Production of fungal spores by solid-state fermentation

Abstract

A method for producing fungal spores using solid-state fermentation (SSF), comprising: combining fungal spores and a solid substrate in a solid-state fermentor to produce a fungal culture; subjecting the fungal culture in the solid-state fermentor to solid-state fermentation conditions, which include conditions for growth of the fungal cells for the targeted production of additional fungal spores by the fungal cells; and harvesting the additional fungal spores.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 399,460, filed August 19, 2022, which is incorporated herein by reference.

[0002] The present disclosure is directed to methods for producing fungal spores by solid-state fermentation. The present disclosure is further directed to targeted production of fungal spores relative to the growth of fungal cells. [Background technology]

[0003] The production of fungal spores is industrially important. For example, fungal spores can be used for their insecticidal properties. Fungal spores have traditionally been produced by submerged fermentation (SmF) in aqueous media or by solid-state fermentation (SSF). Solid-state fermentation generally involves providing fungal cells in a moist environment with no or minimal free-flowing water. However, large-scale SSF operation remains challenging. Summary of the Invention [Problem to be solved by the invention]

[0004] There remains a need in the art for improved methods of producing fungal spores. [Means for solving the problem]

[0005] In one aspect, a method for producing fungal spores using solid-state fermentation (SSF) includes combining fungal spores and a solid substrate in a solid-state fermentor to produce a fungal culture; subjecting the fungal culture in the solid-state fermentor to solid-state fermentation conditions, which include conditions for growth of the fungal cells for the targeted production of additional fungal spores by the fungal cells; and harvesting the additional fungal spores.

[0006] The advantages of the present disclosure will be better understood with regard to the following description, appended claims, and accompanying drawings. [Brief explanation of the drawings]

[0007] [Figure 1] 1 is a graph showing spore productivity results for Scopulariopsis brevicaulis spores on various solid substrates including potato dextrose agar (PDA) adjusted to pH 10 under light and dark conditions on various solid substrates.

[0008] [Figure 2] 1 is a graph showing the results of spore yield of Scopulariopsis brevicaulis spores under submerged fermentation (SmF) conditions compared to solid-state fermentation (SSF) conditions.

[0009] [Figure 3] 1 is a graph showing spore productivity results for Scopulariopsis brevicaulis spores at high pH and high salt conditions.

[0010] [Figure 4] 1 is a graph showing spore yield results for Scopulariopsis brevicaulis spores at alternative high pH conditions.

[0011] [Figure 5] 10 is a graph showing spore yield results for Scopulariopsis brevicaulis spores at additional alternative high pH conditions.

[0012] [Figure 6] 1 is a graph showing spore yield results for Scopulariopsis brevicaulis spores under alternative high salt conditions.

[0013] [Figure 7] 1 is a graph showing spore yield results for Scopulariopsis brevicaulis spores on solid substrates of various sizes.

[0014] [Figure 8] 10 is a graph showing further spore yield results for Scopulariopsis brevicaulis spores on solid substrates of various sizes. DETAILED DESCRIPTION OF THE INVENTION

[0015] Aspects of the present disclosure are directed to improved methods for producing fungal spores by solid-state fermentation (SSF). The production of fungal spores by solid-state fermentation, in which fungal cells are grown in a moist environment with minimal or no free-flowing water, is now recognized to have certain advantages over submerged fermentation in aqueous media. For example, solid-state fermentation production of fungal spores can have lower energy requirements, less water consumption, and greater suitability for using solid waste and by-products as feedstocks / substrates compared to submerged fermentation.

[0016] However, prior to the implementation of the present disclosure, traditional solid-state fermentation production methods still face challenges. For example, certain layered solid substrates tend to have limited and unevenly distributed porosity or void space within them, making it difficult to provide sufficient oxygen to support the respiration of high concentrations of healthy, growing fungal cells throughout one or more packed beds of the solid substrate. Therefore, one or more embodiments of the present disclosure utilize larger components, such as large soybean hull pieces, as a solid substrate, which can allow for better convective airflow through one or more packed beds of the solid substrate. Furthermore, by enabling better oxygen supply to the fungal cells and substrate, spore productivity and yield can be improved by the implementation of the present disclosure. Advantageously, in one or more embodiments, large soybean hull particles can serve as both the sole food source and the sole support for the fungal cells; such a support may be referred to as a physical or structural support. Furthermore, the use of large soybean hull particles allows for SSF to be carried out in a column or tower, compared to traditional shallow trays or bags.

[0017] It may also be desirable to achieve targeted production of spores relative to fungal cell growth. That is, while sporulation generally occurs naturally upon the onset of nutrient starvation of fungal cells, one or more aspects of the present disclosure induce sporulation by fungal cells prior to nutrient starvation. In other words, sporulation occurs when the fungal cells still have an adequate nutrient supply for their growth. Conditions that result in this sporulation are referred to herein as targeted production or selective factors for spores relative to fungal cell growth. As further described herein, these conditions include one or more of pH and salinity (i.e., osmolality). This form of sporulation generally results in higher spore productivity and yield compared to waiting for sporulation to occur naturally at the onset of nutrient starvation.

[0018] Improved spore productivity and yield is also advantageous for end-use applications of the spores. In this regard, one or more aspects of the present disclosure are directed to utilizing spores collected in cementitious materials such as concrete for internal self-healing cracks.

[0019] Solid-state fermentation (SSF) involves depositing a solid substrate on one or more beds, such as inside a solid-state fermenter. Before or after depositing the solid substrate, the solid substrate is inoculated with microorganisms, water, and other components of the culture, which may be referred to as a culture medium, such as salts. In one or more embodiments, the culture comprises a fungus, and thus the culture may be referred to as a fungal culture. In one or more embodiments, the initial inoculum of the culture comprises fungal spores. In one or more embodiments, the initial inoculum of the culture comprises fungal cells. In one or more embodiments, the initial inoculum of the culture is substantially devoid of or devoid of fungal cells. In these embodiments, the fungal cells are believed to be produced from the germination of fungal spores.

[0020] If the initial inoculum of the culture comprises fungal spores, an exemplary initial inoculum size is about 2.5 x 10 per gram of solid substrate. 5 Another suitable initial inoculum is about 1 x 10 fungal spores per gram of solid substrate. 4 From 1×10 6 pieces, or approximately 1 x 103 From 1×10 8 pieces, or approximately 1 x 10 5 From 5 x 10 5 If the initial inoculum of the culture contains fungal spores, the fungal spores subsequently produced by the fungal cells may be referred to as additional fungal spores.

[0021] Although much of this disclosure focuses on suitable fungi and fungal spores, it is possible that certain other microorganisms, such as bacteria and bacterial spores, may be utilized in the solid-state fermentation aspects disclosed herein.

[0022] As part of the SSF process, the deposited culture containing the solid substrate will have a relatively low moisture content in the substrate. Furthermore, certain conditions of the solid-state fermentation process, such as temperature, humidity, light, feedstock to culture ratio, and pH, can be controlled to achieve effective solid-state fermentation.

[0023] As alluded to above, in one or more embodiments, the solid substrate can be characterized by size. In one or more embodiments, the solid substrate has a size of less than 600 μm. In other embodiments, the solid substrate has a size of about 600 μm to 8 mm. In one or more embodiments, the solid substrate has a size of about 600 to 850 μm, or about 850 μm to 2 mm, or about 2 to 5.6 mm, or about 1 mm to 8 mm, or about 1.5 mm to 6 mm, or about 1 mm to 4 mm, or about 3 mm to 6 mm. In one or more embodiments, the solid substrate has a size of at least 0.5 mm, or at least 1 mm, or at least 2 mm, or at least 3 mm, or at least 4 mm. In one or more embodiments, larger substrates may provide certain improvements, such as by providing greater porosity throughout the bed of the solid substrate to allow air to pass through it. The size dimension generally refers to the length of the solid substrate, which is generally the largest dimension of the solid substrate. The particle size of a solid substrate can be determined by a standard ASTM mesh. That is, the size of a solid substrate can be measured by passing the solid substrate through a standard ASTM mesh. Particles of the solid substrate that can pass through a higher size mesh but not a lower size mesh can define a range for the size of the solid substrate, where the mesh size refers to the distance from wire to wire in the mesh. For example, an upper range of 5.6 mm for one or more embodiments refers to ASTM Mesh No. 3 1 / 2.

[0024] Exemplary suitable solid substrates include soybean husks, rice husks, barley husks, wheat husks, grain husks, grain husks, corn husks, coconut husks, bean pods, other agricultural biomass, and mixtures thereof. These may contain small pieces of these materials (e.g., small pieces of corn husks, small pieces of coconut husks, small pieces of bean pods). Larger sized substrates of these substrates are particularly preferred, with large soybean husks being especially preferred.

[0025] When large soybean hull pieces are utilized, these large soybean hull pieces generally create large voids within the SSF substrate volume. These large voids allow for several advantages, including one or more of: allowing good convective airflow through the substrate bed for effective oxygen supply; allowing effective removal of heat generated by cellular metabolism; and allowing control and adjustment of moisture content within the substrate bed based on humidity control of the incoming air. These large soybean hull pieces generally have a curved shape, which further aids in the creation of voids. Large soybean hull pieces as a solid substrate can also absorb necessary water and other soluble compounds and nutrients, if present.

[0026] A solid substrate can be characterized by a porosity, which may be referred to as initial porosity. This porosity generally refers to the overall porosity of the bed of the solid substrate. In one or more embodiments, the initial porosity of the bed of the solid substrate can be about 90%, or about 85%, or about 75%, or about 65%. In one or more embodiments, the initial porosity of the bed of the solid substrate can be at least 65%, or at least 75%, or at least 85%, or at least 90%. As noted above, these porosities may be initial percentages, but the porosity may also be characterized by subsequent percentages. As cells grow, the size of the bed may tend to shrink due to compression from the weight and entanglement of the growing cells. Thus, in certain embodiments, the subsequent porosity of the bed of the solid substrate can be about 90%, or about 85%, or about 75%, or about 65%. In one or more embodiments, the subsequent porosity of the bed of solid substrate can be at least 65%, or at least 75%, or at least 85%, or at least 90%. The initial porosity may be designed to maintain the subsequent porosity at or above a desired value.

[0027] As described above, the deposited culture containing the solid substrate will contain some initial moisture content in the solid substrate. In one or more embodiments, the initial moisture content can be designed for the desired sporulation rather than cell growth. That is, the initial moisture content can be tailored to obtain the highest spore yield. In one or more embodiments, the ratio of water to solid substrate can be about 0.5:1 to 3:1, or about 1:1 to 2.5:1, or about 1.25:1 to 2.25:1, or about 1.5:1 to 2:1, where this ratio refers to mL of water per gram of solid substrate. In one or more embodiments, the ratio of water to solid substrate can be about 0.5:1, or about 1:1, or about 1.5:1, or about 2:1, where this ratio refers to mL of water per gram of solid substrate.

[0028] In one or more embodiments, the solid substrate (e.g., large soybean hull particles) can serve as both a food source and the sole physical support. That is, the solid fermentation tower or column may be substantially devoid of any additional food source other than the solid substrate. The food source may also be referred to as an organic matter source or a carbon source. In one or more embodiments, the solid fermentation tower or column may be substantially devoid of any additional inert support or carrier other than the solid substrate.

[0029] In one or more embodiments, solid-state fermentation can be carried out in a column or tower. That is, the solid-state fermenter can be a column or tower. In other words, combining the fungal cells with the solid substrate under solid-state fermentation conditions can be carried out in a column or tower. Those skilled in the art generally know how to tailor the properties of a column or tower to achieve suitable results. Exemplary properties include tray type, number of trays, tray volume, bed height, column height, column diameter, and overall volume.

[0030] In one or more embodiments, the column or tower can contain a single bed of stacked culture. In other embodiments, particularly when taller columns or towers are desired, the column or tower can contain a series of trays, each tray generally containing a stacked culture. The trays can be made of a variety of materials, such as metal and plastic. The trays typically have an open top and a perforated bottom and are typically stacked one on top of the other with a space between each pair of trays to increase air availability to the culture. The trays are stationary beds, meaning they generally will not mix. Air can be provided within the column or tower and circulated around the trays at controlled humidity and temperature. The column or tower is generally at ambient pressure.

[0031] The addition of air may also be referred to as supplying a convective airflow upward through the solid substrate, thereby supplying oxygen to the fungal cells of the stacked culture. As mentioned, the convective airflow can have a predetermined humidity percentage to control the humidity of the solid-state fermenter at a target humidity for solid-state fermentation conditions. This humidity control can include mixing the humidified stream with ambient air at an adjustable flow ratio based on the target humidity. The humidified stream can include a humidity of about 90% to 100%, or about 90% to 95%, or about 95% to 100%. The humidified stream can include a humidity of about 100%, or about 99%, or about 95%. The humidified stream can include a humidity of at least 95%, or at least 98%, or at least 99%. The humidity of the humidified stream can be achieved by passing the stream through a humidity column. The desired moisture content can be different for cell growth and for sporulation. Therefore, having the ability to control and regulate the moisture content relatively uniformly using air streams with different humidity levels would be highly advantageous.

[0032] In a solid-state fermentation process, such as in a tower or column, the culture is subjected to fermentation conditions such that the solid substrate of the culture is consumed by the fungal cells of the culture. As described herein, this consumption and conditions are generally intended to target sporulation rather than cell growth. In one or more embodiments, the solid substrate is nearly completely consumed by the fungal cells in the solid-state fermentation process before spores are harvested therefrom. In one or more embodiments, at least 90%, or at least 95%, or at least 99% of the initial solid substrate is consumed by the fungal cells, after which spores are harvested therefrom. In one or more embodiments, 80% to 100%, or 90% to 100%, or 90% to 95% of the initial solid substrate is consumed by the fungal cells, after which spores are harvested therefrom.

[0033] The solid-state fermentation process should be provided with nutrients suitable for the deposited culture. In one or more embodiments, these nutrients can be provided to the solid substrate via an inorganic nutrient solution prior to combining with the fungal spores. In one or more embodiments, the solid substrate itself can contain appropriate or sufficient nutrients for cell growth and spore production.

[0034] An example of a nutrient solution for addition to a solid substrate is 0.02 g / L (NH4)2SO4, 0.01 g / L K2HPO4, 0.0025 g / L CaCl2·2H2O, 0.0025 g / L MgCl2·6H2O, and 0.002 g / L FeSO4·7H2O, based on 1 L of nutrient solution. Another example of a nutrient solution for addition is 14 g / L (NH4)2SO4, 1.4 g / L urea, 3.1 g / L KH2PO4, 1.8 g / L MgSO4·7H2O, 0.6 g / L CaCl2·2H2O, and 1 mL / L of trace element solution. The trace element solution may have the following composition (per L of trace element solution): 2.5 g / L FeSO4·7H2O, 0.8 g / L MnSO4·4H2O, 0.7 g / L ZnSO4·7H2O, and 1 g / L CoCl2·2H2O. As mentioned above, depending on the solid substrate used, it may not be necessary to add all or any of the above nutrients, but it may contain nutrients appropriate or sufficient for cell growth and spore production in SSF.

[0035] As described herein, to target sporulation, the method can include inducing spore production when the cells still have an adequate nutrient supply. This aspect of fungal cells with an adequate nutrient supply can be referred to as a method lacking or substantially lacking a nutrient starvation step. However, during cultivation, the cells may naturally deplete certain nutrients that were initially provided. In any case, the goal of the solid-state fermentation process disclosed herein is the targeted production of spores.

[0036] In one or more embodiments, the solid-state fermentation process lacks or substantially lacks an additional carbon source other than the solid substrate, in one or more embodiments, the solid-state fermentation process lacks or substantially lacks supplemental glucose, in one or more embodiments, the solid-state fermentation process lacks or substantially lacks an additional nitrogen source other than the solid substrate.

[0037] Regarding the species of microorganisms and spores to be used in the solid-state fermentation processes disclosed herein, suitable microorganisms and spores can be screened and selected for the characteristics of the solid-state fermentation processes disclosed herein. That is, suitable microorganisms and spores can be screened and selected for enhancing sporulation rather than fungal cell growth. The suitability of certain microorganisms and spores may also be selected based on the end use, such as where the spores will be utilized in the cementitious material for its self-repair. While much of this disclosure focuses on suitable fungal spores, it is also possible that certain bacterial spores can be utilized in accordance with the functions disclosed herein.

[0038] In embodiments of the present disclosure, suitable species of fungal spores include alkaliphilic and / or alkali-tolerant fungi. In embodiments of the present disclosure, suitable species of microorganisms and fungal spores include Scopulariopsis brevicaulis, Purpureocillium lilacinum, Myrothecium verrucaria, Aspergillus nidulans, and combinations thereof. Referring to the USDA-ARS Culture Collection (NRRL), examples include Aspergillus nidulans NRRL 187, Scopulariopsis brevicaulis NRRL 1100, Myrothecium verrucaria NRRL 2003, and Purpureocillium lilacinum NRRL 895.

[0039] In one or more embodiments, the solid-state fermentation process can be subjected to the targeted production of spores relative to the growth of fungal cells. This targeted production of spores can also be referred to as subjecting the solid-state fermentation process to one or more selection factors that target and even induce spore production. This targeted production of spores can also be referred to as inducing sporulation. Exemplary selection factors, which can also be referred to as sporulation conditions or selection influences, include high pH and high salt / osmolality. The particular microorganism and spores utilized can result in specific sporulation conditions to enhance sporulation in the solid-state fermentation process.

[0040] In one or more embodiments, the selection factor for the solid fermentation process includes subjecting the culture to a high pH. In one or more embodiments, the selection factor for the solid fermentation process includes subjecting the culture to a pH of about 10 to about 11, or about 9 to about 11, or about 9 to about 10. In one or more embodiments, the selection factor for the solid fermentation process includes subjecting the culture to a pH of about 9, or about 10, or about 11. In one or more embodiments, the selection factor for the solid fermentation process includes subjecting the culture to a pH of greater than 9 or greater than 10.

[0041] In one or more embodiments, these elevated pH values may refer to the initial medium before the initial medium is combined with the solid substrate of the culture. This may be referred to as an initial pH adjustment. That is, the initial medium may be an aqueous solution containing a certain amount of base to achieve a desired pH in the initial medium. The initial medium may then be combined with the solid substrate and other components of the culture. The conditions of the SSF process may also be adapted to generally maintain these pH values, or other desired pH values, for the desired duration of the SSF process. In one or more embodiments, the pH may be adjusted during SSF to maintain or achieve these elevated pH values. As noted above, in one or more embodiments, the pH may be adjusted with a base solution, either initially or subsequently. Suitable base solutions include sodium hydroxide (NaOH) solution or potassium hydroxide (KOH) solution. An exemplary base solution is 0.1 M NaOH.

[0042] In one or more embodiments, selection factors for solid-state fermentation processes include subjecting the culture to a high salt concentration, which may also be referred to as osmolality or osmolality. Salt concentration may be given in g / L NaCl, which can be adapted to other salts that may be used or measured. In one or more embodiments, selection factors for solid-state fermentation processes include subjecting the culture to a salt concentration of about 10 g / L NaCl to about 25 g / L NaCl, or about 10 g / L NaCl to about 20 g / L NaCl, or about 15 g / L NaCl to about 20 g / L NaCl. In one or more embodiments, selection factors for solid-state fermentation processes include subjecting the culture to a salt concentration of about 10 g / L NaCl, or about 15 g / L NaCl, or about 20 g / L NaCl. In one or more embodiments, selection factors for the solid-state fermentation process include subjecting the culture to a salinity of about 10 g / L NaCl, or about 15 g / L NaCl, or about 20 g / L NaCl, these salinity values generally being measured at the temperature and pressure of the solid-state fermentation process.

[0043] In one or more embodiments, these high salinity values can refer to the initial medium before combining the initial medium with the solid substrate of the culture. This may be referred to as initial salinity adjustment. The conditions of the SSF process may also be adapted to maintain these high salinity values for the desired duration of the SSF process. In one or more embodiments, the salinity may be adjusted during SSF to maintain or achieve these high salinity values. In one or more embodiments, the salinity may be adjusted, either initially or subsequently, with a salt solution. Suitable salts for the liquid solution include sodium chloride (NaCl), potassium chloride (KCl), sodium nitrate (NaNO), potassium nitrate (KNO), sodium sulfate, potassium sulfate, sodium phosphate, and potassium phosphate.

[0044] Solid-state fermentation processes can be characterized based on spore productivity (e.g., in units of spores produced per L of SSF bed / reactor volume per day) and / or yield (e.g., in units of spores produced per g of solid substrate used).

[0045] In one or more embodiments, the solid-state fermentation process produces at least 2 x 10 spores. 9 , or at least 1 × 10 10 , or at least 5 × 10 10 In one or more embodiments, the solid-state fermentation process achieves a spore productivity of about 2 x 10 spores / L-day. 9 From 8 x 10 10 , or approximately 5 × 10 9 From 5 x 10 10 , or approximately 1 × 10 10 From 5 x 10 10 Achieve spore productivity of spores / L-day.

[0046] In one or more embodiments, the solid-state fermentation process comprises culturing at least 5×10 spores. 8 , or at least 8 × 10 8 , or at least 1 × 10 9 In one or more embodiments, the solid-state fermentation process achieves a yield of about 5 x 10 spores / g solid substrate. 8 From 2 x 10 9 , or approximately 8 × 10 8 From 1×10 9 , or approximately 1 × 10 9 From 2 x 10 9 A yield of 1000pg / g solid substrate is achieved.

[0047] After fungal cells grow in a fungal culture on a moist solid substrate under solid-state fermentation (SSF) conditions, fungal spores produced therefrom can be harvested. As mentioned above, the harvested fungal spores may be referred to as additional fungal spores, since the initial culture may contain initial fungal spores. In one or more embodiments, the specific step or steps for harvesting the spores generally do not include adding additional water. Harvesting the spores from the SSF solid substrate can include combining a hydrophobic liquid with the SSF solid substrate to harvest the spores therefrom. Many suitable substances and mixtures can be used for the hydrophobic liquid. Exemplary materials for the hydrophobic liquid include oils, free fatty acids, and melted fats. These include solutions and mixtures thereof.

[0048] After adding the hydrophobic liquid to the SSF solid substrate, mixing, which can include creating shear, can be used to liberate the spores from the fungal biomass. The liberated spores will generally distribute within the oil phase. After mixing is stopped, the larger, non-hydrophobic solids (i.e., the remaining substrate and biomass) can be removed. This removal can be done either by filtration (e.g., through a screening mesh) or by allowing these materials to settle to the bottom. If these materials are allowed to settle to the bottom, a first collection can be made on these larger materials without collecting many spores. The spores will be smaller than these larger materials and therefore will settle much slower than any remaining larger pieces of substrate and biomass. A second collection can then be made on the spore-containing oil phase. Some smaller particles of cells and substrate debris may remain in the collected oil phase, which may be suitable for certain end uses. In other embodiments, these smaller particles of cells and substrate debris may be further separated from the spores.

[0049] After collecting the product containing spores in the hydrophobic liquid, the collected product can be further concentrated in terms of spore concentration. This can include allowing the spores in the collected product to further settle to the bottom, which can be referred to as allowing the collected product to settle. The settling step can be performed under non-mixing conditions, which can also be referred to as a mixing step followed by a settling step. After settling, the upper layer of the hydrophobic liquid from which the spores settled, which can be referred to as a clear oil, can then be removed. This leaves behind a lower liquid (e.g., oil) with a higher spore concentration. Whether and to what extent settling is used can depend on the desired concentration of the spore suspension for the intended use of the product.

[0050] As mentioned above, one suitable end use for the collected spores is in cementitious materials such as concrete. The spores can be provided in a porous matrix, and the porous matrix containing the spores can be present in the cementitious material. The spores present in the cementitious material can germinate to return to vegetative growth as vegetative cells, and then form a solid deposit by biomineralization to repair one or more cracks in the cementitious material.

[0051] Although aspects of the present disclosure are discussed above, certain exemplary aspects are now provided.

[0052] Aspect 1. A method for producing fungal spores using solid-state fermentation (SSF), comprising: combining fungal spores and a solid substrate in a solid-state fermentor to produce a fungal culture; subjecting the fungal culture in the solid-state fermentor to solid-state fermentation conditions, the solid-state fermentation conditions comprising conditions for growth of the fungal cells for the targeted production of additional fungal spores by the fungal cells; and harvesting the additional fungal spores.

[0053] Embodiment 2. The method of embodiment 1, wherein the fungal cells are provided by a fungal culture, and the fungal cells and spores are of a species selected from Scopulariopsis brevicaulis, Purpureoscillium lilacinum, Myrothecium verrucaria, Aspergillus nidulans, and combinations thereof.

[0054] Embodiment 3. The method of any of the above embodiments, wherein the conditions for the targeted production of additional fungal spores comprise subjecting the fungal culture to a pH of about 10 to about 11.

[0055] Embodiment 4. The method of any of the above embodiments, wherein the solid-state fermentation conditions comprise fungal cells having an appropriate nutrient supply for their growth.

[0056] Embodiment 5. The method of any of the above embodiments, wherein the conditions for the targeted production of additional fungal spores comprise subjecting the fungal culture to a salinity of greater than 10 g / L NaCl.

[0057] Embodiment 6. The method of embodiment 5, wherein the salinity is from about 10 g / L NaCl to about 20 g / L NaCl.

[0058] Embodiment 7. The method of any of the above embodiments, wherein the solid substrate is selected from soybean hulls, rice hulls, barley hulls, wheat hulls, grain skins, grain husks, corn husks, coconut husks, bean pods, and mixtures thereof.

[0059] Embodiment 8. The method of embodiment 7, wherein the solid substrate is soybean hulls, and the soybean hulls have a size of about 2 mm to about 5.6 mm.

[0060] Embodiment 9. The method of embodiment 7, wherein the solid substrate is soybean hulls, and the soybean hulls have a size of about 850 μm to about 2 mm.

[0061] Embodiment 10. The method of any of the above embodiments, wherein the solid-state fermentation conditions comprise supplying a convective airflow upward through the fungal culture, thereby supplying oxygen to the fungal cells.

[0062] Aspect 11. Approximately 5 x 10 spores 8From 2 x 10 9 The method of any of the above embodiments, wherein the method achieves an increased fungal spore yield per gram of solid substrate.

[0063] Embodiment 12 The method of any of the above embodiments, wherein the solid substrate has a porosity of at least 75%.

[0064] Aspect 13. The method of any of the above aspects, wherein the solid-state fermentor is a column or tower.

[0065] Embodiment 14. The method of embodiment 4, wherein the adequate nutrient supply is provided by an inorganic nutrient solution.

[0066] Embodiment 15. The method of embodiment 10, wherein the convective airflow has a humidity of about 95% to 100% to provide humidity to the solid-state fermentor.

[0067] Embodiment 16. The method of any of the preceding embodiments, wherein the solid-state fermentor is substantially devoid of any additional inert support or carrier other than the solid substrate, the solid-state fermentor is substantially devoid of any additional carbon source other than the solid substrate, and the solid-state fermentor is substantially devoid of any additional nitrogen source other than the solid substrate.

[0068] Embodiment 17. The method of any of the above embodiments, wherein the solid substrate comprises additional water, provided in a ratio of additional water (in mL) to solid substrate (in grams) of about 1:1 to about 2.5:1.

[0069] Aspect 18. The fungal culture contains about 1 x 10 fungal spores per gram of solid substrate. 4 From 1×10 6

[0023] The method of any of the above aspects, comprising a concentration of

[0070] Embodiment 19 The method of any of the above embodiments, wherein the solid substrate is substantially completely consumed by the fungal cells prior to the collecting step.

[0071] Aspect 20. The method of any of the above aspects, further comprising adding additional fungal spores from the collecting step to the cementitious material to repair one or more cracks in the cementitious material.

[0072] In light of the foregoing, it should be appreciated that the present invention advances the art by providing an improved method for producing fungal spores by solid-state fermentation. While particular embodiments of the invention have been disclosed in detail herein, it should be understood that the invention is not limited thereto or thereby, so long as variations thereon will be readily apparent to those of skill in the art. The scope of the invention is to be understood from the claims which follow. [Example]

[0073] Scopulariopsis brevicaulis spores in the dark and at pH 10 Scopulariopsis brevicaulis cultures were maintained on 9 cm Petri dishes containing approximately 25 ml of 40 g / L potato dextrose agar (PDA). Petri dishes were also used to evaluate soybean material as a growth substrate. Experiments were conducted at room temperature. One system served as a control, growing S. brevicaulis on 40 g / L PDA at pH 7. Cells were grown and sporulated in a constantly lit laboratory. The other six systems were divided into three pairs, with one system maintained in light and one system maintained in a dark drawer. The three pairs were Petri dishes containing (1) 15 g / L plain agar and 40 g / L soybean hulls (SH), (2) 15 g / L plain agar and 40 g / L soybean molasses (SM), and (3) 40 g / L PDA adjusted to pH 10. After inoculation, the plates were allowed to grow and sporulate for 14 days. Spores were then harvested. Spore productivity of these lines was determined by dividing 1 cm of the surface area of the agar plate by 100%. 2The results are reported as the number of spores produced per 1000 cells. The spore productivity results are summarized in Figure 1. Darkness did not affect spore productivity in all three comparisons. The pH 10 condition gave significantly higher spore productivity than the pH 7 system in which cells were grown and sporulated on PDA. The same conclusion was confirmed in SSF flasks containing SH as a substrate, as described herein below, demonstrating improved spore productivity when a high pH was used to promote sporulation. [Example]

[0074] S. brevicaulis spores under solid-state fermentation (SSF) conditions compared to submerged fermentation (SmF) conditions Submerged fermentation (SmF) was carried out in triplicate in 500 mL Erlenmeyer flasks containing 80 mL of water, 10 g of SH, 2 g of glucose, 0.2 g of (NH4)2SO4, 0.1 g of K2HPO4, 0.025 g of CaCl2·2H2O, 0.025 g of MgCl2·6H2O, and 0.02 g of FeSO4·7H2O. The flasks were covered with cheesecloth sheets, autoclaved at 121 °C for 20 min, and cooled to room temperature before inoculation with 2.5 × 10 spores. 5 The spores were inoculated at 1000 spores / g SH. Cultures were grown on a shaker operating at 250 rpm at 25°C. Samples were taken from each flask on days 2, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 21 for spore counting. Each sample was counted in triplicate. Spore production profiles in SmF were generated accordingly.

[0075] SSF experiments were performed in 21 Erlenmeyer flasks (250 ml) containing 15 ml of water, 10 g of SH, and 10% of all other components in the SmF experiments described above. After autoclaving and cooling, 2.5 × 10 spores were added to the SSF system in the same manner as for the SmF flasks. 5 Three flasks were taken as sacrificial samples on days 2, 4, 6, 8, 10, 12, and 14, and spores were counted following the same procedure as for the SmF system.

[0076] The time profiles of S. brevicaulis spore yield (number of spores / g SH) obtained in these SSF and SmF systems with SH as substrate are shown in Figure 2. The SSF system produced no spores in the first 2 days, then produced spores in an approximately linear fashion between days 4 and 8, after which it began to plateau. The spore yield between days 2 and 8 was determined by the best-fit linear equation: spore yield = -4.54 × 10 8 +(1.99×10 8 ) × time (days) (R 2 = 0.98). The maximum spore yield reached on day 14 was (1.33 ± 0.02) × 10 spores. 9 The SmF line did not produce spores for approximately 4 days, after which it produced spores in a nearly linear fashion. The spore yield between days 6 and 21 was calculated using the equation: spore yield = -7.62 x 10 7 +(4.39×10 7 ) × time (days) (R 2 = 0.98), the daily spore production in SmF is 4.39 × 10 spores. 7 spores / (g SH-day) in SSF, i.e., 1.99 x 10 8 The spore yield from SmF was (5.49±0.07)×10 spores / (g SH-day) on the 14th day. 8 spores / g SH, and on the 21st day, spores were (8.27±0.09) × 10 8 spores / g SH, and the number of spores reached by SSF on day 8 was (1.17±0.01) × 10 9 This was much lower than the number of particles / g SH.

[0077] From the above, it can be concluded that SSF provides higher S. brevicaulis spore production than SmF. [Example]

[0078] S. brevicaulis spores at high pH and high salt conditions The positive effects of high pH and high salt on S. brevicaulis spore production were demonstrated in an example using Petri dish agar plates. In the agar plate experiment, four systems were compared. One system was a control with 40 g / L PDA at pH 7. Two other systems were also produced with 40 g / L PDA, but one was supplemented with 15 g / L NaCl (approximately half the salinity level of seawater), and the other was adjusted to pH 10 with 0.1 M NaOH (no added NaCl). All of these systems were kept in a constantly lit laboratory. A fourth system had the same PDA at pH 10 but was kept in a dark drawer. The spore productivity values of these four systems are compared in Figure 3. The high pH of 10 and, particularly, the high salt conditions resulted in significantly higher spore production. [Example]

[0079] S. brevicaulis spores in alternative high pH conditions The positive effect of high pH on S. brevicaulis spore production was demonstrated using SSF flasks. The SSF flask system was prepared in the same manner as described in Example 2 above, except for the pH adjustment. The effect of pH on spore yield was evaluated for SSF flasks. Aqueous solutions of glucose and salt were adjusted to pH 5.5 (control) and pH 10, respectively, using 0.1 M NaOH, then added to SH and autoclaved. Spore yields were measured and compared at 4, 8, and 14 days, and the results are shown in Figure 4. [Example]

[0080] S. brevicaulis spores in further alternative high pH conditions In a further example, the SSF flask system was prepared as described in Example 4, except that the aqueous solution pH was adjusted to 5.5 (control), 9, and 11, respectively, and spore yield was measured only on day 14. The spore yield results are shown in Figure 5. [Example]

[0081] S. brevicaulis spores in alternative high salt conditions The positive effect of high salt on S. brevicaulis spore production was demonstrated using SSF flasks. SSF flask systems were prepared similarly to those described in Example 2, except for the salt adjustment. Regarding the effect of NaCl addition evaluated in SSF flasks, systems were compared without NaCl addition (control) and with the addition of 0.15 g of NaCl (equivalent to a 10 g / L concentration in 15 ml of added water). The spore yields measured on day 14 from these two systems are shown in Figure 6. Although the mean spore yield from the 10 g / L NaCl system was higher than that from the system without added NaCl, the difference between the two systems was not statistically significant (p = 0.17 > 0.05), which is due in part to the large standard deviation of spore yield in the 10 g / L NaCl system. On the other hand, the effect of 15 g / L NaCl was highly significant in experiments performed with PDA in Petri dishes (as discussed above in Example 3 and shown in Figure 3). The results showed that high salt concentrations can significantly improve SSF spore production, with the preferred salt concentration to add being higher than 10 g / L, with 15 g / L in Example 3 (approximately half the concentration in seawater) showing a more significant improvement. [Example]

[0082] S. brevicaulis spores on solid substrates of various sizes The effect of SH particle size on spore production was analyzed for two batches of experiments conducted in SSF flasks (similar to Example 2 above). The resulting SH particles were separated into four different size groups using standard test sieves: fine, less than 600 μm; small, 600 to 850 μm; medium, 850 μm to 2 mm; and large, 2 mm to 5.6 mm. The initial sample containing mixed SH particles was determined to be 38% (by weight) fine particles, 14% small particles, 41% medium particles, and 7% large particles.

[0083] In one batch of SSF flask experiments, three lines were evaluated in three different SH size groups: small, medium, and large, and spore yields were measured and compared at days 5, 9, and 14. The results are shown in Figure 7.

[0084] In another batch of SSF flask experiments, four systems were evaluated: mixed / initial, small, medium, and large SH particles, but spore yields were measured only on day 14. These results are shown in Figure 8.

[0085] Results from both batches of experiments showed that after 14 days, SSF spore yields from large and medium SH particles were comparable and higher than those from small and mixed SH particles. The better spore yields were attributed to the larger void space created by the larger particles, allowing faster and more uniform oxygen transfer from the headspace to all locations in the bed of solid substrate, supporting cell growth and sporulation. [Example]

[0086] Column-based SSF system SSF spore production was scaled up from the small flasks described above (containing 10 g of SH) to a column SSF system containing 50 to 75 g of SH. SH particles were mixed with a nutrient solution containing soluble nutrients / compounds to achieve a designed moisture and nutrient / chemical composition and autoclaved. After cooling, the SH was inoculated with spore seeds and loaded into the column. An upward airflow with a specific humidity was introduced from the bottom of the column for oxygen supply and metabolic heat removal. The flow rate of the incoming air was measured and controlled by a flow meter. Air humidity could be adjusted by mixing approximately 100% humidified airflow with low-humidity air (i.e., ambient air) through the humidification column at different flow rates. Preliminary experiments were performed to measure the moisture content of the SH in the column. Results showed that approximately 48 hours after the introduction of the humidified airflow, the SH moisture content stabilized and remained relatively constant (i.e., equilibrated with the humidity of the airflow).

[0087] Two sets of experiments were performed with SH of different particle sizes (and from two different SH suppliers). In one set of experiments, approximately 75 g of SH from one supplier with an initial mixed particle size (93% by weight, particle size <2 mm, including 38% <0.6 mm) was loaded to a bed height of approximately 34 cm in a 5.1 cm diameter column. The spore yield measured after 14 days was (7.5 ± 1.6) × 10 spores. 8 This yield was (11.0±0.5) x 10 spores / g SH obtained in the shallow flask in Example 7 for the mixed / original SH particles. 8 yields of pcs / g SH (Figure 8), indicating the adverse effects associated with the use of deeper beds, even when convective airflow is introduced through the column.

[0088] In the other set of experiments, the SH used was from a different supplier, and the SH particles had a much larger size (83% by weight ranging from 2 to 5.6 mm). The larger particles had a curved shape and even larger size, creating larger voids within the SSF volume. Only 48 g of SH (less than the 75 g SH above) could be loaded into the same size column (5.1 cm diameter) to an initial bed height of approximately 42 cm. The bed height dropped to 37 cm after 24 hours. The spore yield measured after 14 days was (7.0 ± 1.2) x 10 spores. 8 The yield was 6.1±1.3×10 spores / g SH. 8 This was not statistically different from the day 14 yield obtained in shallow flask experiments from this batch of SH, which was 14 pieces / g SH. Unlike the above use of mixed / small SH particles, there were no adverse effects noted associated with the use of deeper beds when these large SH pieces were used as the solid substrate.

[0089] Of note is the (larger) spore yield from this batch of SH (i.e., spores (6.1 ± 1.3) × 10 8 spores / g SH) is the yield from the other batches of (smaller) SH utilized above (i.e., 11.0±0.5)×10 8spores / g SH), which was due to differences in the SH itself. The hulls of various soybean varieties may have quite different compositions and structures. S. brevicaulis was found to grow more slowly and sporulate more slowly when grown on this batch of larger SH. When grown on other batches of smaller SH, S. brevicaulis spore production was found to plateau after about 8 to 10 days. However, when grown on this batch of larger SH, spore production increased after 14 days; (6.1±1.3) x 10 spores at 14 days. 8 spores / g SH on the 21st day (9.4±1.0) × 10 8 The number of SH particles increased to 1 / g SH.

[0090] The large air gap allowed good convective airflow through the bed to effectively supply oxygen, remove heat generated by cell metabolism, and control and regulate moisture content within the bed.

[0091] Various modifications and alterations that do not depart from the scope and spirit of this invention will be apparent to those skilled in the art. This invention is not to be duly limited to the illustrative embodiments set forth herein.

Claims

1. 1. A method for producing fungal spores using solid state fermentation (SSF), comprising: combining the fungal spores and the solid substrate in a solid state fermentor to produce a fungal culture; subjecting the fungal culture in the solid-state fermentor to solid-state fermentation conditions; the solid-state fermentation conditions include conditions for growth of fungal cells for the targeted production of additional fungal spores by the fungal cells; collecting the additional fungal spores. A method comprising:

2. 2. The method of claim 1, wherein the fungal cells are provided by the fungal culture, and the fungal cells and spores are of a species selected from Scopulariopsis brevicaulis, Purpureocillium lilacinum, Myrothecium verrucaria, Aspergillus nidulans, and combinations thereof.

3. 10. The method of any of the preceding claims, wherein the conditions for the targeted production of additional fungal spores comprise subjecting the fungal culture to a pH of about 10 to about 11.

4. 10. The method according to any of the above claims, wherein the solid fermentation conditions comprise the fungal cells having an appropriate nutrient supply for their growth.

5. 10. The method of any of the preceding claims, wherein the conditions for the targeted production of additional fungal spores comprise subjecting the fungal culture to a salinity of greater than 10 g / L NaCl.

6. 6. The method of claim 5, wherein the salinity is from about 10 g / L NaCl to about 20 g / L NaCl.

7. 10. The method according to any of the above claims, wherein the solid substrate is selected from soybean husks, rice husks, barley husks, wheat husks, grain skins, grain husks, corn husks, coconut husks, bean pods, and mixtures thereof.

8. 8. The method of claim 7, wherein the solid substrate is the soybean hulls, and the soybean hulls have a size of about 2 mm to about 5.6 mm.

9. 8. The method of claim 7, wherein the solid substrate is the soybean hulls, and the soybean hulls have a size of about 850 μm to about 2 mm.

10. 10. The method of any of the preceding claims, wherein the solid-state fermentation conditions comprise supplying a convective air current upward through the fungal culture, thereby supplying oxygen to the fungal cells.

11. Approximately 5 x 10 spores ８ From 2 x 10 ９ 10. The method of any preceding claim, wherein the method achieves a yield of said additional fungal spores of 100 / g solid substrate.

12. 10. The method of any of the preceding claims, wherein the solid substrate has a porosity of at least 75%.

13. 10. The method according to any of the preceding claims, wherein the solid state fermentor is a column or tower.

14. 5. The method of claim 4, wherein the adequate nutrient supply is provided by a mineral nutrient solution.

15. 11. The method of claim 10, wherein the convective airflow has a humidity of about 95% to 100% to provide humidity to the solid-state fermentor.

16. 10. The method of any of the preceding claims, wherein the solid-state fermentor is substantially devoid of any additional inert support or carrier other than the solid substrate, the solid-state fermentor is substantially devoid of any additional carbon source other than the solid substrate, and the solid-state fermentor is substantially devoid of any additional nitrogen source other than the solid substrate.

17. 10. The method of any of the above claims, wherein the solid substrate comprises additional water provided in a ratio of about 1:1 to about 2.5:1 additional water (in mL) to the solid substrate (in grams).

18. The fungal culture contains about 1 x 10 fungal spores per gram of the solid substrate. ４ From 1 x 10 ６ 10. The method of claim 9, wherein the concentration of the saturation region is 0.1 or 1.

2.

19. 10. The method of any of the preceding claims, wherein the solid substrate is substantially completely consumed by the fungal cells prior to the collecting step.

20. 10. The method of any of the preceding claims, further comprising adding the additional fungal spores from the collecting step to the cementitious material to repair one or more cracks in the cementitious material.

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