Three-dimensional biodegradable shaped materials from mycelium-colonized substrate pulp and methods for production thereof

Abstract

The present invention relates to a method for obtaining a three-dimensional molded fungal pulp composite, the method comprising the steps of: b) providing a mycelium-colonized substrate pulp slurry; c) molding said slurry into a particular 3-dimensional shape to obtain a molded mycelium-colonized substrate pulp slurry; d) compressing said pulp slurry in order to remove water from the slurry, thereby obtaining the molded mycelium-colonized substrate pulp composite; and e) demolding the so obtained product. The present invention further relates to a blended mycelium substrate slurry, obtainable in step b) of the method of the present invention. The present invention further relates to a molded mycelium-colonized substrate pulp composite obtainable according to the method of the present invention.

Description

[0001] Three-dimensional biodegradable shaped materials from mycelium-colonized substrate pulp and methods for production thereof

[0002] Field of the invention

[0003] The present invention relates to a method for obtaining a three-dimensional mycelium-colonized substrate pulp composite, the method comprising the steps of: b) providing a mycelium-colonized substrate pulp slurry; c) molding said slurry into a particular 3-dimensional shape to obtain a molded mycelium-colonized substrate pulp slurry; d) compressing said pulp slurry in order to remove water from the slurry, thereby obtaining the molded mycelium-colonized substrate pulp composite; and e) demolding the so obtained product. The present invention further relates to a blended mycelium-colonized substrate slurry, obtainable in step b) of the method of the present invention. The present invention further relates to a molded mycelium-colonized substrate pulp composite obtainable according to the method of the present invention.

[0004] Background of the invention

[0005] The pervasive issue of plastic pollution infiltrating the environment is widely recognized, yet there remains a scarcity of widely applicable biodegradable alternatives that are truly scalable. One promising solution lies in mycelium-based materials, which have been proposed as viable substitutes of plastic. One such mycelium-based material, the mycelium-substrate composite, often referred to as mycelium foams or mycelium composites, show much potential to replace plastic across various applications, particularly in single-use custom shaped expanded polystyrene packaging-replacements. These mycelium-substrate composites benefit from the rapid growth capability of mycelium-forming fungi. Presently, the common method of producing these mycelium-substrate composites involves utilizing the interconnected network of mycelial cells as a living binder, linking discrete substrate particles into a composite. However, this approach has limitations, as the highest achievable level of detail is constrained by the size of substrate’s particles. Typically, the longest axis of said particles must often extend 10 mm, and this size-constraint arises from the requirement for creating void spaces within the substrate to ensure proper aeration. Moreover, each shaped composite necessitates its individual incubation in its respective mold container to achieve its form. This method of production is associated with increased costs for mold-procurement and cleaning. Consequently, there is a demand for alternatives that facilitate continuous production of composite materials regardless of the quantity of mold containers available.

[0006] I n the current economic environment, there are vast amounts of lignocellulosic side streams that are either burned or used in biogas plant for biogas production, which, considering their exact content, may be considered as a waste of these resources. Many mycelium-materials producers are adopting these resources for use as substrates. Nevertheless, any use thereof is again limited by their particle size (longest axis < 10 mm, as noted above). Therefore, clearly a process capable of using these resources to produce mycelium-based materials is needed. Furthermore, many culinary mushroom producers are faced with the problem of disposing of their spent substrate / fruiting blocks after the harvesting of fruiting bodies. This is yet another mostly unused resource of a mostly lignocellulosic side stream. Also, fungal ascorbic acid producers have vast amounts of fungal mycelium waste material.

[0007] In the patent EP3891266A1 by Mogu, a process employing blended fungal pulp is outlined for the manufacture of fungal mats suited for soft goods like textiles or leather substitutes. The resulting material is almost entirely made from mycelium cells with minimal substrate content. Furthermore, this document does not teach in any way achieving three-dimensional shapes. Instead, it focusses only on planar materials and their preparation. Moreover, the fungal mats as disclosed by Mogu are being generated in a process that requires individual cultivation containers, wherein the fungal pulp is grown therein.

[0008] Molded pulp or molded fiber packaging is a type of packaging typically made from recycled cardboard and / or paper made into a pulp and shaped to create custom-shaped packaging or shaped into standardized trays and containers. Typical use-cases for this type of packaging are egg-cartons or household electronics packaging. The shaping of the packaging is typically achieved by thermoforming or transfer molding. In the process of transfer molding a shaped wire mesh is used to shape the pulp, wherein typically a vacuum is used to draw the free water from the pulp to bond the individual fibers together into a thin walled (typically 2.5 mm thick) packaging material. The process for thermoforming the pulp involves heated forming molds that densify the product. The processes for molded pulp are easy to scale and automate, do not require much space and can achieve a high throughput. Even though molded pulp packaging may be considered as an environmentally friendly packaging solution, it also poses environmental challenges, including deforestation, as 93% of raw materials used in the production of paper are sourced from trees. Additionally, the pulp and paper industry emits harmful gases, contributing to atmospheric pollution and exacerbating environmental problems. For instance, in the U.S., pulp and paper operations accounted for 20% of total industrial releases of toxic waste into the air in 2015 (Tiseo L. Global paper industry-statistics and facts. Published 2021. Accessed February 25, 2024. Available at: https: / / www.statista.com / topics / 1701 / paper-industry / ). Manufacturing paper may release nitrogen dioxide, sulfur dioxide, and carbon dioxide, leading to pollution-related issues and greenhouse gas emissions, which contribute to climate change and global warming. Moreover, significant volumes of wastewater discharged from various stages of the paper production process may pose risks to aquatic ecosystems, potentially causing elevated levels of biochemical oxygen demand (BOD) and chemical oxygen demand (COD) and endangering aquatic life (Xiaoyu W. Study on environmental impact and countermeasure of wastewater discharge from paper mill. China Acad J Electron Publ House. 2016; 200331 :255). Clearly, there is a need for a more environment friendly solution for producing pulp-based packaging.

[0009] Pure fungal pulp is created by separating the individual hyphae of a mycelium or fruiting body into discrete hyphal fibers. A recent study by Nakauchi et al. (2023) from the Shinshu University presents a novel method to extract mycelial fibers from mushroom fruiting bodies while preserving their structure, resulting in a micrometer-sized hyphal pulp with good deformability. It is hypothesized by the authors that it may be putatively applicable for a wide range of material applications across one to three dimensions. However, the presented mycelium pulp product has yet been only tested on micrometer-scale. Also, this fungal pulp requires the cultivation of mushroom fruiting bodies, which is a time- and space-consuming process, which demands precise control over the incubation conditions during the colonization of the substrate and after that during the step to induce fruiting of the mushrooms, making economic high- throughput industrialized single-use applications unlikely.

[0010] Finally, there is a need for a scalable, biodegradable and sustainable material that can leverage the vast growth-potential of mycelium, can utilize substrates with a small particle size and the production of which does not require segmented incubation in individual molds for each product, thereby significantly reducing time, space, and costs associated with its production. Also, it is felt that the production of said material can benefit from the scalable methods adjacent to those employed in the production of molded pulp packaging materials, while mitigating the aforementioned hazardous and often unavoidable by-products of modern pulp and paper production processes. Furthermore, there is a need for the conversion / up- cycling of spent fruiting blocks from culinary mushroom production, to enable another possible step for value-creation from said side-stream. Furthermore, document WO 2024 / 062136 discloses certain means and methods for the preparation of a mycelium-colonized substrate. Said methods rely on the use of an intermediate product, comprising mycelium colonized substrate and a synthetic granulometry regulator, in the preparation of said mycelium colonized substrate. Furthermore, said methods focus on the production of standard mycelium composites which depend on the interconnectedness of the mycelium network to bind the discrete particles into the final composite and are individually cultivated within a mold-cavity in order for the mycelium network to connect the particles in the shape of the mold. Moreover, this document does not focus on the described product per se, but describes in detail methods for obtaining a mycelium-substrate composite, in particular focusing on methods which rely on the use of synthetic granulometry regulators, which act by increasing the void spaces in the substrate, thus allowing for more gas exchange (i.e., better aeration of the substrate) and thus allowing higher colonization speeds. In contrast, present invention does not rely in any way on the use of such synthetic granulometry regulators.

[0011] Summary of the invention

[0012] It was an object of the invention to provide a method to produce 3-dimensionally shaped mycelium-based materials, that do not require the step of incubation inside individual molds to create the desired geometry. It is further an object of the present invention to provide a method such that said method does not require the addition of large lignocellulose substrate particles to create void space in the substrate for the duration of the incubation of the mycelium-forming fungi.

[0013] It was further an object of the invention to provide a method for producing a new type of mycelium-based material, the 3-dimensionally shaped molded mycelium-colonized substrate pulp composite (abbreviated as “MMcSPC” or “molded m-c-s pulp composite”), which may also be referred to as fungal molded pulp composite.

[0014] One of the main benefits of the mycelium, having grown on the substrate is the softening, fibrillation and swelling of the substrate by the digestion-processes by the mycelium, next to the increased fiber-count (hyphal fibers and separation of the substrate into multiple strands) and the increased amount of proteins and biological components that act as an “glue”.

[0015] Furthermore, it is an object of the invention to provide a simple process that leverages the vast-growth capability of mycelium to enable value-creation and to enable the economic conversion / up-cycling of spent fruiting blocks from culinary mushroom production. According to the present inventors the fungal molded pulp composite of the invention has the following advantages:

[0016] - Coloration of the Material without infringing on the biodegradability is easily achieved;

[0017] - Substrate particle sizes may be smaller during the step of colonization of the substrate. As there is no need for larger and more void cells inside the substrate for in-mold-incubation;

[0018] - No In-mold-incubation is needed, therefore the time and space needed (and therefore cost) to produce a product is greatly reduced;

[0019] - More complex product geometries are possible because the product shape is not limited by the possible mold-geometries for in-mold incubation;

[0020] - A shorter cycle-time for molding / shaping of the product is possible to achieve;

[0021] - The ability to create easily storage-stable pulp allows for the separation of the molding / forming step from the incubation, therefore allowing for easy distribution of storage-stable pulp to other production sites, where only a molding / forming machine is needed.

[0022] The projected low price and high and fast potential production-throughput of the molded mycelium- colonized substrate pulp composite allows for the entering of new markets. The following Table is to be seen as non-limiting list of potential applications for pulp-based material applications:

[0023] Packaging Design Construction Other

[0024] - Custom-made - Tabletops - Wall-elements - Molds for standard MSC-

[0025] Packaging - Electrical- - MDF-replacement production that become

[0026] - Corner protectors appliances housings - Formwork panels part of the final product.

[0027] - Inlays - Cup-warmers - Thermal insulation - PCBs

[0028] - Cut-to-size sheets - Laptop- stands - Etc. - Arts and Crafts supply

[0029] - Etc. - Toys material

[0030] - Etc. - Etc.

[0031] The invention will be summarized in the following embodiments.

[0032] In a first embodiment, the present invention relates to a method for obtaining a three-dimensional mycelium-colonized substrate pulp composite, the method comprising the steps of: b) providing a mycelium-colonized substrate pulp slurry; c) molding said slurry into a particular 3-dimensional shape to obtain a molded mycelium- colonized substrate pulp slurry; d) compressing said pulp slurry in order to remove water from the slurry, thereby obtaining the molded mycelium-colonized substrate pulp composite; and e) demolding the so obtained product.

[0033] In a second embodiment, the present invention relates to a blended mycelium-colonized substrate pulp, as provided in step b) of the method for obtaining a three-dimensional mycelium-colonized substrate pulp composite of the present invention.

[0034] In a third embodiment, the present invention relates to a molded mycelium-colonized substrate pulp composite obtainable according to the method for obtaining a three-dimensional mycelium-colonized substrate pulp composite of the present invention.

[0035] Definitions

[0036] Unless explicitly indicated to the contrary, the following definitions apply throughout the description.

[0037] As understood herein, a “fiber” is a particle with a length-to-width-ratio of its cross-section through its longest and shortest axis, respectively greater than 4:1 (the plane of said cross-section is understood herein as being coincident with the fiber-length and fiber-width).

[0038] As understood herein, a “substrate” is a mixture of nutritive and non-nutritive components in discrete particle and / or fiber-form capable of supporting the growth of the chosen mycelium-forming fungal strain*s (possibly cocultured with other microorganisms), of which the majority of the components are lignocellulosic.

[0039] As understood herein, a “substrate composition” is a specific selection of and ratios between the dry components and the ratio between the dry substrate-components and water in the substrate. Preferably, said ratio is provided as w / w ratio.

[0040] As understood herein, a mycelium-substrate composite is a composite material comprising (mostly lignocellulosic) substrate material and a mycelium, wherein the substrate, preferably the particles or portions of substrate, are bound by the mycelium network, and preferably wherein said mycelium network has been processed so that it is no longer alive and no longer capable of further growth. Such processing may occur for example by dehydration and / or by denaturation. As understood herein, a “grain” is an individual particle or fiber of a substrate. Accordingly, “grain size” is understood as the length of the grain measured along its longest axis. It is to be understood that the terms “particle” and “grain” can herein be used interchangeably.

[0041] As understood herein, a “projected fiber length” is the length of a given fiber, with all subtractions from its fiber-length due to fiber deformation, such as: folds; compressions; micro-compressions; knees.

[0042] As understood herein, a „fiber-length” is the length of the fiber “stretched out”, therefore the projected- length without all the fiber-deformation-subtractions.

[0043] As understood herein, a “particle” refers to a particle with a length-to-width-ratio of its cross-section through its longest axis smaller than 4:1 .

[0044] As understood herein, a “pulp” is a composition comprising (preferably a mixture of) substrate and mycelium particles and fibers, (wherein the mycelium preferably has been previously grown on / onto said substrate) with or without added liquid, in particular free water or aqueous solution. The term pulp may be used interchangeably with the term “slurry”, which is understandable to the skilled person, and the term pulp slurry. Preferably, said pulp or said slurry comprises at least 30% w / w of water (or aqueous solution), more preferably at least 40% w / w of water (or aqueous solution), even more preferably, at least 50% w / w of water.

[0045] It is also understood that, the water or aqueous solution may also be substituted by using any other suitable liquid, such as oils.

[0046] As understood herein, a “pulp formation” is the step of reducing the particle-size and or fiber length by means of blending, crushing, grinding and the like, preferably increasing the fiber count of the resulting pulp in the process. As understood herein, the fiber count preferably refers to the amount / number of fibers in a substrate, i.e. of particle that has a length-to-width-ratio of more than 4:1 . The increase in fiber count typically happens during most of the pulp-formation processes, as the lignocellulose substrate separates where the (mostly in a parallel-fashion oriented) plant cellulose fibers come into contact.

[0047] As understood herein, the term “storage-stable” preferably refers to being capable of being stored under the respective storage conditions for a time period greater than 4 (four) months. As understood herein, the term “molding” and “shaping” are used herein interchangeably.

[0048] As understood herein, the term “strain-substrate fit” is the efficacy / ability of a given fungal strain to grow on a given substrate.

[0049] As understood herein, “exploration rate”, also referred to as “colonization rate” is a rate to which a given fungal strain has grown onto a given substrate.

[0050] Further definitions applicable in the present invention are also introduced in the detailed description of the invention.

[0051] Brief description of figures

[0052] The invention is further illustrated by the following Figures. These serve merely illustrative purposes and in no way are to be construed as limiting the scope of protection, which is defined by the hereto appended claims.

[0053] Fig. 1 shows an example of mycelium-colonized substrate pulp with a 5x5 mm grid-pattern overlayed to give a frame of reference for the size of the particles / fibers of the pulp.

[0054] Fig. 2 shows an example of mycelium-colonized substrate pulp viewed under a 40x magnification microscope, wherein the fibers / particles of the substrate and hyphal strands are visible.

[0055] Fig. 3 shows a flowchart of the main process steps to create a composite of the invention.

[0056] Fig. 4 shows the Process-Flowchart. The preferred method is drawn in solid arrows; alternative methods are drawn in dashed arrows. Process-steps are shown in gray boxes; intermediates in black boxes; educts and products in black boxes with a white frame. The steps of the process are explained in the following.

[0057] Fig. 5 shows schematic example of a pulp-formation-process, wherein a high-speed blender and the addition of water, aqueous solution or a liquid are used to reduce the particle size of the pieces of mycelium-colonized substrate. (1.) liquid / water / aqueous solution; (2.) mycelium-colonized substrate; (3.) Blending container; (4.) Rotating blending blade; (5.) mycelium-colonized substrate pulp slurry.

[0058] Fig. 6 shows a schematic example of a press-molding setup and the steps performed with such a setup for compressing and shaping the pulp. (A.) Press-mold walls (B.) Mycelium-colonized substrate pulp, (C.) Press-mold baseplate, (D.) Press-mold top-part, (E.) Molded mycelium-colonized substrate pulp composite.

[0059] Fig 7 shows a schematic example of an injection-shaping setup for compressing and shaping the pulp (A.) Injector; (B.) Mycelium-colonized substrate pulp; (C.) Mold-chamber-part A; (D.) Moldchamber-part B.

[0060] Fig. 8 shows a schematic example of a centrifugation-shaping setup for compressing and shaping the pulp : (A.) Injectors / Fluid connection; (B.) Central pulp supply; (C.) mold-chamber-parts A (D.) Injection-mold-chamber-parts B; (E.) Mycelium-colonized substrate pulp. Fig. 9 shows schematic example of the shaped-mesh-based shaping setup and the steps performed with such a setup for shaping the pulp. (A.) Shaped mesh, (B.) Deposited layer of pulp; STEP I Mesh before dipping, STEP II Mesh with deposited layer after dipping, STEP III Separation of mesh and shaped pulp.

[0061] Fig. 10 shows a schematic example of a shaped-mesh-based shaping setup. (A.) Vat / Bath / Container; (B.) Mycelium-colonized substrate pulp; (C.) Shaped mesh.

[0062] Fig. 11 shows a picture of a molded mycelium-colonized substrate pulp composite, molded using the press-molding method.

[0063] Fig. 12 shows a picture of an example for mycelium-colonized substrate pulp, wherein the step of pulpformation has been performed using an high-speed blender and the ratio of water / aqueous solution-to-mycelium-colonized substrate is 4:1 w / w.

[0064] Fig. 13 shows picture of an example for storage-stable granulated mycelium-colonized substrate pulp, wherein the step of pulp-formation has been performed using an high-speed blender and the ratio of water / aqueous solution-to-mycelium-colonized substrate is 4:1 w / w and wherein the granulation has been performed using a tumbler-dryer.

[0065] Fig. 14 shows classification of different components of the substrates to be used in the methods of the present invention.

[0066] Fig. 15 shows the process according to Example 1 .

[0067] Fig. 16 shows a close-up explanation of the pulp formation process according to the invention, wherein three steps that preferably occur are indicated, i.e., (i) separation of the mycelium-colonized substrate into the individual overgrown particles, (ii) separation of the overgrown substrate particles into large fibers, and (iii) separation of the larger fibers into smaller fibers, i.e., cellulose and hyphal fibers. Accordingly, as described herein, the pulp formation involves reducing the particle-size and or fiber length by means of, e.g., blending, crushing, grinding and the like, preferably blending, which preferably leads to increasing the fiber count of the resulting pulp in the process. Detailed description of the invention

[0068] As mentioned before, in one embodiment, the present invention relates to a method for obtaining a three- dimensional molded mycelium-colonized substrate pulp composite, the method comprising the steps of: b) providing a mycelium-colonized substrate pulp slurry; c) molding said slurry into a particular three-dimensional shape to obtain a molded mycelium- colonized substrate pulp slurry; d) compressing said pulp slurry to remove water from the slurry, thereby obtaining a molded mycelium-colonized substrate pulp composite; and e) demolding the so obtained product.

[0069] As it is immediately apparent to the skilled person, while the present invention relates to a method for obtaining a three-dimensional molded mycelium-colonized substrate pulp composite, said composite is demolded in step e) of the method. Thus, insofar the method involves the step of demolding, said method for obtaining a three-dimensional molded mycelium-colonized substrate pulp composite may also be referred to as a method for obtaining a three-dimensional mycelium-colonized substrate pulp composite.

[0070] Accordingly, the method for obtaining a three-dimensional molded mycelium-colonized substrate pulp composite (or preferably a three-dimensional mycelium-colonized substrate pulp composite, as understood herein) comprises step b) of providing a mycelium-colonized substrate pulp slurry. Preferably, the mycelium-colonized substrate slurry is a blended mycelium-colonized substrate slurry. Thus, in other words, step b) may be seen as the step of forming a pulp from mycelium-colonized substrate.

[0071] As understood herein, the term “three-dimensional” requires that the geometric expansion of an object is substantially expressed in each spatial dimension. Accordingly, when reference is made to three dimensional object or material, planar or substantially planar; or longitudinal or substantially longitudinal, objects or materials are therewith excluded. Preferably, the term “three-dimensional” requires that each dimension of the object is at least 1 cm, preferably at least 2 cm, more preferably at least 3 cm, even more preferably at least 4 cm, still more preferably at least 5 cm.

[0072] As understood herein, a mycelium-colonized substrate pulp slurry is a composition comprising particles of substrate attached to mycelium, wherein the mycelium is present bound to the particles / fibers or portions of substrate and physically connected thereto, Preferably, at least 50% of particles of substrate are connected to mycelium (hyphal particles). Further preferably, not more than 5% of particles of substrate are connected with each other by the mycelium. Accordingly, preferably not all particles of substrate (even more preferably, none of the particles of substrate) are connected to each other and all previously established connections / bounds by the mycelium-network of the mycelium-colonized substrate are torn / disconnected during the step of pulp-formation. Alternatively, or in other words, the mycelium-colonized substrate pulp slurry is understood to be obtainable in the process of blending / micronizing the mycelium-colonized substrate, as referred to herein. Thereby, the majority of connections between substrate particles through mycelium do not prevail the process of blending / micronizing.

[0073] The mycelium-colonized pulp slurry further comprises at least 20% w / w water, preferably at least 40% w / w water, more preferably at least 50% w / w water. In the mycelium-colonized substrate pulp slurry, the mycelium may capable of further growth. However, it is not necessary and also by the definition encompassed are embodiments wherein the mycelium comprised in said mycelium-colonized substrate pulp slurry is no longer capable of further growth. Thus, the material described as mycelium-substrate composite pulp slurry is not only obtainable starting from the mycelium-colonized substrate, but it also can be obtained by blending / micronization of mycelium-substrate composite, wherein preferably the mycelium is no longer alive. Alternatively, said pulp / slurry can also be obtained from waste / spent fruiting blocks from culinary mushroom producers.

[0074] In one embodiment, the method for obtaining a three-dimensional molded mycelium-substrate composite pulp composite comprises step b’) of pulp formation. Pulp formation is as described herein. Preferably, the method for obtaining a three-dimensional molded mycelium-substrate composite pulp composite comprises step b’) of blending mycelium-colonized substrate, optionally in the presence of a liquid, preferably a polar liquid such as water or an aqueous solution (which will be collectively referred to as aqueous solution) to obtain a blended mycelium-colonized substrate slurry. As referred to herein, the terms pulp and slurry (or pulp slurry, as applicable) may be used interchangeably. The presence of liquid (such as water or aqueous solution) is optional, and accordingly the blending can be performed with said liquid present. Alternatively, said blending can be performed in the absence thereof. It is to be understood that the method predicates on availability of mycelium-colonized substrate, to be used in the method of the present invention.

[0075] Accordingly, step b’), which is one preferred embodiment of step b), as described herein (i.e., is comprised in one preferred embodiment of step b), may be the step of blending mycelium-colonized substrate, in the presence of a liquid, preferably a polar liquid such as water or an aqueous solution (which will be collectively referred to as aqueous solution) to obtain a blended mycelium-colonized substrate slurry. Alternatively, step b’), as described herein, may be the step of blending mycelium-colonized substrate, without the addition of any further liquid.

[0076] Accordingly, this initial step b) in the formation of the pulp is the separation of the individual substrate- particles / grains from each other of the mycelium-colonized substrate, thereby breaking the bond created by the mycelium-network. Ideally the process of pulp-formation increases the surface-roughness, fibrillation, fiber-count and swelling of the individual pulp-particles / fibers for thereafter. This step can be accomplished for example by using a blender, grinder, mill, hammer mill, geared chipper, shredder, ultrasonic bath, steam explosion or similar or any combinations thereof.

[0077] As apparent to the skilled person, mycelium-colonized substrate pulp slurry, as referred to herein, is preferably a slurry comprising discrete fibers and particles. It is to be understood that in said slurry preferably majority of the cells are dead, in particular if the slurry is obtainable in the process of blending the mycelium colonized substrate. Accordingly and preferably, in the mycelium-colonized substrate pulp slurry up to 20% of the initial mycelium cells by weight are not dead (initial referring to the total number of cells before the step of blending, which is understood to be based on the determination of the number of colony forming units). More preferably, in the mycelium-colonized substrate pulp slurry, up to 10% of mycelium cells by weight are not dead. It is preferably to be understood herein that by a not dead cell (or non-dead cell), a cell capable of growth is meant. As further understood herein, the percentage of cells that are not dead can be estimated by estimating the amount of colony-forming units (CFUs) before and after, for example, blending, that is used for obtaining the mycelium-colonized substrate pulp slurry.

[0078] Thus, the mycelium-colonized substrate pulp slurry, as referred to herein, is structurally and compositionally different from the compositions disclosed in WO 2024 / 062136, wherein myceliumsubstrate composite may be homogenized. As apparent to the skilled person, the process of homogenizing is different from the pulp formation process (for example, blending process), as described herein, as the pulp formation process, for example done by blending, as described herein, focuses on the separation of particles into individual fibers (similar to paper pulp) i.e. splitting / separating of particles (e.g. substrate particles) into fibers such as (mainly) cellulose and hyphae and leads to the destruction of many fungal cells, which is manifested through cell death and overall reduction in the number of cells that are non-dead. The pulp-formation process, as described herein, seperates the mycelium-colonized substrate first into the individual substrate particles and then into fibers as can be seen in Fig. 16. As is apparent to the skilled person this process is similar to the process of paper pulp making. The mycelium aids in delignifiying the lignocellulose raw material (i.e. substrate), similar to the chemical process used for delignification in the paper industry (e.g. using peracetic acid). The now more delignified myceliun- colonized substrate is the blended for example using a blender until the mycelium-colonized substrate pulp is obtained.

[0079] As disclosed in WO 2024 / 062136, upon breaking of the mycelium-substrate composite in the homogenization process, the mycelium, present in the mycelium-colonized substrate, is intended to regrow in a more resilient way, resulting in a stronger material and a denser mycelium network. Furthermore the homogenization-step as disclosed in WO 2024 / 062136 focuses on separating the mycelium-colonized substrate into discrete particles while keeping the mycelium, which has grown onto the particles, intact and alive. This constitutes, tearing apart the connection through the mycelium between discrete substrate particles such that a similar separation to the starting substrate is achieved. In other words, the effect of said homogenization is boosting colonization of the substrate by the mycelium and strengthening the bonds between particles of the substrate by the mycelium.

[0080] Accordingly and preferably, the mycelium-colonized substrate pulp slurry is characterized by the separation of particles into individual fibers such as (mainly) cellulose and hyphae. The so obtained product is structurally similar to paper pulp obtainable in the process of pulping. As preferably encompassed by the present invention, the process of the formation of the mycelium-colonized substrate pulp slurry, according to the present invention, to be performed for example by blending, as described herein, involves (i) separation of the mycelium-colonized substrate into the individual overgrown particles, (ii) separation of the overgrown substrate particles into large fibers, and (iii) separation of the larger fibers into smaller fibers, i.e., cellulose and hyphal fibers (see Figure 16). In other words, as described herein, the pulp formation preferably involves reducing the particle-size and or fiber length by means of, e.g., blending, crushing, grinding and the like, preferably blending, which preferably leads to increasing the fiber count of the resulting pulp in the process.

[0081] As understood herein, step b’) may involve, instead of blending, other processes such as crushing or grinding. As understood herein, a mycelium-colonized substrate relates preferably to a composition comprising mycelium and substrate, wherein the mycelium is present bound to the particles / fibers or portions of substrate and physically connected thereto, wherein the mycelium is capable of further growth and wherein the particles / fibers or portions of substrate are connected by the mycelium which has grown thereon. Preferably at least 20% of particles or portions of substrate are connected with each other by the mycelium in a mycelium colonized substrate. Even more preferably, at least 30% of particles or portions of substrate are connected with each other by the mycelium in a mycelium colonized substrate. Preferably, the % value refer to at least 10% or 20%, respectively, of weight of the substrate to be involved in being bound through the mycelium.

[0082] As apparent to the skilled person, the mycelium-colonized substrate can be produced, for example, according to the production flow presented hereinbelow: a) Engineering of the substrate to fit the chosen fungal strain*s and material-properties-target b) Mixing of the dry components to obtain a homogeneous dry substrate mixture c) Hydrating the dry substrate mixture to obtain a hydrated substrate mixture d) Reducing the inherent bioburden of the substrate to preferably obtain an low-bioburden intermediate substrate e) Cooling and / or chemically neutralizing the low-bioburden intermediate substrate to obtain a prepared substrate f) Inoculation of the substrate with the chosen fungal strain*s utilizing an optimized spawn-rate to obtain a inoculated substrate g) Incubation of the chosen fungal strain*s on the prepared substrate to obtain colonized substrate h) Optionally performing measures to obtain a storage-stable colonized substrate.

[0083] As understood herein, the process of blending is apparent to the skilled person and involves mixing and micronization of a solid composition or a solid, optionally in presence of a liquid. The product of such blending may be referred to as slurry. Accordingly, the slurry, as preferably referred to herein, is a composition comprising liquid and particles and / or fibers of a solid, wherein the average particle diameter of said particle (preferably understood as weight-average particle diameter) is at least 1 micrometer. The liquid may be added during blending. However, in certain embodiments, the mycelium-colonized substrate may include sufficient water content so that addition of water or aqueous solution to the pulp is not necessary. Preferably, blending in step b’) is performed by using a hammer mill, geared chippers, shredders, steam explosion, ultrasonic bath or a high-speed blender.

[0084] As understood herein, a hammer mill is a mill whose purpose is to shred or crush aggregate material into smaller pieces by the repeated blows of small hammers. Such a hammer mill is known to the skilled person and broadly applicable in industry.

[0085] As understood herein, geared chippers are machines capable of chipping wood into smaller pieces (i.e., wood chips). As further understood herein, shredders are similar machines, but mostly designed for processing the material cut from a tree. Fundamentally a shredder is designed to deal with all the material cut from a tree or bush, a chipper is designed first and foremost to process the waste wood. In other words, chippers and shredders are used for reducing the size of organic material. Chippers are specialized for processing larger branches into uniform wood chips, whereas shredders are more versatile and can handle a wider variety of materials by shredding them into smaller pieces. Chippers typically work by utilizing a rotating cutting tool, for example a blade, while shredders typically have interlocking blades / teeth / gears.

[0086] As apparent to the skilled person, ultrasonic bath allows for applying ultrasound to the material of choice.

[0087] Accordingly, as provided by the present invention, blending can be performed for example by using a high-speed blender. Alternatively, blending may also be achieved by mixing using a litter mixer or another mixing element known to the skilled person. It is preferred that blending in step b) is performed by using a hammer mill.

[0088] Furthermore, as is apparent to the skilled person, steam explosion of the mycelium-colonized substrate is also possible, wherein said substrate is exposed to a high-pressure environment, in presence of steam and wherein a sudden release of pressure is used to drive the individual fibers of the substrate apart.

[0089] Alternatively, the particularly preferred method for pulp-formation in step b’) is using a high-speed blender with the addition of water to the mycelium-substrate composite in a 4:1 water-to-mycelium-substrate composite-ratio (w / w). In the method of the invention, the w / w ratio between the mycelium-colonized substrate to the water or aqueous solution is less than 4:1 . At the same time, preferably said ratio is more than 1 :2. Alternatively, it is preferred that said ratio is more than 1 :4.

[0090] Further alternatively, pulp may also be obtained by beating using a pulp-beater similar to those used in the production of paper pulp. Examples of such machines may be a Lampen mill or a PFI mill. The step of pulp-beating increases the conformability of the pulp.

[0091] The process of pulp formation can be aided by adding a liquid, for example by adding water or an aqueous solution to the mycelium-colonized substrate, this also facilitates the transfer of the pulp as it improves the rheological properties of it. This results in a smoothie-like gel-like slurry / paste, which is preferred in the method of the present invention. The improved flowing-properties also allow for the molding / shaping of more complex geometries for example by using an injection-molding process. It is also conceivable that a non-polar liquid can be used for this purpose such as oils.

[0092] During the step of pulp-formation the pulp may also be heated which helps with the softening of the fibers / particles of the pulp and also denatures the proteins contained in it, preferably also increasing the “glueing-properties / the stickiness” of them due to the heating. Temperatures applied may range between 1 and 250°C, but are typically at room temperature or at 60-120°C.

[0093] Alternatively, step b) may comprise a step b”) comprising blending mycelium-substrate composite or mycelium-containing fruiting block, to obtain a blended mycelium-colonized substrate slurry. Due to the intrinsic water content of said fruiting block or said mycelium-substrate composite, the process is preferably performed without addition of any further liquid.

[0094] In the method of the present invention, in principle two types of pulp can be produced. Fine pulp is characterized by a mean particle / fiber-size / length of < 1.5 mm. Rough pulp is characterized by a mean particle / fiber-size / length of > 1.5 mm < 25 mm. The preferred pulp-type is the fine pulp type. As understood herein, the mean particle / fiber-size / length is measured along the longest axis of the particle / fiber. Preferably, rough pulp is generated in step b”), as described herein.

[0095] In step (c) of the method for of the present invention, molding of the obtained slurry into a particular 3- dimensional shape to obtain a molded mycelium-colonized substrate pulp slurry takes place. Accordingly, step c) is in other words the step of pulp moulding, or in other words forming or shaping. The goal of shaping the molded mycelium-colonized substrate pulp composite is to achieve a bonding effect between the individual particles of the pulp. For this to happen, they must be compressed under a force. If the pulp has been created by blending, the water or aqueous solution is typically pressed / squeezed out during this process.

[0096] It is also possible to perform the molding step c) in multiple steps: where pulp is introduced into a shaping tool in multiple “batches” or “fractions”, which can be of a different particle size, different substrate, different additives and or different fungal strain (different physiochemical properties). This can be referred to as fractional forming. The main reason for fractional forming is to engineer the material properties of the resulting product and is most often achieved by the particle-size distribution, wherein for example a rougher core of the molded mycelium-colonized substrate pulp composite may be created and a finer outer layer, with a higher detail-resolution may be applied atop of the core layer(s), or to have an outer layer which has better integrity and / or better abrasive resistivity.

[0097] The step of molding is predetemined by the predefined mold geometry. The goal of the mold geometry is to define the shape of the molded mycelium-colonized substrate pulp slurry, thereby determining the final shape of the molded mycelium-colonized substrate pulp composite product. In order to achieve high quality mycelium composite products certain factors must be taken into account. The pulp is forced into the shape of the mold. The walls of the mold may be covered with some 3D pattern, for example with a grid pattern, resulting in a higher surface area thereof. Said 3D pattern will be present in the final product. The skilled person is able to select the right material for the making of the mold / tool.

[0098] In the step of molding, filling of the mold with the obtained slurry is crucial as it requires to reproduce the shape of the mold. Filling can be done for example by filling and compressing, by hand or by injection, preferably by filling and compressing or by injection. Before filling, the mold may be disinfected, preferably with a disinfecting agent comprising ethyl alcohol. The step of disinfection of the mold is necessary if the mycelium is intended to be kept alive, for example to maintain its further growth. Otherwise, this step is not necessary.

[0099] The treatment with the disinfection agent may also be combined with the treatment with a mold release agent. Herein, a mold-release agent can be sprayed onto the mold to facilitate releasing the molded mycelium-colonized substrate pulp composite material from the mold later on. The so achievable faster and easier demolding leads to lower chance of breaking the molded mycelium-colonized substrate pulp composite product. Accordingly, it may also allow for the creation of more complex geometries of the obtained mycelium-substrate composites.

[0100] According to the method of the present invention, it is also possible to insert functional components such as motors, connectors, conduit, etc. into the material during the molding step. It may also be beneficial for some applications to insert a stiff skeleton, textile, fibers or fiber mats to improve the products strength and or toughness. It is further of note that the pulp of the present invention, made of mycelium-colonized substrate, is capable of binding to veneers of which the binding-surface is porous enough. An application of veneers is especially interesting for applications which demand a high abrasive resistivity of the product.

[0101] The method of the present invention further includes step d) in which compressing said pulp slurry in order to remove water from the slurry takes place. Thereby, the molded mycelium-composite substrates pulp composite is obtained.

[0102] The pulp is molded at a temperature from 1 to 250°C. Higher temperatures induce protein denaturation and / or cauterization of the fungal-cell residue increasing material integrity. Typically the molding / forming step for rough pulp also is performed at temperatures between 60-120°C, higher temperatures are possible.

[0103] In a preferred embodiment of the invention, step c) is performed by using injection-molding.

[0104] According to the present invention, injection molding can be performed as described in the following. A shaped cavity is filled with the fluid-transfer system, that allows for applying exact doses of the pulp under compression such as a syringe-pump (“the injector”). This process is similar to the injection molding of thermoplastic. The injector has a nozzle that is connected to the shaped cavity, to allow for transfer of the pulp from the injector into the cavity. The injector is capable of generating pressures sufficient to compress the pulp into a shaped fungal-molded pulp composite, while expelling the solution from it. This is either achieved by gaps of the seems of the parts of the mold or perforations in the tool*s, which are wide enough for water to flow freely, but tight enough to keep the fibers and particles inside. By thoughtfully positioning the fluid drainage the flow of the pulp may also be controlled allowing for better definition of textures and detailed object features. The tool is preferably a multi-part tool to allow easy retrieval of the final product. The tools may or may not be heated during this step. The tool used to shape the mycelium-colonized substrate slurry and expel the water or aqueous solution may be heated to more than 50°C, preferably more than 65°C, more preferably more than 80°C even more preferably more than 95°C. The process of injection molding is also shown in Fig. 7.

[0105] The process of centrifugation molding, according to the present invention, is described in the following. The pulp is transferred into a shaped cavity. The step of filling may also occur before centrifugation. The centrifugal force, achieved by rotating the setup at speed sufficient to compress the pulp. This is not a preferred method according to the present invention. The tools may or may not be heated during this step. Preferably, the tool used to shape the mycelium-colonized substrate slurry and expel the water or aqueous solution is heated to more than 50°C, preferably more than 65°C, more preferably more than 80°C even more preferably more than 95°C.

[0106] Another alternative method for molding is mesh-transfer molding, as shown in Fig. 9 and 10. This process involves the usage of a large vat / container into which a shaped mesh / sieve (STEP I) is dipped, in order to deposit a layer of the blended particles / fi bers onto the mesh (STEP II). Similar to the production process of molded paper pulp. This layer may further be pressed or put into a centrifuge, but is typically just dripped off or a vacuum is used to drive the fluid out of the pulp. After this the shaped product is desiccated and removed from the mesh (STEPIII). It also possible to dip the mesh multiple times, to achieve a thicker wall.

[0107] Fine pulp tends to bind easier to itself, therefore lowering the needed pressure during the molding / forming process. Fine pulp also profits the most of the addition of water or an aqueous solution, as it typically also is blended. Fine pulp also profits most from the addition of binders, etc. as it provides the most surface area due to the smaller particles size. The particles of fine pulp consist of separated substrate-fibers, substrate-particles, detached hyphal fibers, and particles / fi bers of substrate, with hyphae physically attached thereto.

[0108] Rough pulp typically needs higher compression during the molding / forming process for it to bind, resulting in denser materials. Typically rough pulp is only molded / shaped using press-molding. Also typically during the molding of rough pulp, using press-molding, the mold is heated to 60-200°C

[0109] The method of the present invention further includes step d) of compressing said pulp slurry in order to remove water from the slurry, thereby obtaining the molded mycelium-colonized substrate pulp composite. Preferably, the step of compressing is done by the so called press molding. Accordingly, in the pressmolding step, a shaped cavity (the mold), with parts, that can move relative to each other (the tool*s) is used to create the desired geometry of the pulp. The movement of the parts of the tool allow for compressing of the mycelium-colonized substrate pulp and also allows for easy removal of the final product. Due to the compression of the pulp, the water or aqueous solution is pressed from it and its fibers are bound. The tool is also designed to allow for the water to flow out of the cavity, but the fibers to remain inside the cavity. This can be achieved by leaving tolerances between the intersections of the different parts of the tool or by perforations in the tool*s, which are wide enough for water to flow freely, but tight enough to keep the fibers inside. By thoughtfully positioning the fluid drainage the flow of the pulp may also be controlled allowing for better definition of textures and detailed object features.

[0110] In the step of press-molding, the tools may or may not be heated during this step. The tool used to shape the mycelium-colonized substrate slurry and expel the water or aqueous solution may be heated to more than 50°C, preferably more than 65°C, more preferably more than 80°C even more preferably more than 95°C. The press-molding-technique can be used for all types of pulp (fine, rough, with or without fluidaddition). The minimal pressure for fine pulp is at 4.5 kPa. The minimal pressure for rough pulp is at 0.9 MPa. As preferably understood herein, the compression of the mycelium-colonized substrate slurry is great enough to expel more than 70%, preferably more than 80%, more preferably more than 90% of the water or aqueous solution. As understood herein the compression of the overgrown substrate pulp or pulp-slurry is sufficient to expel more than 80%, preferably more than 90%, more preferably more than 95% of the added water or aqueous solution and is preferably even capable of expelling more than 10% of the water inherent in mycelium-overgrown substrate, preferably more than 30%, more preferably more than 50%, even more preferably more than 70% of the water inherent in mycelium-overgrown substrate. The compression and expelling of water is chosen such that the molded mycelium-colonized-substrate pulp has bound to itself enough to not loose its form after having been removed from the mold / tool.

[0111] As understood herein, the terms “mycelium-colonized substrate” and “mycelium-overgrown substrate” can be used interchangeably.

[0112] As also apparent from the foregoing, the steps of molding, i.e. step c), and of compressing, i.e. step d), may also be performed together, for example by the virtue of injection molding.

[0113] As it is apparent to the skilled person, the compressing process described in step d) of the method of the present invention is distinct from the process described in WO 2024 / 062136, wherein filling of the mold can be done by fill stamp, hand or by injection, which however does not involve any step of compressing the pulp slurry included in the mold in order to remove water from the slurry and bind / interlock the fibers of the slurry. Accordingly, the process described in WO 2024 / 062136 corresponds to filling of the mold rather than to removing water from the composite through compression. Further, WO 2024 / 062136 discloses the pressing step that is aimed at forming a board-like material to increase its strength, and WO 2024 / 062136 stays completely silent about pressing the material with an aim to dehydrate it.

[0114] Of note, while WO 2024 / 062136 discloses that the mycelium-substrate composite may be further processed by, among others, pressing, said pressing is disclosed to occur solely after skin-growth or inmold incubation and is performed as an alternative to drying step. Furthermore, the material obtained in the pressing step disclosed in WO 2024 / 062136 is different from that obtainable in the compressing step, as in step (d) of the method of the present invention. Accordingly, the material obtained in WO 2024 / 062136 features a connected mycelium that is responsible for keeping the mycelium-composite together. In contrast, in the case of the three-dimensional molded mycelium-colonized substrate pulp composite (or preferably a three-dimensional mycelium-colonized substrate pulp composite, as understood herein) of the present invention, the material is kept together by the interlocking of the individual fibers. As immediately apparent to the skilled person, this is similar to the structure of paper.

[0115] Alternatively, instead of step d), a step d1) of evaporative bonding can be performed. In this process the pulp typically is not compressed under a lot of force, but rather just poured / injected into a cavity and the water or aqueous solution is let to be evaporated. A vacuum can be employed to aid with the drawing of the solution during this step. Besides the bonding effect achieved by forcing the individual particles together another bonding effect is the dried intracellular mycelium compounds. This process benefits from the addition of binders. Also this process can achieve materials with a lower density than with the process using compression.

[0116] In the final step (e) of the method of the present invention, demolding the so obtained product takes place. In other words, in the final step (e) demolding of the molded mycelium-colonized substrate pulp composite takes place. Thereby, the (three dimensional) mycelium-colonized substrate pulp composite is obtained.

[0117] Basically, in the demolding step, the molded mycelium-colonized substrate pulp composite is removed from the mold. This can be done in any way that preserves the integrity of said composite and that is apparent to the skilled person. For example, the obtained molded mycelium-colonized substrate pulp composite objects can be removed from the molds by vibrating the molds and pushing / slamming out the objects or by using the object’s momentum. As it is further apparent to the skilled person, the addition of a mold-release agent aids with demolding and results in less failed products, as well as the ability to create higher-resolution molds without running the risk of damaging the final product during demolding. It is further noted that demolding may depend on the mold material and, accordingly, the more flexible mold materials like e.g. silicon may aid demolding.

[0118] As understood herein, in the method of the present invention a three-dimensional molded mycelium- colonized substrate pulp composite (or preferably a three-dimensional mycelium-colonized substrate pulp composite, as understood herein) is obtained. However, as apparent to the skilled person, said product further undergoes the step of demolding, and thus can also be referred to as a three-dimensional mycelium-colonized substrate pulp composite.

[0119] The method of the present invention preferably further comprises step a) of inoculating and cultivating at least one fungal strain onto a solid medium to obtain a mycelium-colonized substrate. Preferably, said solid medium comprises a lignocellulosic material.

[0120] It is to be understood that in step a) first the mycelium inoculated substrate is formed, which is then transformed into mycelium-colonized substrate, in the process of further growing the mycelium used for inoculation on said substrate.

[0121] As understood herein, a mycelium inoculated substrate relates preferably to a composition comprising mycelium and substrate, wherein the mycelium may be bound to the particles or portions of substrates and physically connected thereto, but preferably does not connect different particles or portions of substrate with each other. Preferably not more than 10% of particles or portions of substrate are connected with each other by the mycelium in a mycelium inoculated substrate. Even more preferably, not more than 5% of particles or portions of substrate are connected with each other by the mycelium in a mycelium inoculated substrate. Preferably, the % value refers to the weight of the substrate to be involved in being bound through the mycelium. In other words, typically and preferably, the particles or portions of substrate are not connected by the mycelium. Typically and preferably, said mycelium may be attached to individual particles or portions of said substrate. However, the mycelium used for inoculation may also be not attached to the particles or portions of said substrate.

[0122] Preferably, a mycelium inoculated substrate relates to a composition comprising an inoculum and substrate, wherein the inoculum may be a liquid suspension of spores, liquid suspension of mycelium and or hyphae, grain spawn, sawdust spawn, a tissue sample taken from a fruiting body, a slurry-type spawn (a suspension of substrate and water or an aqueous solution) and the like. The amount of inoculum used varies between the different kinds of inoculum chosen, but is chosen such that it is economically optimized and achieves the preferred exploration-rate of the chosen strain on the chosen substrate at the chosen time-point.

[0123] Preparation of mycelium-inoculated substrate may be performed by using the discrete particle spawn. Accordingly, the method of the present invention may further encompass the step of the preparation of a mycelium colonized substrate, further comprising the step of preparing the mycelium-inoculated substrate, wherein the mycelium-inoculated substrate is prepared by mixing mycelium comprised in a form of discrete particles with the substrate. Accordingly, the particles that act as “mycelium capsules” are added to the substrate in order to inoculate it. This together with mixing of the substrate upon inoculation with mycelium allows achieving homogeneous distribution of mycelium within the substrate volume. Furthermore, discrete particle spawn may also include valuable nutrients that may be added therewith to the substrate. Depending on the nature of said particles, addition of such particles, according to the present inventors, is likely to improve the aeration by creating void spaces in the substrate (i.e. mycelium-inoculated substrate) volume.

[0124] If inoculation of the substrate is to be done in the reactor adapted to solid state fungal culture, said inoculation of the substrate is achieved over one of the ports. The inoculum can be transferred into the vessel by an auger, syringe or can by a stream of sterile air (following the venturi jet principle). The agitator is turned on for typically around 3 min to incorporate and evenly distribute the inoculum throughout the substrate.

[0125] Preferably, the inoculating in step a) is performed by using spores, grain spawn, sawdust spawn, liquid inoculum, spore suspension, slurry inoculum (a mixture of solid media and an aqueous solution), agar wedge, fruiting body tissue sample or a combination thereof. More preferably, the inoculating in step a) is performed using grain-spawn, liquid inoculum or slurry inoculum.

[0126] As understood herein spores refer herein to a fungal cell adapted to resist high temperatures, humidity and other negative environmental conditions, produced by the fungus to defend itself. The spore is a unit reproduction that may be adapted for dispersal and for survival, often for extended periods of time, in unfavorable conditions. Spores form part of the life cycles of many plants, algae, fungi and protozoa. For these reasons, spores can be used to inoculate substrate, as provided in the present invention. Spores may be used in their isolated form. However, spores can also be resuspended in a liquid, for example in a suitable growth medium, and used as spore suspension.

[0127] Grain spawn, as apparent to skilled person, is mushroom mycelium grown into certified organic rye or millet grain which is ideal for inoculating sterilized or pasteurized substrates. Grain spawn is also an industry standard for high volume commercial production.

[0128] Sawdust spawn, as apparentto the skilled person, is obtainable by inoculating sawdust with live mycelium which will then colonize the substrate. Once fully colonized, the sawdust spawn can be used to inoculate other substrates or fruiting blocks. It is to be understood herein that preferably the term substrate-spawn also encompasses a sawdust spawn.

[0129] Liquid inoculum is a liquid composition comprising the growth medium and living mycelium cells.

[0130] Slurry inoculum refers preferably to a composition comprising solid media colonized with a mycelium and an aqueous solution. Such composition can be combined with further solid media, for example by pouring onto and mixing, so that further solid media becomes inoculated.

[0131] As understood herein, inoculation using agar wedge means using a wedge (or, more general, a fragment of said agar), includes combining the colonized piece of agar with the substrate to be colonized / i noculated .

[0132] As it is to be understood to the skilled person, the substrate, as described hereinabove, before it is subjected to the optional pretreatment steps and before it is afterwards subjected to the step comprising inoculation with mycelium, undergoes suitable preparation.

[0133] As understood herein, a substrate is not particularly limited in the method of the present invention. Accordingly, the skilled person is in position of selecting a substrate that would support the growth of the mycelium to an extent necessary for the method of the present invention, which would be acceptable as part of the future final product, i.e., molded mycelium-colonized substrate pulp composite product.

[0134] The substrate may comprise a variety of different components which may influence the final material properties of the resulting composite object. Different lignocellulosic and non-lignocellulosic components influence the growth of the mycelium and the material properties of the resulting material. The skilled person is able to provide a substrate-composition suitable for the cultivation of the chosen fungal strain*s and for the tuning of the material properties. As mentioned previously, it is preferred that the substrate comprises lignocellulosic material. As apparent to the skilled person, and as preferably to be understood herein, the lignocellulosic material is any material that comprises cellulose, hemicellulose and lignin. It is further preferred, in the method of the present invention, that the lignocellulosic material comprised in the substrate is characterized by a total water content of 40 to 90 % w / w. In other words, or alternatively, the substrate comprising the lignocellulosic material is characterized by a total water content of 40 to 90 % w / w. Alternatively or additionally, it is further preferred, in the method of the present invention, that the substrate is characterized by a total water content of 40 to 90 % w / w

[0135] Thus, preferably and according to the present invention, the dry substrate mixture comprises preferably at least of one lignocellulose component. The heap density and the heap porosity of the overall substrate and its individual components is to be understood as non-limiting. It may have a dry heap density reaching from 80 to 1000 g / Liter and a void space volume from 80-10%. The grain-sizes may be up to 60mm but are preferably in the range between 1-25mm.

[0136] Preferably, as used in the method of the present invention, the lignocellulosic material is selected from oil production discharge, apple pomace, hemp blossoms, natural aroma extraction discharge, cocoa hulls, coffee skins, wheat bran, straw, hemps stalks, corn stalks, corn cobs, nut waste, wheat milling filter dust, sawdust, beer mash, threshing residues, sun flower stalks, hemp leaves, bean stalks, tobacco production residues, wood chips from pencil production, dried waste herbs, cork, vertical farming root-waste, sheep wool, and paper waste. More preferably the lignocellulosic material is selected from oil production discharge, apple pomace, natural aroma extraction discharge, coffee skins, wheat bran, hemps stalks, corn stalks, wheat milling filter dust, sawdust, beer mash, sun flower stalks, bean stalks, tobacco production residues and wood chips from pencil production.

[0137] In advance of preparing the mycelium-inoculated substrate, the substrate is homogenized, for example by mixing using a litter mixer or another mixing element known to the skilled person. In such a process of homogenization or mixing, large lumps of substrate are broken down and substantially uniform distribution of grain / particle sizes within the substrate mass can be achieved. Accordingly, such a homogenization / mixing of the substrate together with an even distribution of inoculum will allow for the following even, homogeneous colonization with mycelium. Accordingly, the goal of homogenizing the substrate is to have the same growth conditions throughout the entirety of the substrate. Substrate components can be segmented on the basis of how they affect the mycelium-growth and material properties. Shown in Figure 1 is a hierarchical relationship-diagram of the different componentclasses. The different components are also described in the following Table.

[0138] Filler and Structural main substrate components can be selected from chopped hemp-, corn-, bean-, tobacco- or other stalks; corncobs; rice-, wheat-, rye- or other husks; wood chips from hard- or softwoods; wood shavings from hard- or softwoods; saw dust from hard- or softwoods; bean-hulls (such as cocoa hulls); juice production residues (such as apple pomace); natural oil production residues; aroma-extract- production residues; straw; nut-waste and shells; coffee-production residues; paper waste; bread waste; cereal bran; flour; brewing mash; wool; hair waste; biofiber- based clothing waste; hemp blossoms; coffee skins; milling filter dust; threshing residues; plant leaves (such as hemp leaves); herbs; biodegradable foams such as used for vertical farming; roots (such as hydroponic farming root waste). This list is however not meant to be in any way limiting, and any further components known to the skilled person may be used.

[0139] It is noted that often a component may serve multiple purposes, for example calcium hydroxide makes the substrate more caustic but also changes the inner friction of the substrate acting as a “dry lubricant between the particles and aids in fire-resistance of the final material, as it leads to calcification of the mycelium. It is note that most calcium-rich ingredients aid in the calcification of the mycelium and therefore aid in fire-resistivity and material stiffness.

[0140] Furthermore silica-based additives such as glass-shards or minerals such as foamed minerals (perlite) aid in fire-resistance of the final material. The substrate may be preconditioned before sterilization for example with caustic chemicals, autothermal heating (using composting) or enzymes. This typically facilitates the nutrient-uptake of the mycelium.

[0141] The substrate may be pelletized, i.e. present in a form of pellets. As known to the skilled person, palletization of the substrate facilitates its transport and improves its long term storage.

[0142] The substrate as provided by the present invention may further include mycelium-colonized substrate and / or mycelium-substrate composite, obtained for example from previous production processes of the mycelium-substrate composite. As is conceivable to the skilled person it is also possible to obtain said mycelium-colonized substrate from culinary mushroom producers, for whom the mycelium-colonized substrate is a waste product after harvesting the fruiting bodies. The pulp may also be obtained from culinary waste fruiting blocks or from the production of mycelium-substrate composite production runs, in particular those that have not been brough to completion.

[0143] The goal for the choice of the right substrate-composition is to allow the mycelium to grow in and on it, while minimizing contamination of the substrate with other microorganisms. This is achieved by making the conditions of the substrate favorable towards the fungal strain and less favorable towards other microorganisms. This can be achieved by adding “protective additives” to the substrate, such as, but not limited to turpentine, calcium hydroxide or tree sap.

[0144] The substrate may be washed beforehand and its grain-size and geometry adjusted, for example by way of a wood-chipper, a mill and / or a sieving-system.

[0145] The components may be chipped if necessary to achieve a distribution of particle sizes and shapes, favored by the mycelium. Many side streams have a small mean particle-size, which typically for the production of standard MSCs needs to be compensated with larger particles, that typically are higher in economic value, such as hemp-stalks. This is due to the need of void-cells inside the substrate for better aeration of the substrate. This is especially important during in-mold growth of larger products with a small aeration-area. But since only the step of colonization of the substrate is required and no in-mold growth is needed for the production of molded mycelium-colonized substrate pulp composite, the colonization can take place inside an actively aerated bed or vessel, to compensate for the lack of void-cells.

[0146] The substrate may be hydrated with water or an aqueous solution, for example to achieve a total waterpercentage (w / w) of 40-90%. The load of microorganisms / contamination of the substrate may be reduced via thermal, radiative, chemical or other means, to allow the mycelium to grow without the need or less of a need to compete with other organisms for the substrate.

[0147] Depending on the method employed to reduce the bioburden, the substrate may be cooled or chemically neutralized, to ensure that the mushroom inoculum chosen is not damaged during inoculation.

[0148] In order to obtain a mycelium-colonized substrate, the mycelium inoculated substrate is incubated under conditions favoring the growth of the chosen strain / strains and if other microorganisms are co-cultured also the conditions of those, until the substrate is colonized. Colonization is achieved preferably when the boundaries of the concentric growth-spheres of the mycelium touch the boundaries of other growthspheres and therefore the entirety of the substrate has been explored. As it is conceivable to the skilled person, a full, i.e. complete, colonization may not be necessary to proceed to the next steps to obtain a molded mycelium-colonized substrate pulp composite.

[0149] The colonized substrate may be stabilized for storage for the usage of it at a later point in time. This can be achieved, by removing the moisture from it, freezing it, pelletizing or similar means. If a means is chosen, that allows the mushroom to enter a stasis, the mycelium may be reactivated at a later time point, for example if a mycelium-network-support or mycelium-skin is envisaged in the final shaped product.

[0150] The substrate may be incubated by the mycelium with or without free water present in the substrate. The substrate may or may not be agitated during the incubation period. Also fed-batch and continuous fermentation setups, are encompassed by the invention.

[0151] The incubation may take place in plastic grow bags, trays, or a bioreactor / fermenter.

[0152] It is understood that additives can be added to the substrate, during inoculation, after colonization, during pulp-formation or before molding / shaping

[0153] As provided by the present invention, any mycelium-forming fungal species can be used in the production of a mycelium-colonized substrate pulp composite material of the present invention. It is preferred that the fungal species is not toxic to humans, so that the so obtained product can be safely used by humans. Preferably, in the method of the present invention, the mycelium originates from a Basidiomycetes strain. More preferably, the fungal species is selected from the genera Trametes, Fomes, Ganoderma, Pycnoporus, and Pleurotus. Even more preferably, the fungal species is selected from the genera Trametes (such as Trametes versicolor], Ganoderma (such as Ganoderma lucidum) and Fomes (such as Fomes fomentarius). Thus, accordingly, and preferably, the fungal species is Fomes fomentarius, Ganoderma lucidum or Trametes versicolor. Thus, preferably, the fungal species may be Trametes versicolor. Alternatively, the fungal species may be Fomes fomentarius. In one embodiment, the fungal species may be Ganoderma lucidum.

[0154] The method of the present invention further comprises step b1) of adding a binder to the slurry.

[0155] Preferably, said binders are selected from polymerizing oils, natural glues, Agar-agar, sugars, proteins (such as soy protein), chitosan, gelatin, kappa carrageenan, starch, glycerol, casein, pectin, lignin, wheatpaste, gum arabic, lactic acid, lignin, rosin, shellac, biodegradable polymers.

[0156] Preferably, the binder is selected from whey, protein, agar, alginate and natural glue.

[0157] Step b1) may further comprise adding the linker additives, preferably selected from formalin formaldehyde-based additives, alcohols, acetone, genipin, glutaraldehyde, transglutaminase, tannins, epoxidized vegetable oils, and citric acid.

[0158] Step b1) may further comprise adding the fire-proofing additives, for example selected from minerals, in particular foamed minerals, such as perlite, silica-based additives (such as glass waste, diatomaceous earth), ash, charcoal, and kaolin clay.

[0159] Accordingly, in step b1) added may be additives that increase bonding between the particles / fibers, preferably selected from:

[0160] - polymerizing oils such as linseed oil

[0161] - natural glues

[0162] - sugars

[0163] - proteins

[0164] - soap (which helps to break up the cell walls of the hyphae)

[0165] - denaturing agents such as sodium dodecyl sulfate or sodium laureth sulfate

[0166] - gelling-agents such as agar or kappa carrageenan and histological fixatives such as formalin, alcohols, acetone

[0167] Histological agents typically penetrate the particles and stabilize their structure by cross-linking proteins, nucleic acids, and other cellular components. This process effectively "fixes" the pulp in its current state. Formalin, for example, reacts with amino groups in proteins, creating a stable protein network. Additionally, formaldehyde may act as a dehydrating agent, removing water from the tissue, which further helps to stabilize the cellular structures. Similarly, alcohol acts as a dehydrating agent, removing water from tissues and preventing microbial growth. Alcohol likewise denatures proteins by disrupting their native structure. This denaturation alters the conformation of proteins, rendering them insoluble and thus preserving the integrity of the resulting composite. Typically if histological agents are used the step of molding / forming is performed in a short time after pulp-formation.

[0168] Alternatively or additionally, in step b1) added may be additives that change the material properties (typically make it more flexible), for example selected from:

[0169] - Glycerin

[0170] - Oils, and

[0171] - Plasticizers

[0172] Alternatively or additionally, in step b1) added may be additives that increase fire-resistance, preferably, selected from:

[0173] - Foamed minerals such as perlite

[0174] - Silica-based particles such as glass waste

[0175] - Mineral particles, and

[0176] - Ash

[0177] Furthermore, in step b1) chemicals may be added to the pulp forming process to increase delignification, similar to those used during the production of paper pulp. Examples include Ca(0H)2, NaOH, and hydrogen-peroxide. However, such an addition is not preferred in the method of the present invention as it may impair the sustainability of the produced material.

[0178] It is also conceivable to add fresh substrate to the pulp, even though this is not preferred. Also natural fibers (such as jute or wool) may be added at this step. It is also possible to add synthetic fibers to the pulp, this is also not preferred as it impairs the bio-degradability of the final product. Step b1) may be performed directly after step b). Alternatively, step b1) may be performed during step b) or directly before. In other words, during the step of pulp-formation pulp-additives may be added to it. These additives, as for example described herein, change the final composite integrity, change its appearance, change its fire-resistance-properties, density and the like.

[0179] In one embodiment, the pulp obtainable upon step b) (or step b) combined with / followed by step(s) b1), as applicable), can be stabilized for storage and not necessarily used directly in step c) of the method of the present invention.

[0180] For stabilization of the pulp, the pulp may undergo pelletizing, drying, cooling, and / or freezing.

[0181] Pelletizing is the preferred method, as it decreases the heap density of the material and is better suited for shipment between facilities or to customers.

[0182] Drying of the pulp means removal of water from the pulp by evaporation, and is preferably achieved / performed by using a rotating drum type drier. This typically results in round coagulated pulp pieces. This is the preferred method for keeping the mycelium in a state of stasis, allowing for reactivation of the mycelium, by supplying quickly available nutrients such as simple hydrocarbons (sugars, starches, etc.) and water. It is noted that such a step of mycelium-reactivation only works if water / aqueous solution or a natural oil has been used during the pulp-forming process.

[0183] Should a particular color of the pulp (or the resulting pulp composite) be desired, a particular additive that gives raise to the color of the pulp may be added to said pulp, preferably before the molding step. Such an additive may include any colorant, dye or pigment that is known to the skilled person.

[0184] To reform the pulp from the dried / pelletized pulp, fluid is added to it and it is mixed. For cooled or frozen pulp it is just allowed to warm up to operational temperatures.

[0185] Accordingly, the method of the present invention may further include steps b2) of stabilizing the pulp for long term storage, storing the pulp, and reforming the pulp, as described herein.

[0186] As apparent to the skilled person, the fungal molded-pulp composite do not need to be homogeneous. Accordingly, in one embodiment of the invention, steps b) to d) are repeated in order to create a layered molded mycelium-colonized substrate pulp composite, wherein different layers are formed by using different mycelium-colonized substrates (to be understood by using pulps obtained starting from different mycelium-colonized substrates) or the same mycelium-colonized substrate at a different mean particle size (i.e., in other words, the same pulp reduced to different mean particle sizes).

[0187] The fungal-molded pulp composite obtainable (or obtained) in the method of the present invention may further undergo different steps of post-processing.

[0188] In one embodiment, the method further comprises the step of further incubation of the obtained molded mycelium-colonized substrate pulp composite in order to grow a reinforcing mycelium network. Accordingly, for additional material strength and or a protective mycelium skin, the shaped pulp, in particular after step c) but before step d), may be incubated. To achieve this, the pulp must not be dried after shaping and is put into an environment with conditions suitable for the growth of the mycelium. Typically to achieve this readily available nutrients such as simple hydrocarbons are added to the pulp. The shaped objects are then incubated for a period sufficient for the mycelium to form a network inside and around the object. It is preferably to be understood that in this case, the mycelium grows in the entire volume of the shaped object. It is however preferred that the so obtained product is not further reinforced. In other words, it is preferred that upon step c), the obtained product is not further incubated in order to grow the mycelium (preferably in other words, to grow a reinforcing mycelium network). Accordingly, it is preferred that step d) is performed directly after step c). In other words, as already mentioned, there is preferably no incubation step between steps c) and d). Thus, it is immediately apparent to the skilled person that the process disclosed in WO 2024 / 062136 and involving in-mold incubation of the molded composite, is very distinct from the process of the present invention, as described herein.

[0189] However, in one embodiment of the present invention, the method of the present invention further comprises step d) of incubating the molded pulp obtained in step c). In one embodiment, in step d) the skin-growth occurs.

[0190] Alternatively or additionally, upon demolding in step e), an additional step, referred to as step e1), comprising (or consisting of) incubating the product of step e) to allow for the protective skin growth around the object may be performed. As is apparent to the skilled person further incubation of the molded pulp occurs outside of a mold and is therefore different from in-mold incubation and is preferably referred to as skin-growth.

[0191] As understood herein, said protective skin may also be referred to as a protective mycelium layer. Alternatively or additionally, the fungal-molded pulp composite may be reinforced chemically. For example, for reinforcement a mixture of linseed oil (or any other polymerizing oil) and turpentine (or any other suitable solvent) may be used, that is applied such that the mixture penetrates into the product. The solvent (turpentine) aids with the penetration of the product. Due to the polymerization of the oil, the entire product is reinforced. As is conceivable to skilled person any other thin binder or glue may be used for this purpose.

[0192] According to the present invention, the molded mycelium-substrate pulp composite may be coated (i.e., surface-coated). For coatings the coatings selected from bee wax, alginate, PLA (poly-lactic acid), milk paint, and natural glue, may be used. Alternatively, the molded / shaped pulp-composites can be submerged in a bath of fixative such as histologic fixatives (same list as the one used for the processdescription for the pulp-formation-process) in order to achieve the reinforcing effect. In one embodiment, the present invention comprises the step of reinforcing the product by treating it with a polymerizing agent.

[0193] The method of the present invention in one embodiment further comprises step f) of drying and / or sanding the obtained product. Often during the shaping process, a mold-line stays visible on the product. This mold line can be removed using multiple processes, such as sanding, tumbling the products in a tumbler, stamping, etc.

[0194] The finished molded mycelium-colonized substrate pulp composite may be further mechanically engraved with for example a CNC-router or may be thermally / energetically etched for example using a “branding iron” or a laser-engraver. Also the pulp may be chemically bleached, for example with hydrogen peroxide. These steps may have an aesthetic purpose or may also be functional, for example by engraving the finished pulp, or creating pockets, holes or other functional geometries may be added to the product. Different veneers may also be added / glued atop the finished molded mycelium-colonized substrate pulp composite. These veneers may be of any material.

[0195] Furthermore, it is apparent to the skilled person that multiple molded mycelium-colonized substrate pulp composite products may be assembled into one single product, by gluing them together, a mechanical connection or using an mycelium network-growth-process in order to fuse different molded- mycelium- colonized substrate pulp composite parts together. Furthermore, further elements, like e.g. motors, or a functional component such as a switch or a bracket, can be included in such products, as described in the foregoing. In one embodiment of the invention, the method of the present invention may further involve step f) of desiccating the product obtained in step e) of the method of the present invention. Desiccating is herein understood as partial or complete (or substantially complete) removal of water.

[0196] In one embodiment, the present invention relates to a blended mycelium substrate pulp slurry, as defined herein, as referred to in step b) and as obtainable in step b’) or b”) of the method of the present invention. Said blended mycelium substrate pulp slurry may also be referred to as blended mycelium-colonized substrate slurry, as understood herein. The present invention further relates to the blended mycelium substrate pulp slurry, which has been further processed in the step of pelletizing, drying and / or freezing, preferably drying and / or pelletizing.

[0197] In the method of the present invention, in principle two types of pulp (or pulp-slurries) can be produced. Fine pulp is characterized by a mean particle / fiber-size / length of < 1.5 mm. Rough pulp is characterized by a mean particle / fiber-size / length of > 1 .5 mm < 25 mm.

[0198] As mentioned before the blended mycelium substrate pulp slurry is obtainable in the blending process which renders the cells in the composite subjected to blending to be dead. Accordingly, it is preferred that in the blended mycelium-colonized substrate pulp slurry up to 20% of mycelium cells by weight are not dead. More preferably, in the blended mycelium-colonized substrate pulp slurry up to 10% of mycelium cells by weight are not dead. Thus, it is immediately apparent that the blended mycelium substrate pulp slurry is different from the homogenized mycelium substrate composite disclosed in WO 2024 / 062136.

[0199] As it is further apparent to the skilled person, the number of mycelium cells that are not dead may be directly influenced by a specific method used for obtaining the blended mycelium substrate pulp slurry. For example, high-speed blender is likely to lead to higher number of not dead cells than methods relying on high compression.

[0200] The present invention further relates to molded mycelium-colonized substrate pulp composite, obtainable according to the method of the present invention.

[0201] It is preferred that said composite is a self-supporting molded mycelium-colonized substrate pulp composite, characterized in that the w / w-percentage of remaining non-metabolized lignocellulosic material is greater than 30%, preferably more than 60%, more preferably more than 75% of the obtained product. Further, the product of the invention is characterized by its cohesion. In other words, the cohesion of the obtained product is given by the interlocking and surface interactions of the individual particles / fi bers of the pulp and by the metabolic products of the fungi cultivated on the lignocellulosic material acting as a binding agent. Preferably, the composite of the present invention is characterized by a tri-dimensional shape, as defined herein. Preferably, at least 50% of substrate particles in the composite of the invention are connected to fungal mycelium (or its dried, denatured form).

[0202] Further aspects and embodiments of the invention are summarized in the following numbered items.

[0203] 1 . A method for obtaining a three-dimensional molded mycelium-colonized substrate pulp composite, the method comprising the steps of: b) providing a mycelium-colonized substrate pulp slurry; c) molding said slurry into a particular 3-dimensional shape to obtain a molded mycelium- colonized substrate pulp slurry; d) compressing said pulp slurry in order to remove water from the slurry, thereby obtaining the molded mycelium-colonized substrate pulp composite; and e) demolding the so obtained product.

[0204] 2. The method of item 1 , wherein the step b) is step b’), defined as follows: b') blending mycelium-colonized substrate, optionally in the presence of aqueous solution to obtain a blended mycelium-colonized substrate slurry.

[0205] 3. The method of item 1 or 2, further comprising the step a) inoculating and cultivating at least one fungal strain onto a solid medium comprising a lignocellulosic material to obtain a mycelium- colonized substrate.

[0206] 4. The method of item 3, wherein the lignocellulosic material is selected from oil production discharge, apple pomace, hemp blossoms, natural aroma extraction discharge, cocoa hulls, coffee skins, wheat bran, straw, hemps stalks, corn stalks, corn cobs, nut waste, wheat milling filter dust, sawdust, beer mash, threshing residues, sun flower stalks, hemp leaves, bean stalks, tobacco production residues, wood chips from pencil production, dried waste herbs, cork, vertical farming root-waste, sheep wool, and paper waste.

[0207] 5. The method of item 3 or 4, wherein the lignocellulosic material is characterized by a total water content of 40 to 90 % w / w.

[0208] 6. The method of any one of items 3 to 5, wherein the inoculating is performed by using spores, grain spawn, sawdust spawn, liquid inoculum, spore suspension, slurry inoculum (a mixture of solid media and an aqueous solution), agar wedge, fruiting body tissue sample or a combination thereof.

[0209] 7. The method of any one of items 1 to 6, wherein the mycelium originates from a Basidiomycetes strain.

[0210] 8. The method of any one of items 1 to 7, wherein fungal species is selected from the genera Trametes, Fomes, Ganoderma, Pycnoporus, Pleurotus.

[0211] 9. The method of any one of items 1 to 8, wherein the fungal species is Fomes fomentarius or Trametes versicolor.

[0212] 10. The method of any one of items 2 to 9, wherein the step b) is performed by using a hammer mill, geared chippers, shredders, ultrasonic bath or a high-speed blender.

[0213] 11 . The method of any one of items 1 to 10, further comprising step b1 ) of adding an additive, preferably a binder, to the slurry.

[0214] 12. The method of item 11 , wherein the binder is selected from whey, protein, agar, alginate and natural glue.

[0215] 13. The method of any one of items 1 to 12, wherein the step c) is performed by using injection-molding or centrifugation, preferably by using injection molding.

[0216] 14. The method of any one of items 1 to 13, wherein the step d) is performed by using the press-mold.

[0217] 15. The method of any one of items 1 to 14, wherein the steps b) to d) are repeated in order to create a layered molded fungal pulp composite, wherein different layers are formed by using different mycelium-colonized substrates.

[0218] 16. The method of any one of items 1 to 15, comprising the step of further incubation of the obtained molded fungal pulp composite in order to grow and reinforce mycelium network.

[0219] 17. The method of any one of items 1 to 15, further comprising the step f) of drying and / or sanding the obtained product.

[0220] 18. The method of any one of items 1 to 17, comprising the steps of reinforcing the product by treating it with a polymerizing agent.

[0221] 19. A blended mycelium substrate slurry, obtainable in step b’) of the method of any one of items 2 to 18.

[0222] 20. The blended mycelium substrate slurry, which has been further processed in the step of pelletizing, drying and / or freezing.

[0223] 21 . A molded fungal pulp composite obtainable according to the method of any one of items 1 to 18.

[0224] 22. The molded fungal pulp composite of item 20, further supported by the presence of a mycelium network.

[0225] The invention will be illustrated using the following examples. These, however, are not meant to limit the scope of protection in any way but serve merely illustrative purpose.

[0226] Examples

[0227] Pulp Material & Process Fact Sheet

[0228] Water-addition and Weight-evolution of the pulp and composite

[0229] Before pressing

[0230] The blended mycelium-colonized substrate slurry (BMcSS) consists of 4 / 5 water w / w and 1 / 5 w / w colonized substrate (4:1). The initial water-content of the colonized substrate is 40-65% w / w.

[0231] Typically the range of the relationship between colonized substrate and added water is around 1 :4 to 10:1 (water : colonized substrate). After pressing

[0232] During the pressing / shaping-step 65-80% w / w of the original weight of the BMcSS in the form of removed water is lost. (Also some water-soluble molecules are removed during this step as a side effect)

[0233] After drying

[0234] 8-10% of the initial BMcSS-weight remains and form the final molded mycelium-colonized substrate pulp composite-product.

[0235] Pulp particle sizes

[0236] The mean particle is around 1 mm for the standard Pulp, this is a very basic particle size-analysis and does not take into account the sizes of the fines (i.e., the smallest particles).

[0237] This means that the mean particle-size most likely is shorter than 1mm.

[0238] The particle size distribution is very broad from smaller than 0.1 mm up to 7mm.

[0239] (The particle size is measured as the longest axis)

[0240] In other batches, where greater pressures and heating of the pulp were applied, it was possible to make pulp with a mean particle-size of around 3mm (with the largest particles being 12.5mm long) work. (Here it is senseful to differentiate between the pulp used for high-temperature -and pressure molding / forming and the one used for low-temperature -and pressure molding / forming)

[0241] Temperatures and Pressures applied during the molding / Forming step

[0242] Temperature

[0243] The McSP (mycelium colonized substrate pulp) may can be pressed at room temperature or at “high temperature”: This results in two material types. Typically the high temperature pulp has a lower moisture content and a larger grain size than the low temperature pulp (rough pulp), but high-moisture-and-low- grain-size-pulp (fine pulp) can also be pressed at higher temperatures.

[0244] Finally a temperature-range of 1-220°C is possible. The highest possible temperature is depending on the substrate composition used.

[0245] Pressure

[0246] Lowest pressure possible for fine pulp 4.5 kPa

[0247] Highest tested pressure for fine pulp 15.7 MPa

[0248] Lowest pressure possible for rough pulp 0.9 MPa

[0249] Highest tested pressure for rough pulp 15.7 MPa

[0250] Example 1 - Production of a molded mycelium-colonized substrate pulp composite sample using a highspeed blender and an aqueous solution for the step of pulp-formation OBTAINING A MYCELIUM-COLONIZED SUBSTRATE

[0251] A substrate made of :

[0252] • 11.5 %w / w Spruce sawdust collected from a local saw-mill

[0253] • 11.5 %w / w Fir sawdust collected from a local saw-mill

[0254] • 22 %w / w Chopped hemp stalks collected from a local CBD-producer

[0255] • 55 %w / w Tap water (municipality BS CH) is mixed in a ribbon-blender for t=12min, to retrieve a mixed substrate.

[0256] The mixed substrate is then treated against the inherent bioburden, using an autoclave at T=121 °C for t=40min. Following this step, the substrate is cooled down to temperatures below 40°C and is inoculated with Trametes Versicolor grain-spawn at a spawn-rate or SR=3%w / w.

[0257] The substrate is mixed in order to evenly distribute the grain-spawn.

[0258] Following this step the mycelium is incubated on the substrate for t=96-120 hours, at T=26-28°C, RH=60- 95% & C02>20’000ppm in an incubator (modified Heraeus Instruments cytoperm 2 was used) to obtain a mycelium-colonized substrate.

[0259] PULP-FORMATION

[0260] The mycelium-colonized substrate is manually torn into roughly 2 cmA3-sized pieces.

[0261] M=120g of the torn mycelium-colonized substrate together with m=480g tap water (municipality BS, CH) is added to the blender (Blendtec commercial was used).

[0262] The constituents are blended for t=200s to form the pulp.

[0263] MOLDING / SHAPING

[0264] N=3 Steel tubes (inner-diameter d=63mm, height h=100mm) are filled withl 60g of the Pulp.

[0265] N=3 CNC-cut shaped aluminum stamps (diameter d=62mm, height h=30mm) are placed into / atop the filled steel tubes.

[0266] (The tubes together with the stamps are herein referred to as the mold.)

[0267] The filled molds are placed below the hydraulic press (Metallkraft WPP15T was used).

[0268] The aluminum stamps are pressed into the tubes, thereby compressing the pulp and driving the water out of it, by increasing the pressure slowly and waiting for t= 10s between each increase in pressure, to give the water enough time to evacuate. This process is repeated until the stamps reach 35mm depth relative to the top of the steel tube and / or until the pressure inside the mold reaches P=2MPa - 4.2 MPa.

[0269] The pressure is released and the molded mycelium-colonized substrate pulp composites are released from the molds. DESSICATION

[0270] The obtained MMcSPC’s (molded mycelium-colonized substrate pulp composite) are let to dry inside a convective air dehydrator (Klarstein food dehydrator was used) at T=70°C for t=2-6h.

[0271] OPTIONAL POST-PROCESSING

[0272] The dried MMcSPC’s may be sanded using a belt-sander or manually using sand paper if an undesired mold-line is left visible and sample information may be energetically etched into it by using a Laserengraver (a PLH3D-series OptLasersGrav engraver-attachment for a M1000-series stepcraft CNC was used).

[0273] Example 2 - Lab-scale pulp-formation using a hammer-mill

[0274] OBTAINING A MYCELIUM-COLONIZED SUBSTRATE

[0275] A substrate made of :

[0276] • 3 %w / w coffee skins, collected from a local coffee roastery

[0277] • 10 %w / w Spruce sawdust collected from a local saw-mill

[0278] • 10 %w / w Fir sawdust collected from a local saw-mill

[0279] • 3 %w / w filter dust extract collected from a local grain-mill

[0280] • 9 %w / w carob bean pulp collected from a local aroma-extract producer

[0281] • 10 %w / w Chopped hemp stalks collected from a local CBD-producer

[0282] • 55 %w / w Tap water (municipality BS CH) is mixed in a ribbon-blender for t=1 Omin, to retrieve a mixed substrate.

[0283] The mixed substrate is then treated against the inherent bioburden, using an autoclave at T=121 °C for t=45min. Following this step, the substrate is cooled down to temperatures below T=40°C and is inoculated with Trametes versicolor grain-spawn at a spawn-rate or SR=3%w / w.

[0284] The substrate is mixed in order to evenly distribute the grain-spawn.

[0285] Following this step the mycelium is incubated on the substrate for t=96-120 hours, at T=26-28°C, RH=60- 95% & C02>20’000ppm in an incubator (modified Heraeus Instruments cytoperm 2 was used) to obtain a mycelium-colonized substrate.

[0286] PULP-FORMATION

[0287] The mycelium-colonized substrate is manually torn into roughly 2 cmA3-sized pieces. M=120g of the torn mycelium-colonized substrate together with m=120g tap water is added to a mortar or a mortar grinder (such as the mortar grinder RM 200 by Retsch) and is ground until the desired grain / fiber-size is reached. This process has the benefit, that the particles of the mycelium-colonized substrate are rather separated into individual fiber, than being cut, when using for example a blender. Depending on the wanted flow-properties more water or aqueous solution may be added after this step.

Claims

CLAIMS1. A method for obtaining a three-dimensional mycelium-colonized substrate pulp composite, the method comprising the steps of: b) providing a mycelium-colonized substrate pulp slurry; c) molding said slurry into a particular three-dimensional shape to obtain a molded mycelium- colonized substrate pulp slurry; d) compressing said pulp slurry to remove water from the slurry, thereby obtaining the molded mycelium-colonized substrate pulp composite; and e) demolding the so obtained product.

2. The method of claim 1 , wherein step d) is performed directly after step c), preferably wherein after step c), the obtained product is not further incubated in order to grow the mycelium.

3. The method of claim 1 or 2, wherein step b) of providing a mycelium-colonized substrate pulp slurry comprises step b’), defined as follows: b') blending mycelium-colonized substrate, optionally in the presence of aqueous solution to obtain a blended mycelium-colonized substrate slurry.

4. The method of any one of claims 1 to 3, further comprising step a) inoculating and cultivating at least one fungal strain onto a solid medium comprising a lignocellulosic material to obtain a mycelium-colonized substrate.

5. The method of claim 4, wherein the lignocellulosic material is selected from oil production discharge, apple pomace, hemp blossoms, natural aroma extraction discharge, cocoa hulls, coffee skins, wheat bran, straw, hemps stalks, corn stalks, corn cobs, nut waste, wheat milling filter dust, sawdust, beer mash, threshing residues, sun flower stalks, hemp leaves, bean stalks, tobacco production residues, wood chips from pencil production, dried waste herbs, cork, vertical farming root-waste, sheep wool, and paper waste.

6. The method of claim 4 or 5, wherein the solid medium comprising a lignocellulosic material is characterized by a total water content of 40 to 90 % w / w.

7. The method of any one of claims 4 to 6, wherein the inoculating is performed by using spores, grain spawn, sawdust spawn, liquid inoculum, spore suspension, slurry inoculum (a mixture of solid media and an aqueous solution), agar wedge, fruiting body tissue sample or a combination thereof.

8. The method of any one of claims 1 to 7, wherein the mycelium originates from a Basidiomycetes strain9. The method of claim 8, wherein fungal species is selected from the genera Trametes, Fomes, Ganoderma, Pycnoporus, and Pleurotus.

10. The method of claim 9, wherein the fungal species is Fomes fomentarius, Trametes versicolor or Ganoderma lucidum.11 . The method of claim 3 or any one of claims 4 to 10, insofar dependent on claim 3, wherein step b’) is performed by using a hammer mill, geared chippers, steam explosion, shredders, ultrasonic bath, high-speed blender or any combination thereof.

12. The method of any one of claims 1 to 11 , further comprising step b1 ) of adding an additive to the slurry.

13. The method of claim 12, wherein the additive is a binder.

14. The method of claim 13, wherein the binder is selected from whey, protein, agar, alginate and natural glue.

15. The method of any one of claims 1 to 14, wherein step c) is performed by using injection-molding or centrifugation, preferably by using injection molding.

16. The method of any one of claims 1 to 15, wherein step d) is performed by using a press-mold.

17. The method of any one of claims 1 to 16, wherein steps b) to d) are repeated to create a layered molded fungal pulp composite, wherein different layers are formed by using different mycelium- colonized substrates or the same pulp reduced to different mean particle sizes.

18. The method of any one of claims 1 or 3 to 17, comprising step of further incubation of the obtained molded fungal pulp composite in order to grow a reinforcing mycelium network.

19. The method of any one of claims 1 to 18, further comprising step e1), which comprises incubating the product of step e) to grow a protective skin around the mycelium-colonized substrate pulp composite.

20. The method of any one of claims 1 to 19, further comprising step f) of drying and / or sanding the obtained product.21 . The method of any one of claims 1 to 20, further comprising a step of reinforcing the product by treating it with a polymerizing agent.

22. A blended mycelium substrate slurry, obtainable in step b’) of the method of claim 3 or any one of claims 4 to 21 , insofar dependent on claim 3.

23. The blended mycelium substrate slurry of claim 22, which has been further processed by a step of pelletizing, cooling, drying and / or freezing.

24. A mycelium-colonized substrate pulp composite obtainable according to the method of any one of claims 1 to 21.

25. The mycelium-colonized substrate pulp composite of claim 24, further supported by the presence of a mycelium network.

26. The mycelium-colonized substrate pulp composite of claim 25, wherein the mycelium network is present in the form of a protective mycelium layer on the outside of the composite.

27. A three-dimensional mycelium-colonized substrate pulp composite.

28. The three-dimensional mycelium-colonized substrate pulp composite of claim 27, characterized in that it contains more than 30% w / w of lignocellulosic material.

29. The three-dimensional mycelium-colonized substrate pulp composite of claim 27 or 28, that is characterized by its cohesion.

30. The three-dimensional mycelium-colonized substrate pulp composite of claim 29, wherein the cohesion of the three-dimensional mycelium-colonized substrate pulp composite of claim is given by the interlocking and surface interactions of the individual particles / fibers of the mycelium- colonized substrate pulp and by the metabolic products of the fungi cultivated on the lignocellulosic material acting as a binding agent.

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