WO1995025163A1

WO1995025163A1 - Methods for the production of fungal spores and compositions thereof

Info

Publication number: WO1995025163A1
Application number: PCT/CA1995/000094
Authority: WO
Inventors: Robert Duncan Carmichael; Catherine Brigetta Kuiack
Original assignee: Philom Bios Inc
Current assignee: Philom Bios Inc
Priority date: 1994-03-15
Filing date: 1995-02-24
Publication date: 1995-09-21
Anticipated expiration: 1996-09-15
Also published as: AU1703995A

Abstract

Methods are disclosed for a multi-stage two-phase fungal fermentation process for the production of large quantities of fungal spores which can be used as active ingredients in commercial compositions. The first phase of the fermentation process preferentially stimulates the growth of fungal mycelium, and the volume of mycelial biomass produced during this phase can be considerably increased by successive serial transfers of the mycelial biomass to larger vessels. The second phase, i.e., final phase, of the fermentation process preferentially stimulates fungal sporulation and spore production. The fungal spores produced with this invention can be processed into concentrated slurries or dried powders. Commercial compositions that can be prepared with these fungal spore products as active ingredients, include dry and wettable powders, liquids and granules.

Description

METHODS FOR THE PRODUCTION OF FUNGAL SPORES AND COMPOSITIONS THEREOF

Technical Field

Relates to methods for fermentation product; on of fungal spores which can then be used as the active ingredients in compositic .. of biological input products.

Types of biological input products which can be formulated with the fungal spore active ingredients include plant fertility products such as fertilizers and supplements, and pest control products such as mycoherbicides, mycoinsecticides and mycofungicides. Background Art

A major impediment to the successful commercialization of fungal products has been the difficulties encountered in the development of cost- efficient commercial-scale processes that enable the production of large quantities of fungal propagules that can be formulated as active ingredients into commercial compositions, and which will retain the biological properties of the active ingredients, i.e., fungal propagules, for extended periods of time (Charudattan, R. , 1991, IN Microbial Control of Weeds. (D.O. TeBeest, Ed.) Chapman and Hall, New York. p. 44) (Roberts, D.W., J.R. Fuxa, R. Gaugler, M. Goettel, R. Jaques and J. Maddox, 1991, IN CRC Handbook of Pest Management in Agriculture (D. Pimental, Ed.) CRC Press, Boca Raton, pp. 243-278) .

Traditionally, commercial industrial fermentation of fungi focused on the production of secondary metabolites rather than sporulation and the production of fungal propagules. Therefore, technical expertise and commercial success in large-scale production of fungal spores and other propagules is scant (Roberts, D.W. and A.E. Hajek, 1991, IN Frontiers in Industrial Mycology (G.F. Leatham, Ed.) Chapman and Hall, pp. 144 - 159) . Typical prior art previously disclosed for submerged- liquid fermentation production of fungal propagules, includes single-phase liquid-based processes which may, but not necessarily, include multiple stages (e.g., Anderson and Walker, 1987, U.S. Patent 4,715,881; Tabachnik, 1989, U.S. Patent 4,837,155; Templeton, 1976, U.S. Patent 3,999,973; Walker, 1983, U.S. Patent 4,419,120) . Furthermore, the prior art teaches that the final products of the various single-phase liquid fermentation processes, i.e., a mixture of mycelia and spores referred to as fungal propagules, are not separated but rather, are used directly in selected applications or are incorporated into various compositions. Analyses of the prior art suggest that the disclosed processes are typically expensive, provide low productivities with poor stability of the final products and consequently, have poor commercial potential and limited application.

In particular, Walker, U.S. Patent 4,419,120, and Tabachnik, U.S. Patent 4,837,155, teach that processes disclosedly previously for the production of fungal propagules are laboratory processes and do not produce sufficient amounts of material in large-enough volumes in short-enough time to be commercially viable. Furthermore, Walker, U.S. Patent 4,419,120, and Tabachnik, U.S. Patent 4,837,155, teach individually and separately, that economic large-scale production of fungal propagules is based on single-phase liquid fermentation processes which may, but not necessarily consist of multiple stages. Fungal propagules produced by previously disclosed processes tend to be comprised of mixtures containing mycelia and spores, and tend to be very unstable and have very short shelf lives. Although Daniel, Templeton and Smith, U.S. Patent 3,849,104, teach the production of fungal spores in liquid submerged culture, their process is a small-volume laboratory process and the spores produced, completely lose viability within only 2 to 4 days under refrigeration.

A recent study assessed fungal spore productivity in a variety of media using different production methods including submerged liquid culture (Stilman, R.W., T.C. Nelson and R.J. Bothast, 1991, Comparison of culture methods for production of Colletotrichu truncatum spores for use as a mycoherbicide. FEMS Microbiol . Letts. 79: 69-74) . Based on the numbers of spores produced per millilitre (i.e., mL) of culture, they concluded their results did not enable selection of nor prediction of a reliable cost-efficient process for spore production. Disclosure of the Invention

This invention relates to the discovery of a process which enables the production of very large quantities of fungal spores in submerged liquid fermentations. This process comprises the simultaneous manipulation and maintenance of three key components of fermentations i.e., (a) the carbon: nitrogen ratios of the media, (b) the growth-rate-limiting concentrations of the nutrient components of liquid fermentation media and/or adjustment of the physical fermentation parameters such as aeration or agitation so that the fungal growth rates are affected, and (c) the addition or deletion of sporulation inhibiting or inducing agents into the media, such that two distinct phases of fungal growth occur, i.e., first the production of mycelial biomass and then, sporulation and spore production. These three fermentation components may be manipulated individually or in combination at each stage of the fermentation process so that the onset and duration of the two fungal growth phases are precisely controlled through out the duration of the fermentation process.

This process comprises preparing a fungal inoculum stock which is used to inoculate a fermenter vessel containing a nutrient-balanced liquid medium with a low carbon:nitrogen nutrient ratio. A fermentation is conducted in this vessel to produce primarily mycelial biomass. If more biomass is desired, then the mycelial culture produced in the first fermentation can be serially transferred to one or more larger fermenters containing the low carbon:nitrogen ratio liquid medium. After sufficient mycelial biomass has been generated with the low carbon:nitrogen ratio liquid medium, the mycelial biomass is transferred to a final fermenter vessel containing a liquid medium with an altered carbon:nitrogen nutrient ratio, said carbon:nitrogen ratio altered such that further mycelial production is limited, but such that spore production is stimulated and optimized. A further fermentation is conducted in the final fermenter to produce the desired spores. Thereafter, the final product consisting of mycelial biomass and spores, is removed from the vessel and the spores are separated from the mycelial biomass. The resulting spore suspension is concentrated into a slurry. At this point, the spore slurry can be used as an active ingredient for compositions comprised of liquids, powders or granules. Alternatively, the spore slurry can be dried prior to its use as an active ingredient in compositions.

The fermentation medium used during the first phase of fungal growth, i.e., production of mycelial biomass, is nutrient-balanced and contains a low carbon:nitrogen ratio so that residual nitrogen levels remaining in the medium after the carbon sources have been exhausted, will inhibit sporulation. In addition, the low carbon:nitrogen ratio medium may also contain sporulation-suppressing medium components to further minimize sporulation during the phase of mycelial growth and development . The fermentation medium used during the second phase of fungal growth, i.e., sporulation, has an altered carbon:nitrogen ratio relative to the medium used for mycelial growth, and does not contain any sporulation- inhibiting medium components but rather, may contain sporulation inducing medium components.

The spores produced during the final fermentation are preferably separated from the mycelial biomass by screening, straining, or sieving. The resulting spore suspension is preferably concentrated into a slurry by centrifugation or filtration, for subsequent processing. At this point, the spore slurry may be then packaged in a water- and gas-impermeable container, or added as an active ingredient into commercially useful compositions. Alternatively, the spores may be stabilized by the addition of a stabilizing agent to the spore slurry prior to packaging or use as an active ingredient in compositions. For optimum stability of commercial compositions, it is preferable to add a stabilizing agent to the spore slurry prior to preparing the compositions.

Furthermore, the spore slurry may be dried, preferably by spray-drying, freeze-drying, or air-drying. The spores are preferably stabilized prior to drying by the addition of a stabilizing agent to the spore slurry or alternatively, by adjusting the relative humidity of the dried spores to a constant, preferably in a range between 12% to 33% moisture content. For optimum stability of commercial compositions, it is preferable to add a stabilizing agent to the spore slurry prior to drying, or to adjust the relative humidity of the dried spores to a constant in the range of 12% to 33% moisture content.

This invention also relates to compositions which are comprised of fungal spores as the active ingredients in the form of spore slurries, and carriers thereof.

Compositions containing concentrated spore slurries, and compositions containing dried, stabilized spores, are preferably packaged in sealed water- and gas-impermeable containers, and preferably stored frozen.

Compositions comprised of spore slurries and powders, and spore slurries and granules, are preferably packaged in sealed water-impermeable containers, and can be stored at temperatures in the range of -85C to 25C. Compositions comprised of dried spores and powders, and spore slurries and granules, are preferably packaged in sealed water-impermeable containers, and can be stored at temperatures in the range of -85C to 25C.

As used herein, the term "single-phase" fermentation refers to fermentation processes in which fungal propagules consisting of mixtures of mycelia and spores,^' are produced by growing the cultures in a liquid medium until the nutrient components are exhausted such that no further growth occurs. This term refers to single-phase fermentations which are harvested after one complete growth period in a fermenter vessel, and also to cultures which are produced by expansion through serial transfers from a nutrient-exhausted vessel into a larger vessel containing fresh liquid medium.

As used herein, the term "two-phase" fermentation process refers to all fermentation processes wherein the manipulation of the carbon:nitrogen ratio and the growth- rate-limiting nutrient concentration of the fermentation medium enables precise control over the onset and duration of mycelial biomass growth and development, and over the stimulation and optimization of sporulation. As used herein, the term "primary-phase" fermentation refers to the production of primarily mycelial biomass in a fermenter vessel containing a nutrient-balanced low carbon:nitrogen ratio liquid medium such that carbon sources are completely depleted from the medium before the nitrogen sources are exhausted.

As used herein, the term "multi-stage" refers to serial transfers of mycelial biomass produced during primary-phase fermentation, to successively larger fermenter vessels, so that large quantities of mycelial biomass are produced.

As used herein, the term "final-phase" fermentation refers to the production of large quantities of spores in a fermenter vessels containing a nutrient-balanced liquid medium containing a balanced or high carbon:nitrogen ratio, such that the nitrogen sources are depleted at the same rate or more quickly, than the carbon sources. Best Modes For Carrying Out the Invention

We have surprisingly found that the physiological stages of the growth and development of fungal mycelia in submerged liquid culture and also, the onset of fungal sporulation in liquid media can be precisely controlled by simultaneously manipulating: (a) the overall "carbon-to- nitrogen" (i.e., C:N) ratio, and (b) the "growth-rate- limiting nutrient concentrations" of the liquid media. We further surprisingly found that very large quantities of fungal spores can be economically produced with a "two- phase" liquid fermentation process in which the C:N ratios and growth-rate-limiting nutrient concentrations of the media are manipulated in a manner that firstly, enables the production of large quantities of mycelial biomass while minimizing spore production, and secondly, enables immediate cessation of mycelial expansion and facilitates the onset of sporulation.

C:N ratios are used to define and describe the nutrient availability in fermentation media, and are typically referred to as "high", "low", or "balanced". For example, growth of a fungal culture in a fermentation medium with a "high" C:N ratio will result in a residual level of carbon remaining in the medium after the nitrogen sources have been completely exhausted. In a "low" C:N ratio medium, excess nitrogen will remain in the medium after the microbial culture has completely depleted the carbon sources. In a "balanced" C:N ratio, both carbon and nitrogen sources are depleted simultaneously. The growth rate of a microbial culture in a fermentation medium is primarily determined by nutrient concentration. When all required nutrients are available in excess, fungal growth occurs at a maximal rate. However, when the supply of an essential nutrient (e.g., carbon) is limiting fungal growth to less than the maximal rate, that nutrient, e.g., carbon, is referred to as the growth-rate-limiting nutrient.

Typically, the primary carbon sources in fermentation media are from sugars or other carbohydrates . The primary carbon sources typically do not contain nitrogen. Nitrogen sources are typically supplied as "defined" compounds comprised of nitrate or ammonium ions, organic compounds such as amino acids, or complex materials such as yeast extract. Although, some of the primary nitrogen sources may contain carbon, the C:N ratio of the nitrogen compounds is typically very low.

It should be noted that the definition and concentration limits of "high", "low", and "balanced" C:N ratios in fermenter media will vary considerably with the nutritional requirements of different microorganisms. For example, for Colletotrichum gloeosporioides f.sp. malvae ( Cgm) grown in liquid medium where sucrose is the primary carbon source, and yeast extract is the nitrogen source, the medium is C:N balanced when the ratio of sucrose:yeast extract is 6:1 (mass:mass) . In this case, the growth rate of Cgm will be at a maximum when the concentrations of sucrose and yeast extract are at the 6:1 ratio, e.g., 10 g/L sucrose and 1.67 g/L yeast extract. A C:N ratio less than 6:1 (e.g., 5.5:1) will result in the medium defined as a "low C:N medium" for Cgm, even if 55 g/L sucrose and 10 g/L yeast extract are supplied, because residual nitrogen would remain in the medium after the carbon is depleted. Similarly, a C:N ratio greater than 6:1 (e.g., 6.5:1) would be considered a "high" C:N medium for Cgm. As additional example, a liquid fermentation medium for Fusarium heterosporum is considered "balanced" , when the C:N ratio is 14:1., i.e., 10.5 g/L glucose and 0.75 g/L yeast extract . A key feature of our invention is the precise control during the fermentation process, over the physiological change of the fungal culture from mycelial biomass production to spore production. It should be noted that in order for fermentation production of fungal spores to be commercially viable, a number of transfers of the mycelial biomass, while it is in a state of vegetative growth, to successively larger fermenter vessels is required in order to achieve the necessary volume of mycelial biomass needed to produce the target spore yields in the final fermentation step. During the first fermentation phase, i.e., the primary phase, a low C:N ratio fermentation medium is preferentially used to produce large quantities of mycelial biomass. If the culture is maintained beyond the point of carbon depletion in one of the stages of primary-phase fermentation, sporulation will be suppressed by the residual nitrogen remaining in the exhausted low C:N ratio medium. Fungal sporulation processes are triggered by transferring the mycelial biomass into a fermenter vessel containing an enriched liquid medium with a high C:N ratio to ensure that high levels of carbon are available for further enabling sporulation to continue after the nitrogen sources are exhausted. A further discovery according to this invention is that it is also possible to further increase spore yields in the final-phase fermentation by raising the C:N ratio in the last stage of the primary- phase fermentation. However, a sporulation inhibitor must be included with the high C:N ratio medium in this step to enable maximal mycelial biomass yields while minimizing sporulation.

In the commercial process of the present invention, the large-scale production of fungal spores includes a multi-staged two-phase liquid fermentation process. The first phase, i.e., the primary phase, may consist of multiple stages to increase the amount of mycelial biomass produced during the fermentation process. The mycelial biomass and any spores produced during the primary-phase fermentations are not recovered, but are used to inoculate the next stage of primary-phase fermentation. The final phase of production, i.e., final-phase fermentation, is where the spores to be used as the active ingredient of commercial compositions, are produced and subsequently harvested.

The production process is preferably initiated by using a fungal spore stock to inoculate agar plates, shake flasks or fermenter vessels as appropriate. Fungal spore stocks are produced on a suitable solid agar medium such as sucrose-yeast-extract agar (SYE) or potato dextrose agar (PDA) . After sufficient mycelial growth and sporulation have occurred, the spores are harvested by flooding the surfaces of the agar medium with sterile distilled water and gently agitating the culture. The resulting spore suspension is quickly removed from the agar medium, suspended in a sterile glycerol solution and either used immediately, or stored at -40C to -85C. Fungal inoculum stocks may also be produced from fungal cultures grown on suitable agar media by excising part or all of the culture, which may contain mycelia or mycelia plus spores, from the media, and then homogenizing the excised culture in sterile liquid medium. The second type of fungal inoculum stock, i.e., prepared from mycelia or mycelia plus spores, is used immediately after preparation. The primary-phase fermentation is initiated by inoculating an appropriate fungal stock into a liquid medium which is nutrient-balanced but contains a low C:N ratio. In order to optimize the nutrient composition of low C:N ratio fermenter media and to maximize production efficiencies, the ratio of C:N which is balanced for the specific fungal organism and the growth-rate-limiting concentrations of the carbon and nitrogen sources must be determined. For example, a low C:N ratio medium for Cσlletotrichum sp . such as Colletotrichum gloeosporioides f.sp. alvae ( Cgm) could contain, but is not restricted to, sucrose (10 g/L) , yeast extract (5 g/L) , mono- potassium phosphate (5 g/L) , ammonium sulfate (10 g/L) with the pH adjusted to 6. In this particular medium, the ammonium sulfate will inhibit sporulation by Cgm mycelia if the fermentation in any primary-phase vessel is allowed to continue after the carbon sources are exhausted. An example of a low C:N ratio primary-phase fermenter medium for Penicillium sp . such as Penicillium bilaii may contain, but is not restricted to, glucose (20 g/L) , tryptone (10 g/L) , malt extract (10 g/L) , KH₂P0₄ (5 g/L) with the pH adjusted to 6. The primary-phase fermentations are performed under controlled conditions which may include, but are not restricted to, airflow between 0.9 - 30 1pm, agitation between 50 - 800 rp , temperature maintained between 18 - 35C, pH maintained at 6.0 +_. 0.5, back-pressure maintained between 0 - 0.5 bar, dissolved oxygen levels maintained above 50%. Culture foaming can be controlled by the addition of sterilized commercial anti-foaming agent (e.g., 10% Sigma Emulsion A) . Mycelial biomass production during primary-phase fermentation can be greatly increased by incorporating multiple stages of primary-phase fermentation. For example, the mycelial biomass produced during the first primary-phase fermentation (i.e., stage 1) , is aseptically transferred into a stage-2 fermenter vessel containing the low C:N ratio liquid medium, with the stage-2 vessel being 10 times larger than the stage-1 vessel. Subsequently, the mycelial biomass produced during the stage-2 primary- phase fermentation can be transferred to a stage-3 primary-phase vessel containing the low C:N ratio liquid medium, with the stage-3 vessel being 10 times larger than the stage-2 vessel.

The primary-phase fermentations are continued until sufficient mycelial biomass has been generated to inoculate a final-phase fermenter vessel which is typically 10 times larger than the immediately preceding primary-phase vessel. The time-period required for completion of primary-phase fermentation will range between 2 - 14 days, depending on the number of transfers to larger vessels required to generate the preferred colume of mycelial biomass required to transfer into the final-phase fermenter vessel.

After the primary-phase fermentation has been completed, the final-phase fermentation is initiated by inoculation of the final-phase liquid medium with the preferred volume of primary-phase culture. The final- phase medium has an altered C:N ratio relative to the primary-phase medium and does not contain sporulation- suppressing compounds, such that sporulation and spore production are optimized. Typically, a final-phase medium for Colletotrichum sp . may contain, but is not restricted to, sucrose (5-30 g/L) , yeast extract (0-5 g/L) , mono- potassium phosphate (1-5 g/L) , tryptic soy broth (0 - 30 g/L) . An example of a final-phase medium for Penicillium sp . may include, but is not restricted to, sucrose (5 - 40 g/L) , mono-sodium glutamate (0 - 5 g/L) , MgS0₄.7H₂0 (0 - 2 g/L) , KH₂P0₄ (0 - 5 g/L) , Ca (N0₃)₂.4H₂0 (0 - 10 g/L) . Antibiotic compounds may be added to the primary- phase and final-phase media to ensure that the biological purity of the fungal cultures is maintained during the fermentation process. For example, Penstrep (Sigma cat. no. P0906) may be added such that the media contains 50 UI/mL penicillin and 0.05 mg/mL streptomycin.

The final-phase fermentations are performed under controlled conditions for airflow, agitation, temperature, pH, back-pressure, and dissolved oxygen which may include, but are not restricted to, airflow between 0.9 - 40 1pm, agitation between 50 - 800 rpm, temperature maintained between 10 - 35C, pH maintained between 4 - 7, back¬ pressure maintained between 0 - 0.5 bar, dissolved oxygen levels maintained above 50%. Culture foaming can be controlled by the addition of sterilized commercial anti- foaming agent (e.g., 10% Sigma Emulsion A) .

The final product of the final-phase fermentation consists of mycelial biomass and spores, and typically occurs within 60 - 72 hours, with spore yields in the range of 2 X 10⁷ to 5 X 10⁸ spores/mL of culture. The spores are separated from the sieving, straining, or screening. Alternatively, the final fermentation product consisting of mycelia and spores may be homogenized, then centrifuged to remove the mycelial debris from the spores . The resulting spore suspension is concentrated into a slurry by centrifugation or filtration. The concentrated spore slurry may be directly incorporated into liquid, powder or granular compositions which are then packaged in water- and gas-impermeable containers. Alternatively, the spore slurry may be stabilized by the addition of a stabilizing compound such as glycerol, prior to incorporation into compositions which are then packaged in water- and gas-impermeable containers. The spore slurry may also be dried, preferably using a process such as spray-drying, freeze-drying or air-drying, and then incorporated into liquid, powder or granular compositions which are then packaged in water- and gas-impermeable containers. Dried spores may be stabilized by adjusting their final moisture content to a constant in the range of 12 - 33%, and incorporated into liquid, wettable powder or granular compositions which are then packaged in water- and gas-impermeable containers.

Further specific embodiments of this invention are illustrated by the following, non-limiting examples. EXAMPLE 1:

The following example is of a small-scale, three- stage, two-phase fermentation process for the production of Colletotrichum gloeosporioides f.sp. malvae [ Cgm) spores.

(A) Primary-phase fermentation, i.e., Phase-1 fermentation: A Cgm production inoculum stock culture was plated onto soil-yeast-extract agar and grown at room temperature for 5 days. Spores produced were suspended in sterile culture medium and transferred into 5 vessels, each containing 150 mL of Cgm primary-phase fermentation medium which consisted of sucrose (10 g/L) , yeast extract (5 g/L) , mono-potassium phosphate (5 g/L) , ammonium sulfate (10 g/L) with the pH adjusted to 6 (stage 1) . Ammonium sulfate served as the sporulation-suppressing component in this medium. After 3-days' growth under controlled qonditions in the stage-1 vessels, the stage-1 primary- phase mycelial biomass cultures were pooled and transferred into a vessel containing 16 L of the Cgm primary-phase fermentation medium and grown under controlled conditions (stage 2) .

(B) Final-phase fermentation, i.e., Phase-2 fermentation:

After 2-days' growth in the stage-2 vessel, the stage-2 primary-phase mycelial biomass culture was transferred into a vessel containing 170 L of Cgm final- phase fermenter medium which consisted of sucrose (30 g/L) , yeast extract (5 g/L) , mono-potassium phosphate (5 g/L) , with the pH adjusted to 6 (stage 1) , and then grown under controlled conditions (stage 3) . After a 60-hour fermentation in the stage-3 vessel, the titre of spores produced was 3 X 10⁷ spores/mL.

(C) Processing:

After the 60-hour fermentation in the final-phase medium was completed, the spores produced were separated from the mycelial biomass by sieving. The resulting spore suspension was concentrated by centrifugation. The resulting concentrated spore slurry was air-dried in trays. The viability of the dried spores was 2.0 X 10⁹ cfu/g (i.e., colony-forming units per gram of product) .

The relative humidity of the dried spores was adjusted to a constant by placing the dried spore product into a sealed chamber containing an atmosphere equilibrated with a saturated LiCl solution. The stabilized spores were packaged in gas- and water-impermeable containers. EXAMPLE 2:

The following example is of a large-scale, four-stage two-phase fermentation process for the production of Cgm spores. (A) Primary-phase fermentation:

A Cgm production inoculum stock culture was plated onto SYE agar and grown at room temperature for 6 days. Spores produced .were suspended in sterile culture medium and transferred into 5 vessels, each containing 150 mL of Cgm primary-phase fermentation medium which consisted of sucrose (10 g/L) , yeast extract (5 g/L) , mono-potassium phosphate (5 g/L) , ammonium sulfate (10 g/L) with the pH adjusted to 6 (stage 1) . After 3-days' growth in the stage-1 vessels, the stage-1 primary-phase Cgm mycelial biomass cultures were pooled and transferred into a vessel containing 16 L of the primary-phase fermentation medium and then grown under controlled conditions (stage 2) . After 2-days' growth in the stage-2 vessel, the Cgm mycelial biomass was transferred into a vessel containing 170 L of the primary-phase fermentation medium and then grown under controlled conditions (stage 3) . (B) Final-phase fermentation:

After a 1-day growth period in the stage-3 vessel, the stage-3 primary-phase Cgm mycelial biomass culture was transferred into a vessel containing 1000 L of Cgm final- phase fermenter medium which consisted of sucrose (30 g/L) , yeast extract (5 g/L) , mono-potassium phosphate (5 g/L) , with the pH adjusted to 6 and then grown under controlled conditions (stage 4) . After a 72-hour fermentation in the stage-4 vessel, the titre of Cgm spores produced was 2.2 X 10⁷ spores/ml. (C) Processing:

After the 72-hour fermentation in the final-phase medium, the Cgm spores produced were separated from the Cgm mycelial biomass by sieving. The resulting spore suspension was concentrated by centrifugation. The resulting concentrated spore slurry was stabilized by the addition of sucrose, then tray-dried. The viability of the dried spores was 2.9 X 10⁸ cfu/g. The stabilized spores were packaged in gas- and water-impermeable containers. EXAMPLE 3 :

The following example is a laboratory model of a large-scale, three-stage two-phase fermentation process for the production of Penicillium bilaii (Pb) spores. (A) Primary-phase fermentation: A frozen Pb production inoculum stock culture (1 X10¹⁰ spores) was thawed at room temperature, and then inoculated into a vessel containing 10 L of " Pb primary- phase medium #1" which consisted of glucose (20 g/L) , tryptone (10 g/L) , malt extract (10 g/L) , KH₂P0₄ (5 g/L) with the pH adjusted to 6 with NaOH, and then grown under controlled conditions (stage 1) which consisted of airflow at 2 1pm for 24 hours and then adjusted to 10 1pm for 24 hours, agitation at 600 rpm, temperature maintained at 25C, pH maintained at 6.0 ± 0.1 with 2N H₂S0₄ or 2N NaOH as required. Culture foaming was controlled by the constant addition of a 10% Sigma Emulsion A anti-foam agent. The C:N ratio of the primary-phase medium was 3:1 (mass:mass) . After a 48-hour fermentation in the stage-1 vessel, 1 L of mycelial biomass was transferred into a vessel containing

9 L of "primary-phase medium #2" which consisted of sucrose (10 g/L) , mono-sodium glutamate (6 g/L) , MgS0₄.7H₂0 (2 g/L) , KH₂P0₄ (1 g/L) with the pH adjusted to 6, and then grown under controlled conditions (stage 2) which consisted of airflow 10 1pm, agitation at 600 rpm, temperature maintained at 25C, pH maintained at 6.0 +_. 0.1 with 2N H₂S0₄ or 2N NaOH as required. Culture foaming was controlled by the constant addition of a 10% Sigma

Emulsion A anti-foam agent. The C:N ratio of this medium was 13.6:1. (B) Final-phase fermentation:

After a 24-hour fermentation in the stage-2 vessel, the pH of the fermenter culture was lowered to 4.5 after which, the mycelial biomass was transferred into a vessel containing 9 L of final-phase medium which consisted of sucrose (18 g/L), mono-sodium glutamate (1 g/L) , MgS0₄.7H₂0 (0.2 g/L) , KH₂P0₄ (0.5 g/L) , Ca (N0₃)₂.4H₂0 (8.9 g/L) with the pH adjusted to 4.5 with NaOH, and then grown under controlled conditions (stage 3) which consisted of airflow

10 1pm, agitation at 600 rpm, temperature maintained at 25C, pH maintained at 4.5 ± 0.1 with 2N H₂S0₄ or 2N NaOH as required. Culture foaming was controlled by the constant addition of a 10% Sigma Emulsion A anti-foam agent. The C:N ratio of the final-phase medium was 7.5:1. After a 70-hour fermentation in the stage-3 vessel containing the final-phase medium, the titre of spores produced was 1.14 X 10⁸ spores/ml. EXAMPLE 4 : The following example is a large-scale, three-stage two-phase fermentation process for the production of Penicillium bilaii (Pb) spores. (A) Primary-phase fermentation:

Two hundred and fifty mL of a frozen Pb production inoculum stock culture (2.2 X 10¹¹ cfu) were thawed under running water at 25C, and then inoculated into a 300-L fermenter vessel containing 220 L of " Pb primary-phase medium #1" which consisted of glucose (20 g/L) , casein peptone (10 g/L) , malt extract (10 g/L) , KH₂P0₄ (2.5 g/L) , plus 110 mL of a trace elements solution consisting of boric acid (4 g/L) , cupric sulfate (0.4 g/L) , ferric chloride (2 g/L), manganous chloride (0.4 g/L) , sodium molybdate (0.4 g/L), zinc chloride (4.4 g/L) , and P-2000 anti-foam agent (0.005%) with the pH maintained between 5.8 - 6.0 with NaOH and H₂S0₄. The primary-phase culture was then grown under controlled conditions (stage 1) which consisted of airflow at 14 1pm for 24 hours and then adjusted to 130 1pm for 16 hours, agitation at 400 rpm, temperature maintained at 25C, pH between 4.5 and 6.0 with 2N H₂S0₄ or 2N NaOH as required. The C:N ratio of the primary-phase medium was 3:1. After a 36-hour fermentation in the 300-L stage-1 vessel, the entire 220- L of mycelial biomass was transferred into a 3,000-L stage-2 primary-phase fermentation vessel containing 2,200 L of " Pb primary-phase medium #2" which consisted of sucrose (10 g/L), mono-sodium glutamate (6 g/L) , MgS0₄.7H₂0 (2 g/L) , KH₂P0₄ (1 g/L) plus 2 L of the trace elements solution and P-2000 antifoam agent (0.0005%) with the pH maintained between 5.8 and 6.0 with NaOH or H₂S0₄. The second-stage primary-phase culture grown under controlled conditions which consisted of airflow 1.3 1pm, agitation at 200 rpm, temperature maintained at 25C, pH maintained at 6.0 ± 0.1 with 2N H₂S0₄ or 2N NaOH as required. The C:N ratio of the second Pb primary-phase fermentation medium was 13.6 :1.

(B) Final-phase fermentation: After a 18-hour fermentation in the 3,000-L stage-2 vessel, the entire 2,200 L of mycelial biomass was transferred into a 30,000-L stage-3 fermentation vessel containing 22,000 L of final-phase medium which consisted of sucrose (18 g/L) , mono-sodium glutamate (1 g/L) , MgS0₄.7H₂0 (0.2 g/L) , KH₂P0₄ (0.5 g/L) , Ca (N0₃) ₂.4H₂0 (6.2 g/L) plus 9.9 L of the trace element solution and P-2000 anti-foam agent (0.0005%) , with the pH adjusted to 4.5 ±0.05 with H₂S0₄. The final-phase culture was then grown under controlled conditions (stage 3) which consisted of airflow 11 1pm for 60 hours after which is was reduced to 5 1pm for 12 hours, agitation at 100 rpm, temperature maintained at 25C, pH maintained at 4.7 ± 0.1 with 2N H₂S0₄ or 2N NaOH as required. The C:N ratio of the final-phase medium was 7:1. After a 72-hour fermentation in the stage-3 vessel containing the final-phase medium, the titre of Pb spores produced was 2.1 X 10⁸ spores/ml. EXAMPLE 5:

Penicillium bilaii ( Pb) spores produced in EXAMPLE 4 were separated from the mycelial biomass by passing the final-fermentation product through a 48" vibratory screen. The resulting spore suspension was concentrated into a slurry by continuous centrifugation. The concentrated spore slurry was collected in 55-gal drums. Prior to drying, the spore slurry was stabilized by the addition of sucrose (1 g/L) and mon-sodium glutamate (1 g/L) into the drums and well-mixed. Then, the stabilized slurry was pumped from the 55-gal drums into a feed line which sprayed the spore slurry into 7.5-foot spray drier. The inlet temperature was maintained in a range of 128C - 132C, while the outlet temperature was maintained between 48C to 52C. Spray-dried spores were collected in double-walled plastic bags. The viability of the Pb spores after drying, was 5.2 X 10⁸ (± 2.98X10⁸) cfu/g (i.e., colony-forming-units per gram) of dried spores . The dried, stabilized spores were packaged and sealed in plastic-lined aluminum foil pouches that were water- and gas-impermeable. After three-months of storage at -20C, the viability of the Pb spores packaged in the aluminum foil pouches was 2.4 X 10⁸ cfu/g. EXAMPLE 6:

Penicillium bilaii ( Pb) spores produced with the large-scale two-phase fermentation process outlined in EXAMPLE 4 were separated from the mycelial biomass by passing the final-fermentation product through a 48" vibratory screen. The resulting spore suspension was concentrated into a slurry by continuous centrifugation. The concentrated spore slurry was collected in 55-gal drums.

The concentrated Pb spore slurry was diluted with sterile distilled water and then stabilized by the addition of glycerol (final glycerol concentration in the composition was 10% v/v) . A green food dyestuff was also added to the composition (13% w/v) . The liquid spore concentrate composition was then packaged and sealed in plastic-lined aluminum foil pouches. After 1 week of storage at -20C, the Pb titre in the liquid composition was 1.89 X 10⁹ (± 1.4X10⁸) . After 3 months of storage at -20C, the Pb titre in the liquid composition «as 1.33 X 10⁹ (+ 2.1X10⁷) . EXAMPLE 7:

Fifty two grams of finely ground (300 mesh) dark sedge peat were packaged and sealed in plastic bags, and then sterilized with 5.5 megarads of gamma irradiation. Then, each bag of irradiated peat was aseptically inoculated with 30 L of a Penicillium bilaii (Pb) spore suspension using a sterilized syringe connected to a peristaltic pump, such that a total of 2.6 X 10⁷ cfu of Pb were added to each bag. The inoculated peat was "cured" for 3 weeks at 30C. At the end of the curing period, the Pb titre in the inoculated peat was 8.21 X 10⁸ cfu/g (colony-forming-units per gram) . After 1-month storage at 20C, the Pb titre was 8.41 X 10⁸ cfu/g, and 8.35 X 10⁸ cfu/g after 3 months of storage. EXAMPLE 8 :

A sample of Penicillium bilaii ( Pb) concentrated spore slurry produced with the large-scale two-phase fermentation process outlined in EXAMPLE 4 was used to prepare a granular composition.

An aliquot of concentrated Pb spore slurry containing a total of 1 X 10¹⁰ viable Pb spores was dispersed in sufficient room-temperature tap water so that the final volume of the diluted Pb spore suspension was 1.7 L. The Pb spore suspension was then sprayed onto 10 kg of calcined clay granules (Agsorb 10/20 LVM-MS) that were being tumbled in a rotary blender. The target concentration of Pb colony-forming-units per gram of granules was 1 X 10⁶ cfu. After the coating process was completed, the PJ -coated calcined clay granules were dispensed into plastic bags (1 kg/bag) , tightly sealed, and then stored at 20C. The moisture content of the Pb- coated granules was 17%. The titre of Pb on the clay granules was determined immediately after coating, and subsequently at 4, 8 and 22 weeks. The data are recorded in Table 1.

Table 1 : Effects of storage on the titre of PJ -coated calcined clay granules .

Storage time Log cfu/g* (± SD)

(week :s)

0 6.25 +. 0.14

4 6.06 ± 0.04

8 6.06 ± 0.00

22 6.06 ± 0.44

* Means of three replicates.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it v.-;: il be apparent that various changes and modifications may be practised within the scope of the appended claims.

Claims

Claims :

1. A method for large-scale production of a commercially useful fungal product om the form of spores, said method comprising the steps of: (a) preparing a fungal inoculum stock,

(b) inoculating a fermentation vessel containing a primary-phase fermentation medium with said fungal inoculum, said primary-phase fermentation medium having a low carbon:nitrogen ratio to maximize mycelial biomass production,

(c) conducting a primary-phase fermentation in said vessel to produce primarily mycelial biomass essentially free of spores,

(d) transferring the mycelial biomass from the primary-phase fermentation to a final-phase fermentation in a vessel containing a medium which contains no sporulation-suppressing component and has an altered carbon:nitrogen ratio relative to the carbon:nitrogen ratio of the primary-phase fermentation medium, to thereby limit further mycelial biomass production,

(e) conducting a final-phase fermentation to stimulate and optimize spore production with a yield of at least 2 X 10⁷ spores/mL,

(f) separating said spores from the mycelial biomass, and

(g) concentrating said spores into a slurry.

2. A method according to claim 1 wherein the fungal inoculum consists of spores.

3. A method according to claim 1 wherein the fungal inoculum consists of mycelia.

4. A method according to claim 1 wherein the fungal inoculum consists of spores and mycelia.

5. A method according to any one of claims 1-4 wherein the primary-phase fermentation medium contains an antibiotic component or components.

6. A method according to any one of claims 1-5 wherein the primary-phase fermentation medium contains a sporulation-suppressing component or components.

7. A method according to any one of claims 1-6 wherein the primary-phase fermentation is conducted in multiple stages which comprises inoculating a first vessel with said fungal inoculum, said primary-phase fermentation medium having a low carbon:nitrogen ratio to maximize mycelial biomass production, conducting a primary-phase fermentation in said vessel to produce primarily mycelial biomass substantially free of spores, transferring the mycelial biomass to at least one further vessel containing said primary-phase fermentation medium and continuing the primary fermentation in said at least one further vessel while maintaining the culture in the exponential growth phase to produce primarily mycelial biomass substantially free of spores, and then transferring the mycelial biomass from the primary-phase fermentation to a final-phase fermentation in a vessel containing a medium which contains no sporulation-suppressing component and has a high carbon:nitrogen ratio relative to the carbon:nitrogen ratio of the primary-phase fermentation medium.

8. A method according to claim 7 wherein the volume of the primary-phase fermentation is serially expanded by transfer to a larger vessel or vessels.

9. A method according to claim 7 wherein the mycelial biomass produced during the last stage of the primary-phase fermentation is preconditioned for optimal sporulation in the final-phase fermentation, by altering the composition of the fermentation medium used in the last stage of primary-phase fermentation by simultaneously increasing the carbon:nitrogen ratio relative to the carbon:nitrogen ratio of the primary-phase fermentation medium used in the preceding stages and adding a sporulation-suppressing component or components.

10. A method according to any one of claims 1-9 wherein the final-phase fermentation medium includes a sporulation-inducing component or components.

11. A method according to any one of claims 1-10 wherein the final-phase fermentation has controlled airflow in the range of 5 - 300 lvm, agitation in the range of 50 - 800 rpm, temperature in the range of 10 - 35C, pH in the range of 4.5 - 7.5, back-pressure in the range of 0-0.5 bars, and dissolved oxygen above 50%.

12. A method according to any one of claims 1-11 wherein the spores produced during fermentation are separated from the mycelial biomass by a process selected from sedimentation, screening, sieving, straining, filtration or centrifugation.

13. A method according to claim 12 wherein the separated spores are concentrated into a slurry by a process selected from centrifugation or filtration.

14. A method according to any one of claims 1-13 wherein the concentrated spore slurry is stabilized by the addition of a stabilizing component or components to the spore slurry.

15. A method according to any one of claims 1-14 wherein the separated spores are dried by a process selected from spray-drying, freeze-drying or air-drying.

16. A method according to claim 15 wherein dried spores are stabilized by adjusting their final moisture content to a constant in the range of 12% to 33%.