Chitin aerogels and mineralized aerogels derived from biomass

Abstract

Disclosed herein are methods for processing biomass and materials obtained therefrom. In an aspect, the disclosure relates to methods of processing fungal matter to obtain chitin, chitin aerogels, and mineralized derivatives thereof.

Description

CHITIN AEROGELS AND MI NERALIZED AEROGELS DERIVED FROMBIOMASSACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

[0001] This invention was made with government support under Grant No. 2137663 awarded by the National Science Foundation. The government has certain rights in the invention.CROSS-REFERENCE TO RELATED APPLICATED

[0002] This application claims the benefit of U.S. Provisional Application 63 / 587,849, filed on October 4, 2023, the contents of which are hereby incorporated in its entirety.FIELD OF THE DISCLOSURE

[0003] The disclosure relates to methods for processing biomass and materials obtained therefrom. In an aspect, the disclosure relates to methods of processing fungal matter to provide chitin, chitin aerogels, and mineralized derivatives thereof.BACKGROUND

[0004] The polysaccharide chitin constitutes the framework of various biomineralized tissues, including the exoskeletons of arthropods, crustaceans, and coralline algae, the radula of chitons, mother-of-pearl, and cuttlefish bone. In many biological systems, chitin is closely associated with hard minerals that enhance the mechanical properties of the fibrous organic matrix. Chitin is the main polysaccharide in the fruit bodies of cultivated mushrooms, imparting rigidity and shape to fungal cell walls. In contrast to the exoskeletons of crustaceans, crystalline chitin nanofibers in fungi are embedded in an amorphous P-1,3 glucan matrix.

[0005] The remains a need for improved systems and methods for isolation of chitin from natural sources. There remains a need for improved systems and methods for the chemical modification and / or mineralization of chitin.BRIEF DESCRIPTION OF THE FIGURES

[0006] Figure 1 depicts a depiction of a deacetylation, ring-opening, and oxidation sequence.

[0007] Figure 2 depicts SEM micrograph of chitin scaffolding at 500x magnification with (a) no mineralization; (b) HA control; (c) CaCO3control; (d) 50,000 g / mol PAA HA; (e) 5,000 g / mol PAA HA; and (f) TEMPO-mediated CaCO3.

[0008] Figure 3 depicts SEM micrograph of chitin scaffolding at 2,000x magnification with (a) no mineralization; (b) HA control; (c) CaCO3control; (d) 50,000 g / mol PAA HA; (e) 5,000 g / mol PAA HA; and (f) TEMPO-mediated CaCO3.

[0009] Figure 4 depicts FI-IR spectra obtained after each step of the chemical extraction process, starting with pristine mushrooms (bottom-most curve), post NaOH (second from bottom), post HCI (second from top), and post bleaching (top curve).

[0010] Figure 5 depicts chemical characterization of chitin aerogels. A) FT-IR absorbance spectra indicate the isolation of a- or y-chitin . B) 13C CP / MAS NMR spectrum indicates isolation of a- or y-chitin. C) XRD pattern of scaffold collected with Cu X-ray source and indexed as a-chitin. D) Thermogravimetric Analysis. First weight loss at about 130° C is characteristic of the y-chitin polymorph.

[0011] Figure 6 depicts Scanning Electron Micrographs from critical-point dried chitin scaffolds show the hierarchical organization, fibrous morphology, and multi-scale porosity. Scale bars correspond to 50pm (A), 10pm ( B), 5pm (C), and 5pm (D).

[0012] Figure 7 depicts X-ray nanotomography from critical-point dried chitin aerogels. A) Reconstructed tomography with 325nm voxel size. Cube edge length in A-C corresponds to 650pm. B) Segmentation based on linear absorption coefficient provides the void-to-aerogel volume ratio. C) Volume thickness map displays local variations in wall thickness coded by color. D) Histogram gives the number distribution of the wall thickness and the mean volume thickness of 0.96 ± 1.14pm. E) X-ray nanoCT projections indicate preferred orientation of chitin fibers running horizontally in the paper plane. F) Typical histogram shows the preferred orientation of image features calculated by Fourier components analysis. G) With an average fiber direction of -9.5 ± 2.7°, the preferred fiber orientation is relatively consistent across the projections of the tomographic image stack, spanning over 185pm in distance.

[0013] Figure 8 depicts A) a photo of conospherical nanoindenter approaching critical-point dried chitin aerogel. Scalebar corresponds to 500pm. B) Typical force-time curve showing both loading and unloading response. C) Typical depth-load hysteresis curve of the elasticplastic deformation. The stiffness S is calculated from the slope of the unloading curve. D) The elastic and plastic deformation energy can be calculated from the area under the loaddisplacement curve.DETAILED DESCRIPTION

[0014] Before the present methods and systems are disclosed and described, it is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0015] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another embodiment includes- from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0016] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0017] Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps. "Exemplary" means "an example of" and is not intended to convey an indication of a preferred or ideal embodiment. "Such as" is not used in a restrictive sense, but for explanatory purposes.

[0018] Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0019] Disclosed herein are methods of preparing a chitin aerogel from a fungal fruiting body including the step of subjecting the fruiting body to a non-degradative alkaline extraction. In certain implementations, the fungal fruiting body is also subjected to a non- degradative acidic extraction. The extractive step(s) produce a chitin scaffold, which is then bleached to provide a bleached chitin scaffold. In some implementations, the bleached chitin scaffold is dehydrated to provide a chitin aerogel. In some implementations, the bleach chitin scaffold is chemically modified and / or mineralized. In some implementations, the fungal fruiting body is of the Agaricus family. In certain implementation the fungal fruiting body is an agaric fruiting body. In certain implementations the fungal fruiting body is Agaricus bisporus.

[0020] Due to the mild nature of the processes disclosed herein, chitin aerogels (and other chitin products as disclosed herein) can be prepared while retaining the gross physical shape of the fungal fruiting body subjected to the process. Because fruiting bodies can be grown in controlled shapes, the disclosed processes provide ready access to chitin aerogels and mineralized chitin of any desired shape and size.

[0021] In certain implementations the fungal fruiting body is subjected to a non- degradative alkaline extraction step, and a non-degradative acidic extraction step is not performed. In other implementations, the fungal fruiting body is subjected to both a non- degradative alkaline extraction step and a non-degradative acidic extraction step. In certain implementations, the non-degradative alkaline extraction step is performed prior to the non-degradative acidic extraction step. In other implementations, the non-degradative acidic extraction step is performed prior to the non-degradative alkaline extraction step. The product of the extractive step(s) is designated herein a chitin scaffold.

[0022] In some implementations, the non-degradative alkaline extraction step is performed by contacting a fungal fruiting body with an alkaline aqueous solution. In some implementations, the fungal fruiting body is combined with an alkaline aqueous solution in a suitable vessel. In some implementations the non-degradative extraction is performed using continuous-flow techniques. Suitable bases for the alkaline aqueous solution include metal hydroxides, for example LiOH, NaOH, KOH, Mg(OH)2, Ca(OH)2, or a combination thereof. In certain implementations the base is NaOH. In certain implementations, the non-degradative alkaline extraction step may be carried out in an alkaline aqueous solution having a pH greater or equal than about 10, greater or equal than about 11, greater or equal than about 11, or greater or equal than about 13. In certain implementations the alkaline aqueous solution has a pH from 10 to 15, from 11 to 15, from 12 to 15, from 13 to 15, from 10 to 14,from 11 to 14, from 12 to 14, from 13 to 14, from 10 to 13, from 11 to 13, from 11 to 12, or from 12 to 13.

[0023] The non-degradative alkaline extraction step may be carried out at various temperatures. In certain implementations, the non-degradative alkaline extraction step is carried out at a temperature below room temperature, i.e., 0-18°C. In certain implementations, the non-degradative alkaline extraction step is carried out at room temperature, i.e., 19-28°C. In certain implementations, the non-degradative alkaline extraction step is carried out at a temperature greater than room temperature. In certain implementations the non-degradative alkaline extraction step is carried out at a temperature of at least 35°C., at least 45°C., at least 55°C., at least 65°C., at least 75°C., at least 85°C., or at least 95°C. In some implementations, the non-degradative alkaline extraction step is carried out at a temperature from 40 to 95°C., from 40 to 80°C., from 40 to 60°C., from 45 to 55°C., from 50 to 80°C., from 60 to 90°C, or from 75 to 95°C.

[0024] The non-degradative alkaline extraction step may be carried out for various lengths of time. In some implementations, the non-degradative alkaline extraction step can be carried out for at least 6 hours, for at least 12 hours, for at least 18 hours, for at least 24 hours, for at least 36 hours, for at least 48 hours, or for at least 72 hours. In certain implementations, the non-degradative alkaline extraction step can be carried out for a period from 6 to 96 hours, from 6 to 72 hours, from 12 to 72 hours, from 12 to 48 hours, from 12 to 36 hours, from 12 to 24 hours, from 18 to 36 hours, or from 24 to 48 hours.

[0025] The non-degradative alkaline extraction step may be carried out while agitating the alkaline aqueous solution / fungal fruiting body mixture. Exemplary means of agitation include stirring, shaking, rotating, or a combination thereof. In some implementations, the extracted product may be separated from the alkaline aqueous solution and optionally rinsed. In some implementations, the alkaline aqueous solution may be directly converted to an acidic aqueous solution by addition of a suitable acid.

[0026] In some implementations, the non-degradative acidic extraction step may be carried out in an acidic aqueous solution. In some implementations, the fungal fruiting body is combined with an acidic aqueous solution in a suitable vessel. In some implementations the non-degradative extraction is performed using continuous-flow techniques. Suitable acids include mineral acids, for example HCI, HBr, HI, H2SO4, HNO3, H3PO4, or a combination thereof. In certain preferred implementations the acid is HCI. In certain implementations, the non-degradative acid extraction step may be carried out in an acid aqueous solution having a pH less than about 3, less than about 2, less than about 1, or less than about 0. Incertain implementations the acidic aqueous solution has a pH from -1 to 3, from -0.5 to 3, from -0.5 to 2, from -0.5 to 1.0, or from -0.5 to 0.5.

[0027] The non-degradative acidic extraction step may be carried out at various temperatures. In certain implementations, the non-degradative acidic extraction step is carried out at a temperature below room temperature, i.e., 0-18°C. In certain implementations, the non-degradative acidic extraction step is carried out at room temperature, i.e., 19-28°C. In certain implementations, the non-degradative acidic extraction step is carried out at a temperature greater than room temperature. In certain implementations the non-degradative acidic extraction step is carried out at a temperature of at least 35°C., at least 45°C., at least 55°C., or at least 65°C. In some implementations, the non-degradative acidic extraction step is carried out at a temperature from 10 to 50°C., from 10 to 30°C., from 10 to 20°C., from 20 to 50°C., from 20 to 40°C., from 20 to 30°C., from 30 to 50°C., or from 40 to 50°C.

[0028] The non-degradative acidic extraction step may be carried out for various lengths of time. In some implementations, the non-degradative acidic extraction step can be carried out for at least 6 hours, for at least 12 hours, for at least 18 hours, for at least 24 hours, for at least 36 hours, for at least 48 hours, for at least 72 hours, or for at least 96 hours. In certain implementations, the non-degradative acidic extraction step can be carried out for a period from 6 to 96 hours, from 6 to 72 hours, from 12 to 72 hours, from 12 to 48 hours, from 12 to 36 hours, from 12 to 24 hours, from 18 to 36 hours, or from 24 to 48 hours.

[0029] The non-degradative acidic extraction step may be carried out while agitating the alkaline aqueous solution / fungal fruiting body mixture. Exemplary means of agitation include stirring, shaking, rotating, or a combination thereof.

[0030] Following the non-degradative extraction step(s), the chitin scaffold can be subjected to a bleaching step by contacting the chitin scaffold with a bleaching solution to provide a bleached chitin scaffold. In certain implementations the oxidant is NaCIO. The bleaching solution can include NaCIO at a concentration from 0.1-5%, from 0.1-2.5%, from 0.1-1.5%, from 0.1-1%, from 0.1-0.5%, from 0.25-0.75%, from 0.5-1%, or from 1-2.5% (w / v). In certain implementations the bleaching solution can include an organic acid, for example acetic acid, propionic acid, formic acid, or a combination thereof. In preferred implementations, the bleaching solution includes acetic acid. The bleaching solution can include the organic acid at a concentration of from 0.1-5%, from 0.1-2.5%, from 0.1-1.5%, from 0.1-1%, from 0.1-0.5%, from 0.25-0.75%, from 0.5-1%, or from 1-2.5% (v / v).

[0031] The bleaching step may be carried out for various lengths of time. In some implementations, the bleaching step can be carried out for at least 6 hours, for at least 12 hours, for at least 18 hours, for at least 24 hours, for at least 36 hours, for at least 48 hours, or for at least 72 hours. In certain implementations, the bleaching step can be carried out for a period from 6 to 96 hours, from 6 to 72 hours, from 12 to 72 hours, from 12 to 48 hours, from 12 to 36 hours, from 12 to 24 hours, from 18 to 36 hours, or from 24 to 48 hours.

[0032] The bleaching step may be carried out at various temperatures. In certain implementations, the bleaching step is carried out at a temperature below room temperature, i.e., 0-18°C. In certain implementations, the bleaching step is carried out at room temperature, i.e., 19-28°C. In certain implementations, the bleaching step is carried out at a temperature greater than room temperature. In certain implementations the bleaching step is carried out at a temperature of at least 35°C., at least 45°C., at least 55°C., at least 65°C., at least 75°C., at least 85°C., or at least 95°C. In some implementations, the bleaching step is carried out at a temperature from 40 to 95°C., from 40 to 80°C., from 40 to 60°C., from 45 to 55°C., from 50 to 80°C., from 60 to 90°C, or from 75 to 95°C.

[0033] In certain implementations the bleached chitin scaffold can be dehydrated to provide a chitin aerogel. In some implementations, the bleached chitin scaffold can be dehydrated by spray drying, critical point drying, lyophilization, or a combination thereof. In certain implementations, the bleached chitin scaffold is subjected to a stepwise ethanol dehydration sequence. In some implementations, the bleached chitin scaffold is sequentially soaked in solutions containing increasing concentrations of ethanol, for example (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and finally 100% ethanol). The bleached chitin scaffold can be immersed in each solution for at least 0.5 hours, at least 1 hour, or at least 2 hours in each solution. After soaking in the 100% ethanol solution, the bleached chitin scaffold can be subject to critical point drying.

[0034] In some implementations, the chitin scaffold, bleached chitin scaffold, and / or chitin aerogel can be subjected to a deacetylation step to convert the chitin to a chitosan. The deacetylation step can be conducted using a strongly basic alkaline solution under conditions sufficient to deacetylate the compound. Suitable bases include metal hydroxides, for example LiOH, NaOH, KOH, Mg(OH)z, Ca(OH)z, or a combination thereof. In certain implementations the base is NaOH. In certain implementations, the deacetylation solution includes the strong base at a concentration of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% (w / v). In certain implementations thedeacetylation solution includes the strong base at a concentration of from 10-40%, from 10- 30%, from 15-30%, from 20-30%, or from 22.5-27.5% (w / v).

[0035] The deacetylation step may be carried out at various temperatures. In certain implementations, the deacetylation step is carried out at a temperature below room temperature, i.e., 0-18°C. In certain implementations, the deacetylation step is carried out at room temperature, i.e., 19-28°C. In certain implementations, the deacetylation is carried out at a temperature greater than room temperature. In certain implementations the deacetylation step is carried out at a temperature of at least 35°C., at least 45°C., at least 55°C., at least 65°C., at least 35°C., at least 75°C., at least 85°C., or at least 95°C. In some implementations, the deacetylation step is carried out at a temperature from 40 to 95°C., from 40 to 80°C., from 40 to 60°C., from 45 to 55°C., from 50 to 80°C., from 60 to 90°C, or from 75 to 95°C.

[0036] The deacetylation step may be carried out for various lengths of time. In some implementations, the deacetylation step can be carried out for at least 2 hours, for at least 4 hours, for at least 6 hours, for at least 8 hours, for at least 10 hours, for at least 12 hours, for at least 18 hours, or for at least 24 hours. In certain implementations, the deacetylation step can be carried out for a period from 2 to 12 hours, from 4 to 12 hours, from 6 to 12 hours, from 8 to 12 hours, from 8 to 16 hours, or from 12 to 24 hours. The resulting chitosan product may be filtered from the deacetylating solution and rinsed.

[0037] In certain implementations, the deacetylation step converts at least 10% of the acetamide groups to secondary amine groups. In some implementations at least 25%, at least 35%, at least 50%, at least 75%, at least 80%, at least 90%, or at least 95% of the acetamide groups are converted to secondary amines. The degree of conversion can be assessed using NMR spectroscopy and other analytical techniques.

[0038] In some implementations, the deacetylated product can be subjected to a ringopening step by oxidizing the 1,2 amino alcohol group into aldehydes. The aldehydes may be further oxidized to dicarboxylates, reduced to primary alcohols, reductively aminated, or otherwise functionalized as desired. In certain implementations, the 1,2 amino alcohol can be directly oxidized to the dicarboxylate.

[0039] In certain implementations, the chitosan product may be placed in a ring-opening solution to convert the 1,2 amino alcohol group into aldehydes. In certain implementations, the ring-opening solutions can include sodium periodate or similar oxidant. In certain implementations, the ring-opening step is carried out at a temperature below room temperature, i.e., 0-18°C. In certain implementations, the ring-opening step is carried out atroom temperature, i.e., 19-28°C. In certain implementations, the ring-opening is carried out at a temperature greater than room temperature. In certain implementations the ringopening step is carried out at a temperature of at least 35°C., at least 45°C., at least 55°C., at least 65°C., at least 75°C., at least 85°C., or at least 95°C.

[0040] The ring-opening step may be carried out for various lengths of time. In some implementations, the ring-opening step can be carried out for at least 2 hours, for at least 4 hours, for at least 6 hours, for at least 8 hours, for at least 10 hours, for at least 12 hours, for at least 18 hours, or for at least 24 hours. In certain implementations, the ring-opening step can be carried out for a period from 2 to 12 hours, from 4 to 12 hours, from 6 to 12 hours, from 8 to 12 hours, from 8 to 16 hours, or from 12 to 24 hours. The resulting ring-opened product may be filtered from the ring-opening solution and rinsed to give a dialdehyde product.

[0041] In certain implementations, the dialdehyde product may be further oxidized to a dicarboxylate product. In certain implementations, the dialdehyde is converted to the dicarboxylate by treatment with 2,2,6,6-tetra methylpiperidin-l-yl)oxidanyl ("TEMPO") and NaCIO. The product may be designated a dicarboxylate scaffold. In certain implementations, at least 10% of the carbohydrate residues can be converted to the dicarboxylate residue. In some implementations, at least of 15% of the residues, at least 20% of the residues, at least 30% of the residues, at least 40% of the residues, at least 50% of the residues, at least 60% of the residues, or at least 70% of the residues are converted to the dicarboxylate. In some implementations, from 1-70% of the residues, from 10-70% of the residues, from 25-70% of the residues, from 50-70% of the residues, from 1-10% of the residues from 10-25% of the residues, or from 25-50% of the residues are converted to the dicarboxylate.

[0042] In certain implementations, the bleached chitin scaffold can be mineralized, for instance with calcium, iron, or titanium. In certain implementations, the dicarboxylate can be mineralized, for instance with calcium. In certain implementations the bleached chitin scaffold and / or the dicarboxylate may be combined with an aqueous solution including a calcium salt (e.g., CaCI2) and exposed to carbon dioxide. In certain implementations, the dicarboxylate may be combined with a polyanion to mediate mineralization. In certain implementations the dicarboxylate scaffold is combined with polyacrylic acid. In certain implementations, the polyacrylic acid has a MW from 1,000 - 500,000, from 1,000 - 250,000, from 1,000 - 100,000, from 2,500 - 100,000, from 2,500- 50,000, from 5,000 -100,000, from 5,000 - 50,000, from 5,000 - 25,000, from 25,000 - 50,000, from 25, DOO- 75, 000, or from 50,000 - 100,000.

[0043] In certain implementations, the bleached chitin scaffold and / or the dicarboxylate scaffold may be treated with a hydroxyapatite source. In certain implementations the bleached chitin scaffold and / or the dicarboxylate scaffold can be treated with a calcium salt and a phosphate salt, e.g., CaCI2and K2HPO4, to provide a hydroxyapatite mineralized scaffold. In certain implementations the calcium and phosphate salts are first combined in solution, and the bleached chitin scaffold and / or the dicarboxylate scaffold is immersed in the solution.

[0044] In certain implementations, the chitin aerogels and mineralized scaffolds disclosed herein may be included in a bone substitute or bone graft product. The chitin aerogels and mineralized scaffolds are biocompatible and exhibit favorable mechanical strength to weight ratios.EXAMPLES

[0045] The following examples are for the purpose of illustration of the invention only and are not intended to limit the scope of the present invention in any manner whatsoever.Example 1 - Generation of 3D chitin scaffolds from mushrooms

[0046] The fruit bodies, including stalks and caps, of white button mushrooms were washed in deionized water and sliced into 4 cm x 4 cm mushroom cores using a sterile steel blade. The mushroom samples were fully submerged in a 2% w / v NaOH solution under constant stirring to remove the glucans and alkali-soluble proteins found in mushrooms cell walls. The solution was maintained at 50° C for 24h, then the samples were placed in deionized water for 12h until the pH of the solution was neutral. Then, the NaOH-treated samples were fully submerged in a 2M HCI solution to remove acid-soluble minerals and proteins. The solution was maintained at room temperature for 24h, then, the samples were placed in deionized water for 12h until the pH was neutral. To remove remaining pigments, HCI-treated samples were bleached in a solution containing 0.5% w / v NaCIO and 1% v / v AcOH. The solution was maintained at 50°C for 5 hours under constant stirring. After bleaching, the 3D chitin scaffolds were washed in deionized water for 12h until the pH of the solution was neutral.Example 2 - Critical point drying

[0047] The water-immersed chitin scaffolds were subjected to an ethanol dehydration series. To do so, the scaffolds were soaked in increasing concentrations of ethanol (0%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% ethanol) at intervals of 2-12 hours, dependent on the size of the sample being dehydrated, and stored in 100% ethanol until use. Samples were critical point dried using a Leica EM CPD300 critical point dryer following a 2h protocol. The CO2 influx speed was set to a medium at 15° C. The heating speed was set to slow down at 40° C. Critical point drying yielded white, free-standing aerogels that resembled polyurethane foams (Styrofoam™) in appearance. Tailoring the drying process allowed us to retain the porous nature of the scaffold and minimize shrinkage during processing. We conclude that the white color results from a complete removal of melanin and other pigments during the bleaching step. The produced chitin aerogels are optically opaque, most likely because of light scattering by air-filled micropores.Sample CharacterizationFourier-Transform Infrared Spectroscopy (FT-IR)

[0048] Fourier-Transform Infrared Spectra (FT-IR) were obtained from scalpel cut cuttings with a Thermo Scientific Nicolet iSlO Infrared spectrometer equipped with attenuated total reflection (ATR). FT-IR spectra were acquired from 400-4000cm1with 120 scans at 2cm-1spectral resolution using the OMNIC Series Software and the data were displayed and analyzed using the Origin 2020b software. The spectral raw data were baseline-corrected using atmospheric correction and subsequently normalized against the highest corrected absorbance. Spectra are depicted in Figure 4.X-ray Powder Diffraction (XRD)

[0049] X-ray powder diffraction patterns were collected from ground composite samples using a Siemens D5000 Diffractometer equipped with a Cu X-ray source (X = 1.5406 A) over the angular range 20 = 5-60°. Prior to XRD data acquisition, samples were dehydrated in a desiccator and crushed into power using a mortar and pestle. 15-20mg were used for each analysis. XRD profiles were baseline corrected using the OriginPro 2023b software package.

[0050] Major Bragg peaks were detected at 20 = 8.59° (020), 12.71° (101), 19.33° (110), 21.44 (120), 26.33 (130), 34.81 (013), and 38.55 (210), indicating regular chain packing. The most intense diffraction peak at 20 = 19.33° (110) is an excellent agreement with literature values for y-chitin. Chitins prepared from different sources exhibit similar X-ray diffraction patterns but differ slightly in peak positions and relative peak ratios. Overt differences in crystal structure and polymorphic configuration arise from the specific molecular chain packing as well as intra- or intermolecular hydrogen bonding within the chitin nanofibril. In previous studies, the (020) crystalline reflection was detected at diffraction angles of 9.46°, 8.59° and 9.35° in a-, - and y-chitin, respectively. In the present study, the (020) reflectionappears at 8.59°, hinting towards the presence of p-chitin in the sample. In accordance with X-ray powder diffraction profiles from other natural sources, a-chitin displays the most intense Bragg peak at 19.54°, whereas p-chitin gives rise to a main reflection at slightly higher angles, specifically 19.62°. However, minor Bragg peaks, such as the clearly resolved reflection at 26.33°, are consistent with the presence of chitin organized in the a- or y- isomer, which exhibited Bragg peaks at similar angles in previous studies, 26.42° and 26.67°, respectively. The background is probably attributed to amorphous portions of the chitin microfibrils, as the FT-IR data did not imply the presence of large amounts of residual glucans. Furthermore, the absence of additional diffraction peaks suggests that the material is free from other crystalline phases, confirming the chemical purity and homogeneity of the chitin scaffold.Solid-state13C Nuclear Magnetic Resonance Spectroscopy (13C CP-MAS NMR)

[0051] Prior to NMR data collection, samples were dehydrated in a desiccator and crushed into powder using a mortar and pestle. ~150mg sample was used for each NMR analysis. Cross-Polarization Magic-Angle-Spinning Solid-state Nuclear Magnetic Resonance Spectra were collected at 500 MHz using a Bruker AVIII HD WB500 spectrometer equipped with a 4.0mm HXY CP-MAS probe with high power3H decoupling (HPDEC).13C CP / MAS NMR spectra were collected with sample spinning rates of 10kHz and 12kHz. At 10kHz, weak peaks were detected at 253ppm, 128ppm, 95.2ppm, which turned out to be artefacts from the spinning side band when comparing to NMR spectra collected at 12kHz. NMR data were processed and plotted with the Bruker Topspin software package. Overlapping peaks were deconvoluted into a sum of mixed Lorentzian and Gaussian line shapes using TopSpin. The deconvolution result, i.e. peak positions, line widths and integrals, were reported.

[0052] The use of solid-state NMR technique for chemical and structural analysis provides several advantages such as it does not require deuterated solvents or extensive sample preparation, allowing us to preserve the pristine chitin conformation. The13C NMR spectrum displays 8 major resonances attributed to the 8 carbon atoms of N-acetyl glucosamine residues, as shown in Figure 3B. Measured chemical shifts and relative peak integrals are summarized in Table 2 in comparison with Solid-State CP-MAS13C NMR spectral bands of a- chitin, (3-chitin, and y-chitin from the literature. Pure a-chitin would be characterized by a double peak pertaining to C-5 (75.2 ppm) and C-3 signal (73.1 ppm) in the glucosamine, whereas p-chitin would display a single C-3 / 5 peak at 73 ppm. In this study, we observed a small doublet split at 73.9 ppm and 73.1 ppm, suggesting a mixture of a-chitin and p-chitin. At a sample spinning rate of 12kHz, a broad resonance with multiple maxima at around 73.6ppm, 73.5 ppm, and 74.6 ppm was observed, indicating a mixture of different chitin polymorphs. The carbonyl functionality is associated with a peak at 173.4 ppm. The absence of peaks at 174 ppm, 146 ppm, and 117 ppm confirms the complete removal of protein from the organic matrix. Moreover, C-4 (68.5 ppm) and C-3 (85.7 ppm) peaks pertaining to chitin- associated glucan were not detected. The CHs moiety of the acetyl group gives rise to an intense signal at 22.5 ppm, which means that the amine groups are predominantly acetylated in the mushroom-derived chitin scaffolds. The degree of N-acetylation (NA), which is defined as the mole fraction of acetylated units in the polymer chain, was determined using the peak ratio of methyl carbon divided by summing up the integrals of carbon atoms in the D-glucopyranosyl ring Ci-C5that have chemical shifts around 650-105 ppm. Hence, our experimental data confirms that the mushroom-derived chitin scaffold remained completely acetylated after the chemical extraction protocol.NA = 100100 X 13.81 / (% X 76.36) = 108%DA results calculated by13C CP-MAS NMR are in line with DA values calculated by FT-IR spectroscopy, as discussed above.Thermogravimetric Analysis (TGA)

[0053] For thermogravimetric analysis, samples were crushed into a powder using mortar and pestle and dehydrated in a desiccator. A Perkin Elmer Simultaneous Thermal Analyzer (STA) was used to measure weight loss and the rate of weight change under a nitrogenous atmosphere as a function of temperature using a heating rate of 10.00° C / min and a nitrogen gas flow of 20.0ml / min. The TGA analysis was run in triplicate using 15-20mg sample. The first weight loss at about 130° C is characteristic of the y-chitin polymorph and can be attributed to the evaporation of water absorbed in the material. Previous reports stated that a- and (3-chitin polymorphs start degrading at a much higher temperature of 205° C. Residual amorphous glucan is thought to increase the water uptake capabilities. Major mass loss events occur between 130-400° C, with initial changes occurring between 130-290° C, indicative of the thermal decomposition of y-chitin. At temperatures above 300° C, rapid weight loss is attributed to the degradation of polymeric structure, e.g., saccharide ring dehydration and deterioration of acetylated chitin units. It was previously reported that glucan-containing chitin fiber composites derived from mushrooms exhibit a lower degradation onset temperature than chitin composites fabricated from crustaceans, which start degrading at temperatures above 280° C. However, FT-IR data presented here do not indicate major amounts of bound glucan.Scanning Electron Microscopy (SEM)

[0054] SEM images were collected with a COXEM EM-30N tabletop SEM. Scalpel cuttings of the chitin composites were placed onto double-sided carbon tape and mounted onto aluminum specimen stubs. Samples were sputter coated with lOnm layers of platinum using a MicroNanoTools MNT-JS1600 plasma sputter coater to decrease charging of the sample surface. SEM images were collected at acceleration voltages of 9-15kV using secondary electron (SE) and backscattered electron detectors (BSE) under high vacuum conditions.X-ray nanotomography (nanoCT):

[0055] Critical-point dried samples were cut into <lmm thick slabs and placed into Kapton™ tubes, which were sealed with clay. X-ray nanotomographies were collected with a Rigaku nano3DX nano-computed tomography scanner with parallel beam geometry combined with an ultra-bright 1200W Cu anode (8keV) X-ray source that enhances the contrast of soft materials. Using a high-resolution 20x lens, a voxel size of 325nm was achieved. The reconstructed stack of 2D slices was imported into the 3D visualization and analysis software Dragonfly (Object Research Systems Inc, Montreal, Canada, 2020, Version 2020.2) for Windows. Otsu's method was used for automatic image thresholding. The fiber thickness was quantified and visualized in color-coded mappings using Dragonfly's volume thickness tool. The preferred fiber orientation was quantified across the image stack using the Directionality plugin in ImageJ (Version 1.54g).

[0056] The extracted material consists of high-order microfibers that reflect the inherent macromolecular organization of chitin. Chitin microfiber bundles make up an interconnected, entangled network that extends over the entire tissue sample, enclosing micro- and macrovoids of different sizes. Such multiscale porosity is innate to many biological materials, but often hard to achieve in synthetic materials. Cross-sectional SEM images (Figure 6) show thin-walled chamber-like structures in which parallel stacks of lamellar sheets are spaced at distances of tens of micrometers. Fibers appear to be continuous, spanning hundreds of micrometers in length, while measuring roughly a micron in thickness. The characterization of the surface morphology was complemented by X-ray nano-computed tomography (nanoCT) as a non-destructive imaging technique that resolves fine interior structural features. Based on nanoCT data from multiple regions of interest, mushroom-derived aerogels appear to be structurally homogeneous at the sub-micrometer length scale. To glean more quantitative structural information, we applied Otsu's method to the reconstructed image stack (Figure 7A). This unsupervised technique of automatic threshold selection segments regions that differ in X-ray absorption coefficient, here aerogelvs. air (Figure 7B). Using image segmentation, we quantified the chitin foam's void space volume to be 55.8%, which exceeds the porosity in many classical polymer foams used for thermal insulation. It is worth mentioning that the porosity calculation is limited by the 325nm voxel size, which does not account for smaller voids in the nanometer size regime. Therefore, the surface area and porosity were characterized using the Brunauer-Emmett- Teller (BET) method. Based on N2physisorption isotherms, the BET surface area was quantified to be 79.5 m2 / g. Upon fitting the isotherms using a non-local density functional theory (NLDFT) model, the total pore volume amounted to 0.248 cm3 / g, including a volume of 0.0084 cm3 / g that pertained to small micropores with a diameter below lnm, which are far below the resolution of X-ray nanoCT. We further observed that the BET adsorptiondesorption curve resembles a type IV isotherm with hysteresis loop, which is typically associated with capillary condensation and evaporation in mesopores. Evidently, the hierarchical porous architecture of the chitin aerogel with pore sizes ranging from the nano- to the macroscale corresponds to a relatively large inner surface area and low specific density of 0.060 ± 0.007 g cm'3. The ultralow density is among the critical parameters governing the mechanical performance of porous foams. Volume thickness maps (Figure 7C) translate the local wall thickness at each voxel location to a false color value. We plotted the number-averaged distribution of the wall thickness in a histogram (Figure 7D) whose maximum corresponds to a mean thickness of 0.96 ± 1.14pm, which is in excellent agreement with the fiber thickness measured with SEM. Judging from 2D X-ray projections, the chitin fibers appear to be anisotropically oriented throughout the volume of the aerogel (Figure 7E-G). This visual impression is backed by an in-depth analysis of the preferred fiber orientation across the image stack using the Directionality plugin in ImageJ (Figure 7G). This tool based on Fourier components analysis calculates a Fourier transform of the image, whose direction is compared to the input image. The computed histograms indicate the number of structures in each direction, whereby a 0° angle is defined as a vector pointing eastwards, with positive numbers referring to a counterclockwise direction of a specific feature. In the 2D projections, the texture distribution peaks on average around -9.5 ± 2.7°, corresponding to fibers lying more or less horizontally with respect to the paper plane (Figure 7G). Our microstructural characterization backs our visual observation that the extracted chitin scaffolds retained the unidirectional fiber arrangement and entangled fibrillar network previously observed in native mushroom mycelium. It is noteworthy that the local fiber orientation is a microstructural feature that has a major impact on the mechanical properties of fiber-reinforced composites and biological tissues alike. Forinstance, the supramolecular order and fibrillar organization is a fundamental design principle in chitinous tissues across the biosphere, such as the lobster cuticles, mantis shrimps, and arrow worms. The fiber dimension, aspect ratio, supramolecular order and interconnectedness of the chitin fibers affect the accessibility of internal glycosidic bonds and the mechanical behavior of the aerogel.BET surface area and porosity analysis-.

[0057] The textural properties of chitin aerogels were characterized using a Micromeritics ASAP 2020 Plus MP surface area and porosity analyzer. N2 physisorption was performed at 77 K for relative pressures ranging from 0.001 to 0.998. The material was first degassed under vacuum (100 mmHg) for 2h at 100” C. Using the isotherm data and the Brunauer- Emmett-Teller (BET) method, the specific surface area was calculated using P / Povalues ranging from 0.05 to 0.25. The micropore and total pore volumes were calculated using Non- Local Density Functional Theory (NLDFT). Frequent leak tests ensured that the instrument had no leaks.Nanoindentation testing:

[0058] Flat CPD-dried chitin samples with a thickness > 1.5mm were prepared with a razor blade and glued on plastic petri dishes. Nanoindentation tests were conducted at ambient conditions using a Bruker Hysitron BioSoft nanoindentation system equipped with a conospherical diamond probe with a nominal radius of 50pm. Load-controlled forcedisplacement curves were collected from ten replicates. The approaching load was set to 50nN. Typical load functions included loading with 3000pN displacement at 150pN / s and unloading to -3000pN displacement at -150pN / s. The probe was retracted in displacement control mode with -2pm / s for 10s. The Young's modulus was calculated from the initial linear elastic region of the stress-strain curve using the Hertz model, which assumes a small contact area and sample deformation, an isotropic and homogenous material, and frictionless surface. Average values and their standard deviations are reported.

[0059] Nanoindentation enables mechanical measurements of small sample geometries or heterogeneous materials with high spatial resolution. Due to their high porosity and biological origin, the mushroom-derived aerogels are soft and compliant such that they can be deformed by hand without excessive force, as illustrated by a typical depth-load hysteresis curve in Figure 8. The nanoindentation test consisted of elastic-plastic loading followed by elastic unloading (Figure 8B). When the load was removed from the indenter, the chitin aerogel partially regained its original shape, yet a residual impression was left on the surface of the specimen due to plastic deformation, hp. The distance heis thedisplacement associated with the elastic recovery during unloading. The contact stiffness at the onset of the unloading curve was used to extract the mechanical properties of the test samples using the Hertz model, which relates the contact force P with a nonlinear power function of penetration, h, and the radius of the spherical indenter, R:4P = - Erh3 / 2R' / 23rMechanical properties, such as the Young's modulus (Er), were calculated from the experimental stress-strain curves. The stiffness (S = dF / dh), as defined as the initial slope on the unloading branch, is given by the following relationship:

[0060] The stiffness is related to the reduced elastic modulus (Er) and the area of the tip's sample. There is a general trend that the elastic properties of bio-based aerogels, for instance the Young's modules, scale with the density. At a density of 0.060 ± 0.007 g cm'3, we measured an elastic modulus of 6.39 ± 1.46MPa, which is a typical value of a soft aerogel material or lightweight polymer foam. For instance, this value exceeds the Young's modulus measured for commercially available, closed-cell polyurethane foams, which displayed a modulus of 0.08-0.93 MPa at a similar density of 0.09 g cm'3in static compression tests. The deformation energy, which is determined as the area under the contact force-displacement curve, is an excellent proxy for the energy absorption and impact resistance of a material, rendering it suitable as cushioning material, which dissipates impact energy while reducing the stress levels. Our experiments indicate a moderately high recovery after unloading, as shown by 60.6 ± 11.4% elastic strain, resulting from the hierarchical structure of the three- dimensional microfiber network. However, when a substantial indentation load was applied, the 3D structure was permanently damaged, resulting in plastic deformation and densification. We conclude that the mechanical properties result from the microstructure of the chitin aerogels, which consist of an interconnected and entangled network of intersecting microfibers.

[0061] In another data collection, FT-IR spectra were collected from samples at different stages of chemical extraction, starting with pristine mushrooms, through post NaOH and HCL treatments, and finally post bleaching, resulting in chemically pure three-dimensional chitin scaffolds. FT-IR spectra exhibited characteristic absorption bands associated with functional groups present in the N-acetylglucosamine repeating unit. We detected major changes in the infrared absorption bands as the mushroom samples are processed. For example, we detected a decreasing peak intensity in the broad OH stretching band around 3000-3600cm' 1, starting from the original mushroom sample through the different stages of chemical processing. The FT-IR bands are consistent with the partial removal of the organic matrix. The FT-IR band at 895cm'1is associated with the glycosidic linkage. Infrared signals at 1022cm'1and 1154cm'1are attributed to skeletal and bridged C-O-C stretching, whereas the vibrational band at 1376 cm'1corresponds to C-H symmetric bending. This spectral signature reflects the FT-IR bands expected from constituent N-acetylglucosamine carbon rings. We further observed FT-IR peaks characteristic of the anticipated secondary amides at 1315cm-l (amide III vC-N + oNH), 1542cm'1(amide II C-N-H), 1635cm'1(amide I C=O). FT-IR peaks at 2847-2916cm'1correspond to C-H stretching. Based on the FT-IR data, it can be inferred that the bleached chitin scaffolds are principally comprised of N-acetylglucosamine units, as found in chitin. In a-chitin, the 1635 cm'1absorption band corresponds to C=O groups of the six chitin molecules on the fiber surface that do not fully participate in intermolecular hydrogen bonding or some p-chitin residues.

[0062] We analyzed the thermal decomposition of bleached chitin scaffolds using thermogravimetric analysis (TGA). Gradual weight loss up to 130° C can be attributed to the evaporation of water absorbed in the material. Major mass loss events occur between 130- 400° C, with initial changes occurring between 130-290° C, indicative of the rapid decomposition of chitin. At temperatures above 300° C, rapid changes in weight loss could be attributed to the continuous decomposition of chitin. Overall, the bleached chitin samples follow the weight loss curve expected for chitin.

[0063] X-ray powder diffraction was used to examine the crystalline structure and phase composition of the samples. The major diffraction peak is detected at 20 = 19.4°, which is characteristic of the (110) reflection of a-chitin. a-chitin adopts an orthorhombic unit cell with dimensions a = 0.474 ± 0.001 nm, b = 1.886 ± 0.002 nm, and c = 1.032 ± 0.002 nm, whereby the c-axis corresponds to the long fiber axis, a-chitin is characterized by a higher degree of hydrogen bonding between the chitin molecules compared to p-chitin. The broad, yet well-defined diffraction peaks suggest some degree of crystalline order. The absence ofadditional diffraction peaks suggests that the sample is free from impurities or other crystalline phases, indicating the chemical purity and homogeneity of the chitin material used in scaffold fabrication.Example 3 - Oxidation of chitin scaffolds

[0064] Water-immersed bleached chitin scaffolds were suspended in a 25% w / v NaOH solution (w / v) under constant stirring at 80° C for 10 hours, then washed in DI water. The deacetylated product was placed in 0.1M sodium acetate buffer with 0.01M sodium periodate. The reaction vessel was covered in aluminum foil and stirred continuously for lOh at room temperature to convert the 1,2 amino alcohol groups into aldehydes. After lOh had elapsed, 5 mL of ethylene glycol were added to stop the oxidation and the reaction mixture was stirred for 0.5h. Samples were washed with deionized water, then suspended in deionized water with TEMPO (0.1 mM), NaBr (0.1M), and NaCIO (6mM), for the conversion of aldehydes to carboxylates. The TEMPO solution was adjusted to pH = 10 by adding 0.5M NaOH solution while monitoring the pH. Once the pH value was stable, 0.5 mL of ethanol was added to the solution to quench the oxidation. The dicarboxylates were washed in deionized water and left in deionized water for another 12h.Example 4 - Functionalization of chitin scaffolds with polyacrylic acid and hydroxyapatite mineralization

[0065] 9mM CaCL and 4.2 mM K2HPO4 solution were prepared in TRIS-buffered saline (pH = 7.4) at 37° C to form a solution of precursor hydroxyapatite minerals. Separately, lOOmg / L polyacrylic acid ("PAA") were dissolved in water. Two separate PAAs were tested, one having average molecular weight 5,000 g / mol and the other 50,000 g / mol. The PAA polymer was dissolved in phosphate standard solutions (ImL = 0.05mg P) to mediate hydroxyapatite mineral formation. The PAA solution was added to the mineral solution in a 1:1 (v / v) ratio and the chitin scaffolds were submerged for 72h at 37° C under constant stirring to allow for hydroxyapatite mineral formation. Afterward completing the reaction, the mineralized composites were washed in DI water to remove excess solution.

[0066] The mineralization with hydroxyapatite (HAp) resulted in additional FT-IR peaks. The bending of the POA3' group gives rise to intensive FT-IR absorption bands at v4 = 592 cm1and v3 = 1073 cm'1, which are in accordance with literature values. The vl peak of HAp at 960 cm'1, which is attributed to PO ' stretching, might be obfuscated by the organic matrix, which is present in greater amount. FT-IR peaks centered at 872 cm'1and 710 cm'1reflect the substitution of tetrahedral phosphate with carbonate groups in HAp mineral. Theinfrared bands match the literature values for B-type HAp, confirming the formation of HAp mineral within the scaffold. We observed more pronounced HAp bands in the PAA-modified samples versus the unmodified controls, thus, confirming the successful incorporation of HAp mineral into chitin scaffolds.

[0067] Based on the thermogravimetric analysis of the HAp samples, thermal decomposition takes place in two steps, with major weight loss occurring from 250°C to 400°C and from 550°C to 750°C. The weight loss curve coincides with literature data of hydroxyapatite crystals deposited in chitosan hydrogel. The TGA results for the 50,000 M.W. PAA HA functionalized scaffolds suggest the highest incorporation of HA. In the 50,000 M.W. PAA HAp samples, we observe less decomposition between 200-400°C and greater decomposition above 550°C, when compared to the bleached chitin scaffold without PAA, indicating a larger amount of HA mineral deposition in the polyanionic functionalized scaffolds. As expected, the thermal characterization is consistent with higher thermal stability of PAA-functionalized chitin compared with pristine chitin.

[0068] The XRD pattern from HAp mineralized scaffolds revealed Bragg peaks characteristic of the a-chitin matrix and hydroxyapatite mineral phase. The chitin scaffold mineralized with HAp exhibited sharp diffraction peaks at approximately 20 = 23.64°, 26.06°, 37.57°, 30.59°, 32.22°, and 40.73°, which are in good agreement with literature values for HAp. The presence of hydroxyapatite suggests successful mineralization and incorporation of the mineral phase into the structure for both control and PAA-functionalized samples. Judging from the sharpness of the Bragg peaks, we conclude that HAp crystals were highly crystalline and on the order of micrometers. Taken together, the XRD analysis confirms the successful integration of hydroxyapatite mineral.Example 5 - Calcium carbonate mineralization of chitin scaffolds

[0069] TEMPO-oxidized chitin scaffolds and pristine chitin scaffolds were submerged in 0.02M CaCI2solutions and placed in a closed desiccator with 0.20g of solid (NH zCOs at the bottom for 72h to allow CaCOs mineralization to occur via a gas phase reaction. Afterwards, the mineralized composites were washed in DI water to remove excess solution.

[0070] The incorporation of CaCOa was associated with a new infrared absorption band at vl = 1073cm1, which is attributed to the symmetric stretching of the carbonate group. The formation of calcite is confirmed by sharp and strong absorption peaks at v4 = 710 cm'1and v2 = 872 cm'1, corresponding to in-plane and out-of-plane bending of the carbonate group. The FT-IR results confirmed the formation of the CaCCb polymorph calcite. Similar to theHAP mineralized samples, the calcite FT-IR signals were stronger in the TEMPO-oxidized samples than in the controls, suggesting a successful integration of CaCO3.

[0071] The thermal decomposition of the CaCO3mineralized samples resembles those of HAp mineralized samples. Major weight loss events occur in two steps. The first step at 200- 400°C is associated with chitin decomposition. Further decomposition occurs between 700°C and 800°C, where CaCO3decomposes to the mineral limestone CaO and gaseous CO2. We observed a relatively higher weight loss in TEMPO-mediated CaCO3scaffoldings above 700°C and less at 200-400°C, when compared to CaCO3mineralized controls, which infers a larger amount of CaCO3mineral deposition in the polyanionic functionalized scaffolds.

[0072] We investigated the surface morphology, physical state and microstructure of the filamentous chitin scaffold and the degree of mineralization in both control and functionalized samples for HAp and CaCO3using Scanning Electron Microscopy. As previous research suggested, chemically unaltered mushrooms are characterized by a network of polysaccharide filaments with smooth surface morphology. Chitin matrices furnished with polyanionic groups through PAA modification or TEMPO oxidation exhibited more homogenous and larger amounts of mineral formation. Mycelial networks were apparent in the bleached chitin scaffold, which were derived from the mycelia of pristine white button mushrooms, as previously observed in the literature. The oriented mycelia fiber network constituted an oriented framework for HAp and CaCO3mineralization. In Figures 2 and 3, we observed the formation of minerals within the mycelial chitin networks, showing varying degrees of overall mineral formation depending on the mineral precipitated and the polyanionic functionalization used. The SEM micrographs in Figures 2 and 3b revealed layers of HAp crystals. More homogeneous and overall extensive mineralization was observed in PAA mediated HAp mineralization (Figure 2d, e and Figure 3d, e), resulting in complete coverage of the underlying dicarboxylate matrix. Based on SEM micrographs, PAA mediated mineralization helped us achieve more extensive and homogeneous mineralization, when compared to the unfunctionalized control samples.

[0073] Example 6 - Control samples

[0074] Bleached chitin scaffolds mineralized with HA and CaCO3served as controls for the polyanionic functionalized mineralized composites. CaCO3control samples were generated by submerging chitin scaffolds in a 0.02M CaCI2solution and placed in a desiccator with 0.20g of solid (NH4)2CO3for 72 hours to allow for CaCO3mineralization to occur via a gas phase reaction with CO2. Mineralized composites were washed in DI water to remove excess solution. For HA control samples, 9mM CaCI2and 4.2 mM K2HPO4solution was prepared inTRIS-buffered saline at pH = 7.4 and 37°C. Chitin scaffolds were submerged for 72 hours at 37° C under constant stirring to allow for hydroxyapatite mineral formation. Afterwards, mineralized composites were washed in DI water to remove excess solution.

[0075] The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims and any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term "comprising" and variations thereof as used herein is used synonymously with the term "including" and variations thereof and are open, nonlimiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of" and "consisting of" can be used in place of "comprising" and "including" to provide for more specific embodiments of the invention and are also disclosed. Other than in the examples, or where otherwise noted, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches

Claims

CLAIMSWhat is claimed is:

1. A method of processing a fungal fruiting body, comprising subjecting the fungal fruiting body to an alkaline aqueous extraction step, optionally in combination with an acidic aqueous extraction step, to provide a chitin scaffold, followed by subjecting the chitin scaffold to a bleaching step to provide a bleached chitin scaffold.

2. The method according to claim 1, wherein the process comprises:• first subjecting the fungal fruiting body to an alkaline aqueous extraction step, followed by an acidic extraction step; or• wherein the process does not include an acidic extraction step.

3. The method of any preceding claim, wherein the alkaline extraction step comprises contacting the fungal fruiting body with an alkaline aqueous solution.

4. The method of any preceding claim, wherein the alkaline aqueous solution has a pH from10 to 15, from 11 to 15, from 12 to 15, from 13 to 15, from 10 to 14, from 11 to 14, from 12 to 14, from 13 to 14, from 10 to 13, from 11 to 13, from 11 to 12, or from 12 to 13.

5. The method of any preceding claim, wherein the alkaline aqueous solution comprises a metal hydroxide.

6. The method of any preceding claim, wherein the alkaline aqueous solution comprises LiOH, NaOH, KOH, MgfOH) , Ca(OH)2, or a combination thereof.

7. The method of any preceding claim, wherein the alkaline aqueous solution has a temperature from 40 to 95°C., from 40 to 80°C., from 40 to 60°C., from 45 to 55°C., from 50 to 80°C., from 60 to 90°C, or from 75 to 95°C.

8. The method of any preceding claim, wherein fungal fruiting body is contacted with the alkaline aqueous solution for a period from 6 to 96 hours, from 6 to 72 hours, from 12 to 72 hours, from 12 to 48 hours, from 12 to 36 hours, from 12 to 24 hours, from 18 to 36 hours, or from 24 to 48 hours.

9. The method of any preceding claim, wherein the fungal fruiting body is contacted with the alkaline aqueous solution with continuous agitation.

10. The method of any preceding claim, wherein the constant agitation comprises stirring, shaking, rotating, or a combination thereof.

11. The method of any preceding claim, wherein the acidic extraction step comprises contacting the fungal fruiting body with an acidic aqueous solution.

12. The method of any preceding claim, wherein the alkaline acidic solution has a pH from -1 to 3, from -0.5 to 3, from -0.5 to 2, from -0.5 to 1.0, or from -0.5 to 0.5.

13. The method of any preceding claim, wherein the alkaline acidic solution comprises a mineral acid.

14. The method of any preceding claim, wherein the acidic aqueous solution comprises HCI, HBr, HI, H2SO4, HNO3, H3PO4, or a combination thereof, preferably HCI.

15. The method of any preceding claim, wherein the acidic aqueous solution has a temperature from 10 to 50°C., from 10 to 30°C., from 10 to 20°C., from 20 to 50°C., from 20 to 40°C., from 20 to 30°C., from 30 to 50°C., or from 40 to 50°C.

16. The method of any preceding claim, wherein fungal fruiting body is contacted with the acidic aqueous solution for a period from 6 to 96 hours, from 6 to 72 hours, from 12 to 72 hours, from 12 to 48 hours, from 12 to 36 hours, from 12 to 24 hours, from 18 to 36 hours, or from 24 to 48 hours.

17. The method of any preceding claim, wherein the fungal fruiting body is contacted with the acidic aqueous solution with continuous agitation.

18. The method of any preceding claim, wherein the constant agitation comprises stirring, shaking, rotating, or a combination thereof.

19. The method of any preceding claim, wherein the bleaching step comprises contacting the chitin scaffold with a bleaching solution.

20. The method of any preceding claim, wherein the bleaching solution comprises NaCIO.

21. The method of any preceding claim, wherein the bleaching solution has a temperature from 40 to 95°C., from 40 to 80°C., from 40 to 60°C., from 45 to 55°C., from 50 to 80°C., from 60 to 90°C, or from 75 to 95°C.

22. The method of any preceding claim, wherein chitin scaffold is contacted with the bleaching solution for a period from 1 to 36 hours, from 3 to 36 hours, from 3 to 24 hours, from 3 to 12 hours, or from 3 to 8 hours.

23. The method of any preceding claim, wherein the bleached chitin scaffold is dehydrated by spray drying, critical point drying, lyophilization, or a combination thereof, to provide the chitin aerogel.

24. A chitin aerogel prepared according to any preceding claim.

25. The method of any preceding claim, further comprising deacetylating the chitin scaffold, bleached chitin scaffold, or chitin aerogel by contact with a deacetylating solution to give a deacetylated chitin scaffold or deacetylated bleached chitin scaffold.

26. The method of any preceding claim, wherein the deacetylating converts at least 25%, at least 35%, at least 50%, at least 75%, at least 80%, at least 90%, or at least 95% of the acetamide groups into secondary amine groups.

27. The method of any preceding claim, wherein the deacetylating solution comprises aqueous alkaline hydroxide.

28. The method of any preceding claim, wherein the deacetylating solution comprises at least 25 wt.% of the alkaline hydroxide.

29. The method of any preceding claim, wherein the chitin scaffold, bleached chitin scaffold, or chitin aerogel is deacetylated by contact with aqueous alkaline hydroxide at a temperature from 40 to 95°C., from 40 to 80°C., from 40 to 60°C., from 45 to 55°C., from 50 to 80°C., from 60 to 90°C, or from 75 to 95°C.

30. The method of any preceding claim, wherein the chitin scaffold, bleached chitin scaffold, or chitin aerogel is deacetylated by contact with aqueous alkaline hydroxide for a period from 1 to 36 hours, from 3 to 36 hours, from 6 to 24 hours, from 9 to 24 hours, or from 9 to 18 hours.

31. The method of any preceding claim, further comprising contacting the deacetylated chitin scaffold or deacetylated bleached chitin scaffold is subjected to ring-opening to provide a ring-opened chitin scaffold or ring-opened bleached chitin scaffold.

32. The method of any preceding claim, wherein the ring-opened chitin scaffold is prepared by a first oxidation comprising NaIC to give a dialdehyde product, followed by a second oxidation comprising TEMPO to give a dicarboxylate product.

33. The method of any preceding claim, further comprising combining the bleach chitin scaffold, dialdehyde product and / or dicarboxylate product with a mineral source to provide a mineralized chitin scaffold.

34. The method of any preceding claim, wherein the mineral source comprises an iron salt, a calcium salt, or a combination thereof.

35. The method of any preceding claim, wherein the mineral source comprises CaCI2, and a second compound selected from (NH zCCh, gaseous CO2, or dimethylcarbonate.

36. The method of any preceding claim, wherein the mineral source comprises CaCI2, and K2HPO4.

37. The method of any preceding claim, wherein the mineral source comprises iron oxide, titanium oxide, or a combination thereof.

38. The method of any preceding claim, wherein the fungal fruiting body comprises an agaric fungus.

39. The method of any preceding claim, wherein the fungal fruiting body comprises Agaricus bisporus.

40. A mineralized chitin scaffold, prepared according to any preceding claim.

41. A bone substitute comprising a chitin scaffold prepared according to any preceding claim.

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