Production of high levels of dha in microalgae using modified amounts of chloride and potassium.
Abstract
Methods for production of highly unsaturated fatty acids by marine microorganisms, including the heterotrophic marine dinoflagellate Crypthecodinium, using low levels of chloride ion are disclosed. Specifically, methods of increasing production of highly unsaturated fatty acids by marine microorganisms while growing in low chloride media by manipulating sodium ion and potassium ion levels. The invention also relates to methods of production of highly unsaturated fatty acids by marine organisms at low pH levels, and includes methods for generation of low pH tolerant strains.

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19 claims: 4 independent, 15 dependent
- 1REIVINDICACIONES 1,- Un método para cultivar una microalga heterótrofa en un medio de cultivo, que comprende cultivar la microalga en un medio que comprende:(a) ión cloruro a una concentración menor que o igual a 2 g/L y (b) en donde ¡ón potasio a una concentración mayor que o igual a 0.5 g/L;la microalga produce por lo menos 53% de grasa como un por ciento en peso de la biomasa.
- 2- El método de conformidad con la reivindicación 1, en donde microalga es de la clase Dinophyceae.
- 3- El método de conformidad con la reivindicación 1, en donde microalga es del género Crypthecodinium. donde la donde la donde donde donde
- 4- El método de conformidad con la microalga es de la especie Crypthecodinium cohnii.
- 5- El método concentración de
- 6- El método la concentración de
- 7- El método 1, en de ión de ¡ón de conformidad con la reivindicación 1, en cloruro es menor que o igual a 0.3 g/L. conformidad con la reivindicación 1, cloruro es menor que o igual a 1 g/L. conformidad con la reivindicación 1, una fuente de ¡ón potasio comprende sulfato de potasio.
- 8- El método de conformidad con la reivindicación 1, en en en la concentración de ¡ón potasio es mayor que o igual a 0.8 g/L.
- 9- Una biomasa producida mediante el método de conformidad con la reivindicación 1, en donde dicha biomasa conti n por lo menos 60% de grasa como un por ciento en peso de la biomasa. INSTITUTO Μ£ΧΚ2ΛΝ<) Di M fltÜWWAr INPUSTUAl
- 1010, - El método de conformidad con la reivindicación 1, en donde la microalga produce por lo menos 0.10 g de ácido docosahexaenoico (DHA) por 10 9 células.
- 1111, - El método de conformidad con la reivindicación 1, en 5 donde la microalga produce por lo menos 0.20 g de DHA por 10 9 células.
- 1212, - El método de conformidad con la reivindicación 1, que comprende también recuperar de la microalga un lípido que contiene DHA. 10
- 13- El método de conformidad con la reivindicación 1, en donde el medio comprende también ión sodio a una concentración de 1 g/L hasta 8 g/L.
- 1414,- El método de conformidad con la reivindicación 13, en donde la concentración de ión sodio es 1.5 g/L hasta 5 g/L.
- 1515 15,- El método de conformidad con la reivindicación 13, en donde una fuente de ión sodio comprende sulfato de sodio.
- 16- El método de conformidad con la reivindicación 1, en donde el medio de cultivo tiene un pH menor de 6.
- 17- El método de conformidad con la reivindicación 16, en 20 donde el pH es menor que o igual a 5.5.
- 1818, - El método de conformidad con la reivindicación 16, en donde el pH es menor que o igual a 5.
- 1919, - El método de conformidad con la reivindicación 16, en donde el pH es igual a 4.5.
Independent claims19
480 paragraphs in 54 sections, as filed
(54) Title: PRODUCTION OF HIGH LEVELS OF DHA IN MICROALGAE USING MODIFIED AMOUNTS OF CHLORIDE AND POTASSIUM.
(54) Title: PRODUCTION OF HIGH LEVELS OF DHA IN MICROALGAE USING MODIFIED AMOUNTS OF CHLORIDE AND POTASSIUM.
(57) Summary
The present invention relates to a method of cultivating a heterotrophic microalgae in a culture medium, which comprises cultivating the microalgae in a medium comprising: (a) chloride ion at a concentration less than or equal to 2 g / L (b ) Potassium ion at a concentration greater than or equal to 0.5 g / L; wherein the microalgae produces at least 53% fat as a weight percent of the biomass.
(57) Abstract
Methods for productlon of highly unsaturated fatty aclds by marine microorganisms, includlng the heterotrophlc marine dlnoflagellate Crypthecodinium, uslng low levels of chlorlde Ion are dlsclosed. Speclflcally, methods of increaslng production of highly unsaturated fatty acids by marine microorganisms while growing in low chloride media by manipulating sodlum Ion and potassium Ion levels. The Invention also relates to methods of production of highly unsaturated fatty aclds by marine organisms at low pH levels, and included methods for generatlon of low pH tolerant strains.
PATENT TITLE NO. 338455 ___SE___ ^ ECÍLIIARÚ I heard KCWMÍÁ
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Mexican Institute of Industrial Property
Het Overloon 1, 6411 The Heerlen, NETHERLANDS
<img file="MX338455B_D0001.tif" />
PRODUCTION OF HIGH LEVELS OF DHA IN MICROALGAE USING MODIFIED AMOUNTS OF CHLORIDE AND POTASSIUM.
lnt.CI.8: C12P7 / 64
PAULW. BEHRENS; JOHN M. THOMPSON; KIRK APT; JOSEPH W, PFEIFER III; JAMES P. WYNN; JAMES CASEY LIPPMEIER; JAOUAD FICHTAU; JON HANSEN
REQUEST
International filing date:
MX / a / 2012/012139 October 2004
Divisional Patent Number: 304474
PRIORITY
Country:
Date:
Number:
US October 2003
60/508,505
Validity: Twenty years
Expiration Date 1 or
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i® lustrial.
Reference patent s »grants foundation« η I
In accordance with article 23 of the Law of the Prop counted from the date of provision of the request rights. i- J
Who subscribes to the present title of the Ace based on the 'artientós 8 * fractions III and 7 ° bis 2 of the ey of Industrial property (Dlar ^ Official d ^ ta Federation (DOF) 06/27/1991, refermaife on 02/08/1994, 10/25/1996, 12/26/1997, 1JJ5 / 1999,
Ifaction IV, 6th fraction III, and 59 of the Property Law I present this patent has a validity of twenty unpayable years, to and mat subject to the payment of the fee to maintain the actions V subsection a). 4th and 12th fractions I and III of the Regulation of the Mexican Institute of Industrial Property (DOF 12/14/1999, amended on 07/01/2002, 07/15/2004. 07/28/2004 and 09/07/2004 2007); 1st articles. 3rd. 4th. 5th fraction V subsection a). 16 Fractions I and III and 30 of the Organic Statute of the Mexican Institute of Industrial Property (DOF 12/27/1999, amended on 10/10/2002, 07/29/2004, 08/04/2004 and 09/13/2007) ; 1, 3 and 5 subsection a) of the Agreement that delegates powers to the Deputy Directors General, Coordinator, Divisional Directors, Heads of Regional Offices, Divisional Deputy Directors, Departmental Coordinators and other subordinates of the Mexican Institute of Dggtrial Property. (DOF 12/15/1999, amended on 04/02/2000, 07/29/2004, 08/04/2004 and 09/13/2007).
<img file="MX338455B_D0003.tif" />
Issue Date: April 18, 2016
THE DIVISIONAL DIRECTOR OF PATENTS
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NAHANNY CANAL REYES
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Arenal No. 550. Floor 1, Col. Pueblo Santa María Tepepan.
Xochímilco, CP 16020,
Mexico City
Tei. (55) 53 34 07 00 ww impi jzob.mx
338HSS
PRODUCTION OF HIGH LEVELS OF DHA IN MICRoUgIP I '...............—. ..........- , ,.,.., ------------------------------' INSTITUTE MEXIC? '. ,, .-—, ··, - ......— .-, ... -, -, · ......-......... ...... ........................ '.. MBXICANl INSTITUTE>
OF INDUSTRIAL PROPERTY USING MODIFIED AMOUNTS OF CHLORIDE AND POTASSIUM
FIELD OF THE INVENTION
This invention relates generally to methods for the production of highly unsaturated fatty acids by marine microorganisms using modified amounts of ion chloride and potassium in the culture medium. More specifically, the invention is directed to a process for producing high levels of docosahexaenoic acid (DHA) by cultivating marine microalgae, including the heterotrophic marine dinoflagellate Crypthecodinium, in thermostats under non-corrosive conditions, including cultivation in a environment low in chloride ion and low in potassium ion. This invention also relates to methods for the production of highly unsaturated fatty acids, having DHA, by marine microorganisms at low pH levels.
BACKGROUND OF THE INVENTION
The beneficial effects of increased dietary absorption of long-chain omega-3 fatty acids in humans has been well documented, these include the reduction of cardiovascular and inflammatory diseases (i.e. arthritis and atherosclerosis), reduction or depression, length
<img file="MX338455B_D0006.tif" />
IMPI i
MEXICAN INSTITUTE CL '*'
OF THE PROPERTY
INDUSTRIAL increased gestation in the third trimester, and inhibition of tumor growth. Several marine microorganisms have been found to produce high levels of these essentials, including those of the genus
Chen, Process Biochemistry 35 (2000) important fatty acids
Crypthecodinium (Jiang and
1205-1209; Jiang and Chen,
Journal of Industrial Microbiology & Biotechnology, (1999) Vol.
23,508-513; Vazhappilly and Chen, Journal of the American Oil Chemists Society, (1998) Vol. 75, No. 3 p 393-397; Kyle, US Patent Number 5,407, 957; US Patent Number 5,397,591; US Patent Number 5,492,938; and US Patent No. 5.71 1,983).
Crypthecodinium cohnii is one of the most desirable organisms to use for the production of DHA (C22: 6n-3), one of the most important long-chain omega-3 fatty acids. C. cohnii is advantageous because DHA is the only polyunsaturated fatty acid (PUFA) produced by this organism in appreciable amounts. Other organisms produce two or more polyunsaturated fatty acids (PUFAs) in their lipids, and the complexity of their lipid profile may limit the use of their oils in some foods and pharmaceutical applications (for example, due to the presence of other undesirable PUFAs). in the oil, or due to proportions of the different PUFAs that fall outside the desirable range for the specific application). In the marine environment, Crypthecodinium cohnii is usually found in marine waters with full salinity, and as such is adapted to grow in a
- 3 MEXICAN INSTITUTE <sup>1</sup>
DE LA I'ROPIEDAL · environment with a high concentration of chlorine. In fact, crop yields in published research sobi * e— & ~~ connn show that DHA growth and production is better in salinities greater than about 20% seawater (Jiang and Chen). The chloride ion concentration equivalent to 20% of seawater is approximately 3,870 ppm chloride ion or 3.87 g / L chloride ion (Horne 1969).
Tuttle and Loeblich (1975) developed an optimal growth medium for C. cohnii. The discovered medium contained a sodium chloride concentration of 342 millimolar (mM). The equivalent grams per liter of sodium ion and chloride ion in a solution of 342 mM sodium chloride are 7.86 g / L of sodium ion and 12.12 g / L of chloride ion.
Beach and Holz (1973) reported that when C. cohnii was grown in a range of NaCI concentrations (0.3%, 1.8% and 5.0% (1. 82 g / 1, 10.9 g / l and 30.3 g / l of chloride ion, respectively)), lipid production (expressed as mg per 10<sup>9</sup> cells) decreased as NaCI concentrations decreased. Lipid production with 0.3% NaCI was approximately one third of that obtained with 5.0% NaCI.
More recently, Jiang and Chen (1999) determined the effects of salinity on cell growth, and DHA content with three strains of Crypthecodinium cohnii, and in all cases it was found that optimal growth rates for cells, and DHA productions were between 5 g / L and 9 g / L of
- 4 MEXICAN INSTITUTE
D £ THE PROPERTY
INDUSTRIAL sodium, which corresponds to 3.0 and 5.5 g / L of chloride ion, respectively.
The natural chloride concentration of seawater (19,353 ppm, or 19.35 g / l chloride ion) (Horne 1969, page 151) promotes corrosion in stainless steel heaters. For example, of the two common grades of stainless steel used in manufacturing heaters, 304 stainless steel is susceptible to corrosion when the chlorine level exceeds 300 ppm (0.3 g / L chloride ion), and stainless steel 316 is susceptible to corrosion when the chloride level exceeds 1000 ppm (1 g / L chloride ion). There are other grades of stainless steel that are more resistant to chlorine corrosion, but are extremely expensive and are generally used only in fermentation equipment used in the production of very expensive compounds.
Although it can be predicted that minimizing corrosion of stainless steel thermostats can be achieved by lowering chlorine concentrations in the culture medium, in practice this is not an easy task. Marine microalgae, which are derived from the sea, generally require a certain amount of chloride ion, preferably as sodium chloride, to maintain growth and lipid production when grown in culture.
However, attempts to date to cultivate marine microalgae in low concentrations of chloride while maintaining production levels of unsaturated omega-3 poly- 5 INDUSTRIAL INSTITUTE OF PROPERTY C> m INDUSTRIAL, such as DHA , have been unsuccessful. Jian and
Chen (1999) were unable to demonstrate significant DA productions at NaCl levels less than 5 g / L, corresponding to a chloride level of approximately 3033 ppm or 3 g / L.
US Patent No. 6,410,281 issued June 25, 2002, to Barclay, provides a method for cultivating euryhaline organisms, such as Thraustochytrium sp. and Schizochytrium sp. in low chloride media, substituting non-chloride sodium salts to replace lost sodium when sodium chloride levels are decreased.
There is a need for a process that can enable the production of a high yield of Crypthecodinium cohnii DHA, which at the same time inhibits or prevents corrosion in most desirable production vessels, stainless steel culture warmers. This process would have to allow the effective growth of the microorganism in a medium that preferably contains less than 300 ppm of chloride. Three hundred ppm of chloride represents a level of 10 to 18 times less than the lowest chloride levels demonstrated by Jiang and Chen (1999) as the best for the production of Crypthecodinium strains.
Another desirable feature of microbial fermentations is the ability to grow cells with low pH (less than or equal to about pH = 5.0) to inhibit the growth of bacteria in fungal fermentations. However, the literature
- 6 MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY indicates that Crypthecodinium grows best at a neutral pH (approximately a pH of 7). Tuttle and Loeblich in Phycologia Vol. 14 (1) 1-8 (1975), describe that the optimal pH for the Crypthecodinium growth is 6.6, growth being "very slow" below a pH of 5.5. There is a need for strains and / or methods for growing Crypthecodinium at low pH, while maintaining normal growth and DHA production.
BRIEF DESCRIPTION OF THE INVENTION
By attempting to minimize sodium chloride levels in culture medium for Crypthecodinium, where sodium chloride leads to the problem of corrosion of thermostats, the inventors have surprisingly discovered that sodium chloride levels can be reduced by manipulation. of the sodium and preferably potassium salts in the culture medium, to compensate for the decrease in chloride ion (below 300 ppm or 0.3 g / L chloride ion) while maintaining DHA production similar to that obtained at approximately 4.5 g / L NaCl (corresponding to .73 g / L of chloride ion).
The present inventors have identified culture conditions that allow Crypthecodinium to be grown in medium with substantially decreased chloride levels (down to about 0.3 g / L chloride ion) without adversely affecting the
- 7 1 JV1 Jl 1
MEXICAN INSTITUTE
PROPERTY industrial dry weight, fat content or DHA content when compared to growth in a normal "high chloride" medium. Achieving comparable DHA production was not merely a matter of replacing the sodium chloride in the medium with other sodium salts. In fact, replacing sodium chloride with an equivalent amount of sodium from other sodium salts (i.e. sodium sulfate) did not result in DHA production comparable to the high chloride control case, but actually resulted in a further decrease in culture DHA production. Instead, the current inventors surprisingly found that the best DHA production was obtained when the potassium concentration (relative to that of seawater at 4.5 g / L NaCI or 17% seawater) increased significantly. It is unexpected that a substantial decrease in the amount of sodium and an increase in the concentration of potassium may be effective in compensating for the reduction in the chlorine content of the medium.
In one embodiment, the present invention includes a method of producing docosahexaenoic acid (DHA) by cultivating heterotrophic microalgae of the Dinophyceae class in a culture medium. The medium contains a chloride ion in a concentration less than or equal to approximately 2 g / L, and a potassium ion in a concentration greater than or equal to approximately 0.25 g / L. In this modality, the microalgae produce at least approximately 0.04 g of DHA per liter of culture of 7 days. A 7 day crop
- 8 generally has approximately x 10<sup>6</sup>
<img file="MX338455B_D0007.tif" />
MEXICAN INSTITUTE IS THE INDUSTRIAL PROPERTY
<img file="MX338455B_D0008.tif" />
cells / ML or about 5x10<sup>9</sup> cells per liter. Consequently, a culture that has approximately 0.2 g / L of DHA in 7 days contains approximately 0.04 g of DHA / 10<sup>9</sup> cells.
in a preferred embodiment, the microalgae are
A more preferred microalgae is of the genus Crypthecodinium.
Crypthecodinium cohnii.
Preferably, the chloride ion concentration is less than or equal to about 1 g / L, even more preferable is less than or equal to about 0.3 g / L. Preferably, the potassium ion is greater than or equal to 0.4 g / L, and even more preferable is greater than or equal to 0.8 g / L. Preferably, the source of the potassium ion is potassium sulfate. In a preferred embodiment, the medium further contains a sodium ion source such that the sodium ion concentration is from about 1 g / L to about 8 g / L. More preferably, the sodium ion is in a concentration from about 1.5 g / L to about 5 g / L. A preferred source of sodium ion is sodium sulfate. A biomass produced by this method is included in the present invention.
In another embodiment, the present invention includes a method of producing DHA by cultivating heterotrophic Microalgae of the Dinophyceae class in a culture medium. The medium contains chloride ion in a concentration less than or equal to approximately 2 g / L, potassium ion in a concentration greater than or equal to approximately 0.25 g / L, and sodium ion present in a proportion
- 9 less than or equal to approximately 27: 1 of
I Μ PI
MEXICAN INSTITUTE
Df. THE INDUSTRIAL PROPERTY weight: sodium weight: potassium. In this modality, the microalgae produce at least approximately 0.2 g of DHA per liter of culture in 7 days, or 0.04 g of DHA / 10<sup>9</sup> cells, in a preferred embodiment, the microalgae are of the genus Crypthecodinium. A more preferred microalgae is
Crypthecodinium cohnii. Preferably the chloride ion concentration is less than or equal to about 1 g / L, even more preferably, less than or equal to about 0.3 g / L. Preferably, the potassium ion is greater than or equal to about 0.4 g / L, and even more preferably, it is greater than or equal to about 0.8 g / L. Preferably, the potassium ion source is potassium sulfate. The medium also contains a source of sodium ion such that the sodium ion is present in the medium at a ratio of less than 27 times (by weight) the weight of the potassium ion (expressed as 27: 1 sodium: potassium weight: weight) . In a preferred embodiment, the sodium: potassium ratio is less than about 15: 1. More preferred is a sodium: potassium ratio of about 4: 1. A preferred source of sodium ion is sodium sulfate. A biomass produced by this method is included in the present invention.
The present inventors have also identified the conditions of the culture medium and the strains that allow Crypthecodinium to be cultivated in medium with substantially decreased pH levels, while still maintaining a practical growth rate and lipid production from the commercial point of view,
- 10 IMPIí
<img file="MX338455B_D0009.tif" />
MEXICAN INSTITUTE THE INDUSTRIAL PROPERTY including DHA. In another embodiment, the present invention includes a method of producing DHA by cultivating heterotrophic Microalgae of the Dinophyceae class in a culture medium, where the culture medium has a pH of less than about 6, and where the microalgae produce at least about 0.04 g of DHA / 109 cells. The medium may additionally contain chloride in a concentration less than or equal to approximately 2 g / L, potassium in a concentration greater than or equal to approximately 0.25 g / L and sodium present in a proportion less than or equal to approximately 27 : 1 sodium: potassium weight: weight. In this modality, the microalgae produce at least approximately 0.04 g of DHA / 10<sup>9</sup> cells. In a preferred embodiment, the microalgae are of the Crypthecodinium genus. A more preferred microalgae is Crypthecodinium cohnii. In a preferred embodiment, the pH is less than or equal to about a pH of 5.5, more preferably is less than or equal to about 5.0, and even more preferably less than or equal to about 4.5. In a preferred embodiment, the medium further contains a chloride concentration of less than or equal to about 2 g / L, preferably less than or equal to about 1 g / L, even more preferably less than or equal to about 0.3 g / L. The medium also contains potassium ion in concentrations greater than or equal to approximately 0.25 g / L, greater than or equal to approximately 0.4 g / L, and even more preferably greater than or equal to approximately 0.8 g / L. Preferably, the source of potassium is potassium sulfate. In
<img file="MX338455B_D0010.tif" />
MEXICAN INSTITUTE «'J
OWNERSHIP a preferred embodiment, the medium further contains a sodium fWe ^ H'e ióTr— sodium such that the ion concentration sorttcr t * y - approximately 1 g / L to approximately 8 g / L. More preferably, the sodium ion has a concentration of from about 1.5 g / L to about 5 g / L. A preferred source of sodium ion is sodium sulfate. A biomass produced by this method is included in the present invention.
The present invention also includes a method for the selection of low pH tolerant heterotrophic microalgae of the class Dinophyceae, comprising subculturing said microalgae in low pH medium until the DHA production is greater than or equal to approximately 0.04 g of DHA / 10<sup>9</sup> cells. In a preferred embodiment, pH is less than or equal to about 6, is less than or equal to about 5, is less than or equal to about 4.5. Microalgae and biomass produced by this method are included in the present invention.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graphical representation of a duplicate DHA production time course of C. cohnii strain T-HF grown with a pH of 6.3 and 3 g / L chloride ion (indicated as SSM at pH 6.3) , and of the C. cohnii T-HF strain, adapted to low pH, cultivated with a pH of 5 and 1 g / L of chloride ion (indicated
- 12 MEXICAN INSTITUTE -rr \ ria Dnm »irr, .r
INDUSTRIAL PROPERTY as LCSSI with pH 5.0).
Figure 2 is a graphical representation of a DHA production time course for duplicates of the C. cohnii T-HF strain grown with a pH of 6.3, and 3 g / L of chloride ion (indicated as SSM with pH 6.3. ) and the C. cohnii T-HF strain, adapted to low pH, grown with a pH of 4.5 and 1 g / L of chloride ion (indicated as LCSSI with pH 4.5).
DETAILED DESCRIPTION OF THE INVENTION
The present invention solves the problem identified above of the corrosion of the heaters caused by the high levels of sodium chloride used for the cultivation of marine microalgae of the Dinophyceae class. The inventors have discovered components of culture media that allow commercially viable growth levels of marine microalgae of the Dinophyceae class, and production of DHA under conditions low in sodium chloride, using modified amounts of chloride ion and potassium ion in the culture medium. . More specifically, the inventors have discovered that the loss of sodium caused by reducing sodium chloride to non-corrosive levels can be at least partially compensated for by increasing potassium levels in the culture medium.
The present invention also solves the problem
- 13 MEXICAN INSTITUTE
OF THE PROPERTY
INDUSTRIAL previously identified to allow the cultivation of a marine microalga of the Dinophyceae class where the growth of bacteria is simultaneously discouraged. More specifically, the present invention provides methods for culturing marine organisms such that they become tolerant to low pH. The present invention also provides strains of these microorganisms that are tolerant to low pH. The low pH tolerant strains provided by the inventors can grow, at low pH levels, to cell densities and achieve DHA production levels comparable to those achieved by strains growing at more neutral pH levels. This is only an example of the technology encompassed by the invention, since the concepts of the invention can be easily applied to other production organisms and other desired PUFAs, as described in detail below.
An embodiment of the present invention includes a method of producing docosahexaenoic acid (DHA) by culturing heterotrophic Microalgae of the Dinophyceae class in a culture medium that includes the following components: chloride ion at a concentration of less than about 2 g / L, and ion Potassium in a concentration greater than approximately 0.25 g / L, where the microalgae produce at least approximately 0.2 g of DHA per liter of culture of 7 days. The cultivation of 7 days, generally |
it has 5 x 106 cells / mL, resulting in approximately 0.04 g of DHA / 10<sup>9</sup> cells. In preferred embodiments,
<img file="MX338455B_D0011.tif" />
<sup>, Νϊτ</sup>θ<sub>Γ</sub><sup>υ</sup>™ <sup>W £ x |</sup>Only heterotrophic microalgae produce at least approximately ^ F'tf.CM of DHA / 10<sup>9</sup> cells, at least about 0.12 g DHA / 'IU * cells, at least about 0. 14 g DHA / 10<sup>9</sup> cells at
<td colspan="4">minus about 0.16 g</td><td colspan="2">from DHA / 10<sup>9</sup> cells,</td><td>to the</td><td>less</td>
<td>approximately</td><td> 0.18</td><td> 9</td><td>of</td><td>DHA / 10<sup>9</sup></td><td>cells,</td><td>to the</td><td>less</td>
<td>approximately</td><td> 0.20</td><td>g</td><td>of</td><td>DHA / 10<sup>9</sup></td><td>cells,</td><td>to the</td><td>less</td>
<td>approximately</td><td> 0.22</td><td>g</td><td>of</td><td>DHA / 10<sup>9</sup></td><td>cells,</td><td>to the</td><td>less</td>
<td>approximately</td><td> 0.24</td><td>g</td><td>of</td><td>DHA / 10<sup>9</sup></td><td>cells,</td><td>to the</td><td>less</td>
<td>approximately</td><td> 0.26</td><td>g</td><td>of</td><td>DHA / 10<sup>9</sup></td><td>cells,</td><td>to the</td><td>less</td>
<td>approximately</td><td> 0. 28</td><td>g</td><td>of</td><td>DHA / 10<sup>9</sup></td><td>cells, or</td><td>to the</td><td>less</td>
about 0.30 g of DHA / 10<sup>9</sup> cells. As used herein, the reference to a nutrient concentration in a culture medium refers to the concentration of nutrients in the medium at the beginning of the culture step, including any nutrients transferred from previous stages in the process, such as preparation of an inoculum.
Suitable microorganisms for the present invention include heterotrophic microalgae, which include members of the Dinophyceae class (dinoflagellates). A preferred member of this class is a member of the genus Crypthecodinium. a preferred member of the genus Crypthecodinium is C. cohnii. Crypthecodinium cohnii is a heterotroph that requires a reduced carbon source for growth, and contains a fatty acid profile in which DHA is the only polyunsaturated fatty acid present in appreciable amounts. The appropriate organisms
<img file="MX338455B_D0012.tif" />
can be obtained from a number of publicly available sources, including collection from the example environment, the American Type Culture Collection currently lists forty-five available strains of Crypthecodinium cohnii, identified as ATCC numbers 30021, 30334-30348, 3054130543, 30555-30557, 30571, 30572, 30772-30775, 30812, 40750, 50050-50060, and 50297-50300. As used herein, any microorganism, or any specific type of organism, includes natural, mutant, or recombinant strain types.
Aside from sodium, the concentrations of chloride and potassium that are the subject of the present invention and are discussed more fully below, other media components of the present invention may be any components known in the art that promote growth and DHA production at commercially practicable levels, and include components such as those described in U.S. Patent No. 5,130,242, U.S. Patent No. 5,407,957, US Patent Number 5,397,591, US Patent Number 5,492,938, and US Patent Number 5,711,983, all of which are incorporated by reference herein in their entirety. More specifically, a carbon source such as glucose, various starches, molasses, ground corn, and the like can be used. An assimilable organic or inorganic nitrogen source is also included in the culture medium. Nitrogen sources can
- 16 include nitrate, urea, amino acid ammonium salts and the like.
A source of microbial growth factors is also provided, which are unspecified or specified compounds that enhance the heterotrophic growth of single-celled microorganisms, and may include yeast or other extracts, soil extracts, and the like. Specific examples of growth media for C. cohnii and related organisms, for example, can also be found in Jiang and Chen, Process
Biochemistry 35 (2000) 1205-1209; Jiang and Chen, Journal of
Industrial Microbiology & Biotechnology, (1999) Vol. 23,508-513; Vazhappilly and Chen, Journal of the American Oil Chemists Society, (1998) Vol. 75, No. 3 p 393-397. Specific examples of preferred media for use with the present invention can be found, for example, in the Examples section of this document below.
In one aspect of the media of the present invention, chloride ion concentrations are present in a concentration less than or equal to about 2000 ppm or about 2
<td colspan="2">grams per liter of culture,</td><td>plus</td><td>preferably,</td><td>less</td><td> 0</td><td>same</td><td>than</td>
<td>about 1.9</td><td>g / L,</td><td>plus</td><td>preferably</td><td>less</td><td> 0</td><td>same</td><td>than</td>
<td>about 1.8</td><td>g / L,</td><td>plus</td><td>preferably</td><td>less</td><td>or</td><td>same</td><td>than</td>
<td>about 1.7</td><td>g / L,</td><td>plus</td><td>preferably</td><td>less</td><td> 0</td><td>same</td><td>than</td>
<td>about 1.6</td><td>g / L,</td><td>plus</td><td>preferably</td><td>less</td><td> 0</td><td>same</td><td>than</td>
<td>about 1.5</td><td>g / L,</td><td>plus</td><td>preferably</td><td>less</td><td> 0</td><td>same</td><td>than</td>
<td>about 1.4</td><td colspan="2">g / L, more</td><td>preferably i</td><td>less</td><td> 0</td><td>same</td><td>than</td>
<td>approximately</td><td> 1.3</td><td>g / L,</td>
<td>approximately</td><td> 1.2</td><td>g / L,</td>
<td>approximately</td><td> 1.1</td><td>g / L,</td>
<td>approximately</td><td> 1.0</td><td>g / L,</td>
<td>approximately</td><td> 0.9</td><td>g / L,</td>
<td>approximately</td><td> 0.8</td><td>g / L,</td>
<td>approximately</td><td> 0.7</td><td>g / L,</td>
<td>approximately</td><td> 0.6</td><td>g / L,</td>
<td>approximately</td><td> 0.5</td><td>g / L,</td>
<td>approximately</td><td> 0.4</td><td>g / L,</td>
more preferably more preferably more preferably more preferably more preferably more preferably more preferably more preferably more preferably and more preferably
MEXICAN INSTITUTE ¿i less oq less oi (JU3l qtre — less than or equal to less than or equal to less than or equal to less than or equal to less than or equal to less than or equal to less than or equal to less than or equal to about 0.3 g / L.
In alternative modalities, the minimum chloride concentration is at least 0.025 g / L, at least about 0.05 g / L, or at least about 0.1 g / L. The chloride ion component of the medium is preferably derived from a chloride salt, with a preferred salt being sodium chloride. Other sources of chloride in the media include potassium chloride and calcium chloride. Chloride ion sources can include more than one chloride-containing compound in the medium, and can include hydrochloric acid that can be used to adjust the pH of the media, as well as MnCl<sub>z</sub> and FeCI<sub>3</sub>.
In another aspect of the media of the present invention, the potassium ion concentration is greater than about 0.25 g / L. Potassium ion is generally present at low levels in seawater, being approximately 0.38 g / L of seawater. Culture media known in the art for the
- 18 MEXICAN INSTITUTE cultivation of marine microalgae, closely follow the coW | á ^ f? Á? Ón <^^ «^ seawater, with equal or lower potassium ion levels. For example, Tuttle and Loeblich (1975) describe 9 mM KCI, which is the equivalent of approximately 0.35 g / L potassium ion, In Handbook of Phycological Methods (Janet R. Stein, Ed., Cambridge University Press, 1973), the potassium ion in the medium is described to be 9.83 mM as potassium chloride, which is the equivalent of about 0.36 g / L potassium ion. In one embodiment, the present invention includes potassium ion in a concentration of more than about 0.39 g / L. The present inventors have found that once the potassium ion is greater than a threshold level, crops are relatively insensitive to the precise concentration of potassium ion, grow well, and produce commercially viable DHA levels, in a range of concentrations of potassium ion. Preferably, the lower potassium ion concentration range is at least about 0.2 g / L, at least about 0.25 g / L, at least about 0.3 g / L, at least about 0.35 g / L, at least about 0.4 g / L, at least about 0.45 g / L, at least about 0.5 g / L, at least about 0.6 g / L, and at least about 0.7 g / L. Preferably, the upper range of potassium ion concentration is at most about 10 g / L, at most about 6 g / L, at most about 4 g / L, at most about 3 g / L, at most
- 19 IMPI
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY about 2. 8 g / L, at most about 2.6 g / L,
<img file="MX338455B_D0013.tif" />
<td>as much</td><td>of</td><td>about 2.4</td><td>g / L, as</td><td>much</td><td>of</td>
<td colspan="2">about 2</td><td>.2 g / L, at most</td><td colspan="2">about 2</td><td>g / L,</td>
<td>as much</td><td>of</td><td>about 1.9</td><td>g / L, as</td><td>much</td><td>of</td>
<td colspan="2">about 1.</td><td>8 g / L at most</td><td colspan="2">about 1.7</td><td>g / L,</td>
<td>as much</td><td>of</td><td>about 1.6</td><td>g / L, as</td><td>much</td><td>of</td>
about 1.5g / L, and at most about 1g / L. The most preferred potassium ion concentrations are approximately 0.75 g / L, 0.8 g / L, 0.85 g / L, 0.9 g / L, and 0.95 g / L. The preferred ranges for the potassium ion are between about 0.45 g / L and about 1.5 g / L; more preferably between about 0.5 g / L and about 1.2 g / L; more preferably between about 0.6 g / L and about 1 g / L; even more preferably between about 0.7 g / L and about 0.9 g / L; and much more preferably about 0. 8 g / L.
The potassium ion source can be from any potassium salt compatible with cell culture and microalgae of the Dinophyceae class in particular. The potassium ion can be derived from a mixture of salts in the medium. Preferred potassium salts include potassium chloride, potassium sulfate, potassium acetate, potassium bicarbonate, potassium phosphate, among others. A preferred source of potassium ion is potassium sulfate.
In one aspect of the present invention, the amount of DHA production from the crops at harvest is greater than the
- 20 I ÍVS P!
MEXICAN FNSTITUTE
OF THE PROPERTY
INDUSTRIAL production amount of DHA not grown in media of the present invention. In one embodiment, DHA production using low chlorine concentrations by processes of the present invention is at least 0.2 grams DHA per liter culture days, or 0.04 g DHA / 10<sup>9</sup> cells.
In another aspect of the present invention, the media also contains additional sources of sodium ion other than sodium chloride. The present inventors have found that sodium ion levels are not critical to the present invention. The marine organism cultures of the present invention are relatively insensitive to the precise concentration of sodium ion, perform well, and produce commercially viable levels of DHA at a range of sodium ion concentrations. Many different sources of sodium are combattable with the present invention, including sodium sulfate, sodium carbonate, hydrogenated sodium carbonate, and sodium acetate. A preferred source of additional sodium ion is sodium sulfate. In a preferred embodiment, the medium contains at least about 1 g / L of onion sodium to about 8 g / L of onion sodium. At the low end of the scale, the preferred sodium ion concentration is at least about 1 g / L, at least about 1.5 g / L, at least about 2 g / L, and at least about 2.5 g / L. Preferably, the upper range of the sodium ion concentration is at most about 15 g / L, at most about 12 g / L, at most about 10
1Μ PT
INSTITUTO MEXICANO Κ, ξ ™ g / L, at most about 9 g / L, comd<sup>)The</sup>'1iA<sup>R</sup>Jt<sup>,</sup>cfe® effe · about 8 g / L, at most aprnYimadamenu 7 gil— at most about 6 g / L, at most about 5.5 g / L, at most about 5 g / L, at most about 4.5 g / L, at most about 4 g / L. Most preferred concentrations of sodium ion are approximately 2.75 g / L, 3 g / L, 3.25 g / L, 3.5 g / L, and 3.75 g / L. The preferred ranges for the sodium ion are between about 1.5 g / L and up to about 7.5 g / L, even more preferred from about 2.0 g / L to about 6 g / L, and even more preferred is from about 2.5 g / L up to about 5 g / l. In the most preferred embodiments, the sodium ion is at least from about 3 g / L to about 3.5 g / L. The most preferred sodium level is approximately 3.25 g / L. As described above, crops are relatively insensitive to precise sodium levels, and therefore even higher levels can be used. However, once sodium levels above about 8 g / L are used, crop yields begin to drop slightly.
In another embodiment, the present invention includes a method of producing DHA by cultivating heterotrophic Microalgae of the Dinophyceae class in a culture medium. The medium contains chloride ion in a concentration less than or equal to approximately 2 g / L, potassium ion in a concentration greater than or equal to
- 22 IMPI
MEXICAN INSTITUTE OF PROPERTY approximately 0.25 g / L, and sodium ion present in a p r'djSWc i ó
<img file="MX338455B_D0014.tif" />
less than or equal to about 27: 1 SDGiu.pOta-ei - ^ - weight: weight. In this modality, the microalgae produce at least approximately 0.2 g of DHA per liter of culture of 7 days, or 0.04 g of DHA / 10<sup>9</sup> cells. In this embodiment, the culture medium contains sodium in a ratio with potassium ion, less than or equal to approximately 27: 1, weight / weight. In seawater, the ratio of sodium ion to potassium ion is approximately 27.3: 1. In other words, the amount of sodium ion is approximately 27.3 times greater than the amount of potassium ion, in the present invention, the inventors have found that increasing the potassium ion relative to the sodium ion increases the DHA production of the culture. A preferred ratio of sodium ion to potassium ion less than or equal to about 27: 1, less than or equal to about 25: 1, less than or equal to about 23: 1, less than or equal to about 21: 1, less than or equal that approximately 19: 1. The most preferred ratios are less than or equal to about 17: 1, less than or equal to about 15: 1, less than or equal to about 13: 1, less than or equal to about 11: 1. Even more preferred ratios are less than or equal to about 9: 1, less than or equal to about 7: 1, or less than or equal to about 5: 1. A preferred ratio is approximately 4: 1.
In another embodiment, the present invention includes a
- 23 IMPI
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY method for producing DHA by cultivating heterotrophic microalgae of the Dinophyceae class in a culture medium, where the culture medium has a pH of less than about 6, and where the microalgae produce at least about 0.2 DHA per liter of 7-day culture or 0.04 g DAH / 109 cells. In a preferred embodiment, the pH is less than or equal to about 5.5, and more preferably less than or equal to about 5. In a preferred embodiment, the pH is less than or equal to about 4.5. In a preferred embodiment, the medium further contains a chloride ion concentration of less than or equal to about 2 g / L, preferably less than or equal to about 1 g / L, even more preferably less than or equal to 0.3 g / L. The medium also preferably contains potassium ion in concentrations greater than or equal to approximately 0.25 g / L, greater than or equal to approximately 0.4 g / L, and even more preferably it is greater than or equal to approximately 0.8 g / L. Preferably, the potassium ion source is potassium sulfate. In a preferred embodiment, the medium further contains a sodium ion source which is potassium sulfate. In a preferred embodiment, the medium further contains a sodium ion source such that the sodium ion concentration is from about 1 g / l to about 8 g / L. More preferably, the sodium ion is from about 1.5 g / l to about 5 g / L. A preferred source of sodium ion is sodium sulfate. In this modality a biomass produced by this method is included.
<img file="MX338455B_D0015.tif" />
<img file="MX338455B_D0016.tif" />
OF THE PROPERTY
In another embodiment, the present invention method for the preparation of pll-low tolerant strains of species of the Dinophyceae class and strains thus produced. Methods include preparing low pH media and subculturing the desired Dinophyceae species until the culture produces a desired amount of DHA. Subcultures can be carried out as follows. An inoculum of the desired Dinophyceae species is placed in the low pH medium, and allowed to grow for a defined amount of time, preferably 7 days. The amount of time is not critical, but it should be chosen in such a way that the strain has enough time to grow, but before it reaches senescence. DHA production of the culture is calculated. If it is less than the desired amount, an additional subculture is performed as follows. Fresh medium with low pH is prepared and inoculated with the culture grown in low pH, and incubated for an appropriate amount of time. DHA production of the culture is calculated. If DHA production is less than the desired amount, subculture is repeated until the desired amount of DHA is achieved. A preferred pH to select for tolerance is about 6 or less, more preferably about 5.5 or less, even more preferably about 5 or less, and still more preferable of about 4.5 or less. The means in which to carry out this method are any means known in the art, with the pH adjusted to the desired levels. A preferred medium in
- 25 ρ s * »„
INSTITUTO MEXICANO DE LA PRONEDa INDUSTRIAL, which carry out sub-cultivation is the medium described in example 1.
The present invention also includes a biomass produced by one of the methods of the invention.
Culture conditions consistent with the organisms and methods of the present invention can be accomplished by methods known in the art, and include the methods described in U.S. Patent No. 5,130,242, in U.S. Patent No. 5,407,957, in U.S. Patent No. 5,397,591, in US Patent Number 5,492,938; and in US Patent No. 5,711,983, and optimal conditions can be readily determined by those skilled in the art. Briefly, cultivation can be accomplished in any appropriate heater, preferably either in a stirred tank heater, or in an air-shaker heater, which provide a source of oxygen for the microorganisms. The agitation of the microorganism must be maintained at a level such that while the dissolved oxygen concentrations are sufficient to support the growth of the culture and the production of DHA, the agitation does not cut or otherwise harm the microorganisms. Preferred dissolved oxygen levels are at least 10% of the saturation level in air. More preferably, dissolved oxygen levels are maintained from about 10% to about 50% of saturation levels in the air.
The cultivation can be carried out at any temperature
- 26 that provides life support.
<img file="MX338455B_D0017.tif" />
Generally, microorganisms will grow at temperatures ranging from about 15 ° C to about 34 ° C.
Preferably the temperature is maintained at about 20 ° C to about 28 ° C.
Organisms can be grown by conventional means, familiar to those of ordinary skill in the art, such as centrifugation, flocculation, or filtration, and can be processed immediately, or dried for future processing. In either case, lipid can be removed. As used herein, the term "lipid" includes phospholipids, free fatty acids, fatty acid esters, thiacylglycerols, diaz I g I icé dos, monoacylglycerides, lysophospholipids, soaps, phosphatides, sterols and esters of sterol, carotenoids, xanthophylls ( for example, oxycarotenoids), hydrocarbons, and other lipids familiar to a person skilled in the art. As is well understood by a person skilled in the art, the DHA to which the present invention refers, can take the form of these various lipids, and is not limited to free fatty acid. Different types of components can be extracted, depending on the extraction technique used. Lipids can be extracted with an effective amount of solvent. Appropriate solvents can be determined by those skilled in the art. Polar lipids (eg, phospholipids) are generally extracted with polar solvents (eg, chloroform / methanol), and neutral lipids (eg, triacylglycerols) are generally extracted with non-solvents.
- 27 IΜ PI
MEXICAN INSTITUTE OF PROPERTY
INDUSTRIAL '7 ^ polar (eg hexane). A preferred solvent is pure hexane. An appropriate ratio of hexane to dry biomass is approximately liters of hexane per kilogram of dry biomass. Hexane is preferably mixed with the biomass in a stirred reaction vessel at a temperature of about 50 ° C for about 2 hours. After mixing, the biomass is filtered and separated from the oil-containing hex.
Hexane is separated from the oil by distillation techniques familiar to those skilled in the art.
Conventional oilseed processing equipment is suitable for filtering, separating and distilling.
Additional processing steps, familiar to those of ordinary skill in the art, can be performed if required or desirable for a particular application. Alternative methods for lipid recovery are described in the following references, which are incorporated herein by reference in their entirety: TCP Publication WO 0176715, entitled Method for Fractionation of Natural Oil-Containing Raw Materials and Polar Lipids; TCP Publication WO 0176385, entitled Method for Fractionation of Natural Oil-Containing Raw Materials and Polar Lipids Using Alcohol and Centrifugation; TCP publication WO 0153512, entitled Solvent-free extraction process.
The present invention, while described in terms of specific organisms and methods, is intended to include all those
IMPI
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY methods and strains obtainable and useful in accordance with the teachings described herein, including all substitutions, modifications and optimizations of this type, which may be available, convenient for those skilled in the art. The following examples and test results are provided for the purpose of illustration and are not intended to limit the scope of the invention.
EXAMPLE 1
This example describes the preparation of Standard Detection Medium (SSM) with 4.5 g / L NaCl. To prepare the media, the first step includes adding the following compounds to distilled water up to 90% of the desired final volume as shown in Table 1. All compounds are available from Sigma Aldrich, St. Louis, MO.
TABLE 1. QUANTITIES AND FINAL CONCENTRATIONS OF
MEANS BEFORE SUBMITTING AN AUTOCLAVE.
<td>Compound</td><td>Final concentration</td><td>Amount of chloride ion added (g / l)</td><td>Amount of potassium ion added (g / l)</td><td>Amount of added sodium ion (g / l)</td>
<td>CaCI<sub>2</sub>-2H<sub>2</sub>OR<sup>1</sup></td><td>0.3 g / l</td><td> 0.09</td><td></td><td></td>
<td>MgSO<sub>4</sub>-7H<sub>2</sub>OR</td><td>1.25 g / l</td><td></td><td></td><td></td>
<td>NaCl</td><td>4.5 g / l</td><td> 3</td><td></td><td> 1.5</td>
<img file="MX338455B_D0018.tif" />
MEXICAN INSTITUTE
FROM LA PROriEDAL> INDUSTRIAL
<img file="MX338455B_D0019.tif" />
<td rowspan="2">Compound</td><td rowspan="2">Concentration final</td><td rowspan="2">Amount of chloride ion added (g / l)</td><td rowspan="2">Amount of potassium ion added (g / l)</td><td>Amount of</td>
<td>- “'ícTnsódio added (g / l)</td>
<td>MONTH</td><td>10.7 g / l</td><td></td><td></td><td></td>
<td>MSG</td><td>1.5 g / l</td><td></td><td></td><td></td>
<td>Tastona 154</td><td>0.5 g / l</td><td></td><td></td><td></td>
<td>KH<sub>2</sub>PO<sub>4</sub></td><td>0.014 g / l</td><td></td><td> .004</td><td></td>
<td>KCI</td><td>0.14 g / l</td><td> 0.067</td><td> 0.073</td><td></td>
<td>Cu7SO<sub>4</sub>-5H<sub>2</sub>OR</td><td>0.15X10 '<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>CoCI<sub>2</sub>-6H<sub>2</sub>OR</td><td>0.3X10 '<sup>3</sup> g / l</td><td>Insignificant</td><td></td><td></td>
<td>h<sub>3</sub>bo<sub>3</sub></td><td>10X10 '<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>MnCI<sub>2</sub>-4H<sub>2</sub>OR</td><td>4.5X10<sup>3</sup> g / l</td><td>Insignificant</td><td></td><td></td>
<td>ZnSO<sub>4</sub>-7H<sub>2</sub>OR</td><td>0.3X10 '<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>NaOH (to adjust the pH to 6.3)</td><td>1.16 g / l</td><td></td><td></td><td> 0.67</td>
<td>FeCI<sub>2</sub><sup>2</sup></td><td>6X10 '<sup>3</sup> g / l</td><td>Insignificant</td><td></td><td></td>
<td>Thiamine<sup>3</sup></td><td>1X10 '<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>Biotin<sup>3</sup></td><td>2X10 '<sup>6</sup> g / l</td><td></td><td></td><td></td>
<td>Glucose<sup>4</sup></td><td>50 g / l</td><td></td><td></td><td></td>
<td>Total of each ion</td><td></td><td> 3.16</td><td> 0.08</td><td> 2.17</td>
<sup>1</sup> calcium chloride dihydrate is 244 g / mol with 28.7% chloride.
<img file="MX338455B_D0020.tif" />
MEXICAN INSTITUTE <sub>?</sub> FROM PROPERTY the stock solution was autoclaved by ^ epara and added in a sterile form to the media after autoelewo; · made fresh every two weeks.
<sup>3</sup> the filter of the mother solution was sterilized through a 0.2 micron filter; stored at 4 ° C in the dark. It was added sterile to the media after autoclaving.
<sup>4</sup> the stock solution was autoclaved separately.
It was added sterile to the media after autoclaving.
Autoclaved media was brought up to 100% volume with sterile water. For detection experiments, 35 mL of SSM medium was added to 250 mL sterile Erlenmeyer flasks. 1 mL of inoculum per flask was added for an initial cell concentration of 1 x 10<sup>5</sup> cells per mL. The inoculum is cultivated from 5 to 6 days old. Cultures are grown at 26.5 ° C on a rotary shaker at 135 rpm.
EXAMPLE 2
This example describes the preparation of Detection Medium (SSM) with 1000 ppm of chloride ion, with 1.41 g / L of NaCl (which together with calcium chloride and potassium chloride results in approximately 1000 ppm, 1 g / L of with chloride). To prepare the media, the first step includes adding the following compounds to deionized distilled water up to 90% of the desired final volume, as shown in the Table
2.
compounds are available from Sigma Aldrich, St. LthjIS, IVIO "
TABLE 2. FINAL AMOUNTS AND CONCENTRATIONS OF
MEANS BEFORE SUBMITTING AN AUTOCLAVE
<td>Compound</td><td>Final concentration</td><td>Amount of chloride ion added (g / l)</td><td>Amount of potassium ion added (g / l)</td><td>Amount of added sodium ion (g / l)</td>
<td>CaCI<sub>2</sub>-2H<sub>2</sub>OR</td><td>0.3 g / l</td><td> 0.09</td><td></td><td></td>
<td>MgSO<sub>4</sub>-7H<sub>2</sub>OR</td><td>1.25 g / l</td><td></td><td></td><td></td>
<td>NaCI</td><td>1.41- g / l</td><td> 0.85</td><td></td><td> 0.047</td>
<td>MONTH</td><td>10.7 g / l</td><td></td><td></td><td></td>
<td>MSG</td><td>1.5 g / l</td><td></td><td></td><td></td>
<td>Tastona 154</td><td>0.5 g / l</td><td></td><td></td><td></td>
<td>KH<sub>2</sub>PO<sub>4</sub></td><td>0.014 g / l</td><td></td><td> 0.004</td><td></td>
<td>KCI</td><td>0.14 g / l</td><td> 0.067</td><td> 0.073</td><td></td>
<td>What<sub>4</sub>-5H<sub>2</sub>OR</td><td>0.15X10 '<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>CoCI<sub>2</sub>-6H<sub>2</sub>0</td><td>0.3X10 '<sup>3</sup> g / l</td><td>Insignificant</td><td></td><td></td>
<td>h<sub>3</sub>bo<sub>3</sub></td><td>10X10 '<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>MnCI<sub>2</sub>-4H<sub>2</sub>OR</td><td>4.5X10 '<sup>3</sup> g / l</td><td>Insignificant</td><td></td><td></td>
<td>ZnSO<sub>4</sub>-7H<sub>2</sub>OR</td><td>0.3X10 '<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>NaOH (to adjust the pH to 6.3)</td><td>1.6 g / l</td><td></td><td></td><td> 0.67</td>
- 32 MEXICAN INSTITUTE PE LA PROPIEDAD V * ·
<td rowspan="2">Compound</td><td rowspan="2">Concentration final</td><td rowspan="2">Amount of chloride ion added (g / l)</td><td rowspan="2">Amount of potassium ion added (g / l)</td><td>Quantity<sup>IAL</sup></td>
<td>added (g / l)</td>
<td>FeCÍT</td><td>6X10``g / l</td><td>Insignificant</td><td></td><td></td>
<td>Thiamine<sup>2</sup></td><td>1 XW<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>Biotin<sup>2</sup></td><td>2X10 '<sup>6</sup> g / l</td><td></td><td></td><td></td>
<td>Glucose<sup>3</sup></td><td>50 g / l</td><td></td><td></td><td></td>
<td>Total of each</td><td></td><td> 1.00</td><td> 0.08</td><td> 1.14</td>
<td>ion</td><td></td><td></td><td></td><td></td>
<img file="MX338455B_D0021.tif" />
<sup>1</sup> the stock solution was autoclaved separately, and added in a sterile form to the media after the autoclave; it gets fresh every two weeks.
<sup>2</sup> the filter of the mother solution was sterilized through a 0.2 micron filter; stored at 4'C in the dark. It was added sterile to the media after autoclaving.
<sup>3</sup> the stock solution was autoclaved separately.
It was added sterile to the media after autoclaving.
Autoclaved media was brought up to 100% volume with sterile water. For detection experiments, 35 mL of SSM medium was added to 250 mL sterile Erlenmeyer flasks. 1 mL of inoculum per flask was added for an initial cell concentration of 1 x 10<sup>5</sup> cells per mL. The inoculum is cultivated from 5 to 6 days old. Cultures are grown at 26.5 ° C on a rotary shaker at 135 rpm.
- 33 IMP
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY
<img file="MX338455B_D0022.tif" />
EXAMPLE 3
This example describes the preparation of Detection Medium (SSM) with 300 ppm chloride ion with 0.211 g / L NaCI (which together with calcium chloride and potassium chloride results in 0.3 g / L chloride ion). To prepare the media, the first step includes- adding the following compounds to deionized distilled water up to 90% of the desired final volume, as shown in Table 3. All compounds are available from Sigma Aldrich, St. Louis, MO.
TABLE 3. QUANTITIES AND FINAL CONCENTRATIONS OF MEDIA BEFORE SUBMITTING THEM TO AUTOCLAVE
<td>Compound</td><td>Final concentration</td><td>Amount of chloride ion added (g / l)</td><td>Amount of potassium ion added (g / l)</td><td>Amount of added sodium ion (g / l)</td>
<td>CaCI<sub>2</sub>-2H<sub>2</sub>OR</td><td> 0.3</td><td> 0.09</td><td></td><td></td>
<td>MgSO<sub>4</sub>-7H<sub>2</sub>OR</td><td> 1.25</td><td></td><td></td><td></td>
<td>NaCI</td><td> 0.211</td><td> 0.13</td><td></td><td> 0.07</td>
<td>MONTH</td><td> 10.7</td><td></td><td></td><td></td>
<td>MSG</td><td> 1.5</td><td></td><td></td><td></td>
<td>Tastona 154</td><td> 0.5</td><td></td><td></td><td></td>
<td>KH<sub>2</sub>PO<sub>4</sub></td><td> 0.014</td><td></td><td> 0.004</td><td></td>
<img file="MX338455B_D0023.tif" />
<td>Compound</td><td>Final concentration</td><td>Amount of chloride ion added (g / l)</td><td>Amount of potassium ion added (g / l)</td><td>Ion quantity<sup>1</sup> sodium -added (g / l)</td>
<td>KCI</td><td> 0.14</td><td> 0.067</td><td> 0.073</td><td></td>
<td>What<sub>4</sub>-5H<sub>2</sub>OR</td><td> 0.15</td><td></td><td></td><td></td>
<td>CoCI<sub>2</sub>-6H<sub>2</sub>0</td><td> 0.3</td><td>Insignificant</td><td></td><td></td>
<td>h<sub>3</sub>bo<sub>3</sub></td><td> 10</td><td></td><td></td><td></td>
<td>MnCI<sub>2</sub>-4H<sub>2</sub>OR</td><td> 4.5</td><td>Insignificant</td><td></td><td></td>
<td>ZnSO<sub>4</sub>-7H<sub>2</sub>OR</td><td> 0.3</td><td></td><td></td><td></td>
<td>NaOH (to adjust the pH to 6.3)</td><td> 1.16</td><td></td><td></td><td> 0.67</td>
<td>FeCI<sub>2</sub><sup>1</sup></td><td> 6</td><td>Insignificant</td><td></td><td></td>
<td>Thiamine<sup>2</sup></td><td> 1</td><td></td><td></td><td></td>
<td>Biotin<sup>2</sup></td><td> 2</td><td></td><td></td><td></td>
<td>Glucose<sup>3</sup></td><td> 50</td><td></td><td></td><td></td>
<td>Total of each ion</td><td></td><td> 0.30</td><td> 0.08</td><td> 0.74</td>
<sup>1</sup> the stock solution was autoclaved separately, and added in a sterile form to the media after autoclaving; it gets fresh every two weeks.
<sup>2</sup> the filter of the mother solution was sterilized through a 0.2 micron filter; stored at 4 ° C in the dark. It was added sterile to the media after autoclaving.
<sup>3</sup> the stock solution was autoclaved separately.
- 35 IMPI
MEXICAN INSTITUTE
OF THE PRUFIF.DAt »C'— JSií INDUSTRIAL
It was added sterile to the media after autoclaving.
Autoclaved media was brought up to 100% volume with sterile water. For detection experiments, 35 mL of SSM medium was added to 250 mL sterile Erlenmeyer flasks. 1 mL of inoculum per flask was added for an initial cell concentration of 1 x 10<sup>5</sup> cells per mL. The inoculum is cultivated from 5 to 6 days old. Cultures are grown at 26.5 ° C on a rotary shaker at 135 rpm.
EXAMPLE 4
This example describes the procedure for culture and harvesting of Crypthecodinium cohnii in SSM with pH 6.3.
Media as described in one of Examples 1 to 3 were prepared, depending on which medium was to be tested. Additional media components were prepared and added to a medium as described in Examples 1 to 3. All steps prior to harvesting were carried out under sterile conditions.
To prepare the inoculum culture, the following procedures were used. To a 250 mL Erlenmeyer flask, 49 mL of SSM (described in Example 1) was added. 1 mL of 5-day-old C. cohnii strain T-HF culture was added to the Erlenmeyer flask (the T-HF strain identifies the ATCC 40750 organism that has
<img file="MX338455B_D0024.tif" />
IMPI
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY has been repeatedly cultivated). The flask with the culture was placed on a rotary shaker at 135 rpm in an incubator at 27 ° C without lights. After three days of development, the culture was moved to a sterile hood and 1 mL was taken out and counted using a Coulter counter (Coulter Z2 Particle Count and Size Analyzer, obtained from Beckman Coulter, Inc.). The cell count was used to calculate the amount of the inoculum culture that had to be used to start a new 50 mL culture with a cell density of 1.0 x 10<sup>5</sup> cells per mL.
To test the different media components, appropriate media were prepared as described below, and placed in a 250 mL sterile Erlenmeyer flask. The amount of inoculum, as previously calculated, was transferred to the culture flask containing the media prepared in the Erlenmeyer flask. The culture flask was placed on a rotary shaker at 135 rpm in an incubator at 27 ° C without lights. After seven days of growth, the culture was collected as follows.
A 50 mL centrifuge tube (obtained from VWR Scientific) was labeled for each culture, and weighed. Another 50 mL centrifuge tube was marked, but not weighed, for each culture. The culture was emptied into the labeled 50 mL tube. Volumes were recorded and cell counts were performed with the Coulter Z2 Particle Count and Size Analyzer. PH was measured.
Half the culture was emptied into the 50 mL tarred tube, and a 70% alcohol solution was added
- 37 MEXICAN INSTITUTE OF PROPERTY C isopropyl (IPA, for acronym in English) for friction, to ^ ^ Have total volume in the tube up to 50 mL. The culture was mixed by inverting the tube two to three times. The culture was then centrifuged at 4000 rpm for 5 minutes using a Sorvall General Purpose RC-3 centrifuge. The supernatant was removed, The other half of the culture was emptied onto the granulate, and the steps were repeated starting with the 70% IPA solution. The pellet was then washed twice with 39% IPA using the following procedure: To the cell pellet, 35 mL of 39% IPA is added, the tube is vortexed (using a VWR Scientific Genie-2 Vortex) at speed total for 10 seconds; after harvesting, the granulate was freeze dried for at least hours.
The tube containing the granulate (biomass) was weighed and the dry weight of the biomass was calculated. The dry weight was calculated as follows: the weight of the tube containing the biomass minus the tare weight of the tube is determined. This number is divided by the volume of cultivation recorded at the time of collection, divided by 1000.
Fatty acid composition (and% DHA) can be determined according to procedures described in Morrison and Smith, Preparation of Fatty Acid Methyl Esters and Dimethylacetals from Lipids with Boron Fluoride-Methanol, Journal of Lipid Research, Vol. 5, 1964, and the official methods of the American Oil Chemist's Society used to quantify long-chain fatty acids and eicosapentaenoic acid (EPA) and DHA in
- 38 marine oils (Celb-89 Method).
<img file="MX338455B_D0025.tif" />
Briefly, samples are mixed with standard amounts of oil (internal standards), saponified with 0.5N methanolic sodium hydroxide, and derived with boron trifluoride / methanol. The fatty acid methyl esters are extracted and analyzed on a gas chromatograph with a flame ionization detector (Hewlett Packard 5890 Series II Plus gas chromatograph, using a 30 mx 0.25 mm x 0.25 pm Restek FAMEWAX # 12497 column).
EXAMPLE 5
This example describes the cultivation of C. cohnii and the production of DHA at low NaCI levels, using prior art means.
One liter of SSM containing no NaCI was made and autoclaved. Four stock solutions of concentrated NaCI (135 g / L, 90 g / L, 45 g / L and 22.5 g / L) were prepared. To each shake flask containing 48.75 mL of SSM minus NaCI, medium and 1.25 mL of the appropriate NaCI stock solution were added. Two controls were established: 4.5 g / L NaCI using normal SSM as described in Example 1, and whether NaCI using SSM without added NaCI. Duplicates of each level of NaCI were used.
Cultivation and harvesting was performed as described in Example 4. Table 5 describes the results of this example.
All quantities are given as an average of two crops.
<img file="MX338455B_D0026.tif" />
TABLE 5. BIOMASS,% DHA,% FAT, AND ρΊΓρΗΑ 'PRODUCTION FOR C. cohnii CULTIVATED IN SSM WITH DECREASED AMOUNTS OF NaCl
<td>NaCl g / l</td><td>Ion chloride g / l</td><td>Biomass dry weight (g / l)</td><td>% DHA in fat (weight / weight)</td><td>% of fat in biomass (weight / weight)</td>
<td> 4.5</td><td> 2.73</td><td> 3.53</td><td> 51.63</td><td> 52.45</td>
<td> 3.38</td><td> 2.05</td><td> 3.66</td><td> 51.55</td><td> 47.83</td>
<td> 2.25</td><td> 1.37</td><td> 3.85</td><td> 52.19</td><td> 48.40</td>
<td> 1.73</td><td> 0.68</td><td> 2.73</td><td> 54.65</td><td> 54.59</td>
<td> 0.56</td><td> 0.34</td><td> 2.70</td><td> 55.48</td><td> 48.81</td>
<td> 0</td><td> 0</td><td> 1.99</td><td> 51.00</td><td> 34.19</td>
<sup>1</sup> Reflects the amount of chloride ion (0.20 g / L) of sodium chloride only. See examples 1 to 3.
Table 5 shows biomass production,% fat, and DHA production for C. cohnii grown in SSM containing decreased amounts of NaCl. It can be seen that as the amount of NaCl added to the culture decreases, both biomass production and fat levels decrease, resulting in decreased DHA production.
- 40 This example describes the production of DHA achieved with
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MEXICAN INSTITUTE OE INDUSTRIAL PROPERTY
<img file="MX338455B_D0027.tif" />
EXAMPLE 6
4.5 g / L of NaCl in the culture medium described in Example 1.
The cultures were grown as described in Example 4. Table 6 shows the results of this example.
TABLE 6.
BIOMASS,% DHA,% FAT AND DHA PRODUCTION
BY C. cohnii CULTIVATED IN SSM FROM EXAMPLE 1
<td>Sodium chloride (g / i)</td><td>Sodium sulfate (g / i)</td><td>Chloride ion<sup>1</sup>(g / i)</td><td>Sodium ion (g / i)</td><td>Fat DHA% (w / w)</td><td>% of fat in biomass (weight / weight)</td><td>Biomass dry weight (g / i)</td>
<td> 4.3</td><td></td><td> 2.73</td><td> 1.77</td><td> 53.9</td><td> 65.03</td><td> 3.1</td>
<sup>1</sup> Reflects the amount of sodium chloride chloride ion only.
EXAMPLE 7
This example describes the enhanced growth of C. cohnii and the production of DHA in low chloride medium using various concentrations of potassium ion and sodium ion in the form of potassium sulfate and sodium sulfate.
The low chloride SSM was prepared as described in Example 3, using 0.18 g / L of acetate
IMPI ^ a
INSTITUTO MEXICani) OF THE INDUSTRIAL PROPERTY of calcium and omitting calcium chloride and chloride concentrations of K<sub>2</sub>SW<sub>4</sub> of
0.16 g / L, 0.80 g / L, 1.6 g / L, 3.2 g / L, and
4.8 g / L, tested against Na concentrations<sub>2</sub>SW<sub>4</sub> of
4.9 g / L, 9.8 g / L, 14.7 g / L,
19.6 g / L, and 24.5 g / L using a matrix cultures were grown as
7.
TABLE 7. BIOMASS COMPARISON,% DHA,% FAT, AND
DHA PRODUCTION OBTAINED FOR C. cohnii CULTIVATED IN
MEDIA WITH VARIABLE CONCENTRATIONS OF SULPHATE OF
POTASSIUM AND SODIUM SULPHATE
<td>Flask</td><td>K<sub>2</sub>SW<sub>4</sub>(g / D</td><td>Ν32θθ4 (g / D</td><td>Sodium ion<sup>1</sup>(g / D</td><td>Potassium ion (g / D</td><td>DWg / l</td><td>Fat DHA% (w / w)</td><td>% of fat in biomass (weight / weight)</td>
<td> 1</td><td> 0.16</td><td> 4.90</td><td> 1.77</td><td> 0.07</td><td> 2.55</td><td> 57.97</td><td> 60.80</td>
<td> 2</td><td> 0.16</td><td> 9.8</td><td> 3.35</td><td> 0.07</td><td> 1.53</td><td> 52.39</td><td> 41.45</td>
<td> 3</td><td> 0.16</td><td> 14.70</td><td> 4.93</td><td> 0.07</td><td> -</td><td> -</td><td> -</td>
<td> 4</td><td> 0.16</td><td> 19.60</td><td> 6.53</td><td> 0.07</td><td> 0.75</td><td> 42.88</td><td> 13.28</td>
<td> 5</td><td> 0.16</td><td> 24.50</td><td> 8.11</td><td> 0.07</td><td> .71</td><td> 41.46</td><td> 12.11</td>
<td> 6</td><td> 0.80</td><td> 4.90</td><td> 1.77</td><td> 0.36</td><td> 3.79</td><td> 56.76</td><td> 63.19</td>
<td> 7</td><td> 0.8</td><td> 9.80</td><td> 3.35</td><td> 0.36</td><td> 4.03</td><td> 55.11</td><td> 64.96</td>
<td> 8</td><td> 0.80</td><td> 14.70</td><td> 4.93</td><td> 0.36</td><td> 3.66</td><td> 55.14</td><td> 64.39</td>
<td> 9</td><td> 0.80</td><td> 19.60</td><td> 6.52</td><td> 0.36</td><td> 3.07</td><td> 56.88</td><td> 58.12</td>
<img file="MX338455B_D0028.tif" />
<td>Flask</td><td>K<sub>2</sub>SW<sub>4</sub>(g / l)</td><td>Na<sub>2</sub>SW<sub>4</sub>(g / i)</td><td>Sodium ion<sup>1 </sup>(g / i)</td><td>Potassium ion (g / i)</td><td>DWg / l</td><td>Fat DHA% (w / w)</td><td>% of fat in biomass (weight / weight)</td>
<td> 10</td><td> 0.80</td><td> 24.50</td><td> 8.11</td><td> 0.36</td><td> 2.91</td><td> 57.37</td><td> 53.65</td>
<td> 11</td><td> 1.60</td><td> 4.90</td><td> 1.77</td><td> 0.72</td><td> 3.74</td><td> 55.90</td><td> 63.46</td>
<td> 12</td><td> 1.60</td><td> 9.80</td><td> 3.35</td><td> 0.72</td><td> 3.83</td><td> 55.00</td><td> 65.43</td>
<td> 13</td><td> 1.60</td><td> 14.70</td><td> 4.93</td><td> 0.72</td><td> 3.49</td><td> 56.48</td><td> 60.09</td>
<td> 14</td><td> 1.60</td><td> 19.60</td><td> 6.53</td><td> 0.72</td><td> 3.18</td><td> 54.71</td><td> 54.92</td>
<td> 15</td><td> 1.60</td><td> 24.50</td><td> 8.11</td><td> 0.72</td><td> 2.83</td><td> 54.82</td><td> 49.02</td>
<td> 16</td><td> 3.20</td><td> 4.90</td><td> 1.77</td><td> 1.44</td><td> 3.51</td><td> 54.42</td><td> 63.99</td>
<td> 17</td><td> 3.20</td><td> 9.80</td><td> 3.35</td><td> 1.44</td><td> 3.36</td><td> 55.40</td><td> 61.12</td>
<td> 18</td><td> 3.20</td><td> 14.70</td><td> 4.93</td><td> 1.44</td><td> 3.40</td><td> 55.61</td><td> 59.34</td>
<td> 19</td><td> 3.20</td><td> 19.60</td><td> 6.53</td><td> 1.44</td><td> 3.07</td><td> 57.07</td><td> 59.44</td>
<td> 20</td><td> 4.80</td><td> 24.50</td><td> 8.11</td><td> 1.44</td><td> 2.77</td><td> 57.00</td><td> 57.07</td>
<td> 21</td><td> 4.80</td><td> 4.90</td><td> 1.77</td><td> 2.15</td><td> 2.82</td><td> 54.94</td><td> 57.43</td>
<td> 22</td><td> 4.80</td><td> 9.80</td><td> 3.35</td><td> 2.15</td><td> 2.81</td><td> 53.97</td><td> 58.12</td>
<td> 23</td><td> 4.80</td><td> 14.70</td><td> 4.93</td><td> 2.15</td><td> 2.94</td><td> 54.26</td><td> 58.75</td>
<td> 24</td><td> 4.80</td><td> 19.60</td><td> 6.52</td><td> 2.15</td><td> 2.92</td><td> 55.53</td><td> 56.88</td>
<td> 25</td><td> 4.80</td><td> 24.50</td><td> 8.11</td><td> 2.15</td><td> 2.50</td><td> 57.02</td><td> 53.00</td>
<sup>1</sup> Reflects the amount of sodium chloride chloride ion only.
The results shown in Table 7 indicate that increased potassium levels caused growth and
<td></td><td>IMPI ^ MEXICAN INSTITUTE t> E LA RRORIEDAD CVu, ·. · ...; INDUSTRIAL </td>
<td>production</td><td>of DHA by C. cohnii comparable to that achieved with</td>
high levels of chloride, the improvement effect of this example appeared at 0.8 g / L potassium sulfate, the second lowest level tested, and was thereafter relatively
<td>insensitive</td><td>to the amounts of potassium sulfate. On the levels</td>
Highest potassium sulfate tested, 4.8 g / L, appeared to have a slight decrease in performance. DHA growth and production also appeared relatively insensitive to the amount of sodium sulfate used, however growth and production fell slightly as increasing amounts of sodium sulfate were used, starting at approximately
19.6 g / L sodium sulfate. The best combinations based on the amount of DHA in g / L were those that used: 0.8 g / L of K2SO4, and 9.8 g / L of Na2SO4, representing a 5x increase in potassium and a 2x increase in sodium over SSM normal low in chloride (described in example 3); and 1.6 g / L K2SO4 and 9.8 g / L Na2SO4, representing a 10x increase in potassium and a 2x increase in sodium over normal low chloride SSM (described in Example 3).
EXAMPLE 8
This example demonstrates the enhancement of C. cohnii growth and DHA production using media containing a range of potassium sulfate, 0.32 g / L, 0.64 g / L, 0.96 g / L, 1.28 g / L, 1.60 g / L and 1.9 g / L, and sodium sulfate at 4.9 g / L and 9. 8 g / L.
- 44 The low chloride SSM was prepared in the manner described in Example 7, and all cultures were grown as described in Example 4. The results are presented in Table 8.
<img file="MX338455B_D0029.tif" />
TABLE 8. COMPARISON OF BIOMASS,% DHA,% FAT AND DHA PRODUCTION OBTAINED FOR C. cohnii CULTIVATED IN MEDIA WITH VARIABLE CONCENTRATIONS OF POTASSIUM SULPHATE AND SODIUM SULPHATE
<td>Flask</td><td>K<sub>2</sub>SW<sub>4</sub>(g / l)</td><td>Na<sub>2</sub>SW<sub>4</sub>(g / i)</td><td>DWg / l</td><td>% DHA in fat (weight / weight)</td><td>% of fat in biomass (weight / weight)</td><td>Sodium ion<sup>1 </sup>(g / i)</td><td>Potassium ion (g / i)</td>
<td> 1</td><td> 0.32</td><td> 4.90</td><td> 3.22</td><td> 57.76</td><td> 75.22</td><td> 1.77</td><td> 0.14</td>
<td> 2</td><td> 0.32</td><td> 9.80</td><td> 3.05</td><td> 57.61</td><td> 66.15</td><td> 3.35</td><td> 0.14</td>
<td> 3</td><td> 0.64</td><td> 4.90</td><td> 3.49</td><td> 58.66</td><td> 61.45</td><td> 1.77</td><td> 0.29</td>
<td> 4</td><td> 0.64</td><td> 9.80</td><td> 3.47</td><td> 58.50</td><td> 63.22</td><td> 3.35</td><td> 0.29</td>
<td> 5</td><td> 0.96</td><td> 4.90</td><td> 3.43</td><td> 58.45</td><td> 59.98</td><td> 1.77</td><td> 0.43</td>
<td> 6</td><td> 0.96</td><td> 9.80</td><td> 3.66</td><td> 51.91</td><td> 58.03</td><td> 3.35</td><td> 0.43</td>
<td> 7</td><td> 1.28</td><td> 4.90</td><td> 3.51</td><td> 58.72</td><td> 58.67</td><td> 1.77</td><td> 0.57</td>
<td> 8</td><td> 1.28</td><td> 9.80</td><td> 3.67</td><td> 56.93</td><td> 75.09</td><td> 3.35</td><td> 0.57</td>
<td> 9</td><td> 1.60</td><td> 4.90</td><td> 3.32</td><td> 57.16</td><td> 65.76</td><td> 1.77</td><td> 0.72</td>
<td> 10</td><td> 1.60</td><td> 9.80</td><td> 3.57</td><td> 56.89</td><td> 62.11</td><td> 3.35</td><td> 0.72</td>
<td> 11</td><td> 1.90</td><td> 4.90</td><td> 3.36</td><td> 56.15</td><td> 59.95</td><td> 1.77</td><td> 0.85</td>
<td> 12</td><td> 1.90</td><td> 9.80</td><td> 3.54</td><td> 54.74</td><td> 60.42</td><td> 3.35</td><td> 0.085</td>
- 45 1 Includes sodium ion added in 0.45 g / L of chloride
<img file="MX338455B_D0030.tif" />
sodium or 0.18 g / L sodium ion.
The results shown in Table 8 showed that optimal DHA production occurs with K2SO4 concentrations at 1.28 g / L and 9.8 g / L Na2SO4. The results shown in Table 8 indicate that the effect of additional potassium can be seen at potassium sulfate levels as low as 0.32 g / L and appear relatively constant through 1.90 g / L. Growth and production are relatively insensitive to sodium sulfate levels of 4.9 g / L or 9.8 g / L.
EXAMPLE 9
The following example describes the subculture of C. cohnii to obtain a strain that is adapted to grow at a pH of 5.
The C. cohnii T-HF strain was grown in shake flasks, in the manner described in Example 4, in the media described in Example 1, except that the pH of the media at the start of culture was a pH of 5 After 7 days, a culture inoculum was used to start a new culture with a pH of 5, under the same conditions. Initially, growth at pH 5 was slow, but after multiple transfers, DHA production began to improve and over time has approached
- 46 production observed in crops that
<img file="MX338455B_D0031.tif" />
INSTITUTO MEXICANO grow with a ρΉΚ'αβ '^ δ.> · Resulting in a low pH strain. See Figure 1 ........ So it was noted that the pH of the culture at the end of day Ί of the growth period was 5.4. Attempts to adapt the strain using citrate, maleate, acetate, and lactate pH regulators were unsuccessful due to the toxic effect of those pH regulators on the T-HF strain.
The low pH strain was then grown on the pH 5 media described above, but the pH was maintained at 5.0. The adapted low pH strain grew equally well at pH 5 and pH 5.4.
EXAMPLE 10
The following example describes a comparison of DHA yields of C. cohnii T-HF strain grown with a pH of 6.3 and 2730 ppm of chloride ion medium, and the low pH strain of pH 5 in 1000 ppm of medium with chloride ion.
C. cohnii strain T-HF was cultured as described in Example 4, in media as described in Example 1. C. cohnii strain was cultured in low chloride medium as described in Example 2, with added potassium sulfate and sodium sulfate. Each experiment was run in duplicate, and all flasks were collected daily to determine the kinetics of DHA production. The results are shown in
- 47 nvii- 'l
INSTITUTO MEXICANO D £ LA? F; O? IEDA D and INDUSTRIAL Figure 1. Figure 1 shows that the kinetics of the production of
DHA were almost identical under the two conditions with different means.
EXAMPLE 11
The following example describes the efforts to adapt the C. cohnii T-HF strain to grow it with a pH of
4.5 in 2370 ppm of medium with chloride ion.
The T-HF strain of C.
cohnii was grown in the manner described in Example 10, with the exception that the media were adjusted to a pH of 4.5, and half of the
MSG was replaced with lysine, while maintaining a constant level of organic nitrogen in the media.
After repeated cultivation, DHA productions of approximately one third of the observed productions were obtained with a pH of 5 or with a pH of 6.3.
EXAMPLE 12
The following example describes efforts to define conditions for C. cohnii cultivation and DHA yields at a pH of 4.5 by manipulating potassium concentrations.
A. Factorial experiments were carried out with a pH of
4.5 to 2.73 g / L chloride ion, to evaluate the effect of the potassium ion
- 48 (0.16 g / L up to 3.2 g / L).
IMPI «
MEXICAN INSTITUTE industrial
The results showed that the highest levels of potassium ion increased the production of DHA to approximately two thirds of the productions obtained for C.
cohnii with a pH of 6.3 with the media described in Example 1.
B. A factorial experiment was run as described in part A, above, with the exception that chloride ion levels were kept constant at 1.0 g / L. The results showed that the higher potassium ion levels increased the production of DHA to approximately two thirds of the productions obtained by C. cohnii cultivated with a pH of 6.3 with the media described in example 1.
DHA productions
<img file="MX338455B_D0032.tif" />
obtained in 2.73 g / L of chloride ion (described in part A, above), and in 1.0 g / L of
Chloride ion, they were comparable.
EXAMPLE 13
This example describes an ongoing experiment that compares the DHA yields obtained using the pH 4.5 strain described in Example 12 and the T-HF strain with a pH of 6.3, 20 1.0 g / L chloride ion.
The pH 5 strain of Example 12 was grown in low chloride medium, pH 4.5, as specified in Table 9, in shake flasks, then in the manner described in Example 4. Strain T C. cohnii-HF was grown in the manner described in Example 4 using the media described in Example 1.
<img file="MX338455B_D0033.tif" />
<img file="MX338455B_D0034.tif" />
MEXICAN INSTITUTE OF INDUSTRIAL PROPERTY prepared inoculum for the experiment at pH 4.5, and estimated amounts of inoculum due to agglomeration of cells ~ erruK pH 4.5.
TABLE 9. LOW CHLORIDE MEDIUM, WITH pH 4.5
<td>Compound</td><td>Final concentration</td><td>Amount of chloride ion added (g / l)</td><td>Amount of potassium ion added (g / l)</td><td>Amount of added sodium ion (g / l)</td>
<td>CaCI<sub>2</sub>-2H<sub>2</sub>OR</td><td>0.3 g / l</td><td> 0.09</td><td></td><td></td>
<td>MgSO<sub>4</sub>-7H<sub>2</sub>OR</td><td>1.25 g / l</td><td></td><td></td><td></td>
<td>NaCI</td><td> 1.41</td><td> 0.86</td><td></td><td> 0.55</td>
<td>MONTH</td><td>10.7 g / l</td><td></td><td></td><td></td>
<td>MSG</td><td>0.75 g / l</td><td></td><td></td><td></td>
<td>Tastona 154</td><td>0.5 g / l</td><td></td><td></td><td></td>
<td>Lysine-HCI</td><td> 0.37</td><td></td><td></td><td></td>
<td>KH<sub>2</sub>PO<sub>4</sub></td><td>0.014 g / l</td><td></td><td> 0.004</td><td></td>
<td>K<sub>2</sub>SW<sub>4</sub></td><td> 0.15</td><td></td><td> 0.07</td><td></td>
<td>Na<sub>2</sub>SW<sub>4</sub></td><td> 3.46</td><td></td><td></td><td> 1.12</td>
<td>What<sub>4</sub>-5H<sub>2</sub>OR</td><td>0.15X10 '<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>h<sub>3</sub>bo<sub>3</sub></td><td>10X10 '<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>MnCI<sub>2</sub>-4H<sub>2</sub>OR</td><td>4.5X10 '<sup>3</sup> g / l</td><td>Insignificant</td><td></td><td></td>
<td>ZnSo<sub>4</sub>-7H<sub>2</sub>OR</td><td>0.3X10 '<sup>3</sup> g / l</td><td></td><td></td><td></td>
<img file="MX338455B_D0035.tif" />
<img file="MX338455B_D0036.tif" />
IΜ ΡI
MEXICAN INSTITUTE 2 ___nc ί α unfuiénA ri____ y.TL · .__ * “Ί« ί +
<td rowspan="2">Compound</td><td rowspan="2">Final concentration</td><td rowspan="2">Amount of chloride ion added (g / l)</td><td>Amount of potassium ion</td><td>O'SMdBd d ^ sodium ion</td>
<td>added (g / l)</td><td>added (g / l)</td>
<td>NaOH (to adjust the pH to 6.3)</td><td>1.16 g / l</td><td></td><td></td><td> 0.67</td>
<td>FeCÍT</td><td>6X10 '<sup>3</sup> g / l</td><td>Insignificant</td><td></td><td></td>
<td>Thiamine<sup>2</sup></td><td>1 X10<sup>3</sup> g / l</td><td></td><td></td><td></td>
<td>Biotin<sup>2</sup></td><td>2X10 '<sup>6</sup></td><td></td><td></td><td></td>
<td>Glucose<sup>3</sup></td><td> 50</td><td></td><td></td><td></td>
<td>Total of each ion</td><td></td><td> 0.30</td><td> 0.08</td><td> 0.74</td>
Flasks were collected daily to determine the kinetics of DHA production. The results of the experiment (Figure 2) indicated that DHA production with a pH of 4.5 was always lower than that obtained with a pH of 6.3, but the rate of increase in DHA production as a function of time was approximately the same with each pH. This suggests that at pH 20 of 4.5, the culture is capable of accumulating DHA at the same rate as the culture at pH 6.3, however, there was a delay in DHA production in cultures grown with a pH of 4.5. compared to a pH of 6.3.
This result shows that given overtime, i.e. 25 approximately 24 hours, the production of DHA with a pH of 4.I5
- 51 was the same as with a pH of 6.3. No this
<img file="MX338455B_D0037.tif" />
MEXICAN INSTITUTE OF PROPERTY industrial clear whether the delay was caused by a delay in the accumulation of DHA with the pH of
4.5, and as a result, culture with a pH of
4.5 always had a
<img file="MX338455B_D0038.tif" />
DHA production that was lower than the culture with pH 6.3 of the same age, or if the delay was caused because the culture with pH 4.5 did not receive an equivalent amount of inoculum.
At a pH of 4.5, the cells of the T-HF strain agglomerate, such that it is not possible to obtain an exact cell count from the culture, and the amount of inoculum to be used has to be estimated. Therefore, it is possible that the culture with a pH of 4.5 received less
<img file="MX338455B_D0039.tif" />
I inoculate, and therefore cause an apparent delay in the kinetics of DHA production.
Despite this, these data indicated that using the C. cohnii strain adapted to low pH, and the culture medium present with a pH of 4.5, the same DHA production can be achieved as with the culture medium with pH of 6.3, if the cultivation time is extended.
EXAMPLE 14
Further optimization of the ion concentrations described in Example 13 above, and further subculturing of the C. cohnii T-HF strain, which has been adapted to pH 5, was carried out using the techniques described in Example 10, to 25 improve delay time, resulting in productions of
- 52 DHA in 7 days, with a pH of 4.5, which are comparable to the productions obtained by C. cohnii grown with a pH of 6.3 with the media described in Example 1.
<img file="MX338455B_D0040.tif" />
The principles, preferred embodiments and modes of operation of the present invention have been described in the preceding specification.
The invention which is intended to be protected herein, however, should not be construed as limited to the particular forms described, since these are to be considered as illustrative rather than restrictive. The connoisseurs of the matter can make variations and changes, without
<img file="MX338455B_D0041.tif" />
depart from the spirit of the present invention. Accordingly, the foregoing best mode of carrying out the invention should be considered exemplary in nature, and not as limiting the scope and spirit of the invention as set forth in the appended claims.
<img file="MX338455B_D0042.tif" />
I ° <sup>L</sup> /TO<sup>or</sup>’’<sup>, fd</sup>* d
INDUSTRIAL
<img file="MX338455B_D0043.tif" />
Contents54
45 sheets
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77 members in 13 offices
Priority claims3
| Document | Office | Kind | Date |
|---|---|---|---|
| 60508505 | United States of America | – | |
| 50850503 | United States of America | P | |
| 2004032383 | United States of America | W |
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Numbers
- Publication
- 338455
- Application
- 2012012139
Titles2
- Spanish
- PRODUCCION DE ALTOS NIVELES DE DHA EN MICROALGAS USANDO CANTIDADES MODIFICADAS DE CLORURO Y POTASIO.
- English
- PRODUCTION OF HIGH LEVELS OF DHA IN MICROALGAE USING MODIFIED AMOUNTS OF CHLORIDE AND POTASSIUM.
Classification
- CPC, 8
- C12P7/6434
- C12N5/0018
- A61K36/02
- C12P7/6472
- C12N1/12
- C12N2500/00
- C12P7/6409
- C11B1/10
- IPC, 3
- C12P7 64
- C12P7 6434
- C12P7 6472