WO2023023857A1

WO2023023857A1 - Method and system for culturing filamentous organisms

Info

Publication number: WO2023023857A1
Application number: PCT/CA2022/051281
Authority: WO
Inventors: Jean SAAYMAN; Ali GULAMHUSEIN; Marieta Marin Bruzos; Chand JAGPAL
Original assignee: Albert Labs Inc
Current assignee: Albert Labs Inc
Priority date: 2021-08-24
Filing date: 2022-08-24
Publication date: 2023-03-02
Anticipated expiration: 2024-02-24

Abstract

A method and system for propagating fungi or other filamentous organisms. Liquid media, inoculum and an envelope system in a vessel are agitated to distribute nutrientswithin the liquid media, and to remove waste from the liquid media. Agitation of the liquid media results in shear. The envelope system provides a physical barrier between the filamentous organism and the liquid media, mitigating shear forces on filamentous organism within the envelop system. The barrier may include walls of one or more propagation envelopes making up the envelope system. Inside each propagation envelope is a propagation chamber in fluid communication with liquid media in the vessel through an aperture in the propagation envelope. The aperture may include a valve or other feature to mitigate flow of mycelia, other filamentous propagating material, or other biological structures out of the propagation chambers, facilitating propagation of the filamentous organism within the propagation chambers.

Description

METHOD AND SYSTEM FOR CULTURING FILAMENTOUS ORGANISMS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Patent Application claims priority from United States Provisional patent Application No. 63/236,360, filed August 24, 2021 and entitled METHOD AND SYSTEM FOR CULTURING FUNGI, the entirety of which is hereby incorporated by reference.

FIELD

[0002] The present disclosure relates to a method and system for culturing filamentous organisms.

BACKGROUND

[0003] Fungi have been propagated in liquid culture. When fungi are propagated in the liquid culture, fluid flow provides and distributes nutrients and other important inputs to and throughout the liquid culture. The fluid flow also eliminates waste products from the liquid culture. Propagation of fungi is facilitated by greater fluid flow. Fluid flow results in shear forces, and shear forces of a sufficient magnitude may damage mycelia or other fungal material.

SUMMARY

[0004] In view of the shortcomings of previous approaches to culturing fungi and other filamentous organisms, there is motivation to provide a method and system for culturing fungi that provides strong yields of a consistent output.

[0005] Herein provided is a method and system for culturing fungi and other filamentous organisms. The method applies, and the system includes, an envelope system within a bioreactor tank, flask, cistern or other suitable bioreactor vessel for containing liquid media. The envelope system includes one or more propagation envelopes accessible to inoculum and within which mycelia or other structures of the filamentous organism are able to propagate. The propagation envelopes provide an environment for propagation and cultivation of the filamentous organism with mitigated exposure to shear forces through liquid media in the bioreactor vessel.

[0006] Liquid media may be inoculated with spores, mycelia or other fungal propagating material in the bioreactor vessel. The bioreactor vessel may include features to provide or facilitate mechanical agitation, turbulent agitation, forced fluid flow into the bioreactor vessel, pressurized introduction of oxygen or other gases into the vessel, or other means of inducing agitation for providing fluid flow into and through the bioreactor vessel. In aerobic applications, the fluid flow may facilitate provision of oxygen and other nutrients into and throughout the liquid media in the bioreactor vessel. In aerobic applications, the fluid flow may facilitate removal of CO2 and other waste products resulting from propagation of fungi throughout the liquid media in the bioreactor vessel. In anaerobic applications, the oxygen may be a waste product, and N2, CO2 or other gas may be a nutrient. The agitation may fluidize the propagation envelopes within the liquid media. Fluidization of the propagation envelopes may facilitate exposure of the propagation envelopes to fresh nutrients and oxygen, and removal of waste and CC^from the propagation envelopes, separately from the effects of agitation other than through fluidization.

[0007] Inoculum, which may include mycelia or other propagating material, may be provided to the vessel prior to, following or concomitantly with the propagation envelopes. The inoculum may localize to one or more propagation envelopes located within the bioreactor vessel. The propagation envelopes each include an envelope body, which includes a propagation chamber defined therein. The propagation chamber is accessible through at least one aperture in the envelope body. The aperture sequesters mycelia that propagate from the inoculum, facilitating propagation of the mycelia within the propagation chamber. The envelope body may be manufactured from any material that allows containment of a propagation chamber suitable to provide a matrix, space or other suitable environment for the mycelia to propagate with mitigated exposure to shear forces in the liquid media. The envelope body may also be manufactured from material with an appropriate density, relative to the volume and surface area of the propagation envelope, to facilitate fluidization of the propagation envelopes within the liquid media (e.g. polytetrafluoroethylene (“PTFE”), polypropylene, etc.). [0008] Propagation of the mycelia within the propagation chamber facilitates protection of the mycelia from shear forces in the liquid media. The envelope body provides a resilient and durable boundary between the mycelia and the liquid media. The aperture provides fluid communication between the propagation chamber and the liquid media while mitigating shear within the propagation chamber. The shear may result from fluid flow within the bioreactor vessel that is applied to oxygenate the liquid media, provide and distribute nutrients throughout the liquid media, remove waste products from the liquid media and otherwise facilitate propagation of the mycelia.

[0009] The method and system provided herein may provide advantages in some applications. The method and system may facilitate culturing fungi with efficient and effective biomass growth rates. The method and system may provide a low-cost and small footprint approach that allows for continuous flow and batch extraction. The method and system may facilitate providing metabolites from mycelium, avoiding production of basidiocarps. Concentrations and compositions of metabolites in basidiocarps may have greater variability than in mycelia. The method and system provided herein may provide a consistent controllable process that may be applied to manufacturing material consistently with good manufacturing practice (“GMP”) standards. Effectively growing any kind of mycelia in a liquid culture reactor with the method and system described herein may provide advantages in yield of target substances and other efficiencies. The target substances may be particular compounds, mixtures of related compounds or mixtures of unrelated compounds.

[0010] The system may be used in a method applied to liquid culture of fungi where fluid flow within the bioreactor vessel is sufficient to provide nutrients and oxygen to the fungi, and sufficient to remove CO2 and other waste from the bioreactor, while the envelope system protects the mycelia within the propagation envelopes from damage induced by shear forces resulting from fluid flow. The system may be applied to commercial scale culturing of mycelia, extracting metabolites from the mycelia, purifying the metabolites, and manufacturing a drug product, natural health product, nutraceutical product, food supplement, psilocybin product or other class of product from the metabolites. [0011] Extraction and purification methods described herein may be applied to secondary metabolites from Psilocybe cubensis, other psilocybin-producing fungi, Basidiomycetes or other organisms to produce a harmonized, consistent and safe substance to GMP standards for use in pharmaceutical drug products, natural health products, nutritional supplements or other products intended for use in humans. Variability in any active pharmaceutical ingredient (“API”) from batch to batch may result from variation in execution of extraction techniques, including in some cases extraction that do not stabilize the target substances. Variability in the API from batch to batch may result from natural variation of the target substances or other metabolites as between different geni, species, strains, and between individual basidiocarps. The variability between individual basidiocarps is mitigated by focusing on extraction from mycelia using the method and system described herein.

[0012] The method and system may be applied to many species of fungi that biosynthesize psilocybin, aeruginascin, baeocystin, norbaeocystin, psilocin, norpsilocin, 4-hydroxytryptamine, other 4-substituted tryptamines, betacarbolines or other metabolites (e.g. Gymtiopilus spectabilis, Panaeolus cyanescens, Psilocybe atrobrrmnea, Psilocybe azurescens, Psilocybe baeocystis, Psilocybe caernlipes, Psilocybe cubensis, Psilocybe cyanescans, Psilocybe pelliculosa, Psilocybe semilanceata, Psilocybe strictipes, Psilocybe subaeraiiginasceizs, Psilocybe subcaeriilipes, Stropharia aeruginosa, Stropharia semiglobata, etc.). Any of the above species may be used in commercial applications of the method and system provided herein.

[0013] The method and system may be applied to any species of fungi or other filamentous organism in which target substances may be found in a mycelial or other filamentous structure that may be cultivated in liquid media. Where optimization of an application of the method and system disclosed herein is directed to production of a particular metabolite, a particular combination or metabolites or other end goal related to the compounds found in an end product, a single species and strain that provides optimal results may be cultured and continually propagated to ensure a harmonized and consistent substance results from each separate batch of the species or strain that is being propagated using the method. [0014] In a first aspect, herein provided is A method and system for propagating fungi or other filamentous organisms. Liquid media, inoculum and an envelope system in a vessel are agitated to distribute nutrients within the liquid media, and to remove waste from the liquid media. Agitation of the liquid media results in shear. The envelope system provides a physical barrier between the filamentous organism and the liquid media, mitigating shear forces on filamentous organism within the envelop system. The barrier may include walls of one or more propagation envelopes making up the envelope system. Inside each propagation envelope is a propagation chamber in fluid communication with liquid media in the vessel through an aperture in the propagation envelope. The aperture may include a valve or other feature to mitigate flow of mycelia, other filamentous propagating material, or other biological structures out of the propagation chambers, facilitating propagation of the filamentous organism within the propagation chambers.

[0015] In a further aspect, herein provided is a method for propagating a filamentous organism in a vessel comprising: providing liquid media, inoculum of the filamentous organism and an envelope system into the vessel; and agitating the liquid media for providing nutrients to the filamentous organism inside the envelope system and for removing waste from the filamentous organism inside the envelope system. The envelope system comprises at least one propagation envelope for receiving the inoculum and the filamentous organism, and for propagating the filamentous organism in liquid media received within the envelope system. Agitating the liquid media results in shear forces within the liquid media. Each of the at least one propagation envelopes comprises a physical barrier between the filamentous organism and the liquid media for mitigating shear forces on the filamentous organism within the envelope system.

[0016] In some embodiments, the filamentous organism comprises a fungus. In some embodiments, providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the liquid media to the vessel prior to providing the inoculum to the vessel. In some embodiments, providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the inoculum to the vessel prior to providing the envelope system to the vessel. In some embodiments, providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the envelope system to the vessel prior to providing the inoculum to the vessel. In some embodiments, providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the envelope system to the vessel prior to providing the liquid media to the vessel. In some embodiments, providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the envelope system to the vessel prior to providing the liquid media or the inoculum to the vessel. In some embodiments, providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the liquid media to the vessel prior to providing the inoculum to the vessel. In some embodiments, agitating the liquid media comprises agitating the liquid media sufficiently to fluidize the envelope system in the liquid media. In some embodiments, agitating the liquid media comprises agitating the liquid media sufficiently to induce a circulation time of two minutes or less in the liquid media. In some embodiments, agitating the liquid media comprises application of mechanical mixing to the liquid media. In some embodiments, the mechanical mixing comprises rotary stirring of the liquid media. In some embodiments, agitating the liquid media comprises providing a gas to the liquid media. In some embodiments, agitating the liquid media comprises providing circulating fluid flow to focus fluid flow throughout the liquid media within the vessel. In some embodiments, agitating the liquid media comprises applying axial mixing of the liquid media within the vessel. In some embodiments, agitating the liquid media comprises applying radial mixing of the liquid media within the vessel. In some embodiments, the nutrients comprise oxygen. In some embodiments, the waste comprises CO2. In some embodiments, the method includes elevating the temperature of the liquid media in the presence of the filamentous organism to inactivate a heat-labile enzyme in the filamentous organism. In some embodiments, elevating the temperature of the liquid media in the presence of the filamentous organism comprises elevating the temperature of the liquid media to between 90 and 100 °C. In some embodiments, the method includes adjusting the pH of the liquid media away from physiological pH for the filamentous organism to inactivate a pH-labile enzyme in the filamentous organism. In some embodiments, adjusting the pH of the liquid media in the presence of the filamentous organism comprises raising the pH. In some embodiments, raising the pH comprises raising the pH to 10.0 or higher. In some embodiments, adjusting the pH of the liquid media in the presence of the filamentous organism comprises lowering the pH. In some embodiments, lowering the pH comprises lowering the pH to 4.0 or lower. In some embodiments, the method includes separating the envelope system from the liquid media, wherein the filamentous organism is within the envelope system. In some embodiments, separating the envelope system from the liquid media comprises draining the liquid media from the vessel. In some embodiments, the method includes adding extraction solvent to the vessel for extracting a substance from the filamentous organism within the envelope system. In some embodiments, the method includes combining an extraction solvent with the envelope system for extracting a substance from filamentous organism within the envelope system. In some embodiments, combining the extraction solvent with the envelope system comprises combining the extraction solvent with the envelope system in an extraction vessel separate from the vessel. In some embodiments, the extraction solvent comprises methanol, ethanol, water, acetone, consisting of acetonitrile, diethyl ether, trifluoroethanol or a combination thereof. In some embodiments, the extraction solvent is CO2. In some embodiments, the method includes elevating the temperature of the extraction solvent in the presence of the filamentous organism. In some embodiments, elevating the temperature of the extraction solvent in the presence of the filamentous organism comprises elevating the temperature of the extraction solvent to a temperature of between 1 and 25 °C below the boiling temperature of the extraction solvent. In some embodiments, the method includes dismantling the propagation envelopes for exposing the filamentous organism to the extraction solvent and facilitating extraction of the substance from the filamentous organism. In some embodiments, the method is operated in a batch process. In some embodiments, the method is operated in a continuous process.

[0017] In a further aspect, herein provided is a method for propagating a filamentous organism in a vessel comprising: providing liquid media and inoculum of the filamentous organism to the vessel; and agitating the liquid media for providing nutrients to the filamentous organism and for removing waste from the filamentous organism. The method is characterized in the method comprises providing an envelope system into the vessel; the envelope system comprises at least one propagation envelope for receiving the inoculum and the filamentous organism, and for propagating the filamentous organism in liquid media received within the envelope system; agitating the liquid media provides nutrients to the filamentous organism inside the envelope system and removes waste from the filamentous organism inside the envelope system; agitating the liquid media results in shear forces within the liquid media; and each of the at least one propagation envelopes comprises a physical barrier between the filamentous organism and the liquid media for mitigating shear forces on the filamentous organism within the envelope system.

[0018] In a further aspect, herein provided is a propagation envelope for culturing a filamentous organism, the propagation envelope comprising: an envelope body for receiving a liquid culture; a propagation chamber defined within the envelope body; and at least one aperture defined on the envelope body for providing fluid communication between the propagation chamber and an environment external to the propagation chamber.

[0019] In some embodiments, the envelope body comprises a defined shape selected from the group consisting of a sphere, cylinder, tetrahedron, cube, octahedron, decahedron, dodecahedron, icosagon, and any other shape that is defined by a plurality of surfaces, faces, vertexes, apexes or a combination thereof. In some embodiments, the plurality of vertexes, apexes and faces is repeated regularly and symmetrically. In some embodiments, the at least one aperture comprises an apex aperture defined on an apex of the envelope body. In some embodiments, the at least one aperture comprises a vertex aperture defined on the envelope body along a vertex of the envelope body. In some embodiments, the vertex aperture comprises a vertex midpoint aperture defined on the envelope body at a midpoint of the vertex. In some embodiments, the at least one aperture comprises a face aperture defined on the envelope body on a face of the envelope body. In some embodiments, the face aperture comprises a center face aperture defined on the envelope body at a center of the face. In some embodiments, the envelope body includes a textured inside surface for facilitating adherence of a filamentous organism to the textured inside surface. In some embodiments, the textured inside surface comprises grooves defined in the envelope body. In some embodiments, the textured inside surface comprises ridges extending from the envelope body. In some embodiments, the textured inside surface comprises a roughened inside surface. In some embodiments, the envelope body around the propagation chamber comprises at least one protrusion extending into the propagation chamber for providing additional surface area to the envelope body within the propagation chamber to support propagation upon the envelope body within the propagation chamber. In some embodiments, the envelope body is manufactured from a material with a specific gravity of between 0.5 and 8.0. In some embodiments, the material comprises a polymer material. In some embodiments, the material comprises a metal. In some embodiments, the material comprises a ceramic. In some embodiments, the material comprises a glass. In some embodiments, the at least one aperture comprises a valved aperture, the valved aperture comprising a valve for restricting particle flow from the propagation chamber to the environment. In some embodiments, the at least one aperture comprises a tapered aperture, the tapered aperture comprising a greater inside diameter at an outside mouth of the tapered aperture facing the environment than at an inside mouth of the tapered aperture facing the propagation chamber for restricting particle flow from the propagation chamber to the environment. In some embodiments, the at least one aperture comprises at least two apertures. In some embodiments, the propagation chamber comprises at least two separate portions; the at least one aperture comprises at least two apertures; at least one aperture is in fluid communication with each of the at least two separate portions; and each of the at least two separate portions are isolated from every other separate portion other than by way of at least two apertures and the external environment. In some embodiments, a flow path between the at least two apertures includes a barrier, a flow restriction, non-linear flow features or other features for disrupting a direct flow path between the at least two apertures, for mitigating shear forces within the propagation chamber relative to shear forces that would result from a direct flow path between the at least two apertures.

[0020] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

[0021] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures. In the attached Figures, features sharing a common final pair of numerals with a different first digit or digits correspond to equivalent features across embodiments shown in different figures (e.g. the propagation envelope 50, 150, 250, 350, 450, 550, 650, 750, 850, 950, etc.).

[0022] Fig. 1 shows a schematic of a system including an envelope system received within a liquid media culture tank;

[0023] Figs. 2A, 2B and 2C each show a propagation envelope having one aperture defined in a body of the propagation envelope;

[0024] Figs. 3A, 3B and 3C each show a propagation envelope having two apertures defined in a body of the propagation envelope;

[0025] Figs. 4A, 4B and 4C each show a propagation envelope having three or more apertures defined in a body of the propagation envelope;

[0026] Fig. 5 shows a schematic of a system including an envelope system received within a liquid media culture tank;

[0027] Fig. 6 shows the system of Fig. 5 in operation during sterilization and inoculation;

[0028] Fig. 7 shows the system of Fig. 5 in operation during propagation;

[0029] Fig. 8 shows the system of Fig. 5 in operation during draining of liquid media for extraction from mycelia;

[0030] Fig. 9 shows the system of Fig. 5 in operation during filling with solvent for extraction from mycelia; and

[0031] Fig. 10 shows a flow chart of a method for culturing fungi;

[0032] Fig. 11 shows a flow chart of a method for culturing fungi; and

[0033] Fig. 12 shows a flow chart of a method for culturing fungi.

DETAILED DESCRIPTION

[0034] Generally, the present disclosure provides a method and system for culturing fungi. In view of the shortcomings of previous approaches to culturing fungi, there is motivation to provide a method and system for culturing fungi that provides strong yields of a consistent output.

[0035] Mycelia may contain valuable primary metabolites, secondary metabolites, biosynthetic intermediates, or other organic byproducts or other constituents of biomass. Mycelia may also be cultured to process contaminates and purify a process feed stream through bio catabolism. In natural environments, most fungi thrive on stationary solid substrates. However, stationary solid substrates are not ideally suited for large scale fluid culture of fungi. Fungi can grow in mobile liquid cultures that are stirred under low liquid shear. Gentle conditions at low-liquid shear rates mitigate damage to the fungi and certain structural changes associated with damage, each of which limit growth and yield of compounds from the fungi. Damage to mycelia inhibits growth because the organism needs to divert resources to repair itself. High shear damages hyphae filaments or other structural features of filamentous or other organisms. This damage prompts repair, decreasing potential growth. A second effect is that hyphae filaments become clumped or pelleted instead of freely dispersed. After clumping together due to the external shear forces the hyphae start growing inward. As a result of the hyphae growing inward, a tightly-packed pellet eventually forms. Cells closer to the center of the pellet may have poor oxygenation and other complications (Amanullah et al., 2000).

[0036] Many stirred tank reactors have high shear rates as a result of the stirring that is applied to liquid cultures in the tank. The high shear is a result of stirring with a sufficient force to effectively move oxygen and other nutrients throughout the liquid media, delivering oxygen and other nutrients to mycelia being cultured in the reactor. Certain fungi may be cultured in a high-shear stirred tank reactor. High shear conditions are those agitation and other conditions that result in a pellet with a morphology number greater than 0.7 (Cairns et al. (2019)). Conditions in the vessel, including the viscosity of the liquid media, the rpm and radius of a mechanical mixer, reactor geometry, aeration rates and other factors, all contribute to the morphology number of the pellet.

[0037] Culturing fungi in a stirred tank reactor may have drawbacks related to efficiency, growth and use with a particular species of fungus. High shear rates may result in a protective response by mycelia by forming tightly-grown pellets of mycelia, with the tight growth resulting in tightly-packed pellets. Oxygen and nutrients have poor access to the center of the tightly-packed pellets, which may limit the health and growth of the fungi (Zacchetti et al. (2018)).

[0038] Low-shear stirred tank reactors may be provided, such as stirred tank reactors that apply liquid flow to stir the liquid culture. The liquid flow may be cross-flow, upflow, downflow or any suitable flow induced by mechanical stirring, bubbling gas through the liquid media or turbulent flow induced within the liquid media by use of a venturi jet or other fluid flow system that induces flow within the liquid media. Low-shear stirred tank reactors may provide a more hospitable environment for mycelia. Low-shear stirred tank reactors may be applied to most species of fungi that are likely to be cultured in large scale processes. Low-shear stirred tank reactors may however result in poor transfer of nutrients and waste, and poor mixing, potentially limiting growth of the mycelia. Low-shear stirred tank reactors do not fully eliminate shear, and the shear that is present may inhibit growth to a lesser degree than is found with high-shear tank reactors, but may not mitigate the effects of shear on growth entirely. The shear values may be selected to define a level of shear that minimizes shear damage to mycelia while optimizing aeration. [0039] Some liquid culture approaches include immobilizing mycelia onto a support. Immobilized mycelial cultures may be applied for removal of contaminants from a liquid feed. Immobilized mycelia in a liquid feed are less likely to be used to grow mycelia for fruiting or for directly harvesting the mycelia. In a liquid reactor that includes immobilized mycelia, low-shear trickle flow may be applied. In a trickle-bed reactor, liquid trickles down as a thin film across immobilized mycelia. Air flow is concurrent (downflow) or counter-current (upflow). Immobilized mycelia may be propagated for removal of pollutants from a liquid stream (e.g. using white rot fungus, etc.). The fungus may be immobilized on large wire mesh structures, using a polymer sponge material, using a foam-like material or other suitable immobilization techniques (see e.g. Castillo-Carvajal et. al. (2012), Pedroza-Rodriguez & Rodriguez-Vazquez (2013), Musoni et. al. (2015)).

[0040] Immobilizing the mycelia may offer the mycelia some protection from shear forces. However, immobilized reactors typically use fungus to consume pollutants out of the liquid media. In this case, the fungus is used as an absorber of the pollutants and the goal of these methods are not to produce mycelial biomass for extraction. This immobilization technique has been applied to methods where the goal is growing biomass and producing metabolites excreted by the fungi (Musoni et al. (2015)). The support scaffold for the immobilization may occupy a significant amount of the bioreactor volume available for propagation, which may be particularly relevant to harvesting of basidiocarps or mycelia for extraction rather than sequestering pollutants from media that is external to the fruiting bodies or mycelia, and an immobilized approach may have reduced effectiveness where the goal is generating biomass for extraction rather than removing toxins from the liquid media.

[0041] The method and system described herein apply propagation envelopes inside a bioreactor. A propagation chamber is defined inside each of the propagation envelopes. Material inside the propagation chambers has mitigated exposure to shear forces inside the bioreactor relative to material outside the propagation chamber and outside the propagation envelopes. As a result of fluid flow between the aperture or other channels providing fluid communication between the bioreactor and the propagation envelope, material inside the propagation chamber facilitating exchange of nutrients and waste between the liquid media and the propagation chambers within the propagation envelopes.

[0042] The method and system described herein may be applied to propagating and cultivating mycelia of fungi or other shear-sensitive filamentous organisms, and to extracting metabolites from the mycelia or other biomass from shear-sensitive filamentous organisms. The method and system described herein may also be applied to propagating and cultivation other organisms that are not shear sensitive within the propagation envelopes. Any applications or other references described herein relating to propagation of fungi also apply to propagation of other shear-sensitive filamentous organisms, and to cultivation of fungi or other shear-sensitive filamentous organisms. The method and system described herein may also be applied to anaerobic or other systems in which oxygen is not a nutrient, but is either absent or is a waste product. In either case, the method and system described herein facilitate provision of nutrients to the filamentous organism and removal of waste from the filamentous organism, while protecting the filamentous organism from the effects of shear forces.

[0043] Metabolites from propagated fungi may be purified and used for manufacturing drug products, natural health products or other products. The fungi metabolites may be harmonized and made consistently between batches, and through application of good manufacturing practices (“GMP”) standards for universally recognized sanitary and quality control techniques, providing harmonized drug substances with consistent compositions following extraction from mycelia, other filamentous biomass or other biomass. The harmonized and consistent drug substances manufactured to GMP standards may be used as active pharmaceutical ingredients (“APIs”) in drug products, and subject to the safety of the APIs, manufactured in a manner that provides safe drug products or other therapeutic products.

[0044] When applying the method and system described herein, liquid media may be inoculated with spores, mycelia or other fungal propagating material in the bioreactor vessel. The bioreactor vessel may include systems for applying mechanical agitation, turbulent agitation, forced fluid flow into the bioreactor vessel, pressurized introduction of oxygen or other gases into the vessel, or other means of inducing agitation for providing fluid flow into and through the bioreactor vessel. When cultivating most fungi and other aerobic organisms, the fluid flow may facilitate provision of oxygen and other nutrients into and throughout the liquid media in the bioreactor vessel. The fluid flow may facilitate removal of CO2 and other waste products resulting from propagation throughout the liquid media in the bioreactor vessel. The greater the fluid flow, the greater extent to which administration and distribution of nutrients, and removal of CO2 and other waste, may be accomplished. However, the greater the fluid flow, the greater the shear forces to which organisms propagating within the bioreactor vessel will be exposed.

[0045] Agitation may fluidize the propagation envelopes in the liquid media. Onset, intensity and other aspects of fluidization are a function of parameters including density differences between the propagation envelopes and the liquid media, aeration of the liquid media, and superficial gas flow. Superficial gas flow is aeration gas velocity divided by the open cross sectional area of the bioreactor vessel. Aeration gas velocity is defined as actual gas flow rate of gasses within the vessel.

[0046] Mechanical agitation may be applied intermittently while pressurized introduction of oxygen or other gases into the bioreactor vessel is applied continuously, or other patterns of agitation may be applied. Some methods of applying agitation, such as pressurized introduction of oxygen or other gases into the vessel, may aerate the liquid media directly. Other methods of aeration, such as mechanical mixing or turbulent fluid flow, may indirectly provide aeration through providing fluid flow at an upper surface or other interface between the liquid media and the atmosphere within the bioreactor vessel. [0047] Agitation may turn over the volume of the liquid media in the bioreactor vessel regularly to provide advantages in terms of facilitating culture of the fungi in the envelope system by further providing nutrients and oxygen, and removing waste and CO2 from the propagation envelopes. For example, agitation may be applied at a rate sufficient to turn over at least half the volume of the bioreactor vessel every minute, or a circulation time of two minutes or less. Circulation time is the time it takes for a differential volume of liquid to circulate through the entire vessel volume, and one turnover of the volume of the vessel occurs during the circulation time. (e.g. 1 L of fluid would be turned over every minute in a 2 L bioreactor vessel, 10 L of fluid would be turned over every minute in a 20 L bioreactor vessel, etc.).

[0048] Inoculum, which may include mycelia or other propagating material, may be provided to the vessel prior to, following or concomitantly with the propagation envelopes. Providing the inoculum after providing the propagation envelopes facilitates sterilizing the propagation envelopes and the liquid media before adding the inoculum. The inoculum may localize to one or more propagation envelopes located within the bioreactor vessel. The propagation envelopes each include an envelope body, which includes a propagation chamber defined therein. The propagation chamber is accessible through at least one aperture in the envelope body. The aperture may sequester the inoculum and any mycelia that propagates from the inoculum prior to sequestration within the propagation chamber. The aperture may sequester mycelia that propagate from the inoculum, facilitating propagation and cultivation of the mycelia within the propagation chamber and retention of mycelia within the propagation chamber, where the mycelia benefit from provision of oxygen and other nutrients, and removal of CO2 and other waste waste, as a result of fluid flow caused by agitation, but exposure of mycelia to shear forces is mitigated, while maintaining fluid communication with the liquid culture in the bioreactor vessel.

[0049] The propagation envelopes may include a single aperture. The aperture may include features to facilitate entry and mitigate exit from the aperture. The aperture may include a valve, variable inside diameter or other mechanical approach to facilitate entry and mitigate exit. The variable inside diameter may be shaped to provide a narrower inside diameter at an outside surface of the envelope body relative to the greater inside diameter at an inside surface of the envelope body. The variable inside diameter may vary gradually as the aperture progresses from the outside surface of the envelope body to the inside surface of the envelope body. Where more than one aperture provides access to the propagation compartment, the apertures may be offset or otherwise staggered relative to one another, or otherwise positioned to minimize flow-through into a first aperture and directly out of a second aperture. Flow-through of the liquid media into a first aperture and out of a second aperture with a relatively straight and direct flow path may increase shear within the propagation container, which may in turn result in damage to mycelia.

[0050] An inside surface of the envelope body facing the propagation chamber may be smooth or textured. A textured inside surface of the envelope body may facilitate adhering of mycelia or other filamentous structures to the envelope body within the propagation chamber. The envelope body may be manufactured from any material that allows containment of a propagation chamber suitable to provide a matrix, space or other suitable environment for the mycelia to propagate with mitigated exposure to shear forces in the liquid media. Examples of suitable material include autoclavable, thermally stable, inert, polymer materials, metals, ceramics and glass (e.g. textured PTFE, textured polypropylene, polyethylene, roughened stainless steel, silicon carbide, silicon aluminum oxynitride, alumina based oxides, carbide, borides, nitrides, silicides, composite materials, particulate reinforced, fiber reinforced, combination of oxides and non-oxides, silicate glass, borosilicate glass, etc.).

[0051] The propagation envelopes may be manufactured from a material with a surface that is textured, tacky when wet with liquid media, or that is otherwise facilitates adherence to the inside surface of the envelope body.

[0052] Where the propagation envelopes will be crushed by grinders or otherwise dismantled prior to extraction, then materials that are simpler to grind, crush or otherwise dismantle the propagation envelopes may be used to manufacture the envelope body rather than stronger materials. For example, stainless steel may not facilitate crushing or grinding compared with polymers or glass materials.

[0053] Propagation of the mycelia within the propagation chamber facilitates protection of the mycelia from shear forces in the liquid media as the liquid media is mixed through agitation in the bioreactor vessel. Agitation may be through application of mechanical agitation, turbulent agitation, forced fluid flow into the bioreactor vessel, pressurized introduction of oxygen or other gases into the vessel, or other means of inducing agitation for providing fluid flow into and through the bioreactor vessel. The envelope body provides a resilient and durable boundary between the mycelia and the liquid media. The aperture provides fluid communication between the propagation chamber and the liquid media while mitigating shear within the propagation chamber. The shear may result from fluid flow within the bioreactor vessel that is applied to oxygenate the liquid media, provide and distribute nutrients throughout the liquid media, remove CO2 and other waste products from the liquid media and otherwise facilitate propagation of the mycelia.

[0054] A solid propagation envelope with a greater density than the liquid media could be fluidized with a high gas flow rate or other higher agitation. A solid propagation envelope with a similar or lower density than the liquid media could be fluidized with a low gas flow rate or other lower agitation.

[0055] The propagation envelope may be manufactured from a substance that has a specific gravity of between 0.5 and 8.0. Examples of suitable material include autoclavable, thermally stable, inert, polymer materials, metals, ceramics and glass (e.g. textured PTFE, textured polypropylene, polyethylene, roughened stainless steel, silicon carbide, silicon aluminum oxynitride, alumina based oxides, carbide, borides, nitrides, silicides, composite materials, particulate reinforced, fiber reinforced, combination of oxides and non-oxides, silicate glass, borosilicate glass, etc.). The liquid media would typically have a specific gravity of between 0.97 and 1.4. Selecting a density of the propagation envelopes close the density of the liquid media facilitates suspension and fluidization of the propagation envelopes within the liquid media by application of agitation to the liquid media to affect buoyant flow or other bulk fluid flow during the expected level of agitation that will be applied to fluidize the propagation envelopes, and facilitate propagation by aerating the liquid media where propagating fungi or other aerobic organisms, providing fresh liquid media, and removing CO2 where propagating fungi or other aerobic organisms, and removing other waste. Manufacturing the envelope body from a materials with greater density than the liquid culture increases the amount of agitation required to fluidize the propagation envelopes. There may be advantages to manufacturing the envelope body from materials that have a density close to the density of the liquid media in terms of lowering the amount of agitation that is required to fluidize the envelopes and facilitate propagation of the filamentous organism.

[0056] The method and system provided herein may provide certain advantages for culturing fungi, extracting metabolites from the fungi, purifying the metabolites, and manufacturing a drug product, natural health product, nutraceutical, nutritional supplement, other therapeutic product or other product from the metabolites. The method and system may facilitate culturing fungi with efficient and effective biomass growth rates. The method and system may provide a low-cost and small footprint approach that allows for continuous flow and batch extraction. Concentrations and compositions of metabolites in basidiocarps may have greater variability than in mycelia. The method and system provided herein may provide a consistent controllable process may be applied to manufacturing material consistently with GMP standards. The method and system may be applied to recovery of tryptamines production from Psilocybe species or other tryptamines containing species of the phylum Basidiomycota.

[0057] Fig. 1 shows a system 10 in use for culturing fungi. The system 10 includes a bioreactor vessel 20. A liquid growth media 70 is within the vessel 20. An envelope system 60 is within the vessel 20. The envelope system 60 includes a plurality of propagation envelopes 50, each with an aperture 62. A propagation chamber (e.g. the propagation chamber 154 of Fig. 2A, etc.) is defined within each propagation envelope 150. The propagation chambers within the propagation envelopes 50 are in fluid communication with the liquid media 70 in the vessel 20 through the apertures 62. The vessel 20 includes a mechanical mixer 26 or other means to induce buoyant flow or other bulk fluid flow. The buoyant flow or other bulk fluid flow results in dispersal throughout the liquid media 70 of oxygen, other nutrients or other additives added to the liquid media 70, and removal of CO2 and other waste material that is produced by culturing fungi in the liquid media 70. The buoyant flow or other bulk fluid flow may also result in fluidization of the propagation envelopes 50 within the liquid media 70. An input, output and conditioning system 40 is in fluid communication and thermal communication with the vessel 20 and may otherwise interact with the vessel 20 to control the conditions within the vessel 20 and the liquid media 70. The input, output and conditioning system 40 may also include a heat exchanger 51 to change the temperature of liquid media 70 within the vessel 20.

[0058] A method may be applied using the system 10 for culturing fungi and extracting metabolites from the fungi. The metabolites may be purified, and drug products or other products manufactured from the metabolites. The method may facilitate production of harmonized, consistent and safe APIs including one or more metabolites from fungi. The method may be applied to large-scale propagation of mycelia and biosynthesis of valuable metabolites. Production of harmonized, consistent and safe preparations of metabolites by extraction from fungi may follow.

[0059] The propagation envelopes 50 each include one aperture 62. Other designs of propagation envelopes 50 may also be applied to the method and the system 10.

[0060] Fig. 2A shows a propagation envelope 150 having an aperture system 168, which includes one aperture 162 defined in an envelope body 152 of the propagation envelope 150. A propagation chamber 154 is defined within the envelope body 152. The aperture system 168 provides fluid communication with the propagation chamber 154, facilitating fluid flow to provide nutrients to the propagation chamber 154 and remove CO2 and other waste from the propagation chamber 154. The propagation chamber 154 and the aperture 162 are positioned to mitigate flow of mycelia out of the propagation chamber 154. The relative surface area of the aperture 162 is smaller than the surface area of the propagation chamber 154, providing a form of valve that facilitates ingress from outside the propagation envelope 150 into the propagation chamber 154 through the aperture 162, while limiting egress from the propagation chamber 154 out from the propagation envelope 150 through the aperture 162. Liquid shear that impacts the outside of the envelope body 152 is mitigated within the propagation chamber 154. The propagation chamber 154 provides an environment for mycelia to grow as loosely packed clumps with mitigated exposure to liquid shear outside the propagation envelope 150.

[0061] The propagation chamber 154 may be sized large enough such that mycelia can grow unimpeded for a period selected with reference to the ratio of mycelium-growth to target metabolite production. The propagation chamber 154 may be sized small enough such that the amounts of oxygen and other nutrients available, and the amount of CO2 and other waste material present, throughout the propagation chamber 154 are at or above acceptable concentrations along a gradient that may establish throughout the propagation chamber 154. The gradient may provide greater levels of oxygen and other nutrients, and lower levels of CO2 and other waste material, as the gradient progresses toward the aperture 162 from within the propagation chamber 154. The gradient may provide greater levels of CO2 and other waste material, and lower levels of oxygen and other nutrients, as the gradient progresses from within the propagation chamber 154 toward the aperture 162. The size of the propagation chamber 154 may be selected based on the fungi expected to be propagated in the propagation chamber 154.

[0062] The propagation envelope 150 may be sized, and the envelope body 152 may be prepared from material selected, to be fluidizable or have a buoyancy that allows the propagation chamber 154 to have a propagation chamber 154 sized large enough to be uniformly distributed in liquid media during agitation of the liquid media to fluidize the propagation envelope 150, and to facilitate propagation of fungus within the propagation chamber 154 by providing fresh liquid media, aerating the liquid media, and removing CO2 and other waste from the liquid media.

[0063] The envelope bodies 152 are manufactured from material that will not be consumed by the mycelia, will not be damaged by the liquid media or the extraction solvent, and that does not damage, change or otherwise impact the composition of the mycelia or the liquid media. Internal support in the hollow shape may be added and may be composed of a material that aids to the growth of the mycelium.

[0064] Fig. 2B shows a propagation envelope 250. The aperture system 268 defined in the envelope body 252 includes one aperture 262 for providing fluid communication with the propagation chamber 254. The aperture 262 has a greater surface area relative to the size of the propagation envelope 250 compared with the aperture 162 relative to the size of the propagation envelope 250.

[0065] Fig. 2C shows a propagation envelope 350. The aperture system 368 defined in the envelope body 352 includes one aperture 362 for providing fluid communication with the propagation chamber 354. The envelope body 352 has a cylindrical shape rather than the spherical shape of the envelope body 152. [0066] Fig. 3A shows a propagation envelope 450. The aperture system 468 defined in the envelope body 452 includes a first aperture 462 and a second aperture 463 for providing fluid communication with the propagation chamber 454. A first flow path 456 provides fluid communication between the first aperture 462 and the propagation chamber 454. A second flow path 457 provides fluid communication between the second aperture 463 and the propagation chamber 454. The first flow path 456 and the second flow path 457 are positioned relative to one another in a flow path that does not follow a straight line, includes comers or is otherwise adapted to mitigate flowthrough between the first aperture 462 and the second aperture 463, for mitigating any shear that may result from direct flowthrough between the first aperture 462 and the second aperture 463. Alternatively, a barrier (not shown), such as a baffle, or a flow restriction (not shown) may be located between the first flow path 456 and the second flow path 457 to mitigate flowthrough from the first aperture 462 to the second aperture 463.

[0067] Fig. 3B shows a propagation envelope 550. The aperture system 568 defined in the envelope body 552 includes the first aperture 562 and the second aperture 563 for providing fluid communication with the propagation chamber 554. The envelope body 552 has an irregular shape rather than the spherical shape of the envelope body 452.

[0068] Fig. 3C shows a propagation envelope 650. The aperture system 668 defined in the envelope body 652 includes two apex apertures 664 for providing fluid communication with the propagation chamber 654. The envelope body 652 has a prismatic shape rather than the spherical shape of the envelope body 452. The apex apertures 664 are at the apexes of three sides of the prismatic envelope body 652.

[0069] Fig. 4A shows a propagation envelope 750. The aperture system 768 defined in the envelope body 752 includes a first aperture 762, a second aperture 761 and a third aperture 763. The first aperture 762 is for providing fluid communication with the first propagation chamber 754. A second aperture 761 is for providing fluid communication with the second propagation chamber 753. A third aperture 763 is for providing fluid communication with the first propagation chamber 755. None of the first propagation chamber 754, the second propagation chamber 753 or the third propagation chamber 755 are in fluid communication with each other. [0070] Fig. 4B shows a propagation envelope 850. The aperture system 868 defined in the envelope body 852 includes two apex apertures 864, one face aperture 865 and two vertex midpoint apertures 866 for providing fluid communication with the propagation chamber 854. The envelope body 852 has a prismatic shape rather than the spherical shape of the envelope body 752. The apex apertures 864 are at the apexes of three sides of the prismatic envelope body 852. The face apertures 865 is in the centerpoint of a face of the prismatic envelope body 852. The vertex midpoint apertures 866 are at the midpoint of a vertex of the prismatic envelope body 852.

[0071] Fig. 4C shows a propagation envelope 950. The aperture system 968 defined in the envelope body 952 includes a cluster of apertures 957 for providing fluid communication with the propagation chamber 954.

[0072] The propagation envelopes of different designs may be manufactured to various sizes, resulting in various densities and other material properties that are relevant to fluid flow into the aperture, fluid flow through the propagation chamber, fluidization, crushing and other features of the propagation envelopes that may be optimized for a given application.

[0073] One example propagation envelope may include a cylindrical cup manufactured from PTFE. A cup that is roughly 2.5 cm in diameter and 2.5 cm in height provides a volume of about 12.3 cm³ and a total surface area of about 50 cm². With a material density of about 2.2 g/cm³, the density of the empty cup would be about 0.53 g/cm³. These propagation envelopes may be design similarly to the propagation envelope 350 shown in Fig. 2C.

[0074] Another example propagation envelope may include a spherical hollow ball manufactured from polypropylene. A sphere that is about 2 cm in diameter provides a volume of about 4.2 cm³ and a total surface area of about 24 cm². With a material density of about 0.91 g/cm³, the density of the empty hollow ball would be about 0.24 g/cm³. These propagation envelopes may be design similarly to the propagation envelope 150 shown in Fig. 2A or the propagation envelope 950 in Fig. 4C.

[0075] Fig. 5 shows a schematic of the system 10. The system 10 includes the vessel 20 and the input, output and conditioning system 40 in fluid communication with the vessel 20 and otherwise communication with the vessel 20 to control the conditions within the vessel 20.

[0076] The vessel 20 includes a vessel body 22. A media chamber 24 is defined within vessel body 22. The media chamber may include liquid media 70 (e.g. as shown in Figs. 6 and 7) or extraction solvent 78 (e.g. as shown in Fig. 9). The mechanical agitator 26 may provide agitation in the media chamber, resulting in fluid flow within the media chamber 24. Fluid flow within the media chamber 24 facilitates delivery of nutrients to the mycelia propagating within the media chamber 24, aeration by delivery of oxygen to the mycelia propagating within the media chamber 24, and removal of CO2 and other waste form the mycelia propagating within the media chamber 24. As a result of facilitating aeration and other exchange of nutrients and waste, fluid flow within the media chamber 24 facilitates propagation of mycelia within the media chamber 24. A gas distributor line 28 may also provide agitation for fluid flow within the media chamber 24. The gas distributor line 28 may also provide oxygen or other gasses that facilitate propagation of the mycelia. Fluid flow within the media chamber 24, whether as a result of mechanical agitation by the mixer 26 or introduction of oxygen or other gasses by the gas distributor line 28, also fluidizes the propagation envelopes 50.

[0077] A venturi jet (not shown) or other approach to recirculating liquid media within the vessel body 22 may also be applied, for example application of an angled nozzle (not shown) located tangential to the width (or in the case of a cylindrical vessel body, the circumference) of the vessel body 22, which may induce fluid flow and agitation. The venturi jet or angled nozzle near the bottom of the vessel body 22 to facilitate agitation and fluidization of the propagation envelopes 50 to distribute the envelope system 60 throughout the vessel body 22.

[0078] The vessel 20 may have ports for feed lines, product lines, aeration lines, sample lines, and process condition measurement and will be sanitized prior to inoculation.

[0079] A media inlet 30 is in fluid communication with the media chamber 24 and may be used to provide liquid media, nutrients or other material to the media chamber 24. [0080] An inoculation port 32 is in fluid communication with the media chamber 24 and may be used to provide inoculum to the media chamber 24. [0081] A gas input 34 is in fluid communication with the gas distributor line 28 for supplying gasses to the media chamber 24. The gas input 34 may be positioned proximate a bottom of the vessel body 22 for dispersing gas bubbles throughout the liquid media, agitating the liquid media for exchange of nutrients and waste, aeration, and fluidization of the propagation envelopes 50.

[0082] A drain port 35 may be in fluid communication with the media chamber 24 for draining liquid media, extraction solvent or other fluid from the media chamber 24. The drain port 35 may be positioned proximate a bottom end of the vessel body 22 for facilitating draining of the media, extraction solvent or other fluid from the media chamber 24. A filter 41 may be applied to filter mycelia or other material from the media being drained. The filter 41 may be opened to allow unfiltered material to pass through (e.g. during application of the grinder 46 during extraction; see Fig. 8).

[0083] A venting port 36 with a gas filter 44 may be in fluid communication with the media chamber 24 and may be used to allow intake oxygen, and to allow venting of CO2 or other gases that result from propagation of mycelia. The gas filter 44 may be applied to filter the gases of any particles selected for filtration from the gases.

[0084] A screened liquid overflow port 38 may be in fluid communication with the media chamber 24 and may be used to allow liquid media to flow out of the media chamber 24 when a level of the liquid media exceeds a threshold height. The screened overflow port 38 may be include a screen with an aperture sized to sequester any mycelia or other suspended material in the media chamber 24.

[0085] A plurality of auxiliary ports 48, including instrument ports, sample ports or other ports may also be included in the vessel body 22. The auxiliary ports 48 may be applied to provide communication between the media chamber 24 and any suitable probe, filter or other system (e.g. pH probe, temperature probe, dissolved oxygen probe, other probes, side-stream recirculating filter, other filter, sampling system, etc.).

[0086] The removal and conditioning system 40 includes a gas-liquid mixer 42, which may include a static mixer, for providing gasified liquids into the media chamber 24 using a gas-liquid pump 43 through a recirculated media inlet 49. The gas-liquid static mixer 42 may combine gasses with overflow media that is drained out of the screened liquid overflow port 38. The removal and conditioning system 40 includes a grinder 46 in line with a grinder pump 45 and a ground material input 47 for facilitating extraction by grinding mycelia and propagation envelopes 50 prior to extraction in the media chamber 24. Any other suitable approach to crushing or otherwise dismantling the propagation envelopes 50 other than grinding may also be applied. Alternatively, the grinder pump 45 may be used to deliver ground mycelia and propagation envelopes 50 to a separate extraction vessel for extraction (not shown).

[0087] The vessel 20 may be operated in both recirculation (batch) or continuous flow and can also be used for extraction processes. The vessel 20 will be designed in such a way to allow for process intensification and dedicated continuous flow to downstream process steps such as dewatering of cultures, extraction, and chemical workup such as purification.

[0088] Fig. 6 shows the system 10 in operation during sterilization and inoculation. The vessel 20 is designed to facilitate process intensification by allowing a number of process steps to conducted without transferring the propagation envelopes 50 of the envelope system 60 out of the vessel 20. For example sterilization, inoculation, propagation and extraction may all be completed in the same vessel 20 without removing the propagation envelopes 50. This approach may improve batch-to-batch consistency. [0089] The liquid media 70 is provided to the media chamber 24 through the media inlet 30. Inoculum 72 is provided to the liquid media through the inoculation port 32. The liquid media 70 may be selected to provide the fungi of the inoculum 72 with all the required nutrients to propagate the mycelia. The inoculum 72 may be disbursed throughout the liquid media 70 by agitation of the liquid media 70 with the mechanical mixer 26, through flow of gasses through the gas distributor line 28, or by other suitable agitation devices. The inoculum 72 may enter the propagation envelopes 50 and propagate to mycelia within the propagation envelopes 50. The inoculum 72 may also propagate to mycelia in the liquid media 70 outside the propagation envelopes 50, and the mycelia may enter the propagation envelopes 50 of the envelope system 60.

[0090] The liquid media 70 may be any suitable liquid media for propagating fungi. The liquid media 70 may include glucose, maltose and potentially other simple and usable carbon sources, ammonium succinate, L-tryptophan, potassium dihydrogen phosphate, glycine, yeast extract, thiamine hydrochloride, ammonium heptamolybdate tetrahydrate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate, zinc sulphate heptahydrate, manganese (II) chloride tetrahydrate, iron (II) sulphate heptahydrate, copper (II) sulphate pentahydrate, magnesium sulphate heptahydrate, acetic acid, hydrochloric acid, potassium hydroxide and a pH buffer.

[0091] Fig. 7 shows the system 10 in operation during propagation of mycelia. The venting port 36 and filter 44 are open to the atmosphere to allow effluent gasses 71 to vent from the media chamber 24. As the liquid media 70 drains out of the screened liquid overflow port 38, oxygen or other gases required by the mycelia are added to the liquid media 70, providing gasified liquid media 76, which is provided to the media chamber 24. Gasses 74 are also added to the media chamber 24 from the gas distributor line 28, which provides gasses 74 to facilitate propagation of the fungi by agitation, aeration and exchange of nutrients and waste. Gasses 74 are also added to the media chamber 24 from the gas distributor line 28, which also results in fluid flow and agitation of the liquid media 70, and may also contribute to fluidizing the propagation envelopes 50 within the liquid media 70.

[0092] The vessel 20 may be sized, and the number and size of propagation envelopes 50 selected, to meet production needs of mycelia of fungi being propagated. The liquid media 70 may be agitated by mixing the liquid media 70 with the mechanical mixer 26, by application of a liquid fluidized column of bubbles of the gas 74 and the gasified liquid media 76 from the gas distributor line 28, or both. The liquid media may be saturated with oxygen or other gasses to an appropriate dissolved oxygen level or dissolved level of other gases. Additional oxygen or other gases may be added to the media chamber 24 and dispersed throughout the media chamber 24 by the mixer 26 or the gas distributor line 28. The liquid overflow port 38 and any other outlet may use a sieve, filter, cyclone or other particle separator to prevent the propagation envelopes 50, or larger portions of free mycelium, from being removed from the vessel 20 with liquid media 70 that is removed from the vessel 20 through the overflow port 38 or other outlet. Samples may be taken, and data otherwise acquired, through the auxiliary ports 48.

[0093] Both batch processes and continuous processes may be applied to the system 10. In batch processes, the vessel 20 may be drained and sterilized, and may be filled with extraction solvent, as further described herein. For operation in continuous processes, some of the liquid media 70 would be aseptically drained and some of the propagation envelopes 50 would be aseptically removed. Sterilized liquid media 70 and sterilized propagation envelopes 50 would be added to the vessel 20 aseptically to replace the liquid media 70 and propagation envelopes 50 that were removed. Propagation envelopes 50 may be colour coded or otherwise distinguishable between batches or lots within a continuous process to ensure adequate propagation time on each envelope.

[0094] The liquid feed temperature may be controlled to maintain an optimal growth temperature. The temperature within the vessel 20 may be controlled by the heat exchanger 51 (shown in Fig. 1 ) by fitting electrical heating elements or tube bundles, or jacketing the vessel 20 with a temperature controlled heat exchange medium. The heat exchanger 51 may be integrated into the vessel 20 or may be external and in conductive communication with the vessel 20 for changing the temperature within the vessel 20.

[0095] Before draining the liquid media 70 as shown in Fig. 8, the temperature of the liquid media 70 within the vessel 20 may be increased to a point just below 100 °C (e.g. 90, 91 , 92, 93, 94, 95, 96, 97, 98 or 99 °C) for a period of time (e.g. 1 , 2, 3, 4 or 5 minutes, etc.). A heat exchanger, such as the heat exchanger 51 shown as part of the input, output and conditioning system 40 in the system 10, may be used to modulate the temperature within the vessel body 20.

[0096] Increasing the temperature of the liquid media 70 over the period of time may facilitate denaturation of enzymes that may degrade psilocybin or other metabolites selected for recovery from the mycelia. The PsiK enzyme dephosphorylates psilocybin to psilocin. Psilocin is unstable relative to psilocybin. Heating to between 90 °C and 100 °C over a period of 1 , 2, 3, 4 or 5 minutes or more may denature PsiK present in mycelia. Alteration to pH may also be applied to denature an enzyme. For example, adding acetic acid to lower the pH may denature PsiK, mitigating conversion of psilocybin to psilocin by PsiK. In other cases, the pH of the liquid media 70 may be increased or decreased to denature other enzymes that catalyze reactions that deplete valuable metabolites. Denaturing enzymes that may break down selected metabolites may preserve metabolites that would normally be degraded by the enzymes during the extraction process, increasing recovery of metabolites produced by the mycelia. Denaturing enzymes through heat, changes in pH or other changes in the environment of the liquid media 70 may be completed after propagation of the mycelia has concluded as the change in conditions to denature the enzymes is likely to also impair or terminate propagation of mycelia.

[0097] Fig. 8 shows the system 10 in operation after draining the liquid media 70 for extraction of metabolites from mycelia in the envelope system 60. The grinder pump 45 may be used to pump the liquid media 70 out of the media chamber 24. The vessel body 22 may be drained of liquid media 70, and gas may be pumped into the media chamber 24 to dry the propagation envelopes 50. The propagation envelopes 50 and the mycelia that propagated within the propagation envelopes 50 remain in the vessel body 22. Once the propagation envelopes are partially dried 50, target substances may be separated from the propagation envelopes 50 and extracted from mycelia that propagated within the propagation envelopes 50th.

[0098] Fig. 9 shows the system 10 in operation during filling with an extraction solvent 78 for extraction of target substances from mycelia that is located within the propagation envelopes 50. The extraction solvent 78 may be selected to provide an extraction solvent that is favourable for dissolving the target substances from within the propagation envelopes 50 and also favourable for stabilizing the target substances. Extraction of the target substances from mycelia propagated within the propagation envelopes 50 may be initiated once the mycelia has reached a target concentration. The target concentration may be a target concentration defined in terms of expected yield for one or more target substances. For extraction of tryptamines, other alkaloids or other target substances with comparable chemistry, the extraction solvent 78 may be any suitable liquid or compatible mixtures therefore (e.g. methanol, ethanol, water, acetone, acetonitrile, diethyl ether, trifluoroethanol, etc.), or any suitable supercritical fluid such as CO₂.

[0099] The vessel 20 containing the propagation envelopes 50 may be filled with an extraction solvent 78. The vessel 20 may then be operated at an elevated temperature for a period under mixing to dissolve the target substance located on the propagation envelopes 50. The mechanical mixer 26, the gas distributor line 28, or both, and in either case in addition to or instead of other apparatus (e.g. high-shear mixer, liquid grinder, cavitation mixer, ultrasonic waves, etc.) may be used to apply high-shear mixing to the extraction solvent 78, improve extraction yield, reduce timelines of extraction or both. The extraction solvent 78 loaded with the target substance may be drained and pumped to an evaporator to recover the extraction solvent 78 and precipitate the target substance. The extraction solvent 78, in some cases along with the propagation envelopes 50 as well, may be pumped through the grinder 46 to break up any mycelia that was recovered from the propagation envelopes 50 for extraction. This process may be undertaken in three steps.

[0100] In step 1 of the process shown in Fig. 9, the vessel body 20 containing the propagation envelopes 50 may be filled with the extraction solvent 78. After filling with the extraction solvent 78, the vessel 20 may be operated at an elevated temperature for a period of time to facilitate dissolution of the target substance located in the propagation envelopes 50, and to denature any remaining enzymes that may break down the target substance as described above in relation to Fig. 7. Agitating the extraction solvent may also facilitate dissolution of the target substances. The temperature of the extraction solvent 78 within the vessel body 22 may be increased to a point just below 60 °C for methanol, which has a boiling point of 66 °C (e.g. 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58 or 59 °C) for a period of time (e.g. 1 , 2, 3, 4 or 5 minutes, etc.). Depending on the solvent being used, different temperatures may be applied where the temperature is increased to a point that is between 1 and 20 °C below boiling point for that solvent (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 °C below the boiling point, etc.).

[0101] A heat exchanger, such as the heat exchanger 51 shown as part of the input, output and conditioning system 40 in the system 10 of Fig. 1 , may be used to modulate the temperature within the vessel body 20.

[0102] In step 2 of the process shown in Fig. 9, the extraction solvent 78 may be drained through the filter 41 to sequester the propagation envelopes 50, and any mycelia or other biomass that was propagated outside the propagation envelopes 50, within the vessel body 22. The extraction solvent 78 may be filtered by recirculation through grinder 46 to break up any mycelia or other biomass that passed through the filter 41. Alternatively during step 2, the extraction solvent 78 may be drained through the drain port 35 with the filter 41 open to allow mycelia or other biomass, and the propagation envelopes 50, to pass through the drain port 35. The extraction solvent 78, along with mycelia or other biomass, and the propagation envelopes 50, may be recirculated through grinder 46 to break up the mycelia or other biomass, and also the propagation envelopes 50, before or after filtering through the filter 41 . Applying the grinder 46 to the propagation envelopes 50 may facilitate exposing any mycelia within the propagation envelopes 50 to the extraction solvent 78 other than through the apertures 62. Recirculation and filtration may be followed by a temperature increase to evaporate the extraction solvent and precipitate the target substance.

[0103] In step 3 of the process shown in Fig. 9, recirculation and filtration from step 2 may be followed by a temperature increase or pressure decrease to evaporate the extraction solvent 78 and precipitate the target substance, or by a temperature decrease to lower solubility of the target substance in the extraction solvent 78 and induce precipitation. The separated solvent 78 may be pumped to an evaporator (not shown) to evaporate the extraction solvent 78 and precipitate the target substance. The extraction solvent 78 may be centrifuged, filtered or otherwise processed to separate mycelia and other biomass from the extraction solvent 78.

[0104] All of the steps shown in Fig. 9 may also be undertaken in an extraction vessel separate from the vessel 20 (not shown). Where a separate extraction vessel is used, the propagation envelopes 50 including the mycelia may be provided to the separate extraction vessel after the propagation envelopes 50 are ground by the grinder 46 for facilitating extraction prior to extraction in the separate extraction vessel. Alternatively, the propagation envelopes may be provided to the separate extraction vessel without use of the grinder pump 45, and either ground within the separate extraction vessel or extracted in the separate extraction vessel without grinding.

[0105] Fig. 10 shows a method 80 of culturing mycelia of fungi and extracting target substances from the mycelia using a system, such as the system 10, including a including a bioreactor vessel, such as the bioreactor vessel 20. The method 80 may be applied manually. The method 80 may be automated through use of sensors that detect whether the steps shown in the method 80 are complete. Upon completion of a step in the method 80, a system controlled by automation would either proceed automatically to the next step or notify an operator that a step has been completed and prompt the operator to confirm and advance the method 80 to the next step.

[0106] The method 80 includes eight steps. A loading step 81 is applied to fill the vessel with liquid media, such as the liquid media 70, and an envelope system, such as a plurality of the propagation envelopes 50. A sterilizing step 82 is applied to sterilize, then allow cooling of, the liquid media, the propagation envelopes and the vessel. An inoculation step 83 is applied to introduce inoculum, such as the inoculum 72, to the liquid media. A propagation step 84 is applied to agitate the liquid media, facilitating propagation of the fungi by accelerating provision of oxygen and other nutrients to the mycelia, and removal of CO2 and other waste from the mycelia through greater circulation of the liquid media. Agitation of the liquid media may also fluidize the propagation envelopes within the vessel 20, as shown in Figs. 1 , 6 and 7. A termination step 85 is applied to stop propagation of the mycelia and inactivate any enzymes that may break down target substances in the mycelia. A draining step 86 is applied to remove the liquid media 70. A grinding step 87 is applied to grind and dismantle the propagation envelopes, leaving mycelia exposed outside of the propagation envelopes. An extraction step 88 is applied to extract target substances from the mycelia.

[0107] The loading step 81 , the sterilizing step 82 and the inoculation step 83 are shown for the system 10 in Fig. 6.

[0108] During the loading step 81 , the vessel is loaded with liquid media and an envelope system, such as a plurality of propagation envelopes. Sensors may be applied to the system for detecting whether the vessel has been filled with the liquid media, and whether the propagation envelopes are also present in the liquid media. The liquid media may be added to the vessel prior to adding the propagation envelopes to the vessel, or the propagation envelopes may be added to the vessel prior to adding the liquid media to the vessel.

[0109] During the sterilizing step 82, the vessel is heated to allow raise an internal temperature of the media inside the vessel to a sterilizing temperature suitable for eliminating organisms and other pathogens, which may be 121 °C or any suitable temperature for (e.g. 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140 °C, etc.). The sterilizing temperature is held by the system for a period of time sufficient to kill any organisms that may be propagating inside the vessel, (e.g. 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 minutes, etc.). Once the sterilizing temperature has been held for a sufficient period of time to eliminate any organisms within the vessel, a cool down period begins. The sterilizing step 82 also includes the cool-down period to allow the temperature of the vessel to return to a temperature that is hospitable for the inoculum. Heating and cooling may be accomplished through a heat exchanger, such as the heat exchanger 51. Once the sterilizing step 82 is complete, the vessel includes sterile culture media and sterile propagation envelopes, and is at a temperature that is hospitable to the inoculum. The sterilizing step 82 may include maintaining a suitable temperature for propagation of bacteria, fungi or other organisms that sterilization eliminates to ensure that no such contaminating organisms remain the liquid media upon completion of the sterilizing step 82.

[0110] During the inoculation step 83, the inoculum is added to the vessel. The inoculum may be added through the inoculation port 32 where the vessel 20 is being used in the method 80.

[0111] The propagation step 84 and the termination step 85 are shown for the system 10 in Fig. 7. During the propagation step 84, the temperature and other conditions are maintained while liquid media is agitated. Agitation of the liquid media fluidizes the propagation envelopes and facilitates propagation of the mycelia. The termination step 85 is applied to stop propagation of the mycelia and inactivate any enzymes that may break down target substances in the mycelia. The termination step 85 includes application of heat, a change in pH of the liquid media, or both. After the termination step 85, the liquid media, the propagation envelopes and the mycelia within the propagation envelopes remain in the vessel but there is no longer significant enzymatic activity within the mycelia and no longer significant propagation of the mycelia.

[0112] The draining step 86 is shown for the system 10 in Fig. 8. During the draining step 86, the liquid media is drained out of the vessel and removed from the system, leaving the propagation envelopes and the mycelia within the propagation envelopes. Airflow may be applied to the media chamber to dry the propagation envelopes. [0113] The grinding step 87 and the extraction step 88 are shown for the system 10 in Fig. 9. The grinding step 87 is applied to grind and dismantle the propagation envelopes. During the grinding step, the extraction solvent is added to the vessel, facilitating flow and, under agitation, fluidization, of the propagation envelopes. The propagation envelopes are may transported in flow to a grinder to grind and dismantle the propagation envelopes, leaving mycelia exposed outside of the propagation envelopes and within the extraction solvent. During the extraction step 87, the extraction solvent is exposed to the mycelia to recover the target substances within the mycelia, and the extraction solvent is then recovered with the target substances from the mycelia, which may then be purified for working with the target substances.

[0114] Fig. 11 shows a method 180 of culturing mycelia of fungi and extracting target substances from the mycelia using a system, such as the system 10, including a including a bioreactor vessel, such as the bioreactor vessel 20. The method 180 may be applied manually. The method 180 may be automated through use of sensors that detect whether the steps shown in the method 180 are complete. Upon completion of a step in the method 180, a system controlled by automation would either proceed automatically to the next step or notify an operator that a step has been completed and prompt the operator to confirm and advance the method 180 to the next step.

[0115] The method 180 includes eight steps. A loading step 181 is applied to fill the vessel with liquid media, such as the liquid media 70, and an envelope system, such as a plurality of the propagation envelopes 50. A sterilizing step 182 is applied to sterilize, then allow cooling of, the liquid media, the propagation envelopes and the vessel. An inoculation step 183 is applied to introduce inoculum, such as the inoculum 72, to the liquid media. A propagation step 184 is applied to agitate the liquid media, facilitating propagation of the fungi by accelerating provision of oxygen and other nutrients to the mycelia, and removal of CO2 and other waste from the mycelia through greater circulation of the liquid media. Agitation of the liquid media may also fluidize the propagation envelopes within the vessel 20, as shown in Figs. 1 , 6 and 7. A draining step 186 is applied to remove the liquid media 70. A termination step 185 is applied to stop propagation of the mycelia and inactivate any enzymes that may break down target substances in the mycelia. A grinding step 187 is applied to grind and dismantle the propagation envelopes, leaving mycelia exposed outside of the propagation envelopes. An extraction step 188 is applied to extract target substances from the mycelia.

[0116] The loading step 181 , the sterilizing step 182 and the inoculation step 183 are shown for the system 10 in Fig. 6.

[0117] During the loading step 181 , the vessel is loaded with liquid media and an envelope system, such as a plurality of propagation envelopes. Sensors may be applied to the system for detecting whether the vessel has been filled with the liquid media, and whether the propagation envelopes are also present in the liquid media. The liquid media may be added to the vessel prior to adding the propagation envelopes to the vessel, or the propagation envelopes may be added to the vessel prior to adding the liquid media to the vessel.

[0118] During the sterilizing step 182, the vessel is heated to allow raise an internal temperature of the media inside the vessel to a sterilizing temperature suitable for eliminating organisms and other pathogens, which may be 121 °C or any suitable temperature for (e.g. 105, 106, 107, 108, 109, 110, 111 , 112, 1 13, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140 °C, etc.). The sterilizing temperature is held by the system for a period of time sufficient to kill any organisms that may be propagating inside the vessel, (e.g. 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 minutes, etc.). Once the sterilizing temperature has been held for a sufficient period of time to eliminate any organisms within the vessel, a cool down period begins. The sterilizing step 182 also includes the cool-down period to allow the temperature of the vessel to return to a temperature that is hospitable for the inoculum. Heating and cooling may be accomplished through a heat exchanger, such as the heat exchanger 51. Once the sterilizing step 182 is complete, the vessel includes sterile culture media and sterile propagation envelopes, and is at a temperature that is hospitable to the inoculum. The sterilizing step 182 may include maintaining a suitable temperature for propagation of bacteria, fungi or other organisms that sterilization eliminates to ensure that no such contaminating organisms remain the liquid media upon completion of the sterilizing step 182. [0119] During the inoculation step 183, the inoculum is added to the vessel. The inoculum may be added through the inoculation port 32 where the vessel 20 is being used in the method 180.

[0120] The propagation step 184 is shown for the system 10 in Fig. 7. During the propagation step 184, the temperature and other conditions are maintained while liquid media is agitated. Agitation of the liquid media fluidizes the propagation envelopes and facilitates propagation of the mycelia.

[0121] The draining step 186 and the termination step 185 are shown for the system 10 in Fig. 8. During the draining step 186, the liquid media is drained out of the vessel and removed from the system, leaving the propagation envelopes and the mycelia within the propagation envelopes. Airflow may be applied to the media chamber to dry the propagation envelopes.

[0122] After the draining step 186, the termination step 185 takes place. The termination step 185 is applied to stop propagation of the mycelia and inactivate any enzymes that may break down target substances in the mycelia. The termination step 185 includes application of heat, a change in pH of the liquid media, or both. After the termination step 185, the propagation envelopes and the mycelia within the propagation envelopes remain in the vessel but there is no longer significant enzymatic activity within the mycelia and no longer significant propagation of the mycelia.

[0123] The grinding step 187 and the extraction step 188 are shown for the system 10 in Fig. 9. The grinding step 187 is applied to grind and dismantle the propagation envelopes. During the grinding step, the extraction solvent is added to the vessel, facilitating flow and, under agitation, fluidization, of the propagation envelopes. The propagation envelopes are may transported in flow to a grinder to grind and dismantle the propagation envelopes, leaving mycelia exposed outside of the propagation envelopes and within the extraction solvent. During the extraction step 187, the extraction solvent is exposed to the mycelia to recover the target substances within the mycelia, and the extraction solvent is then recovered with the target substances from the mycelia, which may then be purified for working with the target substances.

[0124] Fig. 12 shows a method 280 of culturing mycelia of fungi and extracting target substances from the mycelia using a system, such as the system 10, including a including a bioreactor vessel, such as the bioreactor vessel 20. The method 280 may be applied manually. The method 280 may be automated through use of sensors that detect whether the steps shown in the method 280 are complete. Upon completion of a step in the method 280, a system controlled by automation would either proceed automatically to the next step or notify an operator that a step has been completed and prompt the operator to confirm and advance the method 280 to the next step.

[0125] The method 280 includes six steps. A loading step 281 is applied to fill the vessel with liquid media, such as the liquid media 70, and an envelope system, such as a plurality of the propagation envelopes 50. A sterilizing step 282 is applied to sterilize, then allow cooling of, the liquid media, the propagation envelopes and the vessel. An inoculation step 283 is applied to introduce inoculum, such as the inoculum 72, to the liquid media. A propagation step 284 is applied to agitate the liquid media, facilitating propagation of the fungi by accelerating provision of oxygen and other nutrients to the mycelia, and removal of CO2 and other waste from the mycelia through greater circulation of the liquid media. Agitation of the liquid media may also fluidize the propagation envelopes within the vessel 20, as shown in Figs. 1 , 6 and 7. A draining step 286 is applied to remove the liquid media 70. An extraction step 288 is applied to extract target substances from the mycelia.

[0126] The loading step 281 , the sterilizing step 282 and the inoculation step 283 are shown for the system 10 in Fig. 6.

[0127] During the loading step 281 , the vessel is loaded with liquid media and an envelope system, such as a plurality of propagation envelopes. Sensors may be applied to the system for detecting whether the vessel has been filled with the liquid media, and whether the propagation envelopes are also present in the liquid media. The liquid media may be added to the vessel prior to adding the propagation envelopes to the vessel, or the propagation envelopes may be added to the vessel prior to adding the liquid media to the vessel.

[0128] During the sterilizing step 282, the vessel is heated to allow raise an internal temperature of the media inside the vessel to a sterilizing temperature suitable for eliminating organisms and other pathogens, which may be 121 °C or any suitable temperature for (e.g. 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140 °C, etc.). The sterilizing temperature is held by the system for a period of time sufficient to kill any organisms that may be propagating inside the vessel, (e.g. 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 minutes, etc.). Once the sterilizing temperature has been held for a sufficient period of time to eliminate any organisms within the vessel, a cool down period begins. The sterilizing step 282 also includes the cool-down period to allow the temperature of the vessel to return to a temperature that is hospitable for the inoculum. Heating and cooling may be accomplished through a heat exchanger, such as the heat exchanger 51. Once the sterilizing step 282 is complete, the vessel includes sterile culture media and sterile propagation envelopes, and is at a temperature that is hospitable to the inoculum. The sterilizing step 282 may include maintaining a suitable temperature for propagation of bacteria, fungi or other organisms that sterilization eliminates to ensure that no such contaminating organisms remain the liquid media upon completion of the sterilizing step 282.

[0129] During the inoculation step 283, the inoculum is added to the vessel. The inoculum may be added through the inoculation port 32 where the vessel 20 is being used in the method 280.

[0130] The propagation step 284 is shown for the system 10 in Fig. 7. During the propagation step 284, the temperature and other conditions are maintained while liquid media is agitated. Agitation of the liquid media fluidizes the propagation envelopes and facilitates propagation of the mycelia.

[0131] The draining step 286 is shown for the system 10 in Fig. 8. During the draining step 286, the liquid media is drained out of the vessel and removed from the system, leaving the propagation envelopes and the mycelia within the propagation envelopes. Airflow may be applied to the media chamber to dry the propagation envelopes.

[0132] The extraction step 288 is shown for the system 10 in Fig. 9 wherein only the solvent 78 flows through the filter 41 and the ground material input 47, and the envelope system 50 remains in the extraction solvent 78. During the extraction step 287, the extraction solvent is exposed to the mycelia within the propagation envelopes to recover the target substances within the mycelia, and the extraction solvent is then recovered with the target substances from the mycelia, which may then be purified for working with the target substances.

[0133] Example

[0134] An example application of the method and system disclosed herein were carried out. In the example, a two liter double-lined glass reactor was used as a bioreactor vessel to culture P. cubensis fungi for the production and extraction of psilocybin. One vessel was used per experimental group. The vessel was equipped with a distributor line connected to a supply of filtered air and a 0.5 inch (1.25 cm) paddle impeller mixer for agitating fluid in the vessel, such as liquid media.

[0135] The vessel was equipped with an inoculation port assembly. The inoculation port assembly included a quick-disconnect, self-closing coupling with 1/4” (6.35 mm) barbed fitting, Altaflo heavy wall virgin high performance Fluorinated ethylene propylene (“FEP”) tubing with 1/4” (6.35 mm) inside diameter and 3/8” (9.525 mm) outside diameter. The FEP tubing was connected to the vessel thought a Cole-Parmer solid color-coded silicone stoppers, standard size 10.5, with a tube hole.

[0136] An aeration assembly was connected to the distributor line and installed on the vessel though a Cole-Parmer solid color-coded silicone stoppers, standard size 10.5, with a tubing-hole, to aerate the liquid media within the vessel. The aeration assembly includes a Cole-Parmer 0.2 pm, 50 mm diameter sterile in-line filter.

[0137] In each example, 1 .8 L of TC-4 liquid media was loaded to the vessel. Table 1 shows the composition of the TC-4 liquid media and Table 2 shows the composition of the 1 ,000x salt solution used to prepare the TC-4 liquid media. The pH was adjusted to 5.5 using HCI/KOH.

Table 1 : TC-4 Liquid Media Composition

Table 2: Salts Solution (1000x) Composition

[0138] The vessel and its contents where autoclaved at 121 °C for one hour to sterilize the liquid media. Condensers were autoclaved separately at 121 °C for one hour, with all ports being plugged with sponge plugs and covered with aluminum foil. Air filters were also autoclaved separately at 121 °C for 15 minutes.

[0139] After allowing the components to cool, the condensers and air supplies were connected to each vessel using proper aseptic technique. The vessels were allowed to cool to ambient room temperature then operated for two days prior to adding inoculum to ensure no contaminating organisms were growing in the liquid culture medium. Inoculum was prepared by grinding a bag of mycelia pellets of the P. cubensis Albino Penis Envy (“APE”) strain using a sterile grinder until the mixture was homogenous. The inoculum was then aseptically transferred to the vessels through an inoculation assembly port in vessels. The liquid culture media were kept between 23 and 28 °C during cultivation of mycelium.

[0140] After sterilization, filtered air was introduced to through the distributor line, the mixer was set to 300 rpm and 18ml of inoculum was left in the vessel to cultivate for ninedays.

[0141] Mycelial biomass was harvested nine days after inoculation. Prior to harvesting, the air supply was deactivated and all connections to the vessel were removed. The culture from each reactor was vacuum filtered and analyzed for media volume, pH, sugar content (% brix), and biomass (g/L). Liquid media was drained from the port and vacuum filtered to collect mycelium biomass. Biomass grown inside the spheres were extracted by scraping the sphere interiors with a small metal spatula. The biomass was then transferred to a metal container and dried at about 35 °C using a 3.8 L forced air convention oven.

[0142] Dried biomass was collected and ground using a mortar and pestle. Methanol was heated to 60°C and added to the biomass at 20 mL/g. The ground biomass in methanol was sonicated for 30 minutes at 60°C. After being left to cool at room temperature for 30 minutes, the extracts were decanted into 10-mL syringes and filtered (13 mm PES 0.22 pm syringe filter attachment). The samples were mixed 1 : 1 with buffer solution (14 mM ammonium formate, 26 mM formic acid) analyzed using an Agilent 1100 HPLC system. Psilocybin yields in the biomass were measured. Extraction with methanol took place in a separate system from the vessel in which the propagation of fungi was completed.

[0143] Two data sets were assessed. In a negative control group, no envelops were loaded into the vessel.

[0144] In a positive control group, thirty polypropylene hollow 1.4” (3.6 cm) diameter spheres were used as envelopes, similar in design to the envelope 950 with the aperture system 968. The spheres include of three evenly spaced 3/8” (9.5 mm) holes along the circumference of the spheres of envelop 950 and a fourth aperture 967 on the top of the envelope sphere 950.

[0145] Compared with the positive control group, the negative control group showed eight times as much mycelial mass. Compared with the negative control group, the positive control group showed sixteen times the amount of psilocybin per mass unit of mycelia. The total yield of psilocybin in the positive control group was double the total yield in the negative control group, notwithstanding a lower yield of mycelial biomass in the positive control group. Results of this example indicate that mycelium grown within propagation envelopes includes elevated concentrations of psilocybin, albeit with a lower biomass compared to mycelium growing freely suspended in liquid media.

[0146] References

[0147] Amanullah A. et al., Bioch Eng J. 5, 109-114 (2000). [0148] Cairns, T C; Zheng, X; Zheng, P; Sun, J; Meyer, V; “Moulding the mould: Understanding and reprogramming filamentous fungal growth and morphogenesis for next generation cell factories" Biotechnology for Bio-fuels. 12(77) (2019)

[0149] Castillo-Carvajal, L., Ortega-Gonzalez, K., Barragan-Huerta, B. E. & Pedroza-Rodriguez, A. M. Evaluation of three immobilization supports and two nutritional conditions for reactive black 5 removal with Trametes versicolor in air bubble reactor. African J. Biotechnol. 11, 3310-3320 (2012)

[0150] Couto, S. R. & Toca-Herrera, J. L. Laccase production at reactor scale by filamentous fungi. Biotechnol. Adv. 25, 558-569 (2007)

[0151] Domingos, M., Souza-Cruz, P. B. de, Ferraz, A. & Prata, A. M. R. A new bioreactor design for culturing basidiomycetes: Mycelial biomass production in submerged cultures of Ceriporiopsis subvermispora. Chem. Eng. Sci. 170, 670-676 (2017)

[0152] Fazenda, M. L., Seviour, R., McNeil, B. & Harvey, L. M. Submerged Culture Fermentation of ‘Higher Fungi’: The Macrofungi. Adv. Appl. Microbiol. 63, 33-103 (2008) [0153] Freitas, A. C. et al. Biological treatment of the effluent from a bleached kraft pulp mill using basidiomycete and zygomycete fungi. Sci. Total Environ. 407, 3282-3289 (2009)

[0154] Galhardi, D. R. V. Study of Temperature variation in Low-Shear Agitated and Aerated Bioreactor for Aplication in Exo-Polysaccharide Production (ESTUDO DA VARIAQAO DE TEMPERATURA EM BIORREATOR AGITADO E AERADO DE BAIXO CISALHAMENTO PARA APLICAQAO NA PRODUQAO DE EXOPOLISSACAR. (UNIVERSIDADE DE SAG PAULO, 2019)

[0155] Gaston, O., Maria, B. C. & Edgardo, A. Artificial spawn generation based on alginate encapsulated mycelium as inoculum for mushroom cultivation. African J. Biotechnol. 16, 1776-1783 (2017)

[0156] Musoni, M., Destain, J., Thonart, P., Bahama, J. B. & Delvigne, F. Bioreactor design and implementation strategies for the cultivation of filamentous fungi and the production of fungal metabolites: From traditional methods to engineered systems. Biotechnol. Agron. Soc. Environ. 19, 430-442 (2015) [0157] Pedroza-Rodriguez, A. M. & Rodriguez-Vazquez, R. Optimization of C/N Ratio and Inducers for Wastewater Paper Industry Treatment Using Trametes versicolor Immobilized in Bubble Column Reactor. J. Mycol. 2013, 1 -11 (2013)

[0158] Pollard, D. J. et al. Scale up of a viscous fungal fermentation: Application of scale-up criteria with regime analysis and operating boundary conditions. Biotechnol. Bioeng. 96, 307-317 (2007)

[0159] Prata, A. M. R., Ferraz, A. L., Domingos, M. & Junior, J. M. da S. Bioreactor with specific agitation and aeration system for the cultivation of adherent and/or shearsensitive cells. Biochem. Eng. J. 5, 109-114 (2000)

[0160] Spina, F., Romagnolo, A., Prigione, V., Tigini, V. & Varese, G. C. A scaling- up issue: The optimal bioreactor configuration for effective fungal treatment of textile wastewaters. Chem. Eng. Trans. 38, 37-42 (2014).

[0161] Vees, C. A., Neuendorf, C. S. & Pflugl, S. Towards continuous industrial bioprocessing with solventogenic and acetogenic Clostridia: challenges, progress and perspectives. Journal of Industrial Microbiology and Biotechnology vol. 47 (Springer International Publishing, 2020)

[0162] Xin, B. et al. A feasible method for growing fungal pellets in a column reactor inoculated with mycelium fragments and their application for dye bioaccumulation from aqueous solution. Bioresour. Technol. 105, 100-105 (2012)

[0163] Zacchetti, B., van Dissel, D., de Ruiter, E., van Wezel, G. P. & Claessen, D. Microencapsulation extends mycelial viability of Streptomyces lividans 66 and increases enzyme production. BMC Biotechnol. 18, 1-8 (2018)

[0164] Examples Only

[0165] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the embodiments. However, it will be apparent to one skilled in the art that these specific details are not required.

[0166] The above-described embodiments are intended to be examples only. Alterations, modifications and variations can be effected to the particular embodiments by those of skill in the art. The scope of the claims should not be limited by the particular embodiments set forth herein, but should be construed in a manner consistent with the specification as a whole.

Claims

1 . A method for propagating a filamentous organism in a vessel comprising: providing liquid media, inoculum of the filamentous organism and an envelope system into the vessel; and agitating the liquid media for providing nutrients to the filamentous organism inside the envelope system and for removing waste from the filamentous organism inside the envelope system; wherein the envelope system comprises at least one propagation envelope for receiving the inoculum and the filamentous organism, and for propagating the filamentous organism in liquid media received within the envelope system; agitating the liquid media results in shear forces within the liquid media; and each of the at least one propagation envelopes comprises a physical barrier between the filamentous organism and the liquid media for mitigating shear forces on the filamentous organism within the envelope system.

2. The method of claim 1 wherein the filamentous organism comprises a fungus.

3. The method of any one of claims 1 or 2 wherein providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the liquid media to the vessel prior to providing the inoculum to the vessel.

4. The method of claim 3 wherein providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the inoculum to the vessel prior to providing the envelope system to the vessel.

5. The method of claim 3 wherein providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the envelope system to the vessel prior to providing the inoculum to the vessel.

6. The method of claim 5 wherein providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the envelope system to the vessel prior to providing the liquid media to the vessel.

- 43 -

7. The method of claim 1 wherein providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the envelope system to the vessel prior to providing the liquid media or the inoculum to the vessel.

8. The of claim 7 wherein providing the liquid media, the inoculum and the envelope system into the vessel comprises providing the liquid media to the vessel prior to providing the inoculum to the vessel.

9. The method of any one of claims 1 to 8 wherein agitating the liquid media comprises agitating the liquid media sufficiently to fluidize the envelope system in the liquid media.

10. The method of any one of claims 1 to 9 wherein agitating the liquid media comprises agitating the liquid media sufficiently to induce a circulation time of two minutes or less in the liquid media.

11 . The method of any one of claims 1 to 10 wherein agitating the liquid media comprises application of mechanical mixing to the liquid media.

12. The method of claim 11 wherein the mechanical mixing comprises rotary stirring of the liquid media.

13. The method of any one of claims 1 to 12 wherein agitating the liquid media comprises providing a gas to the liquid media.

14. The method of any one of claims 1 to 13 wherein agitating the liquid media comprises providing circulating fluid flow to focus fluid flow throughout the liquid media within the vessel.

15. The method of any one of claims 1 to 14 wherein agitating the liquid media comprises applying axial mixing of the liquid media within the vessel.

16. The method of any one of claims 1 to 15 wherein agitating the liquid media comprises applying radial mixing of the liquid media within the vessel.

- 44 -

17. The method of any one of claims 1 to 16 wherein the nutrients comprise oxygen.

18. The method of any one of claims 1 to 17 wherein the waste comprises CO2.

19. The method of any one of claims 1 to 18 further comprising elevating the temperature of the liquid media in the presence of the filamentous organism to inactivate a heat-labile enzyme in the filamentous organism.

20. The method of claim 19 wherein elevating the temperature of the liquid media in the presence of the filamentous organism comprises elevating the temperature of the liquid media to between 90 and 100 °C.

21 . The method of any one of claims 1 to 20 comprising adjusting the pH of the liquid media away from physiological pH for the filamentous organism to inactivate a pH-labile enzyme in the filamentous organism.

22. The method of claim 21 wherein adjusting the pH of the liquid media in the presence of the filamentous organism comprises raising the pH.

23. The method of claim 22 wherein raising the pH comprises raising the pH to 10.0 or higher.

24. The method of claim 21 wherein adjusting the pH of the liquid media in the presence of the filamentous organism comprises lowering the pH.

25. The method of claim 24 wherein lowering the pH comprises lowering the pH to 4.0 or lower.

26. The method of any one of claims 1 to 25, further comprising separating the envelope system from the liquid media, wherein the filamentous organism is within the envelope system.

27. The method of claim 26 wherein separating the envelope system from the liquid media comprises draining the liquid media from the vessel.

- 45 -

28. The method of claim 27 further comprising adding extraction solvent to the vessel for extracting a substance from the filamentous organism within the envelope system.

29. The method of any one of claims 26 or 27 further comprising combining an extraction solvent with the envelope system for extracting a substance from filamentous organism within the envelope system.

30. The method of claim 29 wherein combining the extraction solvent with the envelope system comprises combining the extraction solvent with the envelope system in an extraction vessel separate from the vessel.

31 . The method of any one of claims 28 to 30 wherein the extraction solvent comprises methanol, ethanol, water, acetone, consisting of acetonitrile, diethyl ether, trifluoroethanol or a combination thereof.

32. The method of claims 28 to 30 wherein the extraction solvent is CO2.

33. The method of either of claims 28 to 31 further comprising elevating the temperature of the extraction solvent in the presence of the filamentous organism.

34. The method of claim 33 wherein elevating the temperature of the extraction solvent in the presence of the filamentous organism comprises elevating the temperature of the extraction solvent to a temperature of between 1 and 25 °C below the boiling temperature of the extraction solvent.

35. The method of any one of claims 29 to 34 further comprising grinding the propagation envelopes for exposing the filamentous organism to the extraction solvent and facilitating extraction of the substance from the filamentous organism.

36. The method of any one of claims 1 to 35 wherein the method is operated in a batch process.

37. The method of any one of claims 1 to 35 wherein the method is operated in a continuous process.

38. A method for propagating a filamentous organism in a vessel comprising: providing liquid media and inoculum of the filamentous organism to the vessel; and agitating the liquid media for providing nutrients to the filamentous organism and for removing waste from the filamentous organism; characterized in that the method comprises providing an envelope system into the vessel; the envelope system comprises at least one propagation envelope for receiving the inoculum and the filamentous organism, and for propagating the filamentous organism in liquid media received within the envelope system; agitating the liquid media provides nutrients to the filamentous organism inside the envelope system and removes waste from the filamentous organism inside the envelope system; agitating the liquid media results in shear forces within the liquid media; and each of the at least one propagation envelopes comprises a physical barrier between the filamentous organism and the liquid media for mitigating shear forces on the filamentous organism within the envelope system.

39. The method of claim 38 further comprising the additional features of any one of claims 1 to 37.

40. A propagation envelope for culturing a filamentous organism, the propagation envelope comprising: an envelope body for receiving a liquid culture; a propagation chamber defined within the envelope body; and at least one aperture defined on the envelope body for providing fluid communication between the propagation chamber and an environment external to the propagation chamber.

41 . The propagation envelope of claim 40 wherein the envelope body comprises a defined shape selected from the group consisting of a sphere, cylinder, tetrahedron, cube, octahedron, decahedron, dodecahedron, icosagon, and any other shape that is defined by a plurality of surfaces, faces, vertexes, apexes or a combination thereof.

42. The propagation envelope of claim 41 wherein the plurality of vertexes, apexes and faces is repeated regularly and symmetrically.

43. The propagation envelope of any one of claims 41 to 42 wherein the at least one aperture comprises an apex aperture defined on an apex of the envelope body.

44. The propagation envelope of any one of claims 41 to 43 wherein the at least one aperture comprises a vertex aperture defined on the envelope body along a vertex of the envelope body.

45. The propagation envelope of claim 44 wherein the vertex aperture comprises a vertex midpoint aperture defined on the envelope body at a midpoint of the vertex.

46. The propagation envelope of any one of claims 41 to 45 wherein the at least one aperture comprises a face aperture defined on the envelope body on a face of the envelope body.

47. The propagation envelope of claim 46 wherein the face aperture comprises a center face aperture defined on the envelope body at a center of the face.

48. The propagation envelope of any one of claims 40 to 47 wherein the envelope body includes a textured inside surface for facilitating adherence of a filamentous organism to the textured inside surface.

49. The propagation envelope of claim 48 wherein the textured inside surface comprises grooves defined in the envelope body.

50. The propagation envelope of any one of claims 48 or 49 wherein the textured inside surface comprises ridges extending from the envelope body.

51 . The propagation envelope of any one of claims 48 to 50 wherein the textured inside surface comprises a roughened inside surface.

52. The propagation envelope of any one of claims 40 to 51 wherein the envelope body around the propagation chamber comprises at least one protrusion extending into

- 48 - the propagation chamber for providing additional surface area to the envelope body within the propagation chamber to support propagation upon the envelope body within the propagation chamber.

53. The propagation envelope of any one of claims 40 to 52 wherein the envelope body is manufactured from a material with a specific gravity of between 0.5 and 8.0.

54. The propagation envelope of claim 53 wherein the material comprises a polymer material.

55. The propagation envelope of claim 53 wherein the material comprises a metal.

56. The propagation envelope of claim 53 wherein the material comprises a ceramic.

57. The propagation envelope of claim 53 wherein the material comprises a glass.

58. The propagation envelope of any one of claims 40 to 57 wherein the at least one aperture comprises a valved aperture, the valved aperture comprising a valve for restricting particle flow from the propagation chamber to the environment.

59. The propagation envelope of claims 40 to 58 wherein the at least one aperture comprises a tapered aperture, the tapered aperture comprising a greater inside diameter at an outside mouth of the tapered aperture facing the environment than at an inside mouth of the tapered aperture facing the propagation chamber for restricting particle flow from the propagation chamber to the environment.

60. The propagation envelope of any one of claims 40 to 59 wherein the at least one aperture comprises at least two apertures.

61 . The propagation envelope of claim 60 wherein the propagation chamber comprises at least two separate portions; the at least one aperture comprises at least two apertures; at least one aperture is in fluid communication with each of the at least two separate portions; and

- 49 - each of the at least two separate portions are isolated from every other separate portion other than by way of at least two apertures and the external environment.

62. The propagation envelope of any one of claims 60 or 61 wherein a flow path between the at least two apertures includes a barrier, a flow restriction, non-linear flow features or other features for disrupting a direct flow path between the at least two apertures, for mitigating shear forces within the propagation chamber relative to shear forces that would result from a direct flow path between the at least two apertures.

- 50 -