Mycelium-based biopolymer composites with isocyanate crosslinking and integrated BIO-based fillers

Abstract

A method of forming a mycelium-based biopolymer composite from a mycelium-based biopolymer sheet, panel or slab that has been exposed to deacetylation, utilizes isocyanate chemistry, polyurethane infusion, and / or bio-based fillers to create a robust, hydrophobic, and highly customizable material. The resulting biopolymer composite may be used as a durable mycelium leather alternative, with enhanced flexibility, moisture resistance, and structural integrity.

Description

ECVT.074PCT PATENT MYCELIUM-BASED BIOPOLYMER COMPOSITES WITH ISOCYANATE CROSSLINKING AND INTEGRATED BIO-BASED FILLERSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims the benefit of United States Provisional Patent Application No. 63 / 733,401 filed on December 12, 2024, the contents of which are incorporated herein by reference in their entirety.BACKGROUND OF THE DISCLOSUREField

[0002] The present application relates to methods for treating grown mycelium-based sheet, panel, or slab materials and grown mycelium-based sheet, panel, or slab materials produced thereby.Background

[0003] Grown, mycelium-based materials (e g., sheets, panels, or slabs, such as, for example, grown aerial mycelium-based panels) have demonstrated significant promise in their potential for commercialization of biopolymer-based material alternatives to traditional textile materials, such as animal-based leather, and petroleum-based sheeting (such as foams). Such leather and petroleum-based products are presented with increasing production challenges with respect to environmental sustainability (and associated philosophical / consumer awareness) issues. The availability of seemingly endless amounts of inexpensive animal hides has now been called into question, given the level of natural resources required to support such animal leather and foam production, along with the consumer products incorporating such materials. Traditional leather production methods typically use a large variety of diminishing animal hides, significant volumes of water, and a range of chemical treatments to achieve desired material properties. Furthermore, concurrent changes in climate and consumer purchasing practices may be causing shifts in government priorities and policies towards favoring more environmentally-sustainable materials.

[0004] In contrast, grown mycelium-based materials have demonstrated the potential to perform sought-after textile functionality and performance, with desirable environmentally-friendly and / or sustainable carbon footprints, especially when compared to certain currently-practiced, animal-based or petroleum-based resources. However, despite these advantages, mycelium-based materials continue to present their own performance and manufacturing challenges. In particular, such mycelium-based materials, depending on end product function, may require increases in strength and elasticity based on current consumer expectations for such end product applications. Further, untreated mycelium-based material may lack the durability and water resistance necessary or expected for many consumer and business-to-business (such as construction) product applications. It is to these ends that the current disclosure is directed.SUMMARY OF THE DISCLOSURE[00051 Disclosed herein are methods for modifying the chemical structures of mycelium-based biopolymer materials (aka biopolymer-based materials such as grown mycelial sheet, panel, or slab materials (such as in one instance, grown aerial mycelium sheets, panels, or slabs)) in order to render them more receptive to incorporation of attribute-improving chemistry.

[0006] Also disclosed herein are the chemically modified intermediate, mycelium-based biopolymer materials themselves (aka formed from the grown and chemically modified fungal mycelium sheets, panels, or slabs (e.g., aerially-grown Ganoderma sp.)) that have undergone partial or full deacetylation to expose reactive amino groups. These reactive sites may then later be crosslinked with isocyanates, optionally integrated with polyurethane phases and optionally supplemented with bio-based fillers containing hydroxyl and / or carboxyl groups

[0007] Also disclosed herein are finally produced, mycelium-based biopolymer composites (aka biobased polymer composites or mycelium-based biopolymer composites) which have adopted the previously described, attribute-improving chemistry as a result of such chemical modifications. The resulting composites desirably exhibit improved hydrophobicity, mechanical strength, flexibility, and durability.

[0008] The process for modifying mycelium-based biopolymer materials (aka biopolymerbased materials, such as sheets, panels, or slabs produced from grown mycelium, and in one embodiment, aerially grown mycelium) into higher performance composites is highly tunable, enabling a range of final densities, thicknesses (such as in the case of aerially grown mycelium), and material properties for various end-use product applications.

[0009] In one aspect of the disclosure, a mycelium-based biopolymer composite material is disclosed. The mycelium-based biopolymer composite material comprises: (a) a myceliumbased biopolymer sheet, panel, or slab material, at least partially deacetylated to expose amino groups: (b) one or more isocyanates forming covalent urethane and / or urea linkages with said amino groups to form an isocyanate-crosslinked network; and (c) one or more bio-based fillers covalently integrated into the isocyanate-crosslinked network.

[0010] In another aspect of the disclosure, the bio-based fillers comprise alginate, pectin, chitosan, or a mixture thereof.

[0011] In a further aspect of the disclosure, the mycelium-based biopolymer composite material is derived from Ganoderma species of mycelium. In one aspect, the mycelium may have been grown aerially to produce slabs between about 0.5" and 2" thickness and having a dry density between about 1 and 20 lbs / ft3.

[0012] In still a further aspect of the disclosure, the mycelium-based biopolymer composite material further comprises a polyurethane phase formed in situ by reacting diisocyanates with polyols In yet another aspect of the disclosure, the polyurethane phase is interpenetrated with the mycelium-based sheet, panel, or slab material and the bio-based fillers, enhancing flexibility and tensile strength of the resulting sheet, panel, or slab material, compared to that of an untreated but otherwise similarly constructed mycelium-based biopolymer sheet, panel, or slab material.

[0013] In yet another aspect of the disclosure, the resulting mycelium-based biopolymer composite exhibits improved hydrophobicity, decreased water uptake, and enhanced mechanical properties compared to untreated but otherwise similarly constructed mycelium composites.

[0014] In another aspect of the disclosure, a method of producing a mycelium-based biopolymer composite is disclosed The method comprises: (a) providing a grown myceliumbased biopolymer sheet, panel, or slab material; (b) at least partially deacetylating the grown mycelium-based biopolymer sheet, panel, or slab material to convert chitin to chitosan to expose amino groups; (c) introducing one or more isocyanate compounds that react with the exposed amino and hydroxyl groups to form covalent isocyanate crosslinks in a resulting structure; and (d) integrating one or more bio-based fillers into the isocyanate-crosslinked structure

[0015] In a further aspect of the disclosure, the bio-based fillers comprise alginate, pectin, chitosan, or a mixture thereof. In still a further aspect of the disclosure, the integration of biobased fillers occurs prior to, during, or after the isocyanate reaction to form interpenetrating networks

[0016] In yet another aspect of the disclosure, the mycelium-based biopolymer composite is a stable, moisture-resistant composite.

[0017] In still another aspect of the disclosure, the method further comprises adding a polyurethane prepolymer or polyols during the crosslinking step to form a polyurethane-rich composite.

[0018] In yet another aspect of the disclosure, the resulting mycelium-based biopolymer composite exhibits improved hydrophobicity, decreased water uptake, and enhanced mechanical properties compared to untreated, but otherwise similarly constructed mycelium composites.

[0019] In still another aspect of the disclosure, the mycelium-based biopolymer composite prepared by the method described is suitable as a leather-like material with enhanced abrasion resistance.

[0020] In another aspect of the disclosure, a mycelium-based biopolymer composite is disclosed in which the ratio of isocyanate to total available amino groups can be adjusted to tune the mechanical stiffness and water resistance of the final biopolymer composite product.

[0021] In yet another aspect of the disclosure, a mycelium-based biopolymer composite material is disclosed. The mycelium-based biopolymer composite material comprises: (a) a mycelium-based biopolymer sheet, panel, or slab material, at least partially deacetylated to expose amino groups; (b) one or more isocyanates forming covalent urethane and / or urea linkages with said amino groups to form an isocyanate-crosslinked network, said isocyanate- crosslinked network receptive to receiving additional bio-based filler material through further reactions.BRIEF DESCRIPTION OF THE FIGURES

[0022] FIG. 1 represents a process flow diagram for a method for treating and subsequently modifying the chemical structure of grown mycelium-based sheet, panel, or slab materials in accordance with one embodiment of the disclosure.

[0023] FIG. 2 represents a process flow diagram for an alternative method for treating and subsequently modifying the chemical structure of grown aerial mycelium sheet, panel, or slab materials in accordance with one embodiment of the disclosure.DETAILED DESCRIPTION

[0024] Mycelium-based biopolymer materials can be grown into dense, three-dimensional materials, with structures defined by interwoven fungal hyphae. Such biopolymer materials, for instance, upstanding aerial mycelium-based panels or slabs (aka aerial mycelium-based biopolymer material panels or slabs), often consist of chitin, a naturally occurring polymer containing acetylated amino polysaccharides. To enhance durability, hydrophobicity, and mechanical integrity, it is desirable to modify these materials by converting chitin to chitosan and introducing stable crosslinked networks. Incorporating isocyanate chemistry, polyurethane phases, and bio-based fillers (e.g., alginate, pectin, chitosan) can yield a robust, water-resistant, and flexible biopolymer-based composite suitable as a leather-like material or advanced construction element.

[0025] The following discussion presents detailed descriptions of several embodiments of methods for treating and thereby modifying the chemical structures of grown mycelium sheets, panels or slabs, and in some embodiments aerial mycelium-based panels or slabs (aka aerial mycelium-based biopolymer panel or slab materials) in order to enhance their receptivity to subsequently receiving and incorporating supplemental chemistry into their fungal hyphal matrices or networks, which supplemental chemistries enhance the matrices’ physical attributes beyond those which would normally be provided by untreated mycelium sheet or panel matrices themselves. Such enhancements allow such produced sheets, panels, or slabs to be suitable for use in a wider range of consumer and business-to-business products, they’re having a wider range of available physical attributes, following such chemical treatments and subsequent structural modifications.

[0026] The following discussion also presents detailed descriptions of several embodiments of treated and thereby chemically modified mycelium-based sheets, panels, and slabs, and in embodiment, grown aerial mycelium-based biopolymer panels and slabs (aka grown aerial mycelium biopolymer composites) produced by such chemical modification methods.

[0027] In accordance with the disclosure, mycelium sheets, panels, or slabs are first grown in appropriate growth environments on nutritive substrates (as described below and which may include either liquid state or solid state nutritive substrate), and through one of any of a number of harvest steps, are removed from their supporting nutritive substrates to be treated as described herein, for use as textile material for later incorporation into consumer products.

[0028] The treatments described herein would utilize the reactive nature of various crosslinking agents to establish strong bonds thereby transforming the grown, biobased mycelium material into a durable and versatile material. Use of such enhanced mycelium sheets, panels, or slabs as a consumer product base materials would offer several advantages including (1) sustainability, in that the mycelium sheets, panels, or slabs can be grown rapidly on agricultural waste products, (2) customizability, in that the properties of the grown mycelium sheets, panels, or slabs can be tailored by adjusting growth conditions, (3) ethical production, in that no animals would be harmed in the growth of such materials, and (4) potentially reduced environmental impact, in that the processes described may require less water and energy usage, compared to traditional leather production at least.DEFINITIONS

[0029] The my celia of the present disclosure are growth products obtained from a growth matrix (including nutritive substrate) incubated for a period of time (i.e., an incubation time period) in or on a substrate-supporting surface of a support structure (or tool) in a growth environment, as disclosed herein. For the purposes of this disclosure the following terms are given their respective meanings.

[0030] “Mycelium” as used herein refers to a connective network of fungal hyphae, with mycelia being the plural form of mycelium.

[0031] “Hyphae” as used herein refers to branched filament vegetative cellular structures that are interwoven to form mycelium.

[0032] “ Substrate” or “Nutritive substrate” as used herein refers to a material or surface thereof, from or on which an organism lives, grows, and / or obtains its nourishment. In some embodiments, a substrate provides sufficient nutrition to the organism under target growth conditions such that the organism can live and grow without providing the organism a further source of nutrients. A variety of substrates are suitable to support the growth of an aerialmycelium of the present disclosure. Suitable substrates are disclosed, for example, in U. S. Patent Application Publication US2020 / 0239830A1 to O’Brien et al, the entire contents of which are hereby incorporated by reference in their entirety, to the extent not inconsistent with the content of this disclosure. In some embodiments, the substrate is a natural substrate. Nonlimiting examples of a natural substrate include a lignocellulosic substrate, a cellulosic substrate, or a lignin-free substrate. A natural substrate can be an agricultural waste product or one that is purposefully harvested for the intended purpose of food production, including mycelial-based food production. Further non-limiting examples of nutritive substrates suitable for supporting the growth of mycelia of the present disclosure include soy -based materials, oak-based materials, maple-based materials, corn-based materials, seed-based materials and the like, or combinations thereof. The materials can have a variety of particle sizes, as disclosed in US2020 / 0239830A1, and occur in a variety of forms, including shavings, pellets, chips, flakes, or flour, or can be in monolithic form. Non-limiting examples of suitable substrates for the production of mycelia of the present disclosure include com stover, maple flour, maple flake, maple chips, soy flour, chickpea flour, millet seed flour, oak pellets, soybean hull pellets and combinations thereof. Additional useful substrates for the growth of mycelia are disclosed herein, but may also include liquid state nutritive substrates as are known in the art.

[0033] “ Growth media” or “growth medium” as used herein refers to a matrix containing a nutritive substrate and an optional further source of nutrition that is the same or different than the nutritive substrate, wherein the nutritive substrate, the nutrition source, or both are intended for fungal consumption to support mycelial growth.

[0034] “Growth matrix” as used herein refers to a matrix containing a growth medium and a fungus. In some embodiments, the fungus is provided as a fungal inoculum; thus, in such embodiments, the growth matrix comprises a fungal-inoculated growth medium. In other embodiments, the growth matrix comprises a colonized substrate.

[0035] ‘ ‘Inoculated substrate” as used herein refers to a substrate (or nutritive substrate) that has been inoculated with fungal inoculum. For example, an inoculated substrate can be formed by combining an uninoculated substrate with a fungal inoculum. An inoculated substrate can be formed by combining an uninoculated substrate with a previously inoculated substrate. An inoculated substrate can be formed by combining an inoculated substrate with a colonized substrate.

[0036] “ Colonized substrate” as used herein refers to an inoculated substrate that has been incubated for sufficient time to allow for fungal colonization. A colonized substrate of the present disclosure can be characterized as a contiguous hyphal mass grown throughout the entirety of the volume of the growth media substrate. The colonized substrate may further contain residual nutrition that has not been consumed by the colonizing fungus. As is understood by persons of ordinary skill in the art, a colonized substrate has undergone primary myceliation, sometimes referred to by skilled artisans as having undergone a “mycelium run.” Thus, in some particular aspects, a colonized substrate consists essentially of a substrate and a colonizing fungus in a primary myceliation phase. For many fungal species, asexual sporulation occurs as part of normal vegetative growth, and as such could occur during the colonization process. Accordingly, in some embodiments, a colonized substrate of the present disclosure may also contain asexual spores (conidia). In some aspects, a colonized substrate of the present disclosure can exclude growth progression into sexual reproduction and / or vegetative foraging. Sexual reproduction includes fruiting body formation (e.g., primordiation and differentiation) and sexual sporulation (meiotic sporulation). Vegetative foraging includes any mycelial growth away from the colonizing substrate (such as aerial growth). Thus, in some further aspects, a colonized substrate can exclude mycelium that is in a vertical expansion phase of growth. A colonized substrate can enter a mycelial vertical expansion phase during incubation in a growth environment of the present disclosure. For example, a colonized substrate can enter a mycelial vertical expansion phase upon introducing aqueous mist into the growth environment and / or depositing aqueous mist onto colonized substrate and / or any ensuing extra-particle growth. In some embodiments, the use of aqueous mist can be adjusted, for example, to desired levels and timing, to affect the topology, morphology, density, and / or volume of the growth.

[0037] Any suitable substrate or nutritive substrate can be used alone, or optionally combined with a nutrient source, as media to support mycelial growth. In the case of solid-state platforms (not having a continuous liquid phase throughout portions), the growth media can be hydrated to a final target moisture content prior to inoculation with a fungal inoculum. In a non-limiting example, the substrate or growth media can be hydrated to a final moisture content of at least about 50% (w / w), at most about 95% w / w, within a range of about 50% to about 95%, about 50% to about 90%, about 50% to 85%, about 50% (w / w) to about 80% (w / w), about 50%>(w / w) to about 75% (w / w), within a range of about 50% (w / w) to about 65% (w / w), within a range of about 50% (w / w) to about 60% (w / w), or within a range of about 60% (w / w) to about 70% (w / w). Growth media hydration can be achieved via the addition of any suitable source of moisture. In a non-limiting example, the moisture source can be airborne or non-airbome liquid phase water (or other liquids), an aqueous solution containing one or more additives (including but not limited to a nutrient source), and / or gas phase water (or other compound). In some embodiments, at least a portion of the moisture is derived from steam utilized during bioburden reduction of the growth media. In some embodiments, inoculation of the growth media with the fungal inoculum can include a further hydration step to achieve a target moisture content, which can be the same or different than the moisture content of the growth media. For example, if growth media loses moisture during fungal inoculation, the fungal inoculated growth media can be hydrated to compensate for the lost moisture. Liquid state nutritive substrates may also be used in certain embodiments of the disclosure, as further described.

[0038] Methods for the production of extra-particle aerial mycelium sheets, panels, or slabs (and ultimately, aerial mycelium) disclosed herein can include an inoculation stage, wherein an inoculum is used to transport an organism into a nutritive substrate. The inoculum, which carries a desired fungal strain, is produced in sufficient quantities to inoculate a target quantity of nutritive substrate. The inoculation can provide a plurality of myceliation sites (nucleation points) distributed throughout the nutritive substrate. Inoculum can take the form of a liquid, a slurry, or a solid, or any other known vehicle for transporting an organism from one growthsupporting environment to another. Generally, the inoculum comprises water, carbohydrates, sugars, vitamins, other nutrients, and fungi. The inoculum may contain enzymatically available carbon and nitrogen sources (e.g., lignocellulosic biomass, chitinous biomass, carbohydrates) augmented with additional micronutrients (e.g., vitamins, minerals). The inoculum can contain inert materials (e.g., perlite). In a non-limiting example, the fungal inoculum can be a seed-supported fungal inoculum, a feed-grain-supported fungal inoculum, a seed-sawdust mixture fungal inoculum, or another commercially available fungal inoculum, including specialty proprietary spawn types provided by inoculum retailers. In some aspects, a fungal inoculum can be characterized by its density. In some embodiments, a fungal inoculum has a density of about 0.1 gram per cubic inch to about 10 grams per cubic inch, or from about 1 gram per cubicinch to about 7 grams per cubic inch. A skilled person can modify variables including the nutritive substrate or growth media component identities, nutritive substrate or growth media nutrition profile, nutritive substrate or growth media moisture content, nutritive substrate or growth media bioburden, inoculation rate, and inoculum constituent concentrations to arrive at a suitable medium to support mycelial and in some instances extra-particle aerial mycelial growth. In some embodiments, the inoculation rate can be expressed as a percentage of the target volume of the substrate or growth media (% (v / v)). In some embodiments, the inoculation rate can range from about 0.1% (v / v) to about 80% (v / v). In some embodiments, the inoculation rate is at most about 50% (v / v), at most about 45% (v / v), at most about 40% (v / v), at most about 30% (v / v), at most about 25% (v / v), at most about 20% (v / v), at most about 15% (v / v), at most about 10% (v / v) or at most about 5% (v / v). In some embodiments, the inoculation rate is about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), about 5% (v / v), about 6% (v / v), about 7% (v / v), about 8% (v / v), about 9% (v / v), about 10% (v / v), about 11% (v / v), about 12% (v / v), about 13% (v / v), about 14% (v / v), about 15% (v / v), about 16% (v / v), about 17% (v / v), about 18% (v / v), about 19% (v / v), about 20% (v / v), about 21% (v / v), about 22% (v / v), about 23% (v / v), about 24% (v / v), about 25% (v / v), about 26% (v / v), about 27% (v / v), about 28% (v / v), about 29% (v / v) or about 30% (v / v); or any range therebetween. In some embodiments, the inoculation rate can be expressed as a percentage of the target dry mass of the nutritive substrate or growth media (% (w / w)). In some embodiments, the inoculation rate can range from about 0.1% (w / w) to about 80% (w / w). In some embodiments, the inoculation rate is at most about 50% (w / w), at most about 45% (w / w), at most about 40% (w / w), at most about 30% (w / w), at most about 25% (w / w), at most about 20% (w / w), at most about 15% (w / w), at most about 10% (w / w) or at most about 5% (w / w). In some embodiments, the inoculation rate is about 1% (w / w), about 2% (w / w), about 3% (w / w), about 4% (w / w), about 5% (w / w), about 6% (w / w), about 7% (w / w), about 8% (w / w), about 9% (w / w), about 10% (w / w), about 11% (w / w), about 12% (w / w), about 13% (w / w), about 14% (w / w), about 15% (w / w), about 16% (w / w), about 17% (w / w), about 18% (w / w), about 19% (w / w), about 20% (w / w), about 21% (w / w), about 22% (w / w), about 23% (w / w), about 24% (w / w), about 25% (w / w), about 26% (w / w), about 27% (w / w), about 28% (w / w), about 29% (w / w) or about 30% (w / w); or any range therebetween.

[0039] “ Aerial mycelium” as used herein refers to mycelium obtained from extra-particle aerial mycelial growth, and which is substantially free of growth matrix (i.e. solid particulate material).

[0040] ‘ ‘Extra-particle mycelial growth” (EPM) as used herein refers to mycelial growth from a nutritive substrate particle, which can be characterized in some instances, as being “aerial”.

[0041] “Extra-particle aerial mycelial growth” or “extra-particle aerial mycelium” as used herein refers to a distinct mycelial growth that occurs away from and outward from the surface of a growth matrix (including nutritive substrate having solid particulate material). It has not yet been physically removed from the typically underlying, growth matrix (including particulate nutritive substrate), but has grown so that it extends away from the growth matrix, as opposed to merely between portions of growth matrix. In some embodiments, it extends in a generally vertical orientation, perpendicular to the growth matrix (including nutritive substrate) surface, situated in or upon the support structure, substrate-supporting surface. Extra-particle aerial mycelial growth can therefore exhibit negative gravitropism, in that it grows in a direction opposite that of the direction of gravity. In a geometrically unrestricted scenario, extra-particle aerial mycelial growth could be described as being negatively gravitropic, positively gravitropic, or neutrally gravitropic, aerial, and radial in which growth will expand in all directions from its point source.

[0042] ‘ ‘Positive gravitropism” (or positively gravitropic) as used herein refers to growth that preferentially occurs in the direction of gravity.

[0043] “Negative gravitropism” as used herein refers to mycelial growth that preferentially occurs in the direction away from the direction of gravity. As disclosed herein, extra-particle aerial mycelial growth can exhibit negative gravitropism. Without being bound by any particular theory, this may be attributable at least in part to the geometric restriction of the growth format, wherein an uncovered tool having at least a substrate-supporting surface, supports or contains a growth matrix (including nutritive substrate). With such geometric restriction, growth will primarily occur along the unrestricted dimension(s), which in the scenario is primarily vertically (negatively gravitropic) if the tool is positioned such that its surface or opening (in the case of a four solid-walled open container or flat surfaced tool) is facing vertically upward in orientation (and the fungal organism (e.g. hyphae of mycelium) is attracted to airborne mist).

[0044] Aerial my celia (based on extra-particle aerial mycelial growth) of the present disclosure can be grown into panels or slabs in a matter of weeks or days. This feature is of practical value at least in the production of food ingredients or food products, where time and efficiency are at a premium. Accordingly, the presently disclosed method of making an aerial mycelium panels or slabs (off of extra-particle aerial mycelial growth) comprises incubating a growth matrix (including nutritive substrate) in a growth environment for an incubation time period of up to about 3 weeks. In many instances, such aerial mycelium is upstanding in appearance, that is, stands upright to a fairly significant three-dimensional height, as opposed to being a relatively two-dimensional, flat sheet. In some embodiments, the incubation time period can be within a range of about 4 days to about 17 days. In some further embodiments, the incubation time period can be within a range of about 7 days to about 16 days, within a range of about 8 days to about 15 days, within a range of about 9 days to about 15 days, within a range of about 9 days to about 14 days, within a range of about 8 to about 14 days, within a range of about 7 days to about 13 days, or within a range of about 7 days to about 10 days. In some more particular embodiments, the incubation time period can be about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days or about 16 days, or any range therebetween.

[0045] Advantageously, incubating a growth matrix comprising a colonized nutritive substrate (wherein said colonized nutritive substrate comprises a growth medium previously colonized with mycelium of a fungus) in a growth environment of the present disclosure can result in earlier expression of extra-particle aerial mycelial tissue compared to incubation of a growth matrix (including nutritive substrate) comprising substantially the same or a similar growth medium and a fungal inoculum, wherein the fungal inoculum contains a fungus. Accordingly, a method of making an aerial mycelium (based on extra-particle aerial mycelial growth) of the present disclosure can comprise incubating a growth matrix comprising a colonized nutritive substrate (wherein said colonized nutritive substrate comprises a growth medium previously colonized with mycelium of a fungus) in a growth environment for an incubation time period, and producing extra-particle aerial mycelial growth therefrom, wherein the incubation time period is at least about 1 day, at least about 2 days, at least about 3 days, or at least about 4 days less than the incubation time period for producing extra-particle aerial mycelial growthfrom a growth matrix comprising a growth medium and a fungal inoculum, wherein the fungal inoculum comprises a fungus.

[0046] In some other embodiments, the incubation time period ends no later than when a visible fruiting body forms. In a non-limiting example, the incubation time period can end prior to a karyogamy or meiosis phase of the fungal reproductive cycle. In some other embodiments, the incubation time period ends when a visible fruiting body forms. As disclosed herein, aerial mycelia (based on the extra-particle aerial mycelial growth) of the present disclosure can be prepared without the formation of a visible fruiting body, thus, in some embodiments, an incubation time period can end without regard to the formation of a visible fruiting body. Trial incubation runs can be used to inform the period of time in the growth environment during which sufficient extra-particle aerial mycelial growth product occurs (e.g., aerial mycelial growth of a predetermined thickness) without the formation of visible fruiting bodies.ADDITIONAL DEFINITIONS AND METHODS RELATED TO GROWTH ENVIRONMENT

[0047] U. S. Patent Application Publication 2015 / 0033620 to Greetham et al., the entire contents of which is hereby incorporated by reference in its entirety to the extent not inconsistent with the content of this disclosure, describes techniques for growing a material comprising aerial mycelium, referred to in that application as a “mycological biopolymer.” As described therein, a mycological biopolymer product provided by that disclosed method is characterized as containing a homogenous biopolymer matrix that is comprised predominantly of fungal chitin and trace residues (e.g., beta-glucan, proteins). The mycological biopolymer is up-cycled from domestic agricultural lignocellulosic waste and is made by inoculating the substrate made of domestic agricultural lignocellulosic waste with a selected fungus in a container that is sealed off from the ambient environment external to the container. In addition to the substrate and fungal inoculum, the container contains a void space. A network of undifferentiated aerial mycelium comprising a chitin-polymer grows into and fills the void space of the container. The chitin-polymer-based aerial mycelium is subsequently extracted from the substrate and dried. As further described in US2015 / 0033620, the environmental conditions for producing the mycological biopolymer product described therein, i.e., a high carbon dioxide (CO₂) content (about 3% to about 7% by volume) and an elevated temperature(from about 85°F to about 95°F), prevent full differentiation of the fungus into a mushroom, as evidenced by the absence of a visible fruiting body.

[0048] In one aspect, the present disclosure provides a mycelium, alternatively, extra-particle aerial mycelium (and subsequently harvested aerial mycelium) grown using the described tool apparatus, methods, and systems incorporating the same. In a further aspect, the aerial mycelium (based on extra-particle aerial mycelium) does not contain a visible fruiting body.

[0049] As described in International Patent Publication WO2019 / 099474A1 to Winiski et al., the entire contents of which is hereby incorporated by reference in its entirety to the extent not inconsistent with the content of this disclosure, another method of growing a mycological biopolymer material employs incubation of a substrate with nutritive value inoculated with a fungus in containers that are placed in a closed incubation chamber with air flows passed over each container while the chamber is maintained with a predetermined environment of humidity, temperature, carbon dioxide, and oxygen.

[0050] The aerial mycelia (from extra-particle aerial mycelial growth) of the present disclosure, such as for instance in the form of panels or slabs, are growth products obtained from an inoculated nutritive substrate incubated for a period of time (i.e., an incubation time period) in a growth environment, as disclosed herein.

[0051] In some aspects, a method of making an aerial mycelium (from extra-particle aerial mycelial growth) of the present disclosure comprises placing a growth matrix (containing nutritive substrate) in contact with a tool in the described growth environment. In some aspects, the tool can have a substrate-supporting surface having a surface area. In some embodiments, the surface area can be at least about 1 square inch. In some embodiments, the surface area can be at most about 2000 square feet. In some embodiments, the growth matrix (including nutritive substrate) can be placed in contact with the substrate-supporting surface, e.g., placed directly or indirectly on top of or distributed across the substrate- supporting surface. In some embodiments, the substrate-supporting surface can be a planar surface, or a non-planar surface (as further illustrated below with integrally formed physical spacers). Nonlimiting examples of a tool include a tray, a sheet, a screen, a pan or table, a conveyer belt, a net or a web. In some embodiments, the tool can have at least one side wall and a floor. In other embodiments, the at least one side wall and floor can be solid. In still further embodiments, the tool can have four side walls. In another embodiment, the one or more sidewalls can be porous, perforated, or otherwise open. In some embodiments, the substratesupporting surface (such as a floor) and the at least one side wall can together form a cavity. In other embodiments, the support structure may itself include one or more recesses that form one or more cavities (which cavities include one or more substrate-supporting surfaces). In other embodiments, the support structure may itself include one or more recesses and also includes at least one side wall. In some embodiments, the growth matrix can be placed or packed in the tool cavity or cavities. In some embodiments, the tool can be an uncovered tool. In some other embodiments, the tool can have a lid, the lid having at least one opening, or the tool can be covered at least in part with a perforated barrier. Non-limiting embodiments of a tool having a lid with an opening are disclosed in US2015 / 0033620A1. An uncovered tool, or a tool having a lid with an opening or a perforated barrier, and further having growth matrix (including nutritive substrate) on or within the tool, can allow for aqueous mist to be deposited onto the growth matrix (and nutritive substrate) surface, and / or onto any resulting mycelial growth that may be occurring.

[0052] In some embodiments, the tool may include a perforated material, such as a net, scrimlike material, screen, or mesh situated immediately above the growth matrix, through which mycelial hyphae may grow away from the nutritive substrate. The perforated material in one embodiment includes perforations sized to allow for the easy passage of hyphae away from the growth matrix. Such perforated material is in one embodiment formed from a material that is chemically or biologically inert with respect to the growing mycelium. That is, such perforated material does not have any deleterious effects on the growing mycelium. Such perforated material may, in one embodiment, be fashioned from polymeric, metallic, glass, organic, or ceramic substances. For instance such perforated material, may in one embodiment be formed from a nonwoven material or woven web, such as for instance, from a perforated film, a fibrous nonwoven material (such as a spunbond or meltblown web or a paper-like cellulosic material), alternatively from a fibrous woven material such as a cotton scrim, or similar material. Alternatively, such perforated material may be formed from a metallic screen or such. Such perforated material may, in one embodiment, remain with the growth matrix upon removal of the extra-particle aerial mycelium. In an alternative embodiment, such perforated material may be removed along with the extra-particle aerial mycelium upon harvest, such that it provides some advantageous attribute to the removed extra-particle, aerial mycelium (andharvested aerial mycelium itself). For instance, such included perforated material may provide additional strength to the grown mycelium sheet or panel.

[0053] In a further embodiment, at some beneficial point in the growth cycle of the extraparticle aerial mycelium, a secondary perforated material (such as those previously described), may be placed upon the current upper surface of the growing mycelium. The growing mycelium may then be permitted to continue its growth through the secondary perforated material. Such secondary perforated material may then be used to remove an upper portion of the final grown mycelium sheet or panel, in order to provide multiple attributes to the grown extra-particle aerial mycelium (and resulting aerial mycelium). Such attributes may include one or more of at least: a reduction in brittleness of the final product, a reduction in color variation or other textural variation in the final product (at least along the surface from which the secondary perforated material was removed) and simplified later processing of the grown mycelium sheet or panel.

[0054] “Growth environment” as used herein refers to an environment that supports the growth of mycelia, as would be readily understood by a person of ordinary skill in the art in the mycelial cultivation industry, and which contains a growth atmosphere having a gaseous environment of carbon dioxide (CO₂), oxygen (O₂), and a balance of other atmospheric gases including nitrogen (N₂), and is further characterized as having a relative humidity. In some aspects of the present disclosure, the growth atmosphere can have a CO₂ content of at least about 0.02% (v / v), at least about 5% (v / v), less than about 8% (v / v), less than about 10% (v / v), between about 0.02% and 10%, between about 0.02% and 8%, between about 5% and 10%, or between about 5% and 8%. In some other aspects, the growth atmosphere can have an O₂ content of at least about 12% (v / v), or at least about 14% (v / v), and at most about 21% (v / v). In yet other aspects, the growth atmosphere can have an N₂ content of at most about 79% (v / v). Each foregoing CO₂, O₂, or N₂ content is based on a dry gaseous environment, notwithstanding the growth environment atmosphere relative humidity.

[0055] In some further aspects, a method of making an aerial mycelium (from extra-particle aerial mycelial growth) of the present disclosure comprises incubating the growth matrix (including nutritive substrate) in a growth environment, wherein the growth environment has a temperature that supports mycelial growth. In some embodiments, the growth environment has a temperature within a range of about 55°F to about 100°F, or within a range of about 60°Fto about 95°F. In some more particular embodiments, the growth environment has a temperature within a range of about 80°F to about 95°F, or within a range of about 85 °F to about 90°F throughout the incubation time period. In other embodiments, the growth environment has a temperature within a range of about 60°F to about 75°F, within a range of about 65°F to about 75°F, or within a range of about 65°F to about 70°F. In some embodiments, the growth environment temperature can be tuned to optimize for the growth of a particular fungal genus, species, or strain.

[0056] In some aspects of the present disclosure, the growth environment suitable for the growth of the aerial mycelia (from extra-particle aerial mycelial growth) of the present disclosure can be a dark environment. “Dark environment” as used herein in connection with a growth environment would be readily understood by a person of ordinary skill in the art in the mycelial cultivation industry and refers to an environment without natural or ambient light, and without growing lights.

[0057] Exposing fungi to white light, and especially blue light, has been associated with the induction of fruiting and the enhancement of production efficiency of oyster mushrooms (e.g., see Roshita & Goh, AIP Conference Proceedings 2030, 020110 (2018)), the entire contents of which are hereby incorporated by reference in their entirety to the extent not inconsistent with the content of this disclosure. An aerial mycelium for some genus of the present disclosure, such as Ganoderma, absent visible fruiting bodies, can be prepared by the methods of the present disclosure in the presence of white light, which includes blue light. Aerial mycelium (from extra-particle aerial mycelial growth) prepared in the presence of white light was consistent in yield, thickness, density, morphology and in the absence of visible fruiting bodies when compared to control aerial mycelia produced under the same growth conditions but in a dark environment. Thus, in some embodiments, a growth environment suitable for the growth of the aerial mycelia (from extra-particle aerial mycelial growth) of the present disclosure is not a dark environment. In some embodiments, the growth environment does not exclude light. In some embodiments, the growth environment can include natural light. In some embodiments, the growth environment can include ambient light. In some embodiments, the growth environment can include a growing light.

[0058] As disclosed in US2015 / 0033620, environmental conditions for producing a mycological biopolymer include a CO₂ content of about 3% to about 7% (v / v) to prevent fulldifferentiation of the fungus into a mushroom. Accordingly, in some aspects, the present disclosure provides for methods of producing an aerial mycelium (from extra-particle aerial mycelial growth) in a growth environment comprising a growth atmosphere, wherein the growth atmosphere can have a CO₂ content within a range of about 3% (v / v) to about 7% (v / v), or within a range of about 5% (v / v) to about 7% (v / v). In some embodiments, the growth atmosphere can have a CO₂ content of about 3%, about 4%, about 5%, about 6%, or about 7% (v / v), or any range therebetween.

[0059] Aerial mycelium (from extra-particle aerial mycelial growth) of the present disclosure, such as in the form of panels or slabs, can be produced without visible fruiting bodies under conditions wherein aqueous mist is introduced into a growth environment having a growth atmosphere containing much lower CO₂ content. For example, it has been found that aerial mycelia (from extra-particle aerial mycelial growth) obtained from a growth environment of circulating mist and an atmosphere having a mean CO₂ content of about 0.04% (v / v) over the course of the incubation time period or having a mean CO₂ content of about 2% (v / v) over the incubation time period were similar in yield, thickness, density, and morphology to aerial mycelia (from extra-particle aerial mycelial growth) obtained via growth in an atmosphere having a mean CO₂ content of 5% (v / v) but otherwise identical growth conditions. Furthermore, aerial mycelia (from extra-particle aerial mycelial growth) of increased thickness can be obtained via incubation in a growth environment described herein and characterized as having particular misting profiles. The present disclosure advantageously provides for methods of making aerial mycelia (from extra-particle aerial mycelial growth) of increased thickness, absent visible fruiting bodies, by adopting preselected misting profiles and employing misting deposition methodologies, without requiring a high CO₂ content growth environment. The ability to increase aerial mycelial thickness (from extra-particle aerial mycelial growth), absent visible fruiting bodies, by tuning mist deposition uniformity and rate can also advantageously reduce incubation time periods, thereby allowing more efficient production of aerial mycelia (from extra-particle aerial mycelial growth) and reduced risk of microbial contamination that can occur in high moisture environments.

[0060] Thus, the present disclosure provides for methods of growing aerial mycelia (from extra-particle aerial mycelial growth) in a growth environment comprising a growth atmosphere having markedly reduced CO₂ content and with more uniform deposition of mistand / or control of mist placement, should targeted mist placement be necessary or desirable. Accordingly, in some embodiments, the growth atmosphere CO₂ content can be less than about 3% (v / v). In some embodiments, the growth atmosphere CO₂ content can be no greater than about 2.9% (v / v), no greater than about 2.8% (v / v), no greater than about 2.7% (v / v), no greater than about 2.6% (v / v) or no greater than about 2.5% (v / v). In some further embodiments, the growth atmosphere CO₂ content can be less than 2.5% (v / v). In some embodiments, a growth atmosphere of the present disclosure can have a CO₂ content of at least about 0.02% (v / v). In some embodiments, a growth atmosphere of the present disclosure can have a CO₂ content of at least about 0.03% (v / v). In some further embodiments, the growth atmosphere CO₂ content can approximate ambient atmospheric CO₂ content; for example, the growth atmosphere CO₂ content can be at least about 0.04% (v / v). In some more particular embodiments, the growth atmosphere CO₂ content can be within a range of about 0.02% to about 3% (v / v), about 0.02% to about 2.5% (v / v), about 0.03% to about 3% (v / v), about 0.03% to about 2.5% (v / v), about 0.04% to about 3% (v / v), or about 0.04% to about 2.5% (v / v).

[0061] In other embodiments, the growth atmosphere CO₂ content can be within a wider range. Thus, in some embodiments, the growth atmosphere CO₂ content can be within a range of about 0.02% to about 7% (v / v), within a range of about 0.04% to about 7% (v / v), within a range of about 0.1% to about 7% (v / v), within a range of about 0.2% to about 7% (v / v), within a range of about 1% to about 7% (v / v), or within a range of about 2% to about 7% (v / v); or can be within a range of about 0.02% to about 5% (v / v), within a range of about 0.04% to about 5% (v / v), within a range of about 0.1% to about 5% (v / v), within a range of about 0.2% to about 5% (v / v), or within a range of about 1% to about 5% (v / v). In some more particular embodiments, the growth atmosphere CO₂ content can be about 1%, about 2%, about 3%, or any range therebetween. In yet other embodiments, the growth atmosphere CO₂ content can be a mean CO₂ content over the course of the incubation time period. In some embodiments, the growth atmosphere mean CO₂ content can be less than about 3% (v / v), less than 2.5% (v / v), or no greater than about 2% (v / v) over the course of the incubation time period.

[0062] It is understood that fungal growth requires respiration, which can increase CO₂ content and decrease oxygen (O₂) content in the growth atmosphere, particularly in an enclosed or substantially enclosed growth environment such as an “incubation chamber” or “growth chamber.” In some aspects, the present disclosure provides for a growth environment havinga growth atmosphere that is maintained during the incubation time period by replenishing the growth environment with one or more of the atmospheric gases, such as CO₂, replenishing the growth environment with air having the same composition as the target growth atmosphere composition, venting the growth environment to reduce content of one or more gases, or a combination thereof. In a non-limiting example, if the CO₂ content in a growth chamber is below a target set point, CO₂ gas can be infused into the growth chamber. Conversely, if the CO₂ content exceeds a target set point, then fresh air having the target growth atmosphere composition can be introduced into the growth chamber while venting the chamber to release the existing air having the high CO₂ content. Accordingly, growth chamber atmospheric content can be maintained via CO₂ and fresh air infusion to maintain a target CO₂ set point; as such, O₂ and other atmospheric components are maintained indirectly and fluctuate as a function of fungal respiration. In some other aspects, the present disclosure provides for a growth environment wherein the growth atmosphere CO₂ and O₂ contents are allowed to modulate with fungal respiration, without adjusting the growth atmosphere to maintain preselected CO₂ or O₂ content. Thus, the growth environment can be a closed system. The present disclosure also provides for a growth environment wherein the growth atmosphere CO2 and O2 contents are allowed to modulate with fungal respiration, and further allowing for adjustments to be made to the growth atmosphere under conditions wherein a particular preselected growth atmospheric condition is breached. In a non-limiting example, an aerial mycelium (from extra-particle aerial mycelial growth) can be grown in a growth atmosphere that allows for natural fungal respiration to occur, with a preselected CO2 content ranging from about 0.02% to about 7% CO2 (v / v), wherein the CO2 content is adjusted (e.g., by injection of CO2 into the growth atmosphere) if the CO2 content falls outside the scope of the preselected range.

[0063] A growth environment of the present disclosure can be further characterized as having an atmosphere having a pressure as would be readily understood by a person of ordinary skill in the art in the mycelial cultivation industry. In a non-limiting embodiment, a growth atmosphere of the present disclosure can have an atmospheric pressure within a range of about 27 to about 31 inches of mercury (Hg), can have an atmospheric pressure of about 29 to about 31 inches Hg, or can have an atmospheric pressure of about 29.9 inches Hg. In someembodiments, a growth environment of the present disclosure can be characterized as having an ambient atmospheric pressure.

[0064] In some aspects of the present disclosure, the growth environment suitable for the growth of the aerial mycelia (from extra-particle aerial mycelial growth) of the present disclosure is characterized as having an airflow. In some further aspects, the air composition of the airflow can be substantially the same as the composition of the growth environment atmosphere. In some embodiments, an airflow can be used to direct and / or deposit aqueous mist that is present in the growth environment towards or onto a growth matrix (including nutritive substrate) and / or growing mycelium. The skilled person can adopt various means of directing the flows of air, including baffles, perforated barriers, airflow boxes and / or other tools that can be suitably positioned in the growth environment or in relation to tools (or beds) containing growth matrix (including nutritive substrate) in order to achieve the desired outcome, including a somewhat or substantially homogeneous airflow, with respect to direction and / or velocity, across a plurality of growth matrices (including nutritive substrate(s)) in the growth environment, and / or a somewhat or substantially homogeneous introduction and / or deposition of mist in the growth environment.

[0065] ‘ ‘Horizontal airflow” as used herein refers to flows of air directed substantially parallel to the surface of a growth matrix (including nutritive substrate) and any subsequent extraparticle mycelial growth (aerial or otherwise).

[0066] In some other aspects, the method of preparing an aerial mycelium (from extra-particle aerial mycelial growth) of the present disclosure can include directing an airflow through the growth environment. In some embodiments, the airflow can be a relatively high airflow environment, wherein the airflow can have a velocity of greater than about 250 linear feet per minute (Ifm). In other embodiments, the airflow can be a relatively lower airflow environment, wherein the airflow can have a velocity of less than about 150 Ifm, less than about 125 Ifm, less than about 100 Ifm, or less than about 75 Ifm. In some more particular embodiments, the growth environment can have an airflow, wherein the airflow velocity is less than about 50 Ifm, less than about 40 Ifm, less than about 30 Ifm, or less than about 25 Ifm.

[0067] In some embodiments, the airflow is a substantially horizontal airflow. In some embodiments, the substantially horizontal air flow can have a velocity of no greater than about 350 Ifm, or a velocity no greater than about 300 Ifm. In other embodiments, the substantiallyhorizontal airflow can have a velocity of no greater than about 275 Ifm, a velocity of no greater than about 175 Ifm, a velocity of no greater than about 150 Ifm, a velocity of no greater than about 125 Ifm, or a velocity of no greater than about 110 Ifm. In some further embodiments, the velocity is at least about 5 Ifm, at least about 10 Ifm, at least about 15 Ifm, at least about 20 Ifm, at least about 25 Ifm, at least about 30 Ifm, at least about 35 Ifm, at least about 40 Ifm, at least about 45 Ifm or at least about 50 Ifm. In some more particular embodiments, the substantially horizontal airflow has mean velocity of about 5 Ifm, about 10 Ifm, about 15 Ifm, about 20 Ifm, about 25 Ifm, about 30 Ifm, about 35 Ifm, about 40 Ifm, about 45 Ifm, about 50 Ifm, about 55 Ifm, about 60 Ifm, about 65 Ifm, about 70 Ifm, about 75 Ifm, about 80 Ifm, about 85 Ifm, about 90 Ifm, about 95 Ifm, about 100 Ifm, about 105 Ifm, about 110 Ifm, about 115 Ifm, or about 120 Ifm. In some more particular embodiments still, the substantially horizontal air flow can have a velocity within a range of about 5 Ifm to about 125 Ifm, within a range of about 5 Ifm to about 100 Ifm, within a range of about 5 Ifm to about 75 Ifm, or within a range of about 5 Ifm to about 50 Ifm. In yet more particular embodiments, the substantially horizontal air flow can have a velocity within a range of about 5 Ifm to about 40 Ifm, or within a range of about 5 to about 25 Ifm. In other embodiments, the substantially horizontal air flow can have a velocity within a range of about 40 Ifm to about 120 Ifm. Without being bound to any particular theory, the flows of air can facilitate the distribution of mist throughout the growth environment, can facilitate the distribution of mist onto the growth matrix (including nutritive substrate) surface and / or extra-particle mycelial growth (such as aerial), or both. The air flow and misting methods and associated apparatus, can be tuned in concert to achieve the desired mist deposition rate and / or mean mist deposition rate, and to tune the mycelial tissue morphology.

[0068] In some embodiments, aerial mycelia (from extra-particle aerial mycelial growth) can be prepared by exposing a growth matrix to aqueous mist throughout a portion of the incubation time period (e.g., by introducing mist into the growth environment throughout a portion of the incubation time period). Applicant has measured vertical expansion kinetics of mycelia over the course of an entire incubation period and has characterized the kinetics as having a primary myceliation phase and a vertical expansion phase. The primary myceliation phase included days 1 to 3 of the incubation time period. Introducing aqueous mist throughout a portion of the incubation time period (wherein the portion included the vertical expansionphase), and not introducing aqueous mist on days 1 to 3 of the incubation time period was sufficient to produce aerial mycelium (from extra-particle aerial mycelial growth) having substantially similar characteristics to aerial mycelia (from extra-particle aerial mycelial growth) obtained by depositing mist throughout the entire incubation period.

[0069] The desired airborne mist concentration value, and / or the control of the airborne mist concentration level in response to the mist concentration value, for improved growth may be different during different phases of the growing cycle (including zero). Further, the desired airborne mist concentration value, and / or the control of the airborne mist concentration level in response to the mist concentration value, for improved growth may also be different based on the organism generating the aerial mycelia (from extra-particle aerial mycelial growth). Some aspects of the present disclosure provide for a method of growing an aerial mycelium (from extra-particle aerial mycelial growth) comprising exposing a growth matrix (including nutritive substrate) to a growth environment comprising aqueous mist throughout the incubation time period (e.g., by introducing aqueous mist into the growth environment throughout the incubation time period, i.e., throughout the entire incubation time period). In other aspects, the present disclosure provides for a method of making an aerial mycelium (from extra-particle aerial mycelial growth) comprising exposing a growth matrix (including nutritive substrate) to aqueous mist throughout a portion of the incubation time period (e.g., by introducing aqueous mist into the growth environment throughout a portion of the incubation time period). In some embodiments, a portion of the incubation time period can comprise a vertical expansion phase. In some further embodiments, a portion of the incubation time period can further comprise at least a portion of a primary myceliation phase. In some other embodiments, a portion of the incubation time period can exclude a primary myceliation phase. In yet some other embodiments, a portion of the incubation time period can comprise a vertical expansion phase. Accordingly, in some aspects, introducing aqueous mist into a growth environment throughout a portion of an incubation time period can comprise introducing aqueous mist into the growth environment throughout a vertical expansion phase. In some embodiments, introducing aqueous mist into the growth environment throughout a portion of the incubation time period can comprise introducing aqueous mist into the growth environment throughout a vertical expansion phase and can exclude introducing aqueous mist during the primary myceliation phase. In some embodiments, the portion of the incubation time periodcan terminate at the end of a vertical expansion phase or can terminate at the end of an incubation time period.

[0070] In some other aspects, a portion of an incubation time period can begin during a first day, a second day, a third day or a fourth day of the incubation time period. Accordingly, in some aspects, introducing aqueous mist into a growth environment throughout a portion of an incubation time period can comprise introducing aqueous mist into the growth environment during a first, a second, a third or a fourth day of the incubation time period. In some embodiments, the portion of the incubation time period can terminate at the end of a vertical expansion phase or can terminate at the end of an incubation time period.

[0071] “Dry mass (DM) yield” as used herein refers to the bone-dry mass yield of aerial mycelium (from extra-particle aerial mycelial growth) from a standard mass of solid nutritive substrate. This is representative of the bioefficiency of the organism in converting the solidnutritive substrate components into harvestable aerial mycelium.ADDITIONAL EMBODIMENTS RELATED TO MYCELIUM-BASED BIOPOLYMER COMPOSITE MATERIALS

[0072] The described grown mycelium-based biopolymer sheets, panels, and slabs, which are to be first deacetylated for increased receptivity to further chemical treatments / and subsequent structural modifications, may be provided by any of the preceding described growth methods. Following deacetylation step(s), the deacetylated mycelium-based biopolymer sheets, panels, and slabs may then be exposed to a variety of chemistries in order to take advantage of exposed reactive sites (either amino or hydroxyl) on the chitin / chitosan chemistry in the myceliumbased biopolymer network of the material. Through the incorporation (or interpenetration) of polyurethane, via external incorporation or in situ formation by reaction between the isocyanate and a polyol, into the material and the addition of bio-based fillers, additional binding of the material may be encouraged, so as to tune the produced bio-based composite (mycelium and polyurethane) physical properties further, such as for instance, water resistance, prevention of reswell (or hydrophobicity), density, and other various attributes.

[0073] In FIG. 1 an embodiment of a method for producing mycelium-based biopolymer composites with isocyanate crosslinking and integrated bio-based fillers 5 is described. In such embodiment, a grown mycelium sheet or upstanding aerial mycelial panel (or alternatively aslab) material is first provided 10 for treatment through a subsequent deacetylation step. Such mycelium material may be grown through processes previously described, harvested, and subsequently treated in accordance with the following process steps. The provided grown mycelium sheet, panel, or slab is treated with a strong basic (alkaline) or acidic solution to at least partially convert chitin in the material to chitosan and introduce free amino groups, which will increase site density for subsequent chemical modifications 20. Following this deacetylating step 20, the deacetylated mycelium material (whether it be the sheet, panel, or slab), is exposed to isocyanate compounds (one or more), in order to react them with available amino or hydroxyl groups in the deacetylated mycelium material 30. Following this isocyanate crosslinking step, the isocyanates may be reacted with / in presence of suitable polyols, integrating polyurethanes within the material to form a composite network (or composite). Following this reaction step 40, bio-based fillers are added to interact with the isocyanates to form amide and urethane bonds to the composite material 50. Following the addition of biobased fillers, optional post-processing steps may be initiated to stabilize the crosslinked composite network. Such post-processing steps may impart desired mechanical and / or aesthetic properties to the composite material.

[0074] In an alternative embodiment of an inventive method for producing mycelium-based biopolymer composites with isocyanate crosslinking and integrated bio-based fillers, as illustrated in FIG. 2, a grown aerial mycelium panel or slab material is provided for treatment through a subsequent deacetylation step 70. The provided aerial mycelium sheet, panel, or slab is treated with a strong basic (alkaline) solution to partially at least convert chitin to chitosan in the material, for subsequent chemical modification at free amino group sites 80. Following this deacetylation (or deacetylating) step, the now deacetylated mycelium material is exposed to isocyanate compound(s) to react with available amino or hydroxyl groups in the deacetylated mycelium 90. Isocyanates are reacted with suitable polyols to integrate polyurethanes to form a composite network of the mycelium 100. Following the formation of this network, bio-based fillers are added to the composite network 110, and then optionally further post-processing steps are implemented.

[0075] As previously noted, in various embodiments, a grown mycelium material (such as a sheet or aerial grown panel or slab) is first provided. Such aerial grown mycelium panel or slab may in one embodiment, be less than about 10 inches in thickness, alternatively, be lessthan about 5 inches in thickness, alternatively, be up to about 2 inches in thickness, resulting in a chitin-rich, porous but structurally coherent material. For instance, the thickness may be greater than 0 inches, such as about 0.1 inches or more, such as about 0.2 inches or more, such as about 0.3 inches or more, such as about 0.4 inches or more, such as about 0.5 inches or more, such as about 0.8 inches or more, such as about 1 inch or more, such as about 1.2 inches or more, such as about 1.4 inches or more, such as about 1.5 inches or more, such as about 1.6 inches or more, such as about 1.8 inches or more. The thickness may be about 10 inches or less, such as about 9 inches or less, such as about 8 inches or less, such as about 7 inches or less, such as about 6 inches or less, such as about 5 inches or less, such as about 4 inches or less, such as about 3 inches or less, such as about 2.8 inches or less, such as about 2.6 inches or less, such as about 2.4 inches or less, such as about 2.2 inches or less, such as about 2 inches or less, such as about 1.8 inches or less, such as about 1.6 inches or less, such as about 1.4 inches or less, such as about 1.2 inches or less.

[0076] In one embodiment, the dry density of such aerially grown mycelium panels or slabs can range from about 1 to 20 lbs / ft3, enabling a wide selection of starting materials suitable for various uses. For instance, the dry density may be about 1 lb / ft3or more, such as about 1.2 lb / ft3or more, such as about 1.4 lb / ft3or more, such as about 1.6 lb / ft3or more, such as about 1.8 lb / ft3or more, such as about 2 lb / ft3or more, such as about 2.2 lb / ft3or more, such as about 2.4 lb / ft3or more, such as about 2.6 lb / ft3or more, such as about 2.8 lb / ft3or more, such as about 3 lb / ft3or more, such as about 3.2 lb / ft3or more, such as about 3.4 lb / ft3or more, such as about 3.6 lb / ft3or more, such as about 3.8 lb / ft3or more, such as about 4 lb / ft3or more, such as about 4.2 lb / ft3or more, such as about 4.4 lb / ft3or more, such as about 4.6 lb / ft3or more, such as about 4.8 lb / ft3or more, such as about 5 lb / ft3or more, such as about 5.5 lb / ft3or more, such as about 6 lb / ft3or more, such as about 7 lb / ft3or more, such as about 8 lb / ft3or more, such as about 10 lb / ft3or more, such as about 12 lb / ft3or more, such as about 14 lb / ft3or more, such as about 16 lb / ft3or more, such as about 18 lb / ft3or more. The dry density may be about 20 lb / ft3or less, such as about 18 lb / ft3or less, such as about 16 lb / ft3or less, such as about 14 lb / ft3or less, such as about 12 lb / ft3or less, such as about 10 lb / ft3or less, such as about 8 lb / ft3or less, such as about 6 lb / ft3or less, such as about 5 lb / ft3or less, such as about 4 lb / ft3or less, such as about 3.8 lb / ft3or less, such as about 3.6 lb / ft3or less, such as about 3.4 lb / ft3or less, such as about 3.2 lb / ft3or less, such as about 3 lb / ft3or less, such as about 2.8 lb / ft3or less,such as about 2.6 lb / ft3or less, such as about 2.4 lb / ft3or less, such as about 2.2 lb / ft3or less, such as about 2 lb / ft3or less, such as about 1.8 lb / ft3or less, such as about 1.6 lb / ft3or less, such as about 1.4 lb / ft3or less, such as about 1.2 lb / ft3or less.

[0077] Following the provision of the mycelium-based biopolymer sheet, panel, or slab material, the provided mycelium-based biopolymer sheet, panel, or slab material is deacetylated using either a strong alkaline or acidic solution. For instance, the mycelium sheet, panel, or slab material may be treated with a strong alkaline solution. In one embodiment, the strong alkaline solution may comprise a hydroxide. For instance, the hydroxide may comprise sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonium hydroxide, or a mixture thereof. In one embodiment, the hydroxide may comprise sodium hydroxide. In an alternative embodiment, the strong alkaline solution may comprise a carbonate or bicarbonate, such as sodium carbonate or sodium bicarbonate.

[0078] The alkaline solution may be introduced using various techniques generally known in the art. For instance, it may be introduced using vacuum infusion (e.g., to provide uniform penetration), though passive diffusion (e.g., submerging material without external forces) or pressure impregnation (e.g., applying pressure to force solution into the material). In one embodiment, the alkaline solution may be introduced via vacuum infusion. In one embodiment, the alkaline solution may be introduced through though passive diffusion.

[0079] The deacetylation may be conducted at an elevated temperature in one embodiment. For instance, the temperature may be from about 60-100 degrees C, such as from about 80-100 degrees C. Such deacetylation may at least partially convert chitin in the material to chitosan. This process introduces free amino groups (-NH2) that can increase the site density for subsequent chemical modifications (chemical structure changes). In particular, deacetylation of chitin converts it at least partially into chitosan, exposing more amino groups (-NH2) by removing acetyl groups. This step enhances reactivity by increasing the number of available functional groups for subsequent reactions. Specifically, the reaction changes chitin — chitosan using mechanisms, such as via alkaline hydrolysis (e.g., with NaOH at high temperatures). As a result, ample free amino (-NH2) and hydroxyl (-OH) groups are available for bonding. For instance, and without intending to be limited, the deacetylation may allow for increased accessibility of the hydroxyl groups within the chitin.

[0080] Following the deacetylating step, neutralization may optionally be performed. For instance, the sheet, panel, or slab may be pressed to remove excess alkali. In addition, or alternatively, the sheet, panel, or slab may be rinsed with water. Such pressing and / or rinsing may be conducted to lower the pH.

[0081] In addition, it may be submerged in a weak acid solution, such as a citric acid solution. For instance, the pH may be monitored until about neutral pH is achieved. In certain embodiments, thereafter, a rinse neutralization procedure may follow, involving a rinse, compression to a desired thickness, such as 6 mm, using appropriate means such as a roller press, and re-soaking in water. The process may be repeated, such as 3-4 times, until the sheet, panel, or slab is neutralized. Notably, the number of compression steps applied throughout the process is indicated to have independent positive effects on the final mechanical properties of the material.

[0082] Following the deacetylating step and if conducted, the neutralization step, isocyanate compounds are introduced to react with the available amino (-NH2) and hydroxyl (-OH) groups in the deacetylated mycelium-based biopolymer material. In one embodiment, the isocyanate compounds may react with the amino groups. In one embodiment, the isocyanate compounds may react with the hydroxyl groups. In one embodiment, the isocyanate compounds may react with the amino groups and the hydroxyl groups.

[0083] The isocyanate compounds may include diisocyanate compounds in one embodiment. In one embodiment, the isocyanate compounds may include triisocyanate compounds.

[0084] The isocyanate compounds may include aromatic diisocyanates, aliphatic diisocyanates, polymeric diisocyanates, oligomeric diisocyanates, or a mixture thereof. In one embodiment, the isocyanate compounds may include aromatic diisocyanates. In one embodiment, the isocyanate compounds may include aliphatic diisocyanates. In one embodiment, the isocyanate compounds may include polymeric diisocyanates. In one embodiment, the isocyanate compounds may include oligomeric diisocyanates.

[0085] The aromatic diisocyanates may include, but are not limited to, methylene diphenyl diisocyanate (MDI), toluene diisocyanate (TDI), or a mixture thereof. In one embodiment, the isocyanate compounds, in particular the diisocyanate compounds such as the aromatic diisocyanates, may include methylene diphenyl diisocyanate (MDI). In one embodiment, the isocyanate compounds, in particular the diisocyanate compounds such as the aromaticdiisocyanates, may include toluene diisocyanate (TDI). In one embodiment, the isocyanate compounds, in particular the diisocyanate compounds such as the aromatic diisocyanates, may include a mixture of methylene diphenyl diisocyanate (MDI) and toluene diisocyanate (TDI).

[0086] The aliphatic diisocyanates may include, but are not limited to, hexamethylene diisocyanate (HDI), isophorone diisocyanate (IPDI), or a mixture thereof. In one embodiment, the isocyanate compounds, in particular the diisocyanate compounds such as the aromatic diisocyanates, may include hexamethylene diisocyanate (HDI). In one embodiment, the isocyanate compounds, in particular the diisocyanate compounds such as the aromatic diisocyanates, may include isophorone diisocyanate (IPDI). In one embodiment, the isocyanate compounds, in particular the diisocyanate compounds such as the aromatic diisocyanates, may include a mixture of hexamethylene diisocyanate (HDI) and isophorone diisocyanate (IPDI).

[0087] The isocyanate compounds may be introduced using various techniques generally known in the art. For instance, it may be introduced using vacuum infusion (e.g., to provide uniform penetration), though passive diffusion (e.g., submerging material without external forces) or pressure impregnation (e.g., applying pressure to force solution into the material). In one embodiment, the isocyanate compounds may be introduced via vacuum infusion. In one embodiment, the isocyanate compounds may be sprayed onto the sheet, panel, or slab.

[0088] When introducing the isocyanate compounds, they may be via a solution comprising the isocyanate compounds in a liquid carrier. The liquid carrier may be water, an organic liquid, an alcohol, or a mixture thereof. In one embodiment, the liquid carrier may include water. In one embodiment, the liquid carrier may include an organic liquid, such as acetone. In one embodiment, the liquid carrier may include an alcohol, such as ethanol.

[0089] The isocyanate chemistry linkages are available for crosslinking. Isocyanate compounds contain the reactive -N C=O group. These groups react with nucleophiles to form stable urethane or urea linkages. These reactions occur through the following reaction pathways:(a) with amino groups:■ N=C=O + -NH2→ Urea linkage (–NH–C(=O)–NH–)■ These can form strong covalent bonds form between isocyanates and the amino groups on chitosan and residual chitin.(b) with hydroxyl groups:■ N=C=0 + OH Urethane Linkage(-NH C(=O) O ) * Hydroxyl groups in chitin or chitosan participate in forming additional covalent crosslinks.

[0090] The crosslinking may be conducted at conditions that would promote reaction and crosslinking between the isocyanate compounds and the available functional groups. For instance, this may be at a temperature of from about 40-100 degrees C, such as from about 40-80 degrees C. The crosslinking / reaction time may be any desired time for obtaining the desired crosslink density.

[0091] Without intending to be limited, the reaction may form a stable urethane or urea linkage, creating a hydrophobic, crosslinked network (composite network) that reduces water uptake and enhances overall structural integrity. In this regard, the described crosslinking reactions impart water-resistance as the urea and urethane linkages are hydrophobic and prevent re-swelling. The described crosslinking reactions also impart mechanical strength as the crosslinked networks resist deformation and enhance durability.

[0092] In addition, polyurethane can be formed in situ by reacting isocyanate compounds, such as diisocyanate compounds, particularly those mentioned above, with suitable polyols. This polyurethane phase can interpenetrate the mycelium material and co-crosslink with it, providing improved elasticity, toughness, and dimensional stability. In this regard, the polyurethane, which can be a flexible polymer, can infuse the material.

[0093] The polyols may include low molecular weight polyol compounds, polyester polyols, polyether polyols, or a mixture thereof. In one embodiment, the polyols may include low molecular weight polyol compounds. In one embodiment, the polyols may include polyester polyols. In one embodiment, the polyols may include polyether polyols.

[0094] The polyols, in particular the low molecular weight polyol compounds, may include, but are not limited to, glycerol, sorbitol, mannitol, pentaerythritol, or a mixture thereof. In one embodiment, the polyols comprise glycerol. In one embodiment, the polyols comprise sorbitol. In one embodiment, the polyols comprise a mixture of glycerol and sorbitol.

[0095] The polyols, in particular the polyether polyols, may include polyethylene glycol, polypropylene glycol, polytetramethylene ether glycol, or a mixture thereof. In one embodiment, the polyols may include polyethylene glycol. In one embodiment, the polyolsmay include polypropylene glycol. In one embodiment, the polyols may include polytetramethylene ether glycol.

[0096] The polyols may be introduced using various techniques generally known in the art. For instance, it may be introduced using vacuum infusion (e.g., to provide uniform penetration), though passive diffusion (e.g., submerging material without external forces) or pressure impregnation (e.g., applying pressure to force solution into the material). In one embodiment, the polyols may be introduced via vacuum infusion. In one embodiment, the polyols may be introduced through though passive diffusion.

[0097] When introducing the polyols, they may be via a solution comprising the isocyanate compounds in a liquid carrier. The liquid carrier may be water, an organic liquid, an alcohol, or a mixture thereof. In one embodiment, the liquid carrier may include water. In one embodiment, the liquid carrier may include an organic liquid, such as acetone. In one embodiment, the liquid carrier may include an alcohol, such as ethanol.

[0098] In some embodiments, the polyols may be provided with the isocyanate compounds. For instance, instead of providing the polyols post crosslinking reaction of the isocyanate compounds with the sheet, panel, or slab, the polyols may be provided at the same time. In one embodiment, the polyols may be provided in the same composition or solution. For instance, as indicated herein, the isocyanate compounds may be provided in a solution in a liquid carrier in one embodiment. In this regard, the polyols may also be provided in such solution. As indicated herein, the liquid carrier may be water, an organic liquid, an alcohol, or a mixture thereof. In one embodiment, the liquid carrier may include water. In one embodiment, the liquid carrier may include an organic liquid, such as acetone. In one embodiment, the liquid carrier may include an alcohol, such as ethanol.

[0099] The isocyanate groups in polyurethane can also directly bond with free amino and hydroxyl groups in the mycelium-based biopolymer sheet, panel, or slab bio-polymer material, creating a hybrid network. As a result, polyurethane adds flexibility, durability, and elasticity while reinforcing the hydrophobic network created by the isocyanate crosslinking.

[0100] Following the formation of a polyurethane phase in the mycelium, bio-based fillers may be included in the material. One or more of such fillers, exemplified by alginate, pectin, and / or chitosan (or combinations thereof) can be included in the material. The fillers may comprise alginate, pectin, chitosan, or a mixture thereof. In one embodiment, the fillers maycomprise alginate. In one embodiment, the fillers may comprise pectin. In one embodiment, the fillers may comprise chitosan. In one embodiment, the fillers may comprise a mixture of at least two of alginate, pectin, and chitosan.

[0101] The fillers may be introduced using various techniques generally known in the art. For instance, it may be introduced using vacuum infusion (e.g., to provide uniform penetration), though passive diffusion (e.g., submerging material without external forces) or pressure impregnation (e.g., applying pressure to force solution into the material). In one embodiment, the fillers may be introduced via vacuum infusion. In one embodiment, the fillers may be introduced through though passive diffusion.

[0102] When introducing the fillers, they may be via a solution comprising the fillers in in a liquid carrier. The liquid carrier may be water, an organic liquid, an alcohol, or a mixture thereof. In one embodiment, the liquid carrier may include water. In one embodiment, the liquid carrier may include an organic liquid, such as acetone. In one embodiment, the liquid carrier may include an alcohol, such as ethanol.

[0103] In some embodiments, the fillers may be provided with the isocyanate compounds. For instance, instead of providing the fillers post crosslinking reaction of the isocyanate compounds with the sheet, panel, or slab, the polyols may be provided at the same time. In one embodiment, the fillers may be provided in the same composition or solution. For instance, as indicated herein, the isocyanate compounds may be provided in a solution in a liquid carrier in one embodiment. In this regard, the fillers may also be provided in such solution. As indicated herein, the liquid carrier may be water, an organic liquid, an alcohol, or a mixture thereof. In one embodiment, the liquid carrier may include water. In one embodiment, the liquid carrier may include an organic liquid, such as acetone. In one embodiment, the liquid carrier may include an alcohol, such as ethanol.

[0104] As indicated herein, in some embodiments, the at least partially deacetylated sheet, panel, or slab is first crosslinked using the isocyanate compounds prior to exposure and introduction of the fillers. In some embodiments, the at least partially deacetylated sheet, panel, or slab may first be exposed to the fillers. Thereafter, the sheet, panel, or slab may be exposed to the isocyanate compounds and / or the polyols as mentioned herein. In some embodiments, crosslinking may be conducted while both are present within the sheet, panel,or slab. Without intending to be limited, this may allow for simultaneous formation of a multinetwork, in particular a dual-network, system.

[0105] These chemistries, containing hydroxyl and / or carboxyl functionalities, interact with isocyanates to form urethane and amide bonds, respectively. This secondary biopolymer network can add toughness, control moisture dynamics, and enhance biocompatibility.

[0106] In particular, integration of bio-based fillers may occur with a variety of fillers. Alginate for instance, includes carboxyl (-COOH) reactive groups. Its interaction with isocyanates is illustrated by the following reaction:−N=C=O + −COOH → AmideLinkage(−NH−C(=O)−). Such reaction forms ionic and covalent bonds, enhancing moisture retention and network integrity,

[0107] With the case of pectin, the material includes hydroxyl (-OH) and carboxyl (-COOH) reactive groups. One or more of such groups react with isocyanates in a similar pathway as alginate for amide and urethane formation. Such reactions provide a secondary hydrophilic network for flexibility and water binding.

[0108] With respect to chitosan, such material includes the amino (-NH₂) and hydroxyl (-OH) reactive groups (similar to chitin). With respect to isocyanates, such reactive groups form urea and urethane linkages to integrate chitosan into the network. Such linkages provide structural reinforcement and additional biocompatibility.

[0109] Secondary bio-based networks are essentially formed. The isocyanate crosslinks form the primary network. The bio-based fillers (such as alginate, pectin, and / or chitosan) form the secondary interpenetrating network, stabilized by isocyanate bonds. Benefits of this reinforced network include enhanced mechanical and thermal stability, resistance to moisture while retaining flexibility, and continued biodegradability and sustainability material properties.

[0110] In general, the crosslinking group between two respective available functional groups (e.g., amino group and / or hydroxyl group) of the chitosan in the deacetylated mycelium material may be formed from an isocyanate compound, a polyol, or a combination thereof. In certain aspects, the crosslinking group between two respective groups of the chitosan in the deacetylated mycelium material may be formed from an isocyanate compound. In certain aspects, the crosslinking group between two respective groups of the chitosan in the deacetylated mycelium material may be formed from a polyol, such as those mentioned herein.In certain aspects, the crosslinking group between two respective groups of the chitosan in the deacetylated mycelium material may be formed from an isocyanate compound and a polyol.

[0111] In other aspects, the crosslinking group between two respective groups of the chitosan in the deacetylated mycelium material may be formed from an isocyanate compound and a polyol. For instance, the isocyanate compound may react with a respective group within the deacetylated mycelium material, and thereafter, a polyol may react with the isocyanate thereby forming a urethane bond. An available hydroxyl group may then react with another respective group within the deacetylated mycelium material to form the crosslink. In other aspects, an available hydroxyl group may then react with another isocyanate compound which may then react with another respective group within the deacetylated mycelium material to form the crosslink. In further embodiments, multiple reactions may occur between isocyanate compounds and polyols to form a polyurethane wherein such polyurethane may serve as the crosslinking group between two respective groups within the deacetylated mycelium material. Regardless, in such embodiments, the crosslinking group may be formed from at least one isocyanate compound and at least one polyol.

[0112] In certain embodiments, the bio-based fillers may be incorporated into the crosslinking group. In this regard, in some aspects, the bio-based filler may function as the crosslinking group. For instance, the bio-based filler may include a carboxyl group, a hydroxyl group, or a combination thereof. In this regard, when carboxyl groups are present, such carboxyl groups of the bio-based filler may react with respective available groups of the deacetylated mycelium material to form a crosslink thereby forming amide bonds. In this regard, when hydroxyl groups are present, such hydroxyl groups of the bio-based filler may react with respective available groups of the deacetylated mycelium material to form a crosslink thereby forming amide bonds. In this regard, when carboxyl and hydroxyl groups are present, such carboxyl and hydroxyl groups of the bio-based filler may react with respective available groups of the deacetylated mycelium material to form a crosslink thereby forming amide bonds.

[0113] In other aspects, the crosslinking group may be formed from the bio-based filler and an isocyanate compound, a polyol, or a combination thereof. For instance, it may be formed form an isocyanate compound in one embodiment. In one embodiment, it may be formed from a polyol. In one embodiment, it may be formed from a combination of an isocyanate compound and a polyol.

[0114] For instance, upon reaction of the isocyanate compound with an available group in the deacetylated mycelium material, the bio-based filler may react with the remaining isocyanate group of the isocyanate compound. Then, an available carboxyl or hydroxyl group of the biobased fdler may react with another respective group within the deacetylated mycelium material. Alternatively, the available carboxyl or hydroxyl group of the bio-based filler may react with another isocyanate compound which may react with another respective group within the deacetylated mycelium material.

[0115] In other aspects, in the instance the bio-based fillers include carboxyl groups, such carboxyl groups may react with the hydroxyl groups of the polyol to form an ester bond. In the instance the bio-based fillers include hydroxyl groups, such hydroxyl groups may react with the hydroxyl groups of the polyol to form an ether bond. Such formation of ester bonds or ether bonds may also be incorporated into the crosslinking group, with or without reaction with an isocyanate compound. Thereafter, any respective available carboxyl or hydroxyl group may react with another respective group within the deacetylated mycelium material.

[0116] Following the addition of bio-based fillers, additional post-processing steps may be implemented after reaction completion. For instance, the produced mycelium-based biopolymer composite may be dried and cured in order to stabilize the crosslinked network. Additional treatments, such as mild heating or controlled humidity conditions may be employed to achieve desired mechanical and / or aesthetic properties. In summary, the following groups are available for linkages by using this methodology.A) From Chitin / Chitosan:o Amino (-NH₂)o Hydroxyl (-OH)B) From Isocyanates:o Isocyanate (-N===C=O)C) From Alginate / 'Pectin:o Carboxyl (-COOH)o Hydroxyl (-OH)D) From Polyurethane:o Hydroxyl (-OH) from polyols (pre-polymer stage)o Isocyanate (-N=C=O) groups for further bonding.

[0117] In some embodiments, the sheet, panel, or slab may be exposed to an acid wash. For instance, it may be after introduction of the isocyanate compounds in one embodiment. In one embodiment, it may be after introduction of the polyols. In one embodiment, it may be after introduction of the fillers. In one embodiment, it may be after exposure to the alkaline solution. In one embodiment, it may be after crosslinking.

[0118] The acid wash may be with a weak acid. The weak acid may include, but is not limited to, acetic acid, formic acid, propionic acid, lactic acid, citric acid, tartaric acid, benzoic acid, or a mixture thereof. In one embodiment, the weak acid comprises acetic acid.

[0119] The inventive methods and resulting composition / material may be explained further through the following potential reactions.EXAMPLES OF POTENTIAL REACTIONS DEMONSTRATING EMBODIMENTS OF THE DISCLOSUREExample 1

[0120] A mycelium slab (density ~5 lbs / ft3) may be immersed in a 2% NaOH solution at 90°C for 2 hours to partially deacetylate chitin to chitosan. After rinsing and drying, the slab may be sprayed with a 5% w / w solution of MDI in acetone and heated at 60°C for 4 hours to desirably form urethane / urea crosslinks. The resulting material may desirably demonstrate improved water resistance.Example 2

[0121] A mycelium slab (density ~10 lbs / ft3) may undergo deacetylation as in Example 1. A mixture of TDI (2% by weight) and polyols (glycerol 1%, sorbitol 1%) in ethanol may be infused under vacuum. After curing at 50°C for 6 hours, the mycelium material may desirably form a polyurethane-rich network that increases flexibility and tensile strength.Example 3

[0122] Following the protocol of Example 1, a partially deacetylated mycelium slab may be immersed in an aqueous alginate solution (1% w / v) for 30 minutes and then exposed to 3% w / w MDI. The resulting amide linkages may desirably then form a dual-network system with enhanced toughness and minimal swelling under humid conditions.Example 4

[0123] A mycelium slab (density ~3 lbs / ft3) may be treated with 1.5% NaOH and then infused with a mixture of TDI (3% w / w) and pectin (1% w / v in water). After curing at 70°C for 2 hours, the resulting material may desirably exhibit a smooth surface finish with improved tear strength and moderate flexibility.Example 5

[0124] A thicker 2" mycelium slab, partially deacetylated, may be treated with MDI (2% w / w) and then sequentially sprayed with a solution of alginate (1% w / v) and chitosan (0.5% w / v). The interpenetrating biopolymer network may desirably be locked in place by isocyanate crosslinks and may desirably yield a stable, leather-like material.Example 6

[0125] A high-density mycelium slab (~15 lbs / ft3) may be deacetylated more extensively by increasing NaOH concentration (3%) and time (3 hours). It may then be soaked in a solution of MDI (4% w / w) and glycerol (1%) in acetone. The high crosslink density may desirably result in a stiff, water-resistant panel.Example 7

[0126] A low-density (~2 lbs / ft3) slab may be treated with TDI (1% w / w) and a blend of alginate (0.5% w / v) and pectin (0.5% w / v), followed by a brief acid wash (0.1% acetic acid) to set ionic crosslinks. This may desirably yield a pliable sheet with balanced moisture management and durability.Example 8

[0127] Using MDI (3% w / w) and a polyurethane prepolymer formed from MDI and polypropylene glycol (0.5% w / w), a deacetylated mycelium substrate may desirably gain increased elasticity and tear resistance suitable for flexible footwear components.Example 9

[0128] A partially deacetylated slab may be exposed to TDI (2% w / w) in combination with chitosan solution (1% w / v). The interaction of TDI with both amino and hydroxyl groups may desirably create a tightly crosslinked structure with good abrasion resistance.Example 10

[0129] A mycelium substrate may be processed with NaOH as before and then infused with MDI (2% w / w), pectin (1% w / v), and alginate (1% w / v). The triple-filler system may desirablyyield a gradient network with superior dimensional stability, suitable for protective outerwear or architectural panels.

[0130] Various modifications to the implementations described in this disclosure may be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other implementations without departing from the spirit or scope of this disclosure. Thus, the claims are not intended to be limited to the implementations shown herein but are to be accorded the widest scope consistent with this disclosure, the principles and the novel features disclosed herein.

[0131] While certain embodiments of the disclosure have been described, these embodiments have been presented by way of example only and are not intended to limit the scope of the disclosure. Indeed, the apparatus designs, methods, and systems incorporating such described herein, may be embodied in a variety of other forms. Furthermore, various omissions, substitutions and changes in the apparatus, methods, and systems described herein may be made without departing from the spirit of the disclosure. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the disclosure. Accordingly, the scope of the present disclosure is defined by reference to the appended claims.

[0132] Features, materials, characteristics, or groups described in conjunction with a particular aspect, embodiment, or example are to be understood to be applicable to any other aspect, embodiment or example described in this section or elsewhere in this specification unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The protection is not restricted to the details of any foregoing embodiments. The protection extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process, or system so disclosed.

[0133] The word “exemplary” is used herein to mean “serving as an example, instance, or illustration.” Any aspect or embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or embodiments. Various aspectsof the novel systems, apparatus, and methods are described more fully hereinafter with reference to the accompanying drawings. This disclosure may, however, be embodied in many different forms and should not be construed as limited to any specific structure or function presented throughout this disclosure. Rather, these aspects are provided so that this disclosure will be thorough, and will fully convey the scope of the disclosure to those skilled in the art. Based on the teachings herein one skilled in the art should appreciate that the scope of the disclosure is intended to cover any aspect of the systems, apparatus, and methods disclosed herein, whether implemented independently of, or combined with, any other aspect described. For example, an apparatus may be implemented, or a method may be practiced using any number of the aspects set forth herein. In addition, the scope of the disclosure is intended to cover such an apparatus, method or system which is practiced using other structure, functionality, or structure and functionality in addition to or other than the various aspects of the disclosures set forth herein. It should be understood that any aspect disclosed herein may be embodied by one or more elements of a claim.

[0134] Furthermore, certain features that are described in this disclosure in the context of separate implementations can also be implemented in combination in a single implementation. Conversely, various features that are described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination. Although features may be described above as acting in certain combinations, one or more features from a claimed combination can, in some cases, be excised from the combination, and the combination may be claimed as a subcombination or variation of a subcombination.

[0135] The features and attributes of the specific embodiments disclosed above may be combined in different ways to form additional embodiments, all of which fall within the scope of the present disclosure. Also, the separation of various system components in the implementations described above should not be understood as requiring such separation in all implementations, and it should be understood that the described components and systems can generally be integrated together in a single product or packaged into multiple products.

[0136] Moreover, while operations may be depicted in the drawings or described in the specification in a particular order, such operations need not be performed in the particular order shown or in sequential order, or that all operations be performed, to achieve desirable results. Other operations that are not depicted or described can be incorporated in the example methodsand processes. For example, one or more additional operations can be performed before, after, simultaneously, or between any of the described operations. Further, the operations may be rearranged or reordered in other implementations. Those skilled in the art will appreciate that in some embodiments, the actual steps taken in the processes illustrated and / or disclosed may differ from those shown in the figures. Depending on the embodiment, certain of the steps described above may be removed, others may be added. Furthermore, the features and attributes of the specific embodiments disclosed above may be combined in different ways to form additional embodiments, all of which fall within the scope of the present disclosure.

[0137] For purposes of this disclosure, certain aspects, advantages, and novel features are described herein. Not necessarily all such advantages may be achieved in accordance with any particular embodiment. Thus, for example, those skilled in the art will recognize that the disclosure may be embodied or carried out in a manner that achieves one advantage or a group of advantages as taught herein without necessarily achieving other advantages as may be taught or suggested herein.

[0138] Conditional language, such as “can,” “could,” “might,” or “may,” unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements, and / or steps. Thus, such conditional language is not generally intended to imply that features, elements, and / or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without user input or prompting, whether these features, elements, and / or steps are included or are to be performed in any particular embodiment.

[0139] Conjunctive language such as the phrase “at least one of X, Y, and Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to convey that an item, term, etc. may be either X, Y, or Z. Thus, such conjunctive language is not generally intended to imply that certain embodiments require the presence of at least one of X, at least one of Y, and at least one of Z. Thus, as used herein, a phrase referring to “at least one of X, Y, and Z” is intended to cover: X, Y, Z, X and Y, X and Z, Y and Z, and X, Y and Z.

[0140] The headings provided herein, if any, are for convenience only and do not necessarily affect the scope or meaning of the devices and methods disclosed herein.

[0141] Language of degree used herein, such as the terms “approximately,” “about,” “generally,” and “substantially” as used herein represent a value, amount, or characteristic close to the stated value, amount, or characteristic that still performs a desired function or achieves a desired result. For example, the terms “approximately,” “about,” “generally,” and “substantially” may refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, and within less than 0.01% of the stated amount.

[0142] The scope of the present disclosure is not intended to be limited by the specific disclosures of embodiments in this section or elsewhere in this specification and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non-exclusive.

Claims

1. CLAIMS1. A mycelium-based biopolymer composite material comprising:3.(a) a mycelium-based biopolymer sheet, panel, or slab material, at least partially deacetylated to expose amino groups;4.(b) one or more isocyanates forming covalent urethane and / or urea linkages with the amino groups to form an isocyanate-crosslinked network; and5.(c) one or more bio-based fillers covalently integrated into the isocyanate- crosslinked network.

2. The mycelium-based biopolymer composite material of claim 1, wherein the one or more bio-based fillers comprise alginate, pectin, chitosan, or a mixture thereof.

3. The mycelium-based biopolymer composite material of claim 1 or 2, wherein the mycelium-based biopolymer composite material is derived from Ganoderma species of mycelium.

4. The mycelium-based biopolymer composite material of any of claims 1-3, wherein the mycelium-based biopolymer composite material grown aerially to produce slabs between about 0.5" and 2" thickness and having a dry density between about 1 and 20 lbs / ft3.

5. The mycelium-based biopolymer composite material of any of claims 1-4, further comprising a polyurethane phase formed in situ by reacting diisocyanates with polyols.

6. The mycelium-based biopolymer composite material of claim 5, wherein the polyurethane phase is interpenetrated with the mycelium-based sheet, panel, or slab material and the bio-based fillers, enhancing flexibility and tensile strength of the resulting sheet, panel, or slab material, compared to that of an untreated but otherwise similarly constructed mycelium -based biopolymer sheet, panel, or slab material.

7. The mycelium-based biopolymer composite material of any of claims 1-6, wherein the resulting mycelium-based biopolymer composite exhibits improved hydrophobicity, decreased water uptake, and enhanced mechanical properties compared to untreated but otherwise similarly constructed mycelium composites.

8. A method of producing a mycelium-based biopolymer composite, comprising:13.(a) providing a grown mycelium-based biopolymer sheet, panel, or slab material; (b) at least partially deacetylating the grown mycelium-based biopolymer sheet, panel, or slab material to convert chitin to chitosan to expose amino groups;14.(c) introducing one or more isocyanate compounds that react with the exposed amino and hydroxyl groups to form covalent isocyanate crosslinks in a resulting structure; and15.(d) integrating one or more bio-based fillers into the isocyanate-crosslinked structure.

9. The method of claim 8, wherein the one or more bio-based fillers comprise alginate, pectin, chitosan, or a mixture thereof.

10. The method of claim 8, wherein the integration of the one or more bio-based fillers occurs prior to, during, or after the isocyanate reaction to form interpenetrating networks.

11. The method of claim 8, wherein the mycelium-based biopolymer composite is a stable, moisture-resistant composite.

12. The method of any of claims 8-11, further comprising adding a polyurethane prepolymer or polyols during the crosslinking step to form a polyurethane-rich composite.

13. The method of any of claims 8-12, wherein the resulting mycelium-based biopolymer composite exhibits improved hydrophobicity, decreased water uptake, and enhanced mechanical properties compared to untreated but otherwise similarly constructed mycelium composites.

14. The method of any of claims 8-13, wherein the biopolymer composite is suitable as a leather-like material with enhanced abrasion resistance.

15. A mycelium-based biopolymer composite in which a ratio of isocyanate to total available amino groups can be adjusted to tune the mechanical stiffness and water resistance of the final biopolymer composite product.

16. A mycelium-based biopolymer composite material comprising:24.(a) a mycelium-based biopolymer sheet, panel, or slab material, at least partially deacetylated to expose amino groups;25.(b) one or more isocyanates forming covalent urethane and / or urea linkages with the amino groups to form an isocyanate-crosslinked network, the isocyanate-crosslinked network receptive to receiving additional bio-based filler material through further reactions.

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