EP0954562A1 - Nutrient medium for increasing cell yield in fermentation - Google Patents

Nutrient medium for increasing cell yield in fermentation

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Publication number
EP0954562A1
EP0954562A1 EP97930011A EP97930011A EP0954562A1 EP 0954562 A1 EP0954562 A1 EP 0954562A1 EP 97930011 A EP97930011 A EP 97930011A EP 97930011 A EP97930011 A EP 97930011A EP 0954562 A1 EP0954562 A1 EP 0954562A1
Authority
EP
European Patent Office
Prior art keywords
per liter
medium
fermentation
yield
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97930011A
Other languages
German (de)
French (fr)
Other versions
EP0954562A4 (en
Inventor
Sherry D. Heins
Duane D. Ewing
Pamela G. Marrone
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
AgraQuest Inc
Original Assignee
AgraQuest Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by AgraQuest Inc filed Critical AgraQuest Inc
Priority claimed from PCT/US1997/010343 external-priority patent/WO1998058049A1/en
Publication of EP0954562A1 publication Critical patent/EP0954562A1/en
Publication of EP0954562A4 publication Critical patent/EP0954562A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor

Definitions

  • This invention relates to a novel medium for use in fermentation which provides an increased cell yield compared to that of known media. More particularly, the present invention produces at least a two to three-fold increase in the yield of the fungus Lagenidium giganteum compared to the yield obtained with known media. In addition to increasing yield of cells, L. giganteum grown in novel medium containing lecithin exhibits increased effectiveness against mosquitoes.
  • Fermentation is the process of growing microorganisms or cells in specialized vessels. The cells or organisms may then be purified and used for a variety of purposes. For instance, the fungus Lagenidium giganteum grown in fermenters is used as a biocontrol agent for mosquitoes.
  • Optimal growth of the microorganism during fermentation depends on several factors including available nutrients, oxygen concentration, pH, temperature, and degree of mixing. Nutrients necessary for cell growth are provided in the medium used during the fermentation process. Accordingly, the yield obtained from fermentation depends, in part, on the composition of the medium.
  • Lagenidium giganteum There are several published nutrient media currently used in the fermentation of Lagenidium giganteum. All use deionized water added to a final volume of 1 L, and all are sterilized.
  • One formulation comprises 2.0 g Ardamine pH, 2.0 g glucose, 1 mL corn oil, 0.5 g cholesterol and 2mM Ca2+. (Kerwin, James L. and Washino, Robert K. (1986) "Ground and aerial application of the sexual and asexual stages of Lagenidium giganteum (oomycetes: Lagenidiales) for mosquito control.” J. Am. Mos. Control Assoc.
  • Yet another fermentation medium comprises 1.25 g glucose, 1.25 g peptone, 1.25 g autolyzed yeast extract, 2 g corn oil, 1 g linseed oil, and 0.075 g CaCl 2 2H 2 O.
  • the fourth published medium contains 1.25 g yeast extract, 1.2 g glucose, 3.2 g powdered wheat germ, hemp seed extract to provide 250 mg/L of soluble protein, 1.25 g bactopeptone, 3 g glucose and 1.5 g corn oil.
  • a medium for use in fermentation consisting essentially of 3.6 g per liter peptone
  • This medium provides increased yields of Lagenidium giganteum compared to prior art media, and, yield and infectivity of the organism is further increased when lecithin is included in the medium.
  • DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to an improved medium for fermentation.
  • the medium increases yield at least approximately two to three fold over known media.
  • the invention is useful in large scale production of Lagenidium giganteum, a biocontrol agent for mosquitoes.
  • fertilization refers to the process of growing cells or microorganisms in specialized vessels.
  • Nutrient medium (“medium”) refers to a solid or liquid substrate that will support the growth of an organism.
  • the nutrient medium is prepared as follows:
  • Deionized water is added to a final volume of 1 L and the pH is adjusted to 6.5.
  • the constituents are heated until dissolved and then the medium is sterilized by autoclaving at 121°C, 15 p.s.i., for 30 minutes.
  • this medium increases yield at least two to three fold over known media.
  • the nutrient medium is prepared by adding up to 2.0 g per liter of lecithin to the above formulation.
  • Example 1 The following example is provided only for illustrative purposes, and is not to be construed as limiting the invention in any way.
  • Example 1
  • Medium #1 and Medium #2 yielded approximately the same number of cells per mL of medium in each experiment.
  • the novel nutrient medium consistently increased the number of cells/mL in comparison to either Medium #1 or Medium #2.
  • the average yield of Lagenidium giganteum was increased approximately three and half fold when grown in the novel nutrient medium.
  • Example 1 Having established that the novel medium formulation of Example 1 increases cell yield over known media, the effect of varying amounts of dextrose and yeast extract and adding 1.0 g or 2.0 g lecithin to the basal novel medium was examined. All media were homogenized with a large probe at 70% speed for 10-15 seconds to ensure components were in solution. Using EmReagents color Phast®, the pH of all media was adjusted to 6.5 and sterilized as in Example 1. For each medium, three 250 mL flasks were filled with 50 mL of medium, inoculated, cultured and harvested as described in Example 1. Results are summarized in Table 2 and Table 3 Table 2
  • Lagenidium giganteum was grown in novel media described in Example 2 which contained no lecithin, 0.1000 % by weight lecithin or 0.2000 % by weight lecithin. Culturing conditions were as described in Example 1. The concentration of cells was calculated and their ability to kill mosquitoes measured at concentrations of 5,000; 2,500; 1,250 and 675 cells/mL. Results summarized in Table 3 are averages of duplicate experiments.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

A nutrient medium used in fermentation for increasing the yield of cells or microorganism is provided. The formulation provided increases the yield of the fungus Lagenidium giganteum two-to three-fold over known media.

Description

NUTRIENT MEDIUM FOR INCREASING CELL YIELD IN FERMENTATION
FIELD OF THE INVENTION This invention relates to a novel medium for use in fermentation which provides an increased cell yield compared to that of known media. More particularly, the present invention produces at least a two to three-fold increase in the yield of the fungus Lagenidium giganteum compared to the yield obtained with known media. In addition to increasing yield of cells, L. giganteum grown in novel medium containing lecithin exhibits increased effectiveness against mosquitoes.
BACKGROUND OF THE INVENTION Fermentation is the process of growing microorganisms or cells in specialized vessels. The cells or organisms may then be purified and used for a variety of purposes. For instance, the fungus Lagenidium giganteum grown in fermenters is used as a biocontrol agent for mosquitoes.
Optimal growth of the microorganism during fermentation depends on several factors including available nutrients, oxygen concentration, pH, temperature, and degree of mixing. Nutrients necessary for cell growth are provided in the medium used during the fermentation process. Accordingly, the yield obtained from fermentation depends, in part, on the composition of the medium.
There are several published nutrient media currently used in the fermentation of Lagenidium giganteum. All use deionized water added to a final volume of 1 L, and all are sterilized. One formulation comprises 2.0 g Ardamine pH, 2.0 g glucose, 1 mL corn oil, 0.5 g cholesterol and 2mM Ca2+. (Kerwin, James L. and Washino, Robert K. (1986) "Ground and aerial application of the sexual and asexual stages of Lagenidium giganteum (oomycetes: Lagenidiales) for mosquito control." J. Am. Mos. Control Assoc. 2(2): 182- Another formulation comprises 2.0 g autolyzed yeast extract, 1.0 g proflo, 0.5 g fish meal, 2 mM CaCl22H2O, ImM MgCl26H2O, 0.05 g cholesterol and 2 mL cottonseed oil. (Kerwin, James L. and Washino, Robert K. (1988) "Field evaluation of Lagenidium giganteum (Oomycetes: Lagemdiales) and description of a natural epizootic involving a new isolate of fungus." J. Med. Entomol. 25(6): 452-460) Yet another fermentation medium comprises 1.25 g glucose, 1.25 g peptone, 1.25 g autolyzed yeast extract, 2 g corn oil, 1 g linseed oil, and 0.075 g CaCl22H2O. (U.S. Patent No. 4,687,744). The fourth published medium contains 1.25 g yeast extract, 1.2 g glucose, 3.2 g powdered wheat germ, hemp seed extract to provide 250 mg/L of soluble protein, 1.25 g bactopeptone, 3 g glucose and 1.5 g corn oil. (Lord, Jeffrey C. and Roberts, Donald W. (1986) "The effects of culture medium quality and host passage on zoosporogensis and infectivity of Lagenidium giganteum (Oomycetes: Lagemdiales)," J. Invertebr. Pathol. 48:355-361)
When used in fermentation, the above-referenced published medium formulations all yield approximately the same number of cells and infect susceptible mosquitoes at approximately the same rate. Thus, in order to increase the yield and infectivity of biocontrol agents like Lagenidium giganteum, there is a need for an improved fermentation medium.
SUMMARY OF THE INVENTION A medium for use in fermentation consisting essentially of 3.6 g per liter peptone;
3.0 g per liter autolyzed yeast extract; 3.6 g per liter peptone; 1.5 to 3.0 g per liter autolyzed yeast extract; 1.6 g per liter cottonseed flour, such as ProFlo® (Traders Protein, Memphis, TN); 2.0 to 7.75 g per liter glucose (dextrose); 2.5 g per liter palm oil; 0.2 g per liter cholesterol; 0.6 g per liter CaCl2 • 2H2O; 0.2 g per liter MgCl2 • 6H2O and, optionally, 0.0 to 2.0 g per liter of lecithin. This medium provides increased yields of Lagenidium giganteum compared to prior art media, and, yield and infectivity of the organism is further increased when lecithin is included in the medium. DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to an improved medium for fermentation. The medium increases yield at least approximately two to three fold over known media. The invention is useful in large scale production of Lagenidium giganteum, a biocontrol agent for mosquitoes.
Definitions
As used herein, the term "fermentation" refers to the process of growing cells or microorganisms in specialized vessels. "Nutrient medium" ("medium") refers to a solid or liquid substrate that will support the growth of an organism.
In a preferred embodiment of this invention, the nutrient medium is prepared as follows:
3.6 g per liter peptone;
1.5 to 3 g per liter autolyzed yeast extract; 1.6 g per liter cottonseed flour;
2.0 to 7.75 g per liter glucose (dextrose);
2.5 g per liter palm oil;
0.2 g per liter cholesterol;
0.6 g per liter CaCl2 • 2H2O; and 0.2 g per liter MgCl2 • 6H2O.
Deionized water is added to a final volume of 1 L and the pH is adjusted to 6.5. The constituents are heated until dissolved and then the medium is sterilized by autoclaving at 121°C, 15 p.s.i., for 30 minutes. When used in the fermentation of Lagenidium giganteum, this medium increases yield at least two to three fold over known media.
In another preferred embodiment, the nutrient medium is prepared by adding up to 2.0 g per liter of lecithin to the above formulation.
The following example is provided only for illustrative purposes, and is not to be construed as limiting the invention in any way. Example 1
Shake flask comparison of growth rates of Lagenidium giganteum in different media
Growth rate in the novel nutrient medium was compared with two other media in side by side shake flask experiments. Medium #1:
1.25 g glucose (dextrose)
1.25 g peptone 1.25 g autolyzed yeast extract
2.0 g corn oil
1.0 g palm oil
0.03 g cholesterol
0.4 g CaCl2 .2H2O 0.2 g MgCl2 . 6H2O
Medium #2:
1.2 g peptone
1.2 g autolyzed yeast extract 3.0 g glucose (dextrose) 0.5 g cholesterol
Novel Nutrient Medium:
3.6 g peptone 3.0 g autolyzed yeast extract
1.6 g Proflo cottonseed extract
2.0 g glucose (dextrose)
2.5 g palm oil
0.2 g cholesterol 0.6 g CaCl2 • 2H2O
0.2 g MgCl2 • 6H2O
When preparing each of the media, all ingredients were combined and deionized water was added to a final volume of 1 L. The pH was adjusted to 6.5. Contents were heated in a microwave until dissolved and then sterilized at 121 C°, 15 psi for 30 minutes. For each medium, nine 250 mL flasks were each filled with 50 mL of medium. A disk of Lagenidium giganteum (California strain) taken from a petri dish was used to inoculate each flask. The flasks were shaken at 120 φm, 29 C° in an orbital temperature controlled shaker for 7 days. Cells were harvested by centrifuging the fungal mass at 5,200 m for 20 minutes at 18 C°. The centrifuged cell mass was weighed and cell counts made with a hemacytometer. Mean cell counts were recorded. Results are summarized in Table 1.
Table 1
Medium #1 and Medium #2 yielded approximately the same number of cells per mL of medium in each experiment. The novel nutrient medium consistently increased the number of cells/mL in comparison to either Medium #1 or Medium #2. The average yield of Lagenidium giganteum was increased approximately three and half fold when grown in the novel nutrient medium.
Example 2
Shake flask comparison of novel medium with lecithin added
Having established that the novel medium formulation of Example 1 increases cell yield over known media, the effect of varying amounts of dextrose and yeast extract and adding 1.0 g or 2.0 g lecithin to the basal novel medium was examined. All media were homogenized with a large probe at 70% speed for 10-15 seconds to ensure components were in solution. Using EmReagents color Phast®, the pH of all media was adjusted to 6.5 and sterilized as in Example 1. For each medium, three 250 mL flasks were filled with 50 mL of medium, inoculated, cultured and harvested as described in Example 1. Results are summarized in Table 2 and Table 3 Table 2
Average Cell
Yield (cells/mL) without lecithin:
3.6 x lO5
Average Cell
Yield (cells/mL) with lecithin:
4.63 x 105
As shown in Table 2, for media without lecithin, the average cells /mL yield is 3.6 x 105. With lecithin, yield increases to 4.63 x 105 cells/mL.
Example 3
Infectivity of Lagenidium giganteum grown in various media
Lagenidium giganteum was grown in novel media described in Example 2 which contained no lecithin, 0.1000 % by weight lecithin or 0.2000 % by weight lecithin. Culturing conditions were as described in Example 1. The concentration of cells was calculated and their ability to kill mosquitoes measured at concentrations of 5,000; 2,500; 1,250 and 675 cells/mL. Results summarized in Table 3 are averages of duplicate experiments.
Table 3
These results illustrate that Lagenidium giganteum grown in the novel media killed more mosquitoes than cells grown in media without added lecithin.

Claims

CLAIMS We claim:
1. A medium for use in fermentation, consisting essentially of: (a) 3.6 g per liter peptone; (b) 1.5 to 3 g per liter autolyzed yeast extract;
(c) 1.6 g per liter cottonseed flour;
(d) 2.0 to 7.75 g per liter glucose (dextrose);
(e) 2.5 g per liter palm oil;
(f) 0.2 g per liter cholesterol; (g) 0.6 g per liter CaCl22H2O; and
(h) 0.2 g per liter MgCl26H2O.
2. The medium according to claim 1 , further comprising up to 2.0 g per liter lecithin.
3. The medium according to claim 1, for use in culturing Lagenidium giganteum.
4. The medium according to claim 2, for use in culturing Lagenidium giganteum.
EP97930011A 1997-06-17 1997-06-17 Nutrient medium for increasing cell yield in fermentation Withdrawn EP0954562A4 (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1997/010343 WO1998058049A1 (en) 1996-03-15 1997-06-17 Nutrient medium for increasing cell yield in fermentation

Publications (2)

Publication Number Publication Date
EP0954562A1 true EP0954562A1 (en) 1999-11-10
EP0954562A4 EP0954562A4 (en) 2000-05-10

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EP97930011A Withdrawn EP0954562A4 (en) 1997-06-17 1997-06-17 Nutrient medium for increasing cell yield in fermentation

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EP (1) EP0954562A4 (en)
JP (1) JP2002503090A (en)
KR (1) KR19990087785A (en)
AU (1) AU733497B2 (en)
CA (1) CA2256519A1 (en)

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Publication number Priority date Publication date Assignee Title
JP2015002678A (en) * 2011-10-21 2015-01-08 株式会社カネカ Culturing method of microorganisms, and method for producing pha by microorganisms

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0494592A1 (en) * 1991-01-10 1992-07-15 W.R. Grace & Co.-Conn. A process and method for production and use of pathogenic fungal preparation for insect control

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL7704040A (en) * 1976-04-28 1977-11-01 Merck & Co Inc PROCEDURE FOR PREPARING NEW ANTI-BIOTICS.
US4687744A (en) * 1982-09-30 1987-08-18 The Regents Of The University Of California Artificial culture of the sexual stage of lagenidium giganteum

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0494592A1 (en) * 1991-01-10 1992-07-15 W.R. Grace & Co.-Conn. A process and method for production and use of pathogenic fungal preparation for insect control

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9858049A1 *

Also Published As

Publication number Publication date
KR19990087785A (en) 1999-12-27
CA2256519A1 (en) 1998-12-23
EP0954562A4 (en) 2000-05-10
AU3394297A (en) 1999-01-04
JP2002503090A (en) 2002-01-29
AU733497B2 (en) 2001-05-17

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