WO2023240072A2 - Procédés de fabrication d'un matériau composite à partir de biomasse - Google Patents

Procédés de fabrication d'un matériau composite à partir de biomasse Download PDF

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Publication number
WO2023240072A2
WO2023240072A2 PCT/US2023/067982 US2023067982W WO2023240072A2 WO 2023240072 A2 WO2023240072 A2 WO 2023240072A2 US 2023067982 W US2023067982 W US 2023067982W WO 2023240072 A2 WO2023240072 A2 WO 2023240072A2
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Prior art keywords
biomaterial
solution
plasticizer
fermentation
biomat
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PCT/US2023/067982
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English (en)
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WO2023240072A3 (fr
Inventor
Eric Epstein
Jeremy Weigand
Mike Greene
Jason Quinlan
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The Fynder Group, Inc.
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Publication of WO2023240072A2 publication Critical patent/WO2023240072A2/fr
Publication of WO2023240072A3 publication Critical patent/WO2023240072A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L75/00—Compositions of polyureas or polyurethanes; Compositions of derivatives of such polymers
    • C08L75/04—Polyurethanes

Definitions

  • This invention relates generally to composite materials, and particularly to materials derived from biomaterials, e.g., filamentous fungi, that can be used in textiles and fabrics, such as leather analogs.
  • biomaterials e.g., filamentous fungi
  • a method for forming a biomaterial-containing composite material comprises (a) contacting the biomaterial with a formulation comprising monomers, oligomers, and/or reactive polymers; and (b) polymerizing the monomers, oligomers, and/or reactive polymers to form a composite material comprising the biomaterial and one or more polymeric structures.
  • the method may further comprise crosslinking the one or more polymeric structures to the biomaterial.
  • step (a) and step (b) may be carried out simultaneously.
  • step (b) mav be carried out after stet) (a).
  • the method may further comprise contacting particulates comprising chemically reactive moieties, with at least one of the biomaterial and the formulation; and initiating a reaction of the chemically reactive moieties to cross-link the particulates.
  • the particulates may, but need not, comprise polyurethane and the chemically reactive moieties may, but need not, comprise carboxylic acid functional groups.
  • the particulates may, but need not, comprise at least one of modified cellulose or other waterinsoluble polysaccharides, modified or unmodified clays, metals, metal oxides, minerals or mineral-derived particulates, modified or unmodified silicas, polyesters, polyamides, and modified latex.
  • the formulation may be selected from the group consisting of (i) a solution comprising the monomers, oligomers, and/or reactive polymers as a solute and a fluid solvent, (ii) a colloid comprising the monomers, oligomers, and/or reactive polymers as a dispersed phase and a fluid dispersion medium, and (iii) combinations thereof.
  • Step (a) may, but need not, comprise dispersing particles of the biomaterial in the fluid solvent or fluid dispersion medium to coat the particles with the monomers, oligomers, and/or reactive polymers.
  • the biomaterial may be in a form selected from the group consisting of a paste, a slurry, a semisolid, a solid, a gel, a pulp, a suspension, a foam, and combinations thereof.
  • the biomaterial may, but need not, be produced by a process selected from the group consisting of a submerged fermentation, a surface fermentation, a submerged solid substrate fermentation, a solid substrate fermentation, a membrane fermentation, an airmedium colloid fermentation, and combinations thereof.
  • the biomaterial may comprise at least one material selected from the group consisting of fungal mycelium-derived material, a plant-derived material, a bacteria-derived material, an algal-derived material, and combinations thereof.
  • the biomaterial may, but need not, comprise fungal mycelium, which may, but need not, be produced by a process selected from the group consisting of a submerged fermentation, a surface fermentation, a submerged solid substrate fermentation, a solid substrate fermentation, a membrane fermentation, an air-medium colloid fermentation, and combinations thereof.
  • the method may, but need not, further comprise inactivating the filamentous fungal biomat, and the inactivating step may, but need not, be carried out simultaneously with or after step (a).
  • the formulation may, but need not, comprise a solution and step (a) may, but need not, comprise at least one of soaking the fungal mycelium in the solution, spraying the solution onto a surface of the funeal mvcelium, causing the solution to infiltrate the fungal mycelium via vacuum-assisted solvent exchange and/or vacuum pressure cycling, mechanically pressing the solution into the fungal mycelium, causing the solution to diffuse into the fungal mycelium with heating, causing the solution to diffuse into the fungal mycelium without heating, and injecting the solution into the fungal mycelium.
  • the formulation may, but need not, comprise a solution and step (a) may, but need not, comprise spraying or infusing the solution onto or into the fungal mycelium under at least one of convective heating, radiofrequency-induced heating, microwave heating, ultrasonic heating, and conductive heating.
  • the formulation may, but need not, comprise a solution and step (a) may, but need not, comprise agitating the fungal mycelium with the solution.
  • the formulation may, but need not, comprise a solution, step (a) may, but need not, comprise directly or indirectly heating the fungal mycelium, and the heating may, but need not, produce a thermal gradient within the fungal mycelium.
  • the method may further comprise plasticizing the biomaterial.
  • the plasticizing step may, but need not, be carried out before step (a).
  • the plasticizing step may, but need not, be carried out simultaneously with step (a).
  • the plasticizing step may, but need not, be carried out after step (a) but before step (b).
  • the plasticizing step may, but need not, be carried out simultaneously with step (b).
  • the plasticizing step may, but need not, be carried out after step (b).
  • the method may further comprise contacting the biomaterial with a chemically reactive functionalizing agent; and initiating a reaction of the chemically reactive functionalizing agent with the biomaterial.
  • the chemically reactive functionalizing agent may, but need not, comprise a carboxylic acid and the reaction may, but need not, comprise Fisher esterification of hydroxyl groups in the biomaterial.
  • the chemically reactive functionalizing agent may, but need not, comprise at least one functional group selected from the group consisting of an isocyanate, a silane, an epoxide, a carboxylic acid, a carboxylic ester, an acid anhydride, and a carbodiimide.
  • the chemically reactive functionalizing agent may, but need not, comprise at least one functional group selected from the group consisting of a lactide, a glycolide, and a lactone, and the reaction may, but need not, comprise ring-opening polymerization of the chemically reactive functionalizing agent by hydroxyl groups in the biomaterial.
  • the method may further comprise initiating a chemical reaction between the polymeric structure and the biomaterial.
  • the monomers, oligomers, and/or reactive polymers may be in solid form and the formulation may consist of the monomers, oligomers, and/or reactive polymers.
  • the formulation may comprise a solution of the monomers, oligomers, and/or reactive polymers in an aqueous solvent.
  • the formulation may comprise a solution of the monomers, oligomers, and/or reactive polymers in an organic solvent.
  • the monomers, oligomers, and/or reactive polymers may comprise an epoxide and at least one of an amine, an anhydride, or a carboxylic acid.
  • the monomers, oligomers, and/or reactive polymers may comprise a thiol and an epoxide.
  • the monomers, oligomers, and/or reactive polymers may comprise a thiol and an alkene and the formulation may further comprise a free radical initiator.
  • the monomers, oligomers, and/or reactive polymers may comprise a thiol and an oxidizing agent, whereby the polymeric structure formed in step (b) comprises a polysulfide.
  • the monomers, oligomers, and/or reactive polymers may comprise an amine and an anhydride or cyclic carbonate.
  • the monomers, oligomers, and/or reactive polymers may comprise a Michael acceptor and at least one of a thiol, an amine, an acetoacetate, or a malonate.
  • the Michael acceptor may, but need not, comprise an acrylate.
  • the monomers, oligomers, and/or reactive polymers may comprise an amine and a vegetable tannin.
  • the monomers, oligomers, and/or reactive polymers may comprise an amine and at least one of a carboxylic acid or an ester.
  • the monomers, oligomers, and/or reactive polymers may comprise an alkene and a free radical initiator.
  • a method for forming a multilayer composite material comprises contacting two or more composite materials produced by a process disclosed herein.
  • a multilayer composite material is produced by a process disclosed herein.
  • a flexibility or stiffness of a first layer of the multilayer composite material may be different from a flexibility or stiffness of a second layer of the multilayer composite material.
  • a composite material is made by a method disclosed herein.
  • a composite material comprises a biomaterial; and a polymeric structure, selected from the group consisting of a hydrophobic network, an elastomeric network, a polysulfide network, a polyamide network, a polythioether network, an epoxy-amine network, a carbodiimide-carboxylic acid network, a Diels-Alder polymerized network, a Michael addition polymerized network, a poly(meth)acrylate network, a poly(hydroxy)urethane network, a polyurea network, and a polyester network, wherein the biomaterial and the polymeric structure are intertwined, interlaced, intersecting, or interwoven.
  • a polymeric structure selected from the group consisting of a hydrophobic network, an elastomeric network, a polysulfide network, a polyamide network, a polythioether network, an epoxy-amine network, a carbodiimide-carboxylic acid network, a Diels-Alder polymerized
  • a method for treating a biomaterial comprises (a) contacting the biomaterial with a formulation comprising one or more peptide- binding moieties, wherein the peptide-binding moieties are reactive with one or more amino acids of peptides of the biomaterial; and (b) reacting the peptide-binding moiety with the amino acids to functionalize at least a portion of the peptides of the biomaterial.
  • the peptide-binding moiety may be a crosslinker.
  • the crosslinker may, but need not, be a macromolecule.
  • the peptide-binding moiety may be a dye or pigment.
  • the peptide-binding moiety may impart to the biomaterial at least one functionality selected from the group consisting of hydrophobicity, hydrophilicity, amphiphilicity, cationic charge, anionic charge, and affinity for a selected compound.
  • the peptide-binding moiety may be a surface-functional particulate.
  • the peptide-binding moiety may be an internal plasticizer.
  • the peptide-binding moiety may be a ligand or chelator.
  • the peptide-binding moiety may be a monomer or initiator capable of polymerization after step (b).
  • the peptide-binding moiety may comprise at least one of an epoxy, a maleimide, an iodoacetamide, a succinimidyl ester, a sulfonyl chloride, a cyclic carbonate, a Michael addition acceptor, an alkene, an isocyanate, a carbodiimide, a carboxylic acid (or ester), an anhydride, an amine, an alkyl halide, an aldehyde, a thiol, a polysaccharide, and a peptide.
  • the peptides of the biomaterial may be crosslinked by at least one of covalent bonds, ionic bonds, coordinate bonds, electrostatic interactions, hydrogen bonds, hydrophobic and/or van de Waals interactions, and combinations thereof.
  • a weight ratio of the peptide-binding moiety to the biomaterial may be at least about 0.8, at least about 0.5, at least about 0.2, at least about 0.01, or at least about IO' 6 .
  • the formulation may further comprise a reactant that facilitates the reaction of step (b).
  • the reactant may, but need not, be a carbodiimide.
  • the formulation may further comprise at least one of a pH buffer and a species that promotes the reaction of step (b) at a selected pH.
  • the species may, but need not, comprise side chain amines.
  • the biomaterial may be in a form selected from the group consisting of a paste, a slurry, a semisolid, a solid, a gel, a hydrogel, a solution, a suspension, and combinations thereof.
  • the biomaterial may, but need not, be produced by a process selected from the group consisting of a submerged fermentation, a surface fermentation, a submerged solid substrate fermentation, a solid substrate fermentation, a membrane fermentation, and combinations thereof.
  • the biomaterial may comprise at least one material selected from the group consisting of fungal mycelium, a plant-derived material, a bacteria-derived material, an algae-derived material, and a yeast-derived material.
  • the biomaterial may, but need not, comprise fungal mycelium, which may, but need not, be produced by a surface fermentation process, a membrane fermentation process, an air-medium colloid (AMC) fermentation process, or a combination thereof.
  • the method may, but need not, further comprise inactivating the filamentous fungal biomat, and the inactivating step may, but need not, be carried out simultaneously with or after step (a).
  • the formulation may, but need not, comprise a solution and step (a) may, but need not, comprise at least one of soaking the biomat in the solution, spraying the solution onto a surface of the biomat, causing the solution to infiltrate the biomat via vacuum-assisted solvent exchange and/or vacuum pressure cycling, and mechanically pressing the solution into the biomat.
  • the fungal biomat may, but need not, be grown in the presence of the formulation or a component thereof.
  • the formulation or a component thereof may, but need not, be produced by metabolism of the fungal biomat.
  • the biomaterial may, but need not, comprise two or more filamentous fungi and may, but need not, comprise a filamentous fungus and one or more complementary microorganisms.
  • the formulation or a conwonent thereof mav. but need not, be produced by complementary activity of the filamentous fungus and the one or more complementary microorganisms.
  • the biomaterial may, but need not, comprise fungal mycelium, and at least a portion of the total peptide content of the fungal mycelium may, but need not, be exposed on cell walls of the fungal mycelium.
  • the biomaterial may, but need not, comprise fungal mycelium, and the fungal mycelium may, but need not, be chemically or mechanically processed to expose peptides on a surface of the fungal mycelium.
  • the peptides may be overexpressed or upregulated.
  • the method may further comprise plasticizing the biomaterial using a plasticizer.
  • the plasticizing step may, but need not, be carried out before step (a).
  • the plasticizing step may, but need not, be carried out simultaneously with step (a).
  • the plasticizing step may, but need not, be carried out after step (a) but before step (b).
  • the plasticizing step may, but need not, be carried out simultaneously with step (b).
  • the plasticizing step may, but need not, be carried out after step (b).
  • the plasticizer or a component thereof may, but need not, be produced by metabolism of an organism from which the biomaterial is derived.
  • the formulation may comprise a solution of the peptide-binding moiety in an aqueous solvent.
  • the formulation may comprise a solution of the peptide-binding moiety in an organic solvent.
  • the formulation may comprise a solution of the peptide-binding moiety in a deep eutectic solvent.
  • the method may further comprise, prior to step (b), pre-treating the biomaterial to facilitate the reaction of step (b).
  • the pre-treating step may, but need not, comprise at least one of alkali removal of extracellular polysaccharides, pH increase, lipase treatment, protease treatment, carbohydrase treatment, solvent treatment, decrystallization, acid pretreatment, and reduction of thiols.
  • the biomaterial may be ground or homogenized prior to step (b) and cast as part of a slurry.
  • a treated biomaterial comprises crosslinked peptides and is made by a method disclosed herein.
  • a treated biomaterial wherein at least about 3 wt% of peptides in the biomaterial are crosslinked.
  • a method for plasticizing a biomaterial comprises contacting the biomaterial with a olasticizer comorising at least one selected from the group consisting of (i) a deep eutectic solvent, comprising a hydrogen bond acceptor and a hydrogen bond donor, (ii) a mixture of a compound containing a urea or thiourea functionality with a polyol, and (iii) combinations thereof.
  • the plasticizer may be dissolved in an aqueous or volatile organic solvent, and the method may further comprise evaporating the aqueous or volatile organic solvent after the contacting step.
  • the plasticizer may comprise a mixture of a compound containing a urea or thiourea functionality with a polyol.
  • the polyol may, but need not, be glycerol.
  • the plasticizer may be combined with an amphiphilic compound as a co-plasticizer.
  • the amphiphilic compound may, but need not, be a phospholipid.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond acceptor may be an amino acid.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond acceptor may be an organic acid.
  • the organic acid may, but need not, be a carboxylic acid, which may, but need not, be citric acid.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond acceptor may be a quaternary ammonium salt.
  • the quaternary ammonium salt may, but need not, be selected from the group consisting of betaines, cholinium salts, and l-ethyl-3-methylimidazolium chloride.
  • the quaternary ammonium salt may, but need not, be a cholinium salt selected from the group consisting of choline chloride, choline acetate, and combinations and mixtures thereof.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond acceptor may be a hydrated metal salt.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond acceptor may be an anhydrous or hydrated metal halide.
  • the hydrogen bond acceptor may, but need not, be anhydrous aluminum chloride or aluminum chloride hexahydrate.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond acceptor may be a phosphonium salt.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond donor may be selected from the group consisting of a carboxylic acid, a sugar, an alcohol, an amine, an amino acid, an amide, a thiol, a urethane, a sulfonic acid, a phosphoric acid, and a phosphonic acid.
  • the hydrogen bond donor may, but need not, be selected from the group consisting of ethvlene elvcol. oroovlene glycol, glycerol, and urea.
  • the plasticizer may be dissolved in a solvent and the contacting step may comprise the sub-steps of (i) contacting the biomaterial with the solvent and (ii) evaporating at least a portion of the solvent to leave the plasticizer in contact with the biomaterial.
  • the solvent may, but need not, be water.
  • the plasticizer may be a solution comprising the deep eutectic solvent, the mixture of the compound containing the urea or thiourea functionality with the polyol, or the combination thereof as a solvent and a crosslinker as a solute, and the crosslinker may be reactive with a surface moiety of peptides of the biomaterial.
  • the method may, but need not, further comprise reacting the crosslinker with the surface moiety to crosslink the peptides of the biomaterial.
  • the crosslinker may, but need not, comprise an epoxy.
  • the biomaterial may be in a form selected from the group consisting of a paste, a slurry, a semisolid, a solid, and combinations thereof.
  • the biomaterial may, but need not, be produced by a process selected from the group consisting of a submerged fermentation, a surface fermentation, a submerged solid substrate fermentation, a solid substrate fermentation, a membrane fermentation, and combinations thereof.
  • the biomaterial may comprise at least one material selected from the group consisting of fungal mycelium, a plant-derived material, and a bacteria-derived material.
  • the biomaterial may, but need not, comprise fungal mycelium, which may, but need not, be produced by a process selected from the group consisting of a submerged fermentation, a surface fermentation, a submerged solid substrate fermentation, a solid substrate fermentation, a membrane fermentation, and combinations thereof.
  • the method may, but need not, further comprise inactivating the filamentous fungal mycelium.
  • a plasticized biomaterial is made by a method disclosed herein.
  • a plasticized material comprises a biomaterial; and a plasticizer comprising at least one selected from the group consisting of (i) a deep eutectic solvent, comprising a hydrogen bond acceptor and a hydrogen bond donor, (ii) a mixture of a compound containing a urea or thiourea functionality with a polyol, and (iii) combinations thereof.
  • the polyol may be glycerol or a derivative thereof.
  • the plasticized material may further comprise an amphiphilic coplasticizer.
  • the amphiphilic co-plasticizer may, but need not, be a phospholipid, which may, but need not, be lecithin.
  • the plasticizer may comprise a deep eutectic solvent and a hydrogen bond acceptor of the deep eutectic solvent may be an amino acid.
  • the plasticizer may comprise a deep eutectic solvent and a hydrogen bond acceptor of the deep eutectic solvent may be an organic acid.
  • the organic acid may, but need not, be a carboxylic acid, which may, but need not, be citric acid.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond acceptor may be a quaternary ammonium salt.
  • the quaternary ammonium salt may, but need not, be selected from the group consisting of betaines, cholinium salts, and l-ethyl-3-methylimidazolium chloride.
  • the quaternary ammonium salt may, but need not, be a cholinium salt selected from the group consisting of choline chloride, choline acetate, and combinations and mixtures thereof.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond acceptor may be a hydrated metal halide.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond acceptor may be an anhydrous or hydrated metal halide.
  • the hydrogen bond acceptor may, but need not, be anhydrous aluminum chloride or aluminum chloride hexahydrate.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond acceptor may be a phosphonium salt.
  • the plasticizer may comprise a deep eutectic solvent and the hydrogen bond donor may be selected from the group consisting of a carboxylic acid, a sugar, an alcohol, an amine, an amino acid, an amide, a thiol, a urethane, a sulfonic acid, a phosphoric acid, and a phosphonic acid.
  • the hydrogen bond donor may, but need not, be selected from the group consisting of ethylene glycol, glycerol, and urea.
  • the plasticizer may be a solution comprising the deep eutectic solvent, the mixture of the compound containing the urea or thiourea functionality and the polyol, or the combination thereof as a solvent and a crosslinker as a solute, and the crosslinker may be reactive with a surface moiety of peptides of the biomaterial.
  • the biomaterial may comprise at least one material selected from the group consisting of fungal mycelium, a plant-derived material, and a bacteria-derived material.
  • the biomaterial may, but need not, comprise fungal mycelium, which may, but need not, be produced by a surface fermentation process.
  • “about 750” can mean as little as 675 or as much as 825, or any value therebetween.
  • the terms “about,” “approximately,” etc. when used in relation to ratios or relationships between two or more numerical limitations or ranges, the terms “about,” “approximately,” etc.
  • a statement that two quantities are “approximately equal” can mean that a ratio between the two quantities is as little as 0.9: 1.1 or as much as 1.1 :0.9 (or any value therebetween), and a statement that a four-way ratio is “about 5:3: 1 : 1” can mean that the first number in the ratio can be any value of at least 4.5 and no more than 5.5, the second number in the ratio can be any value of at least 2.7 and no more than 3.3, and so on.
  • Figure 1A is a graph of free fluorescein released from fluorescein-treated biomat samples contacted with protease, chitinase, and buffer solutions.
  • Figure IB is an illustration of fluorescein-treated biomat samples contacted with a buffer solution (left) and a protease solution (right).
  • Figures 2A and 2B are fluorescence microscopy images of the edge and the center, respectively, of a fluorescein-labeled fungal biomat.
  • Figures 3A and 3B are fluorescence microscopy images of the edge and the center, respectively, of a fluorescein-labeled fungal biomat after digestion by a protease enzyme.
  • Figures 3C and 3D are fluorescence microscopy images of the edge and the center, respectively, of a fluorescein-labeled fungal biomat after digestion by a chitinase enzyme.
  • Figure 4 is a graph of water uptake percentage in a fungal biomat as a function of molar quantity of epoxide per gram of epoxide-infused biomass.
  • Figures 5A and 5B are images of a composite material made by casting and curing a fungal slurry on a cotton mesh scaffold.
  • Figure 5C is a microscopy image of the composite material of Figures 5A and 5B.
  • Figure 6 is a graph of the results of flexibility testing of epoxy-amine-polymerized fungal biomats.
  • Figures 7A and 7B are graphs of strain at break and tensile strength, respectively, of epoxy-amine-polymerized fungal biomats.
  • Figure 8 is a graph of the results of flexibility testing of fungal biomats treated with varying compositions of a urea/glycerol plasticizer mixture.
  • aqueous refers to any mixture or solution that includes water. It is to be expressly understood, therefore, that in an “aqueous” solution as that term is used herein, water may be the only solute, one of two or more solutes, the only solvent, or one of two or more solvents.
  • biodegradable refers to a material that, under a given set of conditions (e.g., the conditions specified in ISO 20136:2017, “Leather — determination of degradability by micro-organisms”), biodegrades at least 10% more quickly than “true” (i.e. animal) leather.
  • biomass refers to a mass of a living or formerly living organism.
  • filamentous fungal biomass refers to a mass of a living or formerly living filamentous fungus.
  • Filamentous fungal biomasses may include biomats (as that term is used herein), as well as filamentous fungus produced by submerged fermentation, such as (but not limited to) a mycoprotein paste as described in U.S. Patent 7,635,492 to Finnigan et al. and/or a filamentous fungal biomass described (or produced by the methods described) in U.S. Patent 11,058,137 to Pattillo, or filamentous fungus produced by solid substrate fermentation.
  • biomat refers to a cohesive mass of filamentous fungal tissue comprising a network of interwoven hyphae filaments.
  • Biomats as that term is used herein may, but need not, be characterized by one or more of a density of between about 50 and about 200 erams Der liter, a solids content of between about 5 wt% and about 20 wt%, and sufficient tensile strength to be lifted substantially intact from the surface of a growth substrate (e.g., a liquid growth medium, a solid fungal composite, or a solid membrane or mesh).
  • a growth substrate e.g., a liquid growth medium, a solid fungal composite, or a solid membrane or mesh.
  • Biomats as that term is used herein, may be produced by any one or more fungal fermentation methods known in the art, such as, by way of nonlimiting example, methods described in PCT Application Publications 2020/176758, 2019/099474, and 2018/014004.
  • biomaterial refers to any tissue or other physical material derived from one or more living or formerly living organisms other than animals (e.g., filamentous fungi, plants, bacteria, algae, yeasts, etc.). “Biomaterials,” as that term is used herein, may be biomasses or portions thereof, but may also be materials that are directly or indirectly derived from the organism(s) (e.g., extracts, metabolites, etc.).
  • the term “cohesive” refers to any material that has sufficient structural integrity and tensile strength to be picked up and/or physically manipulated by hand as a solid object, without tearing or collapsing.
  • the term “colloid” refers to a mixture in which particles of one substance (the “dispersed phase”) are dispersed throughout a volume of a different substance (the “dispersion medium”); for example, the dispersed phase can comprise or consist of microscopic or macroscopic bubbles, particles, etc.
  • the dispersed phase and the dispersion medium of a colloid are specifically identified herein, they are separated by a hyphen, with the dispersed phase identified first, e.g, a reference herein to an “oil-water colloid” refers to a colloid in which an oil is the dispersed phase and water is the dispersion medium.
  • the term “degree of swelling” refers to the relative amount of change in the mass of a solid item when the solid is saturated with a liquid.
  • a solid item that has a mass of 200 g when dry and a mass of 300 g when saturated with water has a degree of swelling in water of 50%, or 0.5.
  • the term “degree of swelling” is used herein without explicitly identifying a liquid, the liquid may be assumed to be water.
  • the term “durable” refers to a material that has at least one of a tear strength of at least about 5 N/mm, a tear force of at least about 5 N, and a tensile strength of at least about 1.5 MPa.
  • emulsion refers to a colloid in which both the dispersed phase and the disoersion medium are liquids.
  • examples of emulsions as that term is used herein include but are not limited to butter (when melted), margarine (when melted), mayonnaise and milk.
  • filamentous fungus refers to any multicellular fungus that is capable of forming an interconnected network of hyphae (vegetative hyphae or aerial hyphae, and most commonly both) known as “mycelium.”
  • filamentous fungi as that term is used herein include, but are by no means limited to, fungi of the genera Acremonium, Ahernaria. Aspergillus, Cladosporium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma, and Trichophyton, among many others. It is to be expressly understood that filamentous fungi, as that term is used herein, may be capable of forming other fungal structures, such as fruiting bodies, in addition to hyphae/mycelium.
  • foam refers to a colloid in which the dispersed phase is a gas, and the dispersion medium is a liquid.
  • foams include but are not limited to shaving cream, soap bubbles, and the “head” of a carbonated or nitrogenated beverage.
  • fungal mycelial matter and “fungal mycelial biomass” are interchangeable and each refer to any material that includes at least about 50 wt% fungal mycelium on a dry basis (i.e., disregarding the mass of any water). More specifically, as used herein, unless otherwise specified, the term “consisting essentially of fungal mycelium” refers to any material that includes at least about 95 wt% fungal mycelium on a dry basis.
  • gel refers to a colloid in which the dispersed phase is a liquid, and the dispersion medium is a solid.
  • examples of gels as that term is used herein include but are not limited to agar, hair gel, and opal. Gels, as that term is used herein, may behave as solids or semi-solids and typically have an elastic modulus greater than their dynamic (or loss) modulus, and thus do not readily flow.
  • hide leather and “true leather” are interchangeable and each refer to a durable, flexible material created by tanning the hide or skin of an animal.
  • the term “inactivated” refers to a filamentous fungal biomass in which the fungal cells have been rendered nonviable, or enzymes capable of degrading or causing biochemical transformations within the biomass have been deactivated, or both.
  • the term “inactivation” refers to any method or process by which a filamentous funeal biomass mav be inactivated, such as, by way of non-limiting example, boiling, dehydration, immersion in an organic liquid (e.g., an alcohol, peracetic acid, etc.), irradiation, pressure treatment, rinsing, size reduction, steaming, and temperature cycling.
  • the term “infiltration” refers to the permeation and/or saturation of a solution into a mass of solid material, such that the solution or a portion thereof is distributed in the mass of solid material, such as for example and without limitation, a polymer solution permeating the interstitial spaces in a fungal biomat comprised of mycelia.
  • a solution comprising components such as polymers and plasticizers, results in a textile material having such components distributed in the biomass after the solvent is removed by curing.
  • Such a distribution can be substantially uniformly distributed or not uniformly distributed.
  • liquid aerosol refers to a colloid in which the dispersed phase is a liquid, and the dispersion medium is a gas.
  • loading ratio refers to a weight ratio of fungal biomass to polymer in a fungal textile composition.
  • the term “mass loss upon soaking” refers to the relative amount of mass lost by a solid item after soaking in a liquid, disregarding the mass of liquid absorbed by the solid item.
  • a solid item that has a mass of 100 grams when dry and a mass (disregarding the mass of absorbed liquid) of 95 grams after soaking in water has a mass loss upon soaking in water of 5%.
  • the term “mass loss upon soaking” is used herein without explicitly identifying a liquid, the liquid may be assumed to be water.
  • the term “particle” refers to a small, discrete, localized object to which can be ascribed chemical or physical properties such as volume, density, and/or mass. “Particles,” as that term is used herein, may be microscopic or macroscopic; may be in the gas (e.g., air bubbles), liquid (e.g., droplets of the dispersed phase in an emulsion), or solid (e.g., granules of a powder) phase; and may take any of a variety of shapes (e.g., spheres, oblate spheroids, fibers, tubes, rods, etc.). Particles in the solid phase may be referred to herein as “particulates” or “particulate matter.”
  • sheet refers to a layer of solid material having a generally flat or planar shape and a high ratio of surface area to thickness.
  • sol refers to a colloid in which the dispersed phase is a solid and the disoersion medium is a liauid.
  • sols as that term is used herein include but are not limited to custard (before it is cooked) and jelly (before it is set).
  • solid aerosol refers to a colloid in which the dispersed phase is a solid and the dispersion medium is a gas.
  • solid foam refers to a colloid in which the dispersed phase is a gas, and the dispersion medium is a solid.
  • solid foams include but are not limited to bread, cake, ice cream, and meringue.
  • solid sol refers to a colloid in which both the dispersed phase and the dispersion medium are solids.
  • tannin refers generally to any molecule that forms strong bonds with protein structures, and more particularly to a molecule that, when applied to hide leather, bonds strongly to protein moieties within the collagen structures of the skin to improve the strength and degradation resistance of the leather.
  • the most commonly used types of tannins are vegetable tannins, z.e., tannins extracted from trees and plants, and chromium tannins such as chromium(III) sulfate.
  • Other examples of tannins as that term is used herein include modified naturally derived polymers, biopolymers, and salts of metals other than chromium, e.g., aluminum silicate (sodium aluminum silicate, potassium aluminum silicate, etc.).
  • water uptake refers to the degree of swelling of a solid material when the solid material is saturated with water.
  • the present disclosure provides methods for infusing a biomaterial with a reactive material to improve physical properties e.g., mechanical properties, flexibility, elasticity, thermal properties, optical properties, electrical properties, etc.) of the biomaterial and/or tune interactions of the biomaterial with water.
  • the disclosure further provides biomaterialcontaining composite materials made by these methods.
  • the biomaterial is contacted with a formulation (e.g., a liquid solution) comprising monomers and/or oligomers, and a reaction of the monomers and/or oligomers is initiated to form a composite material containing both a polymeric structure (i.e., polymers formed by reaction of the monomers and/or oligomers) and the biomaterial.
  • a formulation e.g., a liquid solution
  • a reaction of the monomers and/or oligomers is initiated to form a composite material containing both a polymeric structure (i.e., polymers formed by reaction of the monomers and/or oligomers) and the biomaterial.
  • the composite material may have significantly improved physical properties, such as increased tensile strength, increased flexibility, and/or decreased swelling upon contact with water, relative to the untreated biomaterial.
  • the biomaterial may be pre-treated prior to contact with the monomers and/or oligomers to improve the resulting composite material (e.g., inactivated, treated with vacuum or heat to remove moisture, etc.) and/or subjected to various other processing steps before, during, or after contact with the monomers/oligomers and/or formation of the polymeric structure (e.g., plasticizing).
  • the methods of the present disclosure may thus improve the efficiency of infusion of a reactive material into a biomaterial by infusing reactive materials that have relatively low molecular weights (e.g., monomers and/or oligomers) and then polymerizing or otherwise reacting these reactive materials in situ, rather than by attempting to infuse a less reactive or higher-molecular weight material (e.g., a polymeric structure) directly into the biomaterial.
  • reactive materials that have relatively low molecular weights (e.g., monomers and/or oligomers) and then polymerizing or otherwise reacting these reactive materials in situ, rather than by attempting to infuse a less reactive or higher-molecular weight material (e.g., a polymeric structure) directly into the biomaterial.
  • biomasses used as biomaterials in methods according to the present disclosure are filamentous fungal biomasses — that is, biomasses of one or more fungi that produce an interconnected network of hyphae known as mycelium.
  • the filamentous fungal biomass in biomaterials according to the present disclosure may be a fungal mycelial biomass as that term is defined herein (although other filamentous fungal biomasses, such as biomasses that contain a significant quantity of material derived from fruiting bodies of a filamentous fungus, are also contemplated and are within the scope of this disclosure).
  • the fungal mycelial biomasses used in biomaterials according to the present disclosure are cohesive fungal mycelial biomasses, i.e., mycelial biomasses that have sufficient structural integrity and tensile strength to be picked up and physically manipulated by hand without tearing or collapsing; non-limiting examples of cohesive fungal mycelial biomasses that may suitably be used in biomaterials of the present disclosure include fungal mycelial biomasses produced by a liquid surface fermentation process or membrane fermentation process as described in PCT Application Publication 2019/046480 (the entirety of which is incorporated herein by reference) and/or fungal mycelial biomasses produced by a solid-substrate fermentation process as described in, e.g., PCT Application Publication 2016/149002 (the entirety of which is incorporated herein by reference).
  • cohesive fungal mycelial biomasses i.e., mycelial biomasses that have sufficient structural integrity and tensile strength to be picked up and physically manipulated by
  • the infused reactive materials may be completely solid (i.e., provided in the absence of a liquid solvent), as described further throughout this disclosure; this feature may be advantageous for reducing the hazards associated with volatile organic compound (VOC) solvents.
  • the reactive materials may be provided in an aaueous mixture or solvent or in an organic solvent. The reactive materials themselves may covalently bond to the biomaterial without crosslinking, may directly crosslink the biomaterial, or may simply penetrate/infiltrate the biomaterial and then be polymerized to form an independent polymer network within and/or surrounding the biomaterial.
  • In situ polymerization of infused monomers and/or oligomers to form an interpenetrating polymeric network within the biomaterial may, in some embodiments, be a particularly valuable approach because it enables the use of a broad variety of polymers and functionalities that can be incorporated into the biomaterial; by contrast, where a polymeric structure is created or formed prior to contact with the biomaterial, the types of polymers that can be used are generally limited to those that can be dissolved in a solvent that is compatible with the biomaterial.
  • monomers and/or oligomers are more likely to be liquid at room or process temperatures, and/or more readily dissolve in a wide array of solvents, than the polymeric structures formed after polymerization, and may comprise any one or more of a variety of useful chemical functionalities to tailor the properties of the polymerized network.
  • Solutions containing the monomer and/or oligomer may contain any one or more additives such as plasticizers, catalysts, initiators, accelerators, UV or heat stabilizers, dyes, pigments, and the like.
  • biomaterials may be effective to functionalize both whole/raw biomass (e.g. , filamentous fungal biomats) and slurries of biomaterial particles in a fluid matrix (e.g., dispersions of size-reduced filamentous fungal biomats, “wet” fungal biomass derived from submerged fermentation processes, etc.).
  • the particular form in which the biomaterial is provided may thus be selected to obtain certain types of composite materials that would otherwise be very difficult or impossible to obtain using a different form of biomaterial using conventional processes.
  • the biomaterial may be fixed or molded during the step of polymerization or other functionalization to provide a complex shape or a shape memory of the resulting composite material.
  • a first non-limiting example of a method for infusing reactive materials into a biomass or other biomaterial according to the present disclosure is solvent soaking, z.e., soaking the biomaterial in a solution including reactive materials (e.g., monomers, oligomers, prepolymers, and/or plasticizers).
  • reactive materials e.g., monomers, oligomers, prepolymers, and/or plasticizers.
  • This approach allows the solution to infuse into the biomaterial over time and to be polymerized or otherwise reacted in a separate step.
  • the biomaterial may be a cohesive fungal mycelial biomass.
  • the biomats are first deactivated or inactivated, e.g., by boiling in water, and rinsed to remove excess media before the infusion process.
  • it may be beneficial to remove a substantial portion (in some embodiments, the majority) of water from the biomat prior to infusion for example by soaking the biomat in an excess of a low- water content liquid (e.g., at least about 1 mL of ethanol per gram of wet biomat) and agitating (e.g., by sonication, stirring, cycling in and out of solution, etc.) the biomat for a time (e.g. , about 4 hours) for a time sufficient to remove excess growth medium and water from the biomat, then drying the biomat (e.g, under ambient conditions).
  • a low- water content liquid e.g., at least about 1 mL of ethanol per gram of wet biomat
  • agitating e.g., by
  • the biomass or other biomaterial may then be placed in a bath containing a solution of the reactive materials (e.g., crosslinkers and/or other components), with care taken to ensure that the mats remain flat (z.e., are not bunched or folded over).
  • a solution of the reactive materials e.g., crosslinkers and/or other components
  • the solution may contain between about 5 wt% and about 50 wt%, or any subrange thereof, of a crosslinker dissolved in one or more organic solvents (e.g., ethanol), water, or a combination thereof; a nonlimiting example of such a solution includes from about 15 wt% to about 35 wt% (in some embodiments, about 25 wt%) of an epoxy monomer, from about 15 wt% to about 35 wt% (in some embodiments, about 25 wt%) of an amine monomer, and from about 40 wt% to about 60 wt% (in some embodiments, about 50 wt%) of a plasticizer.
  • organic solvents e.g., ethanol
  • a nonlimiting example of such a solution includes from about 15 wt% to about 35 wt% (in some embodiments, about 25 wt%) of an epoxy monomer, from about 15 wt% to about 35 wt% (in some embodiments, about 25 wt%) of an amine
  • Biomats are allowed to soak in this solution, with or without agitation (e.g., by sonication, stirring, cycling in and out of solution, etc.), for sufficient time (e.g., between about 2 hours and about 4 hours), to ensure complete infusion of the reactive species into the biomat.
  • heat, and/or a negative gauge pressure z.e., a sub-atmospheric absolute pressure
  • a positive gauge pressure i.e., a super-atmospheric absolute pressure
  • biomats are removed from the solution and allowed to cure under appropriate conditions (e.g., ambient conditions) for an appropriate period (e.g., about 12 hours).
  • the material may then be further polymerized at an elevated temperature (e.g., about 100 °C for about 2 hours) to improve the mechanical properties and/or hydrophobic character of the resulting composite material relative to “raw” biomats.
  • an elevated temperature e.g., about 100 °C for about 2 hours
  • a second non-limiting example of a method for infusing reactive materials into a biomass or other biomaterial according to the present disclosure is simultaneous infusion and deactivation. Placing biomats in boiling water to deactivate the filamentous fungus densifies the biomat by collapsing at least a portion of its internal structure, which may be detrimental to the biomat’s ability to uptake the reactive materials. To preserve this internal structure and allow for faster and/or more complete infusion, deactivation may thus be carried out during or after infusion.
  • a biomat is removed from its fermentation vessel (e.g., a tray used for surface fermentation) and any excess growth medium is carefully washed away.
  • a solution of reactive materials as described throughout this disclosure is then evenly applied to one or more surfaces of the biomat, after which the biomat is allowed to rest for a sufficient time (e.g., about 12 hours) until no more “bulk” liquid solution is visible on or around the biomat.
  • the biomat is then cured at elevated temperature for a time sufficient to polymerize or otherwise react the monomers or other reactive materials applied to the mat; during curing and/or polymerization, the biomat may also be pressed to flatten any wrinkles, ridges, depressions, etc. in the surface of the biomat and/or to emboss a pattern into the surface of the biomat.
  • a third non-limiting example of a method for infusing reactive materials into a biomass or other biomaterial according to the present disclosure is spray application and infusion via diffusion.
  • biomats are first deactivated and optionally pre-treated with, e.g., ethanol to dehydrate and/or wash the biomat, as previously described.
  • a solution of reactive materials as described throughout this disclosure is then charged into an appropriate spraying device and applied by the spraying device onto one or more surfaces of the biomat as an even coating, with care taken to ensure that the minimum amount of solution is used to coat the desired surface(s); in some embodiments, a brush may be used to ensure even distribution of the solution on the surface(s) and/or a partial vacuum/negative gauge pressure can be applied to an unsprayed side of the biomat to assist in penetration of the solution.
  • the biomat is then cured at elevated temperature for a time sufficient to polymerize or otherwise react the monomers or other reactive materials applied to the mat; during curing and/or polymerization, the biomat may also be pressed to flatten any wrinkles, ridges, depressions, etc.
  • a fourth non-limiting example of a method for infusing reactive materials into a biomass or other biomaterial according to the present disclosure is vacuum bag infiltration.
  • negative pressure may be applied to the biomaterial to aid in the infiltration of reactive materials into the mat.
  • Vacuum-assisted solvent exchange can be much faster and more efficient than the simpler solvent soaking approach discussed above.
  • a filamentous fungal biomat may be fully enclosed and sealed within a flexible film or bag, to which a vacuum is applied to extract air and compress the mat.21hilee maintaining this partial vacuum, a solution of reactive materials as described throughout this disclosure is introduced into the bag or film through a separate valve or port, and the solution Is subsequently pulled through the biomat until the biomat is saturated, whereupon the flow of the solution is stopped while the vacuum is maintained for a time sufficient to ensure infusion of the reactive materials into the mat.
  • the biomat is then cured at elevated temperature for a time sufficient to polymerize or otherwise react the monomers or other reactive materials applied to the mat; during curing and/or polymerization, the biomat may also be pressed to flatten any wrinkles, ridges, depressions, etc. in the surface of the biomat.
  • a fifth non-limiting example of a method for infusing reactive materials into a biomass or other biomaterial according to the present disclosure is vacuum pressure cycling. This method is similar to that described in the preceding paragraph, except that the biomaterial is placed in a vacuum chamber and cycled between atmospheric pressure and a partial vacuum to evacuate the biomat of any trapped gases.
  • a filamentous fungal biomat may be partially dehydrated, placed in a bath of the reactive material solution, placing the bath in a vacuum chamber (optionally at elevated temperature, e.g., about 80 °C), and cycling the vacuum chamber between atmospheric pressure and a partial vacuum (e.g., about -75 kPa gauge pressure) on an appropriate schedule (e.g., cycling the pressure every 30 minutes for three total cycles).
  • the biomat is then cured at elevated temperature for a time sufficient to polymerize or otherwise react the monomers or other reactive materials applied to the mat; during curing and/or polymerization, the biomat may also be pressed to flatten any wrinkles, ridges, depressions, etc. in the surface of the biomat.
  • Vacuum pressure cycling may, in some embodiments, allow for the infusion or infiltration of an increased quantity of reactive material into the biomat and/or infusion or infiltration into the biomat of multiple different reactive materials in a defined manner (e.g, to form separate layers or segments of reactive materials within the biomat).
  • a sixth non-limiting example of a method for infusing reactive materials into a biomass or other biomaterial according to the present disclosure is melt/bulk infusion.
  • Certain reactive materials may be liquids at room temperature and therefore require no additional solvent to diffuse into the biomaterial, and certain other solid or highly viscous reactive materials may suitably be heated to melt and/or reduce viscosity rather than being dissolved in a solvent prior to infusion.
  • the biomaterial may be fully submerged in the bulk liquid reactive material (or mixture of two or more liquid reactive materials), in the absence of a solvent, for a time sufficient to ensure complete infusion of the reactive material(s) into the biomaterial.
  • the biomaterial is then cured at elevated temperature for a time sufficient to polymerize or otherwise react the monomers or other reactive materials applied to the biomaterial; in embodiments in which the biomaterial is a biomat, during curing and/or polymerization, the biomat may also be pressed to flatten any wrinkles, ridges, depressions, etc. in the surface of the biomat.
  • a seventh non-limiting example of a method for infusing reactive materials into a biomass or other biomaterial according to the present disclosure is pressure-assisted infusion. Particularly, to accelerate the rate of infusion of reactive materials into the biomaterial, pressure may be applied to the biomaterial to force the reactive materials into the mat.
  • this method may include spreading a thin layer of a reactive material solution as described throughout this disclosure may be spread onto a first plate and then placing the biomat atop the first plate to wet a bottom surface of the biomat with the solution.
  • Additional solution may then be added to a top surface of the biomat, and the biomat is then pressed between the first plate and a second plate, e.g., using a hydraulic press, to force the solution into the biomat.
  • heat may be applied to accelerate infusion or cure the solution while the biomat remains sandwiched between the plates.
  • the biomat is then cured at elevated temperature for a time sufficient to polymerize or otherwise react the monomers or other reactive materials applied to the mat; during curing and/or polymerization, pressure may continue to be applied to the biomat (by a hydraulic press or other means) to flatten any wrinkles, ridges, depressions, etc. in the surface of the biomat.
  • a mass ratio of infused reactive materials to biomaterial may, upon completion of infusion, range from about 1 :999 to about 999: 1 or any value therebetween, or alternatively may be in any subrange having a lower bound of A:B (where A and B are positive integers whose sum is 1,000) and an upper bound of C:D (where C and D are positive integers whose sum is 1.000 and C is larger than A).
  • a mass ratio of infused plasticizer to biomaterial may, upon completion of infusion, range from about 1 :999 to about 999: 1 or any value therebetween, or alternatively may be in any subrange having a lower bound of E:F (where E and F are positive integers whose sum is 1,000) and an upper bound of G:H (where G and H are positive integers whose sum is 1,000 and G is larger than E).
  • the starting biomaterial e.g., biomass
  • the infused reactive materials and/or one or more plasticizers may each separately and independent make up from about 0.1 wt% to about 99.9 wt%, or alternatively about any tenth of a percent therebetween, of the total mass of the infused material upon completion of infusion.
  • the reactive materials that are infused into biomaterials according to the present disclosure may serve any of numerous purposes. In some embodiments it may be desirable to crosslink portions of the biomaterial itself, as further described below, whereas in other embodiments it may be desirable to form a separate polymeric structure within the biomaterial, add plasticizers to the biomaterial, or chemically modify the biomaterial to improve its toughness, hydrophobic properties, etc.
  • a first non-limiting example of a reaction that may be facilitated by infusion of reactive materials into a biomaterial is functionalization of polysaccharides in the biomaterial to improve hydrophobicity and/or toughness.
  • Many unmodified biomaterials e.g., filamentous fungal mycelium, are highly hydrophilic and, often, brittle.
  • One method of reducing hydrophilicity is to modify hydroxyl groups of polysaccharides within the biomaterial with hydrophobic groups; in turn, one manner of accomplishing this is by infusing a monofunctional carboxylic acid (e.g., stearic acid) and optionally an esterification catalyst into the biomaterial, then heating the infused biomaterial to a temperature of at least about 150 °C to convert hydroxyl groups in the biomaterial to ester groups via Fisher esterification.
  • a monofunctional carboxylic acid e.g., stearic acid
  • an esterification catalyst optionally an esterification catalyst
  • hydroxyl groups in the biomaterial may be reacted with monofunctional isocyanates, silanes, epoxides, or acid anhydrides to produce a similar effect, and/or the hydroxyl groups can act as initiators for ring-opening polymerization of lactides, glycolides, or lactones in the reactive materials.
  • An additional benefit of functionalizing hydroxyl groups with hydrophobic chains is that doing so increases the mobility of the polysaccharide chains, thereby improving the flexibility and fracture toughness of the material.
  • a second non-limiting example of a reaction that may be facilitated by infusion of reactive materials into a biomaterial is Dolvmerization of an interpenetrating polymer network.
  • One way to significantly improve the fracture toughness of a biomaterial, e.g., filamentous fungal mycelium, is to polymerize a second polymer within the biomaterial.
  • the biomaterial may be infused with monomers, oligomers, prepolymers, and/or crosslinkers that are then selectively polymerized such that the network polymerizes around or within the biomaterial without bonding the biomaterial itself, thereby enabling greater control over the composition of the polymer produced.
  • Non-limiting exemplary embodiments of these methods include (1) infusing the biomaterial with acrylate monomers and an initiator (e.g., a photoinitiator or a thermal initiator, such as a peroxide) and polymerizing via free radical polymerization (e.g., upon exposing the biomaterial to actinic radiation or heat); (2) infusing the biomaterial with cyclic monomers (e.g., norbomenes, lactams) and then polymerizing via ring-opening or ring-opening metathesis polymerization; (3) infusing the biomaterial with monomers and/or oligomers and preferentially react these monomers and/or oligomers with each other via step-growth polymerization; (4) infusing the biomaterial with a first monomer selected from amines, anhydrides, and carboxylic acids and a second epoxide monomer and polymerizing to form a strong hydro
  • an initiator e.g., a photoinitiator or a thermal initi
  • a ratio between these two or more monomers, oligomers, etc. may be selected to provide for a desired mechanical property or combination of desired mechanical properties (e.g., any one or more of flexibility, elongation at break, tensile strength, etc.) of the resulting material.
  • a selected ratio between the molar amount of amine and the molar amount of epoxide may be from about 0.17 to about 1, more typically from about 0.33 to about 0.83, and most typically from about 0.5 to about 0.67, which may enable the biomaterial to survive a desired number of flex cycles (e.g., at least about 10,000, at least about 20,000, at least about 30,000, at least about 40,000, etc.) in bally flex testing, provide the biomaterial with a desired tensile strength (e.g., at least about 3 mPa, at least about 4 mPa, at least about 5 mPa, at least about 6 mPa, etc.) while maintaining a desired tensile strength (e.g., at least about 3 mPa, at least about 4 mPa, at least about 5 mPa, at least about 6 mPa, etc.) while maintaining a desired tensile strength (e.g., at least about 3 mPa, at least about 4
  • a third non-limiting example of a reaction that may be facilitated by infusion of reactive materials into a biomaterial is crosslinking the biomaterial with polyfunctional crosslinkers. These embodiments are further described hereinbelow.
  • some embodiments of the methods described herein include a slurry casting procedure, in which discrete particles of biomaterial (e.g., size-reduced filamentous fungal biomats, particles of biomass derived from submerged fermentation processes, etc.) are combined with the reactive materials in a solvent or carrier and the resulting mixture is then cast to a desired shape (typically, but not necessarily, a sheet) and cured.
  • This approach may be particularly useful for incorporating fillers into biomaterial-based composites, especially inorganic fillers that may be very difficult or impossible to incorporate into a whole or intact biomass.
  • the particles of biomaterial may be provided by any suitable method.
  • a whole or intact biomass or portion thereof (which may, but need not, be inactivated and/or partially or completely dried, e.g., by lyophilization or convective drying) may be size-reduced to form the discrete particles by any one or more suitable size-reduction techniques, such as, by way of non-limiting example, grinding (at either room or cryogenic temperature) and milling.
  • “wet” deactivated sheets of biomass may be size-reduced (e.g., using a blade impeller) to a desired fiber length or particle size (e.g., a median fiber length or particle size of less than about 300 pm) and the “cut” sheets may be used in the slurry casting process.
  • a desired fiber length or particle size e.g., a median fiber length or particle size of less than about 300 pm
  • “wet” discrete filaments of mycelium or conidia may be produced by stirred tank fermentation and used to generate a slurry for casting.
  • discrete mycelial filaments or conidia may be spray-dried into a powder for use in slurry casting.
  • the particles of biomaterial in the slurry may have a multi-modal particle size distribution.
  • the fungus may be fermented in a water-in-oil emulsion.
  • the fungus may be fermented as mycelial “shells” on the surface of stabilized air bubbles in a bulk reactor, z.e., by liquid-air interface fermentation, to produce hollow particles.
  • particles of biomaterial, a fluid matrix (c.g, a carrier, dispersant, solvent, etc.), and reactive materials are combined to form a homogeneous flowable mixture (a “slurry”) that is then cast into a specified geometry, typically (but not necessarily) a sheet.
  • This mixture is then cured by a suitable technique (e.g., heating and/or solvent evaporation) such that the biomaterial and the reactive materials interact to form a contiguous solid material.
  • the biomaterial may interact with the reactive materials by any one or more of covalent bonding, electrostatic interactions, hydrogen bonding, physical entanglement, etc.
  • the reactive materials can include any one or more solvents, dispersants, crosslinkers, plasticizers, catalysts, polymers, reactive diluents, etc., as well as any one or more monomers and/or oligomers such as those described above.
  • the slurry of the biomaterial particles, fluid matrix, and reactive materials may be cast onto a solid scaffold prior to curing.
  • the use of a solid scaffold may provide any one or more advantages, and particularly may result in the production of a composite material that incorporates the scaffold to impart improved properties to the material.
  • Non-limiting examples of suitable scaffold structures include nonwoven fabrics, woven fabrics, knit fabrics, continuous fibers (whether oriented or unoriented), chopped or discrete fibers (whether oriented or unoriented), discrete particulates, a separate intact biomaterial (e.g., a filamentous fungal biomat), and a separate biomaterial-containing textile material.
  • fibers or particulates can be added directly to the slurry during mixing. These particulates may provide improved mechanical properties (e.g., tensile strength, tear resistance) or other physical properties (e.g., thermal conductivity, electrical conductivity, shape memory) to the finished composite material.
  • suitable fiber materials include cellulose, plant fibers (e.g., cotton fibers, hemp fibers), polyethylene terephthalate, polypropylene, nylon, carbon fiber, fiberglass, aramid, and metal fibers.
  • suitable particulate materials include clay, talc, carbon black, calcium carbonate, kaolin, carbon nanotubes, chitin whiskers, metal particulates, shape memory alloys, and silica.
  • Particulate materials that may provide a desired degree of mechanical reinforcement to the composite material include, but are not limited to, clay, chopped glass fiber, chopped carbon fiber, rubber particles, talc, alumina, kaolin, carbon black, silica, calcium carbonate, chitin whiskers or flakes, cellulose fibers, cellulose microparticles, cellulose nanoparticles, carbon nanotubes, and nanoparticles of graphene or graphene oxide.
  • PEDOT poly(3,4-ethylenedioxythiophene)
  • Another potential advantage and benefit of the slurry casting techniques disclosed herein is the ability to produce multilayered composite materials, in which one or more layers have substantively different material properties from one or more other layers.
  • Many high-performance mechanical composites are multilayer composites in which layers alternate between soft/flexible layers and stiff/high-strength layers, and the slurry casting techniques disclosed herein enable the creation of multilayer composite materials of this type; in one non-limiting exemplary embodiment, a multilayer composite consists of five layers, in which the first, third, and fifth (i.e..
  • layers of textile supports or discrete particles may be embedded between two or more layers of the biomaterial-containing composite by casting one or more layers of the biomaterial slurry into a container, laying down a layer of textile support or particles, then casting one or more additional layers of biomaterial slurry. In this way, the product of the process after curing is completed can be a multilayer composite material with tuned mechanical properties.
  • the biomaterial slurry can be used as an adhesive to bond two separate substrates together (e.g., textile materials, discrete fibers, other “raw” or processed biomaterials, etc.) and thereby increase the total thickness of the bonded material.
  • a textile material may be laid flat (without wrinkles, folds, etc.) on the bottom of a flat tray and a slurry comprising biomaterial particles, an epoxy monomer, and an amine hardener may be poured onto the textile material in a thin, even layer.
  • the slurry may then be partially cured to become tacky, whereupon a biomat (or other monolithic biomaterial) may be laid atop the slurry (and optionally pressed, e.g., by a roller).
  • This assembly may then be placed in an oven at a sufficiently high temperature to fully cure the adhesive and bond the layers together.
  • biomaterial particles dispersed in a fluid matrix may allow for the manufacture of composite materials having complex geometries.
  • techniques for producing complex geometries from biomaterial-containing slurries include extrusion (reactive or non-reactive), cast molding, and/or injection molding (reactive or non- reactive).
  • the biomaterial-containing slurry may be extruded with one or more thermoplastics (e.g., polyolefins, polylactic acid, nylons, polyesters, etc.) as fibers, pellets, sheets, etc., which may then be injection molded, thermoformed, or the like to make a shaped article.
  • thermoplastics e.g., polyolefins, polylactic acid, nylons, polyesters, etc.
  • biomaterial particles may be swelled in an aqueous solution of monomers, whereby, upon swelling, the monomers penetrate into pores or voids within the particles of biomaterial.
  • the subsequent curing may result in an extremely close and robust interface between the biomaterial particles and the monomers contained within the fluid matrix.
  • the biomaterial-containing slurry may be foamed, which may be advantageous for creating thermally insulating materials or breathable textiles and/or increasing the thickness of the slurry when cast. Foaming of the slurry may be achieved by incorporating porogens, foaming agents, biowine aeents. carbon dioxide, or the like into the slurry prior to curing.
  • the slurry may include a dispersant that allows the slurry to maintain a workable viscosity at biomaterial concentrations in the slurry of at least about 50 wt%, and in some embodiments up to about 85 wt%; in some embodiments, the dispersant may be a supercritical solvent (e.g., supercritical carbon dioxide, supercritical propane, supercritical water, etc.).
  • a supercritical solvent e.g., supercritical carbon dioxide, supercritical propane, supercritical water, etc.
  • the biomaterial-containing slurry may be suitable for use as a topcoat.
  • topcoats which are typically polyurethane-based coatings without biologically-derived components, are applied to improve haptics, appearance, and chemical resistance.
  • particles of biomaterial may be dispersed in a fluid matrix together with topcoat components (e.g., minerals, pigments, dyes, colorants) to form a sprayable topcoat that itself contains a substantial amount of biomaterial.
  • biomaterial is a relatively cohesive mass (e.g., a biomat)
  • this technique may have the added advantage and benefit of enhancing the haptic feel of the composite material after curing/drying and/or reducing shrinkage.
  • the biomaterial may be held in place on the frame by pins, clips, or other similar gripping elements to allow uniform setting of the material.
  • the frame may also contain an open or closed backing pressed against the biomaterial to emboss a pattern or texture to the biomaterial during this step.
  • the biomaterial may be placed onto or into a mold and a stress may be applied (e.g., by clamps, vacuum pump, etc.) to force the biomaterial to take shape of the mold, whereupon heat or UV light is applied to induce reaction of the reactive materials within the biomaterial; after curing, the composite material will thus be “set” in the desired shape.
  • a stress e.g., by clamps, vacuum pump, etc.
  • a formulation comprising the monomers, oligomers, and/or reactive polymers may be selected from the group consisting of (i) a solution comprising the monomers, oligomers, and/or reactive polymers as a solute and a fluid solvent, and (ii) a colloid comprising the monomers, oligomers, and/or reactive polymers as a dispersed phase and a fluid dispersion medium.
  • the contacting of the biomaterial with the monomers, oligomers, and/or reactive polymers may comprise dispersing particles of the biomaterial in the fluid solvent or fluid dispersion medium to coat the particles with the monomers, oligomers, and/or reactive polymers.
  • Biomaterials made according to the methods described above may have advantageous mechanical properties compared to conventional or currently known biomaterials.
  • biomaterials made according to the present disclosure may have a water uptake after 24 hours of no more than about 140%, no more than about 130%, no more than about 120%, no more than about 110%, no more than about 100%, no more than about 90%, no more than about 80%, no more than about 70%, no more than about 60%, no more than about 50%, or no more than about 40%.
  • biomaterials made according to the present disclosure may survive at least about 10,000 flex cycles, at least about 20,000 flex cycles, at least about 30,000 flex cycles, at least about 40,000 flex cycles, at least about 50,000 flex cycles, at least about 60,000 flex cycles, at least about 70,000 flex cycles, at least about 80,000 flex cycles, at least about 90,000 flex cycles, at least about 100,000 flex cycles, at least about 110,000 flex cycles, at least about 120,000 flex cycles, at least about 130,000 flex cycles, at least about 140,000 flex cycles, at least about 150,000 flex cycles, at least about 160,000 flex cycles, at least about 170,000 flex cycles, at least about 180,000 flex cycles, at least about 190,000 flex cycles, or at least about 200,000 flex cycles in bally flex testing.
  • biomaterials made according to the present disclosure may have a strain at break of from about 10 to about 70 percent, or alternatively between a lower bound of any whole number of percent from 10 percent to 70 percent and an upper bound of any other whole number of percent from 10 percent to 70 percent.
  • biomaterials made according to the present disclosure may have a tensile strength of at least about 1 mPa, at least about 2 mPa, at least about 3 mPa, at least about 4 mPa, at least about 5 mPa, at least about 6 mPa, or at least about 7 mPa.
  • Biomaterials made according to the methods described above may particularly have combinations of beneficial properties, such as having both high tensile strength values and the ability to survive a high number of flex cycles in bally flex testing.
  • beneficial properties such as having both high tensile strength values and the ability to survive a high number of flex cycles in bally flex testing.
  • such materials can have a tensile strength of greater than about 3 mPa (or any other tensile strength value referenced above) while also surviving at least about 40,000 flex cycles (or any other number of flex cycles referenced above) in bally flex testing.
  • Volatile organic compounds are organic (ie., carbon-containing) compounds that have a high vapor pressure at room temperature. While there are many biogenic VOCs, anthropogenic sources, including especially fossil fuel combustion and the use of solvents (e.g., aliphatic hydrocarbons, ethyl acetate, glycol ethers, acetone) for coatings, paints, and inks, emit many tens of millions of kilograms of VOCs annually. Many VOCs can cause respiratory, allergic, or immune effects in humans or other animals.
  • solvents e.g., aliphatic hydrocarbons, ethyl acetate, glycol ethers, acetone
  • biomaterials made according to many embodiments of the methods described above is that the monomer and/or oligomer solution has a low content of VOCs and thus represent a lower VOC-related hazard than previous biomaterial-based textiles.
  • the present disclosure also provides methods for functionalizing peptides on the surface of a biomaterial.
  • the disclosure further provides treated biomaterials made by these methods.
  • Many biomaterials e.g., mycelium of filamentous fungi
  • Embodiments of the methods disclosed herein leverage this characteristic to functionalize (e.g., crosslink, modify the structure or composition of, etc.) these surface proteins/peptides and thereby achieve a desired material property of the biomaterial; by way of non-limiting example, the methods may be used to express collagen- like proteins advantageous in high-performance leather analog products and the like.
  • the methods of the present disclosure may instead treat and/or functionalize peptides and/or proteins, thereby simplifying the manufacture of the treated biomaterial, reducing or eliminating the need for harsh chemical treatments, and expanding the universe of functionalizing agents that may be used (e.g., epoxies, which are generally not effective to crosslink or otherwise functionalize polysaccharides).
  • the methods of the disclosure advantageously allow for the engineering of desired peptide/protein chemistries or structures within a treated biomaterial to enhance chemical, mechanical, or physical properties of the treated biomaterial.
  • An additional benefit is that these methods are effective to functionalize both whole/raw biomass (e.g., filamentous fungal biomats) and slurries of biomaterial particles in a fluid matrix (e.s.. disoersions of size-reduced filamentous fungal biomats, “wet” fungal biomass derived from submerged fermentation processes, etc.).
  • polymerizing or reacting a peptide-binding moiety with functional groups already present in the biomaterial may provide an advantage over polymerizing an interpenetrating polymeric structure within the biomaterial (as described above) because it can impart added strength to the biomaterial using smaller quantities of crosslinkers or other reactants.
  • polycarboxylic acids may be used as a peptide-binding moiety to crosslink hydroxyl and primary amine groups within the biomaterial.
  • poly epoxides may be used as a peptide-binding moiety to crosslink hydroxyl, primary amine, and carboxylic acid groups within the biomaterial.
  • polyanhydrides may be used as a peptide-binding moiety to crosslink hydroxyl and primary amine groups within the biomaterial.
  • poly carbodiimides may be used as a peptide-binding moiety to crosslink carboxylic acid groups within the biomaterial.
  • polyisocyanates and polysilanes to further improve the toughness and/or flexibility of the resulting material, it may be beneficial to utilize two or more different types of peptide-binding moiety to crosslink the biomaterial, as doing so can increase the molecular weight between crosslinks; one non-limiting example is using both a polyol and a polycarboxylic acid or polyanhydride.
  • catalysts may optionally be employed to accelerate crosslinking reactions within the biomaterial.
  • the peptide functionalization may be part of a two-step functionalization procedure.
  • Many commercially available crosslinkers are relatively small molecules and so produce a crosslinked material that has increased strength but is also brittle.
  • it can be beneficial to increase the molecular weight between crosslinks while in some embodiments this may be accomplished by infusing functional polymers or high-molecular weight prepolymers into the biomaterial, these materials typically have solubilities and/or viscosities that result in ineffective impregnation of the biomaterial.
  • a first non-limiting example of a two-step functionalization procedure to address these limitations is infusion of a blend of monomers and/or oligomers that preferentially polymerizes with itself but, after this polymerization, retains chemical functionalities that enable reaction with the biomaterial;
  • a blend of monomers and/or oligomers in contact with a biomaterial e.g., an amine/epoxy blend
  • a relatively low temperature e.g., between about 20 °C and about 100 °C
  • the temperature is then raised to a higher temperature (e.g., between about 120 °C and about 200 °C) such that residual unreacted epoxide groups react with hydroxyl and carboxyl moieties in the biomaterial to crosslink the polymeric structure to the biomaterial.
  • a second non-limiting example of a two-step functionalization procedure is infusion of a blend of monomers and/or oligomers that bonds to the mycelium, followed by polymerization and crosslinking of the monomers and/or oligomers; in an exemplary embodiment, the biomaterial is infused with a mixture of a first free radical monomer that does not bind with the biomaterial, a second free radical monomer that binds to hydroxyl groups within the biomaterial (e.g.
  • enzymatic treatment may remove proteins from cell walls of plant or fungal biomaterials to expose functional groups.
  • the biomaterial may have a total protein content of at least about 20 wt%, at least about 30 wt%, at least about 40 wt%, at least about 50 wt%, at least about 60 wt%, or at least about 70 wt%.
  • the peptide-binding moiety may be a crosslinker, e.g., a macromolecule. In some embodiments, the peptide-binding moiety may be a dye or pigment. In some embodiments, the peptide-binding moiety may impart to the biomaterial at least one functionality selected from the group consisting of hydrophobicity, hydrophilicity, amphiphilicity, cationic charge, anionic charge, and affinity for a selected compound. In some embodiments, the peptide-binding moiety may be a surface-functional particulate. In some embodiments the peptide-binding moiety may be an internal plasticizer. In some embodiments, the peptide-binding moiety may be a ligand or chelator.
  • the peptide-binding moietv mav be a monomer or initiator capable of polymerization after the functionalization reaction.
  • the peptide- binding moiety may comprise at least one of an epoxy, a maleimide, an iodoacetamide, a succinimidyl ester, a sulfonyl chloride, a cyclic carbonate, a Michael addition acceptor, an alkene, an isocyanate, a carbodiimide, a carboxylic acid (or ester), an anhydride, an amine, an alkyl halide, an aldehyde, a thiol, a polysaccharide, and a peptide.
  • the peptides of the biomaterial may be crosslinked by at least one of covalent bonds, ionic bonds, coordinate bonds, electrostatic interactions, hydrogen bonds, hydrophobic and/or van de Waals interactions, and combinations thereof.
  • a weight ratio of the peptide-binding moiety to the biomaterial may be at least about 0.8, at least about 0.5, at least about 0.2, at least about 0.01, or at least about 10' 6 .
  • a formulation containing the peptide-binding moiety may further comprise any one or more of (1) a reactant that facilitates the functionalization reaction (e.g., a carbodiimide), (2) a pH buffer, and (3) a species that promotes the functionalization reaction at a selected pH (e.g., side chain amines).
  • the biomaterial may be in a form selected from the group consisting of a paste, a slurry, a semisolid, a solid, a gel, a hydrogel, a solution, a suspension, and combinations thereof.
  • the biomaterial may be produced by a process selected from the group consisting of a submerged fermentation, a surface fermentation, a submerged solid substrate fermentation, a solid substrate fermentation, a membrane fermentation, and combinations thereof.
  • the biomaterial may comprise at least one material selected from the group consisting of fungal mycelium (which may have peptides exposed on cell walls thereof and/or may be chemically or mechanically processed to expose peptides on a surface thereof), a plant-derived material, a bacteria-derived material, an algae-derived material, and a yeast-derived material; the biomaterial may, but need not, comprise materials derived from two or more different species, e.g., biomasses of two or more filamentous fungi, a biomass of a filamentous fungus and one or more complementary microorganisms (which collectively may produce a formulation containing the peptide-binding moiety or a component thereof by complementary activity), etc.
  • the biomaterial may comprise fungal mycelium.
  • the biomat may be produced by a surface fermentation process. Additionally or alternatively, the biomat may be inactivated, whether before, simultaneous with, or after contact with the peptide-binding moiety.
  • a formulation containing the peptide-binding moiety may comprise a solution and the step of contacting the biomat with the peptide-bindine moietv mav comorise at least one of soaking the biomat in the solution, spraying the solution onto a surface of the biomat, causing the solution to infiltrate the biomat via vacuum-assisted solvent exchange and/or vacuum pressure cycling, and mechanically pressing the solution into the biomat.
  • the fungal biomat may be grown in the presence of a formulation containing the peptide-binding moiety, or a component thereof. Additionally or alternatively, a formulation containing the peptide-binding moiety or a component thereof may be produced by metabolism of the fungal biomat.
  • peptides of the biomaterial may be overexpressed or upregulated.
  • the biomaterial may be plasticized using a plasticizer before, simultaneously with, or after contact with the peptide-binding moiety and/or before, simultaneously with, or after the functionalization reaction.
  • a formulation containing the peptide-binding moiety may comprise a solution of the peptide- binding moiety in an aqueous solvent, an organic solvent, and/or a deep eutectic solvent.
  • the biomaterial may be pre-treated prior to the functionalization reaction to facilitate the functionalization reaction, e.g., by at least one of alkali removal of extracellular polysaccharides, pH increase, lipase treatment, protease treatment, carbohydrase treatment, solvent treatment, decrystallization, acid pretreatment, and reduction of thiols.
  • the biomaterial may be ground or homogenized prior to functionalization and cast as part of a slurry. In some embodiments, at least about 3 wt% of peptides in the biomaterial may be functionalized.
  • Biomaterials made according to the methods described above may have advantageous mechanical properties compared to conventional or currently known biomaterials.
  • biomaterials made according to the present disclosure may have a water uptake after 24 hours of no more than about 140%, no more than about 130%, no more than about 120%, no more than about 110%, no more than about 100%, no more than about 90%, no more than about 80%, no more than about 70%, no more than about 60%, no more than about 50%, or no more than about 40%.
  • biomaterials made according to the present disclosure may survive at least about 10,000 flex cycles, at least about 20,000 flex cycles, at least about 30,000 flex cycles, at least about 40,000 flex cycles, at least about 50,000 flex cycles, at least about 60,000 flex cycles, at least about 70,000 flex cycles, at least about 80,000 flex cycles, at least about 90,000 flex cycles, at least about 100,000 flex cycles, at least about 110,000 flex cycles, at least about 120,000 flex cycles, at least about 130,000 flex cycles, at least about 140,000 flex cycles, at least about 150.000 flex cvcles, at least about 160,000 flex cycles, at least about 170,000 flex cycles, at least about 180,000 flex cycles, at least about 190,000 flex cycles, or at least about 200,000 flex cycles in bally flex testing.
  • biomaterials made according to the present disclosure may have a strain at break of from about 10 to about 70 percent, or alternatively between a lower bound of any whole number of percent from 10 percent to 70 percent and an upper bound of any other whole number of percent from 10 percent to 70 percent.
  • biomaterials made according to the present disclosure may have a tensile strength of at least about 1 mPa, at least about 2 mPa, at least about 3 mPa, at least about 4 mPa, at least about 5 mPa, at least about 6 mPa, or at least about 7 mPa.
  • Biomaterials made according to the methods described above may particularly have combinations of beneficial properties, such as having both high tensile strength values and the ability to survive a high number of flex cycles in bally flex testing.
  • beneficial properties such as having both high tensile strength values and the ability to survive a high number of flex cycles in bally flex testing.
  • such materials can have a tensile strength of greater than about 3 mPa (or any other tensile strength value referenced above) while also surviving at least about 40,000 flex cycles (or any other number of flex cycles referenced above) in bally flex testing.
  • biomasses used as biomaterials in the methods described above are filamentous fungal biomasses — that is, biomasses of one or more fungi that produce an interconnected network of hyphae known as mycelium.
  • the filamentous fungal biomass in biomaterials according to the present disclosure may be a fungal mycelial biomass as that term is defined herein (although other filamentous fungal biomasses, such as biomasses that contain a significant quantity of material derived from fruiting bodies of a filamentous fungus, are also contemplated and are within the scope of this disclosure).
  • the fungal mycelial biomasses used in biomaterials according to the methods described above are cohesive fungal mycelial biomasses, ie., mycelial biomasses that have sufficient structural integrity and tensile strength to be picked up and physically manipulated by hand without tearing or collapsing; non-limiting examples of cohesive fungal mycelial biomasses that may suitably be used in biomaterials of the present disclosure include fungal mycelial biomasses produced by a liquid surface fermentation process or membrane fermentation process as described in PCT Application Publication 2019/046480 (the entirety of which is incorporated herein by reference) and/or fungal mycelial biomasses produced by a solid-substrate fermentation process as described in, e.g., PCT Application Publication 2016/149002 (the entirety of which is incorporated herein by reference). Plasticization of Biomaterials Using Deep Eutectic Solvents and Urea/Polyol Mixtures
  • the present disclosure also provides deep eutectic solvents (DESs), methods for using DESs and polyol/urea mixtures as plasticizers for biomaterials, and plasticized biomaterials made by these methods.
  • DESs deep eutectic solvents
  • Non-limiting examples of DESs are described in Yuntao Dai et al.. “Natural deep eutectic solvents as new potential media for green technology,” 766 Analytica Chimica Acta 61 (Mar. 2013), the entirety of which is incorporated herein by reference.
  • such materials can withstand greater than about 10,000 flex cycles, greater than about 20,000 flex cycles, greater than about 30,000 flex cycles, greater than about 40,000 flex cycles, greater than about 50,000 flex cycles, greater than about 60,000 flex cycles, greater than about 70,000 flex cycles, greater than about 80,000 flex cycles, greater than about 90,000 flex cycles, greater than about 100,000 flex cycles, greater than about 110,000 flex cycles, greater than about 120,000 flex cycles, greater than about 130,000 flex cycles, greater than about 140,000 flex cycles, greater than about 150,000 flex cycles, greater than about 160,000 flex cycles, greater than about 170,000 flex cycles, greater than about 180,000 flex cycles, greater than about 190,000 flex cycles, or greater than about 200,000 flex cycles, or any range of flex cycles between about 10,000 and about 200,000.
  • the plasticizers used in these methods have low volatility, and DESs particularly are expected to have a low rate of migration in the finished biomaterial due to their supramolecular complex structure. Additionally, DESs and urea/polyol mixtures have low toxicity and environmental impact.
  • the eutectic nature of DESs may enable the use of two species that are effective to coordinate (e.g., disrupt hydrogen bonding in) polysaccharides of the biomaterial but generally suffer poor mobility due to precipitation and/or crystallization; in the DES system, these species may flow and maintain high mobility, improving the plasticization process.
  • DESs are effective in these methods because they can dissolve both hvdroohilic and hydrophobic compounds and can therefore plasticize both the hydrophobic and hydrophilic parts of a biomaterial (e.g., the hydrophobic bulk and hydrophilic surfaces of fungal mycelium). DESs can also dissolve many hydrophobic species that are frequently used as crosslinkers; thus, dissolving a crosslinker in a DES may enable the use of a single liquid solution as both a plasticizer and a crosslinker. Additionally, and again without wishing to be bound by any particular theory, the present inventors hypothesize that fibers or filaments in biomaterials (e.g. , fungal mycelia and/or hyphae) can be “welded” together using DESs in much the same way as ionic liquid welding, but advantageously using the far more benign chemical components of a DES rather than an ionic liquid.
  • biomaterials e.g., fungal mycelia and/or hyphae
  • the plasticizer may be dissolved in an aqueous or volatile organic solvent, which may be evaporated after contact with the biomaterial.
  • the plasticizer may comprise a mixture of a compound containing a urea or thiourea functionality with a polyol (e.g., glycerol).
  • the plasticizer may be combined with an amphiphilic compound (e.g., lecithin or another phospholipid) as a co-plasticizer.
  • the hydrogen bond acceptor of the DES may be an amino acid, an organic acid (e.g., citric acid or another carboxylic acid), a quaternary ammonium salt (e.g, a betaine, a cholinium salt (choline chloride, choline acetate, etc.), l-ethyl-3-methylimidazolium chloride, etc.), a hydrated metal salt, an anhydrous or hydrated metal halide (e.g anhydrous aluminum chloride or aluminum chloride hexahydrate), or a phosphonium salt.
  • an organic acid e.g., citric acid or another carboxylic acid
  • a quaternary ammonium salt e.g, a betaine, a cholinium salt (choline chloride, choline acetate, etc.), l-ethyl-3-methylimidazolium chloride, etc.
  • a hydrated metal salt e.g anhydrous aluminum chloride or aluminum chloride he
  • the hydrogen bond donor of the DES may be selected from the group consisting of a carboxylic acid, a sugar, an alcohol, an amine, an amino acid, an amide, a thiol, a urethane, a sulfonic acid, a phosphoric acid, and a phosphonic acid.
  • the hydrogen bond donor of the DES may be selected from the group consisting of ethylene glycol, propylene glycol, glycerol, and urea.
  • the plasticizer may be dissolved in a solvent (e.g., water) and contacting the biomaterial with the plasticizer may comprise contacting the biomaterial with the solvent and evaporating at least a portion of the solvent to leave the plasticizer in contact with the biomaterial.
  • a solvent e.g., water
  • the plasticizer may be a solution comprising a DES, a mixture of a compound containing a urea or thiourea functionality with a polyol, or both as a solvent and a crosslinker (e.g., an epoxy) as a solute, wherein the crosslinker is reactive with a surface moiety of peptides of the biomaterial; the plasticizing process may include reaction of the crosslinker with the surface moiety to crosslink the peptides of the biomaterial.
  • a crosslinker e.g., an epoxy
  • the biomaterial mav be in a form selected from the group consisting of a paste, a slurry, a semisolid, a solid, and combinations thereof, and may be produced by a process selected from the group consisting of a submerged fermentation, a surface fermentation, a submerged solid substrate fermentation, a solid substrate fermentation, a membrane fermentation, and combinations thereof.
  • the biomaterial may comprise at least one material selected from the group consisting of fungal mycelium (e.g., a filamentous fungal biomat, which may in some embodiments be inactivated), a plant-derived material, and a bacteria-derived material.
  • Biomaterials made according to the methods described above may have advantageous mechanical properties compared to conventional or currently known biomaterials.
  • biomaterials made according to the present disclosure may have a water uptake after 24 hours of no more than about 140%, no more than about 130%, no more than about 120%, no more than about 110%, no more than about 100%, no more than about 90%, no more than about 80%, no more than about 70%, no more than about 60%, no more than about 50%, or no more than about 40%.
  • biomaterials made according to the present disclosure may survive at least about 10,000 flex cycles, at least about 20,000 flex cycles, at least about 30,000 flex cycles, at least about 40,000 flex cycles, at least about 50,000 flex cycles, at least about 60,000 flex cycles, at least about 70,000 flex cycles, at least about 80,000 flex cycles, at least about 90,000 flex cycles, at least about 100,000 flex cycles, at least about 110,000 flex cycles, at least about 120,000 flex cycles, at least about 130,000 flex cycles, at least about 140,000 flex cycles, at least about 150,000 flex cycles, at least about 160,000 flex cycles, at least about 170,000 flex cycles, at least about 180,000 flex cycles, at least about 190,000 flex cycles, or at least about 200,000 flex cycles in bally flex testing.
  • biomaterials made according to the present disclosure may have a strain at break of from about 10 to about 70 percent, or alternatively between a lower bound of any whole number of percent from 10 percent to 70 percent and an upper bound of any other whole number of percent from 10 percent to 70 percent.
  • biomaterials made according to the present disclosure may have a tensile strength of at least about 1 mPa, at least about 2 mPa, at least about 3 mPa, at least about 4 mPa, at least about 5 mPa, at least about 6 mPa, or at least about 7 mPa.
  • Biomaterials made according to the methods described above may particularly have combinations of beneficial properties, such as having both high tensile strength values and the ability to survive a high number of flex cycles in bally flex testing.
  • beneficial properties such as having both high tensile strength values and the ability to survive a high number of flex cycles in bally flex testing.
  • such materials can have a tensile strength of greater than about 3 mPa (or any other tensile strength value referenced above) while also surviving at least about 40,000 flex cycles (or any other number of flex cycles referenced above) in bally flex testing.
  • biomasses used as biomaterials in the methods described above are filamentous fungal biomasses — that is, biomasses of one or more fungi that produce an interconnected network of hyphae known as mycelium.
  • the filamentous fungal biomass in biomaterials according to the present disclosure may be a fungal mycelial biomass as that term is defined herein (although other filamentous fungal biomasses, such as biomasses that contain a significant quantity of material derived from fruiting bodies of a filamentous fungus, are also contemplated and are within the scope of this disclosure).
  • the fungal mycelial biomasses used in biomaterials according to the methods described above are cohesive fungal mycelial biomasses, ie., mycelial biomasses that have sufficient structural integrity and tensile strength to be picked up and physically manipulated by hand without tearing or collapsing; non-limiting examples of cohesive fungal mycelial biomasses that may suitably be used in biomaterials of the present disclosure include fungal mycelial biomasses produced by a liquid surface fermentation process or membrane fermentation process as described in PCT Application Publication 2019/046480 (the entirety of which is incorporated herein by reference) and/or fungal mycelial biomasses produced by a solid-substrate fermentation process as described in, e.g., PCT Application Publication 2016/149002 (the entirety of which is incorporated herein by reference).
  • a mass ratio between the hydrogen bond donor and the hydrogen bond acceptor, and/or between the urea or thiourea compound(s) and the polyol(s), may be selected to provide for a desired mechanical property or combination of desired mechanical properties (e.g., any one or more of flexibility, elongation at break, tensile strength, etc.) of the resulting material.
  • a selected ratio between the mass of urea and the mass of glycerol may be about 1 :4, about 1 :3, about 1 :2, about 1 : 1, about 2: 1, about 3: 1, or about 4:1 (or any ratio within a range between any two of these values), which may enable the biomaterial to survive a desired number of flex cycles (e.g., at least about 5,000, at least about 10,000, at least about 15,000, at least about 20,000, at least about 25,000, at least about 30,000, at least about 35,000, at least about 40,000, at least about 45,000, at least about 50,000, at least about 55,000, at least about 60,000, etc.) in bally flex testing.
  • a desired number of flex cycles e.g., at least about 5,000, at least about 10,000, at least about 15,000, at least about 20,000, at least about 25,000, at least about 30,000, at least about 35,000, at least about 40,000, at least about 45,000, at least about 50,000, at least about 55,000, at least
  • Biomaterials made according to the present disclosure may be characterized by any one or more advantageous mechanical properties, including but not necessarily limited to the advantageous mechanical properties described elsewhere throughout this disclosure, due to any one or more selected processing conditions, chemical compositions, etc.
  • certain advantageous mechanical properties may be obtained by providing in the biomaterial a selected ratio of a first monomer, oligomer, or reactive polymer (e.g., polyvinyl alcohol, reactive amines, etc.) to a second monomer, oligomer, or reactive polymer (e.g., chitosan, epoxides, etc.), particularly where the first and second monomers, oligomers, or reactive polymers are amenable to differing types of crosslinking reactions (e.g., amidation vs.
  • this ratio (whether a mass ratio or a stoichiometric or molar ratio) may be from about 1 :99 to about 99: 1, or alternatively in any range having a lower bound of any ratio A:B (where A and B are positive integers whose sum is 100) and an upper bound of any ratio C:D (where C and D are positive integers whose sum is 100 and C is greater than A), or alternatively about 20:80, about 40:60, about 50:50, about 60:40, or about 80:20.
  • certain advantageous mechanical properties may be obtained by providing in the biomaterial a selected polymer loading ratio; in some embodiments, this mass ratio may be from about 1 :99 to about 99:1, or alternatively in any range having a lower bound of any ratio E:F (where E and F are positive integers whose sum is 100) and an upper bound of any ratio G:H (where G and H are positive integers whose sum is 100 and G is greater than E), or alternatively about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about 85: 15, about 90: 10, or about 95:5.
  • certain advantageous mechanical properties may be obtained by providing in the biomaterial a selected plasticizer (e.g., polyol) content; in some embodiments, this content may be from about 0.1 wt% to about 60 wt%, or alternatively in any range having a lower bound of any tenth of a weight percent from 0.1 wt% to 60 wt% and an upper bound of any other tenth of a weight percent from 0.1 wt% to 60 wt%, or alternatively about 12.5 wt%, about 25 wt%, about 37.5 wt%, or about 50 wt%.
  • a selected plasticizer e.g., polyol
  • this content may be from about 0.1 wt% to about 60 wt%, or alternatively in any range having a lower bound of any tenth of a weight percent from 0.1 wt% to 60 wt% and an upper bound of any other tenth of a weight percent from 0.1 wt% to 60 wt%, or
  • certain advantageous mechanical properties may be obtained by crosslinking a biomass of the biomaterial using a selected amount of a crosslinker (e.g., an epoxide); in some embodiments, this amount may be at least about 0.1, at least about 0.2 at least about 0.3, at least about 0.4, at least about 0.5.
  • a crosslinker e.g., an epoxide
  • certain advantageous mechanical properties may be obtained by plasticizing a biomass of the biomaterial using a DES or polyol/urea mixture having a selected mass or molar ratio of a first component (e.g., hydrogen bond donor or polyol) to a second component (e.g., hydrogen bond acceptor or urea); in some embodiments, this ratio may be from about 1 :99 to about 99: 1, or alternatively in any range having a lower bound of any ratio I: J (where I and J are positive integers whose sum is 100) and an upper bound of any ratio K:L (where K and L are positive integers whose sum is 100 and K is greater than I), or alternatively about 5:95, about 10:90, about 15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about
  • the biomats were allowed to soak in the ethanol solution with agitation for about 4 hours to remove excess growth medium and water from the biomats.
  • the ethanol-soaked biomats were removed from the ethanol solution and allowed to dry at ambient conditions for 15 minutes. After drying, the mats were light brown in color, semi-rigid, and slightly swollen compared to before the ethanol soak.
  • the biomats were then placed in a bath of a liquid crosslinking solution consisting of 66 wt% ethanol solvent and 34% solute, the solute consisting of 25 wt% epoxy resin (EPONEX 1510), 25 wt% amine curative (EPIKURE 3164), and 50 wt% glycerol.
  • the biomats were allowed to soak in the crosslinking solution for 2 to 4 hours with agitation, or sufficient time to ensure complete infusion of the solution into the biomats.
  • the biomats were removed from the crosslinking solution bath and excess solution was removed from the surface of the mats.
  • the biomats were then heated for sufficient time to polymerize the infused crosslinkers; in an exemplary case, the biomat was first cured at ambient conditions for 12 hours, then heated in a forced-air convection oven at 100 °C for 3 hours. After polymerization, the resulting composite material had significantly improved mechanical properties and a substantially more hydrophobic character relative to untreated biomats.
  • the tensile strength and elongation at break of the polymerized composite material, and the water uptake after 24 hours of soaking in water of both the polymerized composite material and untreated biomats after the ethanol soak, are given in Table 1; tensile strength and elongation at break for the untreated biomats could not be measured because the samples mechanically failed during test preparation.
  • a biomat of Fusarium strain flavolapis having a wet mass of approximately 100 g was inactivated by boiling in water for 30 minutes and then immersed in 150 g of an aqueous solution of 0.9 M ammonium persulfate (APS) and 0.015 M tetramethylethylenediamine (TEMED) at 75 °C for 2 hours. This immersion generated carboxylic acid functional groups within the polysaccharides of the fungal mycelium by oxidation.
  • the biomat was then immersed in a room-temperature water bath to remove residual APS and TEMED, then immersed in an EPONEX/EPIKURE/glycerol crosslinking solution as described in Example 1.
  • the biomat was removed from the crosslinking solution and excess solution was removed from the surface of the mat.
  • the biomat was then heated to 90 °C to selectively polymerize the epoxy/amine mixture.
  • the mat was then heated to 150 °C for 1 hour to crosslink residual epoxide units to the carboxylic acid units on the oxidized fungal mycelium.
  • A4-sized (210 mm x 297 mm) sheets of Fusarium strain flavolapis biomat were immersed in an aqueous solution of 0.5 wt% potassium aluminum sulfate for 60 minutes with light agitation.
  • the pH of the solution was then adjusted to 5.0 using a 10 wt% solution of sodium carbonate, and the biomat sheets were then immersed for a further 60 minutes.
  • the solution was drained and replaced with an aqueous solution containing 10 wt% sucrose, 12.5 wt% sorbitol, 12.5 wt% glycerol, 1 wt% K-carrageenan, and 1 wt% sodium alginate at 60 °C; the sheets were immersed in this solution with light agitation for 120 minutes. The sheets were removed from the solution, excess solution was scraped from the surfaces of the sheets, and the sheets were air-dried under ambient conditions. Throughout the rest of this Example, the resulting material is referred to as “Material 2.”
  • a 4” x 6” (10.2 cm x 15.2 cm) sheet of Fusarium strain flavolapis biomat was immersed in a solution of 23 wt% ethanol in water for 6 hours, then removed from the solution and baked in an oven at 100 °C for 6 hours. Throughout the rest of this Example, the resulting material is referred to as “Material 3.”
  • hydrophilic monomers such as acrylic acid may be polymerized to increase the hydrophilicity of the composite material relative to the untreated biomaterial, whereas more hydrophobic water- soluble monomers, such as the epoxy-amine blend described in this Example or others (e.g., methyl acrylate), may be polymerized to increase hydrophobicity.
  • “Wet” Fusarium strain flavolapis biomats having a moisture content of about 75 wt% were blended with water in a conventional kitchen blender for 60 seconds to form homogeneous pastes.
  • aqueous solutions of polyvinyl alcohol were made by heating PVA in water at 90 °C until the PVA was fully dissolved.
  • PVA solutions chitosan, citric acid, concentrated hydrochloric acid, and glycerol were added, and the solutions were stirred until the chitosan was fully dissolved.
  • These polymer solutions were then added to the fungal pastes and mixed thoroughly to form homogeneous slurries.
  • the homogeneous fungal/polymer slurries were poured into flat-bottomed trays in amounts of 1 to 6 g/cm 2 of tray surface area, depending on the desired thickness of the final material.
  • the slurries were dried in the trays at about 20 °C and 50% relative humidity with mixing air until the cast slurries were sufficiently dried to be removed from the tray as a contiguous sheet.
  • These sheets were then heated at a sufficient temperature and for a sufficient time to obtain a desired extent of crosslinking both on the surface of the sheets and throughout the thickness of the sheet; in an exemplary case, this heating process consisted of placing the dried sheet in an oven at 130 °C for 1 hour.
  • Targeted crosslinking reactions included amidation reactions (between amine groups present in chitosan or mycelial cell wall polysaccharides and carboxyl groups present in the citric acid) and esterification reactions (between hydroxyl groups present in PVA, chitosan, or mycelial cell wall polysaccharides and carboxyl groups present in the citric acid).
  • the loading ratio and the weight ratio of PVA to chitosan were held constant at 70:30 and 50:50, respectively, while the concentration of glycerol in the final dry material was varied.
  • Materials produced in all three runs were assessed for tensile strength (TS), strain at break (SAB), and degree of swelling (DOS) after immersion in water for 24 hours; the results for materials produced in the first, second, and third runs are given in Tables 2, 3, and 4, respectively.
  • Biomats of Fusarium strain flavolapis in “wet” form i.e., without treatment to remove water and thus having a moisture content of about 90 wt%, were inactivated by boiling in water for 30 minutes and added to a sufficient volume of a solution of 95% ethanol to ensure at least 1 mL of ethanol solution per gram of wet biomat.
  • the biomats were allowed to soak in the ethanol solution with agitation for about 4 hours to remove excess growth medium and water from the biomats.
  • the ethanol-soaked biomats were removed and subjected to a partial vacuum (-0.75 bar gauge pressure) at 50 °C for 6 minutes to remove ethanol and residual water, resulting in a 75% reduction in mass.
  • a monomer mixture consisting of 6.0 g triethyl citrate, 6.0 g diethyl adipate, and 16.2 g Priamine 1074, were brushed on one side of a 6.1 g portion of the biomat, which was allowed to rest at room temperature for 16 hours to enable infusion of the monomer mixture.
  • the biomat portion was then cured at 120 °C for 3 hours to partially polymerize a polyamide network interpenetrating the mycelium, and this composite was then further heated to 180 °C for an additional 3 hours to crosslink the interpenetrating polymer network to the mycelium.
  • a biomat of Fusarium strain flavolapis was inactivated by boiling in water and then immersed in a 46.3 pM aqueous solution of fluorescein isothiocyanate (FITC) for 2 hours at room temperature; FITC is an amine-reactive dye that fluoresces when reacted with proteins and thus allows for visualization of proteins within the biomat. After immersion in the FITC solution, the biomat was rinsed repeatedly with water to remove unreacted FITC.
  • FITC fluorescein isothiocyanate
  • fluorescence microscopy images of the FITC- labeled biomat confirm the presence within the biomat of significant quantities of amine groups that are accessible for reaction with small molecules, both at an edge of the biomat ( Figure 2A) and throughout the interior of the mat ( Figure 2B).
  • Figures 3A and 3B are fluorescence microscopy images of a FITC-labeled biomat sample treated with a protease solution
  • Figures 3C and 3D are fluorescence microscopy images of aFITC-labeled mat sample treated with a chitinase solution.
  • protease hydrolyzes the labeled protein while chitinase does not.
  • This Example thus demonstrates that biomats can be enzymatically modified and that proteins throughout the biomat are accessible to large molecules (e.g., protease enzyme molecules) as well as the smaller FITC molecules.
  • Fusarium strain flavolapis biomats were infused with varying amounts of a water- soluble diepoxide (Denacol EX-832, polyethylene glycol diglycidyl ether) and heated for 1 hour at 140 °C, then immersed in water at room temperature for 24 hours, at which point the water uptake was measured.
  • a water- soluble diepoxide (Denacol EX-832, polyethylene glycol diglycidyl ether) and heated for 1 hour at 140 °C, then immersed in water at room temperature for 24 hours, at which point the water uptake was measured.
  • Figure 4 graphs the water uptake percentage as a function of the molar quantity of epoxide per gram of epoxide- infused biomass.
  • each data point is the average of two experiments, and the shaded region represents a 95% confidence interval of the spline fit.
  • a fungal slurry of Fusarium strain flavolapis was made substantially as described in Example 4.
  • a first layer of slurry was cast onto a PTFE sheet between two 4 mm thick rubber strips to control thickness, and spread smoothly to the thickness of the rubber strips using a putty scraper.
  • a sheet of cotton gauze was then placed on the slurry.
  • a second identical slurry was cast onto a second PTFE sheet to the same thickness as the first slurry.
  • the second slurry was then placed on top of the cotton gauze and pressed into the gauze/first slurry by applying pressure to the top PTFE sheet using the putty scraper.
  • the top PTFE sheet was then peeled off the slurry stack.
  • the slurry was then dried at room temperature for 24 hours and then dried in an oven at 100 °C for 75 minutes.
  • FIG. 5 A and 5B a 25cm x 38cm sheet of cast composite material was obtained and had a thickness of about 1 mm.
  • the surface texture had a low roughness, with few air bubbles or other inclusions or impurities.
  • the composite material was observed to tear relatively easily along the perpendicular axes of the cotton mesh, but it was significantly more difficult to tear the sheet at a “bias” (z.e., diagonally relative to the axes of the mesh).
  • the introduction of the mesh was observed to provide a 60% increase in tensile strength relative to a control material produced without the mesh material.
  • Figure 5C is a microscopy image of a sample of this material and clearly illustrates that the fungal slurry material completely surrounds the fibers of the cotton mesh.
  • Fusarium strain flavolapis mats were inactivated by boiling in water for 30 minutes and then immersed for 1 hour in aqueous solutions containing 37.5 wt% of 40:60 urea/glycerol plasticizer and epoxy (Denacol EX-832) and amine (Priamine 1074) crosslinkers in varying amounts to provide for three selected ratios of total hydrogen in the amine to total hydrogen in the epoxide (0, 0.5, and 0.67). Samples of the mats were cured at 110 °C for 3 hours and then punched out to test for durability via bally flex. The results are illustrated in Figure 6; five to seven samples were produced and tested at each amine hydrogen : epoxide hydrogen ratio. These results demonstrate that polymerization by a combination of amine and epoxy crosslinkers results in significantly greater flexibility that use of an epoxy crosslinker alone.
  • Fusarium strain flavolapis biomats were blended with water in a conventional kitchen blender to form a homogeneous paste. The paste was then vacuum-dried to a solids content of 20.4 wt%. Samples of this dried paste were mixed with a plasticizer solution containing (1) a 60:40 (wt) mixture of glycerol and urea, (2) halloysite nanoclay, (3) microfibrillated cellulose, (4) a cationic starch.
  • Foamex 18 defoaming agent and, in some cases, (6) an aqueous solution of epoxy (Denacol EX-832 and/or Denacol EX-614B) and amine (Priamine 1074) monomers at an amine hydrogen : epoxide hydrogen ratio of 1.5; the amine monomer was solubilized by adding 0.5 molar equivalents (relative to one molar equivalent of amine) of acetic acid.
  • the solids content of the combined fungal/plasticizer mixture was 20%, and the relative amounts of components were selected such that the mixture consisted, on a dry basis, of 46 to 47 wt% fungal biomass, 41 to 48 wt% plasticizer (glycerol/urea mixture), 0 or 5 wt% crosslinker (epoxy/amine), 2 to 5 wt% cationic starch, 1 wt% halloysite nanoclay, and 2 wt% microfibrillated cellulose. These slurries were cast on PTFE sheets, dried at ambient temperature for 16 hours, and then cured in a convection oven at 110 °C for 3 hours. The thickness of the resulting dry films was about 0.7 mm.
  • a first composite material referred to in this Example as “Material A,” was made by the following procedure.
  • a Fusarium strain flavolapis biomat was blended with water in a conventional kitchen blender to form a homogeneous paste.
  • the paste was then vacuum- dried to a solids content of 20 wt%.
  • This dried paste was mixed with a plasticizer solution containing (1) a 60:40 (wt) mixture of glycerol and urea, (2) microfibrillated cellulose, (3) a cationic starch, and (4) an aqueous solution of equal molar parts multifunctional carbodiimide (ZOLDINE XL-29SE) and multifunctional carboxylic acid (citric acid) monomers.
  • ZOLDINE XL-29SE multifunctional carbodiimide
  • citric acid multifunctional carboxylic acid
  • the solids content of the combined fungal/plasticizer mixture was 20%, and the relative amounts of components were selected such that the mixture consisted, on a dry basis, of 20 wt% fungal biomass, 37.2 wt% plasticizer (glycerol/urea mixture), 24.8 wt% carbodiimide/carboxylic acid monomers, 10 wt% cationic starch, and 0.8 wt% microfibrillated cellulose.
  • This slurry was cast on a PTFE sheet, dried at ambient temperature for 16 hours, and then cured in a convection oven at 80 °C for 2 hours and 110 °C for 1 hour.
  • a second composite material referred to in this Example as “Material B,” was made by the following procedure.
  • a Fusarium strain flavolapis biomat was blended with water in a conventional kitchen blender to form a homoeeneous paste.
  • the paste was then vacuum- dried to a solids content of 23 wt%.
  • This dried paste was mixed with a plasticizer solution containing (1) a 60:40 (wt) mixture of glycerol and urea, (2) microfibrillated cellulose, (3) a cationic starch, (4) a polyurethane dispersion (Sancure 20051), (5) Foamex 18 defoaming agent, and (6) a multifunctional carbodiimide (ZOLDINE XL-29SE); the waterborne polyurethane dispersion contains surface carboxylic acid groups, for which the carbodiimide may act as a crosslinker.
  • a plasticizer solution containing (1) a 60:40 (wt) mixture of glycerol and urea, (2) microfibrillated cellulose, (3) a cationic starch, (4) a polyurethane dispersion (Sancure 20051), (5) Foamex 18 defoaming agent, and (6) a multifunctional carbodiimide (ZOLDINE XL-29SE); the waterborne polyure
  • the solids content of the combined fungal/plasticizer mixture was 20%, and the relative amounts of components were selected such that the mixture consisted, on a dry basis, of 39.5 wt% fungal biomass, 28.7 wt% plasticizer (glycerol/urea mixture), 26.7 wt% polyurethane dispersion, 2 wt% microfibrillated cellulose, 2.7 wt% carbodiimide, and 0.5 wt% defoaming agent.
  • This slurry was cast on a PTFE sheet, dried at ambient temperature for 16 hours, and then cured in a convection oven at 80 °C for 2 hours and 110 °C for 1 hour.
  • a third composite material referred to in this Example as “Material C,” was made by the following procedure.
  • a Fusarium strain flavolapis biomat was blended with water in a conventional kitchen blender to form a homogeneous paste.
  • the paste was then vacuum- dried to a solids content of 23 wt%.
  • This dried paste was mixed with a plasticizer solution containing (1) a 60:40 (wt) mixture of glycerol and urea, (2) microfibrillated cellulose, (3) a carboxylic acid functional polymer (sodium alginate), and (4) a multifunctional carbodiimide (ZOLDINE XL-29SE), which acts as a crosslinker for sodium alginate.
  • a plasticizer solution containing (1) a 60:40 (wt) mixture of glycerol and urea, (2) microfibrillated cellulose, (3) a carboxylic acid functional polymer (sodium alginate), and (4) a multifunctional carbodiimide (
  • the solids content of the combined fungal/plasticizer mixture was 20%, and the relative amounts of components were selected such that the mixture consisted, on a dry basis, of 45 wt% fungal biomass, 46 wt% plasticizer (glycerol/urea mixture), 2 wt% microfibrillated cellulose, 6 wt% sodium alginate, and 1 wt% carbodiimide.
  • This slurry was cast on a PTFE sheet, dried at ambient temperature for 16 hours, and then cured in a convection oven at 80 °C for 2 hours and 110 °C for 1 hour.
  • Biomats of Fusarium strain flavolapis were deactivated by boiling in water for 30 minutes, immersed in 95% EtOH for 1 hour and then immersed for 1 hour mixtures urea and glycerol in in varying mass ratios at a total of 30 % solids in water. The mats were then removed from the aqueous solutions and baked at 110 C for 3 hours to evaporate water. The resulting plasticized biomats were then subjected to bally flex testing.
  • urea : glycerol mass ratios between about 0.5:0.5 and about 0.8:0.2 result in significant improvements in durability and flexibility, showing a synergistic interaction that is not achieved by using urea alone or glycerol alone.
  • Plasticizing Biomats Using Deep Eutectic Solvents Inactivated biomats of Fusarium strain flavolapis were immersed for 1 hour in aqueous solutions containing (1) 30 wt% of a 1 :4 (mol) mixture of trimethyl glycine to urea or trimethyl glycine to glycerol and (2) a mix of epoxy crosslinkers as shown in Table 6 below. The weight ratio of plasticizer to crosslinker in all cases was 80:20. These biomat samples were then cured at 110 °C for 3 hours (thereby removing water from the mats, leaving behind the deep eutectic plasticizer) and samples were punched out for tensile testing. The results of the tensile testing are given in Table 6 below.
  • a plasticizer solution was made by dissolving 37.5 wt% solids (consisting of Thermolec 200 lecithin as 8 wt% of the overall plasticizer solution, with the remaining 29.5 wt% consisting of a 40:60 (wt) mixture of urea and glycerol) in water.
  • a polymerization solution was made by dissolving 37.5 wt% solids (consisting of an epoxide monomer mixture (a 97:3 (wt) mixture of Denacol EX-832 and Denacol EX-614B) and an amine monomer (Priamine 1074), in relative amounts such that the ratio of total hydrogen in the epoxide to total hydrogen in the amine was 1.5) in a 1 :10 (wt) ethanol/water solvent. These two solutions were combined at a ratio of 74 wt% plasticizer solution, 26 wt% polymerization solution, and an inactivated mycelial biomat of Fusarium strain flavolapis was infused with the resulting mixture for 1.5 hours in a vacuum tumbler.
  • the biomat was then cured for 2 hours at 110 °C, 1 hour at 125 °C, and 1 hour at 140 °C. Samples were then cut from the cured mat and subjected to tensile and bally flex testing. The cured mat had a Young’s modulus of 5.21 mPa, a strain at break of 43.76%, a tensile strength of 1.31 mPa, and survived at least 120,000 flex cycles in bally flex testing before failing.
  • This Example thus demonstrates that the flexibility of biomaterials, and particularly mycelial mats (which tend to have surfaces that are more hydrophobic than the center or bulk of the material), can be improved by incorporating lecithin (which, like many other emulsifiers, dispersants, and/or surfactants, includes a hydrophilic head and a hydrophobic tail) as a co-plasticizer with urea and glycerol.
  • lecithin which, like many other emulsifiers, dispersants, and/or surfactants, includes a hydrophilic head and a hydrophobic tail

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Abstract

L'invention divulgue des matériaux composites qui comprennent un biomatériau, ainsi que des procédés de production et d'utilisation de tels matériaux. Dans certains modes de réalisation, un matériau composite contenant un biomatériau est formé par polymérisation de monomères, d'oligomères et/ou de polymères réactifs en contact avec le biomatériau, de sorte que le matériau composite comprend à la fois un biomatériau et une structure polymère. En outre ou en variante, un biomatériau peut être traité par réticulation de peptides de surface. En outre ou en variante, le biomatériau peut être plastifié par l'utilisation d'un solvant eutectique profond. Dans certains modes de réalisation, le biomatériau est dérivé d'un ou de plusieurs champignons filamenteux.
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