Means and methods for preparing mycelium colonization substrates

Abstract

The present invention relates to a method for preparing a mycelial colonization substrate. The present invention further relates to an intermediate product in the preparation of a mycelial colonization substrate, the intermediate product comprising a mycelial colonization substrate and a synthetic particle size distribution modifier, and a solid-state mycelial bioreactor, which is useful in the method for preparing a mycelial colonization substrate of the present invention.

Description

[Technical Field]

[0001] The present invention relates to a method for preparing a mycelial colonization substrate. The present invention further relates to an intermediate product in the preparation of a mycelial colonization substrate, the intermediate product comprising a mycelial colonization substrate and a synthetic particle size distribution modifier, and a solid-state mycelial bioreactor, which is useful in the method for preparing a mycelial colonization substrate of the present invention. [Background technology]

[0002] As with many biological processes, scaling up presents problems that are ineffective at smaller scales. This also applies when considering scaling up the production process of mycelium composites. Since it is preferably an aerobic solid-state fermentation type process, some of the main problems in scaling up the mass of substrate during the step of initial expansion (colonization) of the mycelium on the substrate are anaerobic conditions inside the substrate, heat accumulation from thermogenesis, insufficient gas exchange inside the substrate mass, and compaction of the substrate by the mass of the substrate above it in the sublayer.

[0003] The paper "Bioreactor designs for solid state fermentation" by A. Durand 2003 provides an overview of the design possibilities for SSF-BR design and its key challenges, especially regarding scaling.

[0004] The most common solution to these problems is to focus on the culinary mushroom production process. When the initial mycelium expansion (colonization) step on the substrate is typically performed inside disposable growth bags, the total volume of the substrate is divided into smaller volumes defined by the size of the growth bag. Because these bags are disposable, this process creates a lot of waste, which is counter to one of the purposes / benefits of the material: reducing plastic waste. Furthermore, the bags are incubated on shelves, taking up a lot of space and making the process difficult and capital-intensive to fully automate (e.g., using warehouse automation robots). In other cases, the substrate is not pre-incubated (colonized) before being filled into molds; rather, the inoculum substrate is placed directly into the molds. These molds are also incubated on shelves. A disadvantage of these two methods is that the mycelium-substrate complex cannot be agitated during incubation. Agitation typically speeds up the incubation process because the mycelium typically forms a stronger and denser network after localized damage has been caused to it, increasing the number of spawn points and creating superior material.

[0005] The paper "A comprehensive framework for the production of mycelium-based lignocellulosic composites" by E. Elsacker et.al. (2020) provides insight into the state of the art of mycelium-based composites.

[0006] Following the initial experimentation with the process by North American artist Philip Ross in his research in the 1990s, and the popularization and commercialization of related processes and products by Ecovative (as shown in their patent based on the process, see below), much effort by researchers in industry and academia has been put into studying the material itself and finding uses for it.

[0007] The article in "Pure culture" by Philip Ross is an art magazine article describing his process for creating his mycelium-matrix composite based artworks.

[0008] Patent WO 2008073489(A2) / EP 2094856(B1) by Ecovative based on a culinary mushroom production process on mycelium-based materials.

[0009] Therefore, there is a need to provide new approaches for the production process of mycelium-matrix composites that focus on the automation and scalability of this process by solving the problems of solid-state fermentation of mycelium-forming fungi using large amounts of substrate, reducing the number of machines required to carry out the process, and providing new cost-saving means for production. These approaches are applicable to the production of mycelium-matrix composites for known applications such as packaging, thermal and acoustic insulation, and engineered products such as furniture. Additional applications can also be envisioned.

[0010] Document WO 2008 / 073489 discloses a particular self-supporting composite material comprising a matrix of discrete particles and a network of interconnected mycelial cells extending through and around the discrete particles and binding them together, as well as a method for making the material.

[0011] Document Chinese Patent No. 102220223 discloses a particular solid-state fermentation material processing device.

[0012] Chinese Patent No. 202089984 discloses a particular multi-functional solid-state fermentation reactor. Summary of the Invention

[0013] The object of the present invention was to provide an improved, scalable method for preparing mycelium-colonized substrates and thus mycelium-substrate composites. A further object of the present invention was to provide such a method with improved aeration of the substrate. Thus, the technical problem of the object of the present invention was to provide a method for preparing mycelium-colonized substrates with improved aeration of the substrate.

[0014] The present invention further provides a means for carrying out the method for preparing a mycelium colonization substrate, which means is the solid-state mycelium bioreactor (SSMB) of the present invention (which may also be referred to as a mycelium culture bioreactor). The present invention also makes it possible to provide new product possibilities from mycelium-substrate composites and mycelium colonization substrates, such as fertilizers / soil conditioners. The design of the SSMB also makes it possible to use, for example, in the production of enzymes, acids, or antibiotics, protein or DNA synthesis, and food production (from and / or for animal use), biocatalysis (e.g., for the paper industry), spawn / inoculant production, or bioremediation.

[0015] The objective technical problem is solved by the embodiments described herein and characterized by the claims.

[0016] Further desirable are methods and / or setups for large-scale production of mycelium-matrix composites. Specifically, methods and setups that do not adversely affect mycelial growth due to intense heat generation caused by mycelial heat production are desirable. Therefore, means and methods that allow for maintaining sterile conditions in large vessels, aerating the substrate during mycelial growth, dispersing humidity during mycelial growth, and dealing with the accumulation of heat and effluent (i.e., mycelial secretions) are desirable.

[0017] The present invention is summarized in the following embodiments.

[0018] In a first embodiment, the present invention relates to a method for the preparation of a mycelium colonization substrate, comprising the step of incubating a mycelium-inoculated substrate to allow mycelium to grow.

[0019] In a second embodiment, the present invention relates to an intermediate product in the preparation of a mycelium colonization substrate, comprising a mycelium colonization substrate and a synthetic particle size distribution modifier.

[0020] In a third embodiment, the present invention relates to a solid-state mycelium bioreactor comprising a reactor body having a cavity, and at least one mixing element disposed within the cavity of the reactor body and rotatable relative to the reactor body about an axis of rotation, the at least one mixing element comprising at least one outlet opening for adding fluids into the reactor body, the at least one outlet opening being fluidly connected to one or more fluid supplies via at least one fluid connection. Preferably, the at least one mixing element is a rotatable spiral paddle.

[0021] Specific embodiments of the present invention will become apparent below. [Brief explanation of the drawings]

[0022] The present invention is further illustrated by the following figures and / or drawings, which are purely for illustrative purposes and are not intended to be construed as limiting unless expressly indicated to the contrary. [Figure 1] It represents a matrix containing structural grains and filler grains. [Figure 2] 1 depicts exemplary synthetic particle size distribution modifier particles within a matrix (mycelium absent / not shown). [Figure 3] Examples of mineral (top) and typical non-digestible (bottom) synthetic particle size distribution modifiers are shown. [Figure 4]1 shows a reactor body according to the present invention having a spiral paddle mixing element including a fluid feed through channels extending through the spiral paddle mixing element, with fluid distribution indicated by grey arrows. [Figure 5-1] The experimental setup used in Example 1 is shown. [Figure 5-2] The experimental setup used in Example 1 is shown. [Figure 6] 1 shows a reactor according to Example 2. [Figure 7] 1 shows a circular hollow synthetic particle size distribution adjuster of Example 3. [Figure 8] Similar to Example 8, a scheme for producing a heat-pressed mycelium-substrate composite chair is shown. [Figure 9] The mixing elements in the reactor according to the present invention are shown herein to include cooling elements in the form of water-cooled channels and / or heat exchange mechanisms. [Figure 10] A prototype SSMB with a rotatable spiral paddle is shown. [Figure 11] 1 shows the contents of the SSMB in the first run of Example 11 (Reference Example). [Figure 12] SSMB contents in a culture run relying on a synthetic particle size distribution regulator, hollow perforated balls, demonstrating complete coverage of the mycelial mat with all areas of fungal growth connected. DETAILED DESCRIPTION OF THE INVENTION

[0023] Embodiments of the present invention are described below, it being understood that all described features may be combined unless expressly stated to the contrary.

[0024] In one embodiment, the present invention relates to a method for the preparation of a mycelium colonization substrate, comprising the step of incubating a mycelium-inoculated substrate to allow mycelium to grow.

[0025] As understood herein, a mycelium-colonized substrate preferably refers to a composition comprising mycelium and a substrate, wherein the mycelium is bound to and physically connected to particles or portions of the substrate, the mycelium is capable of further growth, and the particles or portions of the substrate are connected by the mycelium grown thereon. Preferably, at least 10% of the particles or portions of the substrate are connected to each other by the mycelium in the mycelium-colonized substrate. Even more preferably, at least 20% of the particles or portions of the substrate are connected to each other by the mycelium in the mycelium-colonized substrate. Preferably, the percentages refer to at least 10% or 20% of the weight of the substrate involved in being bound together through the mycelium, respectively.

[0026] When referring to particles or portions of a substrate, we are referring to discrete pieces of the substrate which may include grains, particles, chips, pellets, shreds, etc. Preferably, when referring to particles or portions of a substrate, we are referring to grains.

[0027] As understood herein, a mycelium-inoculated substrate preferably refers to a composition comprising mycelium and a substrate, wherein the mycelium may be bound to and physically connected to particles or portions of the substrate, but preferably does not connect different particles or portions of the substrate to each other. Preferably, 10% or less of the particles or portions of the substrate are connected to each other by mycelium in the mycelium-inoculated substrate. Even more preferably, 5% or less of the particles or portions of the substrate are connected to each other by mycelium in the mycelium-inoculated substrate. Preferably, the percentage refers to the weight of the substrate involved in being bound together through mycelium. In other words, typically and preferably, particles or portions of the substrate are not connected by mycelium. Typically and preferably, the mycelium can be attached to individual particles or portions of the substrate. However, the mycelium used for inoculation may not be attached to particles or portions of the substrate.

[0028] As understood herein, a mycelium-substrate composite material is a composition comprising a substrate material and mycelium, wherein the substrate, preferably particles or portions of the substrate, are bound by a mycelium network, preferably treated so that the mycelium network is no longer viable and no longer capable of further growth. Such treatment can be performed, for example, by dehydration and / or denaturation.

[0029] As understood herein, the substrate is not particularly limited in the method of the present invention. According to the present invention, the substrate preferably comprises at least one structural component and at least one filler component. Therefore, the substrate is considered to preferably comprise particles or portions (e.g., grains) of the structural component and particles or portions (e.g., grains) of the filler component, as shown in FIG. 1.

[0030] According to the present invention, the mix of structural and filler type grains maximizes the rate of mycelial expansion or creates voids in the substrate that control the rate of mycelial expansion, the toughness of individual hyphae, and improve their aeration.

[0031] Preferably, the structural component is characterized by a low stack density of 80-130 g / liter with a significant amount of voids and / or a high porosity of 60-80% v / v voids. Preferably, the structural component is a lignocellulosic material. Preferably, the particles / grains of the structural component are characterized by a diameter of at least 4 mm. Preferably, said dimension is understood as the length of the particle measured along its longest axis. The structural grains are typically and preferably between 4 and 750 mm 3 As described by the inventors, structural components can function as void creators in the substrate. Thus, more abundant and larger voids create a lower overall material density and stronger hyphae. At the same time, too large voids can inhibit mycelial growth, so a size distribution that balances these two properties is required.

[0032] The structural component is made of chopped hemp stalks, corn stalks, bean stalks or other stalks, corn cobs, peanut husks, curled wood chips, grain chaff, rice husks, or other similar components. Thus, as preferred within the scope of the method of the present invention, at least one structural component is selected from chopped hemp stalks, chopped corn stalks, chopped tomato stalks, chopped tobacco stalks, chopped bean stalks, chopped corn cobs, softwood flakes, peanut husks, and straw.

[0033] Within the scope of the present invention, the filler component is characterized by a relatively high stacking density, preferably between 130 and 250 g / liter. The filler component is intended to provide easily accessible nutrients to the mycelium. Therefore, the particles / grains of the filler component are characterized by a diameter of less than 4 mm. Preferably, the diameter is understood as the length of the particle measured along its longest axis.

[0034] The filler grain is typically and preferably 0.001 to 4.0 mm 3 It has a volume of

[0035] Therefore, the filler component may be made from the same components as the filler component, but chopped into finer grains. Therefore, preferably, the filler component is selected from sawdust, mash, coffee grounds, oil press residue, coffee skins, wheat flour, bread waste, and wheat bran. This list is not intended to be limiting, and other materials may also be used. The filler component promotes rapid growth and easily accessible nutrients for the fungus. Preferably, as provided by the present invention, at least one filler component is selected from sawdust, brewer's mash, and paper pulp.

[0036] Consequently, the present invention preferably provides a substrate comprising structural and filler grains, wherein the filler grains have an average volumetric diameter of less than 4 mm and / or the structural grains have an average volumetric diameter of at least 4 mm.

[0037] It should be noted that in one specific embodiment of the present invention, the volume-based average diameter of the filler component grains and the volume-based average diameter of the structural component grains are substantially the same, preferably within 10% of each other, more preferably within 5% of each other, even more preferably within 1% of each other, and even more preferably the same. The term "within % of each other" preferably refers to the ratio of the difference between the two values ​​to the smaller of both values, expressed as a percentage.

[0038] As encompassed by the present invention, the substrate may further comprise adjuvants, preferably selected from calcium sulfate, calcium hydroxide, nitrogen additives, terpenes, lipids, animal hair, natural fibers, charcoal, algae, simple hydrocarbons, and compost.

[0039] Simple hydrocarbons are preferably compounds made up of only C and H atoms, where there are no more than 12 C atoms. Thus, simple hydrocarbons as in the present invention can be straight-chain or branched, cyclic or acyclic, saturated or unsaturated, for example by having at least one carbon-carbon double or triple bond or by containing an aromatic ring system, such as that of benzene or naphthalene.

[0040] As understood herein, nitrogen additives are additives that are relatively rich in protein (relative to the structural and filler components), i.e., have a higher protein content (preferably understood as the amount of protein per unit of weight) than the structural and filler components, such as, but not limited to, peptone, urea, ammonia, potassium nitrate, nutritional yeast and extracts, grape pomace, rye grain, oat and wheat bran, compost, bean waste, or brewer's mash, preferably peptone, urea, ammonia, potassium nitrate, etc. Alternatively, they may be selected from vegetable oils, nutritional yeast and extracts, grape pomace, rye grain, oat and wheat bran, compost, bean waste, or brewer's mash.

[0041] Terpenes, as understood herein, preferably have the general formula (C5H8) n where n is a natural number (e.g., 2 for monoterpenes, 2 for sesquiterpenes, 4 for diterpenes, etc.), and one or more H atoms are optionally replaced with —OH or ═O moieties. An example of a terpene compound is turpentine oil.

[0042] The substrate of the present invention is preferably treated with alkaline chemicals against contamination. Treatment with alkaline chemicals further allows for preconditioning of the substrate. As understood herein, treatment with alkaline chemicals includes treatment with at least one agent selected from calcium hydroxide, sodium hydroxide, and hydrogen peroxide, added in an amount such that the pH of the substrate shifts from slightly acidic to neutral (pH = 7.0 ± 0.5) or even slightly alkaline (pH in the range of 7.5 to 10.0, preferably 7.5 to 9.0). As will be apparent to those skilled in the art, such treatment can enable faster nutrient uptake and build resilience against acidophilic contaminants. According to the inventors, this improvement can be attributed to increased fibrillation of the substrate resulting from the addition of alkaline chemicals. Further contemplated by the present invention is an embodiment in which only a portion of the substrate is preconditioned (e.g., chemically, autothermally, or enzymatically), preferably treated with alkaline chemicals, and then this portion is added to a substrate that has not been preconditioned to obtain the final substrate used in the method of the present invention.

[0043] The substrate can be pelletized, i.e., present in the form of pellets. As known to those skilled in the art, pelletizing a substrate facilitates its transportation and improves its long-term storage. Furthermore, processing the substrate by pelletizing, typically performed at pressures of 1500-5000 bar, allows for particle size control and also increases its bulk density, typically by 2-10 times. As will be apparent to those skilled in the art, the addition of certain binders can improve the toughness of the pellets. Such binders include lignosulfonates, dolomite, starch, potato flour and skins, and certain motor and vegetable oils.

[0044] The substrate as understood herein may be shredded before use. Thus, the particles of the substrate may be shredded to a defined grain size, for example, between 1 and 700 mm, by using suitable devices known to those skilled in the art, such as a hammer mill, a shredder, or a roller chipper. 3 The substrate is chopped to achieve a grain volume in the range of 0.01 to 0.01 mm. As will be apparent to those skilled in the art, chopping can facilitate its transportation and improve its long-term storage properties. Chopping can further contribute to increasing the bulk density of the substrate. As further established by the inventors, chopping the substrate allows for its fibrillation, increasing its active surface and therefore allowing for faster colonization of the substrate by fungi / mycelium. Chopping also results in higher internal friction between discrete particles of the substrate.

[0045] Preferably, before use in the method of the present invention, the substrate is preferably stored under low air humidity conditions (0 to 50% RH) and at a temperature preferably in the range of -5°C to 45°C, more preferably in the range of -5°C to 40°C. The substrate can be stored in different states, for example, pelleted, chopped, chipped, or raw. It can be stored in a container. It can also be stored stacked. Preferably, a substrate characterized by a low water content, for example, less than 20% w / w, is stored as described above. Substrates with a higher water content, for example, more than 20% w / w, are preferably stored frozen or refrigerated, preferably frozen.

[0046] The substrate provided by the present invention may further comprise mycelium-colonized substrate and / or mycelium-substrate composite material obtained from a previous production process of mycelium-substrate composite material. Such products are preferably ground or shredded again and added to a new substrate batch in an amount of up to 90% w / w, preferably up to 80% w / w. According to the inventors, the addition of mycelium-colonized substrate and / or mycelium-substrate composite material from a previous batch.

[0047] As will be appreciated by those skilled in the art, the mycelial colonization substrate can also be obtained from culinary mushroom growers, where the mycelial colonization substrate is a waste product after harvesting the fruiting bodies.

[0048] Preferably, as provided by the present invention, the mycelium inoculation substrate to be incubated according to the present invention contains a synthetic granulometry regulator (SGR). The synthetic granulometry regulator is as described below. The synthetic granulometry regulator is intended to increase the void space in the substrate, thus allowing for more gas exchange and / or a higher colonization rate. It should be understood that according to the present invention, the synthetic granulometry regulator is added to the substrate before the mycelium is inoculated, or according to the present invention, the synthetic granulometry regulator can be added to the mycelium inoculation substrate at the time of inoculation. As will become clear from the following disclosure, the present invention also provides for the combined inoculation of the substrate with mycelium together with the addition of the synthetic granulometry regulator.

[0049] As encompassed by the present invention, SGR can be added before preconditioning the substrate, or SGR can be added during the preconditioning. SGR can also be added before or during the sterilization process. Therefore, it is possible to use SGR only during substrate sterilization, and due to the increased porosity, heat can be transferred more easily throughout the substrate. Therefore, SGR can be used to improve the sterilization process. After the sterilization process, the SGR can be removed again, the substrate can be inoculated, and the resulting inoculated substrate can be directly molded. However, while such a method is encompassed by the present invention, it is not preferred because it may result in an inferior product.

[0050] SGRs can also be added before or during inoculation, for example, as part of the inoculum (where they can function as spawn carriers). SGRs can also be added before or during colonization.

[0051] A conceptual example of a synthetic particle size distribution adjuster incorporated into a matrix of the present invention is shown in Figure 2. Herein, volumetric shapes are added to the matrix to reduce matrix compaction of the underlying matrix layer and to increase airflow by increasing void space. This increased void space, and therefore airflow, also aids in heat removal by increasing evaporative cooling. These shapes can take the form of molded meshes, porous materials, or 3D lattice structures. According to the inventors, shapes with many sharp edges tend to interlock in a favorable manner, while more circular shapes are easier to handle for subsequent processing (e.g., removal of the synthetic particle size distribution adjuster prior to preparation of the molding mix).

[0052] A specific example is shown in Figure 3. Preferably, the material of the synthetic particle size distribution modifier is such that it does not impede the flow of gas, but does impede the presence of substrate or mycelium (e.g., mycelium inoculated substrate) in the volume (or substantially impedes the presence of substrate or mycelium in the volume). Preferably, as understood herein, preventing (or substantially preventing) the presence of substrate or mycelium in the volume of the synthetic particle size distribution adjuster preferably means that at least 50% of the volume of the synthetic particle size distribution adjuster is free of substrate or mycelium, more preferably at least 60% of the volume of the synthetic particle size distribution adjuster is free of substrate or mycelium, even more preferably at least 70% of the volume of the synthetic particle size distribution adjuster is free of substrate or mycelium, even more preferably at least 80% of the volume of the synthetic particle size distribution adjuster is free of substrate or mycelium, even more preferably at least 90% of the volume of the synthetic particle size distribution adjuster is free of substrate or mycelium, even more preferably at least 95% of the volume of the synthetic particle size distribution adjuster is free of substrate or mycelium, even more preferably at least 99% of the volume of the synthetic particle size distribution adjuster is free of substrate or mycelium, and even more preferably the volume of the synthetic particle size distribution adjuster is free of substrate or mycelium. Thus, the synthetic particle size distribution adjuster of the present invention can be made of a non-digestible material (i.e., non-digestible to mycelium), such as plastic, stainless steel, mineral, etc. Alternatively, the synthetic particle size distribution adjuster of the present invention can be made of a digestible (i.e., digestible to mycelium) porous material, such as beeswax-coated peanut-shaped cushioning material. These shapes are digested by the end of colonization, leaving behind voids that can function as ventilation channels.

[0053] Therefore, in the method of the present invention, the synthetic particle size distribution adjuster is preferably selected from plastic mesh, stainless steel mesh, and perlite, and more preferably from plastic mesh and stainless steel mesh. Mesh is preferably understood herein as a three-dimensional shape made of connected strands that can function as a barrier. As understood herein, the term mesh will be clear to those skilled in the art.

[0054] As understood herein, the synthetic particle size distribution adjuster may be a hollow three-dimensional object, the shape of which is not particularly limited, and the object may include one or more cavities (also referred to as openings) on its surface.

[0055] The synthetic particle size distribution adjuster may have an egg-shaped, ball-shaped, or ellipsoid-shaped form. The egg-shaped, ball-shaped, or ellipsoid-shaped form may include one or more cavities or protrusions, preferably cavities (which may also be referred to as openings), on its surface. Furthermore, the egg-shaped, ball-shaped, or ellipsoid-shaped form may be made of plastic mesh, stainless steel mesh, or perlite, and is preferably made of plastic mesh or stainless steel mesh.

[0056] It should be understood that perlite, as preferred herein, can also be replaced by other porous / foamed minerals.

[0057] More preferably, the composite particle size distribution adjuster of the present invention is in the form of a plurality of oval shapes, ellipsoids or balls, more preferably a plurality of balls. The balls preferably contain (i.e., each contains) one or more cavities (or openings) on their surface. As understood herein, the term "plurality" preferably relates to an amount that is an integer greater than 1, preferably greater than 2, even more preferably greater than 10.

[0058] Therefore, and preferably, the composite particle size distribution adjuster is a plurality of hollow three-dimensional shapes that include one or more openings on their surfaces (i.e., each of the three-dimensional shapes includes one or more openings on its surface), which do not impede the flow of gas through its volume. As used herein, the term shape may also refer to an object.

[0059] Therefore, preferably, the composite particle size distribution adjuster is a plurality of hollow three-dimensional objects that include one or more openings on their surfaces (i.e., each of the three-dimensional objects includes one or more openings on its surface), which do not impede the flow of gas through its volume. In other words, the composite particle size distribution adjuster allows the flow of gas through its volume (i.e., is configured to allow the flow of gas through its volume), which should be understood to mean that the composite particle size distribution adjuster preferably allows at least some gas flow through its volume. Thus, the composite particle size distribution adjuster improves aeration within the volume of the substrate. Preferably, the composite particle size distribution adjuster prevents (i.e., is configured to prevent) the presence of mycelium inoculation substrate in its volume (or substantially prevents the presence of substrate or mycelium in its volume). Exemplary embodiments of the composite particle size distribution adjuster are shown in Figure 3, Figure 7, or Figure 12. The material from which the composite particle size distribution adjuster is made is not particularly limited. Preferably, the synthetic particle size distribution adjuster is made of a non-digestible material, ie, a material that cannot be digested by the mycelium, such as plastic or stainless steel.

[0060] Preferably, the SGR occupies a volume that is 200 to 8000 times larger than the average grain volume of the substrate.

[0061] Preferably, the ovoid shape is a sphere having a plurality of evenly spaced ovoid holes. In one exemplary embodiment, the sphere has an outer diameter of 60-80 mm, preferably 72 mm. Preferably, there are 300-500, preferably about 400, more preferably 400 evenly spaced holes on the surface of the sphere, each having a diameter of 1.5-2.5 mm, preferably about 2 mm, more preferably 2 mm. Further embodiments of the SGR of the present invention are described in the Examples, particularly Example 3.

[0062] In one embodiment, a wrinkled stainless steel mesh ball / sphere (similar to a stainless steel scrubber) or a molded stainless steel mesh (similar to a tea strainer) with wrinkled stainless steel mesh and weight inside can be used as the SGR.

[0063] The present inventors have demonstrated the beneficial effects of using SGR, for example, as shown in Example 12 attached hereto.

[0064] As referred to herein, the step of incubating a mycelium-inoculated substrate to grow the mycelium is preferably understood as solid-state incubation or solid-state culturing of the mycelium. This step will be clear to those skilled in the art. In such incubation, liquid medium is substantially absent, and therefore the solid substrate is substantially the main source of nutrients. As will be clear to those skilled in the art, a mycelium-colonized substrate is obtained in the process. Therefore, this process can also be referred to as colonization or colonization of a substrate.

[0065] However, while solid-state incubation of mycelia is preferred, it should be understood that the present invention also encompasses embodiments in which mycelia and substrate are incubated together in liquid suspension, e.g., in a slurry containing particles of substrate and mycelia. As will be apparent to those skilled in the art, the process is, in principle, the same as that described for solid-state fermentation culture, with the difference that the substrate has a higher water content, such that there is also water not bound to the substrate. The water content depends on the field capacity of the selected substrate and is typically in the range of 70% to 95% w / w water. It will be apparent to those skilled in the art that for incubation in liquid suspension, the same types of inoculum, additives, and reactor configurations can be used as for solid-state fermentation culture, except that, in principle, perforated walls cannot be used for this type of culture. It will also be apparent to those skilled in the art that, as in solid-state fermentation culture, higher air exchange rates should also be implemented. The liquid cultures referred to herein may have a number of advantages, including better heat distribution, which may allow for easier sterilization / pasteurization, and / or better agitation, which may lead to more spawn points and increased mycelium growth rates.

[0066] Nevertheless, preferably, the incubation of the mycelium and substrate is carried out as a solid-state incubation, as described above, although incubation of the mycelium and substrate as a liquid suspension is also encompassed by the present invention.

[0067] Mycelium requires a supply of oxygen for its growth. Therefore, it is aerated during its growth (e.g., according to solid-state cultivation). This requires the supply of oxygen / aeration to the entire volume of the substrate in which the mycelium grows. To achieve sufficient aeration of a given volume of substrate, there must be air circulation or exchange over a given area. This can be achieved actively or passively, as known to those skilled in the art. Passive aeration occurs through the dispersion of gases, herein referred to as through the solid substrate. Active aeration is achieved by creating an artificial airflow. Thus, active aeration can also be achieved directly within the volume of the substrate by providing an airflow within the volume of the substrate, for example, by placing air channels within the volume of the substrate. If aeration throughout the volume of the substrate is desired, active aeration must be implemented. However, it is clear to those skilled in the art that even in the case of active aeration, it cannot be excluded that portions of the substrate in the actively aerated substrate are not aerated, or are not aerated during the entire process, or are actively aerated only depending on their exact location or mixing conditions within the reactor. Thus, the term aeration throughout the volume of the substrate does not exclude that the entire substrate is not aerated at all times during the process, as long as preferably all or substantially all of the substrate is aerated at some point during the process.

[0068] Preferably, within the method of the present invention, aeration of the mycelium during growth occurs throughout substantially its entire volume, which can be achieved by using synthetic particle size distribution modifiers as described herein.

[0069] The step of incubating the mycelial inoculum substrate to grow the mycelium can be carried out using a culture tray, for example, a shallow culture tray. Thus, a shallow tray, preferably made of a non-digestible material (e.g., stainless steel), is filled with the mycelial inoculum substrate and closed or sealed with a lid. The mycelial inoculum substrate preferably further comprises a synthetic particle size distribution adjuster, as described herein. The tray can be reusable. Once filled with the mycelial inoculum substrate, preferably comprising a synthetic particle size distribution adjuster, the tray is placed in a chamber with controlled parameters to provide optimal growth conditions for the mycelium. As will be apparent to those skilled in the art, one way to achieve this is to place the tray in a tunnel incubator. Due to the compartmentalized growth, the possibility of cross-contamination is thereby reduced. Depending on the material used to close or seal the lid, and supported by the use of a synthetic particle size distribution adjuster, good aeration can be achieved.

[0070] The step of culturing a mycelium inoculation substrate to grow mycelium can be carried out using a solid-state mycelium bioreactor of the present invention. The solid-state mycelium bioreactor is as described herein. The solid-state mycelium bioreactor allows for aeration and heat exchange of the mycelium during its growth in substantially its entire volume. Furthermore, the solid-state mycelium bioreactor allows for humidity and / or pH control in its entire volume. As will be apparent from the disclosure provided herein, the solid-state mycelium bioreactor allows for the direct provision of gas, herein air or oxygen for aeration, to the solid substrate. Aeration can be further supported by a synthetic particle size distribution regulator present in the mycelium inoculation substrate. Thus, the solid-state mycelium bioreactor provides good aeration throughout the entire volume of the substrate (e.g., mycelium inoculation substrate), making it possible to work with large volumes of substrate and reducing the amount of manual labor required.

[0071] In the solid-state mycelium bioreactor of the present invention, it is further possible to mix and / or agitate the solid mass present in the reactor, herein the mycelium inoculated substrate. Of note, it is known to those skilled in the art that mixing and agitation of a mycelium-inoculated substrate can result in an increase in the number of spawn points and increase the stiffness and toughness of the final material.

[0072] Further parameters that need to be controlled when cultivating mycelium are CO2 concentration, air humidity, oxygen concentration, vibration, addition of additional nutrients, pH, concentration and type of suspended particles in the aeration supply, agitation / mixing temperature, and light / radiation.

[0073] Additionally, it is known to those skilled in the art that mycelial growth benefits from high CO2 concentrations. This can be achieved actively (supplying pure CO2 to the mycelium through an aeration mechanism) or passively (the mycelium produces its own CO2 through cellular respiration). CO2 concentrations ranging from 300 to 100,000 ppm, preferably 20,000 to 60,000 ppm, are preferred. However, the present invention also encompasses embodiments in which the CO2 concentration is greater than 60,000 ppm.

[0074] Considering the possible advantages of electrical stimulation of the mycelium, its incubation rate can be increased. In one non-limiting example, the applied current is about 500 nA. It is considered within the scope of the present invention that the application of current to the mycelium can be achieved, in particular, by using a (rotatable) spiral paddle.

[0075] As will be understood by those skilled in the art, the humidity of the supplied air can be adjusted using specialized devices called humidifiers, such as ultrasonic humidifiers. As the mycelium grows, more water evaporates from the substrate, reducing its absolute moisture content. This process can be supplemented by adding water throughout the cultivation / colonization process. An absolute substrate moisture content in the range of 50-65% w / w is preferred.

[0076] As will be appreciated by those skilled in the art, oxygen concentration is directly related to aeration. Without a supply of fresh oxygen, its concentration decreases as the mycelium grows. Reliable and consistent aeration of the bioreactor contents can be achieved, in particular, by using (rotatable) spiral paddles.

[0077] As known to those skilled in the art, the pH decreases as the mycelium grows, and a higher pH is beneficial against possible microbial contamination.

[0078] Agitation of the substrate during mycelium growth can increase the growth rate by breaking up living mycelium pieces and distributing them throughout the substrate, creating new spawn points throughout the substrate.

[0079] Light is typically associated with mushroom fruiting, but can also be beneficial for mycelium growth.

[0080] The temperature can be controlled over the air supplied to the vessel (e.g., reactor) in which the growth takes place, or over the vessel / reactor itself, or over the agitation mechanism described below. Due to heat production occurring within the substrate, the substrate heats up, which can result in higher temperatures favoring contaminants. At temperatures above 35°C, the risk of contamination increases, but higher temperatures also increase the biological activity of the mycelium. Therefore, one skilled in the art must balance these two factors.

[0081] Preferably, the method for preparing a mycelial colonization substrate of the present invention further comprises the step of preparing a mycelial inoculation substrate, which step is carried out before the step of incubating the mycelial inoculation substrate to grow the mycelium.

[0082] The preparation of the mycelium inoculation substrate can be carried out by using discrete particle spawn. Accordingly, the present invention encompasses a method for preparing the mycelium colonization substrate of the present invention, further comprising the step of preparing the mycelium inoculation substrate, wherein the mycelium inoculation substrate is prepared by mixing mycelium contained in the form of discrete particles with the substrate. Thus, particles acting as "mycelium capsules" are added to the substrate for inoculation. This, along with mixing the substrate during inoculation with the mycelium, allows for uniform distribution of mycelium within the substrate volume. Furthermore, the discrete particle spawn can also contain beneficial nutrients that can be added to the substrate. Depending on the nature of the particles, the addition of such particles may improve aeration by creating voids in the substrate (i.e., mycelium inoculation substrate) volume, according to the inventors.

[0083] As will be appreciated by those skilled in the art, it is also possible to use mycelium colonization substrate from a previous batch as an inoculum* supplement for the step of creating the inoculation substrate. (Typically, this is done together (i.e., in combination) with another type of inoculum, rather than as a separate method for inoculation.)

[0084] It will be appreciated by those skilled in the art that chopped hyphae collected from fruiting bodies may also be used as inoculum.

[0085] The spawn particles are preferably added to the substrate in a ratio of 1 to 20% weight / weight.

[0086] Therefore, and preferably, the mycelium may be contained in grain spawn, sawdust spawn, or synthetic particle spawn. Even more preferably, the mycelium may be contained in grain spawn or sawdust spawn.

[0087] In grain spawning, different types of grains or seeds can be used as spawn and added to the substrate. They are easy to separate, which allows for uniform distribution. Due to their composition, the overall nutrient density in the substrate increases.

[0088] Sawdust spawning is similar to grain spawning and has the advantage of reducing mycelium recovery time if the same substrate is used for spawning as for production, as there is no need to adapt to a new substrate. Alternatively, colonized substrate from a previous production run can be used as inoculum for the next run in a feedback system.

[0089] Synthetic particle spawn makes the most sense in conjunction with a solid-state mycelium bioreactor and can also function as a synthetic particle size distribution adjuster. Therefore, the use of a synthetic particle size distribution adjuster for inoculation provides a vehicle for mycelia to grab onto and be delivered to the substrate. Therefore, and preferably, growing mycelia adhere to such synthetic particles, for example, in a liquid-state pre-culture. The synthetic particles can then be used for inoculation. Preferably, as encompassed by the present invention, synthetic particle spawn can be prepared by using the synthetic particle size distribution adjuster of the present invention. For this purpose, the synthetic particle size distribution adjuster, preferably of an ovoid shape, such as a bole or ellipsoid, having a cavity on its surface, can be used as the synthetic particle spawn of the present invention. Therefore, preferably, the synthetic particle spawn of the present invention is the synthetic particle size distribution adjuster of the present invention.

[0090] Also encompassed by the present invention is a method in which a mycelial inoculation substrate is prepared by mixing a liquid containing mycelium or spores with a substrate. The mycelium or its spores may result from the processing of previously grown mycelium. However, as described above, discrete particles of mycelium, or mycelium contained in a synthetic particle size distribution-adjusting agent, may be suspended in a liquid. Thus, such a liquid suspension containing mycelium (or spores) contained in a synthetic particle size distribution-adjusting agent may be used to prepare a mycelial inoculation substrate according to the present invention. As will be appreciated by those skilled in the art, the spores may be added to the substrate via an air stream. This is preferably carried out while the substrate is being agitated to ensure uniform distribution of the spores throughout the substrate.

[0091] Preferably, the mycelium or spores are suspended in a nutrient solution. Different nutrient solutions can be used to cultivate the mycelium (however, for example, a 4% barley maltose and yeast extract-based nutrient solution should not be treated as limiting in any way). Once the mycelium has grown to a certain extent, it can be blended and / or homogenized, and the solution thus obtained can be added to a substrate to obtain a mycelium-inoculated substrate.

[0092] The present invention further encompasses the use of a slurry inoculant, which is a nutrient composition comprising a substrate (preferably a powdered substrate), water, and optionally a carbohydrate. An exemplary such composition can be prepared by using spruce sawdust (e.g., 4-10% w / w), hemp stalks (4-10% w / w), and water, and blending the resulting composition until a homogenous slurry is obtained.

[0093] The present invention further contemplates, according to the inventors, the combination of different inoculum types, which may result in higher colonization rates. Combining fungal inoculum types at different stages of their life cycles will result in higher colonization rates, likely because inoculum closer to the beginning of the life cycle will be pre-grown and therefore able to grow better on a nutrient-poor substrate, while inoculum further along the life cycle will be able to grow faster. In other words, younger inoculum can fill in the gaps in the pre-digested substrate of older inoculum, resulting in higher colonization rates. As will be appreciated by those skilled in the art, the use of two or more (preferably two) different strains to inoculate a substrate is also contemplated in the present invention.

[0094] The present invention also encompasses the use of symbiotic relationships between bacteria or other microorganisms that can be co-cultured with mycelia. Therefore, the colonization rate in such a setting is higher. Due to the control of the medium, the risk of contamination is lower. The addition of bacteria can act directly by stimulating vegetative growth (e.g., by removing autoinhibitory compounds) or indirectly by inhibiting pathogens. Examples of such bacteria include Pseudomonas, Mycetocola, and Bacillus (velezensis). Also encompassed by the present invention is the co-cultivation of mycelia with cyanobacteria or microalgae (e.g., Spirulina). This can increase the oxygen concentration within the substrate and therefore increase mycelial growth efficiency.

[0095] Preferably, as encompassed by the present invention, the synthetic particle size distribution adjuster is added to the inoculation substrate, i.e., to the prepared mycelium inoculation substrate. The synthetic particle size distribution adjuster may be added to the substrate inoculation, the addition of the synthetic particle size distribution adjuster may constitute inoculation of the substrate, or the synthetic particle size distribution adjuster may be added to the mycelium inoculation substrate.

[0096] Preferably, the method for the preparation of a mycelium-colonized substrate of the present invention further comprises a step of autothermal pretreatment of the substrate, which step of autothermal pretreatment of the substrate is preferably carried out before the step of incubating the mycelium-inoculated substrate, and even more preferably before the step of inoculating the substrate and growing the mycelium.

[0097] More preferably, the method for preparing a mycelium-colonized substrate of the present invention further comprises a step of enzymatic treatment of the substrate and / or a step of chemical treatment of the substrate. Preferably, these pretreatment steps are carried out before the step of incubating the mycelium-inoculated substrate to allow the mycelium to grow.

[0098] In preparing the substrate according to the present invention, several pre-treatment steps may be carried out, which may include enzymatic treatment, chemical treatment, and autothermal pre-pasteurization.

[0099] As will be appreciated by those skilled in the art, physical pretreatments such as hammering the matrix component to fibrillate and expand its grains may also be applied.

[0100] The enzyme treatment preferably refers to treatment with cellulase, xylanase, laccase, lipase, or catalytic RNA. The duration of the enzyme treatment is not intended to be particularly limited, but preferably the enzyme treatment is carried out for 1 minute to 40 hours. Such treatment has been shown to promote nutrient uptake during substrate colonization. Enzymes have a swelling effect on fibers and a fibrillating effect on the substrate (peeling and separation of fiber bundles into multiple pieces, which in turn leads to an increase in reaction / reaction area).

[0101] Chemical treatment involves immersing the substrate in a solution containing sodium hydroxide, calcium hydroxide, and / or hydrogen peroxide. The chemicals are typically added until a solution pH of greater than 7.9 is achieved. The duration of the chemical treatment is not particularly limited. Preferably, the chemical treatment is carried out for a period between 1 minute and 40 hours. Such treatment has been shown to promote nutrient uptake during substrate colonization. In one embodiment, chemical treatment may involve adding turpentine, preferably at 0.1-1.5% w / w, to the substrate before or after its sterilization / disinfection. Turpentine, as known to those skilled in the art, exhibits antibacterial and / or antiviral properties while not broadly inhibiting mycelial growth.

[0102] Autothermal pre-pasteurization, which may also be referred to as thermogenesis, involves aerobic composting of the substrate. This can be accomplished by incubating for a specific time, preferably 2 hours to 10 days, more preferably 1 to 10 days, or until a temperature of 60°C to 80°C is reached, and maintaining this for at least 1 hour, preferably at least 2 hours, more preferably at least 24 hours. Thus, during the thermogenesis step, the temperature of the substrate increases. As will be appreciated by those skilled in the art, thermogenesis can be implemented as part of a substrate preservation routine. Implementing thermogenesis promotes nutrient uptake during substrate colonization, which occurs after inoculation of the substrate with mycelium. Implementing thermogenesis also reduces energy consumption during thermal sterilization.

[0103] It should be noted hereby that heat production may also occur during mycelium incubation in the reactor as well as in the mold due to fungal cellular activity. Therefore, by using the synthetic particle size distribution modifier or solid-state mycelium bioreactor cooling feature according to the present invention, cooling of the substrate during colony formation, for example, may also be assisted or achieved.

[0104] As will be appreciated by those skilled in the art, the substrate undergoes suitable preparation before being subjected to the optional pretreatment steps, as described above, and subsequently to steps including inoculation with mycelium.

[0105] The substrate is homogenized by mixing, for example, using a litter mixer or another mixing element known to those skilled in the art. During such homogenization or mixing, large clumps of the substrate are broken down and a substantially uniform distribution of grain / particle size within the substrate mass can be achieved. Such homogenization / mixing of the substrate thus makes it possible to obtain isotropic, i.e., substantially uniform material properties in all dimensions, which allows for uniform inoculation of the substrate with mycelium and subsequent uniform colonization by the mycelium. Therefore, the purpose of homogenizing the substrate is to have the same growth conditions throughout the substrate.

[0106] In preparation for mycelial growth, the substrate thus prepared, preferably subjected to homogenization, is further subjected to a pasteurization and / or sterilization step. The purpose of pasteurization and / or sterilization is to remove / kill any bacteria or contaminants in the substrate that may harm the mycelial growth. These steps are conventional and therefore feasible for those skilled in the art.

[0107] The process of pasteurization / sterilization of the substrate may involve heat sterilization. Herein, dry heat or moist heat may be used (the use of moist heat is preferred). Thus, heat sterilization may be achieved by using steam / hot water in a double-walled vessel, such as an autoclave or oven, at a temperature of, for example, 60°C to 130°C for a time period of 10 minutes to 24 hours. As will become apparent from the disclosure below, the solid-state mycelium bioreactor of the present invention may be used for this purpose.

[0108] The process of pasteurization / sterilization of the substrate may include radiation sterilization. Thus, ionizing and non-ionizing radiation (e.g., UV, X-rays, etc.) may be used. The advantage of this approach is the rapid penetration of the material, thus avoiding the damage caused by certain types of radiation (i.e., avoiding the damage caused to the initial chemical composition of the substrate by other sterilization / pasteurization procedures (i.e., thermal processes)).

[0109] The process of pasteurization / sterilization of the substrate may involve pulsed magnetic field sterilization, which may also be referred to as PMFS (pulsed magnetic field sterilization), which involves very little change to the starting chemical composition of the substrate, thus allowing improved control of the chemical composition of the substrate.

[0110] The process of pasteurization / sterilization of the substrate may include chemical pasteurization, which may also be referred to as cold pasteurization. Herein, the chemicals used may be selected from lime, hydrogen peroxide, other peroxides, carbendazim, formaldehyde, formalin, etc. Hydrogen peroxide or calcium hydroxide is preferred. Certain chemicals may be neutralized after such pasteurization, for example with other chemicals, to promote colonization of the substrate by mycelium.

[0111] The process of pasteurization / sterilization of the substrate may include pasteurization using oxygen. Here, the substrate is exposed to a high-pressure, high-oxygen atmosphere. Thus, the pressure is set to 1.1 to 12 atmospheres, and the volumetric oxygen content is greater than 20% and up to 100%. As known to those skilled in the art, the advantages of this method include rapid pasteurization and therefore low energy consumption, as well as a higher oxygen concentration during subsequent colonization.

[0112] The process of pasteurization / sterilization of the substrate may involve a combination of the above methods. Particularly desirable is the combination of heat sterilization with oxygen pasteurization, as both require a high-pressure resistant vessel.

[0113] During pasteurization / sterilization (optionally, this can also be done by using a (rotatable) spiral paddle as provided in the present invention), the substrate is cooled so that mycelium can grow thereon. Cooling can be carried out as an active process. Thus, the vessel can be sprayed with cold water or the vessel can be immersed in a cold water bath. A cold air current can also be used for cooling. Alternatively, passive cooling can also be applied. Alternatively, cooling can be carried out using the reactor's built-in cooling system, for example, through the spiral paddle described in the present invention, or by using a water jacket. This process is slower than for active cooling, but its advantage is lower energy consumption. The substrate is considered to be suitably cooled to allow mycelium growth if its temperature does not exceed 40°C.

[0114] Chemical neutralization is necessary if the substrate has previously been pasteurized using a chemical treatment. For example, basic agents can be neutralized with acids, and peroxides can be neutralized with reducing agents, so that when the inoculant is added to such a substrate, it is not damaged by any residual chemicals. Those skilled in the art are in a position to carry out the correct neutralization treatment depending on the previous treatment steps of the substrate.

[0115] Preferably, the method for preparing a mycelium-colonized substrate of the present invention further comprises the step of preparing a molding mix, which is preferably carried out after the step of incubating the mycelium-inoculated substrate to allow the mycelium to grow.

[0116] The mycelial colonized substrate may be subjected to bacteriostatic action, preferably after the colonization step, by cooling, typically to a temperature below 10° C., or by dehydrating the substrate, which allows for long-term storage of the mycelial colonized substrate before further steps for product formulation are carried out.

[0117] Preparation of the molding mix preferably includes the addition of (additional) water and additives to increase the growth rate and improve material properties. Preferably, the mycelium-colonized substrate is characterized by overgrowth when the molding mix is ​​being prepared. The term "overgrowth" preferably describes a situation in which the mycelium's digestive juices have reached all parts of the substrate, and the mycelium's hyphae have also reached all parts of the substrate, meaning that concentric hyphal extensions from each central spawn point have contacted / reached other such points. Thus, there are no areas of the substrate that have not yet been reached by the mycelium. (The process can also be described as complete exploration of the substrate by the mycelium.) The molding mix is ​​mixed until all added nutrients are uniformly dispersed throughout the mixture. Therefore, the process further includes homogenization to ensure uniform distribution of the added nutrients. According to the inventors, homogenization increases the growth rate and toughness of the final material. As known to those skilled in the art, upon disruption in the homogenization process, the mycelium may be able to regrow in a more resilient manner, resulting in a stronger material and a denser mycelial network, depending on the particular strain used.

[0118] If the molding mix is ​​prepared by using a mycelium colonization substrate containing a synthetic particle size distribution adjuster, the synthetic particle size distribution adjuster is removed at this point. Thus, when subjected to molding and preparation of the molding mix, the mycelium colonization substrate no longer contains the synthetic particle size distribution adjuster. Therefore, according to the present invention, the incubation of the mycelium inoculated substrate containing the synthetic particle size distribution adjuster is incubated, and once substrate colonization is performed / achieved, the synthetic particle size distribution adjuster is removed from the mycelium colonization substrate.

[0119] The composite particle size adjuster is removed from the substrate by sieving and stirring / vibration, sieving and blasting with compressed air, or magnetically (if ferromagnetic SGRs are used). Preferably, this process is carried out inside the reactor, with mixing elements stirring the mix on a built-in sieve at the outlet port, and finally, compressed air is sprayed onto the SGRs to remove any remaining substrate from them. This process typically involves splitting the substrate and thoroughly mixing it to allow at least some of the following steps to be carried out simultaneously: SGR removal, addition of molding mix additives, and / or molding mix homogenization. Otherwise, the SGR-mycelium-colonized substrate mixture is transferred out of the SSMB, and the SGR removal, splitting, and molding mix preparation steps are carried out outside the vessel (optionally, such steps can be carried out inside a reactor with built-in means for mixing). In the case of digestible SGRs, they are expected to be incorporated into the substrate and can therefore remain therein; i.e., their removal or separation is not required.

[0120] The preparation of the molding mix can involve the use of a powdered substrate-water slurry or a high-heat-generating additive-water slurry. The powdered substrate-water slurry refers to the same substrate used during the colonization process in powder form (which, according to the inventors, allows for increased nutrient accessibility) and a slurry of water that can be added to the divided (homogenized) mycelium colonization substrate. The use of a powdered substrate-water slurry slightly improves the subsequent moldability and slightly increases fungal growth. The high-heat-generating additive-water slurry (which refers to a high-heat-generating additive, i.e., an additive that can be used as a high-heat-generating nutrient source, such as brewer's mash or a slurry of wheat flour starch (or similar material)) and water can be added to the divided mycelium colonization substrate. The use of a high-heat-generating additive-water slurry improves modability and increases fungal growth. Preferably, 2% w / w to 25% w / w of powdered substrate / water slurry and / or high exothermic additive-water slurry is added to the (homogenized) mycelium colonization substrate (preferably understood as removing the synthetic particle size distribution adjuster).

[0121] As will be appreciated by those skilled in the art, other additives can be added to the molding mix during its formation, such as the addition of additional spawn, gas (e.g., dissolved in water), chemicals, and other organisms such as probiotic bacteria. As will be appreciated by those skilled in the art, chemicals that can change the properties of the material can also be added, such as softeners, aerogels, biodegradable foams, high-calorie carbohydrates (specifically selected from sucrose, dextrose, and starch), beeswax, calcium, enzymes, fats and oils, agar, or cationic organic compounds.

[0122] Preparation of the molding mix may involve the addition of minerals and / or alkaline chemicals. Examples of such minerals and / or alkaline chemicals include calcium hydroxide, calcium sulfate, sodium hydroxide, and lime. During the primary incubation, which converts the mycelium-inoculated substrate into a mycelium-colonized substrate, the pH decreases. Preferably, the pH defined herein is measured by adding water to the dry components of the substrate, as known to those skilled in the art of pH measurement in the food industry. Since most contaminants grow better at an acidic pH, increasing the pH can reduce the risk of contamination. Therefore, the chemicals are added at 0.01% w / w to 2% w / w of the (homogenized) mycelium-colonized substrate (preferably understood as the removal of synthetic particle size distribution adjusters).

[0123] Preferably, the method for preparing a mycelium-colonized substrate further comprises the step of shaping the obtained mycelium-colonized substrate, which step is preferably carried out after the step of preparing a shaping mix, as described above.

[0124] The mold is preferably a container with one of its sides open, typically its top, in which the mycelium colonization substrate can grow according to its predefined shape, i.e. by filling the predefined shape of the mold as defined by the predetermined geometry of the mold.

[0125] The molding process is determined by a predefined mold geometry. The purpose of the mold geometry is to define the final shape of the mycelium composite product. To obtain a high-quality mycelium composite product, certain factors must be considered. The mycelium is forced to take the shape of the mold. On the mold walls, the mycelium tends to grow into a dense network, so that sufficient oxygen supply / ventilation can be provided to those parts further away from the wall. The mycelium is forced to grow flush with the mold walls, so that the mycelial strands that normally grow outward toward the wall are forced to contact each other at the wall.

[0126] Alternatively, the matrix can be packed into standardized blocks, which can later be machined (e.g., by using CNC) to obtain the desired geometry of the final mycelium-matrix composite.

[0127] Mycelium growth within the mold can be influenced by the mold removal angle (which facilitates product removal from the mold), the flexibility of the molding material (flexible materials facilitate product release), the aeration area to volume ratio (the greater the area over which the mycelium has access to fresh air / oxygen supply, the faster a given volume of mycelium-substrate matrix will tend to grow), and the presence / size and resolution of geometric features in the mold (which is strongly correlated with the particle size distribution of the substrate / molding mix).

[0128] The walls of the mold can be coated with some 3D pattern, such as a lattice pattern, resulting in a larger surface area, which can increase the aeration per unit volume of substrate. Therefore, more mycelial surface development can occur and a stronger product can be formed.

[0129] Also encompassed by the present invention is the use of flexible molds made from flexible materials, for example. A suitable flexible material for molds is silicone. Molds made from flexible materials, such as silicone, can be more easily demolded and are more durable.

[0130] In the molding process, it is important to fill the mold with the molding mix, since it is necessary to reproduce the shape of the mold.The filling can be carried out by, for example, a filling stamp, by hand, or by injection, preferably by a filling stamp or by injection.Before filling, the mold is preferably disinfected with a disinfectant containing ethyl alcohol, which is well suited for disinfecting the mold surface.

[0131] Treatment with a disinfectant can also be combined with treatment with a release agent. Herein, the release agent can be sprayed onto the mold to facilitate the subsequent removal of the composite material from the mold. Such a faster and easier release reduces the possibility of destroying the mycelium product. Therefore, this can also enable the production of more complex geometric shapes of the resulting mycelium-substrate composite material.

[0132] The final step in the molding process encompassed by the present invention is sealing, where the mold is sealed so that the (homogenized) mycelium-substrate composite contained in the molding mix grows inside the mold, following the shape of the open side of the mold.

[0133] The mold can be closed with a pop-on lid. In such a case, the mold lid has a protrusion that fits into a recess in the main mold. By popping it on, the two parts are combined, which allows for an effective and quick sealing mechanism. The pop-on lid is intended to apply pressure to the substrate contained in the mold, which allows for achieving a higher mycelium density. Preferably, the pop-on lid as in the present invention is reusable.

[0134] The mold may be closed with a lid that includes an edge-preventing overhang. Here, the lid contacts the mold in a way that makes it less likely that mycelium will form an edge at the mold's border. Thus, fewer mycelium edges are present in the final product.

[0135] The mold can also be closed with a foil or sheet, such as a disposable plastic foil, which is then welded, wrapped or stretched over the mold opening. Alternatively, a multi-use flexible sheet can be placed over and cover the opening.

[0136] The mold closure, i.e., lid, can include vent perforations. To this end, the mold is perforated with holes arranged in a grid-like pattern throughout the mold, sized so that substrate particles cannot pass through. Also included are molds in which the lid and / or body of the mold include vent channels.

[0137] Preferably, the method for preparing a mycelium-colonized substrate further comprises a step of in-mold incubation, which step follows the step of shaping, in which the (homogenized) mycelium-colonized substrate is further grown to the shape of the mold.

[0138] During this process, it is important to ensure that the temperature of the formed mycelium-matrix composite does not rise too much due to increased heat production caused by the mycelial cells reconnecting inside the mold. Therefore, and preferably, the temperature is subjected to a cycle of change in which the temperature increases or decreases, preferably according to a day / night rhythm. Cool ambient air convection around each filled mold ensures that the temperature inside the mold does not rise significantly (perforations in the mold aid this process through the process of evaporative cooling). Cold water vapor can also be used to cool the mold. As understood herein, the main issue with temperature control is downregulating the temperature, not circulating it. For large molds with a small area-to-volume ratio, it is also possible to cool the mold by spraying water directly onto the mold or by having built-in cooling channels within the mold through which a coolant can be pumped.

[0139] Preferably, during the in-mold incubation step, the air humidity is maintained at 40 to 100% RH. Preferably, during the in-mold incubation step, the temperature is maintained or cycled in the range of -5°C to 40°C. Preferably, during the in-mold incubation step, the CO2 content (which may also be referred to as CO2 concentration) is maintained at 5,000 to 100,000 ppm. Preferably, during the in-mold incubation step, the light intensity is maintained at 0 to 10,000 lux. Preferably, during the in-mold incubation step, ventilation is provided.

[0140] As known to those skilled in the art, incubation chambers (e.g., stackable incubation chambers) or similar devices can be used to carry out the in-mold incubation step as described herein.

[0141] Preferably, the in-mold incubation step is carried out until the mycelium has reconnected at least 80% of the particles created during the "splitting" of the mycelium-colonized substrate during the formation of the molding mix.

[0142] As will be appreciated by those skilled in the art, standard industrial incubators pose several challenges when attempting to automate the production process of mycelium-substrate composites. As known to those skilled in the art, it is standard practice to arrange the molds that are filled with the molding mix as trays. These trays are then loaded into racks or incubators.

[0143] In industrial warehouses, pallet-sized stackable units are loaded, filled with identical or similar elements, and stacked; this method is very cost- and space-efficient. Therefore, a simple modularly stackable incubation unit makes sense for the in-mold incubation process. Such a unit would have the same or similar dimensions as a pallet, have multiple vertically arranged tracks holding trays of filled mycelium colony-forming molds, a stacking and stabilization mechanism for vertical stacking, and horizontal support for the individual units. It could also feature fans, sensors, and nozzles for parameter control of each individual unit, which could connect to and transmit data to other units to maintain constant parameters within the unit stack. Controlled parameters include light, CO2, RH (humidity), aeration, and temperature.

[0144] Preferably, once the in-mold incubation is complete, the seal is removed from the mold so that the mycelium-matrix composite can be removed from the mold. For example, a process called pressure removal can be used for this purpose. In pressure removal, compressed gas (e.g., compressed air) is blown into the gap between the seal and the mold. This can also be accomplished by including an opening in the mold for receiving pressurized gas through a suitable adapter. Alternatively, this step can be performed manually. Methods for seal removal include, but are not limited to, other approaches known to those skilled in the art, such as the use of suction cups, separator wedges, air hammers, vibrating tables, or distortion / twisting of the mold, or the use of product momentum to remove it from the mold. Certain disposable molds can also be peeled from the resulting mycelium-matrix composite. However, this is not preferred because it is not sustainable and results in increased waste generation.

[0145] If needed, the edges formed around the periphery of the mycelium-substrate composite are preferably removed. This can be done by filing (the edges are filed away), by using a tumbler (the composite is run through a rotating drum and the edges are broken off), or by cutting / stamping the edges away. Note that depending on the design of the mold, the edge removal step may not be necessary.

[0146] Preferably, the method for preparing a mycelium-colonized substrate further comprises a step of surface growth. The step of surface growth is carried out after the step of in-mold incubation. Before the surface growth is carried out, an additional step of spraying can be carried out to increase the moisture content of the mycelium-substrate composite material. As known to those skilled in the art, an increase in moisture content on the surface of the composite material can result in increased growth during the step of surface growth. The spraying can be carried out, for example, by spraying or atomizing. Thus, water or a composition containing water, preferably an aqueous solution, can be sprayed onto the composite material. Alternatively, the mycelium-substrate composite material can be immersed in water or a composition containing water, preferably an aqueous solution.

[0147] In the surface growth process, a strong, protective mycelium surface is developed around the entire product. Therefore, the mycelium-matrix composite is placed in a high CO2 and high humidity environment (85-100% RH, 50,000-90,000 ppm). This can be achieved by removing the mycelium-matrix composite from the mold and then placing the mold back on the composite, leaving a small gap between the composite and the mold for surface growth. Thanks to this process, each piece of mycelium-matrix composite is individually protected. Because the composite product is segmented, the possibility of cross-contamination is reduced. Furthermore, it should be appreciated that a favorable microclimate is created around the object.

[0148] The surface growth process can be followed by a dehydration process, which can involve surface dehydration and core dehydration. As known to those skilled in the art, a significant amount of water is deposited on the surface of the mycelium-surface composite in the form of droplets. This water must preferably be removed for the next process to be carried out. Therefore, surface dehydration is carried out by using compressed air or by vigorous movement, such as by rotating or shaking the mycelium-substrate composite, so that the water droplets are removed. The surface dehydration process can be followed by the core dehydration process. This core dehydration is carried out by using a dehydrator, in which the object to be dehydrated is incubated in a warm / hot temperature (preferably 35-90°C), high air exchange (preferably 1-90 air exchanges per hour), and low humidity environment. To increase air flow between and around the product, they are stacked to leave sufficient gaps between individual units for air to pass through. Optionally, reduced pressure or vacuum can be applied to the object to be dehydrated. A conveyor-type dehydrator can be used for this purpose. Core dehydration can also be carried out as a slow drying process. The dehydrated composite material can then be stacked on racks and left to dry under normal ambient conditions. Note that because the mycelium is active and alive during this process, material properties such as toughness may also increase, potentially resulting in a very strong composite material. However, the present invention is also believed to encompass embodiments in which the mycelium is no longer alive, i.e., in which at least 50%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 99% of the cells of the mycelium are not viable.

[0149] As will be appreciated by those skilled in the art, it is possible to use the heat generated due to cellular activity during the in-mold incubation and surface growth steps, or other steps, for energy production. It is also conceivable to use CO2-containing air from a solid-state mycelium bioreactor during the surface growth step.

[0150] Preferably, the method for preparing a mycelium-colonized substrate further comprises a step of denaturation, which involves inactivating or killing the mycelia present in the mycelium-colonized substrate.

[0151] As will be appreciated by those skilled in the art, the dehydration and denaturation steps can also be carried out after the step of in-mold incubation, with or without the mold still around the mycelium-matrix composite material.

[0152] The modification can be carried out as a magnetic modification. Thus, after dehydration, the mycelium-substrate composite is placed in a pulsed magnetic field. Preferably, this is carried out on a conveyor belt setup.

[0153] The denaturation can be carried out as a heat denaturation, whereby the product, preferably previously subjected to drying, is placed for a certain time in a high temperature oven, preferably maintained at a temperature between 90°C and 210°C, until the core of the product is denatured.

[0154] The modification can be carried out as radiation modification, in which the mycelium-substrate composite is exposed to a large amount of radiation, which can be achieved by using microwaves.

[0155] Modification can also be achieved by using chemical modification, and therefore this process can be referred to as bleaching and can be associated with a change in the color of the product.

[0156] The denaturation may also involve any combination of the denaturation methods listed above. As will be further known to those skilled in the art, the denaturation may be combined with a step of dehydration.

[0157] Depending on the steps performed within the method for preparing a mycelium-colonized substrate of the present invention, the method may also be referred to as a method for preparing a mycelium-substrate composite material of the present invention, as described herein.

[0158] The mycelium-substrate composite obtained according to the method of the present invention can be further processed by coating, pressing, and / or engraving. As understood herein, pressing occurs after surface growth or in-mold incubation and is performed as an alternative to the drying and firing steps.

[0159] The purpose of coating a composite material is to change the color or other properties of the material. For example, water resistance can be achieved with a suitable coating. These coatings can involve, for example, beeswax coatings, polymerized oils, polyurethanes, SCOBY-based gels, alginates, agar, or thermoplastic materials. Further suitable coatings include biofilms made from carbohydrates, proteins, and / or lipids, plasticized starch, biopolymers, protein-based bioplastics made by amino acid cross-linking (e.g., starting from casein, fibroin, collagen, keratin, gluten, algae, etc.), and PLA (polylactide).

[0160] The purpose of pressing or heat pressing the mycelium-substrate composite is to form it into a plate-like material and increase its strength. This can be achieved through a setup in which two heated plates compress the composite from opposite directions. It should be understood that the term "pressing" as used herein also includes cold pressing, i.e., pressing without heating.

[0161] Preferably, a Basidiomycetes strain is used in the method of the present invention. More preferably, a fungal species from the genera Trametes, Fomes, Ganoderma, Pycnoporus, or Pleurotus is selected. Even more preferably, the fungal species is Fomentarius or Trametes Versicolor. As will be appreciated by those skilled in the art, it is also possible to select a strain from most mycelium-forming saprophytic fungal species for use in the present invention, or to co-cultivate / co-inoculate fungi of different species.

[0162] It should be understood that the present invention also encompasses mycelium-substrate composite materials that can be obtained according to the method for preparing a mycelium-substrate composite material of the present invention or according to the method for preparing a mycelium-colonized substrate of the present invention. In particular, it should be understood that the present invention also encompasses mycelium-substrate composite materials that are directly obtained according to the method for preparing a mycelium-substrate composite material of the present invention or according to the method for preparing a mycelium-colonized substrate of the present invention.

[0163] The present invention further relates to a mycelium-colonized substrate obtainable according to the method for preparing a mycelium-colonized substrate of the present invention or the method for preparing a mycelium-substrate composite material of the present invention. Specifically, the mycelium-colonized substrate referred to herein may comprise the above-mentioned synthetic particle size distribution adjuster. Accordingly, the present invention also relates to an intermediate product comprising the mycelium-colonized substrate defined herein and the synthetic particle size distribution adjuster defined herein.

[0164] The present invention further relates to an intermediate product comprising a mycelium inoculation substrate and a synthetic particle size distribution adjuster as defined herein. The present invention therefore provides such an intermediate, which is characterized by excellent breathability, i.e., can be better aerated than similar intermediate products of the prior art, and which is suitable for use in the method for preparing the mycelium colonization substrate of the present invention.

[0165] Thus, the present invention provides intermediate products in the preparation of mycelial colonization substrates, including mycelial inoculation substrates and / or mycelial colonization substrates as defined herein, and synthetic particle size distribution modifiers as defined herein.

[0166] The substrate is as discussed above.

[0167] Preferably, the substrate in the intermediate product in the preparation of the mycelium-colonized substrate comprises at least one structural component and at least one filler component.

[0168] Preferably, in the substrate in the intermediate product in the preparation of the mycelium colonization substrate, at least one structural component is selected from shredded hemp stalks, shredded corn stalks, shredded tomato stalks, shredded tobacco stalks, shredded bean stalks, shredded corn cobs, softwood flakes, peanut shells, and straw.

[0169] Preferably, in the substrate in the intermediate product in the preparation of the mycelium colonization substrate, at least one filler component is selected from sawdust, brewer's mash, and paper pulp.

[0170] Preferably, the substrate contains 40-70% w / w water. Water is added so that the amount of water in the substrate is similar to the water holding rate (which may also be called the water holding capacity) of the dry ingredients of the substrate.

[0171] Preferably, the substrate in the intermediate product in the preparation of the mycelium colonization substrate further comprises an adjuvant, preferably selected from calcium sulfate, calcium hydroxide, nitrogen additives, terpenes (e.g., turpentine), lipids, simple hydrocarbons, and compost.

[0172] Exemplary applications of the mycelium-substrate composite material of the present invention are illustrated in Examples 8 and 9.

[0173] In a further embodiment, the present invention relates to the solid state mycelium bioreactor of the present invention.

[0174] The solid-state mycelium bioreactor of the present invention comprises a reactor body having a cavity and at least one mixing element disposed within the cavity of the reactor body, the at least one mixing element being rotatable relative to the reactor body about an axis of rotation. The at least one mixing element includes at least one outlet opening for adding a fluid into the reactor body. Further according to the present invention, the at least one outlet opening is fluidly connected to one or more fluid supplies via at least one fluid connection.

[0175] The at least one mixing element also preferably includes a cooling element and / or heat exchange mechanism, preferably the cooling mechanism taking the form of a water-cooled channel, as shown in FIG.

[0176] Preferably, the cavity of the solid-state mycelial bioreactor has a volume of 200 to 360,000 liters. Thus, the solid-state mycelial bioreactor of the present invention can be manufactured and operated over a very wide range of cavity volumes, which differs from prior art solutions in that the prior art solutions are typically operated at smaller volumes.

[0177] The term "reactor" as used herein refers to a device adapted to contain a biological, chemical, and / or physical reaction. A reactor comprises a reactor body having a cavity. The reactor body may substantially enclose the cavity. That is, the reactor body may contain one or more openings through which materials may be added to or removed from the cavity, and / or through which personnel may enter the cavity, e.g., to inspect the reactor. Preferably, therefore, the solid-state mycelium bioreactor further comprises access points within the reactor body that may be opened during operation of the reactor. Such access points may be used for adding materials to the reactor cavity or for withdrawing material samples from the reactor cavity. These openings / ports may also be used to operate the vessel in a fed-batch manner, where additional new substrate or nutrients are added over time during the substrate incubation / colonization process. It should be understood that, preferably, these openings may also be used, e.g., to load and / or unload substrate from the reactor.

[0178] At least one mixing element according to the present invention is configured to rotate around an axis of rotation. Preferably, the axis of rotation is vertical. However, embodiments of the solid-state mycelium bioreactor in which the axis of rotation is horizontal are also encompassed by the present invention. Furthermore, the present invention also preferably encompasses embodiments in which the axis of rotation is inclined, i.e., neither vertical nor horizontal.

[0179] The at least one mixing element according to the present invention is preferably configured to homogenously and / or uniformly mix substances, such as a substrate, a mycelium inoculation substrate and / or a mycelium colonization substrate, which may further comprise a synthetic particle size distribution adjuster contained within a cavity of the reactor body when the at least one mixing element rotates.

[0180] The at least one mixing element is not particularly limited, and any design suitable for the above purpose that is predictable to one skilled in the art can be encompassed by the present invention. For example, the at least one mixing element can include a mixing rod and at least one mixing extension, for example, the at least one mixing extension is attached perpendicular to the mixing rod, and the mixing rod is configured to rotate around the axis of rotation as described above. However, it should be understood that the at least one mixing extension does not have to be attached perpendicular to the mixing rod, and can be attached to it at a different angle, as will be apparent to one skilled in the art.

[0181] Preferably, at least one mixing element is a rotatable spiral paddle. Spiral paddles, as rotating or mixing elements, are known to those skilled in the art. Preferably, a spiral paddle is defined herein as comprising paddle elements arranged along and around an axis of rotation. As encompassed by the present invention, a spiral paddle may comprise any number of turns, may comprise various pitches and / or turn arrangements, and may be arranged at a fixed or variable distance from the axis of rotation as needed for optimal mixing in the solid-state mycelium bioreactor of the present invention. An exemplary embodiment of a spiral paddle is shown in FIG. 4.

[0182] The inventors have demonstrated that a reactor according to the invention, in particular in which the mixing elements are rotatable spiral paddles, provides excellent aeration throughout the entire volume of the reactor contents, as demonstrated, for example, in the growth experiments shown in Example 13 attached hereto.

[0183] As will be appreciated by those skilled in the art, preferably, the reactor body is fixed and at least one mixing element rotates around the axis of rotation. However, since the axis of rotation is defined relative to the reactor body, embodiments are also conceivable in which at least one mixing element is fixed and the reactor body is configured to effectively rotate around the axis defined by the mixing element. Such a configuration may also be referred to as a rotating drum configuration. Particularly preferred is a rotating drum configuration in which the axis of rotation is horizontal or substantially horizontal, at least one mixing element is fixed, and the reactor body is configured to rotate around the axis of rotation. It should be understood that, given the relativity of the rotational motion, such a configuration should also be construed as encompassed by the present invention. Preferably, the vessel is fixed and the mixing element rotates, and the entire volume is uniformly ventilated and preferably also cooled on the mixing element. In vertical vessels, the preferred method is to use hollow spiral mixing elements in addition to baffles on the vessel walls, with both the mixing elements and baffles having perforations, nozzles, or spargers fluidly attached to them to allow forced intra-substrate aeration (see Figure 9). In horizontal vessels, paddle mixers are preferred, and aeration can be addressed similarly to the spiral mixer aeration setup for vertical vessels.

[0184] It is also possible to operate the reactor without any mixing elements that rotate relative to the reactor body. In this configuration, the entire vessel rotates to homogenize the substrate within it. This is similar to the mixing method of a cement / concrete mixer. In this case, the substrate is aerated across the vessel walls, to which baffles-spargers may be attached. These baffles may take the form of inward protrusions / rods similar to the rifling of a gun or inward spiral baffle blades. The vessel in this configuration is horizontal or slightly inclined. As will be appreciated by those skilled in the art, this configuration allows for continuous substrate-mass transfer through the body of the reactor over time. The mycelium-inoculated substrate enters the vessel at one end and exits the vessel at the other end as mycelium-colonized substrate. During its time within the vessel, it moves horizontally along the reactor body. This mass of material is ensured by the vessel's rifling-spiral baffles or by positioning the reactor body at a slight downward angle, thereby configuring the body to allow the mass to move accordingly. The baffling may only partially protrude from the wall of the vessel, or may extend from one wall to the other (similar to an Archimedes screw setup / configuration).

[0185] A continuous mode of reactor operation can also be achieved in a configuration in which a mixing element in the form of a spiral mixer similar to an Archimedes screw is present inside the reactor body. This screw mixing-aerating spiral moves the substrate mass along the axis of rotation as it rotates. As in the whole-vessel rotating configuration, the mycelium inoculation substrate enters at one end of the reactor and the mycelium colonization substrate exits at the other end.

[0186] The at least one outlet opening may refer to an opening provided on the surface of at least one mixing element. For example, the at least one outlet opening may be arranged on the surface of a spiral paddle. In an alternative embodiment, the at least one outlet opening may be arranged on the surface of a mixing rod and / or at least one mixing extension. Preferably, the at least one outlet opening is a plurality of outlet openings. Therefore, preferably, when referring to an outlet opening, it may also be interpreted as referring to a plurality of outlet openings.

[0187] Preferably, the at least one outlet opening comprises a nozzle or sparger, more preferably, the at least one outlet opening comprises a nozzle.

[0188] The term "fluid connection" may refer to, for example, a fluid line or channel that may be provided in at least one mixing rod and / or at least one mixing extension and that connects at least one outlet opening with at least one fluid supply. A fluid connection may include two or more fluid lines or channels. Thus, as referred to herein, the term "fluid connected" means that a fluid may flow between two elements, but the two elements do not necessarily have to be physically connected to one another. As used herein, the fluid preferably refers to a gas (e.g., steam) or a liquid (e.g., water, an aqueous solution), but may also include a suspension or slurry, such as a slurry inoculant composition as described above.

[0189] The at least one outlet opening is configured for the addition of a fluid to the reactor cavity. Preferably, the fluid added through the at least one outlet opening is a gas. More preferably, the fluid added through the at least one outlet opening is air for aeration of the mycelium-colonized substrate.

[0190] As referred to herein, a fluid supply may include a central channel within at least one mixing element. The central channel may extend into the mixing element from an attachment point at the axis of rotation. For example, the central channel may extend along the axis of rotation along the mixing rod, with additional channels branching off from the central channel. These additional channels may branch off onto the surface of at least one mixer rod and / or onto the surface of at least one mixing extension. Alternatively, the central channel may be located within a spiral paddle and sequentially connect to different outlet openings.

[0191] Therefore, and preferably, the fluid connection extends through a channel that extends through the at least one mixing element.

[0192] Thus, the fluid supply may be located outside the reactor body. For example, the fluid supply may include a compressor for supplying a gas, e.g., air, for venting the reactor cavity. The fluid supply is not intended to be particularly limiting, and it is contemplated by those skilled in the art that any other method of providing a fluid, e.g., a gas, for example, by operating a fan, is also encompassed by the present invention.

[0193] Further encompassed by the present invention is a solid-state mycelium bioreactor as described above, wherein the reactor body further comprises at least one outlet opening for adding a fluid into the reactor body. The outlet opening is fluidly connected to one or more fluid supplies via at least one fluid connection. This solution therefore allows a fluid, preferably a gas, more preferably air for venting the reactor cavity, to be supplied to the reactor cavity not only through a fluid supply in the mixing element, but also through a fluid supply in the wall of the reactor body. Preferably, the at least one outlet opening referred to herein comprises a sparger. However, embodiments in which the at least one outlet opening in the reactor body referred to herein comprises a nozzle are also encompassed by the present invention.

[0194] The solid-state mycelium bioreactor of the present invention may also be equipped with a mechanism used for cleaning the interior of the vessel. The process of cleaning the vessel may be carried out by cleaning in place (CIP). For this purpose, detergent may be pumped through a spiral paddle and / or a CIP spray ball is installed inside the vessel. The detergent is sprayed to reach all surfaces of the vessel. The purpose of cleaning is to achieve a clean, substrate residue-free growth environment for the next batch.

[0195] The solid-state mycelium bioreactor of the present invention may comprise perforated walls. Thus, the presence of perforations in the walls may improve aeration during operation of the reactor. Consequently, and preferably within the scope of the present invention, the reactor body may comprise perforated walls configured to allow aeration.

[0196] The solid-state mycelium bioreactor of the present invention may include at least one baffle, which may improve mixing within the reactor body, as known to those skilled in the art.

[0197] Solid-state mycelium bioreactors may allow for monitoring the internal conditions of the reactor and / or providing samples of material placed within the body of the reactor, even during reactor operation. Sample collection may be performed by performing a sample biopsy, i.e., by taking a sample with a large needle through an opening in the reactor body. A variation of this approach involves the use of an archimedes drill to collect samples.

[0198] As will be further known to those skilled in the art, the reactor may be equipped with sensors for measuring standard culture parameters within the reactor. These parameters may be measured, for example, by sensors located within the substrate that measure temperature, pH, humidity, dissolved oxygen levels, and dissolved CO2 levels. As will be known to those skilled in the art, headspace sensors may also be envisioned. These may be used, for example, to measure headspace gas humidity, VOC profiles, dust particle concentrations, or temperature. Furthermore, sensors may be located outside the vessel to measure vessel vibrations. It will be further apparent to those skilled in the art that measurements may also be performed on samples diverted from the reactor (e.g., obtained by collecting them through an opening in the reactor).

[0199] The inventors further propose to install an imaging device arranged on the body of the reactor and configured to provide observation of the interior of the cavity of the reactor. Therefore, preferably, the reactor of the present invention further comprises an imaging device arranged on the body of the reactor and configured to provide observation of the interior of the cavity of the reactor. Preferably, the imaging device is a camera. More preferably, the imaging device, e.g., a camera, is configured to detect mycelia growing on the substrate, preferably without human intervention. According to the inventors, visual growth and contamination recognition can be performed by custom software capable of detecting white mycelia on the substrate.

[0200] The solid-state mycelial bioreactor of the present invention is useful in processes involving the growth or incubation of mycelial biomass. Specifically, the reactor is particularly useful in processes for enzyme production, where yields up to 10 times higher than those achieved with liquid-state fermentation can be achieved. The reactor is also particularly useful in processes for the production of tempeh, fruiting bodies, animal feed, or other mushroom-based products. The reactor can also be used for other applications, such as mycoremediation. The solid-state mycelial bioreactor of the present invention is also particularly useful in the production of mycelium-substrate composites, as described herein.

[0201] Therefore, the solid-state mycelium bioreactor is useful for extracting substances from the metabolic products produced by the organisms / mycelium / fungi on the substrate. Examples of such substances are enzymes, antibiotics, lactic acid, pigments, and solvents.

[0202] The solid-state mycelium bioreactor of the present invention may also be useful in the production of solid fertilizer / soil conditioners, specifically where the mycelium-colonized substrate is used as a carrier for liquid digestate from a biogas production facility. It is contemplated that failed products or side streams may be used for such production of fertilizer-bearing liquid digestate after a step of colonization of the substrate with mycelium (i.e., as a mycelium-colonized substrate or mycelium-substrate composite, either before or after surface growth).

[0203] Although the fertilizer is a solid fertilizer, it is still completely natural and has the advantage of having a higher retention rate in the soil when compared to the direct use of liquid digestate alone; due to the presence of some undigested biomaterial in the fertilizer, the fertilizer can have a positive effect on the soil's microbial diversity and aid in the formation of humus. To produce the fertilizer, a mycelium-substrate-based carrier material is optionally dried and soaked in liquid digestate. The fertilizer can also be used as a slurry-type fertilizer. However, preferably, a pre-dried mycelium-substrate-based carrier is soaked in liquid digestate, after which the slurry-type fertilizer is dried or pelletized. As will be appreciated by those skilled in the art, other types of liquid fertilizers absorbed into mycelium-substrate-based carrier materials can also be used.

[0204] Specifically, in one embodiment of the method of the present invention for preparing a mycelium colonization substrate, the solid-state mycelium bioreactor of the present invention is used. Specifically, the method of the present invention refers to an embodiment in which the solid-state mycelium bioreactor used in the method comprises a reaction body having a cavity, and at least one mixing element disposed in the cavity of the reactor body and rotatable relative to the reactor body around an axis of rotation, the at least one mixing element comprising at least one outlet opening for adding a fluid into the reactor body, the at least one outlet opening being fluidly connected to one or more fluid supplies via at least one fluid connection. Preferably, the at least one mixing element also comprises a cooling element and / or a heat exchange mechanism, preferably a cooling element.

[0205] In the method for preparing a mycelium-colonized substrate of the present invention, a solid-state bioreactor is used for the step of incubating a mycelium-inoculated substrate to grow the mycelium.

[0206] In the method for preparing a mycelium colonization substrate of the present invention, a solid-state bioreactor is used in the step of preparing a mycelium inoculation substrate.

[0207] In the method of the present invention for the preparation of a mycelium-colonized substrate, a solid-state bioreactor is used in the step of autothermal pretreatment of the substrate. A solid-state bioreactor can also be used in the step of pasteurization / sterilization of the substrate.

[0208] In the method of the present invention for the preparation of a mycelium colonization substrate, a solid-state bioreactor is used for the step of chemical and / or enzymatic treatment of the substrate.

[0209] Further examples and embodiments of the present invention are summarized in the following numbered paragraphs. 1. A method for the preparation of a mycelial colonization substrate, comprising the step of incubating a mycelial-inoculated substrate to allow mycelium to grow. 2. The method according to item 1, wherein the mycelium inoculation substrate comprises a synthetic particle size distribution adjuster. 3. The method according to item 2, wherein the synthetic particle size distribution adjuster is selected from a plastic mesh, a stainless steel mesh, and a porous / foamed mineral such as perlite, preferably a plastic mesh and a stainless steel mesh. 4. The method according to item 2 or 3, wherein the synthetic particle size distribution adjuster is in the form of a plurality of balls. 5. The method according to any one of items 1 to 4, wherein aeration of the mycelium during growth occurs over substantially its entire volume. 6. The method according to any one of items 1 to 5, wherein the substrate comprises at least one structural component and / or at least one filler component. 7. The method according to item 6, wherein the at least one structural component is selected from shredded hemp stalks, shredded corn stalks, shredded tomato stalks, shredded tobacco stalks, shredded bean stalks, shredded corn cobs, softwood flakes, peanut shells, and straw. 8. The method according to item 6 or 7, wherein at least one filler component is selected from sawdust, brewer's mash, and paper pulp. 9. The method according to any one of items 6 to 8, wherein the substrate further comprises an auxiliary agent. 10. The method according to item 9, wherein the adjuvant is selected from calcium sulfate, calcium hydroxide, nitrogen additives, terpenes, lipids, simple hydrocarbons, and compost. 11. The method according to any one of items 1 to 10, further comprising the step of preparing a mycelium inoculation substrate, which is carried out before the step of incubating the mycelium inoculation substrate to grow the mycelium. 12. The method according to item 11, wherein the mycelium inoculation substrate is prepared by mixing mycelium contained in the form of discrete particles with the substrate. 13. The method according to item 12, wherein the mycelium is contained in grain spawn or sawdust spawn. 14. The method according to item 11, wherein the mycelium inoculation substrate is prepared by mixing a liquid (or fluid) containing mycelium or spores with the substrate. 15. The method according to any one of items 1 to 14, further comprising a step of autothermal pretreatment of the substrate, which is carried out before the step of incubating the mycelium-inoculated substrate to grow the mycelium. 16. The method according to any one of items 1 to 15, further comprising a step of enzymatic treatment of the substrate and / or a step of chemical treatment of the substrate, which is carried out before the step of incubating the mycelium-inoculated substrate to grow the mycelium. 17. A solid-state mycelium bioreactor comprising: The solid-state mycelium bioreactor comprises a reaction body having a cavity, and at least one mixing element disposed within the cavity of the reactor body and rotatable relative to the reactor body about an axis of rotation; 17. The method according to any one of items 1 to 16, wherein a solid-state mycelium bioreactor is used, wherein at least one mixing element comprises at least one outlet opening for adding a fluid into the reactor body, the at least one outlet opening being fluidly connected to one or more fluid supplies via at least one fluid connection, preferably wherein the at least one mixing element comprises a cooling element and / or a heat exchange mechanism, preferably a cooling element. 18. The method according to item 17, wherein the solid-state bioreactor is used in the step of incubating a mycelium inoculation substrate to grow the mycelium. 19. The method according to item 17 or 18, wherein the solid-state bioreactor is used in the step of preparing a mycelium inoculation substrate. 20. The method according to any one of items 17 to 19, wherein the solid-state bioreactor is used in a step of autothermal pretreatment of a substrate. 21. The method according to any one of items 17 to 20, wherein the solid-state bioreactor is used for a step of chemical and / or enzymatic treatment of a substrate. 22. The method according to any one of items 17 to 21, wherein the solid-state mycelium bioreactor is as described in any one of items 36 to 48. 23. The method according to any one of items 1 to 22, further comprising the step of preparing a molding mix, which is carried out after the step of incubating the mycelium-inoculated substrate to grow the mycelium. 24. The method according to item 23, wherein the step of preparing the molding mix involves the use of a powdered substrate-water slurry or a high-exothermic additive-water slurry. 25. The method according to item 23 or 24, further comprising the step of shaping the obtained mycelium-colonized substrate. 26. The method according to item 25, further comprising a step of in-mold incubation. 27. The method according to item 26, further comprising a step of surface growth. 28. The method according to item 27, further comprising a dehydration and denaturation step, preferably a denaturation step. 29. An intermediate product in the preparation of a mycelial colonization substrate, comprising a mycelial inoculation substrate and a synthetic particle size distribution adjuster. 30. The intermediate product according to item 29, wherein the mycelium substrate comprises at least one structural component and at least one filler component. 31. The intermediate product according to item 30, wherein the at least one structural component is selected from shredded hemp stalks, shredded corn stalks, shredded tomato stalks, shredded tobacco stalks, shredded bean stalks, shredded corn cobs, softwood flakes, peanut shells, and straw. 32. The intermediate product according to item 30 or 31, wherein at least one filler component is selected from sawdust, brewer's mash, and paper pulp. 33. The intermediate product according to any one of items 30 to 32, wherein the substrate further comprises an auxiliary agent. 34. The intermediate product according to item 33, wherein the adjuvant is selected from calcium sulfate, calcium hydroxide, nitrogen additives, terpenes, lipids, simple hydrocarbons, and compost. 35. A solid-state mycelium bioreactor comprising: a reaction body having a cavity; at least one mixing element disposed within the cavity of the reactor body and rotatable relative to the reactor body about an axis of rotation; the at least one mixing element includes at least one outlet opening for adding a fluid into the reactor body; at least one outlet opening is fluidly connected to one or more fluid supplies via at least one fluid connection; Preferably, the solid state mycelium bioreactor comprises at least one mixing element, wherein the at least one mixing element comprises a cooling element and / or a heat exchange mechanism, preferably a cooling element. 36. A solid-state mycelium bioreactor according to item 35, wherein the fluid connections extend through channels that extend through at least one mixing element. 37. The solid-state mycelium bioreactor according to item 35 or 36, wherein at least one outlet opening comprises a nozzle or sparger. 38. The solid-state mycelium bioreactor according to any one of items 35 to 37, wherein the axis of rotation is vertical. 39. The solid-state mycelium bioreactor according to any one of items 35 to 38, wherein at least one mixing element is a rotatable spiral paddle. 40. A solid-state mycelium bioreactor according to any one of items 35 to 39, wherein the fluid added through at least one outlet opening is a gas, preferably air, for aeration of the mycelium colonization substrate. 41. A solid-state mycelium bioreactor according to any one of items 35 to 40, wherein the reactor body comprises perforated walls configured to allow aeration. 42. The solid-state mycelium bioreactor according to any one of items 35 to 41, wherein the reactor body further comprises at least one outlet opening for adding fluid into the reactor body, said outlet opening being fluidly connected to one or more fluid supplies via at least one fluid connection, preferably said outlet opening comprising a sparger. 43. A solid-state mycelium bioreactor according to any one of items 35 to 42, wherein the wall of the reactor body further comprises at least one baffle. 44. A solid-state mycelium bioreactor according to any one of items 35 to 43, wherein the solid-state mycelium bioreactor further comprises an access point within the reactor body that can be opened during operation of the reactor. 45. The solid-state mycelium bioreactor of any one of items 35 to 44, further comprising an imaging device disposed on the body of the reactor and configured to provide observation of the interior of the reactor cavity. 46. ​​The solid-state mycelium bioreactor according to item 45, wherein the imaging device is a camera. 47. A solid-state mycelium bioreactor according to item 45 or 46, wherein the imaging device is configured to detect mycelium growing on the substrate, preferably without human intervention.

[0210] Further examples and / or embodiments of the present invention are disclosed in the following numbered sections: 1. A method for preparing a mycelial colonization substrate, the method comprising the step of incubating a mycelial inoculation substrate, the mycelial inoculation substrate comprising a synthetic particle size distribution modifier, to allow mycelium to grow. 2. The method of paragraph 1, wherein the synthetic particle size distribution adjuster is selected from a plastic mesh and a stainless steel mesh. 3. The method of paragraph 1 or 2, wherein the composite particle size distribution modifier is in the form of a plurality of balls. 4. The method of any one of paragraphs 1 to 3, wherein aeration of the mycelium during growth occurs throughout substantially its entire volume. 5. The substrate in the mycelium-inoculated substrate comprises at least one structural component and / or at least one filler component; Preferably, the at least one structural component is selected from shredded hemp stalks, shredded corn stalks, shredded tomato stalks, shredded tobacco stalks, shredded bean stalks, shredded corn cobs, softwood flakes, peanut shells, and straw; Preferably, the at least one filler component is selected from sawdust, brewer's mash, and paper pulp; Optionally, the substrate further comprises an adjuvant; Preferably, the adjuvant is selected from calcium sulfate, calcium hydroxide, nitrogen additives, terpenes, lipids, simple hydrocarbons, and compost. 6. A step of preparing a mycelium inoculation substrate, which is carried out before the step of incubating the mycelium inoculation substrate to grow the mycelium, Preferably, the mycelial inoculation substrate is prepared by mixing the mycelia contained in the form of discrete particles with the substrate; Preferably, the mycelium is contained in grain spawn or sawdust spawn, or the mycelium inoculation substrate is prepared by mixing a liquid containing mycelium or spores with a substrate; and / or The method further comprises a step of autothermal pretreatment of the substrate prior to the step of incubating the mycelium-inoculated substrate to grow the mycelium; and / or 7. The method of any one of clauses 1 to 6, further comprising a step of enzymatic treatment of the substrate and / or a step of chemical treatment of the substrate, performed prior to the step of incubating the mycelium-inoculated substrate to grow the mycelium. 7. A solid-state mycelium bioreactor comprising: The solid-state mycelium bioreactor comprises a reaction body having a cavity; at least one mixing element disposed within the cavity of the reactor body and rotatable relative to the reactor body about an axis of rotation; at least one mixing element including at least one outlet opening for adding a fluid into the reactor body, the at least one outlet opening being fluidly connected to one or more fluid supplies via at least one fluid connection; Preferably, the solid-state bioreactor is used to incubate a mycelial inoculation substrate to grow the mycelium; and / or the solid-state bioreactor is used to prepare a mycelium inoculation substrate; and / or The solid-state bioreactor is used in a process for autothermal pretreatment of a substrate, and / or 7. The method according to any one of clauses 1 to 6, wherein a solid state mycelium bioreactor is used, wherein the solid state bioreactor is used for the step of chemical and / or enzymatic treatment of a substrate. 8. A step of preparing a molding mix, which is carried out after the step of incubating the mycelium-inoculated substrate to grow the mycelium, Preferably, the step of preparing the molding mix further comprises the step of using a powdered substrate-water slurry or a high-heat-generating additive-water slurry; and / or and / or further comprising the step of shaping the resulting mycelium-colonized substrate. and / or further comprising a step of in-mold incubation and / or further comprising a step of surface growth 8. The method of any one of clauses 1 to 7, further comprising a step of denaturation. 9. An intermediate product in the preparation of a mycelium colonization substrate, comprising a mycelium colonization substrate and a synthetic particle size distribution modifier. 10. A solid-state mycelium bioreactor comprising: a reaction body having a cavity; at least one mixing element disposed within the cavity of the reactor body and rotatable relative to the reactor body about an axis of rotation; the at least one mixing element includes at least one outlet opening for adding a fluid into the reactor body; at least one outlet opening is fluidly connected to one or more fluid supplies via at least one fluid connection; Preferably, the solid state mycelium bioreactor comprises at least one mixing element, wherein the at least one mixing element comprises a cooling element and / or a heat exchange mechanism, preferably a cooling element. 11. The fluid connection extends through a channel extending through at least one mixing element, and / or 11. The solid-state mycelium bioreactor of clause 10, wherein the at least one outlet opening comprises a nozzle or sparger. 12. The axis of rotation is vertical, and / or 12. The solid state mycelium bioreactor of clause 10 or 11, wherein at least one mixing element is a rotatable spiral paddle. 13. The solid-state mycelium bioreactor according to any one of clauses 10 to 12, wherein the fluid added through the at least one outlet opening is a gas, preferably air, for aeration of the mycelium colonization substrate. 14. the reactor body comprises perforated walls configured to allow ventilation; and / or the reactor body further comprises at least one outlet opening for adding a fluid into the reactor body, the outlet opening being fluidly connected to one or more fluid supplies via at least one fluid connection, preferably the outlet opening comprising a sparger; and / or the reactor body wall further comprises at least one baffle; and / or 14. The solid state mycelium bioreactor of any one of clauses 10-13, wherein the solid state mycelium bioreactor further comprises an access point within the reactor body that can be opened during operation of the reactor. 15. An imaging device disposed on a body of a reactor and configured to provide a view of the interior of a cavity of the reactor, comprising: Preferably, the imaging device is a camera, and / or 15. The solid state mycelium bioreactor of any one of clauses 10 to 14, further comprising an imaging device, preferably configured to detect mycelium growing on the substrate, preferably without human intervention.

[0211] This invention is illustrated in the following illustrative examples, which are not intended to be construed as limiting. [Example]

[0212] Example 1 - Production of a mycelium-substrate composite with a protective surface The experimental setup for Example 1 is shown in FIG.

[0213] Storage: Substrate components are stored in large bags in a well-ventilated area, standing on raised bases so that air can circulate throughout them.

[0214] Mixing and Preconditioning: The dry ingredients of the substrate according to Example 6 and boiling water are added into the body of the reactor according to Example 2 through its hinged closure and mixed to create a hot, moist substrate. Hot water is added to germinate the endospores. This hot mix is ​​maintained at a temperature above 60°C for 5-10 hours, after which the substrate is allowed to cool to room temperature ±15°C. After cooling, the substrate can optionally be left undisturbed for another 5-10 hours. During this entire process, the aeration is turned off and the vessel is closed.

[0215] Sterilization / Pasteurization: After preconditioning, the SGR according to Example 3 is added to the mix so that the entire vessel is filled to the surface with SGR and the substrate fills the gaps between the SGR. Hot water at 89°C is circulated throughout the double wall. If a large amount of substrate is used, steam can be blown into the reactor through a port or through the aeration spiral (this helps prevent clogging of the aeration spiral perforations) to accelerate the heat-up time and increase the amount of heat transferred to the substrate. The substrate temperature is maintained above 89°C for 12 hours. During this time, the agitator is operated for 3 minutes every 1-3 hour interval to help distribute heat throughout the substrate. After 12 hours, the substrate is cooled to below 45°C; this process can be accelerated by circulating cold water through the double wall.

[0216] Inoculation: Inoculation is achieved according to Example 2. Specifically, grain spawn of Trametes Versicolor at a 5% w / w wet spawn rate is used.

[0217] Colony formation: The substrate is incubated for 72-120 hours at 24-29°C, 70-95% RH, pH 5-9, preferably 6-8, and a CO2 concentration of 300-100,000 ppm, preferably 20,000-60,000 ppm. Minor adjustments to the incubation parameters are accomplished according to Example 2. If needed, a small amount of crushed and sterilized ground dry waste bread from a local bakery can be added to facilitate growth by the method according to Example 2.

[0218] Forming the molding mix: After the formation of the mycelium colonization substrate, the SGR is removed by sieving using a rod screen. After sieving, autoclaved (121°C for 40 minutes) substrate additive (sterilized and crushed discarded bread from a local bakery or sterilized and crushed dry brewer's mash) and 120% water by the dry weight of the additive are mixed into the substrate. In total, 5-15% w / w of the wet additive is added based on the weight of the colonization substrate.

[0219] Mold filling: Vacuum-formed PETG molds with 2 mm perforations every 4 cm are used. Before filling, the molds are cleaned and disinfected (70% isopropyl-water solution). A release agent such as vegetable oil, mineral oil, or oil-water emulsion can also be sprayed onto the mold to create a thin film on the mold surface for easier demolding.

[0220] The molding mix is ​​added to the mold so that all parts of the mold are filled, and the mold is closed using a vacuum-formed lid, which is also perforated like the mold itself. The closed mold is shaken after closing to aid in even distribution of the substrate throughout the mold.

[0221] In-mold incubation: After the molds are filled, they are incubated for 48 to 96 hours in an incubator at 24 to 29°C, 70 to 95% RH, pH 6 to 8, and a CO2 concentration of 300 to 100,000 ppm, preferably 30,000 to 60,000 ppm.

[0222] Demolding: The mycelium-matrix composite is removed from the mold by vibrating the mold and pushing / knocking the object out or by using the momentum of the object.

[0223] Surface growth: The demolded object is placed in an incubation chamber for the third incubation. This step creates a pure mycelium surface around the object. Prior to incubation, water is sprayed onto the surface of the mold, and the mold is placed back onto the object with a gap (this is achieved by slightly elevating the mold above the object). This creates a microclimate around the object, protecting it. The object is incubated for 48 to 96 hours at 23 to 29°C, 85 to 100% RH, pH 6 to 8, and a CO2 concentration of 50,000 to 90,000 ppm.

[0224] Drying and calcination: After the skin has grown, the water on the surface of the object is blown off and the object is allowed to air dry for 4-48 hours. The object is dried on a metal rack so that air can circulate all around the object. After air drying, the object is baked at 90°C in an air-circulating oven until the core temperature reaches above 60°C.

[0225] Advantages of the described process compared to the segmented bag SSF-based process: Separate machines are required for sterilization / pasteurization for bag-based processes → fewer machines are required • Growth bags are colonized on racks so more space is required for the same mass of substrate → less space is required. The resulting molding mix has adhesive properties that allow for easier mold filling → Easier mold filling Surface growth in a mold with gaps allows for an ideal microclimate for surface growth → better surface growth ●Due to SGR and SSMB, greater aeration can be achieved → better colonization parameters / conditions • The addition of a release agent aids in demolding → fewer failed products and the ability to produce higher resolution molds without the risk of damaging the final product during demolding. Drying the product before firing results in excellent material properties and appears to be without drying.

[0226] Example 2 - Vertical mixed-bed reactor with aeration-mixing spiral Referring to Figure 6, this reactor conforms to that used in Example 1. (The diagram shown is a simplified schematic of the actual vessel.)

[0227] General specifications: -Operation mode: batch or fed-batch - Vessel: A double-walled stainless steel vessel with an internal diameter of 0.63 meters, a rounded bottom, and an overall internal height of 1.48 meters (with space inside the lid). The entire vessel can be rotated along a horizontal axis for easy removal of the vessel contents by rotation and has a hinged closure. The closure has multiple ports for observation, sensors, venting, mass addition, etc. - Agitation and Aeration: The vessel has a geared 400V AC motor for agitating the vessel contents. The shaft of the mixing element is connected to the motor by a shaft key. The mixing element is a 60mm outer diameter stainless steel tubing spiral with 2mm perforations evenly spaced every 40mm. This "aerated mixing spiral" is connected to a central shaft with a 10mm stainless steel rod. The spiral completes 3.5 revolutions and is fluidly connected to an aeration unit on the lid. Air entering the vessel is HEPA filtered and humidified according to controls. - Mass addition: Two of the ports can be used to connect sterilizable augers to the vessel to add mass (such as inoculum or substrate) to the body of the vessel. Alternatively, a hinged closure can be opened.

[0228] Sensors and Controls: Sensors are placed inside the matrix and in the headspace above the matrix. Stainless steel conduit is used to protect the sensor cables inside the matrix. All sensors are connected to a computer for data logging and parameter control.

[0229] The sensors used are: A digital camera (2592 x 1944 resolution) (for recognizing surface mycelial growth) was mounted above the substrate in the headspace. → This data is used to determine agitation intervals and speeds, as well as nutrient / substrate addition (based on visual colonization index). NDIR CO2 ppm sensor. Air from the tank is preconditioned (moisture removed) by passing it through silica gel. → This data can be used to adjust the tank aeration. Combination bandgap and capacitive relative humidity sensors in the headspace of the bath and inside the substrate. → This data is used to adjust the air humidity of the supplied air. This can also be used to add water directly using a peristaltic pump. This can also be used to control the temperature of the water flowing through the double wall. (Resistance temperature sensors can also be used to record the substrate temperature and headspace temperature.) A pH sensor inside the substrate. This data is used to adjust the pH, if needed, by adding an acidic or alkaline solution to the inside of the tank using a peristaltic pump. (Typically, the solutions used are acetic acid solution or lime water, but other solutions can be used.) The data is also an indicator of mycelium growth, as the pH tends to decrease over time due to fungal activity. The display allows nearby personnel to observe the parameters inside the tank, and individuals can manually change the parameters on the UI.

[0230] operation: Filling: The vessel is filled by opening the hinged closure or by adding material through the port. The fill level varies depending on whether SGR is added to the substrate. Without SGR, the vessel is typically filled to 50-80% of its volume. With SGR, the vessel can be filled to 100% of its maximum fill volume. - Preconditioning: The vessel is used as a reaction vessel. Chemicals, hot water, enzymes, or microorganisms can be added to the vessel. The vessel can assist these processes by adjusting agitation, temperature and aeration, and other parameter controls to the ports. - Sterilization: The stainless steel double wall design allows for greater heat transfer to the substrate, especially when combined with agitation. The SGR also aids in rapid heat transfer throughout the substrate, especially when steam is used for sterilization / pasteurization. - Inoculation: Inoculation of the substrate is achieved through one of the ports. The inoculum can be transferred to the vessel by auger, syringe, or by a stream of sterile air (Venturi principle). The agitator is typically turned on for about 3 minutes to incorporate and evenly distribute the inoculum throughout the substrate. - Incubation / colonization: During incubation, the parameters are adjusted by the sensors and control system described above, and additives, air, etc. are added if required.

[0231] Advantages compared to Segmented Growth Bag SSF: The solid state mycelium bioreactor is a one-stop shop: it combines multiple process steps and their required machinery, as well as the transfer step in one machine. - Greater control over growth parameters allows direct adjustment during mycelium growth, which makes it possible to achieve higher efficiencies. - The amount of energy per unit of substrate mass is lower in this process because less space is required during incubation. -Less plastic waste is created. - Lower contamination rates due to early warning systems (cameras) as well as greater parameter control.

[0232] Example 3 - Round and Hollow SGR A round and hollow SGR as considered herein is shown in FIG.

[0233] Outside Diameter: Referring to Figure 7, a stainless steel spherical shell having an outer diameter of 72 mm with 400 evenly spaced holes of 2 mm diameter is shown. The outer diameter of the SGRs is selected so that the non-SGR occupied volume is maximized for a vessel of a particular dimension while still having a sufficient number of SGRs to allow the contents of the vessel to still be distributed throughout.

[0234] Perforation Size: The size of the perforations in the SGR is chosen to be equal to or smaller than the grain size of the smallest grain of a given substrate. However, this limit depends on the adhesion between the small and larger grains. Therefore, it is possible to use a larger hole size than the hole size of the smallest grain size, provided that the smallest grains adhere well enough to the larger grains.

[0235] In another experiment, a spherical SGR with a 10 mm hole was covered with a stainless steel mesh with a mesh size of 1 mm.

[0236] If any *powdered* substrate still gets inside the SGR, it should be cleaned or sterilized / disinfected before next use. Sterilization was achieved with moist heat (autoclaving at 121°C for 20 minutes) and disinfection in a 70% isopropyl bath. Cleaning is done by blowing pressurized air into the bowl, which displaces attached mycelial particles and substrate until they come out with the flow.

[0237] Density and material: To prevent the SGR from separating into two layers from the substrate during agitation of the substrate-SGR composite inside the SSMB, the overall density of the spheres is equal to or greater than the stacking density of the wetted substrate ⇒ so that they remain “below” the substrate.

[0238] Other experiments used metal grains glued (epoxy) to the inside of a PTFE ball with an outer diameter of 60 mm to achieve the desired density.

[0239] Comparison of colony formation with and without SGR: Comparing the colonization of Trametes versicolor on 30 kg of different substrates at a 5% w / w (wet) spawn rate (grain spawn) in a closed, aerated, non-stirred tank (using SSMB according to Example 2 without an aeration spiral) with and without SGR, it was observed that without SGR, fermentation occurred in the lower layers of the substrate due to anaerobic conditions, regardless of the inherent porosity of the substrate. However, with SGR, no fermentation occurred, even when the spawn rate was reduced to 2.5% w / w wet.

[0240] Another benefit of using SGRs is the observed increase in colonization rate of 1.2-1.8 times when compared to colonization of the same mass of substrate in the same tank under otherwise the same parameters and spawning rates.

[0241] Example 4 - Wrinkling the SGR Instead of the hollow SGR described in Example 2, a ball of crinkled stainless steel mesh (similar to a stainless steel scrubber) or a molded stainless steel mesh (similar to a tea strainer) with an internal crinkled stainless steel mesh and weight can be used as the SGR. The shape and size of the SGR are selected to achieve a defined ratio of the SGR-occupied volume to the substrate-occupied volume. The density of the SGR is selected according to Example 2.

[0242] Cleaning of these SGRs is accomplished by rolling them in water for 0.5 to 4 hours and then blowing them with compressed air.

[0243] Mesh-based SGRs deform more under pressure and are more difficult to clean than the SGR described in Example 2, but are more suitable for use as inoculum carriers, as described in Example 5.

[0244] Example 5 - SGR as an inoculum carrier / inoculum The mesh-based SGR described in Example 4 (or to some extent in Example 3) is used as a crystallization point for mycelia during inoculum production using culture broth. A malt extract-based broth was used as the broth (per liter: 40 g malt extract, 2 g nutritional yeast, 1 g calcium sulfate). However, any other carbohydrate-based broth can also be used. The broth is sterilized by autoclaving at 121°C for 20 minutes and inoculated using mycelia from Trametes versicolor or using a mycelium wedge from an agar Petri dish.

[0245] Experiments have shown that this process results in the same or better growth of mycelia. Incubation of the liquid broth is carried out on a shaker incubator, and the temperature is the same as that described in Example 1. During incubation, filtered air is sparged into the broth and / or the broth is agitated. The broth is incubated for 72±24 hours. SGRs are added so that they contact the fill line of the broth.

[0246] After incubation, the result is a mixture of liquid inoculum and synthetic elements inside it, which allows the mycelium to grab hold of it.

[0247] Example 6 - Substrate Composition The substrate composition follows the substrate used in the process described in Example 1.

[0248] The matrix is ​​divided into structural and filler components, and additives. The w / w% and grain size distribution (measured using wet sieve analysis) of the individual components are as follows:

[0249] Structural components: Chopped hemp stalks (collected from a local CBD manufacturer): Grain size distribution: [<0.05mm: 3.4%, >0.05mm: 0.1%, >0.2mm: 7.1%, >2mm: 39.9%, >5mm: 49.5%] Dry substrate ratio: 35% w / w Wood waste (collected from local pencil manufacturers): Grain size distribution: [<0.05mm: 1.9%, >0.05mm: 0.2%, >0.2mm: 3.2%, >2mm: 12.5%, >5mm: 82.2%] Dry substrate ratio: 25% w / w

[0250] Filler Ingredients: Brewing Mash (collected from local breweries): Grain size distribution: [<0.05mm: 6.7%, >0.05mm: 1.5%, >0.2mm: 48.4%, >2mm: 42.5%, >5mm: 0.9%] Dry substrate ratio: 10% w / w Sawdust (collected from a local factory: [20-60% w / w spruce wood, 40-55% w / w beech wood, 10-20% others]): Grain size distribution: [<0.05mm: 3.9%, >0.05mm: 2.5%, >0.2mm: 91.8%, >2mm: 1.8%] Dry substrate ratio: 10% w / w Threshing residue (collected from a local oil mill): Grain size distribution: [<0.05mm: 9.8%, >0.05mm: 0.7%, >0.2mm: 13.7%, >2mm: 60.8%, >5mm: 15.0%] Dry substrate ratio: 5% w / w Rye mill residue (collected from a grain mill near Zurich) Grain size distribution: [<0.05mm: 2.6%, >0.05mm: 2.2%, >0.2mm: 93.1%, >2mm: 2.1%] Dry substrate percentage: 24.8% w / w

[0251] Additives: Ca(OH)2 Dry substrate ratio: 0.2% w / w

[0252] water: Water is added at 55% w / w of the total wet mass of the substrate.

[0253] Example 7 - Fertilizer Product Example Educts: A failed mycelium-substrate composite (failed during demolding) according to the production process described in Example 1 is used as the liquid carrier.

[0254] The liquid to be absorbed is liquid digester residue from a local biogas plant.

[0255] process: The failed composite was crushed to an average particle size of 3 mm (measured along the longest axis of the grain) and left to air-dry under normal conditions until 90% of the moisture content had evaporated. In other experiments, the mycelium-substrate composite was instead calcined / dehydrated at 70°C. The dried grains were placed inside a vessel and covered with liquid digestate (100% v / v). The grains were allowed to absorb the digestate for 4-12 hours, after which the grains were removed from the mixture and allowed to dry by dripping and air-drying or using a dehydrator.

[0256] In another implementation, the dripped grains are pelletized instead of dried, which results in a more compact product with a higher stack density. However, in this method, some of the liquid digestate carries a small portion of the nutrients out of the substrate during pressing, so the final product contains slightly less nutrients from the liquid digestate per mass unit of fertilizer.

[0257] Product: The product is a soil conditioner that retains its nutrients well and slowly releases them into the surrounding soil, plus the mycelial biological mass promotes soil microbial diversity.

[0258] Example 8 - Heat-pressed mycelium-substrate composite chair A surface-protected mycelium-substrate composite produced according to Example 1, except that the fungal species: Pycnoporus sanguineus was used instead of Trametes Versicolor. The geometry of the mold used resembles the final shape of the product, but is elongated in one axis.

[0259] In this case, the chair geometry was the same except that the chair thickness was six times greater than the final chair thickness after pressing. Two stainless steel hollow press dies were used to press the chair. One die resembled the shape of the chair back and the other resembled the shape of the chair front.

[0260] Steam was forced into the hollow cavity of the die to heat it to 80-120° C. The die was mounted on the extended end of a hydraulic press with a press force of 20 metric tons.

[0261] The mycelium-substrate was pressed into its final shape for 10-20 minutes (see Figure 8), after which the chair was left to dry under normal conditions for 10-24 hours.

[0262] Geometric shape: The seat size is 400mm x 400mm and the back panel size is 400mm x 460mm.

[0263] Example 9 - Mycelium-Substrate Composite Cooler for Cold Chain Transportation The mycelium-substrate composite material used for this cooler was made with the production process according to Example 1 and the substrate used according to Example 7.

[0264] Geometric shape: The described cooler is a two-part insulated box with a body and a lid. The lid is held in place with a tongue-and-groove type connection, with a tongue extending from the cooler wall and a groove formed in the lid. The wall thickness is 4 cm. All exterior corners are rounded. The box can be pre-conditioned before use and is typically used with a cardboard box surrounding the main box. The exterior length, width, and height are 600 mm, 400 mm, and 400 mm.

[0265] efficiency: The system can achieve 80-120% of the insulating efficiency of a standard expanded polystyrene cooler of the same size when comparing performance during a temperature profile with the box pre-conditioned to 4°C.

[0266] The following abbreviations are used herein:

[0267] [Table 1]

[0268] Example 10 - Solid-state mycelium bioreactor The same reactor vessel was used for all experiments performed at SSMB. The vessel is a 230 liter (no headspace) pilot-scale reactor with a rounded bottom. The vessel depth (measured from rim to bottom) is 850 mm, the lid depth (measured from rim to top) is 180 mm, and the vessel inner diameter is 630 mm. The reactor is shown in Figure 10.

[0269] [Table 2]

[0270] SOP for SSMB implementation Core methods and measurements for standard SSMB implementation

[0271] -Control bag -For each run in the chamber, a control bag is made using a standard in-bag culture. These bags are used to compare the incubation conditions in the chamber with known functional techniques. This also allows checking if something may be wrong with the substrate. The control bag is considered successful if it meets certain criteria.

[0272] -Flexural strength - To test the characteristics of the resulting material, cylindrical material samples are prepared and tested by a three-point bending test using a UTM.

[0273] -odor -Used as an indicator to determine which microorganisms are incubating on a given substrate and therefore what type of conditions prevail in that substrate (this utilizes the operator's experience)

[0274] -Growth Top layer growth is a good indicator of how well a fungus is performing in a given environment. It is quantified by the percentage area coverage of the mycelial mat on a surface, the density of that mat, and whether the mycelium is competing with other organisms (this is typically indicated by a "front line" where the mycelial "protective secretions" discolor the substrate). If the growth is substantially top layer, the thickness of the top layer is also measured.

[0275] Standard SSMB execution process All experiments described were carried out under the same conditions and using the same procedures and setup (unless otherwise stated). What was varied was the amount and manner in which the mycelium was aerated and incubated in and on the substrate.

[0276] 1. Cleaning and Preparation of the Tank a. Main exam i. Test all supply systems for their functionality according to the guidebook b. Isolation chamber and SSMB cleaning i. Wear the necessary PPE ii. Ensure that all unnecessary equipment, materials, and items are removed. iii. Shut down and disconnect the bioreactor iv. Remove and clean any directly visible dust or small substrate residues v. Use a cleaned and sterilized cloth moistened with disinfectant to wipe down all surfaces in the room, including the outside of the bath, the equipment, and high areas such as the ceiling. vi. If time permits, disinfect the positive air pressure supply (check the binder) vii. Apply disinfectant liberally to all surfaces and allow to sit for the recommended contact time viii. Activate the positive air pressure supply ix. Visually inspect the room to ensure all surfaces are clean and free of visible contaminants. c. Functionality testing i. Testing the tempering unit by heating it to a selected temperature, comparing the temperature achieved to the temperature measured using a calibrated thermometer, and cooling it to another lower selected temperature and comparing the temperature achieved to the temperature measured using a calibrated thermometer. ii. Test the ventilation device used iii. Test the sensors according to their individual test SOPs d. Settings i. Clean all sensors according to SOP ii. All sensors are attached to the tank under positive air pressure. 2. Substrate Preparation a. Homogenization i. Calculate the total for both parts (bath + control bag) ii. First add sawdust to SSMB iii. Add water iv. Premix v. Add the remaining ingredients vi. Mix until homogeneous vii. Collect a portion of the substrate for the control bag b. Control bag production i. Fill the bags and roll them up ii. Add the bag to the autoclave 3. Disinfection of the substrate a. Tank i. Turn on the tempering unit and set it to 89°C ii. Heat disinfection of wet substrate at 89°C for 12 hours b. Control bag i. Autoclave according to standards 4. Inoculation

[0277] [Table 3]

[0278] a. Parameters / Settings i. Bath: Set temperature to 27.5°C (standard procedure) ii. Control bag: Incubate according to standard procedures b.Mixture i. Agitate the substrate in the tank and in the bag at defined intervals as per the standard. c. Growth characterization - tank i. Calculate the percentage of the area of ​​the substrate covered by mycelium ii. Smelling the tank according to standard procedures iii. Note any additional relevant notes d. Growth Characterization—Control Bags i. Check that they are within range according to standard procedures 5. Forming the molding mix a. Adding nutrients i. Add 65% w / w sterilized water and 35% w / w powdered SS to the substrate so that the amount of water and SS added is 15% of the total weight of the substrate. b.Mixture i. Mix the molding additives into the SSMB and the matrix in the bag until homogeneous. c. Substrate removal i. Sanitize sealable boxes ii. Use a sealable box to collect the molding mix 6. Washing a. Tank i. Remove all remaining substrate particles 1. Fill up to the line with water 2. Activate the mixer 3. Drain ii. Clean the bath according to the standard using: 1.Water 2. Alkaline solution 3.Water 4. Acid 5.Water 6. Heat disinfection 7.Mold filling a. Visual test sample (VTS) i. Sanitize the mold according to standard procedures ii. Activate the UV light of the LFH iii. Filling the mold according to standard procedures iv. Fill N=5+ molds b.Flexural test sample (FTS) i. Sanitize the mold according to standard procedures ii. Activate the UV light of the LFH iii. Filling the mold according to standard procedures iv. Fill a minimum of N=10+ molds 8. Demolding and surface growth a. Visual sample i. Manually remove MSCs from the mold ii. Surface growing MSCs according to standard procedures iii. Incubate according to standard procedures iv. Clean the mold according to standard procedures b. Bending test specimen i. Remove the MSCs from the mold using the table fixture ii. Surface growing MSCs according to standard procedures iii. Incubate according to standard procedures iv. Clean the mold according to standard procedures 9. Drying and baking a. Placement i. Place all MSCs into a dehydrator and dry according to standard procedures. ii. Place all MSCs in a denaturing oven and operate according to standard procedures 10.Materials Testing All samples are tested according to standard procedures.

[0279] Example 11 - Cultivation of TV on SS in SSMB under POSA and FOSA Three runs using SSMB were performed, each prepared according to the standard SSMB run procedure as described above, with the difference that the substrate was not agitated. The objective was to rapidly test the feasibility of three types of aeration methods: one run for POSA using passive aeration on micropore tape, and two runs for FOSA using a blower. All three runs were performed without agitating the substrate after inoculation (static). All three runs performed worse than the bag culture control. It was concluded that the settings used for aeration were not sufficient to aerate the volume of substrate used.

[0280] 1st Run - POSA with Micropore Tape setting All ports except those required for the sensors were covered with breathable 3M micropore tape. The ends of the tape were further secured using tesa aluminum duct tape, essentially creating one large growth bag. The air exchange rate of the HEPA air supply unit in the isolation chamber surrounding the SSMB was set to its maximum setting to facilitate gas exchange within the headspace of the tank.

[0281] result

[0282] [Table 4]

[0283] Little to no mycelial growth was visible except around the grains of grain spawn which eventually reached the surface of the substrate and onto small pieces of substrate adhering to the walls of the tank, with some loose, free aerial mycelium being visible.

[0284] [Table 5]

[0285] This method of aerating the SSMB does not appear to provide sufficient air to the substrate.

[0286] Second run - FOSA with blower setting A blower supplying HEPA-filtered air was attached to one of the ports on the lid of the SSMB (the medium-sized port), and a one-way valve was screwed onto the port farthest from the air supply port (the small port) to ensure sufficient air flow and sterile conditions within the headspace. The AXR of air in the headspace was a constant rate of 1 exchange per 3 minutes.

[0287] result

[0288] [Table 6]

[0289] [Table 7]

[0290] [Table 8]

[0291] This method of aerating the SSMB does not appear to provide sufficient air to the substrate.

[0292] Third run - FOSA with higher flow rate blower setting Based on the second run, this run was performed under the same settings except for the AXR: it was increased to 1 exchange per minute.

[0293] result

[0294] [Table 9]

[0295] [Table 10]

[0296] [Table 11]

[0297] This method of aerating the SSMB does not appear to provide sufficient air to the substrate.

[0298] Consideration All three runs performed poorly. The AXR and blower settings of the third run performed best. Also, aeration could be increased by adhering to the agitation standards per the SOP for the SSMB run.

[0299] Example 12 - Cultivation of TV on SS in SSMB using FISA with SGR under static culture and FOSA under mixed culture overview Two runs were performed. The first run was performed in the same manner as FOSA, with one difference: agitation was performed according to standard procedures. The third run, using a higher blower flow rate, served as a comparison for the second run, which performed FISA. During the second run, SGRs in the form of hollow, perforated balls were added to the substrate so that the substrate filled the voids created between the individual SGRs. The run with SGRs resulted in successful colonization of the substrate volume without anaerobic fermentation, despite being incubated under static incubation. The SGR run also yielded the best FTS to date. The mixed culture run yielded better colonization than the third run, FOSA, which used a higher blower flow rate.

[0300] 1st run - no SGR setting - Third run with higher flow blower - same blower as FOSA and AXR -Stay according to the SOP for SSMB execution

[0301] result

[0302] [Table 12]

[0303] Mycelium mat growth was observed during the third run - FOSA, using a higher blower flow rate. The results were better and more uniform throughout the top layer than in the previous example.

[0304] [Table 13]

[0305] [Table 14]

[0306] This method of aerating the SSMB does not appear to provide sufficient air to the substrate, although agitation appears to aid in aeration.

[0307] Second run - with SGR setting The SGRs were previously tested for their thermal stability during the mixing process.

[0308] We also tested how much substrate entered the cavities inside the SGR. Substrate entered the cavities, but only when moved relative to the surface of the SGR. This would theoretically reduce the effectiveness of the SGR as more of the cavities became blocked or filled, thus reducing aeration. For this reason, we decided not to agitate the tanks during colonization. 1. SS was prepared according to the standard but removed before heat disinfection. 2. The tank was filled with SGR up to the fill line. 3. Substrate was added to the SGRs to fill the spaces between the SGRs. 4. Thermal disinfection according to SOP 5. Add the inoculum and stir the entire mixture once to incorporate the inoculum and distribute it evenly.

[0309] We further tested different types of SGRs with openings small enough that the substrate could not enter the cavity inside the SGR. These SGRs also performed well in improving the aeration of the bioreactor contents.

[0310] overview -No agitation during colony formation -Addition of synthetic particle size distribution adjuster - Third run with higher flow blower - same blower as FOSA and AXR -Stay according to the SOP for SSMB execution

[0311] result

[0312] [Table 15]

[0313] All areas of fungal growth were connected (not just the apex)

[0314] [Table 16]

[0315] [Table 17]

[0316] Even though the FTS from the SGR run were not molded under ideal conditions, they were the best-performing samples to date. If they had been molded properly, they could have achieved results similar to or even better than those from the standard bags. This, along with the large incubation results, indicates that FISA is a viable venting technology.

[0317] Example 13 - Cultivation of TV on SS in SSMB under compressed air FISA delivered to the substrate by the mixing spiral (PCS) of the SSMB Two runs were performed according to the SSMB run-SOP. The substrate was aerated using a FISA-aeration system attached to a mixing spiral (PCS). The system consisted of an air tank and a distribution tube. The tube introduced air into the tank.

[0318] A standard air compressor and tank were used. An electric control valve was used to control the aeration interval. The signal to the valve was controlled using a microcontroller. A filter regulator was used to adjust the air pressure and filter it. To distribute the air, a splitter was used to join five tubes to the supply. The splitter had freely rotatable connections so that the mixing spiral could rotate freely.

[0319] A one-way valve was attached to one of the ports of the bath in the lid of the SSMB to release air that had passed through the substrate and prevent pressure buildup within the bath.

[0320] The vent tube was attached to the mixing spiral using a cable clamp block.

[0321] The tubes themselves allow air into the substrate at their ends.

[0322] result

[0323] [Table 18]

[0324] During substrate recovery, it was noticeable that the substrate was dry due to FISA aeration.

[0325] [Table 19]

[0326] [Table 20]

[0327] Consideration The FISA on the spiral worked well. Because the longest tube attached to the spiral did not reach the bottom of the tank, conditions at the bottom became slightly anaerobic, and because each tube only had one opening, colonization was not as homogeneous as it could have been. The air supplied to the tank should be humidified.

[0328] Second run - perforated tubes and more of them setting The setup for this run was the same except that the tube was perforated with holes distributed along it (average every 80 mm). The hole locations and diameters were chosen so that the air pressure was fairly constant and the air supplied to the chamber was humidified to reduce moisture loss from the substrate.

[0329] result

[0330] [Table 21]

[0331] [Table 22]

[0332] [Table 23]

[0333] Consideration This was the best SSMB performance. However, it should be noted that the temperature inside the substrate rose significantly due to autothermal heating caused by mycelial activity. This can be problematic if the substrate is not sufficiently disinfected. Also, humidifying the supplied air helped prevent moisture loss in the substrate.

[0334] overview Based on this experiment, it was confirmed that an aeration mechanism for mycelium cultivation in large volume vessels that relies on the distribution of air through a mixing element, which is a rotatable spiral paddle containing at least one outlet opening for adding fluid into the reactor body, works well.

Claims

1. 1. A method for preparing a mycelial colonization substrate, the method comprising the step of incubating a mycelial inoculation substrate, the mycelial inoculation substrate comprising a synthetic particle size distribution modifier, to allow mycelium to grow.

2. 10. The method of claim 1, wherein the composite particle size distribution adjuster comprises a plurality of hollow three-dimensional objects, each of the three-dimensional objects comprising one or more openings on a surface thereof configured to allow the flow of gas through their volume.

3. 3. The method of claim 2, wherein the synthetic particle size distribution modifier is configured to impede the presence of the mycelium inoculum substrate in its volume.

4. The method according to any one of claims 1 to 3, wherein the synthetic particle size distribution adjuster is selected from a plastic mesh and a stainless steel mesh.

5. The method according to any one of claims 1 to 4, wherein the composite particle size distribution adjuster is in the form of a plurality of balls, preferably each ball comprising one or more cavities on its surface.

6. The method according to any one of claims 1 to 3 or 5, wherein the synthetic particle size distribution adjuster is digestible by the mycelium.

7. 7. The method according to any one of claims 1 to 6, wherein aeration of the mycelium during growth occurs over substantially its entire volume.

8. The method according to any one of claims 1 to 7, wherein the substrate in the mycelium-inoculated substrate comprises at least one structural component and / or at least one filler component.

9. 9. The method of claim 8, wherein the at least one structural component is selected from shredded hemp stalks, shredded corn stalks, shredded tomato stalks, shredded tobacco stalks, shredded bean stalks, shredded corn cobs, softwood flakes, peanut shells, and straw.

10. 10. The method of claim 9, wherein the at least one filler component is selected from sawdust, brewer's mash, and paper pulp.

11. The substrate is an adjuvant, 11. The method of claim 9 or 10, further comprising an adjuvant, preferably selected from calcium sulfate, calcium hydroxide, nitrogen additives, terpenes, lipids, simple hydrocarbons, and compost.

12. 12. The method of any one of claims 1 to 11, further comprising the step of preparing a mycelial inoculation substrate, which step is carried out prior to the step of incubating the mycelial inoculation substrate to grow the mycelium.

13. 13. The method of claim 12, wherein the mycelial inoculation substrate is prepared by mixing mycelia contained in the form of discrete particles with the substrate.

14. 14. The method of claim 13, wherein the mycelium is contained in grain spawn or sawdust spawn and / or the mycelium inoculation substrate is prepared by mixing a liquid containing mycelium and / or spores with the substrate and / or slurry spawn, and / or a mycelium colonization substrate from a previous batch, preferably wherein the mycelium is contained in grain spawn or sawdust spawn and / or the mycelium inoculation substrate is prepared by mixing a liquid containing mycelium and / or spores with the substrate.

15. 15. The method of any one of claims 12 to 14, further comprising a step of autothermal pretreatment of the mycelium-inoculated substrate, performed prior to the step of incubating the substrate to grow the mycelium.

16. 16. The method according to any one of claims 12 to 15, further comprising a step of enzymatic treatment of the substrate and / or a step of chemical treatment of the substrate, carried out prior to the step of incubating a mycelium-inoculated substrate to grow the mycelium.

17. 1. A solid-state mycelium bioreactor comprising: The solid-state mycelium bioreactor comprises a reaction body having a cavity; at least one mixing element disposed within the cavity of the reactor body and rotatable relative to the reactor body about an axis of rotation, 17. The method according to any one of claims 1 to 16, wherein a solid-state mycelium bioreactor is used, comprising: at least one mixing element comprising at least one outlet opening for adding a fluid into the reactor body, the at least one mixing element being a rotatable spiral paddle, and the at least one outlet opening being fluidly connected to one or more fluid supplies via at least one fluid connection.

18. the solid-state bioreactor is used for incubating a mycelial inoculation substrate to grow the mycelium; and / or the solid-state bioreactor is used in the step of preparing the mycelial inoculum substrate; and / or the solid-state bioreactor is used for the step of autothermal pretreatment of the substrate, and / or 18. The method of claim 17, wherein the solid-state bioreactor is used for the step of chemical and / or enzymatic treatment of the substrate.

19. 19. The method according to any one of claims 1 to 18, further comprising the step of preparing a molding mix, which is carried out after the step of incubating a mycelium-inoculated substrate to allow the mycelium to grow.

20. 20. The method of claim 19, wherein said step of preparing a molding mix involves the use of a powdered substrate-water slurry or a high-exothermic additive-water slurry.

21. 21. The method according to claim 19 or 20, further comprising the step of shaping the obtained mycelium colonization substrate or shaping mix, preferably mycelium colonization substrate.

22. The method of any one of claims 19 to 21, further comprising a step of in-mold incubation.

23. 23. The method of any one of claims 19 to 22, further comprising a step of surface growth.

24. 24. The method according to any one of claims 19 to 23, further comprising a dehydration and / or denaturation step, preferably a denaturation step.

25. An intermediate product in the preparation of a mycelial colonization substrate, comprising a mycelial colonization substrate and a synthetic particle size distribution modifier.

26. 26. The intermediate product of claim 25, wherein the composite particle size distribution adjuster comprises a plurality of hollow three-dimensional objects, each of the three-dimensional objects comprising one or more openings on a surface thereof configured to allow the flow of gas through their volume.

27. 27. The intermediate product of claim 26, wherein the synthetic particle size distribution modifier is configured to impede the presence of the mycelium inoculum substrate in its volume.

28. 28. The intermediate product according to any one of claims 25 to 27, wherein the synthetic particle size distribution adjuster is selected from a plastic mesh and a stainless steel mesh.

29. 29. The intermediate product according to any one of claims 25 to 28, wherein the synthetic particle size distribution adjuster is in the form of a plurality of balls, preferably each ball comprising one or more cavities on its surface.

30. 30. The intermediate product according to any one of claims 25 to 27 or 29, wherein the synthetic particle size distribution adjuster is digestible by the mycelium.

31. 1. A solid-state mycelium bioreactor comprising: a reaction body having a cavity; at least one mixing element disposed within the cavity of the reactor body and rotatable relative to the reactor body about an axis of rotation, the at least one mixing element includes at least one outlet opening for adding a fluid into the reactor body; the at least one mixing element is a rotatable spiral paddle; and at least one mixing element, wherein said at least one outlet opening is fluidly connected to one or more fluid supplies via at least one fluid connection.

32. 32. The solid state mycelium bioreactor of claim 31 , wherein said at least one mixing element comprises a cooling element and / or a heat exchange mechanism, preferably a cooling element.

33. 33. The solid state mycelium bioreactor of claim 31 or 32, wherein the fluid connection extends through a channel that extends through the at least one mixing element.

34. 34. The solid state mycelium bioreactor of any one of claims 31 to 33, wherein the at least one outlet opening comprises a nozzle or a sparger.

35. 35. The solid state mycelium bioreactor of any one of claims 31 to 34, wherein the axis of rotation is vertical.

36. 36. A solid state mycelium bioreactor according to any one of claims 31 to 35, wherein the fluid added through the at least one outlet opening is a gas, preferably air, for aeration of the mycelium colonization substrate.

37. 37. The solid state mycelium bioreactor of any one of claims 31 to 36, wherein the reactor body comprises perforated walls configured to allow aeration.

38. 38. The solid state mycelium bioreactor of any one of claims 31 to 37, wherein the reactor body further comprises at least one outlet opening for adding said fluid into the reactor body, said outlet opening being fluidly connected to one or more fluid supplies via at least one fluid connection, preferably said outlet opening comprising a sparger.

39. 39. The solid state mycelium bioreactor of any one of claims 31 to 38, wherein the wall of the reactor body further comprises at least one baffle.

40. 40. The solid state mycelium bioreactor of any one of claims 31 to 39, wherein the solid state mycelium bioreactor further comprises at least one access point within the reactor body that can be opened during operation of the reactor.

41. 41. The solid state mycelium bioreactor of any one of claims 31 to 40, further comprising an imaging device disposed on the body of the reactor and configured to provide a view of the interior of the cavity of the reactor.

42. the imaging device is a camera; and / or 42. The solid state mycelium bioreactor of claim 41, wherein the imaging device is configured to detect the mycelium growing on the substrate, preferably without human intervention.

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