BIOREACTOR PARADIGM FOR THE PRODUCTION OF SECONDARY EXTRA-PARTICLE HYPHAL MATRICES

Abstract

The invention describes a methodology for production of a secondary extra-particle fungal matrix for application as a mycological material, manufactured via a Type II actively aerated static packed-bed bioreactor. A pre-conditioned air stream is passed through a substrate of discrete elements inoculated with a filamentous fungus to form an isotropic inter-particle hyphal matrix between the discrete elements. Continued feeding of the air through the substrate of discrete elements and isotropic inter-particle hyphal matrixes develops an extra-particle hyphal matrix that extends from an isotropic inter-particle hyphal matrix in the direction of airflow into a void space within the vessel.

Claims

1.-11. (canceled)

12. A mycological material comprising: an aerated inter-particle hyphal matrix, the aerated inter-particle hyphal matrix consisting essentially of: a filamentous fungus and a substrate of discrete elements; and an aerated extra-particle hyphal matrix growing from the aerated inter-particle hyphal matrix, wherein the aerated extra-particle hyphal matrix has a higher cell volume density than the aerated inter-particle hyphal matrix, a higher anisotropy than the aerated inter-particle hyphal matrix, and a higher hyphal strand thickness than the aerated inter-particle hyphal matrix.

13. The mycological material of claim 12, wherein the aerated inter-particle hyphal matrix and the aerated extra-particle hyphal matrix are aerated with a preconditioned air.

14. The mycological material of claim 13, wherein the aerated extra-particle hyphal matrix is positively gravitropic.

15. The mycological material of claim 13, wherein the aerated extra-particle hyphal matrix is negatively gravitropic.

16. The mycological material of claim 13, wherein the preconditioned air is horizontal through the aerated inter-particle hyphal matrix and the aerated extra-particle hyphal matrix, thereby causing the aerated extra-particle hyphal matrix to be horizontal and parallel to the flow of the preconditioned air.

17. The mycological material of claim 13, wherein the preconditioned air comprises one or more of: air, a paramorphogen, a volatile compound, or an aromatic compound.

18. The mycological material of claim 17, wherein the paramorphogen is one of: a terpene or an alkyl pyrone.

19. The mycological material of claim 13, wherein the preconditioned air is preconditioned to have a target temperature and a target humidity.

20. The mycological material of claim 13, wherein the preconditioned air flows laminarly from the aerated inter-particle hyphal matrix to the aerated extra-particle hyphal matrix.

21. The mycological material of claim 12, wherein the aerated extra-particle hyphal matrix is a target thickness.

22. The mycological material of claim 21, wherein the target thickness is up to 12 inches.

23. The mycological material of claim 12, wherein the aerated inter-particle hyphal matrix is disposed within a bioreactor, wherein the thickness of the aerated extra-particle hyphal matrix is a function of the temperature of the bioreactor, a volumetric air flow rate of the bioreactor, an incubation time of the mycological material, and a constituency of a preconditioned air passing through the bioreactor.

24. The mycological material of claim 12, wherein the discrete elements of the substrate are lignocellulosic particles.

25. The mycological material of claim 12, wherein the filamentous fungus is Ganoderma tsugae.

26. The mycological material of claim 12, wherein the aerated extra-particle hyphal matrix comprises a second substrate.

27. The mycological material of claim 26, wherein the second substrate is cotton fiber.

28. A mycological foam, meat substitute or biomedical material comprising the mycological material of claim 12.

29. The biomedical material of claim 28, wherein the mycological material mimics vessels of vasculature.

30. A cellular scaffold comprising the aerated extra-particle matrix of claim 12.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] These and other objects and advantages of the invention will become more apparent from the following detailed description taken in conjunction with the drawings wherein:

[0036] FIG. 1A schematically illustrates the direction and pattern of airflow and respiratory effluent through an inoculated substrate in a vessel in accordance with the method of the invention;

[0037] FIG. 1B schematically illustrates the pattern of fungal growth within the inoculated substrate of FIG. 1A in accordance with the method of the invention;

[0038] FIG. 2 schematically illustrates an embodiment wherein the direction of airflow is upward through the inoculated substrate;

[0039] FIG. 3 schematically illustrates an embodiment where the substrate of discrete elements inoculated with a filamentous fungus is separated into two spaced apart sections within the chamber of a vessel and air passed through each section in accordance with the method of the invention;

[0040] FIG. 4 schematically illustrates a vessel as in FIG. 3 disposed in a horizontal manner in accordance with the invention;

[0041] FIG. 5 schematically illustrates a vessel of cubic shape for performing the method of the invention; and

[0042] FIG. 6 schematically illustrates a vessel as in FIG. 1A with bottom void space of a defined geometry in accordance with the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0043] Referring to FIG. 1A, the method of producing a mycological material comprising the steps of providing a vessel 1 having a chamber that can be loaded with a substrate of discrete elements 3 inoculated with a filamentous fungus into the chamber.

[0044] As illustrated, the vessel 1 has a head space 2 at the upper end and a permeable partition 5 within the vessel 1 separating the chamber from a void space 6 below the partition 5.

[0045] Pre-conditioned air (at near-saturation and a defined temperature and gas composition) is fed into the top of the vessel 1 (or head-space 2) and diffuses down and between the discrete substrate elements 3 as indicated by the arrows 4 with the air flow exiting through the permeable partition 5. In this case, the specific gas composition and volumetric air flow rate may be constant or may be modulated dynamically.

[0046] Referring to FIG. 1B, under these conditions, the filamentous fungus expands a contiguous network of filamentous cells (hyphae) between and around the discrete substrate particles 3 forming an isotropic inter-particle hyphal matrix (IPM) 7.

[0047] As air diffuses between the particles 3 and through the IPM 7, a polarized condition develops within the vessel 1 in which air exiting the IPM 7 as laminar flow (as a function of the substrate particle matrix-IPM acting as a plenum) into the underlying void space 6 is of higher concentration of moisture than air entering the vessel (due to re-saturation during passage through IPM) and contains respiratory effluent (CO.sub.2, VOC, other signaling chemicals). Importantly, this creates a single, vertically oriented gradient of moisture and respiratory effluent (a polarized condition) culminating in the underlying void space 6 experiencing the highest concentrations with even, laminar flow of the air and respiratory effluent. Within this polarized condition, an extra-particle hyphal matrix (EPM) 8 extends from the IPM 7 in a positively gravitropic orientation, extending in the direction of airflow within the bottom void-space 6.

[0048] The morphology of EPM 8 is of increased anisotropy as compared to IPM 7 with dominant directionality occurring in the vertical orientation. The EPM 8 is then removed from the permeable partition 5 for utilization as a mycological material per Applications.

[0049] The following example is given with respect to FIG. 1A and FIG. 1B.

Example 1. Production of EPM

[0050] 1. Discrete lignocellulose particles are amended with supplemental nutrition, hydrated to a stage amenable to fungal growth, and combined with the spawn of filamentous fungal species Ganoderma tsugae (i.e., preparation of inoculated substrate) to form discrete particles 3 inoculated with a filamentous fungus. [0051] 2. Inoculated substrate of inoculated particles 3 are loaded into the bioreactor vessel 1 which contains a permeable partition 5 and which consists of a top head-space 2, a chamber to receive the inoculated substrate matrix, and a bottom void-space 6 below the permeable partition 5. [0052] 3. Air is fed into the top (head-space) of the bioreactor vessel at a rate of 0.2 volumes per bioreactor volume per minute, which has been pre-conditioned to an average temperature of 85° F. and a relative humidity (RH) of >90%. This pre-conditioned air diffuses through the inoculated substrate as indicated by the arrows 4 and exits through the permeable partition 5, into the bottom void-space 6, and out of the bioreactor with effluent CO.sub.2 concentration of <3%. These input conditions are maintained for the duration of the growth cycle. [0053] 4. Fungal growth occurs within the lignocellulose particle matrix by development of an isotropic hyphal matrix between and around the discrete lignocellulose particles (i.e., development of IPM). As growth of IPM progresses, the flow of pre-conditioned air per step 3 continues through the IPM, re-saturating the air to approach 100% RH and evacuating respiratory effluent, creating a top-down gradient of RH and respiratory effluent, and laminar flow from the bottom of the lignocellulose particle matrix-IPM into the bottom void-space 6 and out of the bioreactor. [0054] 5. From the IPM, a positively gravitropic extra-particle hyphal matrix (EPM) extends through the permeable partition 5 and into the bottom void-space 6, extending in the direction of airflow. The developed EPM represents a distinct structural morphology from the IPM, with a cell volume density (cell volume per total volume) of 2× that of the IPM, a directional coherency (degree of anisotropy of the hyphal matrix) of 3.2× that of IPM, and oriented hyphal agglomeration (galvanotropism) into strands increasing the average strand thickness to 1.11× that of IPM. [0055] 6. EPM is expanded to a target thickness based on the specific application requirements, then separated from the permeable partition 5 for post-processing dictated by the specific application.

[0056] Referring to FIG. 2, wherein like reference characters indicate like parts as above, the vessel 1 is constructed so that the pre-conditioned air is fed into the bottom of the vessel 1 and diffuses up and between the discrete substrate elements 3 to form isotropic inter-particle hyphal matrixes (IPM) 7 between the elements 3. In this embodiment, the extra-particle hyphal matrixes (EPM) 8 extends from the IPM 7 in the direction of airflow within the upper void-space 6 and development of the EPM is negatively gravitropic.

[0057] The following example is given with respect to FIG. 2.

Example 2. The Procedure of Example 1, Modifying EPM Structural Characteristics by Specific Modification of Input Temperature and Airflow Conditions

[0058] 1. Example 1 steps 1 and 2. [0059] 2. Per Example 2 step 3, with average temperature modified to 90° F., and airflow rate modified to 1.2 volumes per bioreactor volume per minute. [0060] 3. Example 1 step 4. [0061] 4. Example 1 step 5, with EPM morphology modified to a cell volume density of 4.5× that of IPM, a directional coherency of 2.6× that of IPM, and average hyphal strand thickness of 1.29× that of IPM. [0062] 5. Example 1 step 6.

[0063] Referring to FIG. 3, wherein like reference characters indicate like parts as above, the vessel 1 has a pair of permeable partitions 5 at mid-height to form a head space 2 therebetween and a pair of chambers for loading of two separate sections of the substrate of inoculated elements 3 therein.

[0064] In operation, air is input into the head-space 2 in the center of the bioreactor vessel 1 defined by the permeable partitions 5, from which air diffuses both down and up through the substrate particle matrix-IPM, in which laminar flow of the respiratory effluent outputs at both the bottom and top of the substrate particle matrix-IPM, where EPM 6 manifests as both positively and negatively gravitropic growth.

[0065] The following example is given with respect to FIG. 3.

Example 3. The Procedure of Example 1. Modifying EPM Structural Characteristics by Introduction of an Aromatic Compound into the Input Air

[0066] 1. Example 1 steps 1 and 2. [0067] 2. Example 1 step 3, with terpene introduced into the pre-conditioned air prior to introduction to the inoculated substrate. [0068] 3. Example 1 step 4. [0069] 4. Example 1 step 5, wherein the EPM and/or IPM is of reduced density and greater directional coherency as a function of the terpene exposure during IPM/EPM development. [0070] 5. Example 1 step 6.

[0071] Referring to FIG. 4, wherein like reference characters indicate like parts as above, the orientation of the vessel 1 of FIG. 3 is adjusted so that airflow, the gradient of respiratory effluent, and EPM extension occurs in the horizontal direction rather than the vertical direction.

[0072] Referring to FIG. 5, wherein like reference characters indicate like parts as above, the vessel 1 is a 4×4×4fl Type II packed-bed actively aerated bioreactor of cubic shape.

[0073] The following example is given with respect to FIG. 5.

Example 4. Production of EPM Using a 4×4×4fl Type II Packed-Bed Actively Aerated Bioreactor

[0074] 1. The vessel 1 is a 4×4×4fl container. [0075] 2. The permeable partition 5 is placed at a depth of 3 ft, allowing for 1 ft of empty volume 6 below the permeable partition 5. [0076] 3. Inoculated substrate elements 3 are loaded in the top 3 ft of the vessel 1. [0077] 4. Air is fed into the top (head-space) of the bioreactor vessel and through the particle matrix per Example 1 steps 3. and 4. [0078] 5. IPM 7 develops around and between substrate elements 3. [0079] 6. EPM 8 extends in a positively gravitropic orientation into the bottom void space 6 to a given target thickness, e.g., a thickness of up to 12 inches as a function of incubation time. [0080] 7. EPM is separated from the permeable partition 5 and post-processed per Example 1 step 6.

[0081] Referring to FIG. 6, wherein like reference characters indicate like parts as above, the vessel 1 may be made with a base that defines a void space 6 of a selected geometric shape, for example, of an oval cross-sectional shape.

[0082] In operation, EPM 8 expands in a positively gravitropic orientation into the bottom void space 6, producing an EPM of a defined geometry.

[0083] Alternatively, a vessel 1 with a base that defines a void space 6 of a selected geometric shape may have a second low-density substrate positioned in the void space 6 and, during operation, the extra-particle hyphal matrix is allowed to grow around and within the second low-density substrate to form a composite of the extra-particle hyphal matrix and the second low-density substrate.

Example 5. Expansion of EPM into a Secondary Substrate to Form an EPM-Secondary Substrate Composite

[0084] 1. Example 1 step 1. [0085] 2. A secondary substrate consisting of a low-density cotton fiber is loaded into the bottom void-space 6 of the vessel 1 below the permeable partition 5. [0086] 3. Example 1 steps 2-4. [0087] 4. Example 1 step 5, wherein the developing EPM extends through and around the cotton fiber substrate creating a composite EPM-cotton fiber material with a higher tensile strength than the mycelium EPM alone or cotton fiber individually. [0088] 5. EPM is expanded to a target thickness depending on the depth of the void space 6 so as to envelope the cotton fiber adequately, then is separated from the permeable bottom for post-processing as dictated by the specific application.

[0089] The invention thus provides a method of producing a mycological material, i.e., a secondary extra-particle fungal matrix, in a simple inexpensive manner. Further, the invention provides a paradigm to efficiently use a Type II actively aerated static packed-bed bioreactor to manufacture a secondary extra-particle fungal matrix for application as a mycological material.

[0090] The invention provides a paradigm for production of secondary extra-particle hyphal matrices (EPM) as: [0091] A mycological material for replacement of petroleum-based low-density foams, such as polyurethane foams. The simplified paradigm described here, as compared to the prior art, provides an opportunity for direct modification of density and morphological characteristics of the EPM, increases the potential scalability of manufacture and material range of fungal EPM, increasing competitiveness with petroleum-based foams. [0092] A cellular scaffolding, for example the growth of mammalian cells within the EPM. The described invention is a paradigm for allowing for specific modification of EPM density and morphological characteristics by modification of the direct temperature and gas exchange inputs. This may be applied to producing EPM specifically intended for providing a scaffold for mammalian cells for applications such as whole-cut meat substitutes and biomedical applications. For example, EPM porosity and density may be specifically modified for impregnation of mammalian cells of a given size, or the degree of hyphal agglomeration into cords and directional coherency of the hyphal cords may be modified to mimic vessels or vasculature.