Integrated fungal encapsulation and material embedding systems for controlled biotransformation of polymeric and absorbent articles

Abstract

Systems, compositions, and methods for encapsulating, embedding, and activating fungal or microbial inoculants within polymeric, absorbent, and biodegradable articles to enable controlled, moisture-triggered biotransformation. The technology provides alginate hydrogels and protective matrices that preserve inoculant viability during drying, storage, and thermoplastic processing. Encapsulated or native spores are incorporated into polymers, fibers, adhesives, films, and laminates under controlled thermal and shear conditions that maintain biological stasis until environmental activation. Upon hydration, the inoculants emerge, secrete oxidative and hydrolytic enzymes, and initiate degradation of surrounding substrates, including waste polymer materials. Additional embodiments include degradable pouches for timed release and multi-stage successional inoculant systems incorporating fungi, bacteria, algae, and invertebrates for progressive material breakdown. The disclosed platforms enable scalable manufacture of biologically responsive materials compatible with conventional processing equipment, providing articles capable of predictable activation and environmentally driven degradation across a wide range of consumer, industrial, and waste-treatment applications.

Description

[0001] INTEGRATED FUNGAL ENCAPSULATION AND MATERIAL EMBEDDING SYSTEMS FOR CONTROLLED BIOTRANSFORMATION OF POLYMERIC AND ABSORBENT ARTICLES

[0002] Background

[0003] The present disclosure relates to biodegradable and bioactive compositions and systems for managing the controlled activation of living organisms in synthetic, absorbent, or biodegradable materials. It addresses the challenge of maintaining biological viability through industrial processing and storage while providing predictable activation and enzymatic degradation when the materials are exposed to moisture or composting conditions.

[0004] Conventional fungal immobilization and encapsulation techniques, such as alginate bead formation or gel entrapment, have been used to stabilize microorganisms for laboratory or environmental applications. However, these approaches often result in limited shelf life, variable nutrient diffusion, and inconsistent rehydration performance. Standard alginate gels tend to become brittle after drying, and high calcium crosslinking can produce dense, impermeable structures that restrict oxygen and water transport. These limitations hinder their use in large- scale consumer or industrial products that require reproducible activation and performance across wide temperature and humidity ranges.

[0005] Attempts to incorporate biological inoculants directly into polymeric materials have also been constrained by the high temperatures and shear forces typical of melt processing. Spores or cells frequently lose viability during extrusion, molding, fiber spinning, or thermoforming, making it difficult to produce bio-responsive plastics, filaments, or nonwovens using standard manufacturing equipment. Existing microbial additives incorporated into thermoplastics typically activate at unpredictable times, deactivate prematurely, or fail to survive the manufacturing step at all. As a result, there is an unmet need for materials that can survive melt processing and remain dormant until environmental moisture triggers activation.

[0006] Delivery systems that localize and release biological inoculants in a controlled manner further remain limited. Existing pouches, sachets, and pelletized systems are commonly based on synthetic films that persist after release, dissolve too rapidly or too slowly, or lack tunable degradation rates suitable for timed activation. Many commercial water-soluble films were designed for detergent delivery and are not optimized for maintaining fungal viability, balanced hydration, or nutrient buffering.

[0007] Biological degradation of complex waste materials is additionally limited by the absence of structured ecological succession. In natural decomposition, a sequence of fungal and microbial communities participates in staged breakdown of lignocellulosic and polymeric substrates. Industrial biodegradation strategies rarely replicate this temporal structure, instead relying on single-species inoculants or undifferentiated blends that cannot adapt to changing substrate composition. A controlled successional approach, in which distinct inoculants are activated at different times or under different environmental conditions, can improve overall degradation efficiency, stability, and robustness.

[0008] Accordingly, there is a continuing need for integrated materials and processes that (i) preserve the viability of biological inoculants during production and storage, (ii) enable their embedding or encapsulation within polymeric, absorbent, or biodegradable matrices, (iii) provide controlled or delayed release through degradable pouch, capsule, or film systems, and (iv) support multi-stage

[0009] HIRO / 105 / PC 1 or successional activation involving fungi or other organisms capable of polymer degradation. The present disclosure addresses these needs by combining optimized encapsulation chemistry, polymer-processing compatibility, and staged ecological design to achieve reproducible and tunable biotransformation of waste and product substrates.

[0010] Brief Description of the Drawings

[0011] Figure 1 is a schematic illustration of a cellulose- or starch-based biopolymer matrix containing uniformly dispersed alginate-calcium beads, each representing an encapsulated fungal inoculum. Figure 2 is a schematic diagram illustrating the application of an alginate-based fungal inoculum onto a cellulose or fiber substrate by sequential spraying of a pre-gel mixture followed by a divalent-ion solution. The process yields a thin alginate-calcium hydrogel layer containing dispersed fungal inclusions on the surface of the underlying substrate.

[0012] Figure 3 is a schematic illustration of a multilayer biopolymer laminate containing intermediate alginate layers with dispersed encapsulated fungal beads. The drawing shows upper and lower bioplastic sheets bonded around a central functional layer that houses the alginate-calcium bead inclusions, representing a representative laminate configuration.

[0013] Figure 4 is a representative schematic diagram showing a process for producing and reactivating a bio-activated filament as described in Example 9.

[0014] Figure 5A and 5B-D are a cross-sectional illustration and corresponding microscope depiction of a polycaprolactone filament containing embedded native fungal spores.

[0015] Figure 6 is a photographic depiction of fungal emergence from the surface of an extruded PCL filament after incubation on malt-extract agar, demonstrating reactivation of viable spores.

[0016] Figure 7 is a cross-sectional schematic depiction of an adhesive layer containing dispersed fungal inoculum applied to a substrate surface.

[0017] Figure 8 is a set of plots showing emergence performance of Ganoderma A1 alginate-encapsulated inocula embedded in adhesive matrices.

[0018] Figure 9 is a set of plots showing emergence performance of Aspergillus A2 native spores embedded in adhesive matrices.

[0019] Figure 10 is a photographic depiction of fungal emergence from Ganoderma A1 alginate-encapsulated beads embedded in different adhesives, including hot-melt, PVA, eco, and acrylic PSA formulations.

[0020] Figure 11 is a photographic depiction of fungal emergence from Aspergillus A2 native spores embedded within hot-melt and general-purpose plastic adhesives.

[0021] Figure 12 is a schematic perspective view of a laminated polymer film containing dispersed fungal inclusions.

[0022] Figure 13 is a two-panel composite diagram showing fungal emergence performance for Aspergillus A2 spores and Ganoderma A1 inocula embedded in LDPE films.

[0023] Figure 1 is a photographic depiction of LDPE film samples after incubation on malt-extract agar, showing hyphal emergence from film edges and surfaces, confirming post-processing viability of both native spores and encapsulated inocula.

[0024] Figure 15 is a schematic cross-sectional and plan-view representation of a nonwoven or spun bond material containing distributed biological inclusions.

[0025] HIRO / 105 / PC 2 Figure 16 shows fungal hyphae emerging from the surface of processed polycaproiactone (PCL) filaments after incubation. The left panel depicts growth from a melt-fused filament, while the right panel shows emergence from an extruded and woven filament, confirming fungal viability after processing.

[0026] Figure 17 comprises two photomicrographs (40× magnification) showing fungal conidiophores emerging from the surface of a polycaproiactone (PCL) filament after incubation for 10 days. Figure 18 is a schematic diagram illustrating the melt-spinning process for producing bioactive polymer fibers. The figure depicts sequential stages including polymer melting, introduction of fungal inoculum into the molten stream, fiber extrusion through a spinneret, cooling and solidification, and subsequent biological activation upon exposure to moisture or composting conditions.

[0027] Figure 19 shows fungal emergence from polymer samples containing embedded spores after incubation for 0 days: (a) polycaproiactone (PCL), (b) polylactic acid (PLA), and (c) polyethylene terephthalate (PET).

[0028] Figure 20 illustrates a schematic process for producing bioactive polymer fibers containing coated fungal spores. The figure shows the preparation of spores with protective trehalose coating, their introduction into a rotating melt-spinning drum together with polymer feedstock, extrusion and fiber formation, cooling of the resulting filaments, and subsequent incubation to confirm biological reactivation.

[0029] Figure 21 shows polymer samples containing fungal spores under different formulations: polycaproiactone (PCL) with (bottom left) and without trehalose (top left) and polylactic acid (PLA) with (top right) and without trehalose (bottom right), illustrating differences in texture and fiber formation.

[0030] Figure 22 shows fungal emergence from polymer composites containing trehalose-coated spores. The left panel depicts growth from PLA-based samples, and the right panel shows growth from PCL-based samples following incubation.

[0031] Figure 23 shows fungal hyphae and conidia emerging from the surface of a PLA film containing trehalose-coated A2 spores, demonstrating post-processing viability and active regrowth from the polymer surface.

[0032] Figure 24 shows fungal emergence and regrowth from PCL films containing spore-loaded alginate beads, (a) demonstrates visible surface growth after incubation on MEA plates, while (b) and (c) are microscopic images showing conidial emergence from alginate bead residues within the polymer matrix.

[0033] FIG. 25 is a schematic illustration of a two-part additive-fungi interaction system showing the spatial separation of an encapsulated fungal inoculant and an additive-containing substrate prior to activation, and their interaction under environmental conditions that promote enzymatic degradation of the substrate.

[0034] FIG. 26 is a cross-sectional view of a representative multilayer article incorporating a degradable delivery vehicle for fungi, an optional nutrient or control layer, and an additive-doped polymer or material layer configured for staged biological activation and degradation.

[0035] FIG. 27 is a diagram showing the sequence of fungal activation and emergence from an encapsulated inoculum, including stages of dormancy, hydration, bead swelling, emergence, and colonization leading to enzymatic degradation of the surrounding substrate.

[0036] HIRO / 105 / PC 3 FIG. 28 is a cross-sectional diagram of a substrate containing spatially distributed additive zones arranged through the material thickness to control fungal activation and create zones of enhanced or delayed degradation.

[0037] FIG. 29 is a schematic time-sequence diagram illustrating environmental activation of the two- part system, including stages of dry storage, hydration and additive diffusion, enzyme induction, and progressive material breakdown over time.

[0038] FIG. 30 is a schematic illustration depicting the enzymatic depolymerization and hyphal penetration of a fungal inoculant acting on a representative absorbent or polymeric substrate. FIG. 31 is a schematic illustration showing bacterial and algal activity on a partially degraded substrate following fungal colonization, representing the secondary stage of the successional process.

[0039] FIG. 32 is a drawing showing a conceptual cross-section of a waste article with distinct biological zones illustrating fungal colonization fronts, bacterial and algal biofilm formation, and invertebrate penetration areas.

[0040] FIG. 33 is a depiction of an exploded view of a multilayer absorbent article containing spatially arranged inoculant zones, including a fungal inoculant within one layer, a bacterial or algal inoculant in another, and an invertebrate attractant or inoculant close to the outer layer.

[0041] FIG. 34A is a photograph of an experimental observation showing an inoculated sample at the conclusion of a soil burial trial, exhibiting extensive degradation, layer separation, and insect colonization.

[0042] FIG. 34B is a photograph of an experimental observation showing a comparative view of an inoculated sample and an uninoculated control, demonstrating substantial breakdown of the treated sample relative to the intact control.

[0043] FIG. 34C is a photograph of an experimental observation showing an intermediate-stage sample exhibiting concurrent fungal and algal colonization on the substrate surface.

[0044] FIG. 34D is a photograph of an experimental observation showing a later-stage sample with visible soldier-fly larvae and fragmentation of polymeric and absorbent layers.

[0045] FIG. 34E is a photograph of an experimental observation showing a terminal-stage sample exhibiting dark, friable, soil-like residue with minimal remaining polymer structure, corresponding to significant biological transformation of the original material.

[0046] FIG. 35 is a schematic illustration of a representative degradable pouch for storing and releasing biological inoculants.

[0047] FIGS. 36A and 36B show representative Petri-plate assays of degradable pouches containing fungal inoculant A1 after ten days of incubation on malt-extract agar.

[0048] FIGS. 37A and 37B show representative absorbent-article assays in which degradable pouches containing fungal inoculant A1 were positioned within diaper cores and incubated under moist conditions for ten days.

[0049] FIGS. 38A-38H show representative absorbent articles after one month of incubation containing degradable pouches filled with varying amounts of fungal inoculum.

[0050] FIG. 39 is a graph showing the relationship between inoculum mass and fungal growth score for two species, A1 and A4, after one month of incubation within absorbent articles.

[0051] HIRO / 105 / PC 4 Summary

[0052] The present disclosure provides integrated systems and compositions for encapsulating, embedding, and activating biological inoculants such as fungi, bacteria, algae, or other microorganisms within polymeric, absorbent, and biodegradable materials. The disclosed systems enable long-term stabilization of viable inoculants during manufacture and storage and their controlled activation under environmental or composting conditions to promote progressive biotransformation of synthetic and natural substrates.

[0053] Conventional alginate encapsulation methods often yield beads that are either too rigid or too nutrient poor to support vigorous fungal emergence. High crosslink densities and insufficient osmotic balancing can suppress hyphal expansion and delay enzyme secretion, while weak gels may lose mechanical integrity during drying or handling. The present compositions address these deficiencies by incorporating a coordinated suite of additives, including carbohydrates, nitrogen sources, trace mineral cofactors, polymeric structural modifiers, and humectants — that together modulate gel porosity, ionic exchange, and internal hydration potential. The resulting encapsulated inocula demonstrate robust colonization, uniform emergence, and sustained metabolic activity even after desiccation and rehydration cycles.

[0054] In various embodiments, the encapsulated fungal systems described herein are useful for promoting biodegradation within consumer or industrial articles such as absorbent hygiene products, packaging films, fiber matrices, or soil-amendment pellets. Upon exposure to moisture, the hydrogel matrix swells, releasing the immobilized fungi and activating oxidative enzyme cascades that initiate polymer oxidation and depolymerization. The formulations can be customized for particular environments, for example, urine-synthetic systems, saline-rich absorbents, or lignocellulosic substrates, by adjusting alginate concentration, crosslinking ions, carbohydrate osmolyte levels, nitrogen content, and transition-metal cofactor ratios.

[0055] The invention further encompasses methods of manufacturing such compositions, including controlled external gelation of alginate solutions containing nutrient and additive systems, curing within divalent-ion baths for defined time periods, optional post-curing infusion of metal cofactors, and gentle drying protocols that maintain fungal viability. The resulting beads or coatings may exhibit shell thicknesses ranging from about 20 micrometers to about 120 micrometers, swelling ratios between about 1.2× and 2.5×, and emergence indices corresponding to at least 70 percent surface colonization within seventy-two hours of hydration.

[0056] Additional compositions and manufacturing methods for stabilizing, embedding, and activating fungal inocula within polymeric, absorbent, and biodegradable matrices are also disclosed. Optimized formulation and processing strategies that enable fungal spores or mycelial fragments to survive thermoplastic manufacturing conditions and later activate under environmental or composting stimuli are shown. Through this approach, biological inocula can remain dormant during fabrication and storage yet become metabolically active under defined conditions to promote biodegradation, oxidation, or transformation of the host material.

[0057] The invention combines two complementary technological platforms. The first, referred to as encapsulation and stasis chemistry, preserves biological viability during dehydration, heating, and long-term storage. The second, referred to as embedding and material integration, introduces the inoculum into polymeric or fibrous host materials without compromising structural or mechanical integrity. Together, these platforms allow for the scalable manufacture of biologically responsive materials using conventional polymer-processing equipment.

[0058] Encapsulation or protective coating may be achieved through hydrogel or glass-forming formulations containing carbohydrates, polyols, and structural polymers such as alginate,

[0059] HIRO / 105 / PC 5 chitosan, starch, and trehalose. These materials can form microbeads, thin films, or coated spore powders that maintain spore viability after drying and provide resistance to transient heating and shear stress. The ratios of alginate, calcium cross-linker, and optional osmoprotectants can be adjusted to produce compositions that enable rapid fungal emergence and enzymatic activation when hydrated. In other embodiments, the fungal spores are incorporated without encapsulation or protective coating, as native dry powders, provided that the processing conditions — particularly temperature, dwell time, and shear — remain within the tolerance limits for spore survival. This unprotected mode of incorporation enables broader material compatibility and simpler production routes while still achieving survival and reactivation.

[0060] The embedded inoculum may be introduced into a wide range of thermoplastic polymers including, but not limited to, aliphatic and aromatic polyesters such as polylactic acid (PLA) and polycaprolactone (PCL), polyamides, polyolefins such as polyethylene (PE) and polypropylene (PP), polyethylene terephthalate (PET), polyurethane-based materials, styrenic polymers, vinyl polymers, elastomeric thermoplastic blends, and copolymers or composites thereof. Typical processing methods include extrusion, melt-spinning, film casting, compression molding, or adhesive coating. The equipment may operate at temperature setpoints up to about 300 °C, while the inoculum contact temperature, the local temperature experienced by the fungal material, is maintained below approximately 180 °C, and preferably below 160 °C, for short residence times. Under these controlled conditions, the spores or encapsulated inclusions remain viable while the polymer retains its desired physical properties.

[0061] Protective matrices and coatings containing trehalose, sorbitol, starch, or alginate confer significant thermal and mechanical resilience, preserving fungal viability after exposure to temperatures between 100 °C and 160 °C. However, survival is also achievable with native, unprotected spores when residence time and local temperature are minimized or when inoculation is introduced downstream of the hottest melt zone. Once the polymer solidifies, the inoculum remains dormant and stable under ambient storage conditions. Upon hydration or exposure to composting environments, the spores germinate and produce enzymes such as laccases and peroxidases, initiating oxidative and hydrolytic modification of the surrounding matrix.

[0062] The disclosed approach establishes a general manufacturing and materials framework in which living inocula are integrated into polymeric systems that are inert during production and use but biologically responsive under environmental conditions. The technology is compatible with both biodegradable and recyclable polymers, composite structures, and absorbent articles. The resulting materials combine the durability of synthetic polymers with the regenerative and degradative capacity of biological systems. Accordingly, the disclosure encompasses compositions of matter, fabrication methods, and uses directed to bio-activated polymers and hybrid materials capable of controlled degradation, detoxification, or nutrient release in natural or engineered environments.

[0063] In one aspect, the invention provides optimized hydrogel-based encapsulation compositions formulated to preserve the viability of fungal spores and mycelia. These hydrogels incorporate balanced levels of alginate and divalent crosslinking ions together with osmoprotective carbohydrates, nitrogen sources, and structural modifiers that regulate porosity, hydration, and nutrient release. The resulting encapsulated inocula maintain viability during drying and exhibit uniform emergence and enzymatic activity upon hydration. The formulations may be prepared as beads, microcapsules, coatings, or inclusions that can be integrated into a wide range of host materials.

[0064] In another aspect, the disclosure provides embedding and material-integration methods in which spores, coated spores, or encapsulated inclusions are introduced into polymeric matrices such as PLA, PCL, PHA, PET, PE, or PP during extrusion, casting, or fiber formation. Processing

[0065] HIRO / 105 / PC 6 parameters are controlled so that the local temperature experienced by the inoculum remains within fungal survival limits, allowing the production of biologically responsive polymers, fibers, and nonwoven webs using conventional equipment. Upon exposure to moisture or composting environments, the embedded inocula resume metabolic activity and secrete oxidative and hydrolytic enzymes that initiate polymer degradation.

[0066] In further embodiments, the invention provides degradable pouch delivery systems in which encapsulated or immobilized inocula are contained within biodegradable or water-soluble films. These pouches disintegrate or dissolve under composting or moisture conditions, releasing the inoculum to the surrounding substrate in a controlled manner. The pouches can be tuned to degrade over predetermined time intervals, allowing staged activation or sequential release of the contained organisms.

[0067] Additional embodiments relate to successional inoculant systems designed for progressive biotransformation of waste materials. In these systems, multiple biological species with complementary metabolic functions are encapsulated or immobilized within matrices that release each group at defined intervals or environmental triggers. For example, oxidative fungi may be activated first to initiate surface modification of a polymer, followed by hydrolytic or fermentative organisms that complete degradation and mineralization. The approach can include fungi, bacteria, algae, or invertebrates to establish ecological succession that improves overall breakdown efficiency.

[0068] Collectively, the described compositions and methods provide a modular platform for producing shelf-stable, biologically active materials capable of controlled activation and degradation. The technology integrates advances in encapsulation chemistry, polymer processing, and ecological inoculation design to enable scalable manufacturing of biodegradable articles, absorbent products, packaging, and environmental treatment systems with reproducible biotransformation performance.

[0069] Also disclosed are embodiments of biological systems and methods for achieving progressive or sequential biotransformation of waste materials by means of successional inoculants. More particularly, the embodiments concern design and coordinated use of multiple biological species that are applied, encapsulated, immobilized, or otherwise stabilized to activate at distinct times or under different environmental conditions. When combined, the inoculants function cooperatively to reproduce natural ecological succession in a controlled and accelerated form, thereby enabling the breakdown, mineralization, or stabilization of absorbent, polymeric, or fibrous waste materials. Also disclosed are embodiments of a degradable pouch delivery system for storing and releasing biological inoculants, including fungal, bacterial, algal, or mixed consortia. The pouch comprises a biodegradable or water-soluble film material that encloses one or more encapsulated inoculant compositions such as alginate beads or microcapsules. Upon contact with moisture or composting conditions, the pouch dissolves or mechanically disintegrates, releasing the contained inoculum into the surrounding substrate. The disclosure further encompasses methods of manufacturing and deploying such pouches, including filling, sealing, and use within absorbent articles, composting units, or waste-treatment systems. The pouches can be tuned to degrade over controlled time intervals, allowing staged or localized activation of the encapsulated organisms. In one embodiment, a composition includes a biodegradable hydrogel matrix and a fungal inoculum immobilized within the matrix. The matrix includes alginate crosslinked by at least one divalent cation and one or more nutrients or additives useful for one or both of maintaining fungal viability during storage and promoting enzymatic activation upon hydration.

[0070] In yet another embodiment, method of producing an encapsulated fungal composition includes a step of preparing an aqueous solution of alginate and one or more nutrients or additives and then

[0071] HIRO / 105 / PC 7 it with a fungal inoculum to form a pre-gel mixture. A divalent ion solution is added to the pre-gel mixture to initiate crosslinking and form a hydrogel matrix. The hydrogel matrix is dried to form the encapsulated fungal composition that is storage-stable and capable of rehydration and activation.

[0072] In yet another embodiment, an article includes a substrate, a composition with a biodegradable hydrogel matrix and a fungal inoculum. The fungal inoculum is immobilized within the matrix. The matrix further includes alginate crosslinked by at least one divalent cation and one or more nutrients or additives useful for one or both of maintaining fungal viability during storage and promoting enzymatic activation upon hydration. The composition is coated on the substrate or combined with the substrate to form a composite, and the composition is configured to activate upon exposure to moisture to initiate fungal emergence and enzymatic degradation of at least one organic or polymeric substrate.

[0073] In another embodiment, a polymeric composition comprises a polymeric matrix and a fungal inoculum. The polymeric matrix is selected from thermoplastic polymers, biodegradable polymers, and absorbent polymers, and the fungal inoculum is distributed within the matrix. The inoculum comprises viable fungal spores or mycelial fragments in a form that remains dormant under dry storage conditions and are activated when exposed to environmental moisture or composting conditions.

[0074] In another embodiment, a method of manufacturing a biologically activated polymeric composition includes heating a polymer to form a molten or softened polymer matrix and then adding a fungal inoculum into the molten or softened polymer matrix. The fungal inoculum may include fungal spores or mycelial fragments. The conditions maintain an inoculum contact temperature within an approximate range of 80-180°C for a residence time of less than 3 minutes to form a polymeric composition precursor. The polymeric composition precursor is formed and cooled such that the inoculum remains viable and dormant within the matrix.

[0075] In yet another embodiment, a method of initiating biological activation of a polymeric article that includes a fungal inoculum comprises exposing the article to moisture, humidity, or composting conditions that are effective to rehydrate the fungal inoculum, thereby initiating fungal growth or enzyme secretion.

[0076] In yet another embodiment, a polymeric composition is formed by a process including a step of heating a polymer to form a molten or softened polymer matrix and then adding a fungal inoculum into the molten or softened polymer matrix to form a polymeric composition precursor. The fungal inoculum may include fungal spores or mycelial fragments and the conditions maintain an inoculum contact temperature within an approximate range of 80-180°C for a residence time of less than 3 minutes. The polymeric composition precursor is formed and cooled such that the inoculum remains viable and dormant within the matrix. The fungal inoculum remains viable after storage for at least 30 days at ambient temperature.

[0077] The polymeric composition may be used for promoting biodegradation or nutrient release in polymeric or fibrous materials.

[0078] In another embodiment, a process for the progressive biotransformation of a waste material is disclosed. Waste material is exposed to a first inoculant comprising one or more fungal species which thereby initiates enzymatic depolymerization of organic, cellulosic or polymeric components in the waste material and forms partially depolymerized intermediates. The partially depolymerized intermediates are exposed to a second inoculant comprising bacterial or algal species which thereby initiates metabolization of the partially depolymerized intermediates and forms a biotransformed waste material.

[0079] HIRO / 105 / PC 8 In another embodiment, a system for progressive biodegradation of a waste material includes a plurality of biologically distinct inoculant formulations arranged to activate in sequence. A first inoculant includes one or more fungal species contained within a first carrier that is biodegradable, and a second inoculant includes bacterial or algal species contained within a second carrier having a slower hydration or degradation rate than the first carrier. The carriers are arranged or combined such that the inoculants become active successively when the system is exposed to environmental moisture or heat.

[0080] In yet another embodiment, an inoculant system has a first inoculant in a first matrix and a second inoculant in a second matrix. The first inoculant and second inoculant are biologically distinct from one another. The first matrix and second matrix possess different hydration or degradation profiles from one another, which results in sequential activation of the inoculants when exposed to one or both of environmental moisture and temperature conditions. The first inoculant and second inoculant are independently selected from fungi, bacteria, algae, and invertebrates. Each of the first inoculant and second inoculant is capable of initiating degradation, metabolization, or fragmentation of organic or polymeric waste materials.

[0081] In another embodiment, a method of manufacturing the inoculant system may comprise a step of cultivating each inoculant species under predetermined growth conditions, and then stabilizing each inoculant within a respective biodegradable matrix having a predetermined activation profile. The inoculants and matrices may be assembled into a multilayer or multi-compartment configuration configured to provide staged activation during exposure to waste material or environmental conditions.

[0082] In another embodiment, an active degradable pouch includes a biodegradable or water-soluble film forming an enclosure with a biological inoculant composition is positioned within the enclosure. The film material is configured to dissolve, disintegrate, or mechanically rupture upon exposure to moisture, composting, or hydration conditions to release the biological inoculant. In yet another embodiment, a method of initiating biodegradation in a waste or compost substrate includes a step of placing at least one of the pouches in or adjacent to the waste or compost substrate, and then exposing the pouch to moisture. This initiates dissolution or rupture of the film, releasing the biological inoculant, and initiating colonization or enzymatic degradation of the substrate.

[0083] In yet another embodiment, a system for biological degradation of waste includes at least one absorbent article or waste material, at least one embedded fungal or microbial inoculant in the at least one absorbent article or waste material, and at least one of the degradable pouches positioned for co-disposal with the article. The embedded inoculant and biological inoculant composition are configured for sequential or cooperative degradation of the waste material. In another embodiment, an inoculant composition comprises an immobilization matrix and fungal spores encapsulated in the immobilization matrix.

[0084] In another embodiment, a composite material includes a polymeric matrix substrate and fungal spores embedded within the polymeric matrix substrate to form the composite material.

[0085] In another embodiment, a filament composition includes a biodegradable thermoplastic polymer matrix selected from aliphatic polyesters, aliphatic-aromatic copolyesters, or bio-derived polymers having hydroxy- or ester-containing repeating units and a biological inoculant comprising one or more of fungal spores, mycelial fragments, and encapsulated microbial inclusions.

[0086] Fungi that are usable within the disclosed embodiments include members of the genera Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium,

[0087] HIRO / 105 / PC 9 Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.

[0088] Detailed Description

[0089] Among the benefits and improvements disclosed herein, other objects and advantages of the disclosed embodiments will become apparent from the following wherein like numerals represent like parts throughout the figures. Detailed embodiments of a system and methodology of fungal encapsulation, embedding and related products and uses for biotransformation of polymeric and absorbent articles are disclosed; however, it is to be understood that the disclosed embodiments are merely illustrative of the invention that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the invention are intended to be illustrative, and not restrictive.

[0090] Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in some embodiments” as used herein does not necessarily refer to the same embodiment(s), although it may. The phrases “in another embodiment” and “in some other embodiments” as used herein do not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments may be readily combined without departing from the scope or spirit of the invention. As used herein, “based on” is not exclusive and permits being based on additional factors not expressly described unless the applicable context clearly dictates otherwise.

[0091] In addition, as used herein, the term “or” is equivalent to the term “and / or,” unless the context clearly dictates otherwise. The term “based on” is not exclusive and allows for being based on additional factors not described unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of “a,” “an,” and “the” include plural references. The meaning of “in” includes “in” and “on.”

[0092] Further, the terms “substantial,” “substantially," “similar,” “similarly,” “analogous,” “analogously,” “approximate,” “approximately,” and any combination thereof mean that differences between compared features or characteristics is less than 25% of the respective values / magnitudes in which the compared features or characteristics are measured and / or defined.

[0093] Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints and open-ended ranges should be interpreted to include only commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.

[0094] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the inventive subject matter and does not pose a limitation on the scope of the inventive subject matter otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the inventive subject matter.

[0095] Groupings of alternative elements or embodiments of the inventive subject matter disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of

[0096] HIRO / 105 / PC 10 convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0097] As used in the present disclosure, the following terms have the meanings indicated, unless the context clearly indicates otherwise:

[0098] As used herein, the term “biological inoculant” refers broadly to any biological material introduced into a composition, substrate, or article for the purpose of germination, metabolic activation, enzymatic secretion, biodegradation, ecological succession, or related biological activity. Fungal inoculants constitute the preferred and primary form of biological inoculant disclosed herein, although bacterial, algal, and invertebrate forms may also be employed in certain embodiments. Unless otherwise indicated, the terms inoculant, inoculum, biological inoculum, biological agent, inoculant component, inoculated material, inoculum-containing layer, inoculant zone, microbial inoculum, and encapsulated inclusion are used interchangeably and refer to a biological inoculant as defined above.

[0099] The term “fungal inoculant” refers to any fungal propagule or growth form capable of surviving stabilization, processing, and storage and subsequently resuming metabolic activity under appropriate environmental conditions. Suitable fungal propagules include, without limitation, spores, conidia, chlamydospores, sclerotia, resting structures, mycelial fragments, hyphal segments, germ tubes, or cultured biomass, as well as functional equivalents thereof. Representative genera include Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus, together with any isolates, mutants, variants, or recombinant strains exhibiting comparable oxidative, hydrolytic, ligninolytic, or polymer-modifying enzyme activity. The fungal isolates identified in the Examples by FS or A'X' designations are internal reference identifiers used for convenience, and each such designation may correspond to any species within the stated genus or any functional equivalent having similar enzymatic activity or degradation behavior.

[0100] A “native inoculant” refers to a biological inoculant, preferably a fungal inoculant, in an unencapsuiated or minimally processed state, including dry spores, freeze-dried spores, powdered conidia, desiccated mycelial fragments, dried bacterial spores, dried algal cells, or invertebrate eggs. Native inoculants may be incorporated directly into polymer melts, absorbent matrices, fibers, nonwoven structures, films, or coatings, provided that the local thermal and shear conditions permit inoculant survival.

[0101] The term “encapsulated inoculant” refers to a biological inoculant immobilized, enclosed, coated, or otherwise contained within a protective matrix or structure. Suitable protective matrices include alginate hydrogels, chitosan or starch gels, gelatin matrices, cellulose derivatives, polyvinyl alcohol, sugar-glass systems such as trehalose or sorbitol, carbohydrate-polyol blends, and combinations thereof. Encapsulation may take the form of beads, pellets, capsules, microcapsules, coated particles, thin films, pouches, sachets, or matrix-embedded inclusions. The encapsulation may be partial or complete and is configured to permit hydration, swelling, porosity increase, and germination under environmental triggers such as moisture uptake, temperature change, or pH shift. A “protective matrix” refers specifically to the solid or semi-solid phase that reduces thermal, shear, or desiccation stress on a biological inoculant during processing, storage, or activation.

[0102] The terms “embedding polymer,” “host polymer,” and “polymeric matrix” refer to a polymeric material into which a biological inoculant or encapsulated inclusion is incorporated. Suitable polymers include aliphatic polyesters such as polylactic acid (PLA), polycaprolactone (PCL),

[0103] HIRO / 105 / PC 11 polybutylene succinate (PBS), and biodegradable aliphatic polyesters derived from hydroxy¬ functional monomers; aromatic polyesters such as polyethylene terephthalate (PET) and polytrimethylene terephthalate (PTT); polyolefins such as polyethylene (PE) and polypropylene (PP); thermoplastic elastomers, polyurethanes, polyamides, vinyl polymers, styrenic polymers, and any copolymers, blends, composites, laminates, or multilayer structures thereof, provided that the processing conditions maintain inoculant viability.

[0104] The terms “viability” or “viable” refer to the ability of a biological inoculant to germinate, resume metabolism, or produce hyphal outgrowth following exposure to processing or environmental conditions. “Stasis” refers to a dehydrated or dormant condition in which the inoculant maintains viability with minimal metabolic activity. “Activation” refers to the transition from stasis to metabolic activity upon exposure to moisture, nutrients, additives, pH changes, temperature changes, or other environmental stimuli. “Emergence” refers to hyphal outgrowth or visible colonization from the encapsulated or embedded inoculant, typically observed as surface coverage or radial expansion.

[0105] The term “thermal protection” refers to compositional or structural features that reduce thermal stress on an inoculant during polymer processing or melt exposure. Thermal protection may arise from endothermic phase transitions, sugar-glass formation, evaporative cooling, localized water release, microdomain buffering, or heat-modulating encapsulation matrices.

[0106] An “additive” refers to any compound or mixture that influences germination, enzyme expression, metabolic activation, or degradation behavior. Additives may include nutrients, cofactors, metal ions, redox mediators, humectants, mineral salts, sugars, organic acids, polyols, buffering agents, or combinations thereof. An “additive-doped substrate” refers to any polymeric, absorbent, cellulosic, fibrous, nonwoven, composite, or multilayer structure containing additives that modulate inoculant activity.

[0107] An “activation interface” refers to the region where a biological inoculant first contacts moisture, additives, or substrate components following environmental exposure. The activation interface may occur at the surface of a bead, the membrane of a pouch, a laminate boundary, a film interface, a fiber surface, or a junction between layers.

[0108] A “successional inoculant” refers to a biological inoculant that becomes active after or downstream of another inoculant. Successional behavior may result from layer separation, hydration gradients, encapsulation differences, nutrient availability, additive distribution, or environmental staging. While fungi are the primary successional agents described herein, the term also encompasses bacterial, algal, or invertebrate systems capable of functioning in later degradation phases. A “control layer” or “nutrient layer” refers to any layer, coating, deposit, or spatial region that modulates hydration, nutrient release, additive diffusion, or the timing of inoculant activation. A “degradation zone” refers to the region within or adjacent to a substrate in which biological activity has initiated polymer oxidation, hydrolysis, depolymerization, or structural weakening.

[0109] As used herein, the term “layer” refers to any sheet, film, region, zone, coating, or stratum of an article that is compositionally, structurally, or functionally distinct from an adjacent region. Layers may exist in single-layer, multilayer, zoned, patterned, or gradient configurations and may include any combination of inoculants, additives, or matrices as described herein.

[0110] An article according to the present disclosure may comprise one or more layers, and when multiple layers are present, the layers may be arranged in any suitable order. Unless otherwise stated, the terms “upper layer,” “lower layer,” “intermediate layer,” “inner layer,” “outer layer,” or “inoculant-containing layer” refer to relative positioning within the article, and do not require any particular material, thickness, or function. A single layer may contain a fungal inoculant, an

[0111] HIRO / 105 / PC 12 additive, a nutrient zone, or any combination thereof, and the same type of inoculant or additive may appear in more than one layer.

[0112] A “degradation zone” refers to the region within or adjacent to a substrate where biological activity has initiated structural or chemical modification. The degradation zone may expand over time as inoculant activity progresses, enabling staged or successional breakdown.

[0113] All drawings and figures herein show only representative embodiments. Additionally, all drawings herein are schematic and not to scale. Layer thicknesses, spatial relationships, and relative proportions are shown for clarity of illustration rather than dimensional accuracy.

[0114] Unless otherwise indicated, the foregoing definitions apply uniformly across all compositions, articles, delivery vehicles, multilayer assemblies, systems, and methods described in the present disclosure.

[0115] (1) Compositions, systems, and methods for immobilizing and activating fungal inocula within biodegradable hydrogel matrices

[0116] Disclosed herein are compositions, systems, and methods for immobilizing and activating fungal inocula within biodegradable hydrogel matrices. Embodiments relate to nutrient-amended alginate compositions that are configured to encapsulate one or more fungal species and to promote controlled emergence, enzymatic activation, and subsequent biodegradation of organic or polymeric substrates under ambient or composting conditions. The compositions described herein provide a tunable platform for maintaining fungal viability during storage and transport, while enabling rapid initiation of fungal metabolism upon hydration or other environmental triggers. In certain embodiments, the hydrogel matrices may be formed as beads, microcapsules, coatings, or porous structures that contain balanced nutrient and cofactor systems designed to sustain oxidative enzyme production, including laccase, manganese peroxidase, and versatile peroxidase, thereby facilitating the transformation of lignocellulosic or synthetic waste materials. Unless otherwise indicated, all percentages herein are expressed by weight relative to the total formulation, and all concentrations are provided as weight / volume (w / v) or millimolar (mM) values as appropriate. Numerical ranges and ratios are intended to include the stated limits as well as intermediate values. It will be understood that the features of the embodiments described herein may be combined or substituted in any operable manner to achieve similar technical effects within the scope of the invention.

[0117] Hydrogel Matrix Composition

[0118] In one aspect, the invention provides a biodegradable hydrogel matrix that serves as the encapsulation medium for fungal inocula. The matrix is primarily based on alginate, a naturally derived polysaccharide composed of p-D-mannuronic acid and a-L-guluronic acid residues that form ionic crosslinks in the presence of divalent cations. The composition of the hydrogel can be tailored to control porosity, gel strength, and hydration behavior by adjusting the alginate concentration, crosslinking ion type, and curing parameters.

[0119] In certain embodiments, the hydrogel matrix comprises from about 0.5 to about 5 percent by weight sodium alginate, optionally from about 0.8 to about 2.0 percent by weight for typical bead formulations. The crosslinking agent may be selected from calcium, barium, magnesium, zinc, or other divalent metal salts capable of forming stable ionic bridges with the carboxylate groups of the alginate polymer. Calcium chloride is particularly suitable due to its solubility and biocompatibility. Concentrations of the crosslinking agent may range from about 10 millimolar to about 200 millimolar, more typically between about 25 millimolar and about 150 millimolar, depending on the desired gel density and curing time.

[0120] HIRO / 105 / PC 13 In an exemplary embodiment, sodium alginate at 1.0 weight percent is combined with calcium chloride at approximately 50 millimolar to produce uniform spherical beads exhibiting a mean diameter of about 300 to about 400 micrometers. The hydrogel thus formed maintains mechanical integrity during drying while allowing sufficient diffusion of gases and nutrients for fungal viability. The gelation process may be conducted using an external gelation method, in which droplets of the alginate mixture containing fungal inoculum are introduced into a crosslinking bath containing calcium or other divalent ions. Curing times may range from about 1 minute to about 12 minutes, with shorter durations producing softer beads that favor early emergence and longer durations producing denser shells suitable for high-moisture environments. The shell thickness of the resulting beads can range from about 20 micrometers to about 120 micrometers, and the internal porosity can be controlled by varying ion concentration, pH, and the presence of other co¬ additives.

[0121] To further regulate mechanical and transport properties, the hydrogel matrix may include one or more secondary polymers or plasticizers. For instance, polyethylene glycol (PEG) with an average molecular weight of about 300 to about 600 may be incorporated at concentrations from about 1 to about 5 percent by volume to enhance flexibility, reduce brittleness, and maintain internal humidity. In other embodiments, glycerol, propylene glycol, or polyvinyl alcohol may serve as alternative plasticizers or humectants.

[0122] The hydrogel matrix may also incorporate carbonate sources such as calcium carbonate or sodium bicarbonate, which act as pH-stabilizing agents and buffering reservoirs during sterilization and subsequent hydration. These agents may also contribute controlled carbon dioxide release during fungal activation, which can influence hyphal extension and gas exchange. In certain embodiments, the hydrogel may be blended with bio-based fillers such as rice starch, corn starch, cellulose powder, or lignin-derived fibers to provide reinforcement and additional nutrient supply. Such fillers can also serve to modulate water retention and provide microtextural cues that guide fungal colonization after emergence.

[0123] The hydrogel composition can be sterilized using conventional autoclave procedures, for example at 121 degrees Celsius for 10 to 15 minutes, with subsequent cooling before addition of any thermally sensitive additives or fungal inoculum. Following sterilization, the alginate solution may be mixed with the desired fungal culture in a volumetric ratio of about 1:1 to produce the encapsulation slurry. The resulting matrix thus combines structural stability, controlled hydration kinetics, and biocompatibility conducive to maintaining spore or mycelial viability for extended storage periods.

[0124] Additive Systems

[0125] The hydrogel matrix described above may further comprise one or more additive systems selected to sustain fungal metabolism, modify the mechanical or transport properties of the matrix, and regulate water activity and ionic balance during both storage and activation. These additives may be included individually or in compatible combinations, and the amounts may vary depending on the physical form of the encapsulated product — such as beads, films, or coatings — and on the species of fungus selected for encapsulation. In general, the additive systems function cooperatively to maintain fungal viability during desiccation, promote rapid emergence upon hydration, and support enzymatic activity necessary for substrate degradation.

[0126] HIRO / 105 / PC 14 In certain embodiments, the composition includes at least one carbohydrate osmolyte such as sucrose, sorbitol, trehalose, maltose, mannitol, glucose, fructose, inulin, or dextrin. These sugars act as osmoprotectants that moderate internal water potential and provide an immediately available carbon source following rehydration. Suitable concentrations are typically between about 0.5 and 10 percent by weight, preferably between about 2 and 6 percent by weight. Combinations of disaccharides and polyols— for example, about 5 percent sucrose with about 5 percent sorbitol — have been shown to stabilize fungal spores and hyphae, preserving enzymatic potential and enhancing early emergence following rehydration.

[0127] To promote enzyme synthesis and mycelial expansion, the formulation may include one or more organic or inorganic nitrogen sources. Exemplary organic nitrogen sources include yeast extract, malt extract, peptone, casein hydrolysate, soybean hydrolysate, and combinations thereof. Suitable inorganic nitrogen sources include urea, ammonium sulfate, ammonium nitrate, or ammonium phosphate. The total nitrogen concentration is typically adjusted to provide about 0.5 to 10 millimoles of total nitrogen per liter of gel solution. Balanced mixtures of organic sources such as yeast extract and peptone in approximately equal mass ratios are preferred for rapid activation and robust enzyme expression.

[0128] Buffering salts may also be incorporated to maintain internal pH within a range of about 6 to 8 during sterilization and subsequent hydration. Examples include phosphate-buffered saline (10 to 100 millimolar), citrate, acetate, bicarbonate, and HEPES. In one embodiment, a phosphate buffer of approximately 50 millimolar at pH 6.8 ± 0.2 provides optimal ionic strength and gelation control during external calcium-induced crosslinking. These buffers stabilize pH and ionic composition, improving reproducibility and preventing local acidification that may inhibit fungal growth.

[0129] To improve toughness and control porosity, the alginate matrix may further comprise secondary polymers such as chitosan, starch, cellulose, hydroxyethyl cellulose, pectin, carrageenan, guar gum, xanthan gum, or combinations thereof. Typical concentrations range from about 0.5 to 2 percent by weight. Chitosan confers antimicrobial properties and forms cohesive films that reduce bead brittleness, while starch and cellulose derivatives increase water-holding capacity and provide microtextural roughness that facilitates hyphal anchoring and radial emergence during activation.

[0130] Plasticizers and humectants such as polyethylene glycol (PEG 300-600), glycerol, or propylene glycol can be incorporated at levels of about 1 to 5 percent by volume to preserve elasticity during drying and maintain uniform swelling upon rehydration. These agents minimize brittleness and prevent cracking, allowing the hydrogel to rehydrate evenly without compromising fungal viability. In some embodiments, the composition includes trace metal ions that serve as enzymatic cofactors, particularly for oxidative enzymes such as laccases, manganese peroxidases, and versatile peroxidases. Suitable ions include Fe2+, Fe3+, Mn2+, Cu2+, Co2+, Zn2+, and Mo6+, introduced as soluble salts such as sulfates, chlorides, nitrates, or gluconates. The total concentration of such ions may range from about 0.0001 to 0.1 percent by weight, and is preferably maintained between about 0.001 and 0.02 percent by weight. Iron and manganese ions promote peroxidase activity, copper ions are essential for laccase catalysis, and zinc or cobalt ions may act as stabilizers or secondary cofactors supporting enzyme conformation and redox cycling.

[0131] HIRO / 105 / PC 15 Trace vitamins and growth cofactors may also be incorporated at microgram levels to enhance long-term fungal viability. Examples include thiamine (vitamin Bi), riboflavin (vitamin B2), niacin (vitamin B3), pyridoxine (vitamin B6), pantothenic acid, biotin, and folic acid. Typical concentrations are below about 0.001 percent by weight, sufficient to replenish coenzymes lost during storage and rehydration cycles.

[0132] Optional redox mediators and electron shuttles may be included to facilitate electron transfer during oxidative degradation processes. Non-limiting examples include humic acid, fulvic acid, anthraquinone derivatives, ABTS analogs, and riboflavin. Amounts are generally between about 0.001 and 0.1 percent by weight. These compounds may remain partially bound to the gel network or be released gradually during hydration to sustain electron flow between fungal enzymes and substrate surfaces.

[0133] To protect fungal cells and enzymes from oxidative stress during storage, antioxidants and stabilizers such as ascorbic acid, citric acid, a-tocopherol, or related compounds can be included in concentrations from about 0.01 to 0.5 percent by weight. These agents also help preserve color, limit auto-oxidation of phenolics, and contribute additional buffering capacity.

[0134] Minor surfactants and wetting agents can be incorporated to improve dispersion of hydrophobic additives and prevent localized gel aggregation. Examples include polysorbate 80 (Tween 80), lecithin, rhamnolipids, and saponins, typically at concentrations below about 0.5 percent by weight. Naturally derived biosurfactants are preferred to maintain the biodegradability of the encapsulated system.

[0135] Mineral or organic fillers can be employed to control density, porosity, and swelling behavior. Examples include silica gel, perlite, diatomaceous earth, biochar, zeolite, and powdered lignin, generally added at 0.5 to 5 percent by weight. These fillers introduce microchannels that enhance oxygen and moisture transport and can act as secondary nutrient reservoirs during fungal growth. Chelating and pH-adjusting agents such as EDTA, gluconic acid, or citrate at concentrations of about 0.01 to 0.05 percent by weight may be used to regulate the availability of free metal ions and stabilize pH. Alkaline buffering agents such as calcium carbonate, sodium bicarbonate, or magnesium carbonate may also be added to neutralize acids formed during fungal metabolism and to provide gradual carbon dioxide evolution that aids gas exchange within the gel structure. In certain embodiments, the hydrogel may co-encapsulate compatible microorganisms such as bacterial spores, actinomycetes, or enzyme-producing symbionts that assist in substrate degradation or nutrient recycling. The total microbial load is generally less than about 10 percent of the total inoculum volume to preserve fungal predominance while allowing synergistic metabolic interactions.

[0136] Collectively, these additive systems provide a tunable chemical environment within the alginate matrix that promotes spore preservation, accelerates emergence, sustains oxidative metabolism, and enhances the structural and environmental performance of the encapsulated compositions. Process of Manufacture

[0137] The compositions disclosed herein may be manufactured by a variety of encapsulation and gelation techniques designed to immobilize fungal inoculum within a nutrient-amended alginate matrix. Suitable methods include external gelation, internal gelation, hybrid gelation, co-extrusion,

[0138] HIRO / 105 / PC 16 spray-gelation, emulsion templating, and casting, any of which may be selected or combined according to the desired bead size, mechanical strength, or release characteristics.

[0139] In one representative process, an aqueous solution of sodium alginate is first prepared at the desired concentration, typically between about 0.5 and 5 percent by weight, and preferably between about 0.8 and 2.0 percent by weight. The alginate may be hydrated in deionized or buffered water under moderate agitation until a homogeneous viscous solution is obtained. Carbohydrate osmolytes, nitrogen sources, and thermally stable salts or fillers may be incorporated at this stage. The resulting pre-gel mixture can be sterilized by autoclaving at approximately 121 °C for 10-15 minutes, ensuring both sterility and complete polymer hydration. After cooling to below approximately 40 °C, heat-labile components such as polyethylene glycol, vitamins, antioxidants, or transition-metal cofactors are incorporated with low-shear mixing to avoid polymer chain degradation. The cooled solution is then combined with a liquid fungal culture, spore suspension, or fragmented mycelial inoculum, usually in a volumetric ratio of about 1:1. The mixture forms a viscous slurry containing fungal propagules and nutrient additives uniformly dispersed within the alginate matrix.

[0140] In one preferred embodiment, droplets of the inoculated alginate mixture are dispensed into a crosslinking bath containing a solution of a divalent cation, such as calcium chloride, calcium lactate, or barium chloride. Concentrations of the crosslinking solution may range from about 25 millimolar to about 150 millimolar. Droplet formation can be achieved using syringe extrusion, peristaltic pumps, vibrating nozzles, air-assisted atomizers, or encapsulator devices. The droplets are allowed to cure in the ion bath for a period of about 1 minute to about 12 minutes. Shorter curing times yield softer beads that favor rapid fungal emergence, whereas longer times produce denser, moisture-resistant shells. After curing, the beads may be rinsed in sterile buffer or subjected to a second crosslinking bath of differing ion concentration to form gradient shells. Shell thicknesses may range from about 20 micrometers to about 120 micrometers, with average bead diameters between about 200 micrometers and about 3 millimeters.

[0141] In an alternative embodiment, gelation is induced internally by including an insoluble or sparingly soluble calcium source, such as calcium carbonate or calcium sulfate, together with an acid¬ releasing agent. Upon controlled acidification — achieved, for example, by adding glucono-5-lactone (GDL) or an organic acid precursor — the local pH decreases, liberating calcium ions from the carbonate or sulfate source. The calcium ions then crosslink the alginate uniformly throughout the droplet volume, forming a cohesive gel without the need for an external ion bath. This method can yield beads with a more homogeneous internal texture and may be advantageous for forming larger capsules, continuous filaments, or bulk cast gels. The relative proportions of alginate, calcium carbonate, and GDL can be tuned to control gelation rate and bead rigidity. Suitable ratios of calcium carbonate to alginate range from about 0.5: 1 to about 2: 1 by weight, and GDL may be present at about 0.1 to about 2 percent by weight.

[0142] Internal gelation can also be combined with external gelation in a hybrid process, wherein a pre¬ mixed alginate slurry containing calcium carbonate and GDL is extruded into a mild calcium chloride bath. The dual ion sources produce a two-stage gelation, providing a firm shell and a softer core that promotes nutrient diffusion and fungal emergence. The hybrid approach allows improved mechanical control and may be tuned for controlled release of different fungal species or additives.

[0143] HIRO / 105 / PC 17 In certain embodiments, co-extrusion methods are used to form core-shell beads or fibers, wherein an inner core containing the fungal inoculum and nutrients is extruded simultaneously with an outer shell solution of alginate or another polymer. The two fluids are delivered through concentric nozzles and contact a crosslinking stream to form continuous or discontinuous encapsulates. The resulting architecture allows separation of nutrient-rich zones from protective shells and can be designed to release different fungal species or enzymes sequentially.

[0144] Alternative small-scale or high-throughput methods may include spray-gelation, in which a fine mist of alginate solution is sprayed directly into a cloud or mist of calcium-containing solution, or emulsion-templated gelation, wherein the alginate mixture is emulsified as aqueous droplets within a continuous oil phase followed by addition of calcium ions to induce crosslinking. Such methods can produce microspheres ranging from about 10 micrometers to about 200 micrometers in diameter, suitable for coatings, films, or composite articles.

[0145] In another embodiment, the alginate solution containing fungal inoculum and additives may be cast or coated onto a substrate and subsequently exposed to a crosslinking solution or vapor to form thin hydrogel films. These films can be used to coat the surfaces of absorbent cores, packaging materials, or fibrous webs, forming immobilized zones of fungal activity that activate upon exposure to moisture.

[0146] Following gelation, beads, films, or other shapes may undergo optional post-treatment steps such as rinsing with sterile buffer, soaking in solutions of transition-metal cofactors (for example, 0.001-0.02 wt % FeSO4, MnSO4, or CuSO4), or coating with biopolymers such as chitosan or starch to adjust permeability. The encapsulated materials are then dried under mild conditions, typically below 55 °C, or air-dried under controlled humidity to achieve the desired residual moisture content. The dried product may be stored as free-flowing beads, pellets, films, or granules and later rehydrated to restore the hydrogel structure.

[0147] In certain embodiments, the alginate compositions are further processed into multilayered or composite structures by combining them with superabsorbent polymers, cellulose fibers, or biodegradable plastic matrices. Such composites can be integrated into absorbent articles or packaging to provide on-demand biodegradation capability. The manufacturing processes described herein thus provide flexibility in format and allow tuning of physical and biological properties while maintaining fungal viability and enzymatic potential.

[0148] Functional Mechanism

[0149] Without intending to be bound by any particular theory, it is believed that the compositions described herein function through a combination of controlled hydration, ionic exchange, and nutrient activation events that collectively support fungal emergence, enzyme production, and substrate degradation. When the encapsulated compositions contact moisture, the alginate- based hydrogel undergoes rapid ion exchange in which divalent cations within the crosslinked matrix— typically calcium ions— are replaced by monovalent cations such as sodium or potassium present in the surrounding medium. This exchange results in local relaxation of the gel network, allowing the matrix to swell and to create diffusion pathways that facilitate the outward growth of fungal hyphae. The swelling ratio of the beads, typically between about 1.2 and 2.5 times the original dry diameter, is sufficient to permit expansion while maintaining mechanical cohesion. Simultaneously, the rehydrated environment activates internal osmolytes such as sucrose and sorbitol, which stabilize cell membranes and balance osmotic potential during the early stages of

[0150] HIRO / 105 / PC 18 emergence. The carbohydrate additives also serve as readily metabolizable carbon sources, supporting the energy demands of germinating spores or regenerating mycelia until the fungal hyphae establish contact with external organic material. The nitrogen sources incorporated within the matrix, including yeast extract, peptone, or ammonium salts, provide essential amino acids and peptides that enable rapid protein synthesis and enzyme formation during this emergence phase.

[0151] As fungal metabolism resumes, enzymes such as laccase, manganese peroxidase, and versatile peroxidase are secreted into the local environment. These oxidative enzymes initiate depolymerization of complex substrates by generating reactive oxygen species and radical intermediates. The presence of transition-metal cofactors, particularly copper, iron, and manganese ions, within the matrix or supplied from the surrounding environment, acts to activate or stabilize these enzymes through the formation of metal-enzyme complexes. In certain embodiments, the cofactors are gradually released from the gel matrix in response to pH and ionic shifts, providing sustained enzymatic activity over prolonged hydration periods.

[0152] The buffering agents included in the formulation serve to maintain the local pH within an optimal range for both fungal growth and enzyme function, typically between about 5.5 and 7.5. The phosphate or carbonate buffers mitigate acidification resulting from metabolic by-products and from the hydrolysis of organic acids. This buffering capacity ensures that the enzymatic reactions proceed under stable conditions, preventing premature deactivation or structural denaturation of the hydrogel matrix.

[0153] Secondary polymers such as chitosan, starch, or cellulose contribute to the mechanical and diffusional characteristics of the matrix. Chitosan, for example, forms a semi-permeable complex with alginate that enhances structural stability and modulates oxygen permeability. Starch and cellulose derivatives increase internal water retention, which prolongs the hydration window available for fungal colonization. Together, these structural additives enable the formation of microenvironments that balance gas exchange, moisture control, and nutrient availability.

[0154] Antioxidants such as ascorbic acid and tocopherols further stabilize the encapsulated fungi by quenching free radicals generated during oxidative metabolism or by residual peroxidase activity. Their inclusion minimizes auto-oxidative damage to lipids, proteins, and enzymes within the encapsulated biomass. Similarly, redox mediators such as humic acid, fulvic acid, and anthraquinone derivatives act as electron shuttles, coupling enzymatic oxidation of the substrate to extracellular electron transport and thereby extending the effective reaction zone beyond the immediate surface of the hydrogel bead.

[0155] When the encapsulated composition is placed in contact with a degradable substrate, such as a lignocellulosic fiber or a polymeric film, the emerging fungal hyphae physically penetrate the substrate surface and deposit oxidative enzymes that initiate localized degradation. In certain embodiments, the hydrogel matrix remains partially intact, providing continuous nutrient supply and structural support to the growing mycelial network. In other embodiments, the matrix gradually disintegrates as crosslinks are depleted or consumed, leaving behind a colonized region enriched with active fungal biomass.

[0156] The combined function of the carbohydrate osmolytes, nitrogen sources, transition-metal cofactors, buffers, and structural polymers creates a self-regulating biochemical environment capable of autonomous activation. The system transitions from a dormant, storage-stable state to a metabolically active, degradative state upon simple exposure to moisture. This activation

[0157] HIRO / 105 / PC 19 mechanism allows the compositions to be integrated into a wide variety of articles— such as absorbent cores, compostable packaging, fiber mats, and soil-conditioning pellets — where fungal emergence is triggered only under defined environmental conditions. The result is a biodegradable, bioactive material that maintains stability during use and storage but initiates biological breakdown when exposed to moisture, temperature, or pH cues associated with disposal or composting environments.

[0158] In certain embodiments, the compositions described herein comprise a biodegradable alginate-based hydrogel matrix containing defined ratios of alginate, divalent crosslinking ions, nutrient additives, and optional cofactors, formulated to maintain fungal viability during storage and to promote emergence and enzymatic activity upon hydration. The following non-limiting embodiments illustrate representative formulations and manufacturing variations that may be implemented within the scope of the invention.

[0159] In one preferred embodiment, the composition comprises from about 0.8 to about 2.0 percent by weight sodium alginate and from about 25 to about 150 millimolar calcium chloride as the primary crosslinking ion source. The matrix further contains a carbohydrate osmolyte system composed of approximately 5 percent by weight sucrose and 5 percent by weight sorbitol; a nitrogen source consisting of 0.2 percent by weight yeast extract and 0.2 percent by weight peptone; and optional minor salts including 0.05 percent by weight magnesium sulfate heptahydrate, 0.05 percent by weight potassium dihydrogen phosphate, 0.02 percent by weight potassium chloride, and 0.001 percent by weight ferrous sulfate heptahydrate. Polyethylene glycol having an average molecular weight between 300 and 600 may be included at 2 percent by volume as a plasticizer and humectant. The mixture is sterilized, combined with the desired fungal inoculum, and gelled by external calcium crosslinking to form spherical beads of about 300 to 400 micrometers in diameter. The cured beads are dried gently at 50 to 55 °C for 24 hours to obtain a stable, rehydratable inoculant composition exhibiting at least 85 percent viability retention.

[0160] In another embodiment, the hydrogel matrix incorporates structural polymers such as 1 percent by weight rice starch and 1 percent by weight chitosan, producing a reinforced, semiinterpenetrating network that enhances bead strength and water retention. This combination provides a synergistic improvement in emergence and enzyme secretion under high-ionic- strength conditions typical of absorbent articles or compost leachate. The resulting beads display rapid fungal colonization, with visible hyphal coverage exceeding 70 percent of the bead surface within seventy-two hours after hydration.

[0161] In a further embodiment, the composition includes trace transition-metal cofactors selected from ferrous, manganous, and cupric ions, each at concentrations between about 0.001 and 0.02 percent by weight, to support oxidative enzyme activity such as laccase, manganese peroxidase, and versatile peroxidase. These ions may be introduced during mixing as soluble salts or by post¬ curing diffusion into the gel. The combination of these cofactors provides balanced activation of redox enzymes responsible for polymer oxidation and lignin depolymerization.

[0162] Alternative embodiments employ different gelation and curing techniques. For example, an internal-gelation formulation may include sodium alginate at 1.0 percent by weight, calcium carbonate at 1.0 percent by weight, and glucono-5-lactone at 0.3 percent by weight to achieve slow, homogeneous crosslinking throughout the gel mass. This configuration produces uniform texture and is suited to forming monolithic blocks, films, or coated substrates. Hybrid gelation

[0163] HIRO / 105 / PC 20 methods combining internal and external calcium sources may also be employed to yield gradient or multilayer structures with distinct diffusion and mechanical profiles.

[0164] In certain embodiments, the composition is buffered with phosphate-buffered saline at 50 millimolar and pH 6.8, ensuring stability during sterilization and activation. Antioxidants such as ascorbic acid (0.05 percent by weight) and citric acid (0.05 percent by weight) may be added to protect labile nutrients and fungal cells from oxidative damage. The hydrogel may also include biosurfactants such as lecithin or rhamnolipids at concentrations below 0.5 percent by weight to improve mixing uniformity and nutrient dispersion.

[0165] In some embodiments, the hydrogel is integrated into a composite structure with superabsorbent polymers, cellulose fibers, or biodegradable plastic films to form functional inserts or coatings within absorbent hygiene products or packaging materials. Upon exposure to liquid, the encapsulated fungi emerge from the hydrated zones and initiate localized enzymatic degradation of the surrounding material. In other embodiments, the encapsulated inoculum is formulated as pellets, granules, or soil-conditioning beads for agricultural or environmental remediation applications.

[0166] It will be appreciated that numerous variations may be made without departing from the scope of the invention. Any of the additives, curing methods, or cofactor systems described herein can be combined in compatible ratios to obtain a composition optimized for a particular fungal species or environmental condition. The embodiments thus represent illustrative examples of the broader class of nutrient-amended alginate encapsulation systems encompassed by the present disclosure.

[0167] Encapsulation Test Methodology

[0168] The following test procedures were employed to evaluate the performance and stability of the encapsulated fungal compositions described herein. Unless otherwise indicated, all observations were performed under ambient laboratory conditions (approximately 22-25 °C) using sterilized water or buffer for rehydration. Each method may be used independently or in combination to characterize emergence, enzymatic activity, swelling, mechanical behavior, and viability retention of the alginate-based compositions. The test methods are semi-quantitative in nature and designed to provide consistent comparative data among formulations.

[0169] Emergence Index

[0170] Fungal emergence was assessed using a semi-quantitative visual scoring system representing the extent of visible fungal growth across the surface of the hydrogel bead or film after hydration. Beads were incubated for seventy-two hours on a moist substrate or agar surface under controlled humidity. Unless noted otherwise, fungal emergence was evaluated via growth on 2% MEA (2% malt extract agar) plate. Emergence was recorded on a five-point scale, where a score of 1 indicates no visible hyphal growth; 2 indicates sparse or discontinuous emergence covering less than approximately 25 percent of the bead surface; 3 represents moderate emergence covering 25-50 percent of the surface; 4 corresponds to dense coverage over 50-75 percent of the surface with visible radial hyphae; and 5 denotes complete colonization (approximately 75-100 percent coverage) accompanied by dense mycelial growth or sporulation. The emergence index thus provides a reproducible, visual measure of fungal activation and colonization behavior.

[0171] HIRO / 105 / PC 21 Enzymatic Activity (ABTS Oxidation Assay)

[0172] Enzymatic activity, particularly oxidative enzyme expression, was determined by a qualitative ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) oxidation test Hydrated beads or colonies were overlaid or placed in contact with an ABTS-containing medium. The appearance of a green to blue coloration was recorded as evidence of laccase or peroxidase activity. The intensity of coloration was evaluated visually on a scale of 1 to 5, where 1 indicates no visible oxidation and 5 indicates complete and uniform coloration within forty-eight hours. This qualitative assay provides an indicator of enzymatic activation within the encapsulated fungal system and correlates with oxidative degradation potential.

[0173] Swelling Index

[0174] The swelling behavior of the dried hydrogel beads was evaluated using a visual scale that reflects the degree of bead expansion upon hydration. Beads were immersed in deionized water or buffer for twenty-four hours, then examined for dimensional changes. Swelling was scored on a five-point scale, in which a score of 1 represents negligible expansion or visible hardening, 2 indicates partial softening with less than 25 percent size increase, 3 corresponds to moderate expansion of 25-50 percent, 4 indicates 50-100 percent expansion with soft, elastic texture, and 5 denotes full rehydration with an estimated two- to threefold increase in bead diameter relative to the dry state. The swelling index provides a practical measure of rehydration capacity and internal porosity without requiring precise dimensional measurement.

[0175] Shell Thickness and Structural Gradient

[0176] Apparent shell formation and relative gel density were inferred based on curing parameters and supported by known diffusion-gelation models for alginate systems. Shell development correlates with the concentration of divalent cations and the duration of exposure to the crosslinking bath. Beads cured for approximately one minute at low ion concentrations (25-50 millimolar calcium chloride) exhibited soft, thin shells, whereas beads cured for 8-12 minutes at higher concentrations (100-150 millimolar calcium chloride) exhibited denser outer layers. Based on these conditions and accepted gelation kinetics, shell thickness is estimated to range from approximately 20 to 120 micrometers. Qualitative assessment of shell integrity was performed by bisecting hydrated beads and examining cross sections under a stereomicroscope for visible density gradients.

[0177] Viability After Drying

[0178] The retention of fungal viability following drying and storage was evaluated by rehydrating dried beads in sterile buffer and observing subsequent emergence relative to freshly prepared control samples. Viability was expressed as the percentage ratio of emergence index for stored samples to that of controls. Compositions exhibiting relative emergence above approximately 85 percent were considered to have acceptable viability retention. This comparative method provides a reliable indicator of long-term storage stability under moderate temperature and humidity conditions.

[0179] Bead Size and Morphology

[0180] Bead size and surface morphology were determined from optical or digital micrographs taken prior to and following hydration. Average bead diameters were measured from representative images using calibrated imaging software or optical scales. The beads produced under the

[0181] HIRO / 105 / PC 22 described conditions generally exhibited mean diameters between about 200 micrometers and 3 millimeters, depending on nozzle size, extrusion rate, and curing time. Bead morphology was evaluated visually and by stereomicroscopy immediately after drying and following rehydration. The morphology quality index (1-5) reflects bead sphericity, shell smoothness, absence of fissures or pitting, and handling durability. A score of 1 indicates irregular or collapsed beads with poor cohesion; 2 represents partial rounding with visible defects; 3 denotes generally round beads with minor surface roughness; 4 corresponds to well-formed, smooth-surfaced spheres with good mechanical strength; and 5 represents highly uniform, defect-free beads maintaining structural integrity through drying and rehydration cycles. This index provides a practical measure of manufacturing robustness and serves as a secondary quality metric complementing the fungal emergence and enzymatic-activity indices.

[0182] Mechanical Integrity

[0183] Mechanical integrity was evaluated qualitatively by manual compression and visual inspection. Hydrated beads were subjected to gentle pressure between gloved fingertips or between glass slides to assess elasticity and fracture resistance. Beads maintaining their shape without cracking or collapsing were classified as mechanically stable. This test provides an empirical comparison of formulations differing in polymer content, crosslink density, or plasticizer composition.

[0184] Design of Experiments (DoE) Analysis

[0185] Formulation optimization was further analyzed using a fractional factorial design of experiments, wherein the main and interaction effects of multiple formulation variables (alginate concentration, calcium concentration, carbohydrate additives, nitrogen source, polymeric additives, and buffer composition) were assessed relative to the emergence index and enzyme-activity scores. Statistical significance was evaluated by Pareto ranking of factor effects and visualization of interaction trends. Although the scoring was semi-quantitative, the experimental design provided a reproducible framework for identifying critical and synergistic parameters within the formulation space.

[0186] Encapsulation Examples

[0187] The following examples illustrate representative embodiments of the invention and methods for producing and evaluating the compositions described herein. These examples collectively demonstrate the influence of formulation components, curing parameters, and nutrient additives on fungal emergence, enzymatic activity, and mechanical stability. Unless otherwise noted, all percentages are expressed by weight and concentrations by weight / volume (w / v) or millimolar (mM).

[0188] • Example 1 — Baseline Nutrient-Amended Alginate Formulation

[0189] A representative nutrient-amended alginate formulation was prepared by dissolving sodium alginate (1.0 wt %) in deionized water under moderate agitation until a uniform, viscous solution was obtained. During initial formulation screening, calcium carbonate (1.0 wt %) was evaluated as a latent cross-linking source for potential internal-gelation applications; however, for the present embodiment the carbonate was omitted to avoid premature gelation during sterilization and to maintain full control over external cross-linking. Polyethylene glycol 400 (2 vol %) was added as a plasticizer and humectant to improve flexibility of the dried beads. The formulation was fortified with carbohydrate osmolytes consisting of sucrose (5 wt %) and sorbitol (5 wt %),

[0190] HIRO / 105 / PC 23 mineral salts (0.05 wt % magnesium sulfate heptahydrate, 0.05 wt % potassium dihydrogen phosphate, 0.02 wt % potassium chloride, and 0.001 wt % ferrous sulfate heptahydrate), and an organic nitrogen mixture of yeast extract (0.2 wt %), malt extract (0.5 wt %), and peptone (0.1 wt %). The resulting pre-gel solution was sterilized at 121 °C for 15 minutes and cooled to approximately 40 °C before addition of the fungal inoculum.

[0191] A liquid culture of Ganoderma A1 was incorporated at a volumetric ratio of 1:1 with the cooled alginate mixture to form a homogeneous encapsulation slurry. The inoculated solution was extruded dropwise through a peristaltic nozzle Into a 1 % (w / v) calcium chloride bath maintained at ambient temperature. Each droplet gelled immediately upon contact with the calcium ions, producing spherical beads with mean diameters of approximately 500 - 1000 micrometers. The beads were cured for five minutes, rinsed in sterile buffer to remove excess calcium, and dried in a forced-air oven at 50 °C for 24 hours to achieve a free-flowing powder.

[0192] Upon rehydration in sterile water, the beads swelled visibly to roughly twice their dry diameter (swelling index = 3 - 4) while retaining mechanical integrity. Within 48 hours of incubation on moist substrate, hyphal emergence from the bead surface was observed across the majority of particles. After 72 hours, an emergence index of 2 - 3 was recorded according to the defined scoring system, corresponding to moderate surface colonization. Enzymatic activity was confirmed using the ABTS oxidation assay, which produced a moderate green-blue coloration within 48 hours. These results demonstrate that a balanced combination of carbohydrate osmolytes and organic nitrogen sources within the alginate hydrogel sustains fungal viability and supports rapid enzymatic activation following hydration. The composition of this embodiment, summarized in Table 1 as the V2 (Control), served as the baseline formulation for subsequent additive and process optimization studies.

[0193] • Example 2 — Effect of Structural and Nutritional Additives on Fungal Emergence Building upon the baseline formulation of Example 1, additional trials were conducted to evaluate the effect of structural polysaccharides and nutrient variations on bead integrity and fungal activation. The standard alginate matrix (1.0 wt % sodium alginate, 5 wt % sucrose, 5 wt % sorbitol, 0.2 wt % yeast extract, 0.5 wt % malt extract, 0.1 wt % peptone, and 2 vol % polyethylene glycol 400) served as the control composition designated “V2.”

[0194] Two modified systems were prepared. In the first, 1.0 wt % rice starch was incorporated as a filler and slow-release carbohydrate source (“V3 + RS”). In the second, 1.0 wt % chitosan (degree of deacetylation ~ 85 %) was dissolved in 1 % acetic acid, neutralized, and added to the alginate solution prior to sterilization (“V3 + CH”). All formulations were sterilized, cooled, inoculated with Ganoderma A1, and gelled by external cross-linking in 1 % CaCI2for five minutes, following the procedure described in Example 1.

[0195] After drying at 50 °C for 24 hours, beads were rehydrated and incubated on moist agar substrates for 72 hours. The emergence index and visual morphology are summarized in Table 1. Both starch- and chitosan-amended systems produced dense mycelial coverage (emergence index 4-5) with uniform bead expansion (swelling index 4). In contrast, the control formulation without structural additives exhibited partial collapse and slower emergence (index 2-3). Beads containing chitosan displayed slightly firmer texture and reduced fragmentation during handling, whereas rice-starch-amended beads showed greater moisture retention and more even fungal colonization.

[0196] HIRO / 105 / PC 24 Collectively, the data in Table 1 demonstrate that inclusion of structural polysaccharides such as chitosan or rice starch enhances mechanical strength and water-holding capacity of the alginate hydrogel while simultaneously improving fungal emergence. The combination of these additives with balanced organic nutrients therefore provides a synergistic benefit, yielding robust, nutrient-rich beads that maintain structural integrity during drying yet readily reactivate under moist conditions.

[0197] Table 1. Formulations and Fungal Emergence Results

[0198] Parameter V2 (Control) V3 + RS V3 + CH V2 (Sulfate Blank control) Alginate Structural None 1 % rice 1 % chitosan None None Additives starch

[0199] Crosslinker CaCi2(1 %, CaCI2(1 %, CaCI2(1 %, CaSO4(1 %, CaCI2(1 %, (type, cone.) 50 mM) 50 mM) 50 mM) ~ 50 mM 50 mM)

[0200] Ca2+)

[0201] Carbohydrate 5 % sucrose 5 % sucrose 5 % sucrose 5 % sucrose None System + 5 % + 5 % sorbitol + 5 % + 5 %

[0202] sorbitol sorbitol sorbitol

[0203] Nitrogen YE (0.2 %) + YE (0.2 %) + YE (0.2 %) + YE (0.2 %) + None System ME (0.5 %) + ME (0.5 %) + ME (0.5 %) + ME (0.5 %)

[0204] PP (0.1 %) PP (0.1 %) PP (0.1 %) + PP (0.1 %) Other PEG 400 (2 PEG 400 (2 PEG 400 (2 PEG 400 (2 PEG 400 (2 Additives vol %) vol %) vol %) vol %) vol %) Curing Time 1-2 hour 1-2 hour 1-2 hour 1-2 hour 1-2 hour Fungal Ganoderma Ganoderma Ganoderma Ganoderma Ganoderma Species A1 A1 A1 A1 A1 Emergence 2 - 3 4 - 5 4 - 5 1 -2 1 -2 Index (1—5)

[0205] Swelling 3 4 - 5 4 2 3

[0206] Index (1-5)

[0207] Qualitative Moderate High Dense Premature No visible Observation swelling; moisture hyphal layer; hardening; growth;

[0208] partial hyphal retention; improved low nutrientcoverage rapid uniform bead emergence deficient emergence cohesion matrix

[0209]

[0210] Abbreviations: YE ~ yeast extract; ME = malt extract; PP = peptone; CH = chitosan; RS = rice starch.

[0211] Emergence and swelling indices scored visually on a 1-5 scale (1 = none; 5 = complete or full expansion).

[0212] HIRO / 105 / PC 25 Table 2 summarizes the aggregated emergence, swelling, and enzymatic activity results obtained across replicate trials and multiple fungal strains Ganoderma A1, Aspergillus A2, and Trametes A3) from the experimental series. The compiled data confirms that starch- and chitosan-amended formulations consistently outperformed the baseline control in both emergence and oxidative activity, while the sulfate-crosslinked and nutrient-free systems remained inactive. Averaged emergence indices and qualitative ABTS color intensities demonstrate reproducible performance across fungal isolates and validate the use of these additive systems as structural and nutritional enhancers in subsequent optimization studies.

[0213] Table 2. Comprehensive Summary of Formulation Adjustments (Aggregated Across A 1, A2, A3) Formulation Species Mean Swelling ABTS Qualitative

[0214] ID Tested Emergence Index (1- Color Observations Index (1-5) 5) Intensity (+

[0215] to +++++)

[0216] V2 (Control) A1, A2, 2.6 ± 0.4 3 ± 0.2 Moderate swelling;

[0217] A3 partial surface coverage; uneven mycelium

[0218] V3 + RS (1 % A1, A2, 4.8 ± 0.3 4.6 ± 0.2 +++++ High moisture Rice Starch) A3 retention; uniform emergence; intense green-blue ABTS response

[0219] V3 + CH (1 % A1, A2, 4.5 ± 0.3 4.2 ± 0.2 ++++ Dense hyphal layer;

[0220] Chitosan) A3 improved bead cohesion; slightly reduced swelling V2 (Sulfate A1 1.8 ± 0.3 2 ± 0.3 + Premature Control) hardening; opaque shell; limited emergence Blank A1 1.0 ± 0.0 3 ± 0.2 ± Nutrient-deficient; no Alginate (no visible growth nutrients)

[0221] V3 + RS (High A3 5.0 ± 0.0 4.8 ± 0.1 +++++ Complete surface Repetition colonization; rapid A3) reactivation < 48 h V3 + CH A2 4.3 ± 0.2 4.0 ± 0.1 ++++ Maintained shape (Extended after storage; minor Drying A2) fragmentation only

[0222]

[0223] HIRO / 105 / PC 26 • Example 3 — Formulation Optimization

[0224] A multivariable optimization study was conducted to determine the principal and interactive effects of formulation parameters on fungal emergence and enzymatic activity. A fractional factorial 27~3design was selected to efficiently evaluate seven independent variables:

[0225] (A) sodium-alginate concentration,

[0226] (B) calcium-chloride concentration,

[0227] (C) carbohydrate system (presence or absence of sucrose + sorbitol),

[0228] (D) nitrogen source (yeast extract + peptone versus none),

[0229] (E) chitosan addition,

[0230] (F) rice-starch addition, and

[0231] (G) phosphate-buffer inclusion.

[0232] The complete design matrix and coded factor levels are summarized in Table 3, while representative high- and low-performance formulations are listed in Table 4. Each formulation was prepared as described in Example 1 using Ganoderma A1 as the exemplary fungal species. Aspergillus A2 was also used as an exemplary fungal species in certain conditions.

[0233] The inoculated alginate mixtures were extruded dropwise into aqueous calcium-chloride solutions corresponding to the target cross-linker concentrations (25-150 mM). The beads were allowed to cure in the calcium bath for 1 to 2 hours under gentle stirring to ensure uniform ion diffusion and complete network formation throughout the bead volume. After curing, the beads were rinsed with sterile buffer to remove residual calcium and dried at 50 °C for twenty-four hours to yield free- flowing granules suitable for testing.

[0234] Emergence and enzymatic activity were assessed after seventy-two hours of incubation under moist conditions. General analysis of the standardized effects indicated that alginate concentration (Factor A) and calcium concentration (Factor B) were the dominant contributors to mechanical integrity and fungal emergence. The nutrient-related factors — carbohydrates (C) and nitrogen (D) — produced the largest positive influence on metabolic activation. Secondary polymeric additives (E ~ chitosan, F = rice starch) displayed a significant positive interaction term (E x F), demonstrating synergy in maintaining moisture and improving fungal emergence. The interaction between calcium concentration and buffering (B x G) was also statistically relevant, indicating that the pH environment modulates calcium-ion diffusion and cross-linking uniformity during the extended curing process.

[0235] Response-surface defined a broad optimum at 1.0—1.5 wt % sodium alginate and 100-150 mM calcium chloride. Within this domain, formulations containing both the sucrose + sorbitol osmolyte system and the yeast extract + peptone nitrogen source consistently yielded emergence indices of 4-5 and strong ABTS colorimetric responses. Beads produced under these conditions displayed high mechanical stability and rehydration capacity (swelling index 4-5).

[0236] Formulations corresponding to Runs 15 and 16 (Table 3), comprising 1.5 wt % sodium alginate, 150 mM CaCI2, 1 wt % chitosan, 1 wt % rice starch, 5 wt % sucrose, 5 wt % sorbitol, and 50 M phosphate buffer, produced spherical beads of uniform size and smooth surface morphology after 1.5 hours of curing. The extended curing duration promoted complete calcium diffusion and a more homogeneous internal gel network, resulting in improved shape retention and reduced surface pitting during drying. Following hydration, these beads exhibited full fungal colonization (emergence index = 5) and strong oxidative activity as confirmed by ABTS oxidation.

[0237] HIRO / 105 / PC 27 Table 3. Experimental Design Matrix and Results for Fractional Factorial Study (16 Runs) Ru A B C D E F G Emergenc Morpholog n Alginat CaCI Car Nitroge Chitosa Rice Buffe e Index y Quality e (%) 2 b n n Stare r (1-5, (1-5) (mM) h (PBS mean ±

[0238] ) SD)

[0239] 1 -1 (0.5) -1 — — — — 1.5 ± 0.3 1

[0240] (25)

[0241] 2 + 1 (1.5) -1 + + — “ - 2.8 ± 0.4 3

[0242] (25)

[0243] 3 -1 (0.5) + 1 — 4- 4- — — 3.0 ± 0.4 3

[0244] (150)

[0245] 4 + 1 (1.5) + 1 4° 4- — — — 3.9 ± 0.3 4

[0246] (150)

[0247] 5 -1 (0.5) -1 + — — + ““ 2.7 ± 0.2 2

[0248] (25)

[0249] 6 + 1 (1.5) -1 + + + + — 3.8 ± 0.3 4

[0250] (25)

[0251] 7 -1 (0.5) +■ 1 4- 4- — ■ 4- — 3.2 ± 0.3 3

[0252] (150)

[0253] 8 + 1 (1.5) +1 + + + + — 4.8 ± 0.2 4

[0254] (150)

[0255] 9 -1 (0.5) -1 — + __ — + 3.1 ± 0.3 2

[0256] (25)

[0257] 10 + 1 (1.5) -1 4- 4- 4~ 4~ 4.1 ± 0.3 4

[0258] (25)

[0259] 11 -1 (0.5) + 1 4- 4" 4- — 4- 4.3 ± 0.2 4

[0260] (150)

[0261] 12 + 1 (1.5) + 1 + + + ““ + 4.7 ± 0.2 5

[0262] (150)

[0263] 13 -1 (0.5) -1 — — — + + 1.8 ± 0.2 2

[0264] (25)

[0265] 14 + 1 (1.5) -1 4- — 4- 4- 4- 3.9 ± 0.3 2

[0266] (25)

[0267]

[0268] HIRO / 105 / PC 28 15 -1 (0.5) + 1 + + + + + 4.9 ± 0.1 5

[0269] (150)

[0270] 16 + 1 (1.5) + 1 + + + + + 5.0 ± 0.0 5

[0271] (150)

[0272]

[0273] Notes: +1 = high level; -1 = low level for each factor. Emergence index scored on 1-5 visual scale.

[0274] Variable definitions: A = alginate (0.5-1.5 wt %), B = CaCI2(25-150 mM), C = carbohydrate (5 % sucrose + 5 % sorbitol), D = nitrogen (yeast extract + peptone), E = chitosan (1 wt %), F = rice starch (1 wt %), G = buffer (50 mM PBS).

[0275] The fractional factorial design summarized above encompassed sixteen experimental runs that systematically explored the principal formulation variables governing fungal emergence and enzymatic activity. Each factor was evaluated at two coded levels (-1 and +1) representing the low and high ranges described above, while the design matrix was generated using a resolution IV fractional factorial arrangement to identify main effects and two-factor interactions with minimal experimental redundancy. The resulting sixteen combinations covered the practical compositional space for alginate concentration (0.5-1.5 wt %), calcium-ion concentration (25-150 mM), nutrient inclusion, and polymeric modifiers under otherwise constant processing conditions.

[0276] The morphology index reported above in Table 3 provides a comparative measure of bead uniformity, surface continuity, and mechanical robustness across the design space. Bead morphology improved markedly with increasing alginate and calcium-chloride concentrations, reflecting formation of a denser ionic network and enhanced structural cohesion. The inclusion of chitosan and rice starch further stabilized the hydrogel shell, producing smoother, more spherical particles with reduced surface cracking. Formulations containing both structural additives and buffered conditions (Runs 12, 15, and 16) achieved morphology scores of 5, corresponding to highly uniform beads that retained integrity during drying and rehydration. In contrast, low-alginate, low-calcium systems lacking additives (Runs 1 and 13) produced irregular or collapsed beads with fragile shells, confirming the dependence of physical quality on polymer concentration and cross-linker strength.

[0277] Analysis of variance and standardized-effect calculations revealed that alginate concentration (Factor A) and calcium-chloride concentration (Factor B) exerted the strongest influence on mechanical integrity and fungal emergence, accounting for approximately 40 % of total model variance. Nutrient factors, specifically the combined carbohydrate system (Factor C) and nitrogen source (Factor D), produced the largest positive contributions to enzymatic activation, confirming their essential role in metabolic re-initiation following hydration. The secondary polymeric additives, chitosan (E) and rice starch (F), displayed a significant positive interaction term (E x F), indicating synergistic reinforcement of the hydrogel network and enhanced water retention. The buffering factor (G) alone had a modest main effect but interacted favorably with calcium concentration (B x G), consistent with the observed dependence of gel uniformity on pH control during prolonged curing.

[0278] Visual scoring of emergence across all sixteen runs yielded values ranging from 1 to 5, as shown in Table 3. Formulations at the high levels of alginate, calcium, nutrient additives, and both structural polymers (Runs 15 and 16) consistently achieved the highest emergence indices

[0279]

[0280] 5)

[0281] HIRO / 105 / PC 29 and strong ABTS colorimetric responses. Runs lacking either nutrient or structural components (e.g., Runs 1, 7, 13) displayed poor emergence (< 2) and brittle bead morphology. The response¬ surface contours derived from these data define an optimum domain centered near 1.0-1.5 wt % sodium alginate and 100-150 mM CaCI2with inclusion of sucrose + sorbitol, yeast extract + peptone, 1 wt % chitosan, 1 wt % rice starch, and 50 mM phosphate buffer. This design thus established the key compositional interactions that yield reproducible, mechanically stable, and biologically active encapsulated fungal formulations. Parallel validation runs using strain A2 confirmed consistent bead morphology and emergence trends under the high-nutrient, high- cross-linker conditions of Runs 15-16.

[0282] Table 4. Representative Formulations and Results from Design-of- Experiments Optimization Parameter Run 1 Run 8 Run 12 Run 15 Run 16 Run 20 Run (Low (Mid- (High (Optimal (Optimal (Intermedia 24 Baseline) Performin Ca / Formulati Formulati te) (Low- g) Modera on 1) on 2) Nutrie te nt Nutrient Contro s) I) Alginate 0.5 wt % 1.0 wt % 1.5 wt 1.5 wt % 1.5 wt % 1.0 wt % 0.5 wt (A) % % CaCI2(B) 25 mM 25 mM 150 mM 150 mM 150 mM 100 mM 150 mM Carbohydr Absent Present Present Present Present Absent Absen ate (C) (5 % t sucrose

[0283] + 5 %

[0284] sorbitol)

[0285] Nitrogen Absent Present Present Present Present Absent Absen (D) (YE + t PP)

[0286] Chitosan 0 wt % 1 wt % 1 wt % 1 wt % 1 wt % 0 wt % 0 wt % (E)

[0287] Rice 0 wt % 0 wt % 1 wt % 1 wt % 1 wt % 0 wt % 0 wt % Starch (F)

[0288] Buffer (G) None 50 mM 50 mM 50 mM 50 mM None None PBS PBS PBS PBS

[0289] Curing 1-2 h 1-2 h 1-2 h 1-2 h 1-2 h 1-2 h 1-2 h Time

[0290] Funga! Al A1 A1 A1 A1 A2 A2 Species

[0291]

[0292] HIRO / 105 / PC 30 Emergence 1.5 ± 0.3 4.8 ± 0.2 4.7 ± 4.9 ± 0.1 5.0 ± 0.0 3.0 ± 0.3 1.8 ± Index (1-5) 0.2 0.2 Morpholog 1 4 5 5 5 3 2 y Quality

[0293] (1-5)

[0294] ABTS 1 4 4 5 5 3 1 Activity (1- 5)

[0295] Qualitative Fragile Moderate Dense Uniform Equivalen Moderate Fragile Observatio beads; structure; shell; beads; t to Run swelling;

[0296] n poor partial strong maximal 15 uneven nutrien emergen growth activati emergen growth t- ce on ce and limited enzyme

[0297] activity

[0298]

[0299] • Example 4 — Nitrogen Source Screening

[0300] A focused 22screening study was conducted to compare three nitrogen sources against a nitrogen-free control under standardized gelation and curing conditions. All formulations used 1% (w / v) sodium alginate, 100 mM CaCI2as the cross-linker, and a standard cross-linking time of 1- 2 hours, selected to enable direct comparison with prior studies. Four nitrogen conditions were prepared: N-1 (None; 0% w / v), N-2 (Hydrolyzed Protein; 2% w / v), N-3 (Organic Fertilizer; 5% w / v), and N-4 (Potato Peptone; 0.2% w / v). As per the SOP for this series, pre-culture mixes were made at 2* concentration (to be diluted 1:1 upon inoculation), and included the shared mineral set (MgSO4-7H2O 0.10%, K2HPO40.10%, FeSO47H2O 0.002%, KCI 0.04%), rice starch (2%), and PEG-400 (4% added post-sterilization) prior to mixing with an equal volume of blended liquid culture, extrusion, and drying at 130 °F for 24 h.

[0301] During manufacture, N-3 (Organic Fertilizer 5% w / v) caused gelation during sterilization and could not be extruded, consistent with prior observations that fertilizer minerals (e.g., calcium and other multivalent ions) can trigger premature alginate cross-linking. By contrast, N-2 and N-4 produced rounder beads that retained shape after dehydration relative to N-1 (control), which tended to dry flatter and adhere more strongly to silicone mats; upon hydration, all bead sets re-swelled satisfactorily.

[0302] Quantitative emergence scoring confirmed high viability across all nitrogen conditions tested (Table 5). The hydrolyzed-protein formulation (N-2) yielded the strongest combination of emergence and ABTS activity, consistent with the presence of short-chain peptides supporting early enzymatic activation. The potato-peptone system (N-4) produced slightly slower emergence but comparable re-swelling behavior. The nitrogen-free control (N-1) remained viable but showed reduced enzyme intensity, indicating that endogenous reserves can sustain early germination but not maximal oxidative performance.

[0303] HIRO / 105 / PC 31 Table 5. Performance of Nitrogen-Source Formulations Under Standardized External Gelation Conditions

[0304] Parameter N-1 (Control) N-2 (Hydrolyzed N-3 (Organic N-4 (Potato Protein) Fertilizer) Peptone) Nitrogen None Hydrolyzed Organic Potato Peptone Formulation Protein Fertilizer

[0305] Target Cone. 0 % 2 % 5 % 0.2 %

[0306] (final, w / v)

[0307] Pre0 % 4 % 10 % 0.4 % Sterilization

[0308] Load (2x)

[0309] Fixed Alginate 1 % (w / v); Same as N-1 Same as N-1 Same as N-1 Parameters CaCI2100 mM;

[0310] cross-link 1-2 h; rice

[0311] starch 2 %; PEG- 4004 % (poststerilization)

[0312] Manufacture Beads formed; dried Round beads; Internal Round beads;

[0313] Status / flatter; adhered to retained shape gelation during retained shape Notes mat; rehydrated well after drying; sterilization; after drying;

[0314] rehydrated well not extruded rehydrated well Emergence 5 4-5 n / a 3 — 4

[0315] Index (1-5)

[0316] ABTS Activity 3-— 4 4-5 n / a 2-3

[0317] (1-5)

[0318] Swelling 4 4 n / a 4

[0319] Index (1-5)

[0320] Morphology 3 4 n / a 4

[0321] Quality (1-5)

[0322]

[0323] Morphology Quality scale (1-5): 1 = irregular / fragile; 3 = mostly round with minor roughness (N-1, flatter); 4 = round, smooth, robust (N-2 / N-4); 5 = highly uniform, defect-free.

[0324] Rationale: N-2 and N-4 showed superior roundness and shape retention after drying; N-1 beads were serviceable but visibly flatter and more adhesive to the mat, hence the lower morphology score.

[0325] Example 5 — Curing-Time Optimization

[0326] A time-course study was performed to investigate the effect of cross-linking duration on bead morphology and shell formation using the nitrogen-free control formulation (N-1 baseline). All formulations contained 1 % (w / v) sodium alginate and were cured in 100 mM calcium chloride for varying durations to evaluate progressive ion diffusion and gel-network densification. The

[0327] HIRO / 105 / PC 32 objective of this series was to correlate visual opaqueness and tactile hardness of the dried beads with the effective shell thickness produced by different curing times. The six curing intervals tested were 5 minutes (T-1), 20 minutes (T-2), 1 hour (T-3), 2 hours (T-4), 3.5 hours (T-5), and overnight ~ 19 hours (T-6). A “jar-swapping” method was employed, in which aliquots of a single extruded batch were transferred sequentially to fresh CaCI2baths to minimize ion depletion over time. The beads were extruded using aforementioned parameters, rinsed after each time point, and dried at 130 °F (~ 55 °C) for 24 hours. Beads cured for shorter durations were translucent and soft, while those cured for longer times appeared progressively more opaque and rigid. The opaqueness served as a qualitative proxy for increased shell density. After rehydration, all bead sets swelled to comparable sizes, confirming that diffusion-limited curing primarily altered outershell composition rather than total water uptake.

[0328] Table 6. Effect of Cross-Linking Time on Bead Morphology and Fungal Emergence Parameter T-1 (5 min) T-2 (20 min) T-3 (1 h) T-4 (2 h) T-5 (3.5 T-6 19 h) h) Cross-link 5 min 20 min 1 h 2 h 3.5 h ~ 19 h time

[0329] Alginate (% 1 1 1 1 1 1 w / v)

[0330] CaCI2(mM) 100 100 100 100 100 100 Nitrogen None (N-1 Same Same Same Same Same source control)

[0331] Appearance Translucent; Slightly Opaque; Fully Dull Chalky (dry) soft edges opaque; firm opaque; white; white;

[0332] pliable rigid brittle dense surface Relative Very low Low Moderate High Very high Maximum shell

[0333] density

[0334] Swelling 4 4 4 4 4 3 Index (1-5)

[0335] Emergence 5 5 5 5 4-5 3 — 4 Index (1-5)

[0336] ABTS 3 3 4 4 4 3 Activity (1- 5)

[0337] Morphology 3 4 4 5 5 4 Quality (1- 5)

[0338]

[0339] HIRO / 105 / PC 33 Remarks Softest Intermediate; Typical Balanced Standard Overbeads; flexible shell working hardness lab cured; minimal cure; condition; diffusionopacity smooth firm shell limited surface core

[0340]

[0341] Quantitative scoring of emergence and enzymatic activity confirmed that fungai viability remained high across all curing times, with no significant inhibition up to 3.5 hours. Slightly reduced emergence and enzyme coloration after - 19 hours (T-6) indicate diffusion-limited nutrient access in the most heavily cross-linked beads. Morphology improved progressively from T-1 to T-5, plateauing near T-4 - T-5 where shell smoothness and mechanical rigidity were optimal. These results corroborate the trend that increasing curing duration enhances surface density while marginally reducing metabolic activation at very long times.

[0342] In certain embodiments, cross-linking time is adjusted according to the target application. Shorter curing (< 20 min) may be used where rapid hydration and emergence are desired, whereas extended curing (1-3 h) provides denser shells for prolonged storage stability. Very long curing (> 12 h) can produce diffusion-limited cores with reduced metabolic activation but improved mechanical strength. Such control over cross-linking duration allows tuning of activation kinetics and bead hardness across multiple product formats.

[0343] The results of the cross-linking-time series confirm that bead morphology and shell density can be finely tuned by adjusting curing duration under otherwise identical conditions. Shorter cross¬ linking periods produce translucent, soft shells suited for rapid hydration and fungal emergence, whereas extended curing yields opaque, rigid beads that favor long-term storage and mechanical stability.

[0344] These findings establish curing time as an independent process variable that may be modulated alongside composition and nutrient content to balance physical strength, rehydration rate, and biological activation. The optimized 1-2-hour window defined in Example 5 represents a suitable standard condition for subsequent experiments, including viability testing, trace-metal cofactor incorporation, and composite-material integration. The following examples build upon these results to evaluate storage stability, enzymatic activation, and application performance of the encapsulated formulations under representative conditions.

[0345] • Example 6 — Viability After Drying and Storage

[0346] Example 6 is a long-term stability assessment study using optimized encapsulated fungal beads corresponding to the composition identified in Example 3 (1.0 wt % sodium alginate, 100 mM calcium chloride, 5 wt % sucrose + 5 wt % sorbitol, yeast extract 0.2 wt %, peptone 0.1 wt %, 1 wt % chitosan, 1 wt % rice starch, and 50 mM phosphate buffer). The objective of this study was to evaluate the effects of drying method and storage duration on bead integrity, rehydration behavior, and fungal viability under ambient storage conditions.

[0347] Beads were prepared by external gelation in 100 mM CaCI2with a 1.5-hour curing time, rinsed in sterile phosphate buffer, and divided into two drying treatment groups:

[0348] HIRO / 105 / PC 34 (1) Convection-dried at 50-55 °C for 24 hours under controlled airflow, and

[0349] (2) Freeze-dried using a shelf temperature of -40 °C and gradual vacuum desorption to a final pressure of < 0.1 mbar.

[0350] Both sets were stored in sealed polypropylene containers at ambient laboratory temperature (20-25 °C) and moderate humidity (~40-50 % RH). Beads were periodically sampled at 1, 3, 6, and 10 months to assess physical appearance, moisture content, and fungal viability.

[0351] Upon rehydration in sterile water or placement on 2 % malt extract agar (MEA) plates, both drying methods produced rapid swelling and reactivation. The convection-dried beads restored to approximately 90-95 % of their original hydrated diameter within two hours, corresponding to a swelling index of 4-5. Freeze-dried beads exhibited slightly faster water uptake and marginally higher porosity, producing more diffuse colony edges during initial outgrowth. Mechanical testing by gentle compression indicated that both bead types retained shell integrity and elasticity comparable to freshly prepared controls, demonstrating minimal brittleness after extended storage.

[0352] Quantitative emergence assays conducted on MEA plates after 10 months of storage showed emergence indices equivalent to 85-90 % of fresh controls within 10 days of incubation, with visible mycelial coverage across the majority of bead surfaces. ABTS oxidation tests confirmed retention of oxidative enzyme activity, showing characteristic green-blue coloration similar to that of freshly produced beads. No bacterial or contaminant growth was observed, confirming sterility of the stored samples.

[0353] These results demonstrate that the optimized nutrient-amended alginate encapsulates maintain high viability and functional activity following both thermal and freeze-drying processes. Storage stability exceeding 10 months under ambient conditions establishes that the encapsulated fungal propagules remain metabolically competent and structurally protected by the alginate-chitosan- starch composite matrix. The data further indicates that controlled dehydration does not significantly impair fungal emergence or enzymatic function, validating the suitability of this encapsulation format for shelf-stable biological products and downstream composite applications.

[0354] * Example 7 — Transition-Metal Cofactor incorporation and ion Exchange Enhancement

[0355] In a further embodiment, the nutrient-amended alginate formulations described previously were modified to include trace levels of transition-metal ions known to function as cofactors in fungal oxidative enzymes. Incorporation of iron, manganese, or copper ions within the calcium-alginate network is expected to enhance redox enzyme performance and stabilize reactive intermediates by providing localized catalytic centers. Without intending to be bound by theory, it is understood that such ions will partially exchange with calcium ions coordinated to the guluronate blocks of alginate, forming mixed-metal domains that support electron transfer in laccase- and peroxidase-type enzymes.

[0356] Representative formulations were prepared according to the optimized nutrient-amended composition from Example 3 (1.0 wt % sodium alginate, 100 mM CaCI2, 5 wt % sucrose, 5 wt % sorbitol, 0.2 wt % yeast extract, 0.5 wt % malt extract, 0.1 wt % peptone, 1 wt % chitosan, 1 wt % rice starch, and 50 mM phosphate buffer). After standard curing and drying, aliquots of the dried beads were immersed for two minutes in aqueous metal-salt solutions containing FeSO4-7H2O,

[0357] HIRO / 105 / PC 35 MnSO4H2O, or CuSO4-5H2O at concentrations between 0.001 and 0.02 wt %. Excess solution was drained, and the beads were air-dried under mild airflow.

[0358] In prior experiments, it was observed that addition of unchelated, pure metal salts to the alginate bath resulted in pre-gelation due to interactions of metal ions (Fe, Mn, Cu) with the alginate structure. All metal-treated samples using ion exchange exhibited improved handling and completed eliminated tendency toward pre-gelation during preparation compared with untreated controls and prior pre-gelation tests. Bead surfaces remained smooth and non-tacky, and no significant changes to cross-linking or viscosity increase were observed during sterilization, indicating that the trace-ion soak moderated the ionic activity of the calcium-alginate mixture. The resulting beads retained uniform size distribution and opacity comparable to the baseline calciumalginate formulation, confirming that low-level ion exchange did not compromise gel integrity. Studies indicate that Fe2+, Mn2+; and Cu2+act as cofactors for fungal laccases, peroxidases, and oxidases, suggesting that such supplementation will enhance oxidative activity and electron¬ transfer efficiency upon activation.

[0359] Table 7. Transition-Metal Ion Supplementation in Nutrient-Amended Alginate Formulations Parameter M-1 M-2 (Fe2+M-3 (Mn2+M-4 (Cu2+M-5

[0360] (Control) Supplement) Supplement) Supplement) (Combined Fe / Mn / Cu) Metal Salt None FeSO4-7H2O MnSO4H2O CuSO4-5H2O FeSO4-7H2O Source +

[0361] MnSO4H2O +

[0362] CuSO4-5H2O Total Metal 0 0.005 % 0.005 % 0.005 % 0.015 % Cone, (w / w of (total) bead)

[0363] Incorporation None Post-curing Post-curing Post-curing Sequential Method soak in metal soak in metal soak in metal soak (each 2 solution solution solution min) Alginate / 1.0 wt % 1.0 wt % Na- 1.0 wt % Na- 1.0 wt % Na- 1.0 wt % Na- CaCI2Base Na- alginate; 100 alginate; 100 alginate; 100 alginate; 100 alginate; mM CaCI2mM CaCI2mM CaCI2mM CaCI2100 mM

[0364] CaCI2

[0365] Observed Standard Completely Completely Completely Completely Gelation gelation; eliminated eliminated eliminated eliminated Behavior no pre-gelation; pre-gelation; pre-gelation; pre-gelation;

[0366] additive smoother smoother smoother smoother bead surface bead surface bead surface bead surface

[0367]

[0368] HIRO / 105 / PC 36 Predicted Ca2+-only Partial Fe2+«-> Partial Mn2+Partial Cu2+<->■ Mixed-metal Ion-Exchange network Ca2+<-> Ca2+Ca2+coordination Behavior exchange exchange exchange domains Emergence 4 — (expected — (expected — (expected — (expected Index (1-5) unchanged) unchanged) unchanged) unchanged) Remarks Beads Slightly darker Slightly darker Slightly bluish Uniform remained tint; no pretint; no pretint; no preappearance; intact gelation gelation gelation no pregelation

[0369]

[0370] Note: All effects are anticipated based on established catalytic roles of Fe, Mn, and Cu in fungal oxidative enzymes and observed improvements in gelation behavior.

[0371] In certain embodiments, the ion-exchange step may be performed during or after gelation, using either soluble salts or chelated complexes that gradually release transition-metal ions. The extent of exchange can be adjusted by varying the ion concentration or exposure time. Other transition¬ metal ions such as Co2+, Zn2+, Ni2+, or Mo6+may also be employed to tune enzymatic specificity or stability. Such embodiments broaden the applicability of the compositions described herein to diverse enzyme-activated or redox-responsive biodegradable systems.

[0372] Collectively, these findings indicate that controlled introduction of transition-metal ions into the alginate network provides an additional tuning mechanism for redox activity and gel behavior, enabling embodiments in which the encapsulated fungi or enzyme systems participate in accelerated oxidative degradation, selective substrate modification, or redox-triggered activation in composite materials. Such metal-supplemented hydrogel constructs may therefore be incorporated into biodegradable plastics, enzymatically active coatings, soil-amendment granules, wastewater biofilters, or other applications in which enhanced oxidative capacity, prolonged catalytic stability, or tailored ion-exchange properties provide functional advantages beyond those obtainable with calcium-alginate matrices alone.

[0373] • Example 8 — Composite and Coating Applications (Figure 9)

[0374] Building upon the optimized encapsulated fungal formulations developed in Examples 1-5, the next series of embodiments envisions the incorporation of these hydrogel beads or films into composite matrices and coated substrates to create functional, moisture-activated biodegradable materials. These embodiments are designed to translate laboratory-scale hydrogel systems into product forms suitable for absorbent articles, packaging, or surface coatings. Based on prior observations that the dried beads retain structure, viability, and rapid reactivation upon hydration, such systems are expected to enable controlled activation of the fungal inoculum when the surrounding matrix becomes moist.

[0375] In a representative embodiment, the dried nutrient-amended beads described in Example 3 (1.0 wt % alginate, 100 mM CaCI2, sucrose + sorbitol, yeast extract, peptone, chitosan, rice starch) are blended at 5-20 wt % loading into a cellulose-based or starch-filled biopolymer matrix. The composite mixture may be formed by solution casting, extrusion, or compression molding to produce sheets, films, or porous cores. Upon exposure to moisture, the encapsulated hydrogel beads swell and create localized microenvironments conducive to fungal emergence and

[0376] HIRO / 105 / PC 37 enzymatic oxidation. The process thereby enables moisture-triggered biodegradation or odor¬ neutralization functions in biodegradable consumer products.

[0377] In another embodiment, the alginate formulation is applied as a surface coating. A 1 % sodium¬ alginate pre-gel mixture containing the fungal inoculum is cast or sprayed onto a cellulose or fiber substrate and subsequently ion-cross-linked using a mist or vapor of CaCI2or other divalent-ion solutions. The resulting thin hydrogel layer (typically 50-200 pm) forms a continuous, adherent coating that retains fungal viability while remaining permeable to oxygen and moisture. Such coatings may be applied to paperboard, fiber pads, or molded bioplastic surfaces to create active films that initiate fungal growth upon hydration.

[0378] In further embodiments, the composite or coated substrates may include trace transition-metal cofactors (as described in Example 8) to sustain enzymatic activity and improve oxidative degradation of organic residues. The combination of nutrient-amended beads and structural polymer substrates provides both mechanical robustness and biochemical functionality. It is expected that these hybrid materials will demonstrate accelerated moisture-dependent breakdown compared with unmodified biopolymer controls.

[0379] Parameter C-1 (Bead- C-2 (Bead- C-3 (Film C-4 (Biopolymer Cellulose Starch Coating on Laminate) Composite) Composite) Fiber

[0380] Substrate)

[0381] Matrix Cellulose fiber + Corn starch + Cellulose sheet PLA / PBAT blend Material PVA binder glycerol or molded fiber (bioplastic)

[0382] plasticizer

[0383] Bead Loading 10 15 Surface coating 5

[0384] (wt %) (approx. 0.2 mg

[0385] cm~2)

[0386] Cross-Linking CaCI2(100 mM) CaCI2(100 mM) CaCI2mist or CaCI2(in situ) System vapor

[0387] Additives PEG-400 (2 %), PEG-400 (2 %), Chitosan topTrace Fe / Mn / Cu rice starch (1 %), malt extract (0.5 coat (0.5 %) (0.01 wt % total) sucrose (5 %) %)

[0388] Processing Blending + hot Solution casting Spray or dipLayer-by-layer Method pressing + drying coating + lamination cross-linking

[0389] Expected Moisture- Rapid fungal Surface fungal Active film with Functionality activated activation; high growth and slow release of degradation; absorbency oxidation enzymes structural core

[0390]

[0391] Samples of each of Forms C-1 through C-4 were prepared successfully, thus establishing viability of incorporating the alginate / fungi formulations into the various form factors. Schematics of each

[0392] HIRO / 105 / PC 38 form are visualized in Figures 1-3. Emergence and ABTS activity is expected to be high quality based in part on the earlier Examples.

[0393] The compositions and processes described herein may be varied in numerous ways without departing from the scope of the invention. Any of the ingredients, concentration ranges, or manufacturing parameters disclosed in the foregoing examples may be substituted, adjusted, or combined to achieve equivalent technical effects appropriate for a given fungal species, substrate, or end-use application. The alginate concentration, crosslinking ion identity, curing time, and additive composition may be selected to balance mechanical strength, nutrient availability, and activation timing. In certain embodiments, other biopolymers such as pectin, carrageenan, xanthan, gellan gum, or cellulose derivatives may partially or completely replace alginate to achieve distinct textural or degradation profiles. Likewise, alternative divalent or trivalent cations — such as magnesium, zinc, strontium, or aluminum — may be used as partial substitutes for calcium to tailor gelation kinetics and ionic-exchange behavior.

[0394] The encapsulated inocula may incorporate a wide range of fungal species capable of secreting oxidative or hydrolytic enzymes, including Pleurotus, Ganoderma, Lentinula, Trametes versicolor, or Phanerochaete species. Mixed cultures or co-encapsulated bacterial symbionts may be employed to establish synergistic biodegradation or nutrient-cycling interactions. The nutrient and additive systems may likewise be customized to favor specific enzymatic pathways — such as laccase-dominant oxidation for lignin or aromatic polymer breakdown, peroxidase activity for cellulose oxidation, or hydrolase expression for polyester depolymerization.

[0395] The encapsulated compositions can be manufactured as free-flowing beads, pellets, or granules for direct use as soil inoculants or compost accelerators, or as coatings and inserts within biodegradable consumer products. In one embodiment, the beads are blended with absorbent materials such as cellulose fibers, superabsorbent polymers, or starch-based fillers to form hybrid absorbent cores that retain fluid while initiating biological degradation upon disposal. In another embodiment, the alginate-fungal hydrogel is cast or printed as a coating on packaging films, molded articles, or fibrous substrates to provide controlled biodegradation or odor mitigation in waste streams. The encapsulated fungi may also be used in environmental bioremediation, such as in hydrocarbon-contaminated soils or wastewater, where the nutrient-balanced matrix ensures immediate metabolic activation following hydration.

[0396] The invention further encompasses scalable production methods suitable for continuous manufacturing. Droplet generators, vibrating-nozzle encapsulators, or microfluidic systems can be adapted to produce uniform beads or filaments with controlled diameters. Continuous belt casting or 3D printing may be used to create patterned hydrogel films or coatings containing encapsulated inocula. Drying and packaging steps may be conducted under controlled humidity to ensure shelf stability. The compositions can be designed to remain inactive during product use, activating only upon exposure to specific moisture or pH conditions, thus ensuring predictable biodegradation timing.

[0397] Because the compositions rely on biodegradable and biocompatible materials, they are suitable for integration into sustainable product platforms where environmental degradation is desirable after use. Applications include absorbent hygiene articles, paper or fiber-based packaging, filtration media, mulch films, soil conditioners, and controlled-release agricultural carriers. Upon contact with moisture, the hydrogel matrix swells, releasing the encapsulated fungi and initiating enzymatic oxidation and depolymerization of the surrounding organic or polymeric substrates.

[0398] HIRO / 105 / PC 39 The result is an environmentally benign degradation pathway that reduces landfill persistence and supports composting or nutrient recovery.

[0399] FIG. 1 is a schematic representation of a composite encapsulation material according to one embodiment of the invention. The composite hydrogel structure 38 comprises a dispersed phase of alginate beads 40 containing biological inclusions 42 embedded within a continuous cellulose or starch-based matrix 44. The alginate beads provide a localized microenvironment for immobilized organisms, enzymes, or spores while maintaining hydration and protection from environmental stress. The surrounding matrix supports mechanical stability and diffusion of moisture and nutrients, permitting limited exchange through the matrix interface region 46 while preserving separation of the encapsulated inclusions. This configuration enables long-term viability and controlled activation of biological agents during exposure to moisture or degradation conditions, thereby maintaining functional stasis until triggered for activity.

[0400] FIG. 2 is a schematic illustration showing a representative process for forming a thin alginate hydrogel layer containing immobilized fungal inclusions on a cellulose or molded-fiber substrate. A sodium-alginate pre-gel mixture 48 containing a biological inoculum 42 is applied as a fine spray using an application device 74 onto the surface of the substrate 38. A separate spray or vapor of a divalent-ion solution such as calcium chloride 70 is then introduced to crosslink the alginate, producing a cohesive hydrogel layer 72 that encapsulates and stabilizes the fungal material 42. The directional indicator 76 denotes the sequential transition from liquid pre-gel deposition to gelation and formation of the hydrogel coating. The resulting layer maintains the encapsulated fungal inclusions in a hydrated yet dormant state, allowing later reactivation under environmental moisture and temperature conditions.

[0401] FIG. 3 is a schematic representation of a multi-layer composite illustrating the structural arrangement of functional alginate-fungal layers within biopolymer sheets. The assembly comprises a bottom biopolymer layer 78 that provides mechanical strength and moisture regulation, over which a central functional alginate-fungal layer 80 is disposed. The central layer 80 contains immobilized fungal inclusions 42 encapsulated within an ion-crosslinked alginate hydrogel matrix derived from the hydrogel layer 72 formed according to the process of Figure 2. An optional intermediate layer 82 of biopolymer or similar material may be positioned between successive alginate-fungal layers to adjust hydration dynamics or diffusion rates. A second functional alginate-fungal layer 84 may then be applied above the intermediate layer, and the structure is completed with a top biopolymer layer 86 that seals the composite and protects the encapsulated components from desiccation or premature activation. The multi-layer arrangement enables localized biological activity within discrete zones while maintaining overall structural integrity and controllable release of enzymatic or microbial functions upon environmental exposure.

[0402] (2) Embedding and Material integration of Fungal Forms

[0403]

[0404] The compositions described herein comprise one or more of the following components:

[0405] 1. Encapsulating hydrogel phase.

[0406] o Sodium alginate at concentrations between about 0.5 % and 2 % w / v.

[0407] HIRO / 105 / PC 40 o Calcium chloride or calcium carbonate at 25 - 150 mM as the cross-linking agent.

[0408] o Optional structural or rheology modifiers such as chitosan (0.5 - 2 % w / v), rice starch (0.5-2 % w / v), or polyethylene glycol (2 -5 % v / v).

[0409] 2. Nutrient and osmoprotectant system.

[0410] o Carbohydrates and polyols such as sucrose, sorbitol, trehalose, or maltose at 2 - 10 % w / v.

[0411] o Nitrogen and mineral sources such as yeast extract, potato peptone, magnesium sulfate, potassium chloride, and phosphate salts (0.01 - 1 % w / v total).

[0412] 3. Fungal inoculum.

[0413] o At least one strain selected from Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, Rhizopus, or functional equivalents exhibiting ligninolytic or oxidative enzyme activity.

[0414] o Spore loading typically ranges from 104- 108spores mL“1of encapsulation mixture but is not limited to that range.

[0415] 4. Embedding polymer phase.

[0416] o Thermoplastic polymer selected from biodegradable or recyclable polyesters (PLA, PCL, PHA, PBS, PBAT, PET, PTT), polyolefins (PE, PP), or blends thereof. o Processing temperatures maintained between 80°C and 300°C, with residence times under 5 minutes to preserve inoculum viability.

[0417] 5. Optional coatings or compatibilizers.

[0418] o Trehalose, starch, dextran, or mannitol coatings on spores to enhance heat resistance.

[0419] o Surfactants, dispersants, or coupling agents that promote uniform distribution of inclusions within the host polymer.

[0420] Representative compositions may be formed as free alginate beads, coated spore powders, composite filaments, cast films, spunbond or meltblown fibers, or bonded nonwoven fabrics containing distributed viable inocula. The formulation parameters can be tuned to adjust bead integrity, polymer compatibility, or moisture-triggered activation.

[0421] The compositions and articles of the present disclosure may be produced by polymer-processing equipment capable of operating at temperatures up to 300 °C. Fungal viability is maintained by controlling the actual temperature experienced by the inoculum (the inoculum contact temperature) within a defined window and limiting the residence time in the molten or softened polymer. The methods are applicable to both native and protected spores as well as encapsulated inclusions.

[0422] HIRO / 105 / PC 41 A. General Method for Incorporating Fungal Inoculum Into Polymeric Materials

[0423] 1. Preparation of inoculum: the fungal inoculum, which may be unprotected, sugar-coated, or encapsulated, is dried to <10 % moisture. Optional coatings (trehalose, sorbitol, starch, alginate) or microbeads may be used for additional heat stability.

[0424] 2. Polymer softening or melting: the host polymer is processed in standard extrusion, spinning, or molding equipment with setpoint temperatures between 80 °C and 300 °C. The inoculum is introduced at a location where the inoculum contact temperature is maintained between 80°C and 300°C, preferably 9 -160°C, and for short residence times of <5 minutes, preferably <2 minutes.

[0425] 3. Incorporation of inoculum: the inoculum is blended into the molten or softened polymer under low to moderate shear (40-80 rpm screw speed or 100-500 s~1shear rate). Side¬ feeders, vent ports, or downstream injection points are used when processing high- setpoint polymers so that the inoculum enters cooler regions of the melt.

[0426] 4. Formation of articles: the mixture is shaped into filaments, fibers, films, or molded parts using extrusion, film casting, melt-spinning, or compression molding. Examples include:

[0427] o Filaments and fibers produced by melt-spinning or drawing with downstream inoculum injection.

[0428] o Films and sheets produced by casting or lamination; surface temperature at the inoculum plane <160°C.

[0429] o Nonwoven webs produced by spunbond or meltblown methods where inoculum is added in-line or sprayed between die and collector.

[0430] o Adhesive or coating layers produced at <200°C using PEG, PVA, or starch bases.

[0431] 5. Cooling and solidification: the formed article is cooled at a rate >5 °C s-1by ambient or forced air to minimize total thermal dose. Cooling zones or chilled rollers may be used for high-throughput processes.

[0432] 6. Quality control: fragments of the resulting polymer are rehydrated and plated on nutrient agar. Visible colony growth within 2-5 days confirms viability. Viability at polymer- equipment setpoints up to 300 °C demonstrates that controlled dwell and downstream inoculum injection effectively shield the spores from lethal exposure.

[0433] B. Manufacture Using Alginate-Encapsulated Spores or Beads

[0434] Encapsulated spores according to the disclosure are produced by cross-linking alginate solutions containing spores in CaCI2(25-150 mM) to form beads 0.5-3 mm in diameter. Dried beads (<10 % moisture) are metered into molten polymers such as PLA, PCL, PHA, PET, or PBS. The polymer may be processed at equipment setpoints up to 300 °C, while maintaining the bead contact temperature <180 °C by adjusting feed location, throughput, and air cooling. The resulting composites retain bead integrity and spore viability after cooling.

[0435] C. Manufacture Using Coated or Native Spores

[0436] HIRO / 105 / PC 42 Native spores or sugar-coated spores may be blended directly into polymers processed at up to 300 °C, provided that the inoculum experiences transient exposure <180 °C. Controlled feed location, short dwell time, and rapid cooling preserve viability. The resulting polymers contain distributed dormant spores that activate upon hydration or composting.

[0437] D. integration into Fibrous and Nonwoven Structures

[0438] Spores or inclusions may be incorporated before, during, or after fiber formation:

[0439] • Pre-spun blending into pellets prior to extrusion at 80-300 °C setpoints.

[0440] • In-process aerosol injection into the fiber stream; contact temperature typically 100-140 °C.

[0441] • Post-bonding addition followed by low- temperature lamination or adhesive fixation.

[0442] These methods yield nonwoven webs containing viable fungal inoculum with maintained tensile and porosity characteristics.

[0443] 5. Adhesive and Surface Applications

[0444] Adhesives or coatings are prepared at <100 °C with PEG, PVA, or starch matrices. Spores or beads are mixed into the adhesive and applied to surfaces or films. The dried layer retains dormant inoculum that reactivates when moistened.

[0445] 6. Representative Process Parameter Ranges

[0446] Parameter Typical Preferred Notes

[0447] Range Range

[0448] Equipment temperature 80-300°C — Applies to extruders, meltsetpoint spinners, laminators

[0449] Inoculum contact 80-200°C 90-160°C Controlled by feed position, air temperature cooling

[0450] Thermal exposure <5 min <2 min Measured from inoculum (residence) injection to quench

[0451] Shear rate 100-500 s’1Moderate shear to disperse without rupture

[0452] Cooling rate >5 °C s-1— Prevents cumulative thermal stress

[0453] Inoculum loading 0.1-10 wt — Uniformly distributed

[0454] %

[0455]

[0456] Bead diameter 0.5-3 mm 1-2 mm For encapsulated inclusions

[0457] Thermal profiling confirms that spores experience temperatures within the stated inoculum¬ contact window even when the equipment setpoint exceeds 200°C, enabling the use of conventional polymer and fiber-processing machinery without modification.

[0458] Embedding and Incorporation Test Methodology

[0459] The effectiveness of the compositions described herein was evaluated through a series of experiments designed to confirm fungal survival, emergence, and enzymatic activation following processing and storage. These tests demonstrate that the fungal inocula remain viable after

[0460] HIRO / 105 / PC 43 exposure to a wide range of polymer-processing conditions and can later resume metabolic activity when rehydrated or placed in a moist environment. The results provide strong evidence that the same inocula, once activated, are capable of initiating biological transformation or degradation of polymeric and cellulosic substrates under environmental or composting conditions. In the conducted trials, sections of extruded filaments, cast films, molded samples, and nonwoven webs were sterilized externally and placed on malt-extract agar (MEA) or other nutrient media. The samples were maintained at 25 to 30 °C for up to seven days and observed for visible fungal emergence. Growth from the material surface confirmed the survival of the embedded spores. Viability was observed in polymers including PLA, PCL, PHA, PET, and spunbond or meltblown composites. Representative experiments demonstrated survival of both unprotected and encapsulated spores after transient exposure to processing temperatures as high as 80 °C, and in certain cases under equipment setpoints up to 300 °C where the inoculum contact temperature remained below the fungal survival threshold. Comparative controls containing heat-killed inocula or un-inoculated polymers did not exhibit any growth.

[0461] Recovered colonies from viable samples were transferred to indicator media containing ABTS or guaiacol substrates to verify the secretion of oxidative enzymes. The formation of characteristic color halos confirmed active enzyme production, consistent with laccase and peroxidase activity. These enzymes are well recognized in the field as responsible for the degradation of lignin, polyesters, and related synthetic polymers. Accordingly, demonstration of survival and enzyme activity provides functional evidence that the incorporated fungi are capable of contributing to the biodegradation of host materials once activation occurs.

[0462] Samples exposed to high humidity or compost-like conditions (25-40 °C, >95 % relative humidity) exhibited visible emergence of fungal growth within several days, particularly at cut edges or surface imperfections. This confirms that the fungal inoculum remains dormant within the solid article during storage and processing and is activated only under environmental conditions favorable to growth. The ability to achieve controlled activation following processing at industrially relevant temperatures establishes the practical feasibility of incorporating viable fungal inocula directly into thermoplastic and fibrous materials.

[0463] Negative controls consisting of sterilized or un-inoculated materials showed no growth or enzyme activity, while positive controls of unprocessed spores produced rapid emergence within 48 hours. Across the range of tested formulations and polymers, survival rates of 30 to 80 percent were typical, with higher values observed for spores protected by trehalose or encapsulated in alginate microbeads. The results collectively show that fungal inocula can survive polymer manufacturing processes, remain stable during storage, and reactivate when exposed to moisture. Based on these observations and known fungal enzymatic pathways, it is reasonable to infer that the reactivated inocula can contribute to the biodegradation or transformation of the host polymer under composting or natural environmental conditions.

[0464] The inventive embodiments encompass a range of compositions, methods, and articles that incorporate fungal inocula into polymeric and absorbent materials while maintaining biological viability and enabling controlled activation. Preferred embodiments described below illustrate representative configurations, formulations, and process conditions that achieve the desired combination of manufacturability, viability, and post-use biological responsiveness.

[0465] In one preferred embodiment, the fungal inoculum is incorporated into a thermoplastic polymer matrix such as polylactic acid (PLA), polycaprolactone (PCL), polybutylene succinate (PBS), or

[0466] HIRO / 105 / PC 44 polyethylene terephthalate (PET) and its copolymers, including polytrimethylene terephthalate (PTT) and polyethylene terephthalate glycol (PETG). The thermoplastic polymer may be selected from biodegradable aliphatic polyesters including, for example, polylactic acid, polycaprolactone, and other renewable or fermentation-derived polyesters comprising repeat units such as hydroxyalkanoate, hydroxybutyrate, hydroxyvalerate, or related aliphatic hydroxyacid residues. The inoculum may consist of native spores, coated spores, or encapsulated inclusions. The polymer is processed by extrusion, melt spinning, injection molding, or compression molding using equipment with temperature setpoints between 80°C and 300°C, while maintaining the inoculum contact temperature below 200°C, and preferably between 90°C and 160°C. The molten polymer containing the inoculum is formed into filaments, films, or molded articles and rapidly cooled to preserve spore viability. This embodiment allows for the direct manufacture of fungal- embedded polymers, including both biodegradable and conventional thermoplastics, using standard equipment and conditions.

[0467] In another preferred embodiment, the fungal inoculum is encapsulated within a hydrogel bead or microcapsule before incorporation into the polymer. The encapsulation matrix typically contains sodium alginate (0.5-2 wt%) cross-linked with calcium chloride (25-150 mM) and may further include osmoprotective additives such as sucrose, sorbitol, trehalose, chitosan, rice starch, or polyethylene glycol to enhance structural integrity and moisture balance. Beads with diameters of 0.5-3 mm are dried at <55°C to reduce moisture content below 10 % and are subsequently mixed into the polymer melt, resin, or adhesive. The resulting composite retains bead morphology and fungal viability after processing, and the inclusions rehydrate upon environmental exposure to trigger controlled fungal emergence.

[0468] In a further embodiment, fungal spores coated with a protective saccharide or polyol layer, such as trehalose, sorbitol, or starch, are incorporated directly into molten polymers, including PLA, PCL, PHA, PBS, and PET or PETG. The protective coating acts as a transient thermal buffer and osmotic stabilizer, allowing survival at brief temperature exposures above 150 °C. The coated spores are uniformly distributed throughout the polymer and remain dormant during manufacturing and storage, reactivating upon contact with moisture, humidity, or composting environments. This embodiment is particularly suited for extrusion and fiber spinning applications where fine dispersion and consistent viability are required.

[0469] In another embodiment, fungal inocula are incorporated into fibrous or nonwoven materials using meltblown, spunbond, or wet-laid processes. Spores or encapsulated inclusions can be introduced into the polymer stream before extrusion, injected into the fiber plume between the die and collector, or deposited onto the web surface followed by low-temperature lamination or adhesive bonding. The resulting nonwoven structures retain mechanical strength and porosity while embedding viable inocula that can later colonize or degrade the fibers when activated by environmental moisture.

[0470] Additional embodiments include adhesive and coating systems that function as localized carriers for the fungal inoculum. These systems may contain hydrophilic binders such as polyethylene glycol (PEG), polyvinyl alcohol (PVA), or starch processed at <100 °C. Fungal spores or microbeads are suspended in the adhesive and applied as a coating, stripe, or spot pattern on a substrate. After drying, the inoculum remains dormant within the adhesive film and reactivates upon hydration to initiate localized biological growth or transformation.

[0471] HIRO / 105 / PC 45 Preferred fungal species for use in these embodiments include oxidative and ligninolytic fungi such as Aspergillus, Ganoderma, Lentinula, Trametes, and Phanerochaete, as well as functional equivalents exhibiting comparable enzyme systems. The inoculum concentration within the polymer or substrate may range from 0.1 to 10wt%, depending on desired activity and distribution. In certain embodiments, the embedded or encapsulated fungi are combined with nutrients, cofactors, or mineral additives, such as yeast extract, peptone, magnesium sulfate, potassium phosphate, or iron salts, to promote reactivation and enzyme expression after exposure to moisture. In other embodiments, the composition is free of calcium sulfate or other additives that may cause internal gelation during sterilization or drying.

[0472] The compositions can be fabricated into a variety of articles including filaments, films, molded parts, multilayer laminates, and absorbent cores. These embodiments cover both biodegradable and recyclable polymers, as well as durable plastics such as PET, so long as the inoculum contact temperature during processing remains within the fungal survival threshold. The resulting materials are stable during production and use but biologically responsive after disposal, enabling controlled activation, enzymatic oxidation, or degradation when exposed to environmental or composting conditions.

[0473] Embedding and Incorporation Examples

[0474] • Example 9 - Filament Extrusion of Fungal Spores in Thermoplastic Polymers This example demonstrates that native fungal spores can remain viable after incorporation into molten polymers and extrusion into filaments under controlled thermoplastic processing conditions, provided that exposure temperature and residence time are maintained within a defined survivable range.

[0475] FIG. 4 is a schematic representation of a process for embedding fungal forms within a polymer matrix through extrusion or melt-processing techniques. A fungal inoculum 16 is introduced into polymer pellets or resin 14 within a mixing device or hopper 15 of an extrusion system. The combined material is conveyed through a heated screw assembly 17, where it becomes homogenized while maintaining fungal viability under controlled temperature and residence-time conditions. The molten composite is extruded to produce a polymer filament containing dispersed fungal inclusions 21. Upon later exposure to environmental moisture or controlled hydration 20, the encapsulated fungal forms rehydrate and initiate localized growth or enzymatic activity, as represented by surface colonization regions 23. The process provides a means of incorporating biologically active agents into thermoplastic or biodegradable polymers while preserving structural integrity and enabling controlled post-use activation.

[0476] Figure 4 shows, in sequence, blending of fungal spores with molten polymer pellets, extrusion of the mixture through a temperature-controlled barrel and nozzle, cooling and solidification of the filament, maintenance of the embedded spores in a dormant state during storage, and subsequent fungal emergence from the filament surface upon exposure to moisture or composting conditions. As shown, a single-screw laboratory extruder 17 equipped with a cooled take-off was used to process mixtures of polymer pellets 14 and fungal inoculum 16. Two representative polymers were evaluated: polycaprolactone (PCL) and low-density polyethylene (LDPE). Fungal strains A2 and A5, corresponding respectively to Aspergillus and Phanerochaete species, were cultivated on standard nutrient media and harvested after sporulation. Spores were

[0477] HIRO / 105 / PC 46 suspended in sterile distilled water at approximately 107spores per mL or dried to powder form for direct blending.

[0478] For the PCL formulation, the extruder barrel was maintained at 140 °C, and the nozzle at 50 °C, thereby limiting the inoculum contact temperature to below about 160 °C. For LDPE, barrel temperatures of 180-190 °C and nozzle temperatures of approximately 70 °C were used, resulting in inoculum exposure exceeding 180 °C. In both cases, the screw speed was 60-65 rpm, and the residence time was approximately three minutes from inoculum introduction to filament discharge. The inoculum was manually mixed into the softened polymer prior to extrusion to yield continuous strands approximately 1.5 mm in diameter. The inoculum filament is shown generally as reference numeral 18a (post extrusion, elevated temperature) and 18b (cooled). The overall process sequence — blending, extrusion, and air-cooling — is summarized in Figure 4. A representative cross-section of the resulting filament is shown in Figures 5A and Figures 5B-D are photographs showing microscope views of a sample filament with fungal inclusions, where discrete particulate inclusions corresponding to intact spores are visible throughout the polymer matrix. The inclusions remained morphologically distinct, confirming successful embedding without apparent fragmentation or dissolution.

[0479] With reference to Figure 5A, the polymer filament 96 includes embedded fungal inclusions 152 within a surrounding polymer matrix 154. The inclusions are distributed uniformly throughout the filament cross-section and remain morphologically distinct, confirming that the embedding process preserves spore integrity under controlled extrusion conditions. The interfacial boundary region 156 indicates minimal interaction between the spore surface and the polymer melt, ensuring the biological material remains viable and structurally intact. The illustrated configuration represents the as-produced form prior to hydration or environmental activation.

[0480] Table 8. Representative Processing Temperatures, Residence Times, and Fungal Viability Polymer Barrel Nozzle Estimate Reside Inoculu Observ Emerge Tempera Tempera d nee m Type ed nee on ture (°C) ture (°C) Inoculu Time Viability MEA m (min) (Days) Contact

[0481] Tempera

[0482] ture (°C)

[0483] Polycaprola 140 50 < 160 ~3 Native Hyphal 3-5 ctone (PCL) spores emerge days (Aspergill nee

[0484] usA2) observe

[0485] d; viable

[0486] Low-Density 180-190 70 > 180 -3 Native No

[0487] Polyethylen spores visible

[0488] e (LDPE) (Aspergill growth;

[0489] usA2) inoculu

[0490] m

[0491]

[0492] inactivat

[0493] HIRO / 105 / PC 47 ed due

[0494] to

[0495] combine

[0496] d high

[0497] tempera

[0498] ture and

[0499] dwell

[0500] time (Comparative < 180 <2 Native or Expecte expectation) (predicte encapsul d to

[0501] d) ated retain

[0502] spores viability

[0503] under

[0504] limited

[0505] dwell

[0506] time

[0507]

[0508] Representative temperature, residence-time, and qualitative viability data for fungal spores incorporated into molten polymer filaments. Viability is retained when contact temperature remains <160 °C and residence time < three minutes, but is lost under higher melt temperatures and extended exposure.

[0509] Filament sections were surface-sterilized with ethanol and placed on 2 % malt-extract agar (MEA) and ABTS indicator plates. Plates were incubated at 25-30 °C for up to fourteen days. Visible hyphal growth was observed for the Aspergillus inoculum extruded in PCL, confirming survival of a portion of the inoculum and full metabolic recovery upon subculture. In contrast, no growth was observed for LDPE-embedded spores, consistent with thermal inactivation under higher melt temperatures and equivalent residence time.

[0510] The relationship between inoculum contact temperature, residence time, and survival thus establishes practical limits for unprotected spores in molten polymers — contact temperature preferably < 160 °C, not exceeding about 200 °C, and residence time preferably < three minutes. Under these constraints, fungal spores can survive thermoplastic processing and subsequently germinate when hydrated. The biological outcome is displayed in Figure 6, showing hyphal emergence from the surface of an extruded PCL filament containing Aspergillus A2 after incubation on MEA. The images demonstrate that viable spores embedded within the polymer remain dormant after processing but reactivate upon hydration, providing direct visual evidence of post-processing viability.

[0511] These results demonstrate that viable fungi can be embedded in thermoplastic materials using standard polymer-processing equipment when both temperature and exposure duration are carefully controlled, forming the foundation for broader embodiments incorporating coated or encapsulated inocula.

[0512] • Example 10 - Adhesive Embedding of Fungal Spores

[0513] This example evaluates the survival and reactivation of fungal inocula embedded within representative adhesive systems, including both hot-melt and cold-cure chemistry formulations. The objective was to determine whether inoculated adhesives could retain viable spores or

[0514] HIRO / 105 / PC 48 encapsulated inocula after typical coating and curing operations. A representative structural configuration of the adhesive systems described herein is illustrated schematically in Figure 7. The adhesive layer 20 is shown with fungal spores or encapsulated inocula 22 distributed throughout its thickness, representing an adhesive composition capable of retaining viable microorganisms after coating or curing. The lower region depicts a generic underlying substrate, which may include paper, polymeric film, fabric, or other support materials. The upper region illustrates the cured or semi-cured adhesive film containing the embedded biological inclusions. The figure depicts a cross-sectional view of an adhesive layer containing dispersed fungal inclusions positioned on an underlying substrate. The adhesive layer represents either a hot-melt or cold-applied formulation, within which the fungal spores or encapsulated inocula are embedded at varying depths through the film thickness. The inclusions are distributed in a generally uniform manner and remain physically immobilized within the cured or solidified adhesive matrix. The underlying substrate may comprise a polymeric film, paper, fabric, or nonwoven material commonly used in absorbent or packaging applications. Upon hydration or exposure to environmental moisture, the embedded fungal inclusions are capable of germination and hyphal extension through or across the adhesive surface, providing localized biological activation within the material.

[0515] FIG. 7 is a schematic representation of a coated-surface embodiment in which a hydrogel film containing immobilized fungal inclusions is applied to a supporting substrate. The substrate 252 provides mechanical stability and defines the underlying structure of the composite article 250. A hydrogel coating layer 254 including an ion-crosslinked alginate or comparable polysaccharide is deposited over the substrate, forming a conformal layer that follows the surface contours of the underlying substrate material 252. Within this hydrogel layer 254, encapsulated fungal inclusions 256 are distributed evenly to maintain localized viability and enzymatic potential. The interface boundary 258 denotes the region of adhesion and moisture exchange between the hydrogel coating and the substrate. This configuration enables direct integration of biological functionality onto existing biodegradable or partially biodegradable products, allowing the inoculated surface to activate upon exposure to environmental moisture or during post-disposal degradation.

[0516] Two fungal species were tested: Aspergillus A2 (native spores) and Ganoderma A1 (encapsulated in sodium-alginate beads). Each was incorporated into 3-5 commercially typical adhesive films representative of common classes used in consumer, packaging, and absorbent products and representative of a cross-section of thermoplastic and cold-cure chemistries:

[0517] (1) a polyolefin-based hot-melt construction adhesive,

[0518] (2) a styrene-isoprene-styrene hot-melt elastic adhesive,

[0519] (3) a polyvinyl-acetate (PVA) water-borne adhesive,

[0520] (4) an acrylic pressure-sensitive adhesive (PSA),

[0521] (5) a “eco” bio-based tack adhesive, and

[0522] (6) a general-purpose plastic adhesive.

[0523] Adhesive layers of approximately 200-300 pm thickness were prepared on inert liners. For hot-melt systems, coating surface temperatures were maintained < 70 °C to minimize thermal stress on the inoculum. Spores or alginate beads were applied to freshly coated adhesive and gently over-layered to achieve partial embedding. Samples were cured or cooled, then placed onto 2 % malt-extract agar (MEA) and incubated at 25-30 °C for up to 30 days.

[0524] HIRO / 105 / PC 49 Quantitative emergence data for the encapsulated Ganoderma A1 inoculum is provided in Figure 8. The bar plot on the left side shows the percentage of replicates exhibiting visible emergence across the different adhesive types (acrylic PSA glue, eco glue, hot melt construction, hot melt elastic, PVA glue). The scatter plot on the right side shows the number of days until first emergence was observed. As shown, A1 remained viable in both hot-melt and cold-applied adhesives, though growth occurred more rapidly in PVA and acrylic PSA systems (« 10-12 days) and was delayed in hot-melt construction and elastic adhesives (« 20-25 days). The results demonstrate that alginate encapsulation provided sufficient thermal and mechanical protection for spores during coating at < 70 °C.

[0525] Photographs showing A1 growth from different forms of adhesive substrates are shown in Figure 10. Each adhesive system exhibited localized hyphal outgrowth from embedded alginate beads after incubation, confirming reactivation of the encapsulated inoculum. Emergence was uniform across the water-borne adhesives and more variable within the hot-melt films, correlating with the quantitative data.

[0526] Native Aspergillus A2 spores displayed faster emergence across all adhesive systems, as shown in Figure 9. The quantitative plots illustrate consistent 100 % emergence across replicates with visible hyphal growth within ~ 5 days, even for the hot-melt construction and elastic adhesives. Photographic growth results in Figure 11 show dense surface colonization within 3-5 days for both hot-melt and plastic adhesives, confirming that native spores can tolerate brief exposure to elevated coating temperatures and remain fully viable.

[0527] Collectively, the data from Example 10 confirms that fungal inocula, either as native spores or encapsulated beads, can be incorporated into a wide range of adhesive formulations without loss of viability when processing temperatures are maintained < 70 °C. Upon exposure to moisture, the embedded inocula reactivate and produce visible hyphae, providing a direct functional link between adhesive composition and biological activation.

[0528] • Example 11 - Embedding of Fungal Spores and Beads in Polymeric Films Example 11 demonstrates the incorporation and post-processing viability of fungal inocula within low-density polyethylene (LDPE) film substrates under representative lamination conditions. The objective was to determine whether fungal spores and encapsulated inocula could survive the transient heat exposure typical of heat-bonding or extrusion-lamination processes.

[0529] As illustrated schematically in Figure 12, the laminated film 24 configuration comprises a thin polymer substrate layer 164 encapsulating multiple fungal inclusions 28 dispersed within its interior volume. The inclusions are represented as discrete particulate bodies distributed through the film thickness, corresponding to either native fungal spores or encapsulated inocula such as alginate beads. The cross-sectional representation highlights the uniformity of embedding achieved during the lamination process, as well as the maintenance of discrete inclusion boundaries following thermal sealing. This configuration effectively isolates individual fungal propagules while retaining overall film integrity and mechanical uniformity. The structure is representative of laminated polymer films produced under low-density polyethylene (LDPE) processing conditions, in which heat and pressure are applied briefly to encapsulate the inoculum between polymer layers.

[0530] The laminated product shown generally in Figure 12 corresponds to the experimental preparation described herein, in which two LDPE sheets were heat-sealed around the inoculum to produce

[0531] HIRO / 105 / PC 50 bioactive film laminates. The laminated film 24 includes a thin polymer film substrate structure 164 incorporating fungal spores or encapsulated inocula 28 within its thickness. The inclusions are distributed throughout the body of the film and are retained following heat pressing or lamination. The configuration represents a bioactive polymeric film in which the fungal inoculum remains dormant after thermal processing but can later reactivate and extend hyphae upon exposure to moisture or composting conditions. Upon hydration and incubation, fungal emergence occurred through the surface or edges of the laminated film, demonstrating that the embedded spores remained viable after processing. The figure thus provides a structural model for film-based bioactive composites fabricated by lamination, extrusion, or thermal bonding, wherein the inoculum remains immobilized but capable of reactivation under environmental stimuli.

[0532] FIG. 12 illustrates a representative sheet-like composite incorporating dispersed biological inclusions within a continuous biopolymer matrix. The composite sheet 24 is formed from a flexible or semi-rigid biopolymer substrate 164 that provides mechanical support and environmental protection for the inclusions. The encapsulated biological inclusions 28 are embedded throughout the thickness of the sheet in a spatially distributed arrangement that may be uniform or patterned depending on the fabrication process. The upper surface 162 of the composite remains permeable to moisture and gas, allowing environmental interaction and eventual biological activation of the embedded inclusions when the material is exposed to humid or composting conditions. This configuration can be fabricated by casting, lamination, or compression molding and enables integration of inoculant functionality into packaging, absorbent, or molded articles. LDPE films were prepared by sealing two thin sheets around fungal inoculum using a laboratory thermal press. The inocula included Aspergillus A2 (native spores) and Ganoderma A1, prepared as whole alginate beads or ground alginate beads to evaluate the effect of inclusion size on survival. The press temperature was varied between 135 °C and 163 °C, with a dwell time of approximately 10 seconds. After pressing, films were cooled to ambient temperature, surface- sterilized with ethanol, and placed on malt-extract agar (MEA) plates for incubation at 25-30 °C for up to 20 days. Fungal emergence was recorded as visible hyphal growth extending from the surface or edge of each film.

[0533] Figure 13 includes data from both samples, and shows that native spores and ground alginate beads exhibited measurable survival and post-processing emergence. The bar graph on the left side presents the percentage of replicates showing visible emergence for Aspergillus A2 spores and Ganoderma A1 inocula at lamination temperatures of 135-163 °C. The plot on the right side shows the corresponding number of days required for first emergence. A2 spores survived and produced uniform growth within approximately five to twelve days at 135-146 °C, while A1 in ground alginate beads displayed delayed but consistent emergence up to 163 °C. Whole alginate beads, in contrast, failed to show recovery after pressing at 146 °C, indicating that smaller particle size improved heat tolerance.

[0534] Representative qualitative outcomes are summarized in Table 9 below. Native Aspergillus A2 spores retained full viability at temperatures up to 146 °C, producing dense hyphae within twelve days. Ganoderma A1 ground beads exhibited rapid localized growth at 146 °C and reliable emergence at 163 °C, whereas whole beads did not recover under the same conditions.

[0535] HIRO / 105 / PC 51 Table 9. Representative Growth Outcomes for Spores, Whole Alginate Beads, and Ground Beads Embedded in LDPE Films

[0536] inoculum Strain Processing Replicates Average Observation Type Temp (°F) with Days to

[0537] Emergence / Growth

[0538] Total

[0539] Native A2 275 2 / 3 « 5 Rapid growth; no spores (Aspergillus) film discoloration Native A2 295 3 / 3 « 12 Uniform

[0540] spores (Aspergillus) emergence; no color change on indicator media Ground A1 295 2 / 3 « 5 Fast localized alginate (Ganoderma) emergence; beads consistent with smaller particle size

[0541] Ground A1 325 3 / 3 « 12 Strong focal alginate (Ganoderma) growth; minor beads contamination in one replicate Whole A1 295 0 / 3 Contaminant alginate (Ganoderma) colonies only; no beads focal emergence observed

[0542]

[0543] Representative processing temperatures and qualitative emergence outcomes for Aspergillus A2 spores and Ganoderma A1 inocula (whole vs ground alginate beads) embedded in LDPE films. Viability was retained at < 146 °C for native spores and ground beads; whole beads showed poor survival due to slower cooling and internal heat retention.

[0544] Figure 14 includes photographs of regrowth from embedded inocula, showing visible hyphal emergence from the edges and surfaces of laminated LDPE film samples after incubation. The morphological characteristics and absence of contamination in control films confirm that the growth originated from the embedded fungal material.

[0545] The results of Example 11 establish that both native and encapsulated fungal forms can withstand exposure to temperatures encountered during thermoplastic film processing. Reduction in bead size markedly improved survival consistency, likely by enhancing heat dissipation during pressing. The embedded inocula remained dormant during storage and reactivated upon hydration, demonstrating the feasibility of producing polymer films containing viable biological propagules suitable for biodegradable and reactive composite materials.

[0546] HIRO / 105 / PC 52 • Example 12 - Nonwoven Filament and Spunbond Weave Testing

[0547] Example 12 demonstrates that native fungal spores can survive incorporation into polycaprolactone (PCL) filaments processed under melt conditions and subsequently show regrowth after plating, confirming post-processing viability.

[0548] A representative embodiment of the nonwoven or spunbond system described herein is illustrated schematically in Figure 15, including a fibrous matrix of overlapping thermoplastic filaments 32 that have been thermally bonded to form a coherent web structure 36. Fungal spores 34 are distributed both within the individual filament bodies and between adjacent filaments at their crossover or fusion points. During thermal bonding, a portion of the inoculum becomes encapsulated in the polymer matrix of each filament, while another portion remains localized in the interfilament regions where partial melting and fusion occur. The schematic highlights the interlaced orientation of filaments and the embedding of fungal inclusions throughout the web thickness, representing the morphology achieved during simulated nonwoven fabrication. Other discrete biological inclusions, such as fungal spores, bacterial inocula, or microcapsules containing enzymes or microorganisms may be embedded within the fibrous structure. The configuration represents a bioactive nonwoven substrate in which the inclusions remain dormant during fabrication and storage but can later activate under environmental conditions such as moisture, heat, or microbial stimuli.

[0549] FIG. 15 illustrates a representative process and resulting structure for fabricating a fiber- reinforced composite containing immobilized biological inclusions. A fibrous substrate composed of biodegradable or partially biodegradable filament strands 32 is combined with encapsulated inclusions 28 dispersed throughout the interstices of the network. The mixture is subjected to compression in a lamination or pressing device 166. During this step, the fibers partially fuse or bond together to form a consolidated fiber-inclusion matrix 170 corresponding to the structure shown on the right side of the figure. The resulting composite 36 exhibits uniform inclusion distribution within a continuous biopolymer and fiber framework, maintaining porosity for gas and moisture exchange while providing sufficient cohesion for handling and downstream conversion into absorbent or packaging articles. This configuration enables integration of microbial inoculants or enzymes directly into fibrous substrates using standard lamination or compression-molding processes without loss of biological viability.

[0550] Sample PCL filaments containing Aspergillus A2 spores were fabricated and tested as part of a nonwoven filament weaving experiment. Short filament segments were prepared, interwoven to form small braids, and heat-bonded using a surface iron to simulate localized melt fusion during nonwoven consolidation. Each filament or braid segment was placed on 2 % malt-extract agar (MEA) plates and incubated at 25-30°C for up to 28 days.

[0551] Of the processed filaments, those produced at approximately 135-140 °C and briefly melted at the interface retained fungal viability. Growth was first noted around 10 days after plating, with clear colony morphology consistent with the focal strain A2. No growth was observed from control filaments or from samples exhibiting discoloration after excessive melting. These observations confirm that a portion of the inoculum remains viable even after direct melt fusion and cooling within the PCL matrix.

[0552] The results are summarized in Table 4, which consolidates representative processing conditions and qualitative outcomes.

[0553] HIRO / 105 / PC 53 Table 10. Growth Outcome for Simulated Spunbond Processing

[0554] Polymer Strain Processing Emergence Days to Observation Type Temp (°C) Growth

[0555] PCL A2 135-140 Yes « 10 Visible hyphal growth (nonwoven (Aspergillus) days on MEA; morphology filament) consistent with focal strain

[0556]

[0557] On the left side of Figure 16, fungal hyphae can be seen emerging from the surface of a processed PCL filament sample after incubation on MEA plates. The growth pattern matches that of the focal species A2, confirming survival through thermoplastic processing and subsequent reactivation under nutrient and moisture exposure. Hyphal growth extends outward from the filament region, confirming post-processing survival and biological activity of the embedded spores.

[0558] On the right side of Figure 16, fungal hyphae can be seen emerging from a polycaprolactone (PCL) filament that underwent extrusion and weaving but was not subjected to a final melt-fusion step, demonstrating that the incorporated fungal spores remained viable through the mechanical processing sequence and were capable of germination upon incubation.

[0559] At higher magnification, individual fungal structures were observed directly emerging from the polymer surface, confirming localized germination from embedded propagules. Figure 17 presents representative microscopic images captured at 40* magnification showing single conidiophores extending from the surface of a polycaprolactone (PCL) filament following incubation on malt-extract agar. Both panels depict an isolated spore-derived hyphal stalk extending from the filament surface and branching into a conidiophore structure exhibiting characteristic branching morphology. These observations provide direct visual confirmation that viable fungal units were incorporated into the polymer matrix during processing and subsequently reactivated under nutrient and moisture exposure.

[0560] The results of Example 12 demonstrate that even without encapsulation, native fungal spores can endure two temperature extremes through filament extrusion and short-duration melt-fusion steps during nonwoven fabrication of PCL, enabling their direct integration into biodegradable fiber networks. The survival and reactivation observed provide functional support for compositions and methods encompassing bio-activated filaments and nonwoven webs containing viable fungal propagules.

[0561] • Example 13 - Melt Spinning of Polymers with Loose Fungal Spores

[0562] This example demonstrates that fungal spores can be incorporated into synthetic and biodegradable polymer fibers using a simplified melt-spinning analogue, confirming that biological material can survive direct melt fiber formation. The method provides proof of concept for large- scale incorporation of fungal spores into continuous filaments and woven structures without chemical encapsulation.

[0563] This example demonstrates that fungal spores can be incorporated into synthetic and biodegradable polymer fibers using a rotating-drum melt-spinning apparatus, confirming that

[0564] HIRO / 105 / PC 54 biological inocula can survive direct melt fiber formation without encapsulation. The experiment establishes feasibility for scalable production of continuous bio-activated filaments.

[0565] A bench-scale fiber-forming device equipped with a heated rotating drum was used to simulate low-throughput melt-spinning. Polymer pellets including polycaprolactone (PCL), polylactic acid (PLA), and polyethylene terephthalate (PET) were sequentially introduced into the drum and heated to molten viscosity. Dried spores of Aspergillus A2 were dispersed across the molten polymer as the fibers were drawn outward by centrifugal and shear forces. The surface temperature of the rotating chamber ranged between 140 °C and 260 °C, depending on polymer type.

[0566] Based on published thermal residence times for bench-scale melt-spinning, the contact duration between molten polymer and inoculum was estimated at <2 minutes before quenching. The resulting fibers were collected on a stainless-steel mandrel and cooled under ambient air.

[0567] Filament sections were plated on 2 % malt-extract agar (MEA) and incubated at 25-30 °C for up to 28 days. Emergence was defined as visible hyphal growth from filament surfaces or contact zones. Representative qualitative results are summarized in Table 5.

[0568] Table 11. Representative Observations for Melt-Spun Polymer Filaments Containing Fungal Spores

[0569] Polymer Approx. Estimated Emergence Approx. Observation Melt Residence Observed Days to

[0570] Temp Time (min) Growth

[0571] (°C)

[0572] Polycaprolactone 250-260 1-2 4-6 days Distinct hyphal (PCL) (confirmed) emergence at filament contact points; colony morphology consistent with Aspergillus A2. Polylactic acid 210-220 1-2 5-7 days Moderate (PLA) emergence from fiber ends; slower radial growth than PCL.

[0573] Polyethylene 250-260 1-2 5-7 days Emergence only terephthalate after pressing into (PET) agar; limited surface outgrowth observed.

[0574]

[0575] HIRO / 105 / PC 55 As shown in Figure 18, the process includes polymer melting within a rotating drum, inoculum introduction into the molten polymer stream, fiber formation, cooling, and subsequent incubation for biological activation. The photographs in Figure 19 (panels A-C) show representative results for the three polymer types.

[0576] FIG. 18 depicts a representative melt-blown or spunbond process for incorporating biological inclusions into fibrous substrates. Biological inclusions 174 and polymer pellets 176 are introduced into a rotating feed drum or spinneret 172 that delivers a uniform blend into an extrusion or melt-blown nozzle 178. The resulting molten mixture is forced through fine orifices and attenuated by an air-flow or quenching fan 180 to form continuous fibers. The fibers are deposited onto a collection surface to create a fibrous web or mat 182 containing embedded viable inclusions. Subsequent post-processing or bioassay 184 verifies that the inclusions retain structural integrity and metabolic potential after fiber formation. The process enables direct integration of microbial, enzymatic, or spore-based agents into nonwoven materials such as absorbent layers, filters, or biodegradable packaging substrates while maintaining standard industrial processing parameters.

[0577] These observations demonstrate that fungal spores can withstand transient exposure to molten polymer under fiber-forming conditions and reactivate upon hydration. The data confirm that melt¬ spinning and related high-shear processes can produce biologically active fibers suitable for integration into nonwoven, textile, or composite structures while maintaining fungal viability.

[0578] • Example 14 - Coated Spores (Trehalose and Analogs) in Polymers

[0579] This example demonstrates that applying a protective carbohydrate coating to fungal spores improves their survival during melt-processing and polymer embedding. The approach provides structural and functional evidence that protective sugar coatings, such as trehalose or other saccharides, stabilize spores through brief thermal and dehydration stress associated with polymer processing.

[0580] Fungal strain Aspergillus A2 was selected as the representative organism. Spores were suspended in a 10 % (w / v) aqueous trehalose solution, thoroughly mixed, and dried under mild heat (< 50 °C) until a free-flowing powder formed. The resulting coated spores were visually homogeneous and easily dispersed into polymer matrices. Parallel small-batch preparations substituted sucrose, mannitol, dextran, or starch to verify process generality.

[0581] Coated spores were blended into softened or molten polymers including polycaprolactone (PCL), polylactic acid (PLA), and other biodegradable thermoplastic polyesters. For PCL, polymer was softened at ~ 60 °C, close to the glass transition temperature; for PLA and biodegradable polyesters, processing occurred at 210-250 °C within a rotating-drum fiber-forming device (as described in Example 5). Each formulation contained approximately 5 wt % coated spores. Fibers or thin films were drawn onto foil and cooled to ambient temperature.

[0582] Sample fragments were rehydrated on 2 % malt-extract agar (MEA) plates and incubated at 25- 30 °C for up to 28 days. Fungal emergence was recorded as visible hyphal growth extending from the polymer surface. Representative qualitative results are summarized in Table X.

[0583] HIRO / 105 / PC 56 Table 12. Representative Observations for Trehalose-Coated Spores Embedded in Polymers Polymer Approx. Coating Emergence Approx. Observation

[0584] Melt Temp Type Observed Days to

[0585] (°C) Growth

[0586] PLA 230-250 Trehalose y 3-5 Rapid emergence;

[0587] (10 %) coating prevented discoloration.

[0588] PCL 60-70 Trehalose y 2-4 Strong colony formation,

[0589] (10 %) most consistent with A2;

[0590] minimal contamination.

[0591]

[0592] Representative processing conditions and qualitative emergence outcomes for Aspergillus A2 spores coated with trehalose or analogous saccharides and embedded in thermoplastic polymers. Viability was retained after processing at 180-260 °C for short residence times (<2 mins), confirming the stabilizing effect of the carbohydrate coating.

[0593] As illustrated in Figure 20, the process utilizes four sequential stages: coating and drying of spores, polymer softening, embedding and shaping, and post-processing rehydration for viability testing. As shown in the photograph of Figure 21, polymer formulations incorporating fungal spores demonstrated distinct morphological characteristics depending on polymer type and protective additive. Polycaprolactone (PCL) samples containing unprotected spores exhibited a cohesive, gummy consistency, while those containing trehalose-treated spores were more pliable and retained surface tack. In contrast, polylactic acid (PLA) formulations yielded partially spun fibrous structures, with trehalose-containing formulations producing the most continuous and strand-like textures. These differences indicate that polymer chemistry and additive inclusion influence the filamentous character and thermal flow of bioactive composite materials.

[0594] FIG. 20 depicts a process variation of the melt-blown or spunbond method shown in Figure 18, incorporating an upstream coating or encapsulation step for the biological inclusions. Fungal or biological inclusions 174 are first introduced into a preparation vessel or coating reactor 186, where they are combined with stabilizing excipients or encapsulating agents to produce coated inclusions 188. These coated inclusions are then introduced together with polymer pellets 176 into a rotating feed drum or spinneret 172, which delivers a homogeneous mixture into an extrusion or melt-blown nozzle 178. The fibers formed by the nozzle are drawn and cooled by an air-flow or quenching fan 180 before being collected into a fibrous web. Post-processing or viability testing 184 confirms that the pre-coated inclusions retain biological integrity following extrusion. This process configuration improves thermal tolerance and dispersion uniformity of biological inoculants during high-temperature fiber formation, enabling large-scale fabrication of bioactive nonwoven materials.

[0595] The photographs in Figure 22 show hyphal emergence from the surfaces of PLA (left) and PCL (right) fibers, confirming that the trehalose coating preserved spore function under thermal stress. Early replicates exhibited growth within two to three days on the polymers, compared with delayed and less growth points for uncoated controls. Parallel experiments with sucrose, mannitol, and dextran are underway and will provide comparative emergence data once validated.

[0596] HIRO / 105 / PC 57 Figure 23 displays microscopic examination of the PLA composites containing trehalose-coated A2 spores revealed distinct hyphal emergence and conidial formation from the polymer surface following incubation. Filamentous structures extended outward from the embedded inclusions, confirming that the trehalose coating preserved spore viability through the melt-processing step and supported biological reactivation upon exposure to moisture and nutrients.

[0597] These findings demonstrate that sugar- or polysaccharide-coated spores embedded in thermoplastic matrices retain biological viability following short high-temperature exposure and reactivate upon hydration. The results establish predictive support for a broader genus of saccharide-based stabilizing agents that can be interchanged without loss of function, enabling composition-of-matter claims covering polymer-embedded, carbohydrate-coated inocula.

[0598] Example 15 -Alginate Bead-Encapsulated Spores in Polymers

[0599] This example demonstrates the incorporation of alginate-encapsulated fungal spores into thermoplastic polymers and the resulting preservation of biological viability after melt processing. The study further confirms that encapsulated inclusions retain structural integrity within polymer matrices and can release viable fungi upon hydration.

[0600] Fungal strain A2 (Aspergillus) was used as the representative inoculum. Spores were suspended in a 1 % (w / v) sodium alginate solution containing osmoprotectants and structural modifiers including 5 % (w / v) sorbitol, 5 % (w / v) sucrose, and 1 % (w / v) rice starch optimized from Example 3. The suspension was extruded dropwise into a 100 mM calcium chloride solution to form spherical beads approximately 1.0 mm in diameter. The beads were rinsed, air-dried, and further dehydrated at 55 °C for 24 hours to a final moisture content below 10 %. The resulting micro¬ beads were free-flowing, smooth, and mechanically stable.

[0601] The dried alginate beads containing fungal spores were incorporated into polycaprolactone (PCL) by manual blending and molding. Rather than extrusion, the polymer was softened and repeatedly immersed in water maintained at approximately 100 °C to achieve near-melt pliability. During each immersion, alginate beads were pressed and molded into the softened PCL matrix by hand to ensure even distribution throughout the material. The resulting composite was drawn into a thin film and allowed to cool on a silicone mat at ambient temperature.

[0602] Upon cooling, the films were removed and incubated on malt-extract agar (MEA) plates at 25-30 °C. All replicates exhibited robust fungal growth originating from the embedded beads and across the film surfaces, confirming that the fungal inoculum remained viable through the repeated hot- water molding process and subsequent solidification of the PCL matrix.

[0603] Mechanical testing of the polymer composites indicated that inclusion of 1-5 % alginate beads did not significantly affect tensile strength or elongation compared with unfilled controls. The embedded beads also acted as micro-porosity centers, improving localized water permeability upon hydration and facilitating controlled fungal emergence. As shown in Figure 24, fungal emergence was observed from PCL films containing alginate spore beads following incubation on MEA for ten days. Panel (a) depicts macroscopic growth across the film surface, confirming viability of the embedded inoculum. Panels (b) and (c) provide microscopic evidence of conidial emergence from alginate bead remnants and empty bead vacuoles within the polymer matrix. These observations demonstrate that the fungal inoculum survived the near-boiling water molding

[0604] HIRO / 105 / PC 58 process and reactivated under nutrient and moisture exposure, confirming successful biological retention and activation within the polymer structure.

[0605] This example establishes that alginate bead encapsulation provides a reliable means of protecting fungal inocula during thermoplastic processing. The inclusions survive exposure to equipment operating at setpoints of high temperature and can further improve thermal stability at higher temperatures, while maintaining functional viability within a range of polymer hosts including PLA, PCL, PET, and similar thermoplastics. The approach provides a scalable, modular platform for embedding biologically active inclusions into commercial plastics and composites.

[0606] In further embodiments, the biological inoculant is incorporated directly into the raw polymer material used for forming filaments, sheets, or additive-manufacturing feedstock. The inoculant may be blended with pellets, granules, or powders of biodegradable polymers such as polylactic acid (PLA), polycaprolactone (PCL), or other aliphatic or polyester- based materials capable of melt processing and environmental degradation. Suitable polymers include both synthetic and bio-derived thermoplastics having hydroxy-containing repeating units or ester linkages, which soften below about 200 °C. Encapsulated or spore-based forms of the fungi are particularly suitable for dry blending with these raw materials, as they remain dormant under standard storage and compounding conditions. The inoculated polymer mixture can then be processed by extrusion or melt-spinning to form pellets or filaments that retain viable inoculant distributed throughout the polymer matrix.

[0607] The biologically active filament or pelletized feedstock may subsequently be introduced into an additive-manufacturing device, such as a fused-filament-fabrication (FFF) or fused-deposition-modelling (FDM) printer, and extruded layer by layer to produce three-dimensional articles containing viable but dormant fungal inoculants. Because the extrusion and printing conditions of the biodegradable thermoplastics are comparable to those previously described for melt¬ embedding (typically 80 - 180 °C with short residence times), the inoculants remain intact and recoverable after printing. Upon exposure of the printed object to environmental moisture or composting conditions, the embedded fungi or microbial consortia become active, initiating enzymatic degradation of the polymer matrix.

[0608] This additive-manufacturing approach enables direct fabrication of biologically active or selfdegrading structures, including packaging components, test coupons, foams, or lattice structures designed to disintegrate under defined environmental conditions. Incorporation of the inoculant at the raw-material or filament stage provides flexible manufacturing options compatible with standard polymer-extrusion and 3D-printing processes, thereby extending the biological activation concept to scalable additive-manufacturing systems.

[0609] (3) Two-Part Additive-Fungi Interaction System

[0610] A two-part additive-fungi interaction system was also developed, in which a biological inoculant component operates in conjunction with an additive-containing substrate to achieve controlled, environmentally triggered biodegradation of polymeric and absorbent materials. The two components are configured to remain physically and chemically isolated during manufacture and storage, thereby preserving biological viability, and to interact only under specific environmental conditions such as moisture exposure, elevated temperature, or changes in pH.

[0611] The first component of the system is a biological inoculant, typically comprising spores or mycelia of one or more fungal species encapsulated or immobilized within a biodegradable matrix. The

[0612] HIRO / 105 / PC 59 matrix can include alginate, chitosan, starch, gelatin, polyvinyl alcohol, or combinations thereof, and may incorporate osmolytes, humectants, nutrients, or buffering agents that stabilize the inoculum and regulate its hydration rate. The inoculant may be supplied as discrete beads, pellets, coatings, or degradable pouches suitable for integration into films, fibers, nonwoven webs, or molded articles.

[0613] The second component is an additive-containing substrate that forms the material to be degraded. The substrate can be a polymeric, cellulosic, or composite structure such as polyethylene, polypropylene, polyethylene terephthalate, polyiactic acid, polycaprolactone, polybutylene succinate, or pulp-based absorbents, as well as related copolymers, blends, or multilayer constructions. One or more functional additives are incorporated within or upon this substrate to influence fungal metabolism or enzymatic expression when contact occurs. Exemplary additives include nutrient compounds, trace metals, redox mediators, enzyme cofactors, pH modifiers, or small organic molecules that serve as metabolic triggers.

[0614] Upon environmental activation, the encapsulated inoculant becomes hydrated and viable, while soluble or diffusible additives migrate from the substrate into a reaction interface between the two components. The additives act as environmental triggers, promoting fungal germination and inducing the production of oxidative and hydrolytic enzymes that initiate degradation of the substrate. In this way, the substrate functions as both a degradable medium and a biochemical controller that governs the timing, rate, and selectivity of enzyme activity.

[0615] Upon hydration or environmental exposure, additives incorporated within the substrate or discrete additive-doped regions dissolve or diffuse toward the inoculant-containing matrix. This movement establishes an activation interface, defined by the location where moisture, additives, and the biological inoculant first interact. The local concentration of nutrients, metal ions, redox mediators, or other stimulatory compounds at this interface initiates metabolic activation within the inoculant, resulting in germination, enzyme secretion, or increased oxidative or hydrolytic activity. The formation of this interface may be influenced by the spatial arrangement of additives, the hydrophilicity of surrounding layers, and the hydration kinetics of the substrate.

[0616] As the inoculant transitions from stasis to metabolic activity, the organism produces oxidative, hydrolytic, or ligninolytic enzymes that begin to modify the surrounding polymeric or absorbent matrix. These enzymes diffuse outward from the inoculant or emerge from hyphal tips, creating a localized degradation zone characterized by softening, surface etching, porosity development, or early-stage chain scission in susceptible polymer regions. The extent and progression of this zone depend on additive concentration, spatial distribution, moisture availability, and the composition of the polymeric matrix. In multilayer articles or substrates containing patterned additives, enzyme induction and degradation may occur in discrete regions, enabling controlled or directional breakdown of the material.

[0617] In embodiments containing multiple biological inoculants or staged nutrient delivery, the activation of a first inoculant may facilitate the subsequent activation of a second inoculant. For example, partial oxidation or hydrolysis by a primary fungal inoculant may increase porosity, release soluble degradation products, or alter pH in a manner that favors the metabolic activity of bacteria, algae, or invertebrate-associated inoculants included in the article. This successional activation allows for additive or staged degradation behavior, with each biological component contributing to progressive material breakdown at different time points or environmental conditions. Such

[0618] HIRO / 105 / PC 60 systems may be configured using spatial separation of inoculants, differential encapsulation stability, or additives that dissolve or mobilize only after the initial degradation phase begins. In certain integrated embodiments, the article includes both an encapsulated inoculant and an embedded inoculant, arranged in combination with one or more additive-doped regions to produce a coordinated, multi-stage degradation system. For example, an encapsulated fungal inoculant may be positioned within a pouch, bead, or hydrogel inclusion located in a first region of the article, while a second fungal inoculant in native, coated, or dispersed form is embedded directly within a polymeric matrix of a different region. Additives such as nutrients, metal ions, or redox mediators may be localized within adjacent substrate layers or patterned zones, enabling an initial activation of the encapsulated inoculant followed by a secondary activation of the embedded inoculant as the substrate softens, swells, or becomes partially oxidized. In such systems, the spatial arrangement and differing encapsulation stabilities provide successional activation, in which early-stage enzymatic activity from the encapsulated inoculant facilitates later- stage activation of the embedded inoculant, leading to progressive or staged biodegradation of the article under composting or environmental conditions.

[0619] In related embodiments, a first fungal inoculant may initiate oxidation or hydrolysis within an encapsulated matrix, while a second biological component— such as a bacterial or algal successional inoculant — is embedded within or adjacent to a polymeric matrix that becomes more accessible only after the first stage of degradation. Additives may be selected to favor sequential activation, such as nutrients that dissolve only after partial material breakdown or ions that accumulate in localized regions following initial enzyme activity. These combined embodiments allow the coordinated use of multiple inoculant types, encapsulation formats, and substrate architectures to achieve extended or multi-phase material transformation.

[0620] Figures 25-29 illustrate representative embodiments and mechanisms of this two-part system, including the general concept and exemplary depiction (FIG. 25), representative product crosssections (FIG. 26), internal structure of the inoculant and substrate (FIGS. 27-28) and the environmental activation sequence (FIG. 29). Together these drawings demonstrate how complementary encapsulant and substrate chemistries provide a controllable, interdependent platform for environmentally triggered biodegradation of synthetic and natural materials.

[0621] FIG. 25 illustrates the representative activation and degradation mechanism associated with the encapsulated inoculum within a degradable substrate. An encapsulated inoculum 28 remains dormant until exposed to an environmental trigger 190 such as moisture, heat, or a specific nutrient cue. Upon activation, the biological system initiates enzyme production192, releasing hydrolytic or oxidative enzymes into the surrounding medium. These enzymes act upon an additive-containing substrate 194, degrading susceptible regions and initiating local polymer depolymerization, oxidation, or chain-scission reactions. The localized activity results in a degradation zone 196 that expands progressively as enzymatic reactions continue, accelerating the breakdown of the substrate or composite article. This schematic captures the functional basis of the invention — controlled activation of encapsulated biological systems to promote post-use material degradation or biotransformation under defined environmental conditions.

[0622] FIG. 26 illustrates a representative multi-layer composite structure designed for controlled biological activation and subsequent material degradation. The uppermost layer functions as a degradable delivery vehicle 198 housing an encapsulated fungal inoculum 28 within a hydrogel or polysaccharide matrix. Beneath this layer lies an optional nutrient or control layer 200, which

[0623] HIRO / 105 / PC 61 may indude buffering agents, metabolic inducers, or moisture regulators to delay or modulate fungal activation until the article encounters suitable environmental conditions. The lower section of the composite comprises an additive-doped polymer or material layer 202 containing degradable polymer components such as polycaprolactone (PCL), polylactic acid (PLA), or similar biopolyesters blended with pro-oxidant or bioavailable additives. The interface region 204 between the delivery and substrate layers serves as the primary site for enzymatic interaction once the fungal inoculum is activated. This layered configuration enables programmed activation of biological degradation, maintaining product stability during use while ensuring efficient decomposition or biotransformation after disposal.

[0624] FIG. 27 illustrates a representative activation and colonization sequence for an encapsulated inoculum, showing progressive transitions from dormancy to substrate degradation. In the initial dormant state, the inoculum 28 remains quiescent within the encapsulation matrix 198 together with optional additives or nutrients 206 that support subsequent biological activation. Upon exposure to environmental moisture, the hydrophilic or hygroscopic surface 208 facilitates rapid water absorption, initiating the bead swelling stage 210. This hydration softens and expands the encapsulation matrix, increasing internal porosity within the porosity transition zone 216. Once swelling is complete, the inoculum enters the emergence stage 212, during which hyphal or microbial filaments penetrate the softened encapsulation layer and extend toward the surrounding substrate. In the final colonization and enzymatic degradation stage 214, the activated organism secretes hydrolytic or oxidative enzymes that depolymerize adjacent material, progressively degrading the substrate surface. This schematic captures the dynamic behavior of the encapsulated inoculum system, emphasizing the functional coupling between environmental triggers, structural transitions of the encapsulant, and biological activity leading to controlled degradation.

[0625] FIG. 28 illustrates a representative substrate design incorporating a compositional gradient of additives to regulate degradation and biological response. The substrate 194 comprises a biodegradable or partially degradable polymer matrix into which functional additives are distributed in a concentration gradient 218. The direction of gradient formation 220 extends through the thickness of the substrate, typically established during casting, lamination, or co¬ extrusion. The high-additive region 222 contains greater concentrations of pro-degradant or nutrient components, promoting increased hydrophilicity and microbial colonization after exposure to environmental conditions. Conversely, the low-additive region 214 maintains structural strength and barrier performance during use. This configuration enables differential degradation behavior, in which biological activation and polymer breakdown preferentially initiate on the high-additive side and progress inward, offering a tunable balance between stability and controlled disintegration.

[0626] FIG. 29 illustrates a representative sequence of biological activation and material transformation involving an encapsulated inoculum on a gradient-doped substrate. In the first stage 222, the inoculum 28 remains dormant within an encapsulation matrix that contains optional additives 206 for long-term stability. Upon environmental exposure, hydration initiates the second stage 224, during which water absorption causes swelling of the encapsulant and metabolic reactivation of the inoculum. As the structure softens, the third stage 226 begins, characterized by hyphal or microbial filament emergence and initial surface contact with the substrate 194. Interaction with locally enriched regions of the additive gradient 218 accelerates the transition to the fourth stage 228, where enzymatic activity produces localized degradation zones on the substrate surface.

[0627] HIRO / 105 / PC 62 This schematic represents the coupling of temporal and spatial control mechanisms— hydration-induced activation and gradient-based reactivity — that together regulate the onset, rate, and extent of biodegradation in biologically augmented materials.

[0628] In certain embodiments, the disclosed materials and systems employ a two-part structure in which the biological inoculant and the activating additive are physically or chemically separated until the point of use. The inoculant, such as an encapsulated, immobilized, or embedded fungal culture, may be contained within beads, pouches, microcapsules, or polymeric inclusions as described in other embodiments of this disclosure. The substrate, host material, or waste product into which the inoculant is introduced or released contains one or more functional additives configured to influence fungal metabolism, enzymatic expression, or community succession upon contact The combination of these two elements forms an interdependent system in which the substrate functions not only as a degradable material but also as a biochemical controller for activation and metabolic tuning of the inoculant

[0629] The additives can include nutrient sources, trace elements, redox mediators, co-factors, surfactants, or polymer-bound functional groups that are selectively metabolized by target fungi or other microorganisms. Suitable additives may comprise metal ions (for example calcium, manganese, iron, or copper) that activate oxidative enzymes, or organic compounds such as phenolic mediators, carboxylic acids, or amino acid derivatives that stimulate peroxidase and laccase production. Other additives can act as enzyme inducers, pH buffers, or osmolytes that regulate spore germination and hyphal growth. The additives may be formulated as powders, granules, coatings, or polymer-integrated dopants positioned within specific regions of a composite article, such as within the surface or internal layers of a film, fiber, foam, laminate, or molded component.

[0630] In certain embodiments, the additives are incorporated directly into the polymeric or absorbent matrix during compounding or coating, where they remain dormant until environmental conditions such as moisture, temperature, or pH cause partial solubilization or diffusion. When the encapsulated fungal inoculum becomes hydrated and contacts the additive-containing substrate, the released molecules serve as environmental triggers, inducing production of oxidative, hydrolytic, or ligninolytic enzymes in the fungal system without requiring genetic modification of the organism. In some configurations, the additive distribution is spatially patterned to generate zones of enhanced or delayed fungal activation, allowing controlled progression of colonization and degradation through a multilayer structure.

[0631] The two-part system can be implemented across a variety of applications. In absorbent products, additives may be incorporated into superabsorbent polymer layers, fluff pulp, or barrier films to direct activation of encapsulated fungi released from a degradable pouch or coating. In packaging or single-use plastics, additives such as biodegradable esters, organic acids, or trace metal salts can be blended into polyesters or polyolefins so that, following disposal, contact with fungal inoculants triggers enzymatic oxidation and surface erosion. In agricultural or horticultural contexts, nutrient or mineral additives embedded in mulch films, seed coatings, or soil conditioners can interact with inoculated capsules or beads to promote beneficial fungal activity, soil conditioning, or biodegradation of residues. In environmental treatment systems, the same principles can be applied to reactors, filters, or remediation matrices where staged release of additives and inoculants drives biotransformation of organic contaminants.

[0632] HIRO / 105 / PC 63 Through the use of complementary encapsulant and additive chemistries, the system can also be designed to control successional behavior among multiple inoculant species. For example, a first additive layer may favor the growth of oxidative white-rot fungi that initiate depolymerization, while a second additive composition released later favors hydrolytic brown-rot or bacterial species that continue degradation. By engineering both the inoculant formulation and the additive environment, the two-part system provides an additional dimension of control over the timing, rate, and selectivity of biological degradation. This interdependency enables environmentally triggered expression systems and dynamic bio-catalytic responses, allowing passive control of enzymatic pathways through the composition of the substrate itself. The approach is applicable to any degradable or partially degradable product in which a biological inoculant is introduced, including films, coatings, foams, molded goods, nonwoven fabrics, absorbent articles, packaging, filters, construction materials, and composite waste-treatment media.

[0633] FIG. 25 illustrates a representative schematic of an additive-fungi interaction system 100 that forms the basis of the present disclosure. As shown, the system 100 includes a biological inoculant component 110 and an additive-containing substrate 120 that remain physically separated prior to activation and interact only when exposed to environmental conditions such as moisture, temperature change, or pH shift.

[0634] The inoculant component 110 includes one or more encapsulated fungal inocula 112, which may comprise spores, mycelial fragments, or fungal consortia immobilized within a biodegradable matrix 114. Suitable matrices include alginate, chitosan, starch, gelatin, polyvinyl alcohol, or other hydrogel or polymer systems capable of maintaining spore viability during drying and storage. The inoculant component may be configured as discrete beads, microcapsules, pellets, coatings, or a degradable pouch. In some embodiments, the matrix 114 contains osmolytes, nutrients, or structural modifiers that facilitate hydration and fungal emergence once exposed to moisture. The substrate 120 represents the material to be degraded and may include polymeric, absorbent, or composite structures such as polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polylactic acid (PLA), polycaprolactone (PCL), polybutylene succinate (PBS), and other biodegradable aliphatic polyesters of similar composition and thermal processing behavior, as well as pulp-based absorbents, copolymers, blends, and multilayer constructions. The substrate further comprises additives 122 distributed throughout its bulk or on its surface. The additives 122 may include nutrient sources, metal cofactors, redox mediators, organic acids, or other small molecules that modulate fungal metabolism or enzymatic expression upon contact. Additives can be blended into the polymer during compounding, coated onto the substrate, or localized within specific layers or regions to control the spatial pattern of fungal activation.

[0635] Under initial storage conditions the inoculant component 110 and substrate 120 remain isolated, preventing premature biological activity. Upon hydration or environmental exposure (190), water permeates the matrix 114, rehydrating the fungal inoculum 112 and allowing diffusion of the additives 122 from the substrate 120 into a reaction interface region. Within this region, the additives act as environmental triggers that stimulate fungal growth and enzyme production (192), leading to secretion of oxidative and hydrolytic enzymes. These enzymes initiate polymer oxidation and depolymerization within the adjacent portion of the substrate, forming a degradation zone 140 that expands outward as degradation proceeds.

[0636] HIRO / 105 / PC 64 Arrows depicted in FIG. 25 illustrate the sequential events of contact upon hydration, enzyme activation, and substrate degradation, highlighting the interdependency between the inoculant and the additive environment. The schematic shown in Figure 25 therefore conveys the central inventive principle: an engineered system in which the substrate itself functions as a metabolic controller for biological degradation, enabling precise, environmentally triggered activation of fungal enzymes and progressive breakdown of polymeric or cellulosic materials.

[0637] FIG. 26 illustrates a representative cross-sectional configuration of a multilayer article that incorporates the two-part additive-fungi interaction system described herein. The article includes a degradable delivery vehicle for fungi positioned above an optional nutrient or control layer and an underlying additive-doped polymer or material layer. Together, these layers provide both structural support and a controlled biochemical environment for staged activation of the biological inoculant and subsequent degradation of the substrate.

[0638] The degradable delivery vehicle may contain fungal spores, mycelial fragments, or combinations thereof, which are maintained in a dormant or semi-dormant state until environmental activation. In certain embodiments, the spores are embedded within a biodegradable matrix such as alginate, starch, chitosan, gelatin, polyvinyl alcohol, or combinations thereof. The matrix can further include humectants, plasticizers, or buffering agents that preserve viability during drying and storage. Upon exposure to environmental moisture, this upper layer hydrates and gradually releases viable spores or mycelia that migrate toward the layers beneath.

[0639] In alternative embodiments, the delivery vehicle can be configured as an encapsulated or degradable pouch containing a plurality of inoculated beads or microcapsules. The pouch may be fabricated from a biodegradable film or nonwoven material that dissolves, swells, or mechanically ruptures upon hydration, thereby releasing the inoculum into the adjacent layers of the article. The pouch configuration allows for modular placement within absorbent articles, packaging films, compostable products, or other systems where controlled biological activation is desired.

[0640] Immediately below, the optional nutrient or control layer provides supplemental functionality. This layer may contain soluble carbohydrates, amino acids, mineral salts, trace metals, or pH modifiers that facilitate fungal germination and early enzyme expression. In some embodiments, the layer serves as a diffusion barrier that modulates the rate of inoculum migration or additive exposure, thereby establishing a timed sequence of activation between the upper and lower layers.

[0641] The additive-doped polymer or material layer forms the primary substrate to be degraded. This layer may comprise synthetic or biodegradable polymers such as polyethylene, polypropylene, polyethylene terephthalate, polylactic acid, polyhydroxyalkanoates, or pulp-based composites. One or more functional additives are distributed throughout the layer 230, including nutrient compounds, metal cofactors, redox mediators, enzyme cofactors, or organic molecules that influence fungal metabolism and enzymatic activity. The concentration and spatial distribution of the additives can be tailored to create zones of enhanced or delayed degradation and to promote species-specific activation in multi-organism systems.

[0642] During operation, exposure to environmental moisture or composting conditions causes the upper delivery layer or pouch to hydrate and degrade, releasing the fungal spores or mycelia into or through the optional nutrient layer. As the inoculant encounters the additive-doped polymer layer, the additives act as environmental triggers that stimulate enzyme production and fungal colonization. Localized enzymatic activity initiates oxidation, depolymerization, and fragmentation of the polymer matrix. The multilayer configuration depicted in FIG. 2 thus represents an

[0643] HIRO / 105 / PC 65 embodiment of a self-contained, environmentally responsive article that integrates a degradable inoculant delivery system with a reactive substrate, enabling controlled fungal activation and progressive biodegradation of polymeric or absorbent materials.

[0644] FIG. 27 illustrates a representative sequence of fungal activation and emergence from an encapsulated inoculum within the two-part additive-fungi interaction system. The diagram depicts five successive stages — dormant, awaken, bead swells, emergence, and colonize — that occur as environmental moisture is absorbed and enzymatic activity begins.

[0645] In the dormant stage, the inoculum, which may comprise fungal spores, mycelial fragments, or conidia, is maintained in a non-metabolic state within a biodegradable encapsulation matrix. The matrix can include alginate, chitosan, starch, gelatin, or other hydrophilic polymers, optionally combined with nutrients or osmolytes that stabilize the inoculum during storage. Small inclusions within the matrix represent optional additives or nutrients such as sugars, mineral salts, or humectants that support viability and initiate early metabolic activity upon rehydration.

[0646] In the awaken stage, the encapsulated bead or matrix is exposed to environmental moisture, such as water vapor, humidity, or liquid condensation. The surface of the bead becomes hydroscopic or hydrophilic, allowing rapid moisture adsorption and initial hydration of the outer matrix region. This rehydration activates the fungal spores or mycelia, initiating germination and metabolic reactivation.

[0647] In the bead swelling stage, continued moisture uptake causes the encapsulation matrix to swell and become more porous, increasing its permeability to oxygen and dissolved additives. The hydrated structure enables diffusion of soluble nutrients both within the matrix and toward the outer environment. The increased porosity also facilitates the outward growth of germinating hyphae and enhances contact with any external substrate or additive-containing layer.

[0648] In the emergence stage, hyphal filaments penetrate through the softened matrix surface and extend outward, initiating physical contact with the surrounding substrate. At this point, environmental or substrate-borne additives— such as metal cofactors, redox mediators, or nutrient compounds — can act as metabolic triggers, further stimulating enzyme production and colonization.

[0649] Finally, in the colonization stage, the fungal hyphae expand over and into the adjacent substrate, secreting oxidative, hydrolytic, and ligninolytic enzymes that begin enzymatic degradation of the material. The lower insets in FIG. 27 schematically represent moisture absorption and matrix swelling processes that govern this transition from a compact, protective encapsulant to an open, metabolically active structure.

[0650] The sequence depicted in FIG. 27 demonstrates how the encapsulation matrix provides both stasis and activation control, protecting the inoculum under dry storage conditions and enabling rapid reactivation under defined environmental triggers. This controlled emergence mechanism allows the inoculated beads or pouches to remain dormant until use, ensuring reliable performance when incorporated into absorbent articles, polymeric composites, or other degradable materials.

[0651] FIG. 28 illustrates a representative cross-sectional view of a substrate containing spatially distributed additive zones configured to modulate fungal activation and enzymatic degradation within the two-part additive-fungi interaction system. The diagram demonstrates how the spatial

[0652] HIRO / 105 / PC 66 arrangement and concentration gradient of additives within a polymeric or composite substrate can control the rate and localization of biological activity once the encapsulated inoculum becomes hydrated and active.

[0653] In the example shown, the substrate comprises a polymeric or absorbent base material such as polyethylene, polypropylene, polyethylene terephthalate, polylactic acid, polyhydroxyalkanoates, or pulp-based composite. Distributed through the thickness of the substrate are zones of differing additive concentration, schematically represented by varying densities of dots or hatching. The additives may include nutrients, metal cofactors, redox mediators, or small organic compounds that act as metabolic triggers for fungal enzyme production.

[0654] The upper zone, positioned closest to the inoculant source, may contain a higher concentration of readily soluble additives that dissolve quickly upon hydration. This promotes early-stage fungal activation and rapid enzyme induction near the material surface. The intermediate zone may contain a moderate additive concentration that sustains enzymatic activity as fungal hyphae penetrate deeper into the material. The lower zone 430 may contain reduced or delayed-release additives, providing a gradual decline in concentration that stabilizes long-term colonization or ensures residual structural integrity during degradation.

[0655] The gradients or patterned distributions may be formed during manufacturing through coextrusion, multilayer casting, selective coating, or differential additive compounding, producing continuous or discrete concentration profiles through the materials thickness. In some embodiments, the additive patterning may be achieved laterally (for example, in stripes, grids, or localized domains) to create zones of enhanced or delayed degradation across the surface of the substrate.

[0656] When the fungal inoculum contacts the substrate under environmental conditions, the spatially patterned additives influence where and how the enzymatic degradation begins. The upper, nutrient-rich zones favor rapid germination and surface colonization, while deeper, nutrient-limited zones promote slower or sequential breakdown. This configuration allows fine control over degradation kinetics and structural behavior, enabling applications such as timed composting, staged release of materials, or selective weakening of specific product regions.

[0657] The cross-section depicted in FIG. 28 therefore illustrates how the disclosed embodiments enable programmable biodegradation through engineered additive gradients within the substrate, establishing a controllable interface between the encapsulated biological inoculant and its surrounding material environment.

[0658] FIG. 29 illustrates a representative environmental activation sequence for the two-part additive¬ fungi interaction system, showing the progressive transformation of the material through distinct stages of hydration, enzymatic induction, and substrate degradation. The stepwise timeline corresponds to the physical and biochemical events that occur when the encapsulated inoculum and the additive-containing substrate are exposed to environmental conditions such as moisture, elevated temperature, or composting humidity.

[0659] In the first stage (222), the system remains in a dry, dormant state. The encapsulated inoculum contained within a biodegradable matrix or pouch, is physically separated from the additive-doped substrate. The inoculum is stable under storage conditions, with little to no metabolic activity. The additives within the substrate remain immobilized, and no diffusion or enzymatic reaction occurs.

[0660] HIRO / 105 / PC 67 This stage ensures extended shelf life and controlled activation upon disposal or environmental exposure.

[0661] In the second stage (224), the system undergoes hydration and additive diffusion. Environmental moisture penetrates the matrix surrounding the inoculum, causing the encapsulating material to swell and become permeable. Simultaneously, soluble additives within the substrate begin to diffuse toward the interface between the two components. This migration establishes localized regions of increased ionic strength and nutrient availability, creating favorable conditions for fungal germination. Moisture absorption may also lead to partial softening or swelling of the substrate, further facilitating biochemical exchange.

[0662] In the third stage (226), enzyme induction and degradation zone formation occur. The hydrated fungal spores or mycelia become metabolically active and begin to secrete oxidative and hydrolytic enzymes in response to the diffusing additives. The enzymes initiate polymer oxidation and chain scission near the interface, forming a degradation zone characterized by localized color change, porosity increase, or surface erosion. The reaction rate within this zone can be controlled by the additive concentration gradient, the hydration level of the encapsulant, and the composition of the surrounding substrate.

[0663] In the fourth stage (228), the process advances to progressive material breakdown. As enzymatic activity continues, the degradation zone expands deeper into the substrate, resulting in partial or complete structural disintegration of the polymeric or absorbent matrix. The fungal network spreads across and within the material, producing mineralization products such as carbon dioxide, organic acids, and biomass. The remaining material gradually weakens and fragments under physical or microbial stress, leading to final decomposition or composting.

[0664] The stepwise sequence shown in FIG. 29 demonstrates the dynamic interdependence between the encapsulated inoculant and the additive-containing substrate. Each stage transitions naturally to the next through environmental triggers, without external control or genetic modification of the organisms. This design enables predictable, timed biodegradation of products such as absorbent articles, packaging films, or compostable plastics, while maintaining storage stability prior to use. The depiction in FIG. 29 thus provides a clear mechanistic representation of the invention’s functional relationship between environmental activation, enzyme expression, and material breakdown.

[0665] When complex waste materials such as absorbent hygiene products, packaging, or polymeric composites enter the environment, their degradation is typically slow because each layer or component presents different chemical bonds and moisture-access barriers. The disclosed systems address this by combining organisms that specialize in complementary stages of degradation, each altering the substrate chemistry to facilitate the next stage.

[0666] In its general form, the invention provides a process in which a first inoculant, typically comprising a filamentous fungus or other primary decomposer, initiates enzymatic depolymerization and disintegration of the waste matrix. Fungal species such as Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus may be employed, each capable of secreting oxidative enzymes including laccases and peroxidases that act on cellulosic, lignin-like, or polymeric materials. Activation of the first inoculant may occur immediately upon hydration, contact with organic matter, or exposure to environmental humidity. The resulting biochemical and structural changes, including liberation of soluble oligomers and

[0667] HIRO / 105 / PC 68 reduction of mechanical integrity, create conditions favorable for the establishment of secondary microbial populations.

[0668] In the early stage, fungal inoculants initiate oxidative and hydrolytic depolymerization. Fungal hyphae penetrate fiber networks and polymer interfaces, secreting extracellular enzymes such as laccases, manganese peroxidases, lignin peroxidases, cutinases, and esterases. These enzymes catalyze cleavage of ether and ester bonds in cellulose, lignin analogs, polyesters such as PLA and PHA, and synthetic polymer coatings such as PET or PCL. The enzymatic oxidation generates radicals and partially oxidized intermediates (e.g., carboxylic acids, alcohols, and oligomers) that soften the matrix and lower the local pH. Hyphal colonization also physically expands the substrate, increasing porosity and water retention (Figure 30).

[0669] In some embodiments, the fungal inoculant is provided as an encapsulated or immobilized form such as alginate, chitosan, or starch beads that hydrate upon exposure to moisture, such as in some of the above described embodiments. In other embodiments, the fungi may be introduced as grain spawn, myceliated substrate, or raw mycelial tissue directly applied to the waste material. The timing of fungal activation may occur immediately or within several days depending on substrate moisture and temperature.

[0670] Table 13. Representative bacterial and algal genera, enzyme classes, and substrate targets inoculant Representative Primary Enzyme Substrate Mechanistic Roie Type Genera Classes or Target

[0671] Material

[0672] Bacteria Bacillus, Esterases, lipases, Aliphatic Oxidation and Pseudomonas, cutinases, polyesters hydrolysis of shortAzotobacter, dehydrogenases, (PLA, PCL, chain Rhodococcus, oxidases etc.), intermediates; Acinetobacter residual biofilm formation;

[0673] lipids, nutrient recycling cellulose

[0674] oligomers

[0675] Bacteria Comamonas, PET hydrolase Aromatic Cleavage of Sphingomonas, (PETase), polyesters aromatic ester Paenibacillus, monooxygenases, (PET), bonds, ring Streptomyces peroxidases lignin-like hydroxylation,

[0676] residues release of terephthalic acid intermediates Bacteria Bacillus subtilis, Proteases, Protein Degradation of Pseudomonas amylases, adhesives, natural binders putida, cellulases starch and cellulosic Micrococcus binders, material; supports luteus pulp fibers fungal regrowth

[0677]

[0678] HIRO / 105 / PC 69 Algae Chlorella, Carbonic CO2, volatile Assimilation of (microalgae) Scenedesmus, anhydrase, organics, carbon dioxide, Anabaena, peroxidase, microbial oxygenation of Spirulina catalase exudates substrate,

[0679] stabilization of microbial community Cyanobacteria Oscillatoria, Nitrogenase, Nitrogen Nitrogen Nostoc, hydrogenase, fixation, supplementation Synechococcus oxidoreductase organic for subsequent acids microbial growth and enzyme synthesis

[0680]

[0681] As the fungal phase progresses (230), the altered chemical environment supports the activation of secondary inoculants such as bacteria or algae (stage 232). These stages are represented in Figure 31. The bacteria metabolize soluble intermediates released by the fungal enzymes through known pathways including p-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation. Certain bacterial species including but not limited to Bacillus subtilis, Pseudomonas putida, and Azotobacter vinelandii can assimilate aliphatic oligomers and residual starch or cellulose fragments, producing biosurfactants and extracellular polysaccharides that further disrupt polymer surfaces. Concurrently, algal species such as Chlorella and Scenedesmus can colonize exposed surfaces, forming photosynthetic biofilms that produce oxygen and assimilate CO2and volatile organics. This stage accelerates aerobic metabolism and suppresses anaerobic odor generation.

[0682] A second inoculant may subsequently become active, comprising bacterial or algal species that metabolize intermediate breakdown products generated by the fungal phase. Suitable bacterial genera include Bacillus, Pseudomonas, and Azotobacter, while suitable algal genera include Chlorella and Scenedesmus. Activation of this secondary stage may occur naturally as the fungal inoculant alters the substrate pH or nutrient content, or may be engineered through delayed inoculation or use of a slower-degrading carrier matrix such as starch, protein, or lipid hydrogel. In certain embodiments, the successional inoculant may comprise one or more bacterial species configured to activate after the fungal inoculant has initiated oxidation, softening, or partial hydrolysis of the substrate. Bacterial inoculants may be incorporated in dried, spore-forming, or microencapsulated form using stabilization strategies analogous to those described for fungal inoculants, including carbohydrate glass matrices, hydrogel coatings, or microcapsule systems. Bacterial activation typically occurs under elevated moisture and nutrient availability and may contribute to secondary hydrolysis, organic acid production, or localized pH changes that accelerate downstream degradation. The specific bacterial species are not limiting, provided that they exhibit metabolic activity compatible with post-fungal substrate modification.

[0683] Algal inoculants may be incorporated as dried cells, spores, cysts, or desiccated biomass and may activate during later stages of biodegradation when moisture and light penetration increase within the substrate. Stabilization of algal inoculants may be achieved by dehydration,

[0684] HIRO / 105 / PC 70 encapsulation, or embedding within hydrophilic matrices similar to those used for fungal preparations. Algal activation may support tertiary biotransformation by producing organic acids, enzymes, or biomass that alter local porosity or pH. Algal-based succession is particularly relevant in composting or outdoor degradation environments.

[0685] In the tertiary stage (234), invertebrate organisms such as black soldier fly larvae (Hermetia illucens), isopods, or other detritivores interact mechanically and biologically with the partially degraded substrate. Larvae feed on microbial biomass and partially digested material, reducing particle size, mixing residual layers, and increasing oxygen diffusion. The digestive enzymes of these invertebrates include proteases, lipases, and chitinases that contribute to further breakdown of complex residues. The frass and excreted matter from this stage contain stabilized organic compounds and microbial inocula that promote subsequent soil colonization.

[0686] A third inoculant or biological component may be introduced during later stages to accelerate fragmentation, aeration, or nutrient redistribution. In one embodiment, the third inoculant comprises invertebrate organisms such as soldier fly larvae (Hermetia illucens), isopods, or other detritivorous species that feed on the microbial biomass and physically fragment remaining polymeric residues. Activation of this stage may occur passively through natural colonization or by placing encapsulated eggs or pupae within a biodegradable carrier designed for delayed exposure.

[0687] In certain embodiments, a successional inoculant may comprise an invertebrate-associated component, including dried eggs, larvae, pupae, or attractant compounds configured to recruit invertebrates under composting or waste-processing conditions. Although invertebrates are not incorporated into polymer melts, they may be positioned within external layers, pouches, or permeable matrices that remain intact during storage and activate only after fungal or bacterial softening of the substrate. Invertebrate interaction constitutes a late-stage biodegradation mechanism, contributing to fragmentation, aeration, nutrient turnover, or mechanical disruption of the substrate.

[0688] The progression of each stage is guided by environmental indicators such as moisture, temperature, pH, and nutrient gradients. For example, fungal spores may germinate at relative humidity above 80% and moderate temperature (20-35 °C). The fungal metabolism consumes oxygen and releases organic acids (e.g., oxalic, malic), shifting pH toward slightly acidic conditions (pH 4-6). These changes promote bacterial proliferation once easily metabolizable carbon sources appear and pH begins to rise. Invertebrate colonization typically follows when decomposition raises temperature and emits volatile attractants such as ammonia, short-chain fatty acids, and CO2.

[0689] Table 14. Representative environmental triggers and activation windows for successional inoculants

[0690] Stage Representativ Primary Typical Physiological Functional

[0691] e Inoculant Environments Activation or Ecological Outcome in Type I Triggers Window Adaptations Process 1 Fungi High moisture Immediate Spore Depolymerization (Aspergillus, (>70% RH), to -3 days germination; of cellulose, lignin Ganoderma, moderate hyphal analogs, and

[0692]

[0693] temperature growth; biodegradable

[0694] HIRO / 105 / PC 71 Lentinula, (20-35 °C), secretion of polymers;

[0695] Trametes) availability of oxidative porosity increase organic enzymes

[0696] substrate, (laccase,

[0697] oxygen peroxidase,

[0698] presence cellulase)

[0699] 2 Bacteria Availability of 3-10 days Biofilm Metabolism of (Bacillus.. soluble carbon after formation; fungal Pseudomonas, compounds, fungal enzyme byproducts;

[0700] Azotobacter) pH 5-8, initiation secretion continued moisture (esterase, breakdown of >40%, oxygen protease, oligomers and presence lipase); SAP residues surfactant

[0701] production

[0702] 2a Algae / Light exposure, 3-14 days Photosyntheti Stabilization of Cyanobacteria surface c oxygenation; microenvironment (Chlorella, moisture, CO2carbon; oxygen supply Scenedesmus, presence, assimilation; for bacterial Nostoc) elevated nitrogen oxidation oxygen fixation

[0703] 3 Invertebrates Warm 10-30 Attraction to Physical (Hemietia temperatures days microbial disintegration, illucens, (25-40 °C), volatiles; aeration, isopods, odor volatiles, feeding and conversion to Tenebrio spp.) microbial mechanical stabilized frass biomass fragmentation and humic buildup material

[0704] All Environmental Continuou Ecological Self-sustaining stage moisture s feedback succession and s cycling, among final

[0705] nutrient species mineralization depletion, and

[0706] redox

[0707] gradients

[0708]

[0709] The successions! inoculation systems described herein may involve any combination of the foregoing biological categories, where the activation timing is controlled by spatial separation, hydration gradients, encapsulation stability, nutrient availability, or environmental triggers. Although fungal inoculants are preferred for initiating early-stage oxidation or hydrolysis, the subsequent activation of bacterial, algal, or invertebrate components is enabled by the structural and biochemical modifications produced during early fungal colonization. Equivalent successional

[0710] HIRO / 105 / PC 72 systems may be formulated by substituting any biologically compatible species exhibiting the functional activity described herein.

[0711] In some embodiments, these transitions are achieved purely by sequential inoculation, where each group is added after a defined interval (for example, fungal inoculation at time zero, bacterial at 5-7 days, and insect introduction at 15-30 days), in other embodiments, the same sequence may be engineered through differences in matrix composition or protective coating that control moisture uptake or nutrient availability. Suitable carrier matrices may include alginate, starch, gelatin, chitosan, or wax, each with distinct hydration and degradation profiles. Activation intervals can thus be tuned from hours to several weeks depending on composition and environmental exposure, although such activation times are representative and not limiting.

[0712] Figure 32 represents a conceptual cross-section of waste article showing fungal colonization front, bacterial biofilm formation, and insect penetration zones.

[0713] The combined activity of the successional inoculants produces measurable physicochemical changes in the substrate. The fungal stage 232 lowers polymer crystallinity and increases carboxyl group density; the bacterial / algal stage 234 increases oxygen content and microbial biomass; the insect stage 236 results in mechanical fragmentation and conversion to humic-like compounds. Infrared spectroscopy and carbon-nitrogen analysis of similarly degraded materials typically reveal increased carbonyl absorption and C: N ratios indicative of humification. The process thereby converts complex absorbent or polymeric materials into partially mineralized, soil-compatible residues.

[0714] FIG. 33 illustrates an exploded view of a multilayer article 300 that includes a plurality of spatially arranged biological inoculant zones. Specifically, the exemplary multilayer article 300 is shown as a diaper or similar absorbent pad. The article 300 comprises multiple layers 310, 320, 330, and 340, each of which may contain one or more types of biological inoculants or attractants configured for activation under environmental or composting conditions.

[0715] In the illustrated embodiment, one layer 310 includes a fungal inoculant zone, which may contain encapsulated fungal beads 312, spores, mycelial fragments, or other fungal propagules incorporated within or upon the layer. Another layer 320 includes a bacterial or algal inoculant zone 322, represented by spores, microcapsules, or dried cultures that are rehydratable upon exposure to moisture. A further layer 330 contains an invertebrate attractant or invertebrate inoculant 332, which may include attractant materials, nutrient pellets, eggs, or larvae of organisms capable of participating in later-stage biotransformation.

[0716] Additional structural or absorbent layers 340 may be included within the multilayer article to provide mechanical support, fluid management, or functional separation between inoculant zones. The arrangement shown within the magnified region 302 is only one example, and the sequence or positioning of inoculants is not limited to the configuration illustrated. Any layer or sublayer may contain fungal, bacterial, algal, or invertebrate inoculants individually or in combination, depending on the desired activation profile and functional performance of the article. Although illustrated schematically, any of the layers shown may be polymeric, absorbent, composite, or biodegradable as described herein.

[0717] HIRO / 105 / PC 73 This multilayer configuration enables successional or staged activation of different biological systems, in which one inoculant type becomes active prior to or concurrently with another. For example, fungal inoculants may activate first to initiate enzymatic modification of the material, followed by bacterial or algal activity, and ultimately invertebrate interaction or colonization. The spatially distributed arrangement permits controlled or progressive biotransformation of the article as environmental conditions change.

[0718] Table 15. Representative chemical transformations and associated enzyme mechanisms Stage Dominant Key Enzyme Primary Observabl Resulting Inoculant Classes Chemical e Material Functional Type Reactions / Changes Effect Transformati

[0719] ons

[0720] 1. Fungal Filamentous Laccases, Oxidative Softening Enhanced depolymerizat fungi manganese cleavage of and substrate ion phase (Aspergillus, peroxidases, C--0 and C-C swelling of porosity Trametes, lignin bonds in fibers; and Ganoderma) peroxidases, cellulose, decreased accessibilit cellulases, lignin-like polymer y for cutinases, polymers, crystallinity; bacteria esterases PLA, and pH and algae PCL; reduction

[0721] hydrolysis of (acidificatio

[0722] ester linkages; n); increase introduction of in soluble

[0723] carbonyl and organics

[0724] carboxyl

[0725] groups

[0726] 2. Bacterial / Aerobic Esterases, P-oxidation Reduction Continuatio algal bacteria lipases, and of residual n of metabolism (Bacillus, dehydrogenas dehydrogenati oligomers; polymer phase Pseudoman es, oxidases, on of oxidation of degradatio asy, monooxygena oligomers; reduced n; odor microalgae ses, assimilation of carbon; reduction; (Chlorella, nitrogenases organic acids; increased environmen Scenedesmu oxygen oxygen tai s) evolution via concentrati stabilization photosynthesi on and

[0727] s; nitrification microbial

[0728] and nitrogen biomass

[0729] fixation

[0730] 3. Invertebrate Soldier fly Proteases, Mechanical Visible Final fragmentation larvae lipases, shredding and fragmentati stabilization and (Hermetia ingestion of on; and

[0731]

[0732] illucens), biomass; reduction in mineralizati

[0733] HIRO / 105 / PC 74 mineralization isopods, chitinases, enzymatic bulk on of phase beetle larvae carbohydrases digestion of volume; waste;

[0734] residual conversion generation organics; of biomass of nutrienttransformation to granular rich organic of microbial organic material matter to frass residue

[0735] containing

[0736] humic

[0737] precursors

[0738] Integrated Mixed Combination of Sequential Gradual Conversion succession consortia above oxidationcolor of complex (fungi -» reduction darkening, absorbent bacteria / alga cycles; carbon texture and e and nitrogen softening, polymeric invertebrates assimilation; odor materials ) humification neutralizati into

[0739] on stabilized, soilcompatible matter

[0740]

[0741] The described successional techniques are applicable to a wide range of waste materials including absorbent hygiene products such as diapers, training pants, and sanitary napkins, as well as packaging, agricultural residues, and composites containing biodegradable or partially biodegradable polymers such as polylactic acid (PLA), polycaprolactone (PCL), polyethylene terephthalate (PET), and other biodegradable polyesters. The successional inoculants may be incorporated directly into such articles, applied externally during disposal, or introduced into composting, soil, or reactor environments containing the waste material.

[0742] The invention is applicable to absorbent hygiene products, paper pulp composites, compostable packaging, and any material containing cellulose, superabsorbent polymers, or biodegradable polyesters. In one embodiment, fungal inoculants are applied as grain spawn or raw mycelium within the absorbent core, bacterial inoculants are sprayed or blended into outer layers, and invertebrate attractants are placed in peripheral zones or pouches for delayed activation. In another embodiment, the inoculants are co-deployed as powders, beads, or coatings, each responding independently to environmental triggers.

[0743] The disclosed successional inoculant systems reduce the time required for natural decomposition, enhance uniformity of breakdown across multi-material articles, and enable control over biological sequencing through material and process design The systems can be adapted for decentralized composting, industrial waste treatment, or integration into the manufacturing of inherently degradable consumer products. The approach harmonizes ecological principles of succession with engineered control of biological activation, allowing efficient conversion of complex waste materials into stabilized organic matter and recoverable biomass.

[0744] HIRO / 105 / PC 75 The invention provides compositions, systems, and processes that employ successional inoculants across multiple biological taxa to achieve progressive transformation of organic, cellulosic, and polymeric materials. The approach combines early-stage decomposers, secondary microbial metabolizers, and late-stage detritivores in a controllable sequence, either through staged application or through matrices that enable time-delayed activation.

[0745] In one embodiment, the invention provides a process for progressive biotransformation in which a first inoculant comprising one or more fungal species initiates enzymatic oxidation and disintegration of a waste substrate, a second inoculant comprising bacterial or algal species becomes active after a defined interval to metabolize soluble intermediates, and a third inoculant comprising invertebrate species subsequently fragments or mineralizes the remaining residue. The inoculants may be applied sequentially in time or provided together in stabilized forms that activate at different stages depending on moisture, temperature, or pH.

[0746] In another embodiment, the invention provides successional inoculant systems or kits containing two or more biological formulations, each encapsulated or immobilized within a biodegradable carrier having distinct hydration or degradation kinetics. Representative carriers may include alginate, starch, gelatin, chitosan, or wax, chosen to provide activation intervals from hours to several weeks. The inoculant formulations can be supplied as pellets, beads, powders, or pouch inserts, optionally including nutrients or cofactors that promote microbial succession.

[0747] In further embodiments, the invention provides composite waste articles or treatment units incorporating the successional inoculants directly within the structure of the material. Absorbent hygiene products may, for example, contain a fungal inoculant within the cellulose core, a bacterial or algal inoculant in or near the topsheet, and an insect attractant or encapsulated larvae near the backsheet or disposal pouch. Other waste types such as compostable packaging or pulp-based absorbents can be configured similarly.

[0748] Across all embodiments, the invention establishes a flexible platform for coupling biological and material-engineering principles to promote sustained degradation through multiple ecological stages. The inoculants may be selected from fungi such as Aspergillus, Ganoderma, or Trametes: bacteria such as Bacillus or Pseudomonas; algae such as Chlorella; and invertebrates such as Hermetia illucens. Activation times and encapsulation types described herein are representative and may be adjusted according to species, substrate composition, and environmental conditions. The disclosed systems accelerate breakdown of superabsorbent polymers and cellulose composites, reduce odor, enhance aeration, and produce stabilized organic residues suitable for soil reintegration.

[0749] The invention may be embodied in various forms without departing from its scope, and the following description illustrates representative embodiments that demonstrate the principles of successional inoculant systems for progressive waste biotransformation. The following embodiments illustrate representative implementations of the invention and are not intended to limit the scope of the claims. Where specific inoculant species or materials are mentioned, they are provided as examples of classes or functions that may be substituted with other equivalents possessing similar enzymatic or ecological activity. The compositions, methods, and organisms described herein can be adapted to numerous substrate types and deployment formats and are presented as illustrative examples rather than limitations.

[0750] In one embodiment, a waste substrate such as an absorbent hygiene product containing cellulosic pulp, superabsorbent polymers, and polymeric films is treated by a sequence of biological

[0751] HIRO / 105 / PC 76 inoculants that activate at different times. A first inoculant comprising filamentous fungi is introduced onto or into the absorbent core. The fungi may be applied as hydrated spores, as grain spawn, or as fragments of raw mycelium distributed over the surface or embedded within the fibrous matrix. Suitable fungal species are included, but not limited to, Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus. Upon exposure to ambient humidity, the fungal propagules germinate and extend hyphae through the porous substrate. The fungi secrete extracellular oxidative and hydrolytic enzymes such as laccases, peroxidases, cellulases, and esterases, which depolymerize cellulose, lignin analogs, and biodegradable polymers such as polylactic acid (PLA) and polycaprolactone (PCL). As enzymatic oxidation proceeds, the substrate softens, its pH gradually decreases, and soluble intermediates appear, creating a nutrient environment that favors secondary colonization.

[0752] After a preliminary colonization period of several days to one week, a second inoculant becomes active. This stage may be introduced deliberately or occur naturally as bacterial and algal communities develop in response to fungal metabolism. Representative bacterial genera include Bacillus, Pseudomonas, and Azotobacter, while representative algal or cyanobacteria! genera include Chlorella, Scenedesmus, and Nostoc. These microorganisms metabolize the organic acids, oligomers, and partially oxidized compounds released by the fungi, continuing the degradation through enzymatic hydrolysis and oxidation reactions. Bacterial populations secrete esterases, dehydrogenases, and monooxygenases that act upon polymer fragments and lipids, while algal populations contribute oxygen through photosynthetic activity and assimilate carbon dioxide, stabilizing the microenvironment. The bacterial and algal stages collectively reduce odor, promote aerobic conditions, and enrich the substrate with microbial biomass and nitrogenous compounds.

[0753] As the degradation process continues, the microenvironment warms and releases volatile compounds that attract macro-organisms. In the tertiary stage, invertebrate species such as black soldier fly larvae (Hermetia illucens), isopods, or other detritivores colonize the softened substrate. These invertebrates feed on microbial biomass and residual organics, physically fragmenting the remaining material and increasing aeration. Their digestive processes include proteolysis, lipolysis, and chitinolysis, and their activity results in further comminution and conversion of the decomposed matter into granular frass rich in humic precursors. Over several weeks to months, the multilayer absorbent structure becomes unrecognizable, with SAP granules and polymeric films disintegrated into stable, soil-compatible residues. Control substrates that are not inoculated remain largely intact, demonstrating the functional advantage of the successional process.

[0754] In another embodiment, the successional activation is achieved through formulation design rather than sequential inoculation. Each inoculant may be provided in a form that responds differently to environmental cues such as moisture, pH, or temperature. For example, a fungal inoculant can be incorporated into a rapidly hydrating carrier such as an alginate hydrogel or cellulose-based coating that enables immediate colonization upon exposure to moisture. A bacterial or algal inoculant can be formulated within a starch, protein, or lipid matrix that remains inert until partially degraded by fungal enzymes, producing a delayed activation window of several days. A third inoculant, comprising invertebrate eggs or pupae, can be contained in a wax or chitin-based capsule that softens or ruptures after prolonged microbial activity or elevated temperature. When these encapsulated or immobilized formulations are co-deployed within a waste article or

[0755] HIRO / 105 / PC 77 treatment vessel, they activate sequentially, reproducing the ecological progression observed in natural systems but with predictable timing and composition.

[0756] The successional inoculants can also be integrated into the structure of manufactured articles. In one embodiment, an absorbent product is constructed with distinct biological zones: the cellulose core contains a fungal inoculant, the upper or topsheet region contains bacterial or algal microcapsules, and the outer pouch or backsheet includes an attractant or compartment for invertebrates. Upon disposal, environmental moisture initiates fungal colonization, followed by bacterial and algal proliferation, and finally insect participation, allowing the article to undergo self-directed degradation without manual intervention. Similar configurations can be used for compostable packaging or pulp-based composites where sequential activation is beneficial. In some embodiments, the successional inoculant system is prepared by separately cultivating the inoculants and stabilizing them for combined use. Fungal material may be grown on sterilized grain or plant substrate, dried to a controlled moisture level, and optionally milled or blended with biodegradable carriers. Bacterial and algal cultures may be propagated in aqueous media, concentrated, and stabilized through drying or encapsulation methods known in the art. Invertebrate eggs or larvae may be maintained under dormancy and added to the final product shortly before use. The respective inoculants are then arranged in kits, layered pouches, or mixed matrices designed to achieve the desired sequence of activation.

[0757] Across these embodiments, the invention provides a controllable biological system that integrates enzymatic oxidation, microbial metabolism, and macro-organism fragmentation into a continuous degradation process. The biological and chemical interactions among the inoculants convert complex absorbent or polymeric substrates into stabilized organic material suitable for reintegration into soil or composting systems. The process can be tuned through inoculation timing, matrix composition, or environmental parameters, offering a reproducible and scalable platform for waste biotransformation.

[0758] • Example 16: Sequential Biotransformation of Absorbent Hygiene Articles in a Semi-Buried Soil System

[0759] To evaluate the performance of successional inoculant techniques under realistic environmental conditions, a controlled underground trial was conducted using used absorbent hygiene articles as representative multilayer waste substrates. Each article comprised a cellulose pulp matrix, superabsorbent polymer (SAP) granules, and polymeric films typical of commercial diapers. The experiment was performed using partially buried containers designed to permit contact with native soil while maintaining separation among inoculant types.

[0760] Eight two-gallon plastic buckets were prepared by drilling apertures around the lower half of each vessel at intervals of 3-4 cm to allow soil contact and aeration. Approximately 30 diaper sections were placed in each bucket, yielding a total of about 228 articles across seven inoculation groups and one uninoculated control. The diapers were pre-hydrated by immersion for one hour in a 4 % sugar solution to achieve a moisture content of approximately 65 %, then drained prior to inoculation.

[0761] A first inoculant consisting of fungal grain spawn was prepared using spent oyster substrate colonized by fungal strains Pleurotus A6, Lentinus A7, Ganoderma A1, Cordyceps A9, and Penicillium A8. The hydrated diaper materials were inoculated heavily with fungal spawn at 15-20 % (w / w) and packed in alternating layers within the buckets to ensure even distribution. Lids

[0762] HIRO / 105 / PC 78 were applied loosely to maintain humidity while permitting gas exchange. Each inoculated bucket was partially buried to the level of the perforations in open soil, and an additional set of three replicates per species was buried directly without buckets to compare soil-contact conditions. Throughout the four-month test period, the buried units were maintained outdoors under ambient temperature ranging from 20 °C to 35 °C and were periodically rehydrated with the 4 % sugar solution once per month to sustain microbial activity. Monthly inspections and sampling were carried out at one-, two-, and three-month intervals, followed by final evaluation at four months. Within the first week after inoculation, white mycelial growth was observed along the cellulose layers and within the SAP-pulp interface of all fungal treatments. By one month, visible hyphal mats covered the upper surfaces of the diapers, accompanied by softening of the absorbent matrix and partial delamination of the polymer films. In buckets containing fungal species A7 and A1, strong colonization and enzymatic staining were evident, indicating active ligninolytic enzyme secretion.

[0763] Between weeks 3 and 5, bacterial and algal colonization appeared spontaneously in the buckets, producing thin biofilms and green surface patches consistent with Bacillus, Pseudomonas, and Chlorella genera typical of native soil microflora. These organisms utilized organic acids and soluble sugars generated by the fungal stage, sustaining aerobic metabolism and further degrading the softened polymer fragments.

[0764] After approximately six weeks, soldier-fly Hermetia illucens) larvae and small isopods were observed within the inoculated buckets and in the directly buried replicates. The larvae fed on the microbial biomass and decomposed substrate, fragmenting the remaining layers and blending the material with soil. By the end of the four-month period, the contents of the inoculated buckets had darkened to a friable, soil-like consistency with no intact SAP granules or continuous film layers visible. Control buckets lacking fungal inoculum retained their laminated structure and exhibited only minor discoloration.

[0765] These results demonstrate that sequential fungal, bacterial, algal, and invertebrate colonization can be established through a single initial inoculation using fungal grain spawn and that the resulting ecological succession produces complete disintegration of absorbent hygiene articles within a few months under moderate soil conditions. The semi-buried configuration effectively balances aeration and moisture retention, creating a reproducible framework for evaluating multi-species waste biotransformation.

[0766] Representative observations of the inoculated materials at the conclusion of the trial are shown in Figures 34A-34E. The inoculated samples exhibited extensive degradation and partial disintegration of the absorbent matrix, with visible insect colonization and layer separation (Figure 34A), while control samples retained compact, intact layers (Figure 34B). Intermediate-stage samples showed simultaneous fungal and algal colonization on the same substrate (Figure 34C), and later-stage samples demonstrated active soldier-fly larvae and substantial fragmentation of remaining polymeric films (Figure 34D). In the final stage, the inoculated materials were converted into a dark, friable, soil-like residue with only minor remnants of polymer structure, indicating significant biological transformation and near-complete breakdown of the absorbent and cellulosic components (Figure 34E).

[0767] The inventive embodiments described above provide an integrated biological approach for the decomposition and stabilization of complex waste materials containing organic, cellulosic, and

[0768] HIRO / 105 / PC 79 polymeric components. By coordinating multiple inoculant types that activate in sequence, the system reproduces the natural efficiency of ecological succession while introducing engineered control over activation timing and environmental response. Sequential activation of fungal, bacterial, algal, and invertebrate inoculants produces measurable acceleration in material disintegration and mass loss compared with conventional single-organism or passive composting systems. Each inoculant modifies the physical and chemical properties of the substrate in a way that promotes activation of the subsequent stage, thereby creating a self-sustaining progression without external intervention once initiated. The process reduces odor generation and anaerobic zones, enhances aeration and oxygen diffusion, and promotes conversion of synthetic absorbent layers and polymer films into friable, soil-compatible matter. The system can be adapted to a wide range of substrate types and environmental conditions by varying inoculant species, moisture levels, and carrier compositions, offering flexibility across waste-management contexts. In addition, the method supports circular-economy objectives by enabling the biological transformation of post-consumer absorbent hygiene articles and similar materials into stabilized organic matter suitable for reuse in soil or compost systems.

[0769] The embodiments are industrially applicable to waste-management, composting, and materials-recycling processes involving biodegradable or partially biodegradable materials. The system can be used for on-site treatment of absorbent hygiene products, pulp-fiber composites, and packaging materials, as well as for controlled degradation of manufacturing waste and recovery of organic carbon. The successional inoculant compositions may be produced as packaged kits for municipal or industrial composting facilities, incorporated directly into the manufacture of biodegradable goods, or applied in open-air, enclosed, or soil-based treatment systems. The process requires no specialized infrastructure and may operate under ambient outdoor conditions, making it suitable for both centralized and decentralized waste streams. The biological species employed are non-pathogenic and can be cultivated using standard fermentation or substrategrowth methods, ensuring environmental safety and economic feasibility. The invention therefore offers a scalable and cost-effective solution for accelerating biodegradation and converting persistent waste materials into useful organic matter, contributing to sustainable industrial, agricultural, and environmental practices.

[0770] (5) Degradable Pouch Delivery Method & Composition

[0771] The present disclosure further provides degradable pouch delivery systems for storing, transporting, and releasing biological inoculants including fungal, bacterial, algal, or mixed consortia. The pouch is formed from a biodegradable or water-soluble film material such as polyvinyl alcohol (PVA), polyvinyl-alcohol-cellulose blends, starch-based films, regenerated cellulose, water-soluble papers, or other polymers configured to dissolve, disintegrate, or mechanically rupture under composting or hydration conditions. One representative film suitable for pouch fabrication is a water-soluble heat-seal paper having a caliper of about 125 pm and dispersibility of 5-10 seconds in water. The pouch may comprise one or more layers, including heat-sealed seams or biodegradable adhesives that maintain integrity during storage but release upon wetting.

[0772] Each pouch encloses a biological inoculant composition selected from (i) encapsulated alginate or hydrogel beads containing fungal spores or mycelia; (ii) grain-based or solid-state substrates inoculated with fungal cultures; or (iii) dehydrated spore formulations optionally mixed with carriers such as perlite, trehalose, or maltodextrin. The inoculant may include white-rot, brown-rot, or soft- rot fungi as described in hiro103US and their bacterial or algal counterparts used in successional

[0773] HIRO / 105 / PC 80 degradation systems. The internal composition can further contain abiotic amendments such as sodium bicarbonate, pH buffers, oxygen carriers, or micronutrients that facilitate rehydration and early growth of the inoculum. Pouch fill mass may range from 0.1 g to 10 g depending on the target application.

[0774] A representative configuration of the pouch is illustrated schematically in FIG. 35. As shown, the pouch 50 comprises an outer film layer 52 formed of a biodegradable or water-soluble material that encloses an internal cavity 54 containing a biological inoculant composition 56. The pouch is shown as generally square in plan view, with heat-sealed seams 60 along its perimeter, although other shapes such as rectangular, circular, or pillow-type constructions are equally suitable. The internal cavity 54 may include encapsulated beads, grain-spawn granules, or dried spores suspended in a carrier, optionally combined with nutrient or other additive particles 57 that promote viability upon activation, shown generally as reference numeral 58. When the pouch contacts moisture, the film material 52 softens and disintegrates, allowing the inoculant 56 to be released or to extend hyphae through the weakened film. The schematic in FIG. K represents a non-limiting embodiment illustrating the general construction and function of the degradable pouch.

[0775] Upon contact with water, waste fluids, or compost moisture, the film material dissolves or delaminates, releasing the inoculant into the surrounding substrate. The pouch thereby serves as a self-contained deployment unit that protects the inoculum during handling and storage, allows clean addition to waste streams, and initiates degradation only under appropriate environmental conditions. In one embodiment, the pouch is introduced into a disposal bag containing absorbent articles, where the outer film dissolves and releases fungal inoculants that colonize cellulosic, super-absorbent, or polymeric components. In other embodiments, pouches are embedded within packaging waste, industrial compost reactors, agricultural mulch films, or soil conditioners to initiate targeted biological activity.

[0776] The pouch material and construction can be tuned for staged or delayed release by modifying film thickness, coating composition, or incorporation of hydrophobic barriers. Multi-compartment designs may contain separate inoculant types or abiotic stimulants that are released in sequence. For example, an inner sachet may contain a fungal inoculant and an outer envelope may include nutrients or attractants that dissolve first, establishing an environment favorable to fungal activation. Alternatively, different pouches may be co-packaged, each containing a distinct fungal species selected for sequential degradation of high-molecular-weight polymers followed by metabolism of low-molecular-weight intermediates.

[0777] The form of the inoculum can be adapted to the intended processing environment Encapsulated forms such as alginate beads or microcapsules are preferred where moisture control and shelf stability are critical. Grain-spawn forms, including milo, rye, or sorghum grains colonized with fungal mycelium, are suitable for nutrient-rich solid-state systems and exhibit rapid post-release colonization. Spore-only or dehydrated conidial forms provide extended shelf life and tolerance to elevated temperatures. Combinations of these inoculant forms can be employed within a single pouch or among pouch sets to provide immediate and sustained biological activity.

[0778] The disclosed pouches can be manufactured by form-fill-seal, heat-sealing, or wet-molding techniques using standard packaging equipment. The biodegradable film can be cut into sheets, tubes, or pre-formed pockets that are filled with the desired inoculant composition and sealed by heat or pressure. The filled pouches may be enclosed in moisture-barrier overwraps fortransport

[0779] HIRO / 105 / PC 81 and storage. Typical storage conditions include relative humidity between 30 % and 70 % and temperature between 10 °C and 25 °C to maintain dispersibility and biological viability.

[0780] The degradable pouch system thereby provides a modular biological delivery platform that is compatible with a wide range of materials and biological forms. It enables controlled deployment of fungal, bacterial, algal, or mixed inoculants into waste, compost, packaging, absorbent articles, and environmental systems, extending the biological degradation mechanisms taught and claimed in co-owned U. S. Patent Application No. 18 / 835,522 to a scalable, single-use format.

[0781] • Example 17 - Comparative Evaluation of Degradable Pouch Materials Containing Fungal Inoculant

[0782] Two different pouch materials were evaluated for their ability to store and release fungal inoculants under controlled laboratory conditions. The first material was a commercial water-soluble heat¬ seal paper composed primarily of a polyvinyl-alcohol-cellulose blend having a thickness of approximately 125 pm and a dispersibility of about six to eight seconds in water. The second material was a polyvinyl-alcohol (PVA) film of comparable thickness used as a reference material. Each sample pouch was produced by heat-sealing approximately one gram of a component comprising dehydrated, pre-colonized milo grain inoculated with the fungal strain A1. 24 sample pouches were prepared, 12 made from water-soluble paper and 12 made from PVA film.

[0783] To evaluate the ability of each pouch to permit fungal emergence, the pouches were placed on plates containing 2 percent malt-extract agar and incubated at ambient temperature. Emergence of fungal hyphae was recorded daily for a period of ten days. Growth of A1 was observed from both pouch types beginning on day four, with initial hyphal emergence from the water-soluble paper pouches generally occurring on days four to five and from the PVA pouches on days five to six. Quantitative surface-coverage analysis showed average emergence of 90 to 100 percent for the paper pouches and 95 to 100 percent for the PVA pouches by day seven, although three of six PVA replicates displayed delayed outward growth. Minor yeast co-growth occurred in several replicates but did not inhibit expansion of the target fungus. Both film types permitted full emergence of A1; however, the paper-based pouches exhibited slightly faster initial growth and more consistent emergence onto the agar surface, suggesting more rapid hydration and nutrient exchange.

[0784] Parallel tests were conducted by embedding single pouches within the absorbent-core layers of unused infant diapers to simulate a practical application environment. Each diaper received approximately 150 mL of an activation solution containing four percent trehalose and 0.5 percent sodium chloride and was then sealed in a moisture-retaining polymer bag. After seven days of incubation at room temperature, fungal colonization was visible through the absorbent layer for both pouch materials. The paper-based pouches showed more extensive mycelial development and stronger attachment of hyphae to surrounding cellulosic fibers, whereas the PVA pouches exhibited fewer visible hyphae and retained more of their original structure. The paper film softened and disintegrated earlier than the PVA film, indicating faster water uptake and earlier release of the inoculum into the surrounding matrix.

[0785] As shown in FIGS. 36A and 36B, both pouch types supported fungal emergence and growth following incubation for ten days on malt-extract agar. In FIG. 36A, the water-soluble polyvinyl-alcohol-cellulose paper pouches displayed dense, uniform surface colonization by the fungal inoculant A1. In FIG. 36B, the PVA pouches exhibited similar but slightly delayed emergence, with

[0786] HIRO / 105 / PC 82 hyphal coverage visible on ail samples by the end of the incubation period. These images illustrate representative growth behavior used to compare film performance in Example 17. The results show that fungal inoculants can be successfully packaged and activated using multiple film chemistries and that the physical properties of the film, including composition and thickness, can be adjusted to control the rate of release and hydration.

[0787] As shown in FIGS. 37A and 37B, degradable pouches containing fungal inoculant A1 were embedded within the absorbent-core region of infant diapers to evaluate colonization and film disintegration under realistic moisture and substrate conditions. Each diaper received an activation solution containing four percent trehalose and 0.5 percent sodium chloride to simulate a humid waste environment. After incubation for seven days at room temperature, both pouch types exhibited visible fungal colonization within the core region.

[0788] As shown in FIG. 37A, the water-soluble polyvinyl-alcohol-cellulose paper pouches demonstrated extensive mycelial growth and clear attachment of hyphae to the surrounding cellulosic fibers of the absorbent matrix. As shown in FIG. 37B, the PVA pouches likewise supported growth of the inoculant but retained more of their original film structure and showed slower surface colonization. These results illustrate the ability of both pouch materials to maintain inoculant viability and release fungal propagules into absorbent substrates following activation.

[0789] Comparable performance is expected from other biodegradable film types such as starch-based blends, regenerated cellulose, polyvinyl-alcohol-starch composites, and water-soluble polyesters. The same principles are applicable to inoculants provided in other forms, including grain spawn, alginate-encapsulated beads, and dehydrated spores. Based on the enzymatic pathways and degradation behavior described in the related hiro103US application for Aspergillus, Ganoderma, and related species, these pouch materials and inoculant combinations can be applied in absorbent articles, packaging, compostable containers, agricultural mulch films, and environmental-treatment systems to achieve controlled biological release and degradation.

[0790] • Example 18 - Effect of Inoculum Amount on Growth and Emergence in Absorbent Articles

[0791] Two fungal species, Ganoderma A1 and Stropharia A4, were evaluated for their growth and emergence capabilities in absorbent articles as a function of inoculum mass per pouch. The example was used to determine whether increasing the amount of inoculum within each degradable pouch would enhance fungal colonization and visible emergence on the surface of the substrate.

[0792] Degradable pouches were prepared as described previously and filled with dehydrated, pre¬ colonized grain inoculum at four loading levels: 2 g, 5 g, 7 g, and 10 g. Each pouch was placed within the absorbent-core region of a standard infant diaper. Approximately 150 ml_ of synthetic urine solution were added to each diaper, which was then rolled, sealed in a moisture-retaining polymer bag, and stored at room temperature for one month. Five replicates were included for each treatment, giving a total of forty diapers across both species.

[0793] After the one-month incubation period, diapers were unsealed and photographed (see FIGS.

[0794] 38A-38H). Fungal colonization was assessed visually and scored using an emergence index ranging from 0 (no visible growth) to 3 (full surface colonization). Both species exhibited clear positive responses to increasing inoculum mass. ForA1, strong growth occurred at 5 g and above, with most replicates achieving complete colonization of the diaper surface. A4 displayed more

[0795] HIRO / 105 / PC 83 gradual growth, with moderate colonization at 7 g and 10 g but lower coverage at smaller inoculum amounts.

[0796] As shown in FIGS. 38A-38H, absorbent articles containing degradable pouches of different fill weights were incubated for one month to assess fungal emergence and colonization within the diaper matrix. Each pouch was positioned in the absorbent-core region and activated by addition of a nutrient and saline solution. After the incubation period, the diapers were unsealed and photographed to document growth intensity, color, and distribution across the surface.

[0797] FIGS. 38A-38D illustrate results for fungal strain A1 at inoculum fill levels of 2, 5, 7, and 10 grams, respectively. Increasing inoculum mass produced progressively greater surface coverage and mycelial density. At relatively higher fill levels, extensive hyphal networks developed within the absorbent layer, accompanied by localized browning and degradation of cellulosic components. FIGS. 38E-38H show results for fungal strain A4 under the same conditions. A4 exhibited slower emergence and less surface coverage than A1 but displayed consistent colonization across all inoculum levels. Moderate hyphal growth and pigmentation were visible after one month, particularly at the 7 g and 10 g fill levels.

[0798] The observed results demonstrate that both fungal species remained viable and metabolically active during extended incubation within absorbent articles, and that pouch inoculum mass directly influences the rate and extent of visible fungal colonization. These observations correspond to the quantitative emergence index data presented in FIG. 39.

[0799] Quantitative analysis confirmed these visual observations. Mean emergence scores for A1 increased significantly with inoculum amount (F = 31.0, p < 0.000001), while A4 also showed a significant though smaller effect (F = 14.9, p < 0.0001). Linear regression models indicated a growth rate of 0.32 emergence units per gram for A1 and 0.13 units per gram for A4. Quadratic and logistic fits revealed a plateau in A1 growth at approximately 7-10 g, suggesting diminishing returns at higher inoculum levels, while A4 continued to increase gradually without a defined saturation point. These patterns are illustrated in FIG. 39, which presents the growth-score versus inoculum-mass relationships with both linear and nonlinear regression fits.

[0800] The visual and statistical results together indicate that A1 responds more strongly to inoculum loading, achieving near-maximum emergence at moderate pouch fill masses. A4, in contrast, exhibits slower but steady increases with additional inoculum, suggesting species-specific strategies for colonization efficiency and substrate utilization. The observed saturation of A1 around 7-10 g implies that smaller pouch fill levels can provide equivalent biological performance, improving efficiency of inoculum usage. A4’s gradual trend, however, may offer advantages under field conditions with variable inoculum distribution or higher moisture stress.

[0801] In addition, the relationship between pouch fill mass and physical thickness was characterized using prototype pouches prepared with varying quantities of grain-based inoculum. Table 1 summarizes typical measurements obtained for pouches containing 2 - 7 grams of pre-colonized grain substrate. Thickness was measured in both the compressed (“flat”) and uncompressed ("thick”) states. These data illustrate that pouch thickness increases approximately linearly with fill weight, providing a simple means of controlling dose per unit area or per packaged article. The resulting dimensional relationships can guide automated filling, sealing, and storage operations for different inoculum loadings.

[0802] HIRO / 105 / PC 84 Table 16. Representative pouch weight and thickness values

[0803] Grain weight (g) Thickness (flat, mm) Thickness (uncompressed, mm)

[0804] 2 4 7

[0805] 3 4 10

[0806] 4 5 11

[0807] 5 6 13

[0808] 6 7 15

[0809] 7 8 17

[0810]

[0811] These findings demonstrate that both species remain viable and active after one month of confinement in absorbent substrates and that pouch inoculum mass serves as an effective control parameter for tuning growth performance. The results further support the scalability of the degradable-pouch delivery system across different fungal species and inoculum densities.

[0812] • Example 19 - Representative Pouch Dimensions and Fill Weights

[0813] The relationship between pouch fill mass and physical thickness was characterized using prototype pouches prepared with varying quantities of grain-based inoculum. Table 16 summarizes typical measurements obtained for pouches containing 2 - 7 grams of pre-colonized grain substrate. Thickness was measured in both the compressed (“flat”) and uncompressed (“thick”) states. The data illustrates that pouch thickness increases approximately linearly with fill weight, providing a simple means of controlling dose per unit area or per packaged article. The resulting dimensional relationships can guide automated filling, sealing, and storage operations for different inoculum loadings.

[0814] It will be understood that the specific materials, concentrations, and process parameters described herein are representative and may be modified as necessary to accommodate different biological systems or end-use environments. The features and combinations disclosed in the present description may be implemented independently or together to achieve equivalent outcomes. All such variations and modifications are intended to fall within the scope of the invention as defined by the appended claims.

[0815] HIRO / 105 / PC 85

Claims

ClaimsWhat is claimed is:

1. A composition, comprisinga biodegradable hydrogel matrix; anda fungal inoculum immobilized within said matrix.

2. The composition of claim 1, wherein the matrix includes alginate.

3. The composition of claim 2, wherein the alginate is crosslinked by at least one divalent cation.

4. The composition of claim 1, further comprising one or more nutrients or additives useful for one or both of maintaining fungal viability during storage and promoting enzymatic activation upon hydration.

5. The composition of claim 1, further comprising at least one carbohydrate osmolyte selected from a group consisting of sucrose, sorbitol, trehalose, maltose, mannitol, glucose, fructose, inulin, dextrin, and combinations.

6. The composition of claim 5, wherein the carbohydrate osmolyte is present in an approximate amount within a range of 0.5-10.0 percent by weight.

7. The composition of claim 1, further comprising at least one nitrogen source selected from a group consisting of yeast extract, malt extract, peptone, casein hydrolysate, soybean hydrolysate, urea, ammonium sulfate, ammonium nitrate, ammonium phosphate, and combinations.HIRO / 105 / PC 868. The composition of claim 1, further comprising at least one secondary polymeric additive selected from chitosan, starch, cellulose, hydroxyethylcellulose, pectin, carrageenan, guar gum, xanthan gum, and combinations.

9. The composition of claim 1, further comprising at least one plasticizer or humectant selected from a group consisting of polyethylene glycol, glycerol, propylene glycol, and combinations.

10. The composition of claim 1, further comprising at least one transition-metal cofactor selected from a group consisting of Fe2+, Mn2+, Cu2+, Co2+, Zn2+, Mo8+, and combinations.

11. The composition of claim 10, wherein the transition-metal ions are introduced by post-curing ion exchange following gelation.

12. The composition of claim 10, wherein the at least one transition-metal cofactor is present in an amount within an approximate range of 0.0001-0.1 percent by weight.

13. The composition of claim 1, wherein the alginate is present in the matrix in an amount within an approximate range of 0.5-5.0 percent by weight.

14. The composition of claim 13, wherein the alginate is present in the matrix in an amount within an approximate range of 0.8-2.0 percent by weight.

15. The composition of claim 1, wherein the divalent cation is selected from a group consisting of calcium, barium, magnesium, zinc, and strontium.

16. The composition of claim 15, whereinthe divalent ion is calcium chloride, andthe calcium chloride is present in a concentration within an approximate range of 25-150 millimolar.HIRO / 105 / PC 8717. The composition of claim 1, wherein the hydrogel matrix takes the form of one or more of beads, microcapsules, films, coatings, and composite inclusions.

18. The composition of claim 1, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penici ilium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.

19. The composition of claim 1, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Pleurotus, Lentinula, Lentinus, Trametes, Ganoderma, Stropharia, Phanerochaete, and Aspergillus.

20. The composition of claim 1, wherein the fungal inoculum is includes one or more fungal species selected from Pleurotus ostreatus, Ganoderma sessile, Lentinula edodes, Trametes versicolor, Phanerochaete chrysosporium, and Ganoderma oryzae.

21. The composition of claim 1, further comprising one or more vitamin or growth cofactor selected from a group consisting of thiamine, riboflavin, niacin, pyridoxine, pantothenic acid, biotin, folic acid, and combinations thereof.

22. The composition of claim 1, further comprising one or more redox mediator or electron shuttle selected from a group consisting of humic acid, fulvic acid, anthraquinone derivatives, ABTS analogs, riboflavin, and combinations thereof.

23. The composition of claim 1, further comprising one or more antioxidant selected from a group consisting of ascorbic acid, citric acid, a-tocopherol, and combinations thereof.HIRO / 105 / PC 8824. The composition of claim 1, further comprising one or more buffering or pH-adjusting agent selected from a group consisting of phosphate-buffered saline, citrate, acetate, bicarbonate, HEPES, and combinations thereof.

25. The composition of claim 24, wherein the composition has an internal pH within an approximate range of 6-8.

26. The composition of claim 1, wherein the hydrogel matrix further comprises at least one filler or porosity modifier selected from a group consisting of silica gel, perlite, diatomaceous earth, biochar, zeolite, powdered lignin, and combinations thereof.

27. The composition of claim 1, further comprising a chelating agent configured to regulate metal-ion availability selected from EDTA, gluconic acid, and combinations thereof.

28. The composition of claim 1, further comprising compatible microbial consortia comprising bacterial spores or enzyme-producing actinomycetes, wherein the compatible microbial consortia is present in an amount of less than 10 percent of the total inoculum volume.

29. The composition of claim 1, further comprising a coordinated combination of osmolytes, nitrogen sources, polymeric structural additives, metal cofactors, and buffering agents configured to preserve fungal viability, enable controlled emergence, and initiate enzymatic biodegradation upon hydration.

30. The composition of claim 1, wherein the fungal inoculum includes fungal spores.

31. A method of producing an encapsulated fungal composition, comprising(a) preparing an aqueous solution of alginate;(b) mixing the aqueous solution with a fungal inoculum to form a pre-gel mixture;HIRO / 105 / PC 89(c) adding a divalent ion solution to the pre-gel mixture to initiate crosslinking of the alginate and form a hydrogel matrix composite; and(d) drying the hydrogel matrix composite to form the encapsulated fungal composition, whereinthe encapsulated fungal composition is storage-stable and capable of rehydration and activation.

32. The method of claim 31, wherein the aqueous solution also includes one or more nutrients or additives.

33. The method of claim 31, wherein crosslinking occurs via external gelation, internal gelation, or a hybrid of external gelation and internal gelation.

34. The method of claim 33, wherein crosslinking occurs via internal gelation with calcium carbonate and glucono-6-lactone to achieve homogeneous crosslinking.

35. The method of claim 31, further comprising a step of infusing with metal cofactors or polymeric coatings after forming the hydrogel matrix composite.

36. The method of claim 31, wherein the step of drying is performed at a temperature below 55°C and a fungal viability is maintained above 85% relative to an original fungal viability prior to drying.

37. The method of claim 31, wherein the hydrogel matrix composite takes the form of beads, and the step of drying is performed over a duration of 1-2 hours to produce a balanced shell density and emergence properties.

38. The method of claim 37, wherein the beads exhibit a swelling ratio within an approximate range of 1.2-2.5 times and an emergence index of at least 4 within 72 hours after hydration.HIRO / 105 / PC 9039. The method of claim 31, wherein the fungal inoculum includes one or more fungai species selected from a group consisting of Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.

40. The method of claim 39, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Pleurotus, Lentinula, Lentinus, Trametes, Ganoderma, Stropharia, Phanerochaete, and Aspergillus.

41. An article, comprisinga substrate; anda composition comprising a biodegradable hydrogel matrix with a fungal inoculum immobilized within said matrix, whereinthe composition is coated on the substrate or combined with the substrate to form a composite,and the composition is configured to activate upon exposure to moisture to initiate fungal emergence and enzymatic degradation of at least one organic or polymeric substrate.

42. The article of claim 41, wherein the matrix includes alginate and one or more nutrients or additives for maintaining fungal viability during storage, promoting enzymatic activation upon hydration or both.

43. The article of claim 41, wherein the composition comprises a matrix of one or more of cellulose, starch, PLA, PVA, PBAT, and combinations thereof.

44. The article of claim 41, wherein the composition is present in an amount within an approximate range of 1-20 percent by weight relative to the total matrix.HIRO / 105 / PC 9145. The article of claim 41, wherein the composition comprises a coating on the substrate having a thickness within an approximate range of 50-200 micrometers.

46. The article of claim 41, further comprising transition-metal cofactors or redox mediators for enhancing enzymatic activity.

47. The article of claim 41, wherein the encapsulated composition remains dormant during storage and activates upon one or more of hydration, pH change, and exposure to composting conditions.

48. The article of claim 41, wherein the article is in a form of absorbent hygiene products, packaging films, fiber mats, and soil-conditioning pellets.

49. The article of claim 41, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.

50. The article of claim 41, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Pleurotus, Lentinula, Lentinus, Trametes, Ganoderma, Stropharia, Phanerochaete, and Aspergillus.

51. A polymeric composition, comprising:(a) a polymeric matrix selected from one or more of a group consisting of thermoplastic polymers, biodegradable polymers, and absorbent polymers; and(b) a fungal inoculum distributed within the matrix, whereinthe inoculum comprises fungal spores or mycelial fragments in a form that remains dormant under dry storage conditions and are activated when exposed to environmental moisture or composting conditions.HIRO / 105 / PC 9252. The polymeric composition of claim 51, wherein the polymeric matrix comprises a thermoplastic material selected from biodegradable aliphatic polyesters, aromatic polyesters, polyolefins, and copolymers or composites thereof.

53. The polymeric composition of claim 51, wherein the polymeric matrix comprises one or more polymers selected from polylactic acid (PLA), polycaprolactone (PCL), polybutylene succinate (PBS), polyethylene terephthalate (PET), polyethylene (PE), polypropylene (PP), biodegradable aliphatic polyesters of similar composition or thermal behavior, and copolymers, blends, or composites thereof.

54. The polymeric composition of claim 51, wherein the fungal inoculum comprises unprotected native spores incorporated directly into the polymer.

55. The polymeric composition of claim 51, wherein the fungal inoculum is coated with one or more protective agents selected a group consisting of trehalose, sorbitol, maltose, starch, chitosan, and mixtures thereof.

56. The polymeric composition of claim 51, wherein the fungal inoculum is encapsulated within a matrix inclusion selected from a group consisting of a hydrogel, alginate, or cross-linked polysaccharide matrix.

57. The polymeric composition of claim 56, whereinthe inclusion has a diameter within an approximate range of 0.5-3.0 mm, and the inclusion comprises a calcium-cross-linked alginate gel optionally including sorbitol, trehalose, sucrose, starch, or polyethylene glycol.

58. The polymeric composition of claim 51, wherein an elevate inoculum contact temperature during fabrication does not exceed 180°C and the residence time at the elevated inoculum contact temperature is less than 3 minutes.HIRO / 105 / PC 9359. The polymeric composition of claim 51, wherein the inoculum is present in a concentration within an approximate range of 0.1 -10% by weight of the polymeric matrix.

60. The polymeric composition of claim 51, wherein the polymeric matrix is formed as a filament, fiber, film, molded part, coating, adhesive layer, or nonwoven web.

61. The polymeric composition of claim 51, wherein the polymeric matrix is thermoplastic and the fungal inoculum remains viable after processing at an equipment setpoint of up to 300°C.

62. The polymeric composition of claim 51, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.

63. The polymeric composition of claim 51, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Pleurotus, Lentinula, Lentinus, Trametes, Ganoderma, Stropharia, Phanerochaete, and Aspergillus.

64. The polymeric composition of claim 51, wherein the polymeric matrix further comprises nutrients, cofactors, or trace minerals configured to promote post-activation enzymatic activity.

65. A method of manufacturing a biologically activated polymeric composition comprising:(a) heating a polymer to form a molten or softened polymer matrix;(b) adding a fungal inoculum comprising fungal spores or mycelial fragments into the molten or softened polymer matrix under conditions that maintain an inoculum contact temperature within an approximate range of 80- 80°C for a residence time of less than 3 minutes to form a polymeric composition precursor; andHIRO / 105 / PC 94(c) forming and cooling the polymeric composition precursor such that the inoculum remains viable and dormant within the matrix.

66. The method of claim 65, wherein the fungal inoculum is added downstream of a primary melt zone of an extruder or spinneret operating at a temperature setpoint up to 300°C.

67. The method of claim 65, wherein the fungal inoculum is added in the form of unprotected spores, coated spores, or encapsulated inclusions.

68. The method of claim 65, wherein the step of forming comprises extrusion, melt spinning, film casting, compression molding, or adhesive coating.

69. The method of claim 65, wherein cooling is performed by forced air, chilled rollers, or ambient quenching at a rate of at least 5°C per second.

70. The method of claim 65, wherein the polymeric composition is collected as a filament, fiber, film, molded article, or nonwoven web.

71. The method of claim 65, wherein the inoculum is encapsulated in alginate beads formed bycross-linking a sodium-alginate solution containing fungal spores with a calcium¬ chloride solution,drying the beads to less than 10% moisture, andincorporating the beads into the molten polymer.

72. The method of claim 65, wherein the fungal inoculum retains viability as determined by emergence or enzymatic activity following hydration.

73. The method of claim 65, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Ganoderma, Pleurotus, Lentinula, Lentinus,HIRO / 105 / PC 95Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.

74. The method of claim 65, wherein the fungal inoculum includes one or more fungai species selected from a group consisting of Pleurotus, Lentinula, Lentinus, Trametes, Ganoderma, Stropharia, Phanerochaete, and Aspergillus.

75. A method of initiating biological activation of a polymeric article comprising a fungal inoculum, comprising exposing the article to moisture, humidity, or composting conditions effective to rehydrate the fungal inoculum, thereby initiating fungal growth or enzyme secretion.

76. The method of claim 75, wherein activation occurs at a relative humidity of at least 80% or in the presence of aqueous media.

77. The method of claim 75, wherein activation yields enzymatic oxidation or hydrolysis of the polymeric matrix.

78. The method of claim 75, wherein activation occurs within 2-7 days at a temperature within an approximate range of 20-40°C.

79. The method of claim 75, wherein the polymeric article is selected from filaments, films, nonwoven webs, molded parts, and coatings comprising a polymeric composition.

80. The method of claim 75, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.HIRO / 105 / PC 9681. The method of claim 75, wherein the fungal inoculum includes one or more fungai species selected from a group consisting of Pleurotus, Lentinula, Lentinus, Trametes, Ganoderma, Stropharia, Phanerochaete, and Aspergillus.

82. A polymeric composition formed by a process comprising steps of:(a) heating a polymer to form a molten or softened polymer matrix;(b) adding a fungal inoculum comprising fungal spores or mycelial fragments into the molten or softened polymer matrix under conditions that maintain an inoculum contact temperature within an approximate range of 80-180°C for a residence time of less than 3 minutes to form a polymeric composition precursor; and(c) forming and cooling the polymeric composition precursor such that the inoculum remains viable and dormant within the matrix, whereinthe fungal inoculum remains viable after storage for at least 30 days at ambient temperature.

83. The polymeric composition of claim 82, wherein the encapsulated inoculum forms discrete visible inclusions within the polymer matrix.

84. The polymeric composition of claim 82, wherein the polymer melt has a shear rate within an approximate range of 100 s"1to 500 s~1.

85. The polymeric composition of claim 82, wherein the polymeric matrix is biodegradable and the composition exhibits enzymatic degradation under composting conditions.

86. The polymeric composition of claim 82, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.HIRO / 105 / PC 9787. The polymeric composition of claim 82, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Pleurotus, Lentinula, Lentinus, Trametes, Ganoderma, Stropharia, Phanerochaete, and Aspergillus.

88. A use of a polymeric composition of claim 82 for promoting biodegradation or nutrient release in polymeric or fibrous materials.

89. A process for the progressive biotransformation of a waste material, comprising steps of:exposing the waste material to a first inoculant comprising one or more fungal species, thereby initiating enzymatic depolymerization of organic, cellulosic or polymeric components in the waste material to form a partially depolymerized intermediate; and exposing the partially depolymerized intermediate to a second inoculant comprising one or more bacterial or algal species, thereby initiating metabolizing the partially depolymerized intermediates to form a biotransformed waste material.

90. The process of claim 89, further comprising a step of introducing an invertebrate organism after the step of exposing the partially depolymerized waste material to the second inoculant, thereby initiating physical fragmentation or mineralization of the biotransformed waste material to convert it to a stabilized residue under environmental conditions of moisture, temperature, and aeration sufficient to support sequential biological activity.

91. The process of claim 89, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.HIRO / 105 / PC 9892. The process of claim 89, wherein the first inoculant comprises a filamentous fungus selected from species Pleurotus, Lentinula, Lentinus, Trametes, Ganoderma, Stropharia, Phanerochaete, and Aspergillus.

93. The process of claim 89, wherein the first inoculant is applied as grain spawn, raw mycelium, or an encapsulated form that hydrates upon exposure to moisture.

94. The process of claim 89, wherein the second inoculant is a bacterial species selected from species Bacillus, Pseudomonas, Azotobacter, and Rhodococcus.

95. The process of claim 89, wherein the second inoculant is an algal inoculant selected from species Chlorella, Scenedesmus, and Anabaena.

96. The process of claim 90, wherein the invertebrate organism comprises larvae of Hermetia illucens, Tenebrio molitor, or an isopod species.

97. The process of claim 89, wherein the inoculants are introduced sequentially over a period of one to thirty days or are provided in stabilized formulations that activate in sequence through hydration, pH change, or substrate degradation.

98. The process of claim 89, wherein the waste material comprises an absorbent hygiene product, packaging article, or composite containing cellulose, superabsorbent polymer, and a biodegradable or partially biodegradable polyester selected from polylactic acid (PLA), polycaprolactone (PCL), polybutylene succinate (PBS), polyethylene terephthalate (PET), polyethylene (PE), polypropylene (PP), biodegradable aliphatic polyesters of similar composition or thermal behavior, and copolymers, blends, or composites thereof.

99. The process of claim 98, wherein the waste material comprises an absorbent hygiene product, packaging article, or composite containing cellulose, superabsorbent polymer, and a biodegradable or partially biodegradable polyester selected from one orHIRO / 105 / PC 99more of polylactic acid (PLA), polyethylene terephthalate (PET), polyethylene (PE), and polypropylene (PP).

100. The process of claim 89, wherein sequential biological activity results in at least partial disintegration of the absorbent matrix and formation of a friable, soil-like residue within 60-120 days under ambient outdoor conditions.

101. The process of claim 89, wherein the first and second inoculants are non-pathogenic and environmentally safe species cultivated using standard substrate-growth or fermentation techniques.

102. A system for progressive biodegradation of a waste material, comprisinga plurality of biologically distinct inoculant formulations arranged to activate in sequence, whereina first inoculant comprises one or more fungal species contained within a first carrier that is biodegradable,a second inoculant comprises bacterial or algal species contained within a second carrier having a slower hydration or degradation rate than the first carrier, wherein the carriers are arranged or combined such that the inoculants become active successively when the system is exposed to environmental moisture or heat.

103. The system of claim 102, further comprising a third inoculant comprising invertebrate eggs or larvae contained within a third carrier configured for delayed activation having a slower hydration or degradation rate than the second carrier.

104. The system of claim 103, whereinthe first carrier comprises alginate, cellulose, or chitosan,the second carrier comprises starch or protein, andthe third carrier comprises wax or chitin.HIRO / 105 / PC 100105. The system of claim 102, wherein the inoculants are packaged within a multilayer article, pouch, or cartridge configured to permit gas and moisture exchange while maintaining species a separation between the first inoculant and the second inoculant prior to activation.

106. The system of claim 102, wherein the first inoculant and the second inoculant are incorporated within an absorbent hygiene product, packaging article, or composting insert such that sequential activation is initiated after disposal.

107. An inoculant system, comprisinga first inoculant in a first matrix;a second inoculant in a second matrix, whereinthe first inoculant and second inoculant are biologically distinct from one another, the first matrix and second matrix possess different hydration or degradation profiles from one another, thereby resulting in sequential activation of the inoculants when exposed to one or both of environmental moisture and temperature conditions,the first inoculant and second inoculant are independently selected from fungi, bacteria, algae, and invertebrates, andeach of the first inoculant and second inoculant is capable of initiating degradation, metabolization, or fragmentation of organic or polymeric waste materials.

108. The system of claim 107, wherein the first matrix and second matrix independently comprise materials selected from alginate, starch, gelatin, chitosan, wax, and combinations thereof.

109. The system of claim 107, wherein the first inoculant and second inoculant are selected and arranged within the composition to replicate ecological succession such that fungal activity precedes bacterial and algal metabolism, and bacterial and algal metabolism precedes invertebrate fragmentation.HIRO / 105 / PC 101110. The system of claim 107, wherein one or both of the first matrix and second matrix includes nutrients, cofactors, or protective agents that maintain viability during storage and promote activation under ambient environmental conditions.

111. The system of claim 107, wherein the first inoculant and second inoculant collectively convert absorbent, polymeric, or fibrous materials into stabilized organic residues suitable for use as soil amendments.

112. A method of manufacturing a successional inoculant system according to claim 107, comprising steps of:cultivating each inoculant species under predetermined growth conditions; stabilizing each inoculant within a respective biodegradable matrix having a predetermined activation profile; andassembling the inoculants and matrices into a multilayer or multi-compartment configuration configured to provide staged activation during exposure to waste material or environmental conditions.

113. An active degradable pouch, comprising:(a) a biodegradable or water-soluble film forming an enclosure; and(b) a biological inoculant composition positioned within the enclosure; wherein the film material is configured to dissolve, disintegrate, or mechanically rupture upon exposure to moisture, composting, or hydration conditions to release the biological inoculant.

114. The pouch of claim 113, wherein the film material comprises polyvinyl alcohol, a polyvinyl-alcohol-cellulose blend, a starch-based polymer, regenerated cellulose, or a water-soluble polyester.

115. The pouch of claim 113, wherein the biological inoculant composition comprises a solid-state substrate inoculated with one or more fungal, bacterial, or algal species.HIRO / 105 / PC 102116. The pouch of claim 113, wherein the biological inoculant composition includes one or more fungal species selected from a group consisting of Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Penicillium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.

117. The pouch of claim 115, wherein the biological inoculant composition comprises a fungal species selected from white-rot, brown-rot, soft-rot, and combinations thereof.

118. The pouch of claim 115, wherein the biological inoculant comprises a mixture of fungal spores and bacterial or algal cells forming a multi-kingdom consortium.

119. The pouch of claim 115, wherein the substrate comprises grain, perlite, alginate beads, hydrogel microcapsules, or combinations thereof.

120. The pouch of claim 113, wherein the biological inoculant is in a dehydrated or dormant state and becomes metabolically active upon hydration.

121. The pouch of claim 113, further comprising at least one abiotic additive selected from a pH buffer, desiccant, nutrient source, metal cofactor, and oxygen carrier.

122. The pouch of claim 121, wherein at least one of the at least one abiotic additive comprises perlite, sodium bicarbonate, trehalose, or sodium peroxide to modulate moisture retention and oxygen availability.

123. The pouch of claim 113, wherein the pouch is configured for inclusion within an absorbent article, packaging component, compost reactor, or soil conditioning system.

124. The pouch of claim 113, whereinthe pouch is formed by heat-sealing, form-fill-sealing, or wet-molding the biodegradable film, andHIRO / 105 / PC 103the biological inoculant composition is filled to a mass within an approximate range of 0.1-10.0 g.

125. The pouch of claim 113, whereinthe pouch defines multiple compartments,a first compartment of the multiple compartments contains a biological inoculant, anda second compartment of the multiple compartments contains a nutrient or abiotic stimulant configured for sequential release.

126. The pouch of claim 125, wherein the first compartment is a relatively inner compartment and includes a fungal inoculant, and the second compartment is a relatively outer compartment and includes a nutrient formulation that dissolves before the inner compartment.

127. The pouch of claim 113, wherein the film includes a coating configured to dissolve at a predetermined pH threshold between 6.5 and 8.5, thereby delaying activation until environmental moisture exceeds the pH threshold.

128. The pouch of claim 113, wherein the pouch remains inert during storage and activates only upon direct contact with aqueous fluid.

129. The pouch of claim 113, wherein the pouch is polygonal, extends laterally a length within a range of 10-40 mm, and has sealed margins extending laterally a length between 1-3 mm.

130. The pouch of claim 113, wherein the pouch has a thickness that increases proportionally with inoculum mass between about 4 mm and 8 mm in a compressed state, and between 7 mm and 17 mm in an uncompressed state.HIRO / 105 / PC 104131. The pouch of claim 113, further comprising an internal reinforcing mesh or biodegradable nonwoven layer configured to maintain form of the pouch prior to dissolution.

132. The pouch of claim 113, further comprising an indicator dye or tracer configured to provide a visual indication of activation of the pouch upon contact with moisture.

133. A method of initiating biodegradation in a waste or compost substrate, comprising:(a) placing at least one pouch according to claim N in or adjacent to the waste or compost substrate; and(b) exposing the pouch to moisture, thereby initiating dissolution or rupture of the film, releasing the biological inoculant, and initiating colonization or enzymatic degradation of the substrate.

134. The method of claim 133, wherein the step of exposing is performed within a sealed disposal bag including soiled absorbent articles, and moisture from the articles triggers dissolution of the pouch film.

135. The method of claim 133, wherein the step of placing includes placing multiple pouches each containing distinct fungal species to initiate successional degradation of organic and synthetic waste components.

136. A system for biological degradation of waste, comprising:(a) at least one absorbent article or waste material;(b) at least one embedded fungal or microbial inoculant in the at least one absorbent article or waste material; and(c) at least one degradable pouch according to claim N positioned for co-disposal with the article, whereinthe embedded inoculant and biological inoculant composition are configured for sequential or cooperative degradation of the waste material.HIRO / 105 / PC 105137. The system of claim 136, wherein the biological inoculant composition includes a fungal species that colonizes cellulosic or polymeric layers of the absorbent article, and the embedded inoculant comprises a secondary species activated by subsequent degradation by-products.

138. An inoculant composition, comprising:an immobilization matrix; andfungal spores encapsulated in the immobilization matrix.

139. The inoculant composition of claim 138, wherein the immobilization matrix is a hydrogel.

140. The inoculant composition of claim 139, wherein the composition is formed as one or more of free alginate beads, coated spore powders, composite filaments, cast films, and spunbond or meltblown fibers.

141. A composite material, comprising:a polymeric matrix substrate; andfungal spores embedded within the polymeric matrix substrate to form the composite material.

142. The composite material of claim 141, wherein the composite material is in the form of a filament, fiber, pellet, film, molded part, coating, adhesive layer, or nonwoven web.

143. The composite material of claim 141, wherein the polymeric matrix substrate is a thermoplastic.

144. The composite material of claim 143, wherein the thermoplastic is selected from a group consisting of polyethylene (PE), polypropylene (PP), polyvinyl chloride (PVC), polystyrene (PS), polyethylene terephthalate (PET), and nylon.HIRO / 105 / PC 106145. The composite of claim 144, wherein the thermoplastic is a polyester or a polyethylene.

146. The composite of claim 145, selected from Polycaprolactone (PCL) and Low-Density Polyethylene (LDPE).

147. A filament composition, comprising:a biodegradable thermoplastic polymer matrix selected from aliphatic polyesters, aliphatic-aromatic copolyesters, or bio-derived polymers having hydroxy- or ester-containing repeating units; anda biological inoculant comprising one or more of fungal spores, mycelial fragments, and encapsulated microbial inclusions.

148. The filament of claim 147, wherein the filament is configured for use as a feedstock in additive-manufacturing or fused-filament-fabrication processes and the inoculant retains viability after melt-processing.

149. The filament composition of claim 147, wherein the polymer matrix comprises polylactic acid, polycaprolactone, or a blend thereof.

150. The filament composition of claim 147, wherein the inoculant is encapsulated within alginate, hydrogel, or another polysaccharide-based matrix that protect the inoculant during melt extrusion.

151. The filament composition of claim 147, wherein the inoculant remains dormant below 40°C and becomes metabolically active upon exposure to moisture or composting conditions.

152. The filament composition of claim 147, wherein the inoculant comprises a fungal species capable of enzymatic depolymerization of polyesters or cellulosic fibers.HIRO / 105 / PC 107153. The filament composition of claim 147, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Ganoderma, Pleurotus, Lentinula, Lentinus, Trametes, Phanerochaete, Aspergillus, Fusarium, Peniclllium, Chaetomium, Schizophyllum, Bjerkandera, Neurospora, Coprinellus, Cordyceps, Stropharia, and Rhizopus.

154. The filament composition of claim 147, wherein the fungal inoculum includes one or more fungal species selected from a group consisting of Pleurotus, Lentinula, Lentinus, Trametes, Ganoderma, Stropharia, Phanerochaete, and Aspergillus.HIRO / 105 / PC 108

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