AU733497B2 - Nutrient medium for increasing cell yield in fermentation - Google Patents

Description

WO 98/58049 PCT/US97/10343 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, g cholesterol and 2mM Ca2+. (Kerwin, James L. and Washino, Robert K. (1986) "Ground and aerial application of the sexual and asexual stages ofLagenidium giganteum (oomycetes: Lagenidiales) for mosquito control." J. Am. Mos. Control Assoc. 182- 189).

SUBSTITUTE SHEET (RULE 26) WO 98/58049 PCT/US97/10343 2 Another formulation comprises 2.0 g autolyzed yeast extract, 1.0 g proflo, 0.5 g fish meal, 2 mM CaCI 2 2HO, ImM MgCI 2 6H 2 O, 0.05 g cholesterol and 2 mL cottonseed oil. (Kerwin, James L. and Washino, Robert K. (1988) "Field evaluation of Lagenidium giganteum (Oomycetes: Lagenidiales) 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 CaC1 2 2HO2. 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: Lagenidiales)," 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; 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 CaCl 2 2H 2 0; 0.2 g per liter MgCl 2 6H20 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.

SUBSTITUTE SHEET (RULE 26) WO 98/58049 PCT/US97/10343 3 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; to 3 g per liter autolyzed yeast extract; 1.6 g per liter cottonseed flour; to 7.75 g per liter glucose (dextrose); g per liter palm oil; 0.2 g per liter cholesterol; 0.6 g per liter CaCI 2 2H,0; and 0.2 g per liter MgCl 2 Deionized water is added to a final volume of 1 L and the pH is adjusted to The constituents are heated until dissolved and then the medium is sterilized by autoclaving at 121 0 C, 15 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 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.

SUBSTITUTE SHEET (RULE 26)

C

4 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 0 C, 15 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.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the S 15 purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application. o The following example is provided only for illustrative purposes, and is not to be construed as limiting the invention in any way.

.0o$0i *go Example 1 i 25 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 g corn oil 1.0 g palm oil 0.03 g cholesterol 0.4 g CaC1 2 92H 2 0 0.2 g MgC1 2 96H 2 0 Medium #2: 1.2 g 1.2 g g 0.5 g peptone autolyzed yeast extract glucose (dextrose) cholesterol WO 98/58049 PCT/US97/10343 shaker for 7 days. Cells were harvested by centrifuging the fungal mass at 5,200 rpm for minutes at 18 Co. 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 Medium #2 Novel Nutrient Fold Increase Medium in cells/mL when Novel Medium used Exp't#l 1.2 2.0 x 10° cells/mL 1.2-2.0 x 10" cells/mL 4.4 x 10"cells/mL 2.2 fold Exp't #2 6.25 x 10' cells/mL 7.5 x 10'cells/mL 1.38 x 10 cells/mL 1.84-2.2 fold Exp't #3 2.97 x 10' cells/mL 3.3 x 10 cells/mL 4.75 x 10 cells/mL 1.4-1.6 fold Exp't #7 9.77 x 10 cells/mL not done- 9.38 x 10 cells/mL 9.6 fold Exp't #8 1.93 x 10 cells/mL not done 7.30 x 10 cells/mL 3.7 fold 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 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 and sterilized as in Example 1. For each medium, three 250 mL flasks were filled with mL of medium, inoculated, cultured and harvested as described in Example 1. Results are summarized in Table 2 and Table 3 SUBSTITUTE SHEET (RULE 26) WO 98/58049 PCT/US97/10343 Table 2 Dextrose Yeast extract Lecithin Cell Yield Wt Wt Wt (cells/mL) 0.8750 0.1250 0.0000 2.0 x 0.8750 0.1250 0.0000 3.0 x 10' 0.6875 0.3125 0.0000 4.8 x 10' 0.5000 0.5000 0.0000 4.1. x 0.5000 0.5000 0.0000 4.13 x 0.5875 0.3125 0.1000 5.4 x 0.5875 0.3125 0.1000 4.05 x 0.3000 0.5000 0.2000 7.4 x 10' 0.3000 0.5000 0.2000 4.9 x 10' 0.6750 0.1250 0.2000 4.5 x 0.6750 0.1250 0.2000 4.1 x 0.4875 0.3125 0.2000 3.6 x Average Cell Yield (cells/mL) without lecithin: 3.6 x 105 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 10 5 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; SUBSTITUTE SHEET (RULE 26) WO 98/58049 WO 9858049PCTIUS97/10343 7 1,250 and 675 cells/mL. Results summarized in Table 3 are averages of duplicate expeniments.

Table 3 Mortality at Mortality at 1% Mortality at Mortality at cells/mL [2,500 cells/mL 1,250 cells/mL J675 cells/mL Medium without 66 67 61 51 lecithin- Medium with 87 87 89 74 lecithin These results illustrate that Lagenidium giganteum gown in the novel media killed more mosquitoes than cells grown in media without added lecithin.

SUBSTITUTE SHEET (RULE 28)

Claims (3)

1. A medium for use in fermentation, consisting essentially of:

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 CaCl 2 2HO; and 0.2 g per liter MgCI 2 6H 2 O. 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. SUBSTITUTE SHEET (RULE 26)