# Patent

## Title
Enhanced production of lipids containing polyenoic fatty acids by very high density cultures of eukaryotic microbes in fermentors

## Abstract

The present invention provides a process for growing eukaryotic microorganisms which are capable of producing lipids, in particular lipids containing polyenoic fatty acids. The present invention also provides a process for producing eukaryotic microbial lipids.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application is a Continuation of U.S. application Ser. No. 11/745,490, filed 2007 May 8, now U.S. Pat. No. 8,216,812, Ser. No. 11/745,498, filed 2007 May 8, now U.S. Pat. No. 8,288,133, Ser. No. 11/745,500, filed 2007 May 8, now U.S. Pat. No. 8,288,134, Ser. No. 11/745,502, filed 2007 May 8, Ser. No. 11/745,506, filed 2007 May 8, now U.S. Pat. No. 8,124,384, Ser. No. 11/745,511, filed 2007 May 8, now U.S. Pat. No. 8,124,385, Ser. No. 11/745,513, filed 2007 May 8, now U.S. Pat. No. 8,206,956, Ser. No. 11/745,526, filed 2007 May 8, now U.S. Pat. No. 8,133,706, Ser. No. 11/745,531, filed 2007 May 8 now U.S. Pat. No. 8,187,845, and Ser. No. 11/745,533, filed 2007 May 8 now U.S. Pat. No. 8,187,846. U.S. application Ser. No. 11/745,490, filed 2007 May 8, Ser. No. 11/745,498, filed 2007 May 8, Ser. No. 11/745,500, filed 2007 May 8, Ser. No. 11/745,502, filed 2007 May 8, and Ser. No. 11/745,506, filed 2007 May 8 are Continuations of U.S. application Ser. No. 11/352,421, filed 2006 Feb. 10, now U.S. Pat. No. 7,579,174, issued 2011 Dec. 28. U.S. application Ser. No. 11/745,511, filed 2007 May 8, Ser. No. 11/745,513, filed 2007 May 8, Ser. No. 11/745,526, filed 2007 May 8, Ser. No. 11/745,531, filed 2007 May 8, and Ser. No. 11/745,533, filed 2007 May 8 are Divisionals of U.S. application Ser. No. 11/352,421, filed 2006 Feb. 10, now U.S. Pat. No. 7,579,174, issued 2011 Dec. 28. U.S. application Ser. No. 11/352,421, filed 2006 Feb. 10 is a Continuation of U.S. application Ser. No. 10/371,394, filed 2003 Feb. 21. U.S. application Ser. No. 10/371,394, filed 2003 Feb. 21 is a Continuation of U.S. application Ser. No. 09/771,352, filed 2001 Jan. 26, now U.S. Pat. No. 6,607,900, issued 2011 Dec. 28. U.S. application Ser. No. 09/771,352, filed 2001 Jan. 26 is a Non-Provisional of U.S. application No. 60/178,588, filed 2000 Jan. 28.


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 is generally known 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., xanthophyls), ubiquinones, and compounds made from any of these lipids (i.e., secondary metabolites).
Eukaryotic microbes (such as algae; fungi, including yeast; and protists) have been demonstrated to be good producers of polyenoic 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 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 a 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 a 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.
Present inventors have 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 Thraustochyrids which exhibit a 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/hr 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 fermentors.
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 which 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-alcoholic carbon source, and a nitrogen source. Preferably, the carbon source and the nitrogen 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 nitrogen source, and the lipid production stage comprises adding the carbon source without adding the nitrogen source to induce nitrogen limiting conditions which induces 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, protists, and mixtures thereof, wherein the microorganisms are capable of producing polyenoic fatty acids or other lipids which requires molecular oxygen for their synthesis. A particularly useful microorganisms of the present invention are eukaryotic microorganisms which 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. Moreover,
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 second half of the fermentation process.
Another embodiment of the present invention provides a process for producing eukaryotic microbial lipids comprising:

    
    
        (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 a fermentation condition sufficient to allow said microorganisms to produce said lipids; and
        (c) recovering said lipids,
    
    


wherein greater than about 15% of said lipids are polyunsaturated lipids.
Another aspect of the present invention provides a lipid recovery step which comprises:

    
    
        (a) removing water from said fermentation medium to provide dry microorganisms; and
        (b) 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.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a table and a plot of various lipid production parameters of a microorganism versus the amount of dissolved oxygen level in a fermentation medium.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a process for growing microorganisms, such as, for example, fungi (including yeast), algae, and protists. 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; xanthophyls (e.g., oxycarotenoids); hydrocarbons; 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, xanthophyls, ubiquinones, other compounds which 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 which 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 which are capable of producing DHA. 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. Pat. Nos. 5,340,594 and 5,340,742, both issued to Barclay, all of which are incorporated herein by reference in their entirety. 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 nitrogen to a fermentation medium comprising the microorganisms at a rate sufficient to increase the biomass density of the fermentation medium to those described above. This fermentation process, where a substrate (e.g., a carbon source and a nitrogen source) is 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 which does not cause a significant detrimental effect on productivity of microorganisms. 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-alcoholic carbon source, i.e., 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 isopropanol. 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.
A particularly preferred nitrogen source is inorganic ammonium salt, more preferably ammonium salts of sulfate, hydroxide, and most preferably ammonium hydroxide.
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 nitrogen 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 by eukaryotic microorganisms including omega-3 and/or omega-6 polyunsaturated fatty acids. 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 at least about 8% of saturation, and preferably at least about 4% of saturation, during the production stage the dissolved oxygen level 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. 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 process.
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 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 saline (i.e., sodium). More particularly, a significant portion of the sodium requirements of the fermentation are 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 2.0 g/L to about 25 g/L.
Various fermentation parameters for inoculating, growing and recovering microorganisms are discussed in detail in U.S. Pat. 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, 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.
Preferably, 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 a 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 a DHA production rate of at least about 0.2 g of DHA/L/hr, preferably at least about 0.3 g of DHA/L/hr, more preferably at least about 0.4 g of DHA/L/hr, and most preferably at least about 0.5 g of DHA/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.
EXAMPLES
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 following examples only list the amount of DHA, one can readily calculate the amount of DPA produced by using the above disclosed ratio.
Example 1
This example illustrates the affect of oxygen content in a fermentation medium on lipid productivity.
Fermentation results of Schizochytrium at various levels of dissolved oxygen content were measured. The results are shown in FIG. 1, where RCS is residual concentration of sugar, and DCW is dry-cell weight.
Example 2
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-α-tocopheryl acetate was added for nutritional supplementation purposes prior to analysis of the lipid.
Nine fermentations of Schizochytrium were run and the results are shown in Table 1. The dissolved oxygen level was about 8% during the first 24 hours and about 4% thereafter.







TABLE 1







Fed-batch fermentation results for the production of DHA.












Entry
Age (Hrs)
Yield1 (g/L)
DHA (%)
FAME2 (%)
Productivity3















1
100.3
160.7
17.8
49.5
0.285


2
99.8
172.4
19.4
51.3
0.335


3
84.7
148.7
14.4
41.4
0.253


4
90.2
169.5
19.7
53.9
0.370


5
99.0
164.1
12.5
38.9
0.207


6
113.0
187.1
19.7
47.2
0.326


7
97.0
153.5
13.7
41.0
0.217


8
92.8
174.8
16.4
48.6
0.309


Avg
97.1
166.4
16.7
46.5
0.288


4


Std 5
8.4
12.3
2.9
5.4
0.058


CV 6
8.7
7.4
17.3
11.7
20.2


(%)





1actual yield of biomass density.


2total fatty acid methyl esters.


3(grams of DHA)/L/Hr.


4. average.


5. standard deviation.


6. coefficients 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.






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′′×36′′). 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 1 is summarized in Table 2.







TABLE 2







Analysis of lipids from microorganisms of Table 1.










% DHA relative
Total Lipid %


Entry
to FAME1
by wt.












1
36.0
72.3


2
37.8
70.3


3
34.8
61.5


4
36.5
74.8


5
32.1
52.8


6
41.7
67.7


7
33.4
49.9


8
33.7
61.4


Avg
35.8
63.8


Std. 3
3.0
9.1


CV 4 (%)
8.5
14.2





1see Table 1


2. see discussion above


3. standard deviation


4. coefficients 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 (0.951.1); 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 B120.15 mg/L (0.05-0.25) and Ca1/2 Pantothenate 3.2 mg/L (1.3-5.1). In addition, 28% NH4OH solution is used as the nitrogen source.
The ash content of the dried microorganisms is about 6% by weight.
Example 3
This example illustrates the effect of reduced dissolved oxygen level in the fermentation medium on the productivity of microorganisms using G-tank scale.
Using the procedure described in Example 2, a 14,000 gallon nominal volume fermentation was conducted using a Schizochytrium, which can be obtained using isolation processes disclosed in the above mentioned U.S. Pat. Nos. 5,340,594 and 5,340,742. 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 3.







TABLE 3







14,000 gallon scale fermentation of Schizochytrium.



















DHA







% DHA
Productivity



Age


%
rel. to
(g of


Entry
(Hrs)
Yield (g/L)
% DHA
FAME
FAME
DHA/L/hr)
















1
82.0
179.3
21.7
52.4
41.4
0.474


2
99.0
183.1
22.3
55.0
40.5
0.412


3
72.0
159.3
—
—
40.9
—


4
77.0
161.3
—
—
43.2
—


5
100.0
173.0
23.9
53.3
44.9
0.413


6
102.0
183.3
21.6
50.8
42.6
0.388


7
104.0
185.1
23.7
55.0
43.1
0.422


8
88.0
179.3
22.3
52.6
42.4
0.454


9
100.0
166.4
22.5
53.5
42.1
0.374


10
97.0
182.6
22.8
51.6
44.1
0.429


11
87.5
176.5
19.8
45.6
43.5
0.399


12
67.0
170.8
18.8
48.1
39.1
0.479


13
97.0
184.9
23.2
52.7
44.0
0.442


14
102.0
181.9
23.6
52.9
44.6
0.421


15
102.0
186.9
19.9
47.8
41.8
0.365


16
97.0
184.4
19.6
45.5
43.0
0.373


17
98.0
174.7
19.7
45.1
43.7
0.351


18
103.5
178.8
18.3
44.5
41.2
0.316


19
102.0
173.7
15.8
43.1
36.7
0.269


20
94.0
190.4
19.3
46.9
41.1
0.391


21
72.0
172.5
22.8
52.8
43.2
0.546


22
75.0
173.1
21.0
51.7
40.8
0.485


23
75.0
152.7
20.3
50.3
40.4
0.413


24
75.5
172.5
21.9
51.7
42.3
0.500


25
61.0
156.4
17.3
45.7
37.8
0.444


26
74.5
150.6
20.2
50.1
40.2
0.408


27
70.5
134.3
14.8
40.6
36.6
0.282


28
75.5
146.1
21.3
49.7
42.8
0.412


29
82.0
174.3
21.4
50.4
42.5
0.455


30
105.0
182.3
21.7
50.7
42.8
0.377


31
66.0
146.2
16.4
44.6
36.7
0.363


Avg
87.2
171.5
20.6
49.5
41.6
0.409


Std
13.9
14.1
2.4
3.8
2.3
0.061


CV
16.0%
8.2%
11.6%
7.7%
5.5%
15.0%









Example 4
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.
Same procedure as Example 3 in a 41,000 gallon fermentor was performed. Results are shown in Table 4.







TABLE 4







41,000 gallon scale fermentation of Schizochytrium














Age
Yield
%
%
% DHA rel.
DHA Productivity


Entry
(Hrs)
(g/L)
DHA
FAME
to FAME
(g of DHA/L/hr)
















1
75.0
116.1
17.3
46.1
37.4
0.268


2
99.0
159.3
17.4
47.0
37.1
0.280


3
103.0
152.6
16.0
47.2
33.8
0.237


4
68.0
136.8
17.9
45.9
39.1
0.360


5
84.0
142.0
17.5
47.0
37.2
0.296


Avg
85.8
141.4
17.2
46.6
36.9
0.288


Std
15.1
16.6
0.7
0.6
1.9
0.046


CV
17.5%
11.8
4.2%
1.3%
5.2%
15.8%









Example 5
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 3. Two control experiments and two experiments containing extra ammonia (1.15× and 1.25× the normal amount) were conducted. Results are shown in Table 5.







TABLE 5







Affects of extra ammonia on fermentation of Schizochytrium.
















Con-








version





Age
Yield
Biomass
Effi-
DHA
FAME
DHA


(hrs)
(g/L)
Productivity
ciency
Content
Content
Productivity










Sugar target: 7 g/L, Base pH set point: 5.5, Acid pH set point:


7.3, 1.0X NH3













48
178
3.71 g/L/hr
51.5%
10.7%
37.8%
0.40 g/L/hr


60
185
3.08 g/L/hr
46.9%
16.3%
47.2%
0.50 g/L/hr


72
205
2.85 g/L/hr
45.2%
17.4%
47.4%
0.50 g/L/hr


84
219
2.61 g/L/hr
43.8%
17.1%
45.5%
0.45 g/L/hr


90
221
2.46 g/L/hr
44.1%
18.4%
48.9%
0.45 g/L/hr







Sugar target: 7 g/L, Base pH set point: 5.5, Acid pH set point:


7.3, 1.15X NH3













48
171
3.56 g/L/hr
55.6%
12.0%
36.3%
0.43 g/L/hr


60
197
3.28 g/L/hr
54.6%
9.4%
38.4%
0.31 g/L/hr


72
191
2.65 g/L/hr
52.8%
9.4%
40.0%
0.25 g/L/hr


84
190
2.26 g/L/hr
52.5%
10.0%
42.5%
0.23 g/L/hr


90
189
2.10 g/L/hr
52.2%
9.2%
43.3%
0.19 g/L/hr







Sugar target: 7 g/L, Base pH set point: 5.5, Acid pH set point:


7.3, 1.25X NH3













48
178
3.71 g/L/hr
56.4%
11.5%
33.7%
0.43 g/L/hr


60
179
2.98 g/L/hr
48.6%
10.3%
36.0%
0.31 g/L/hr


72
180
2.50 g/L/hr
48.8%
12.0%
37.6%
0.30 g/L/hr


84
181
2.15 g/L/hr
46.1%
13.6%
40.1%
0.29 g/L/hr


90
185
2.06 g/L/hr
45.7%
12.6%
40.7%
0.26 g/L/hr







Sugar target: 7 g/L, Base pH set point: 5.5, Acid pH set point:


7.3, 1.0X NH3













48
158
3.29 g/L/hr
55.7%
13.1%
36.5%
0.43 g/L/hr


60
174
2.90 g/L/hr
48.9%
17.9%
39.2%
0.52 g/L/hr


72
189
2.63 g/L/hr
45.7%
21.0%
39.4%
0.55 g/L/hr


84
196
2.33 g/L/hr
44.1%
22.4
40.1%
0.52 g/L/hr


90
206
2.29 g/L/hr
44.8%
22.1%
40.3%
0.51 g/L/hr









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 were added. As shown on Table 5, the control batches resulted in final DHA levels of 18.4% and 22.1% versus the 9.2% (1.15× ammonia) and 12.6% (1.25× ammonia) for extra nitrogen supplemented batches.

Example 6
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 3. Kinetic profile of the fermentation process is shown in Table 6.







TABLE 6







Kinetic Profile for a 1,000 gallon scale Fed-Batch fermentation of Schizochytrium.














Yield
Biomass
Conversion
% DHA
% FAME
DHA


Age (hrs)
(g/L)
Productivity
Efficiency
Content
Content
Productivity
















24
118
4.92 g/L/hr
78.2%
7.4
18.8
0.36 g/L/hr


30
138
4.60 g/L/hr
60.3%
10.6
30.9
0.49 g/L/hr


36
138
3.83 g/L/hr
46.6%
11.6
36.5
0.44 g/L/hr


42
175
4.17 g/L/hr
49.8%
13.4
41.7
0.56 g/L/hr


48
178
3.71 g/L/hr
45.1%
18.7
52.8
0.69 g/L/hr


 48*
164
3.42 g/L/hr
41.5%
15.3
33.1
0.52 g/L/hr


54
196
3.63 g/L/hr
45.7%
16.6
51.2
0.60 g/L/hr


60
190
3.17 g/L/hr
41.7%
16.9
33.9
0.54 g/L/hr


72
189
2.62 g/L/hr
39.1%
15.6
31.8
0.41 g/L/hr


84
195
2.32 g/L/hr
38.5%
16.4
32.7
0.38 g/L/hr


90
200
2.22 g/L/hr
39.0%
18.8
33.3
0.42 g/L/hr


90
171
1.90 g/L/hr
33.3%
22.2
61.6
 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.






Example 7
This example illustrates effect of the amount of carbon source on productivity.
Three different fermentation processed using the process of Example 3 were conducted using various amounts of carbon source. Results are shown on Table 7.







TABLE 7







Fermentation results for various amounts of carbon source on fermentation


of Schizochytrium.













Age
Yield
Carbon
Conversion
% DHA
% FAME
Productivity


(hrs)
(q/L)
Charge
Efficiency
Content
Content
(g/L/hr)
















90
171
51.3%
33.3%
22.2
61.6
0.42


94
122
40.5%
30.1%
19.1
57.3
0.25


59
73
20.0%
36.5%
11.9
40.8
0.15









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.

## Claims

1. A process for producing microbial lipids comprising conducting a fermentation of microorganisms of the order Thraustochytriales capable of producing at least 15% of lipids as highly unsaturated fatty acids having at least four unsaturated carbon-carbon bonds in a fermentation medium comprising a carbon source and a limiting nutrient source, the process comprising:
a) providing the fermentation medium comprising the carbon source and limiting nutrient source;
b) adding the microorganisms to the fermentation medium;
c) adding an additional amount of the carbon source and limiting nutrient source to a fermentation medium to increase the biomass density of the fermentation medium to at least 100 g/L on a dry cell weight basis; and
d) adding an additional amount of the carbon source while limiting the amount of the limiting nutrient source added to the formulation medium to induce lipid production by the microorganisms.

2. The process of claim 1, further comprising e) recovering at least some of the lipids from the microorganisms.

3. The process of claim 1, wherein the fermentation of the microorganisms produces lipids containing highly unsaturated fatty acids when the biomass density of the fermentation medium is at least 150 g/L on a dry cell weight basis.

4. The process of claim 1, wherein the process produces lipids at an average rate of at least 0.5 grams lipids per liter of fermentation medium per hour fermentation.

5. The process of claim 1, wherein the microorganisms are selected from the group consisting of Thraustochytrium, Schizochytrium, and mixtures thereof.

6. The process of claim 1, wherein the process produces on average at least 0.2 grams docosahexaenoic acid per liter of fermentation medium per hour fermentation.

7. The process of claim 1, wherein the microorganisms produce lipids containing highly unsaturated fatty acids when the biomass density is at least 100 g/L on a dry cell weight basis.

8. The process of claim 1, wherein the highly unsaturated fatty acids are selected from the group consisting of docosahexaenoic acid, docosapentaenoic acid, eicosapentaenoic acid, arachidonic acid and mixtures thereof.

9. The process of claim 8, wherein the highly unsaturated fatty acids are docosahexaenoic acid.

10. The process of claim 1, wherein at least 25% of the lipids is docosahexaenoic acid.

11. The process of claim 1, wherein the process produces lipids at an average rate of at least 0.5 g/L/hr and wherein at least 25% of the lipids is docosahexaenoic acid.

12. The process of claim 1, wherein the carbon source comprises a carbohydrate.

13. The process of claim 1, wherein the limiting nutrient source comprises a nitrogen source.

14. The process of claim 1, wherein the pH of the fermentation medium is about pH 5 to about pH 11.

15. The process of claim 1, wherein the fermentation medium is at a temperature of at least 20° C.

16. The process of claim 1, wherein the microorganisms produce highly unsaturated fatty acids under aerobic conditions.

17. The process of claim 1, wherein dissolved oxygen in the fermentation medium is controlled.

18. The process of claim 1, further comprising: removing water from the fermentation medium to provide dry microorganisms prior to recovering the lipids from the microorganisms, wherein at least 15% of the lipids are highly unsaturated fatty acids.

19. The process of claim 18, wherein the water is removed from the fermentation medium by evaporation without prior centrifugation to provide dry microorganisms prior to recovering the lipids from the microorganisms, wherein at least 15% of the microbial lipids are highly unsaturated fatty acids.

20. A process for growing microorganisms of the order Thraustochytriales by conducting a fermentation of the microorganisms in a fermentation medium comprising a carbon source, and a limiting nutrient source the process comprising:
a) providing the fermentation medium comprising the carbon source and the limiting nutrient source;
b) adding the microorganisms to the fermentation medium;
c) adding an additional amount of the carbon source and limiting nutrient source to the fermentation medium to increase the biomass density of the fermentation medium to at least 100 g/L on a dry cell weight basis, wherein the additional amount of 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 100 g/L;
d) adding an additional amount of the carbon source while reducing the amount of limiting nutrient source added to induce lipid production by the microorganisms; and
e) recovering the lipids from the fermentation medium,
wherein the primary use of the carbon source by the microorganisms is to produce the lipids after induction of lipid production;
wherein the fermentation of the microorganisms when the biomass density is at least 100 g/L on a dry cell weight basis produces lipids containing polyunsaturated fatty acids;
wherein the lipids are at least 20% of the biomass when the biomass density is at least 100 g/L;
wherein the process produces lipids at least at an average rate of 0.5 g/L/hr; and
wherein at least about 15% of the total lipids produced by the microorganisms are polyunsaturated lipids.