isolate microorganisms, typically
a steam-heated drum-dryer is employed. In addition when using the direct drum-dryer
recovery process, the biomass density of the fermentation medium is preferably at
least about 130 g/L, more preferably at least about 150 g/L, and most preferably
at least about 180 g/L. This high biomass density is generally desired for the direct
drum-dryer recovery process because at a lower biomass density, the fermentation
medium comprises a sufficient amount of water to cool the drum significantly, thus
resulting in incomplete drying of microorganisms. Other methods of drying cells,
including spray drying, are well known to one of ordinary skill in the art.
Processes of the present invention provide an average lipid
production rate of at least about 0.5 g/L/hr, preferably at least about 0.7 g/L/hr,
more preferably at least about 0.9 g/L/hr, and most preferably at least about 1.0
g/L/hr. Moreover, lipids produced by processes of the present invention contain
polyunsaturated lipids in the amount greater than about 15%, preferably greater
than about 20%, more preferably greater than about 25%, still more preferably greater
than about 30%, and most preferably greater than about 35%. Lipids can be recovered
from either dried microorganisms or from the microorganisms in the fermentation
medium. Generally, at least about 20% of the lipids produced by the microorganisms
in the processes of the present invention are omega-3 and/or omega-6 polyunsaturated
fatty acids, preferably at least about 30% of the lipids are omega-3 and/or omega-6
polyunsaturated fatty acids, more preferably at least about 40% of the lipids are
omega-3 and/or omega-6 polyunsaturated fatty acids, and most preferably at least
about 50% of the lipids are omega-3 and/or omega-6 polyunsaturated fatty acids.
Alternatively, processes of the present invention provides an average omega-3 fatty
acid (e.g.. DHA) production rate of at least about 0.2 g of omega-3 fatty acid (e.g.,
DHA)/L/hr, preferably at least about 0.3 g of omega-3 fatty acid (e.g., DHA)/L/hr,
more preferably at least about 0.4 g of omega-3 fatty acid (e.g., DHA)/L/hr, and
most preferably at least about 0.5 g of omega-3 fatty acid (e.g., DHA)/L/hr. Alternatively,
processes of the present invention provide an average omega-6 fatty acid (e.g.,
DPAn-6) production rate of at least about 0.07 g of omega-6 fatty acid (e.g., DPAn-6)/L/hr,
preferably at least about 0.1 g of omega-6 fatty acid (e.g., DPAn-6)/L/hr, more
preferably at least about 0.13 g of omega-6 fatty acid (e.g., DPAn-6)/L/hr, and
most preferably at least about 0.17 g of omega-6 fatty acid (e.g., DPAn-6)/L/hr.
Still alternatively, at least about 25% of the lipid is DHA (based on total fatty
acid methyl ester), preferably at least about 30%, more preferably at least about
35%, and most preferably at least about 40%.
Microorganisms, lipids extracted therefrom, the biomass
remaining after lipid extraction or combinations thereof can be used directly as
a food ingredient, such as an ingredient in beverages, sauces, dairy based foods
(such as milk, yogurt, cheese and ice-cream) and baked goods; nutritional supplement
(in capsule or tablet forms); feed or feed supplement for any animal whose meat
or products are consumed by humans; food supplement, including baby food and infant
formula; and pharmaceuticals (in direct or adjunct therapy application). The term
"animal" means any organism belonging to the kingdom Animalia and includes, without
limitation, any animal from which poultry meat, seafood, beef, pork or lamb is derived.
Seafood is derived from, without limitation, fish, shrimp and shellfish. The term
"products" includes any product other than meat derived from such animals, including,
without limitation, eggs, milk or other products. When fed to such animals, polyunsaturated
lipids can be incorporated into the flesh, milk, eggs or other products of such
animals to increase their content of these lipids.
Additional objects, advantages, and novel features of this
invention will become apparent to those skilled in the art upon examination of the
following examples thereof, which are not intended to be limiting.
The strain of Schizochytrium used in these examples
produces two primary polyenoic acids, DHAn-3 and DPAn-6 in the ratio of generally
about 3:1, and small amounts of other polyenoic acids, such as EPA and C20:3, under
a wide variety of fermentation conditions. Thus, while the following examples only
list the amount of DHA, one can readily calculate the amount of DPA(n-6) produced
by using the above-disclosed ratio.
This example illustrates the effect of oxygen content in
a fermentation medium on lipid productivity.
Fermentation results of Schizochytrium ATCC No.
20888 at various levels of dissolved oxygen content were measured. The results are
shown in Figure 1, where RCS is residual concentration of sugar, and DCW is dry-cell
This example also illustrates the effect of low oxygen
content in the fermentation medium on DHA content (% dry weight) of the final biomass
A "scale-down" type experiment was conducted in 250 mL
Erlenmeyer flasks to mimic the effect of low oxygen content on the DHA content in
Schizochytrium sp. cells cultured in large-scale fermentors. Schizochytrium
sp (ATCC 20888) was cultured in 04-4 medium. This culture media consisted of the
following on a per liter basis dissolved in deionized water: Na2SO4
12.61 g; MgSO4•7H2O 1.2g; KCl 0.25 g; CaCl2
0.05 g; monosodium glutamate 7.0 g; glucose 10 g; KH2PO4 0.5
g; NaHCO3 0.1 g; yeast extract 0.1 g; vitamin mix 1.0 mL; PII metals
1.00 mL. PII metal mix contains (per liter): 6.0 g Na2EDTA, 0.29 g FeCl3•6H2O,
6.84 g H3BO3, 0.86 g MnCl2•4H2O,
0.06 g ZnCl2, 0.026 g CoCl2•6H2O, 0.052
g NiSO4•H2O, 0.002 CuSO4•H2O
and 0.005 g Na2MoO4•2H2O. Vitamin mix contains
(per liter): 100 mg thiamine, 0.5 mg biotin and 0.5 mg cyanocobalamin. The pH of
the culture media was adjusted to 7.0 and it was then filter sterilized.
The idea behind this scale-down experiment was to culture
the cells in shake flasks with different volumes of culture media in the flasks
- almost full flasks (e.g. 200 mL in a 250 mL shake flask) would not mix well on
a shake table and therefore as the cells grew, low dissolved oxygen conditions would
be generated. Therefore 4 treatments were established in the experiment, each conducted
in duplicate: (1) 250 mL flasks filled with 50 mL culture medium; (2) 250 mL flasks
filled with 100 mL culture medium; (3) 250 mL flasks filled with 150 mL culture
medium; and (4) 250 mL shake flasks filled with 200 mL culture medium. Each of the
eight flasks was inoculated with cells from a 48 hour old culture of Schizochytrium
cultured in 04-4 medium under the conditions in treatment 1, and at 28°C and
220 rpm on a shaker table. All eight flasks for the experiment were placed on a
shaker table (220 rpm) in a incubator (28°C) and cultured for 48 hours in the
dark. At the end of the experiment, dissolved oxygen (DO) levels in each flask were
measured with a YSI dissolved oxygen meter, pH of the culture medium was also determined,
and the dry weight of cells and their fatty acid content was also measured. The
results of the experiment are outlined in Table 1.
Table 1. Results of scale-down experiment examining effect of low dissolved
oxygen concentrations on the long chain highly unsaturated fatty acid content (DHA
% dry weight) of Schizochytrium sp.
DHA (% dry wt)
DO (% sat)
The results indicate that the lipid content (as % FAME)
and DHA content (% dry weight) were higher for cells cultured at low dissolved oxygen
levels - the lower the dissolved oxygen level the higher the lipid and DHA content.
This is unexpected because oxygen had been generally believed to be necessary to
form desaturated (double) bonds. It is surprising that so much DHA was formed at
low dissolved oxygen level, because DHA is one of the most unsaturated fatty acids.
Although the biomass production decreases as the dissolved oxygen level is decreased,
the DHA content is increased. Therefore, it is advantageous to have a growth phase
with higher dissolved oxygen levels to maximize the formation of biomass and then
lower the dissolved oxygen level to maximize long chain fatty acid production.
This example illustrates the reproducibility of processes
of the present invention.
Microorganisms were produced using fermentors with a nominal
working volume of 1,200 gallons. The resulting fermentation broth was concentrated
and microorganisms were dried using a drum-dryer. Lipids from aliquots of the resulting
microorganisms were extracted and purified to produce a refined, bleached, and deodorized
oil. Approximately 3,000 ppm of d-1-&agr;-tocopheryl acetate was added for nutritional
supplementation purposes prior to analysis of the lipid.
Nine fermentations of Schizochytrium ATCC No. 20888
were run and the results are shown in Table 2. The dissolved oxygen level was about
8% during the first 24 hours and about 4% thereafter.
Table 2. Fed-batch fermentation results for the production of DHA from
1. actual yield
of biomass density.
2. DHA content
as % cell dry weight.
3. total fatty
acid content as % cell dry weight (measured as methyl esters).
4. (grams of DHA)/L/Hr.
6. standard deviation
of variability. Coefficients of variability values below 5% indicate a process which
has excellent reproducibility, values between 5% and 10% indicate a process which
has good reproducibility and values between 10% and 20% indicate a process which
has reasonable reproducibility.
Corn syrup was fed until the volume in the fermentor reached
about 1,200 gallons, at which time the corn syrup addition was stopped. The fermentation
process was stopped once the residual sugar concentration fell below 5 g/L. The
typical age, from inoculation to final, was about 100 hours.
The fermentation broth, i.e., fermentation medium,
was diluted with water using approximately a 2:1 ratio to reduce the ash content
of the final product and help improve phase separation during the centrifugation
step. The concentrated cell paste was heated to 160° F (about 71° C) and
dried on a Blaw Knox double-drum dryer (42"x36"). Preferably, however, microorganisms
are dried directly on a drum-dryer without prior centrifugation.
The analysis result of lipids extracted from aliquots of
each entries in Table 2 is summarized in Table 3.
Table 3. Analysis of the microbial biomass produced in the fed-batch fermentations
outlined in Table. 2.
% DHA relative to
Total Lipid % by
1. see Table 2.
2. see discussion
3. standard deviation.
of variability. Coefficients of variability values below 5% indicates a process
which has excellent reproducibility, values between 5% and 10% indicates a process
which has good reproducibility and values between 10% and 20% indicates a process
which has reasonable reproducibility.
Unless otherwise stated, the fermentation medium used throughout
the Examples section includes the following ingredients, where the first number
indicates nominal target concentration and the number in parenthesis indicates acceptable
range: sodium sulfate 12 g/L (11-13); KCl 0.5 g/L (0.45-0.55); MgSO4•7H2O
2 g/L (1.8-2.2); Hodag K-60 antifoam 0.35 g/L (0.3-0.4); K2SO4
0.65 g/L (0.60-0.70); KH2PO4 1 g/L (0.9-1.1); (NH4)2SO4
1 g/L (0.95-1.1); CaCl2•2H2O 0.17 g/L (0.15-0.19);
95 DE corn syrup (solids basis) 4.5 g/L (2-10); MnCl2•4H2O
3 mg/L (2.7-3.3); ZnSO4•7H2O 3 mg/L (2.7-3.3); CoCl2•6H2O
0.04 mg/L (0.035-0.045); Na2MoO4•2H2O 0.04
mg/L (0-0.045); CuSO4•5H2O 2 mg/L (1.8-2.2); NiSO4•6H2O
2 mg/L (1.8-2.2); FeSO4•7H2O 10 mg/L (9-11); thiamine
9.5 mg/L (4-15); vitamin B12 0.15 mg/L (0.05-0.25) and Calcium Pantothenate
3.2 mg/L (1.3-5.1). In addition, 28% NH4OH solution is used as the nitrogen
The ash content of the dried microorganisms is about 6%
This example illustrates the effect of reduced dissolved
oxygen level in the fermentation medium on the productivity of microorganisms at
the 14,000-gallon scale.
Using the procedure described in Example 3, a 14,000-gallon
nominal volume fermentation was conducted using a wild-type strain Schizochytrium,
which can be obtained using isolation processes disclosed in the above-mentioned
U.S. Patent Nos. 5,340,594
. The dissolved oxygen level in the fermentation medium was about 8% during
the first 24 hours, about 4% from the 24th hour to the 40th
hour and about 0.5% from the 40th hour to the end of fermentation process.
Results of this lower dissolved oxygen level in fermentation medium processes are
shown in Table 4.
Table 4. Results of 14,000-gallon scale fed-batch fermentations of
Schizochytrium at reduced dissolved oxygen concentrations.
%DHA rel. to FAME
(g of DHA/L/hr)
This example illustrates the effect of reduced dissolved
oxygen level in the fermentation medium on the productivity of microorganisms on
a 41,000-gallon scale.
The same procedures as Example 4 were employed except that
the fermentation was conducted in a 41,000-gallon fermentor. Culture media volumes
were increased to maintain target compound concentrations at this scale. Results
are shown in Table 5.
Table 5. 41,000-gallon scale fermentation of Schizochytrium
%DHA rel. to FAME
DHA Productivity (g
of DHA/ L/hr)
This example illustrates the affect of extra nitrogen on
the fermentation process of the present invention.
Four sets of 250-L scale fed-batch experiments were conducted
using a procedure similar to Example 4. Two control experiments and two experiments
containing extra ammonia (1.15x and 1.25x the normal amount) were conducted. Results
are shown in Table 6.
Table 6. Affects of extra ammonia on fermentation of Schizochytrium.
In general, extra nitrogen has a negative effect on fermentation performance, as
significant reductions were observed in the DHA productivity for the two batches
where extra ammonia was added. As shown on Table 6, the control batches resulted
in final DHA levels of 18.4% and 22.1 % of total cellular dry weight versus the
9.2% (1.15x ammonia) and 12.6% (1.25x ammonia) for extra nitrogen supplemented batches.
target: 7 g/L, Base pH set point: 5.5, Acid pH set point: 7.3, 1.0X NH3
target: 7 g/L, Base pH set point: 5.5, Acid pH set point: 7.3, 1.15X NH3
target: 7 g/L, Base pH set point: 5.5, Acid pH set point: 7.3, 1.25X NH3
target: 7 g/L, Base pH set point: 5.5, Acid pH set point: 7.3, 1.0X NH3
This example shows a kinetic profile of a fermentation
process of the present invention.
A 1000-gallon scale fed-batch experiment was conducted
using a procedure similar to Example 4. Kinetic profile of the fermentation process
is shown in Table 7.
Table 7. Kinetic Profile for a 1,000-gallon scale Fed-Batch fermentation
0.42 g/L/hr **
* Two separate
samples were analyzed at 48 hrs.
** This is for
a washed dry-cell weights (DCW) sample. Other reported values are for unwashed samples.
This example illustrates affect of the amount of carbon
source on productivity.
Three different fermentation processes using the process
of Example 4 were conducted using various amounts of carbon source. Results are
shown on Table 8.
Table 8. Fermentation results for various amounts of carbon source on fermentation
This example illustrates the effect of nutrient limitation
on carbon conversion efficiency to biomass, lipid and most specifically DHA.
A continuous culture experiment to investigate the effect
of nutrient limitation was performed by culturing Schizochytrium ATCC No.
20888 in a 2-liter volume Applikon fermentor in basal growth (ICM-2) medium consisting
of the following compounds (nominal concentration): Group I ingredients: Na2SO4
(18.54 g/L), MgSO4•7H2O (2.0 g/L) and KCL (0.572 g/L);
Group II ingredients (each prepared separately): glucose (43.81 g/L), KH2PO4
(1.28 g/L), CaCl2•2H2O (0.025 g/L) and (NH4)2SO4
(6.538 g/L); Group III ingredients: Na2EDTA (6.0 mg/L), FeCl3•6H2O
(0.29 mg/L), H3BO3 (6.84 mg/L), MnCl2•4H2O
(0.86 mg/L), ZnSO4•7H2O (0.237 mg/L), CoCl2•2H2O
(0.026 mg/L), Na2MoO4•2H2O (0.005 mg/L),
CuSO4•5H2O (0.002 mg/L) and NiSO4•6H2O
(0.052 mg/L); and Group IV ingredients: thiamine HCl (0.2 mg/L), Vitamin B12
(0.005 mg/L), Calcium pantothenate (0.2 mg/L). Groups I and II were autoclave sterilized,
while Groups III and IV were filter sterilized prior to adding to the fermentor.
The growth medium was then inoculated with Schizochytrium and grown under
controlled conditions of 30°C, pH 5.5 and dissolved oxygen of 20% of saturation
until maximum cell density was achieved.
A continuous operation mode was then established by simultaneously
pumping sterile ICM-2 feed medium into the fermentor and removing the broth containing
Schizochytrium cells at a flowrate sufficient to maintain a dilution rate
of 0.06hr-1, until a steady state is reached. To investigate the effect
of nutrient limitation, the compound containing the specified required nutrient
is lowered in the ICM-2 feed medium such that this nutrient is depleted in the outlet
cell-containing broth, so that growth of the cells is limited by absence of the
particular required nutrient. Once steady state operation was established for each
condition, final broth dry biomass, residual glucose, and limiting nutrient concentrations,
lipid content of the cell and DHA content of the cells were measured. The conversion
efficiency of glucose to biomass was calculated by dividing the total glucose consumed
by the total dried biomass formed, and expressed on a percentage basis.
The effects of limiting growth by each individual nutrient
were studied by repeating this experiment for each individual nutrient listed in
the following table. Final results are summarized in the following table:
Table 9. Effect of nutrient limitation on the biomass yield, conversion efficiency
(glucose -> biomass), lipid content and DHA content of Schizochytrium
of dry biomass (grams/liter)
2. yield coefficient
(% biomass produced/glucose consumed)
3. residual glucose
concentration in broth (grams/liter)
4. lipid content
of dry biomass (g lipid (as FAME)/g dry biomass)
5. DHA content
of dry biomass (g DHA/g dry biomass)
It is clear from the table that nitrogen limitation resulted
in the highest accumulation of DHA in the cells, followed by phosphate, sodium,
nickel, manganese, glucose(carbon), zinc and iron. This information can be employed
commercially by feeding one or more of these nutrients to a batch fermentation at
a rate sufficient to limit cell growth. In the most preferred case, nitrogen is
fed in a limiting manner to the batch fermentation to maximize the DHA content of
the cells. Other nutrients (or mixtures thereof) can be fed in a limiting manner
to maximize production of biomass or other valuable products. Other biologically
required elements or nutrients that were not evaluated, such as sulfur, could also
be employed as limiting nutrients in this fermentation control strategy.
The present invention, in various embodiments, includes
components, methods, processes, systems and/or apparatus substantially as depicted
and described herein, including various embodiments, subcombinations, and subsets
thereof. Those of skill in the art will understand how to make and use the present
invention after understanding the present disclosure. The present invention, in
various embodiments, includes providing devices and processes in the absence of
items not depicted and/or described herein or in various embodiments hereof, including
in the absence of such items as may have been used in previous devices or processes,
e.g., for improving performance, achieving ease and\or reducing cost of implementation.
The foregoing discussion of the invention has been presented
for purposes of illustration and description. The foregoing is not intended to limit
the invention to the form or forms disclosed herein. Although the description of
the invention has included description of one or more embodiments and certain variations
and modifications, other variations and modifications are within the scope of the
invention, e.g., as may be within the skill and knowledge of those in the
art, after understanding the present disclosure. It is intended to obtain rights
which include alternative embodiments to the extent permitted, including alternate,
interchangeable and/or equivalent structures, functions, ranges or steps to those
claimed, whether or not such alternate, interchangeable and/or equivalent structures,
functions, ranges or steps are disclosed herein, and without intending to publicly
dedicate any patentable subject matter.