Waste management using biological and non-biological compositions

Abstract

An inoculant composition having a first fungal species or strain and a different second fungal species or strain. At least one of the first fungal species or strains is a saprophytic fungus. Each of the first fungal species or strain and the second fungal species or strain is present in the composition in a relative concentration such that the fungal biomass is inoculated on one or more polymeric materials in a mass ratio of no greater than 1: 1. Furthermore, the inoculant may include a first engineered fungal species or strain that identifies the plastic product as a food source and decomposes refractory long chain carbons more effectively than the naturally occurring counterpart of the same species or strain. A first engineered fungal species or strain may be immobilized in a metabolic dormant state. A method for converting long-chain carbon material in plastic waste includes adding an inoculum to a plastic substrate.

Description

Background Technology

[0001] This invention relates generally to the field of degradation, and more specifically to material compositions and methods for enhancing the digestion of recalcitrant long-chain carbon materials (e.g., plastics and other materials present with plastic waste products).

[0002] In recent years, the negative environmental impact of plastic waste has received increasing attention. Traditional plastic materials can take hundreds of years to degrade, leading to environmental pollution and ecological damage. This invention addresses this problem by enhancing the decomposition of plastic materials and other synthetic or organic materials.

[0003] This invention provides a method for enhancing the decomposition of said materials. The method includes using a pre-selected fungal species or strain that has been domesticated to decompose recalcitrant polymers. The selected fungal species are fixed in a manner that maximizes efficiency, effectiveness, and ease of use, and can be combined in a mixture of fungal species to achieve the desired decomposition results.

[0004] When fungi are inoculated into a mixture containing plastic materials, they first utilize their hyphae—a network of linear structures that make up the fungal body—to grow and spread on the surface of the material. This growth and spread of hyphae is called hyphal growth. During hyphal growth, the fungi secrete various enzymes that can break down different types of compounds in the plastic material, such as cellulose and lignin.

[0005] This invention relates to the field of liquid soft plastics, such as films and nonwovens, and product compositions thereof, such as liquid absorbent articles, particularly to the field of diapers and other personal waste disposal articles. This invention relates to the development of novel absorbent materials and structures that can be used in a variety of absorbent products. The performance objectives of absorbent products are to provide improved absorbency, liquid retention, and overall performance, while also ensuring comfort, fit, and ease of use. The methods provided eliminate the need for new materials to manufacture more sustainable products, thus removing the trade-off between these performance indicators and sustainability.

[0006] Absorbent products, such as diapers, typically consist of several layers of material designed to manage and contain human or animal waste. The layers in a diaper may include a topsheet, a saturation layer, an absorbent core, a backsheet, and various other materials such as adhesives, elastomers, and fasteners.

[0007] The top layer is typically a nonwoven material made of PE, PP, bicomponent polymers, or other polymers and designed to come into contact with the skin. The wicking layer is designed to quickly draw liquid away from the skin and into the absorbent core. The absorbent core is typically composed of cellulose fibers and superabsorbent polymers (SAPs) to capture and retain liquid.

[0008] The backing layer is typically a thin film made of PE, PP, or other polymers and is designed to prevent liquid leakage from the diaper. Other materials present in the diaper may include hot melt adhesives for bonding purposes, surfactants and dyes for humidity indicators, and various fastening components such as Velcro and elastomers.

[0009] Different types of polymers present in diapers serve specific purposes in diaper design, such as absorbency, liquid management, and containment. SAP is a key polymer in diapers because it can absorb up to hundreds of times its own weight in liquid, making it highly effective at trapping and retaining liquid. Attached Figure Description

[0010] Figure 1A The chemical structures of some of the most common plastics are shown as background to the disclosed embodiments;

[0011] Figure 1B The graphical equation shows that the disclosed inoculum implementation is effective for inoculating plastic materials when the inoculum:plastic weight ratio is less than 1:1;

[0012] Figure 2 Colorimetric analysis was used to demonstrate the production of lignin peroxidase from species S and laccase and manganese peroxidase [C] from species W.

[0013] Figure 3 SEM images showing the effectiveness of the embodiments disclosed in Example 3 are shown;

[0014] Figure 4 This is a SEM image showing that BSNW was effectively inoculated with species W in Example 3;

[0015] Figure 5 Includes a TGA diagram illustrating the effectiveness of the embodiments disclosed in Example 3;

[0016] Figures 6 to 8 Includes DSC diagrams illustrating the effectiveness of the embodiments disclosed in Example 3;

[0017] Figure 9 Includes FTIR plots illustrating the effectiveness of the embodiments disclosed in Example 3;

[0018] Figure 10 An exemplary adaptation process according to this disclosure is illustrated;

[0019] Figure 11 Includes a carbonyl index diagram of the present disclosure embodiments illustrating the decomposition effect;

[0020] Figure 12 Includes FTIR plots showing significant differences between adapted species and non-adapted controls of the same species according to this disclosure;

[0021] Figure 13 An exemplary adaptation process according to this disclosure is illustrated;

[0022] Figure 14 The illustrated equations show that, under treatment conditions (including other wastes including natural polymer waste), a disclosed embodiment of inoculating plastic waste with an inoculant of engineered fungal species at an inoculation ratio of less than 1:1 with a fungal inoculant to waste ratio is effective.

[0023] Figures 15 to 16 Includes FTIR data showing growth regions of the species on diapers under different conditions according to this disclosure;

[0024] Figure 17 It is a photograph showing fungal growth on a diaper with an inoculum having the disclosed implementation scheme;

[0025] Figure 18 This includes FTIR data comparing implementation schemes of adapted species according to this disclosure with their non-adapted counterparts;

[0026] Figure 19 The implementation scheme of the disclosed method is illustrated;

[0027] Figure 20 An embodiment of the species community of this disclosure for waste degradation is illustrated;

[0028] Figure 21 Includes photographs showing the growth of each inoculum mixture of Example 8;

[0029] Figure 22 Including photographs of the species mixture on the petri dish according to Example 8;

[0030] Figure 23 Carbonyl index data for individual and collective samples of various engineered species are shown;

[0031] Figure 24 Data on the occurrence of beads according to this disclosure and Example 8 are shown;

[0032] Figure 25 illustrates the various shape factors applicable to the disclosed embodiments;

[0033] Figure 26 It is a graphical equation showing the dry inoculum of a contaminated polymer product mixed at a low inoculum rate in the presence of a moist contaminant;

[0034] Figure 27 This is a photograph showing the emergence of fungi from beads of fixed encapsulated mycelium in an exemplary waste environment according to Example 10;

[0035] Figure 28 This is a photograph of a culture dish showing the appearance of a nonwoven fabric-bonded and fixed fungal inoculum according to Example 12;

[0036] Figure 29 Including photographs showing the rehydration process and the appearance of the inoculum according to this disclosure;

[0037] Figure 30 Including photographs showing the presence and vigorous colonization of fungal hyphae on diaper materials according to this disclosure;

[0038] Figure 31 The illustration shows a method for growing filamentous fungi on a polymer scaffold to form a storage-resistant product according to the present disclosure;

[0039] Figure 32 Including photographs showing the appearance of fiber scaffold inoculum of engineered species according to this disclosure;

[0040] Figure 33 Including photographs showing the presence of coated fibrous inoculum of engineered species according to this disclosure;

[0041] Figure 34 The illustration shows a method for manufacturing an absorbent product according to the present disclosure;

[0042] Figure 35 Including photographs showing the emergence of fungal inoculum from engineered species of absorbent products according to this disclosure;

[0043] Figure 36 Includes photographs showing the emergence of fungal inoculum from a film container according to this disclosure;

[0044] Figure 37 Includes photographs showing the results of Example 15;

[0045] Figure 38 This is a photograph showing the absorption results of Example 16;

[0046] Figure 39 The illustration shows the unexpected synergistic absorption effect of combining natural and synthetic hydrogels; and

[0047] Figure 40 This is a diagram of a plastic waste fungal inoculum treatment scheme based on a publicly available implementation plan. Summary of the Invention

[0048] Plastic pollution has become a global problem affecting almost every ecosystem. Due to the practicality, widespread use, and ultimate impact of plastic polymers, research on plastic degradation has become a promising field. The types of plastic polymers in use are numerous, with varying chemical compositions, requiring a wide variety of enzymes to achieve complete decomposition into microbial biomass, water, and carbon dioxide.

[0049] The rate of plastic degradation depends on the properties of the individual plastic, such as hydrophobicity, chemical structure, crystallinity, and molecular weight. Plastics with higher molecular weights and a higher proportion of crystalline regions are more difficult to decompose and degrade. However, the relationship between chemical structure and degradation is not so linear. Broadly speaking, there are two classes of plastic polymers: polymers composed of repeating chains of the same chemical elements (referred to herein as homochain) and polymers composed of a more heterogeneous mixture of elements (referred to herein as heterochain). Homochain polymers (such as polyethylene (PE) and polypropylene (PP)) are more resistant to microbial degradation compared to heterochain polymers (such as polyurethane (PU), polyethylene terephthalate (PET), and polystyrene (PS)). Although PE (30%) and PP (19%) are the most commonly produced plastics annually, PET and PU are the two most extensively studied plastics, indicating a need for further research into degradation pathways. The examples of heterochain polymers, homochain polymers, and crosslinked polymers provided in this article are illustrative and serve to demonstrate the various degradation mechanisms specific to each polymer family. These non-limiting examples are classified based on their respective decomposition mechanisms, and as will be readily understood by those skilled in the art, the described methods are applicable to a wider range of materials within these polymer families.

[0050] As background, Figure 1A The chemical structures of some of the most common plastics are shown. These are the main plastics used in diapers. Cross-linked heterochains composed of polyethylene glycol (PEG) and sodium polyacrylate (NaPA) are components of highly absorbent polymers and are present in all absorbent products. The vast differences in chemical composition are one reason why mixed plastics cannot be recycled together.

[0051] Generally, homogeneous polymers are polymers whose backbone consists only of carbon and hydrogen atoms. This structural simplicity indicates a unified bonding pattern focused on carbon-carbon bonds. For enzymatic degradation, homogeneous polymers require an initial oxidation process before hydrolysis can occur. The lack of heteroatoms in their backbone makes direct hydrolysis less feasible, necessitating an oxidation step to introduce functional groups for enzymatic attack. Oxidation can occur through non-biological (e.g., thermal or chemical) or biological means. Considering their composition and degradation requirements, homogeneous polymers are believed to be inherently more resistant to degradation, potentially extending the time required for complete decomposition.

[0052] Heterochains are characterized by the presence of oxygen and other non-carbon, non-hydrogen elements, such as sodium (Na) and nitrogen (N), within the polymer backbone. This diversity introduces a variety of functional groups and bonding arrangements into the polymer structure. The presence of oxygen and other heteroatoms allows for hydrolysis at the inoculation site without prior oxidation. This structural complexity can facilitate enzyme entry and attack, potentially simplifying the degradation process. Heterochains are believed to be more readily degraded than homogeneous chains due to their varied and less uniform structures.

[0053] A unique ensemble of fungi colonizes plastics (i.e., the plastic acts as a community filter on the substrate); these microbial communities are called plastispheres. Which aspects of the plastisphere act as a community filter, and what specific characteristics allow fungi to overcome this barrier, are not yet clear. Certain fungal properties favor plastic colonization and degradation. The production of hydrophobic proteins facilitates colonization on hydrophobic plastic surfaces by forming aerial structures. Hydrophobic proteins enable hyphae to attach to hydrophobic plastic surfaces, and there is evidence that some hydrophobic proteins significantly increase hydrolysis. The production of extracellular polysaccharides further facilitates adhesion to the matrix surface, creating a fungal-matrix interface for enzyme secretion. Most studies on plastic degradation and / or decomposition have been conducted in highly controlled laboratory settings using “raw” and / or pretreated plastics (i.e., without additives or surface texture variations), rather than consumer-grade plastic products under natural conditions, which limits real-world inferences that can be made. Overall, fungi exhibit a variety of enzymatic capabilities for decomposing and / or degrading conventional plastics, but an effective method to increase the decomposition rate of commercial-grade plastic waste remains needed.

[0054] The disclosed embodiments provide methods for degrading various plastic waste products, including absorbent articles, using a preselected fungus capable of breaking down organic and synthetic recalcitrant long-chain carbon. These embodiments provide for the broad commercialization of this method in intended use areas, such as diapers and absorbent products. The fungus can be stored in a shelf-stable form and added to plastic-containing products during manufacturing or after use, promoting environmental sustainability by reducing plastic waste.

[0055] Also provided is a composition comprising a fixed, storage-resistant fungal inoculum that has been domesticated for targeting and decomposing polymers in polymer-based materials. This composition, containing one or more fungal species or strains, can be encapsulated in a fixed material to ensure its stability, prevent contamination, and promote its storage resistance. Upon activation by exposure to moisture, the encapsulated fungi are able to resume their metabolism and promote the decomposition process, providing an eco-friendly solution for the decomposition of various polymer-based materials.

[0056] This paper also provides a method for the industrial-scale decomposition of polymer-based materials (optionally including organic waste) using preselected fungal inoculants. Waste materials are collected and aggregated from various streams, and the fungal inoculant is introduced. Environmental conditions within an industrial-scale waste treatment system (e.g., a bioreactor) are controlled to optimize fungal activity. Optional processing aids may be introduced to promote the growth and colonization of the rehydrated fungal inoculant. The decomposition process results in the production of biomass, water, carbon dioxide, and other byproducts. The resulting biomass can be used for other applications, such as composting, soil amendment, or as a feedstock for renewable energy production. This method provides an efficient, environmentally friendly, and scalable approach for managing polymer-based and organic waste on an industrial scale.

[0057] In another embodiment, the method includes adding a fixed fungal inoculum to the product before or after use. The fungal inoculum is configured to effectively decompose the product at a significantly increased rate. The inoculation process is flexible, allowing consumers to apply the inoculum in various ways, such as directly onto the used product, in a waste container, or in a dedicated decomposition bag. The inoculum can also be added during the product manufacturing process.

[0058] In another embodiment, the material composition comprises a mixture of adapted fungal species and strains, combined into multiple encapsulated fungal inoculants or other immobilization materials, each bead containing a single fungal species or strain. The mixture of fungal species creates a novel “consortium” not found in nature, which promotes enhanced fungal growth and plastic decomposition, and exhibits tolerance to abiotic stresses, including feces, urine, and other conditions. The fungal species are domesticated to recognize plastic products as a food source and degrade recalcitrant long-chain carbon present in synthetic polymers more efficiently than in their naturally occurring forms.

[0059] This article also provides material compositions comprising immobilized, storage-resistant fungal inoculants that have been domesticated to target and decompose polymers present in polymer-based materials. These compositions, containing one or more fungal species or strains, are encapsulated in an immobilized material to ensure stability, prevent contamination, and promote storage resistance. Upon activation by exposure to moisture, the encapsulated fungi are able to resume their metabolism and facilitate the decomposition process, providing an eco-friendly solution for the decomposition of various polymer-based materials.

[0060] The species used in the disclosed embodiments are selected from the white rot, brown rot, and soft rot categories (species W, B, and S, respectively). The classification of these species and their strains is based on properties observed in nature, their morphology, and their genomic potential. Only a small fraction of the enzymatic potential of these fungal species in degrading plastics has been previously characterized. Therefore, the process of screening and adapting these fungi can upregulate the gene expression of known enzymes and activate the expression of enzymes that have not yet been characterized. This complex mixture of enzymes from single and multiple species enhances the degradation of complex polymers. This process involves an iterative approach, including experimental optimization and system operation. The process of selecting, adapting, and combining these fungal species forms the basis of the inventive embodiments disclosed herein, which are measured through experimental characterization of degradation markers on a polymer substrate.

[0061] This article also provides a method for managing the rehydration and activation of dormant, immobilized fungal inoculants in absorbent products. This process ensures optimal fungal performance, improves the product's biodegradability, and maintains its absorbency. The strictly controlled presence of the fungal inoculant allows it to effectively decompose the absorbent product after use, contributing to more sustainable waste management solutions.

[0062] This article also provides a method for encapsulating and immobilizing preselected fungi in discrete beads or on a continuous substrate, facilitating their use in the decomposition of recalcitrant long-chain carbon materials. The method involves homogenizing and optionally filtering fungal hyphae, mixing them with an alginate polymer solution, forming the mixture into beads or hydrogels, and then dehydrating them. The encapsulated fungi can be coated to increase protection and extend shelf life, applicable to a variety of decomposition environments.

[0063] In another embodiment, the method includes using a fixed fungal inoculum, which is added to the product before or after use and is configured to effectively decompose the product within an improved timeframe (preferably, for example, 6-12 months (not limiting)). The inoculation process is flexible, allowing consumers to apply the inoculum in various ways, such as directly onto the used product, in a waste container, or in a dedicated decomposition bag. The inoculum can be added during the product manufacturing process.

[0064] This article also presents a superabsorbent hydrogel encapsulated with a fungal inoculant, designed for use in absorbent products. This hydrogel exhibits high swelling capacity, rapid hydration rate, and excellent liquid retention, while also providing the unique characteristics of a bioactive fungal inoculant. This combination results in absorbent products with the added benefits of enhanced liquid handling performance and improved biodegradability, contributing to more sustainable and environmentally friendly absorbent products.

[0065] Implementation schemes for methods of incorporating fungal inoculants into the production process of absorbent products to improve their biodegradability are also provided. In one embodiment, the fungal inoculant is prepared into particles similar in size to superabsorbent polymers (SAPs). These particles are mixed with a pulp fiber mixture in a hopper and then form the absorbent core of the product. This process is designed to ensure that most of the fungal inoculant particles do not reach the temperature that kills stagnant fungi.

[0066] In an alternative embodiment, the fungal inoculum is embedded in a nonwoven substrate, cut into fragments, and then applied to a partially assembled absorbent product using a vacuum rotary roller. The fragments are secured with a tackifier, adhesive, or mechanical bonding and covered with another protective layer.

[0067] This paper also provides a method for the industrial-scale decomposition of polymer-based materials (optionally including organic waste) using a pre-selected fungal inoculant. In this embodiment, waste materials are collected and aggregated from various streams, and a fungal inoculant is introduced. Environmental conditions within an industrial-scale waste treatment system (e.g., an anaerobic or aerobic decomposer or a bioreactor landfill) are controlled to optimize fungal activity. Optional processing aids may be introduced to promote the growth and colonization of the rehydrated fungal inoculant. The decomposition process results in the production of biomass, water, carbon dioxide, and other byproducts. The resulting biomass can be used for other applications, such as composting, soil amendment, or as a feedstock for renewable energy production. This novel approach provides an efficient, environmentally friendly, and scalable method for managing polymer-based and organic waste on an industrial scale. Detailed Implementation

[0068] The decomposition of polymers can be described using a first-order kinetic model, which correlates the degradation rate with the remaining amount of material through an exponential decay equation:

[0069] C = CO * e^(-kt)

[0070] Where C is the concentration of the remaining material, C0 is the initial concentration, k is the degradation rate constant, and t is time.

[0071] Based on experimental studies, the estimated degradation times of some common polymer materials are as follows:

[0072] · Superabsorbent polymersThese polymers are designed to be highly stable and may take hundreds of years to degrade. For example, studies have found that superabsorbent polymers in soil have a half-life of about 300 years.

[0073] · PE film The degradation time of PE films in the environment is estimated to be several hundred years. A study on the degradation of PE films in seawater found that after 28 months, the weight loss of the film was less than 3%.

[0074] · PE nonwoven fabric Like PE film, PE nonwoven fabrics may take hundreds of years to degrade in the environment. One study on nonwoven fabric degradation found that after 90 days, PE nonwoven fabrics experienced less than 2% weight loss.

[0075] · PP nonwoven fabric PP nonwoven fabric can take hundreds of years to degrade. One study on the degradation of PP nonwoven fabric in soil found that after 180 days, the material lost less than 5% of its weight.

[0076] · Cellulose fiber Cellulose fibers are biodegradable, degrading much faster than synthetic polymers, typically taking months to years, depending on conditions. One study on the degradation of cellulose fibers in soil found that after 90 days, the fibers had lost approximately 30% of their weight.

[0077] As used herein, “immobilization” refers to fixing fungi or bacteria to appropriate locations within a material to facilitate targeted and controlled degradation. As used herein, “storage resistant” refers to an inoculum prepared in a manner that allows it to remain active for extended periods without requiring refrigeration or other special storage conditions. As used herein, “network-transformation stable” refers to an inoculum maintaining its activity throughout the manufacturing process of an absorbent product, even under stress conditions such as high temperatures. As used herein, “rehydration” refers to the process by which a dehydrated fungal inoculum absorbs moisture to reactivate its catabolic processes. As used herein, “emerge” refers to the process by which a fungal inoculum begins to detach from its encapsulation, begins to reactivate, grows, and recognizes the polymer matrix after rehydration or under favorable environmental conditions. As used herein, “fungus” refers to a microorganism used in the degradation process of polymer-based materials. As used herein, “preselected” refers to fungi selected and screened based on their ability to degrade specific types of polymer materials in the presence of biological and chemical contaminants in the treatment environment and to effectively combat these contaminants. As used herein, “adapted” or “engineered” refers to fungal strains or microbial communities that have been genetically or environmentally modulated to enhance their ability to decompose on a specific substrate. As used herein, “colonization” refers to the ability of fungi or microorganisms to settle and multiply on polymer-based materials, initiating a decomposition process. As used herein, “degradation” refers to the biochemical process by which fungi or microorganisms break down complex polymer chains into simpler, less harmful substances. As used herein, “waste stabilization” refers to techniques used to manage waste by minimizing the leaching of toxins and microplastics into the environment. As used herein, “enzymatic decomposition” or “enzymatic degradation” refers to the process by which fungi produce a series of enzymes (and other metabolites) that chemically break down polymers into simpler compounds. As used herein, “metabolism” refers to the process by which fungi absorb carbon during decomposition and convert it from polymers into energy and cellular material. As used herein, “scalable” refers to a process or technology that can be scaled up from a laboratory scale to industrial applications without losing its function or efficacy. As used herein, “community” refers to a group of different microbial species that work together to decompose polymers more efficiently than a single species alone. As used herein, “community composition” refers to a specific composition of a microbial community, typically measured as a percentage of each species in that community. As used herein, “contamination” refers to contaminated or discarded products used for decomposition, particularly those containing biological waste. As used herein, “treatment environment” refers to the environment or conditions under which polymer-based products are treated and decomposition processes are carried out. As used herein, “inoculum” refers to active fungal or microbial cells introduced into polymeric material to initiate decomposition. As used herein, “inoculum rate” refers to the density of microbial or fungal cells introduced per unit weight or volume of substrate, which is crucial for ensuring efficient and scalable decomposition.As used herein, “inoculation” refers to the act of introducing a microbial or fungal inoculant into a polymer material to initiate a degradation process. As used herein, “absorbency” (in relation to liquids) refers to a material’s ability to absorb and retain liquids, relevant to the design of polymer-based materials such as diapers for degradation. As used herein, “retention capacity” (in relation to liquids) refers to a product’s ability to retain absorbed liquids under pressure, which is crucial for the performance of products such as sanitary napkins and diapers. As used herein, “permeable” (in relation to liquids) describes a material that allows liquids to pass through, which can affect the rate of plastic consumption and the efficiency of microbial inoculants. As used herein, “binding” refers to the process of incorporating a fungal inoculant into a polymer matrix during or after manufacturing to promote degradation. As used herein, “encapsulation” refers to the process of directly binding a fungal inoculant into the structure of a polymer-based material to ensure close contact and effective degradation. As used herein, “controlled time release” refers to a formulation feature that allows for the timed release of a fungal inoculant from a product to synchronize degradation with a waste management program. As used herein, “scaffold” refers to a structural framework in which a fungal inoculant is incorporated to promote uniform distribution and effective colonization for degradation. As used herein, “pretreatment” refers to the treatment of polymeric materials prior to the introduction of an inoculant to enhance their susceptibility to decomposition and / or degradation. As used herein, “bioadditive” refers to a nutrient or other compound added to a substrate to support the growth and metabolic activity of a degrading organism. As used herein, “bead” is a particle of unspecified shape or size. As used herein, “filler” material is a non-fungal biomass material included in embodiments of the disclosed inoculant, including but not limited to alginate, trehalose, and nutrient mixtures. As used herein, “hydrogel” is a naturally derived or synthetic material having a three-dimensional network of hydrophilic polymers crosslinked by chemical or physical means and capable of absorbing water. As used herein, “natural hydrogel” includes, but is not limited to, cellulose, chitosan, collagen, alginate, agarose, hyaluronic acid, gelatin, and fibroin. As used herein, “synthetic hydrogel” includes, but is not limited to, poly(hydroxyethyl methacrylate) PHEMA, polyethylene glycol (PEG) hydrogel, polyacrylic acid (PAA), including superabsorbent polymers (SAP). As used herein, "microcolony" or "microcolonies" refers to isolated populations of fungal cells. In embodiments of the present invention, microcolonies comprise individually encapsulated engineered fungal cells, each possessing different properties and behaviors, enabling multiple inoculation sites and enhancing adaptability and functionality compared to natural fungal colonies.

[0078] Engineered fungi that enhance the degradation of polymer-based materials by utilizing pre-selected fungi and the resulting species combinations

[0079] This paper discloses a method for developing fungal species suitable for decomposing plastic materials and resistant to abiotic stress. The development process includes screening potential fungi and mixtures of fungal inoculants with plastic waste products, followed by subsequent engineering modifications to improve decomposition efficiency.

[0080] First, fungal species with the inherent ability to break down plastic materials are screened. Then, the selected fungi are engineered to accept plastic as a carbon source for their metabolic processes and to tolerate abiotic stress conditions, such as feces, urine, and other challenging environments, which are common in used absorbent materials and other polymer-based commercial staples.

[0081] The engineered fungi were able to grow under challenging conditions and utilize plastics as a carbon source. They were subsequently further optimized, including further engineered to increase the rate of plastic decomposition, enabling them to decompose plastic waste efficiently and faster in a variety of environments.

[0082] The engineered modification methods and the resulting engineered fungal products are highly versatile and scalable, applicable to a wide variety of polymer types commonly found in absorbent products and other polymer-based materials.

[0083] In addition to degrading plastics, the disclosed embodiments also include methods for improving the decomposition of non-plastic, cellulose, and human excrement. The immobilized fungi can be combined with other technologies to eliminate environmental toxins, reduce methane production, and fix carbon.

[0084] Compared to known methods of incorporating enzymes into products, the disclosed embodiments using fungi to degrade plastics represent an improvement because they utilize living organisms engineered to continue growing in the presence of abiotic stressors and other fungal species using polymers as carbon sources at relatively low inoculation rates. Known methods and techniques typically rely on producing enzymes in a laboratory or under controlled conditions, which can be time-consuming, costly, and unsuitable for typical commercial environments.

[0085] In contrast, the disclosed implementation utilizes the natural ability of fungi to produce enzymes and break down plastic materials. Even at low inoculation rates, the fungi are able to grow and spread rapidly, allowing for large-scale applications. Unlike laboratory-produced enzymes, fungi are able to adapt to changing environmental conditions and continue to grow and produce enzymes for extended periods.

[0086] The examples shown and described below demonstrate the effectiveness of the disclosed engineered fungal embodiments in breaking down polymers present in diapers faster than fungi that are not engineered according to the disclosed embodiments. Based on experimental studies, the estimated natural degradation time of ordinary diaper materials (without using the disclosed embodiments) is as described above.

[0087] The disclosed embodiments focus on the degradation of absorbent products (e.g., diapers) and the various material components used in their construction. Specifically, the embodiments demonstrate the ability of fungal species / strains or combinations thereof to target and degrade various types of polymers common in absorbent products, including films, elastomers, nonwovens, heat-fused materials, and superabsorbent materials. As disclosed herein, selected and engineered fungal strains specifically target and degrade these polymers, resulting in highly efficient and effective products and technologies to improve the degradation of absorbent products, even in the presence of abiotic stressors.

[0088] In the presence of human excrement and other abiotic stressors in absorbent products, the ability of fungal strains to target and break down various types of polymers is applicable to other types of absorbent products, including but not limited to adult diapers, feminine hygiene products, and other absorbent products designed for human or animal use. Fungal strains can be engineered to specifically target and break down the polymers used in each type of absorbent product, thus providing an efficient method for degradation and treatment.

[0089] The ability of fungal species / strains or combinations thereof to decompose absorbent products also extends to other product categories, such as plastic bags, packaging, textiles, and other common polymer-based materials. As shown herein, the disclosed methods and compositions are applicable to targeting and decomposing polymers present in these materials, thus providing an efficient and versatile decomposition and treatment approach.

[0090] The disclosed methods and products represent a significant advancement over existing technologies in the field of biodegradable absorbent products. Unlike some published studies, the disclosed embodiments can be operated outside of laboratory settings, enabling consumer-scale commercial applications. Furthermore, the disclosed embodiments have achieved the degradation of materials such as superabsorbent polymers (SAP). Additionally, the disclosed embodiments do not require material pretreatment, making them a more practical and effective solution for plastic waste management. Other advantages of the disclosed embodiments include:

[0091] • The use of engineered fungal inoculants provides a scalable solution for degrading polymer components in absorbent products without altering any plastic materials.

[0092] Compositions engineered to decompose long-chain carbon in plastics by fungal species provide efficient decomposition of a variety of polymer types.

[0093] The disclosed implementation scheme is effective for a broad range of commercially available types of plastic waste (i.e., without a pretreatment step for the plastic).

[0094] The disclosed implementation scheme is engineered to target and break down plastic components within absorbent products, unlike existing research which shows that fungi grow on the natural polymer components of diapers (such as cellulose), but rather actually breaks down the plastic present therein.

[0095] This article also discloses a method for expanding the scale of preselected fungi via hyphal amplification through bioprocesses including bio-fermentation to produce large-scale transportable fungi that enhance the decomposition of recalcitrant long-chain carbon materials.

[0096] A seed inoculum for clonal or direct progeny engineered saprophytic species is provided. This seed inoculum is combined with a nutrient mixture tailored to the specific metabolic needs of the selected fungal species. A bioreactor facilitates maximum mycelial amplification. Once maximum amplification is reached, the resulting fungal biomass is homogenized after a filtration process, if necessary. The homogenized mycelium is then mixed with a polymer solution (e.g., alginate) for further processing. An encapsulation and fixation process is subsequently applied to ensure the stability of the fungal inoculum. The processing technology for engineered saprophytic fungi allows for the transformation of enhanced decomposition and / or degradation methods from laboratory-based concepts into commercially viable, scalable operations.

[0097] The disclosed embodiments relate to the decomposition of absorbent products (e.g., diapers) and various material components used in their construction. Specifically, the embodiments demonstrate the ability of engineered fungal species / strains or combinations thereof to target and decompose various types of polymers present in absorbent products, including films, elastomers, nonwovens, hot-melt materials, and superabsorbent materials.

[0098] Films are commonly used in absorbent products as layers to prevent liquid leakage. These films are typically composed of polyethylene (PE), polypropylene (PP), or other suitable polymers. Elastomers are used in absorbent products to provide stretch and fit, and are typically composed of synthetic rubber or other suitable materials. Nonwoven fabrics are used in absorbent products as top sheets or wicking layers and can be composed of various polymers such as PE, PP, or bicomponent (bico) fibers. Hot-melt materials are used in absorbent products as adhesives and are typically composed of polymers such as polyethylene or polypropylene. Superabsorbent materials are a key component of absorbent products and are typically composed of cross-linked polymers such as sodium polyacrylate.

[0099] By selecting and then engineering fungal strains to specifically target and decompose these polymers, efficient and effective decomposition of absorbable products can be achieved.

[0100] Importantly, the ability of fungal strains to target and break down various types of polymers in the presence of human excrement and other abiotic stressors in absorbent products translates into a wide range of absorbent products. This includes, but is not limited to, adult diapers, feminine hygiene products, and other absorbent products designed for human or animal use. Fungal strains can be engineered to specifically target and break down the polymers used in each type of absorbent product, thus providing efficient decomposition and disposal methods.

[0101] The ability of fungal species / strains or combinations thereof to decompose absorbent products also extends to other adjacent categories, such as plastic bags, packaging, textiles, and other polymer-based materials. The methods and compositions of this invention are applicable to targeting and decomposing polymers present in these materials, thus providing an efficient and versatile decomposition and treatment method.

[0102] Furthermore, the examples demonstrate the feasibility of immobilizing and rehydrating the fungal inoculum to maintain its efficacy. A scaled-up fungal inoculum was prepared using 2% sodium alginate of medium viscosity and homogenized mycelium, with the percentage of alginate ranging from 1-4% (low and medium viscosity). This mixture was used in Examples 1-3 below.

[0103] Example 1: Identification and screening of fungal species used to decompose diaper materials

[0104] The embodiments involve the utilization of fungal species, each of which has been identified and its efficacy studied. These fungi possess saprophytic capabilities adapted to advance sustainable biological processes. Fungi play a vital role in natural ecosystems by decomposing organic matter, including complex polymers present in wood, such as lignin, cellulose, and hemicellulose. The disclosed embodiments utilize the unique saprophytic capabilities of specific fungal species, which have been further identified as: white-rot fungi, brown-rot fungi, and soft-rot fungi (hereinafter referred to as species W, species B, and species S, respectively). The use of fungal species with these unique capabilities in a unique manner forms the basis of the embodiments of the invention disclosed herein, and fungal species are frequently described herein with reference to these species categories.

[0105] White-rot fungi (species W)These fungi are characterized by the production of large amounts of oxidases, enabling them to break down lignin, a complex aromatic polymer found in wood. They possess robust hyphae and secrete hydrophobic protein metabolites, which contribute to their colonization and stability. These species have a moderate emergence lag but grow and colonize rapidly. Taxonomically, they belong to the phylum Basidiomycota, primarily the class Agaricales, including the orders Agaricles, Hymenochaetales, and Polyporales. Specific species used include *Pleurotus ostreatus*, *Pleurotus djamor*, *Dichomitus squalens*, *Trametes versicolor*, *Phanerochaetechrysosporium*, *Lentinula edodes*, and *Phellinus pini*.

[0106] Brown-rot fungi (species B) Brown-rot fungi are known for their high production of hydrolytic enzymes that efficiently break down cellulose and hemicellulose in wood, leaving behind a brown, crumbly lignin residue. These fungi excel at overcoming microbial contaminants and typically have a high emergence lag time, but once established, they exhibit moderate growth and colonization rates. They also belong to the phylum Basidiomycota and class Agaricales, with representative orders including Gloeophyllales and Polyporales. Utilized species include *Inonotus obliquus* and *Fomitopsis spraguei*.

[0107] Soft-rot fungi (species S)Soft-rot fungi thrive in harsh conditions where other fungi might not survive, such as in wood with high moisture content or in environments with extreme temperatures or pH levels. These fungi exhibit good compatibility with other fungal species, making them useful in diverse microbial communities. They have a moderate to rapid emergence lag time, but grow and colonize slowly. Taxonomically, they belong to the phylum Ascomycota, primarily the classes Dothideomycetes and Sordariomycetes, encompassing the orders Hypocreales, Xylariales, and Eurotiales. Specific species used include *Aureobasidium pullullans*, *Pestalotiopsis microspora*, *Aspergillus* sp., *Fusarium* sp., *Aspergillus versicolor*, and *Aspergillus fumigatus*.

[0108] Table 1: Taxonomic and Functional Classification of Species

[0109]

[0110] Table 2: Examples of the pre-selected fungal species

[0111]

[0112]

[0113] Fungal degradation of polymers refers to the extracellular process by which fungi break down complex polymer materials. This process involves several steps: fungi first sense and recognize the polymer as a food source, then colonize it and form a biofilm. Within this biofilm, fungi secrete extracellular enzymes, such as oxidases and hydrolases, which trigger the degradation of the polymer into intermediate compounds. These intermediates are then assimilated by the fungi. Therefore, as used herein, the term "degradation" encompasses the entire sequence of recognizing, colonizing, enzymatically degrading, and assimilating polymer materials.

[0114] Table 3: Fungal decomposition of polymers

[0115]

[0116] The following details the stages of the decomposition process and the relevant engineering processes and procedures used in the disclosed engineering modification implementation schemes for the engineered fungi:

[0117] IdentificationFungi recognize polymers as potential food sources through chemical signals or unique structural components. The engineered modification process disclosed herein involves using previously degraded polymers to induce fungal hyphae to extend toward the polymer, rather than ignoring it. To determine if the recognition phase is occurring, fungal growth toward the polymer can be observed by inoculating nutrient media petri dishes with fungi and placing small pieces of polymer at a distance from the inoculum. In comprehensive field studies of polymer products, fungal colonization on polymer surfaces can be monitored through visual inspection and microscopic observation to confirm polymer-oriented growth.

[0118] colonization After identification, the fungi begin to colonize, attach, and grow on the polymer surface. This stage prepares the polymer for complete degradation by developing fungal hyphae and establishing a stable environment. To determine if colonization is occurring, visual observation and microscopy can be used to detect the attachment and growth of fungal hyphae on the polymer surface. The area of ​​fungal growth can be calculated by measuring the degree of colonization, and the growth rate can be determined by tracking the expansion of the fungal network over time.

[0119] Biological membrane secretion Within biofilms, fungi secrete a variety of extracellular substances, including enzymes and hydrophobic proteins. These adaptations enable fungi to rapidly secrete mixtures suitable for specific polymers. In addition to visual observation and microscopic examination, colorimetric assay kits can be used to detect the presence of enzyme products during fungal colonization on plastics. These techniques confirm biofilm formation and the active participation of fungi in the decomposition process.

[0120] Biological degradation Fungi mechanically weaken polymers via oxidases, forming carbonyl groups. This method also targets crosslinked and amorphous groups in the polymer to promote further decomposition, particularly for homogeneous chains resistant to immediate hydrolysis. Tensile testing can be used to determine changes in polymer strength and elasticity, indicating structural weakening. Furthermore, FTIR can be used to detect changes in functional groups within the polymer structure, particularly identifying oxidation by detecting an increase in the intensity of the OH bands compared to a control.

[0121] Biological decomposition - Hydrolytic enzymes cleave polymer carbon chains, producing intermediates such as oligomers. Fungi are adapted to produce the necessary enzymes to break down polymers into intermediates suitable for assimilation. Biodegradation can be identified using various analytical methods for polymers. FTIR is used for homogeneous chains, where an increase in the carbonyl index indicates the breaking of the polymer backbone. For heterogeneous chains, DSC is used to detect thermal stability and properties such as crystallinity. TGA is best suited for crosslinked polymers (SAP) to identify mass loss in the sample during heating, indicating decomposition and fragmentation.

[0122] Biological assimilation Intermediate products of biodegradation, such as oligomers, are absorbed by fungal cells. These intermediate products are then metabolized, providing energy and building blocks for fungal growth. Fungi possess a unique ability to initiate and catalyze biodegradation and decomposition. Once these steps are identified, fungi can absorb intermediate byproducts. This absorption was demonstrated and observed through continuous metabolism when polymers were inoculated at low inoculum rates, indicating that fungi rely on this carbon source for survival.

[0123] mineralization Hydrolysis products are transferred within the cell wall and converted into microbial biomass. The continuous life cycle and sustained growth on the polymer substrate indicate that the fungi efficiently utilize plastic as a food source and continue all previous processes. Mineralization was determined by the sustained growth on the polymer product at low inoculation rates, demonstrating that the fungi efficiently utilize degraded polymer components as a carbon source for ongoing metabolic activity and further colonization of the substrate.

[0124] Polymer testing

[0125] The disclosed embodiments are primarily, but not exclusively, directed at the field of liquid absorbent products, particularly diapers and other personal waste disposal items. Furthermore, embodiments of novel absorbent materials and structures are disclosed, which can be used in a variety of absorbent products to provide improved absorbency, liquid retention, and overall performance, while ensuring comfort, fit, and ease of use. The methods presented herein do not require new materials to achieve more sustainable products, thus eliminating the trade-off between performance metrics and sustainability.

[0126] The polymers used in the methods and products described herein are categorized into homochain, heterochain, crosslinked polymers, and copolymers. Homochain polymers, such as polyethylene (PE) and polypropylene (PP), are not readily hydrolyzed and require oxidation for decomposition and degradation. These materials were tested in various diaper components, including backing nonwoven fabrics, backing films, and leg cuffs. Heterochain polymers, including polyethylene terephthalate (PET) and polyurethane (PU), are more readily hydrolyzed but have complex structures. These were tested in the acquisition and distribution layer (ADL) and elastic thread, respectively.

[0127] Crosslinked polymers and copolymers also play a crucial role in the disclosed methods and products. The superabsorbent polymer (SAP) used is a hydrogel crosslinked from sodium polyacrylate (NaPA) and approximately 5% polyethylene glycol (PEG). This hydrogel is designed to enhance liquid retention. Furthermore, copolymers (such as acrylonitrile butadiene styrene (ABS) copolymers) are used in adhesive applications, combining the properties of different monomers to improve adhesive performance.

[0128] The materials tested in this paper encompass a wide range of polymer families used in various diaper components. For example, the backing nonwoven material made of polypropylene (PP) and the backing film made of polyethylene (PE) represent homogeneous polymers. The collection and distribution layer composed of polyethylene terephthalate (PET) and the elastic thread made of polyurethane (PU) represent heterogeneous polymers. The adhesives composed of SAP (a crosslinked hydrogel) and ABS (a copolymer) further demonstrate the range of materials used.

[0129] Absorbent products (such as diapers) consist of multiple layers designed for specific functions. The topsheet, typically a nonwoven material made of PE or PP, is in contact with the skin. The wicking layer rapidly draws liquid into the absorbent core, which is composed of cellulose fibers and SAP to trap and retain the liquid. The bottomsheet, also made of PE or PP, prevents leakage of liquid from the diaper. Additional components include adhesives, elastomers, and fasteners to ensure functionality and a proper fit.

[0130] This versatile approach allows these materials to be scaled and adapted to other combinations within absorbent product layers. Furthermore, the methods and materials developed in this invention can be widely applied to a variety of plastic products, providing scalable solutions for waste management (not limited to absorbent products).

[0131] Table 4: Diaper Materials

[0132]

[0133] Example 2: Diaper Material Decomposition

[0134] In Example 2, 30 fungal species from the white-rot, brown-rot, and soft-rot fungal categories were mixed with superabsorbent polymer (SAP) at progressively decreasing inoculation rates, starting at a fungal inoculum:plastic weight ratio of 99:1; then 10:1; then 4:1; and finally 1:1. From this adaptation process, 12 species were selected that effectively recognized and colonized SAP as a food source. The adapted cultures were then applied at a 1:1 inoculation rate to various diaper materials: topsheet nonwoven fabric (PP), bottomsheet nonwoven fabric (PP), core-wrapping material (PP), bottomsheet film (PE), and SAP. Average growth rates were observed over 30 days. Figure 1 A flowchart of Example 2 is provided, along with photographs of petri dishes showing the significant growth of species S and species W, and growth rate data of species W on different plastic materials.

[0135] To confirm biofilm secretion and enzyme production, fungal species and strains were compared with known polymer-degrading proteins to assess their ability to be cultured in TS and gene expression. Qualitative assessment of specific enzyme activities was performed using colorimetric analysis.

[0136] • Laccase: The formation of a dark green halo on a plate supplemented with ABTS indicates that laccase secretion is positive.

[0137] • Lignin peroxidase: The formation of a clear halo on a plate supplemented with azure B indicates a positive result for lignin peroxidase secretion.

[0138] • Manganese peroxidase: The formation of a reddish-brown halo on a plate supplemented with guaiacol indicates a positive result for manganese peroxidase secretion.

[0139] Qualitative analysis showed that enzymes from adapted fungal species were produced in a diverse and abundant manner in the presence of plastic. Figure 2 Colorimetric analysis was described to demonstrate the production of lignin peroxidase from species S and laccase and manganese peroxidase [C] from species W.

[0140] Example 3: Optimization of inoculum for diaper material decomposition

[0141] In Example 3, a 1:1 inoculum-to-plastic weight ratio was used, meaning the weight of the fungal biomass and nutrient compounds was equal to the weight of the plastic waste being treated. This ratio is important because it confirms that the fungi have sufficient resources to effectively colonize and decompose the plastic material. In a preferred embodiment of the inoculum, described in more detail below, inoculating the plastic material with an inoculum-to-plastic weight ratio less than 1:1 is effective, such as... Figure 1B As shown in the representative example.

[0142] The wet inoculum used in Example 3 comprised 4% fungal biomass and 96% cross-linked sodium alginate. Fungal biomass was cultured from three screened and adapted fungal species, species S and W (two species S and one species W). This inoculum was applied to each individual layer of the diaper and tested for 30 days on the following materials: backsheet nonwoven fabric, backsheet film, front ear nonwoven fabric, receiving area, leg liner nonwoven fabric, topsheet nonwoven fabric, adhesive, collection and distribution layer, elastic thread, and SAP. Environmental control samples were tested alongside the fungal-treated samples, and the results of these treatments were compared to the original (bulk) undegraded / uncomposted material. Degradation results for each material layer were analyzed, including bioactivity and polymer characterization. Data showed that these enzymes produced by the adapted focal fungal strains degraded polymers from homo-chain, hetero-chain, and cross-linked polymer families. The data also showed changes in the physical and chemical structure (oxidation) and polymer bond breaking (hydrolysis) of the fungal treatment compared to the original bulk material and environmental control, indicating that the adapted fungal samples successfully identified, colonized, secreted, biodegraded, and biodecomposed.

[0143] Furthermore, the mycelial growth and enzyme activity present on the surface of the respective diaper materials provided evidence of degradation. All tested diaper materials were successfully identified and colonized by one or more adapted species. Figure 3 This is an SEM image showing that TS was effectively inoculated by species S (the thin lines running through the matrix are fungal hyphae). Figure 4 This is a SEM image showing BSNW effectively inoculated with species W (the decomposition of the polypropylene nonwoven material can be seen).

[0144] Thermogravimetric analysis (TGA) revealed that fungal-treated samples containing SAP species W (NaPA crosslinked with PEG) exhibited poorer thermal stability across all test phases compared to the original SAP control and environmental control samples. Figure 5 As shown, the fungal-treated samples exhibited a significant initial weight loss of approximately 95-96% at around 100°C, indicating high hydrophilicity, likely due to increased porosity or surface area resulting from fungal decomposition of the cross-linked structures. Further weight loss occurred between 100°C and 500°C, indicating polymer bond breakdown; smaller, continuous weight losses suggest the decomposition of shorter polymer chains (a result of fungal decomposition) at lower temperatures. In the high-temperature range (500°C–1000°C), the fungal-treated samples left approximately 2.08% residue, the smallest amount found in the samples, indicating complete decomposition of the organic structures. These results clearly demonstrate the poor thermal stability of the fungal-treated samples across all temperature ranges, highlighting the significant impact of fungal decomposition on SAP.

[0145] Furthermore, differential scanning calorimetry (DSC) results showed significant decomposition in the hot melt adhesives exposed to environmental conditions and treated with fungi of species W. Figure 6 As shown, both the environmental control and fungal-treated samples exhibited lower glass transition temperatures (Tg) compared to the original binder, indicating that decomposition was due to a decrease in molecular weight or structural changes. The environmental control sample showed crystallization behavior with an initial temperature of 37.74 °C and a peak temperature of 71.76 °C in the first heating cycle, while fungal treatment significantly altered this behavior, indicating structural changes. Furthermore, the environmental control sample showed reduced crystal content in the second heating cycle, with an initial temperature of 92.53 °C, a peak temperature of 100.09 °C, and a specific energy of 1.712 J / g, while the fungal-treated sample showed no melting peak, indicating significant decomposition and the elimination of crystalline regions. These results collectively suggest that both environmental exposure and fungal treatment decompose the binder, with fungal treatment causing more significant structural changes and a complete loss of crystallinity.

[0146] Similarly, as Figure 7As shown, the DSC results of the elastomers, compared to the original and environmental control samples, highlight the significant decomposition in the fungal-treated (species W) samples. While the original elastomers exhibited minimal decomposition between cycles, indicating a stable structure, the environmental control samples showed increased crystallinity with a higher specific energy (8.129 J / g during the first heating cycle), suggesting that environmental conditions enhanced the polymer's structure, with slight decomposition between cycles. Conversely, the fungal-treated samples exhibited moderate crystallinity (3.921 J / g during the first heating cycle) and significant decomposition compared to the original and environmental controls. The stable structure of the fungal-treated samples between heating cycles indicates that the initial fungal treatment induced significant structural changes, leading to significant decomposition and moderate crystallinity, compared to the other samples.

[0147] Figure 8 The DSC results of the PET nonwoven layer are shown, revealing significant decomposition in the fungal-treated sample (species S) compared to the original sample and the environmental control sample. The environmental control sample showed slight decomposition and a slightly reduced specific energy during the endothermic peak (melting), indicating a slight reduction in crystal content. In contrast, the fungal-treated sample maintained a high specific energy during the endothermic peak, indicating a smaller impact on crystallinity, but exhibited a secondary melting peak, suggesting complex thermal behavior and the possible formation of different crystalline phases or structures due to fungal treatment. These findings suggest that while the original sample exhibits stable crystallinity and thermal properties, the fungal-treated sample displays significant structural changes and more complex thermal behavior.

[0148] Fourier transform infrared spectroscopy (FTIR) was used to analyze diaper materials before and after inoculation with species W, and the results are as follows: Figure 9 As shown. FTIR results indicate that exposure to fungi reduced and oxidized the carbon backbone of the material, providing additional evidence beyond environmental factors that could cause decomposition. (See reference...) Figure 9 Sections A and B respectively show the polyethylene and polypropylene samples corresponding to undecomposed, environmentally decomposed, and fungal decomposed samples. Environmental decomposition is due to UV and heat. In polypropylene and polyethylene, 2900 cm⁻¹ -1 The sharp peak at 1460 cm⁻¹ corresponds to the CH stretching vibration of the methyl group, representing a long carbon backbone. Additionally, at 1460 cm⁻¹... -1 and 1375cm -1 The peaks at 1740 cm⁻¹ represent the CH₂ and CH₃ minor groups on the polypropylene. This is indicated by the reduction of these peaks, and by the peaks at 1740 cm⁻¹. -1 (carbonyl), 1100cm -1 (ethers and alcohols) and 3350cm -1 The new peak formed at (alcohol) indicates evidence of oxidation.

[0149] Example 4: Adaptation of fungi to polymers

[0150] Figure 10 An exemplary adaptation process is illustrated in general, and the original fungal species 100, engineered by adapting to plastic 112 and then further to abiotic stress 114, is described in general. Subsequently, the biomass of the adapted fungal species is recovered from the agar plate and used to form inoculum. In Example 4, species S is adapted to decompose polyethylene (PE), and species W is adapted to decompose polypropylene (PP). In the process of engineering this adaptation, the fungi gradually adapt to using these complex polymers as their primary carbon source by systematically reducing access to simple carbon sources such as glucose and sucrose.

[0151] The adaptation process begins with the preparation of a mixture of preselected polymers (species S using PE and species W using PP) and carbon nutrient food sources. Initially, the fungi utilize the polymers and the additional food source for growth and metabolism. Over several generations, the concentration of the additional carbon nutrients gradually decreases. This forces the fungi to rely more heavily on the polymers as their primary carbon source, thereby increasing the expression of enzymes required to break down the polymers. This systematic process produces engineered fungal species that utilize plastic carbon to grow faster than their naturally occurring counterparts. Fungal species can be further engineered to more readily accept certain types of polymer materials as carbon sources for metabolism via a similar sequential process.

[0152] Each generation was cultured for 2 weeks before being transferred to the next culture (with the carbon concentration in the medium reduced). The contents of each generation in Example 4 were determined as follows.

[0153] • Generation 1 (100% carbon): 5g glucose, 5g malt extract, 2.5g yeast extract, 0.5g MgSO4·7H2O, and 1g KH2PO4 in 500mL of ultrapure water.

[0154] · Second generation (75% carbon): 3.75g glucose, 3.75g malt extract, 1.875g yeast extract, 0.5g MgSO4·7H2O, and 1g KH2PO4 in 500mL ultrapure water.

[0155] ·3rd generation (50% carbon): 2.5g glucose, 2.5g malt extract, 1.25g yeast extract, 0.5g MgSO4·7H2O, and 1g KH2PO4 in 500mL ultrapure water.

[0156] ·4th generation (25% carbon): 1.25g glucose, 1.25g malt extract, 0.625g yeast extract, 0.5g MgSO4·7H2O, and 1g KH2PO4 in 500mL ultrapure water.

[0157] • Generation 4.1 (15% carbon): 0.75g glucose, 0.75g malt extract, 0.375g yeast extract, 0.5g MgSO4·7H2O, and 1g KH2PO4 in 500mL ultrapure water.

[0158] • Generation 4.2 (10% carbon): 0.5g glucose, 0.5g malt extract, 0.25g yeast extract, 0.5g MgSO4·7H2O, and 1g KH2PO4 in 500mL ultrapure water.

[0159] • Generation 4.3: 0.5g glucose and 0.5g yeast extract in 500mL ultrapure water.

[0160] ·5th generation (0% carbon): 0.0g glucose, 0.0g malt extract, 0.0g yeast extract, 0.5g MgSO4·7H2O, 1g KH2PO4 in 500mL ultrapure water.

[0161] Before transferring to the next generation of cultures, the fungal strains were cultured for 2 weeks in each generation with their respective plastic polymer fragments. First, the plastic polymers were removed, all fungal mycelia were scraped off, and the mycelia were dried in a dehydrator at 95°C for 12 hours. FTIR data collected during this process showed an increase in the carbonyl index, which is an indicator of the degradation of PE (species S) and PP (species W) polymers. Figure 11 As shown in the figure, FTIR results indicate that the decomposition activity of the corresponding adapted fungal species increases significantly with the reduction of simple carbon sources, demonstrating the enhanced ability of fungi to decompose complex polymers.

[0162] Example 5: Field Test

[0163] The efficacy of the engineering modification / adaptation was verified through field trials (Example 5). In these trials, 0.3 g of fungal inoculum identified as species S1 and W1 was mixed with 30 g of polymer product. Adapted and non-adapted inoculum conditions were tested. The mixture was placed in an open environment and recovered after six weeks. Film backing (BSF) made of polyethylene (PE) and nonwoven backing material made of polypropylene (PP) were recovered and tested.

[0164] Figure 12 The FTIR spectra revealed significant differences between adapted and unadapted species S1 and W1, respectively. The adapted inoculum exhibited increased oxidation peaks and more significant polymer structure changes. Adapted species S1 and W1 successfully decomposed polyethylene and polypropylene, respectively, highlighting the scalability from fungi maintaining enzyme expression outside of laboratory conditions to real-world treatment environments.

[0165] Example 6: Adaptation to Pollutants

[0166] Example 6 focuses on the adaptation of fungi to abiotic stressors (e.g., urine) to enhance their performance on substrates (e.g., plastics, urea, and feces). This example demonstrates the adaptation of fungi to saline environments, focusing on growth, development, and enzyme activity at different salt concentrations. A diagram illustrating abiotic stress adaptation processes similar to those used in Example 6 is shown below. Figure 13 As shown. Species W adapted according to Example 4 above was cultured on potato dextrose agar (PDA) and liquid medium. The fungus was gradually exposed to salt concentrations of 0.45%, 0.9%, and 1.8% in the liquid medium, with a positive control (salt-free) and a negative control (no inoculum). The cultures were incubated at 30°C and monitored every 3 days for 43 days, measuring biomass and enzyme activity. Every 3 days, 15 mL of liquid was extracted from each wide-mouth bottle for analysis. Parameters (e.g., pH, conductivity, salinity, redox potential (ORP-MV), and total dissolved solids (TDS)) were measured from the liquid culture to monitor environmental conditions and fungal metabolic activity. These parameters indicated that the fungus continued to grow without dying. The fungus adapted well to the increased salinity, exhibiting significant growth and enzyme activity at the highest salt concentration. The engineered fungal species adapted to plastic and abiotic stresses form the basis of the disclosed embodiments and are used in the following examples.

[0167] Example 7: Combining field trials of contaminated products and inoculum

[0168] like Figure 14 As shown, the disclosed implementation of inoculating plastic waste with an inoculum of engineered fungal species at an inoculation ratio of less than 1:1 with fungal inoculum to waste under “on-site” conditions including other wastes including natural polymer waste is effective. Example 7 demonstrates that the inoculum is effective under conditions where the product is completely contaminated and the fungal inoculum remains in the open environment. 650 contaminated diapers of the same composition were collected from daycare centers. The diapers were inoculated with different percentages of fungal inoculum. The type of contamination, whether urine or feces, was recorded. Nine engineered species of each of species W, S, and B were tested, with three replicates of each condition placed in a bucket. The wet inoculum used in Example 7 contained 4% fungal biomass and 96% crosslinked sodium alginate. According to the above examples, fungal biomass was cultured from screened and adapted fungal species and scaled up using liquid fermentation. The mycelial biomass was recovered, uniformly mixed with 2% sodium alginate, and extruded into a calcium chloride solution for crosslinking to obtain beads with a diameter of approximately 3 mm. It is worth noting that the recorded inoculation ratios only include the weight of dry diapers and do not include the weight of contaminated items.

[0169] Samples were collected one month later for FTIR analysis, and each sample was observed and collected three months later for other analyses. Figure 15 The total growth area on the diaper is shown, and the types of contamination (urine, urine, and feces) for all species are illustrated with box diagrams and filament diagrams, demonstrating that the engineered fungi in the inoculum can colonize over biotic and abiotic contaminants.

[0170] Figure 16 The vaccination rates are shown as 40%, 20%, and 10% (vaccination rates are as follows). Figure 14 The total growth area (as shown in the figure) indicates that the fungal inoculant implementation scheme can effectively colonize even at lower inoculation rates.

[0171] One month later, significant fungal growth was observed, and two months later, species W had completely penetrated the diaper tub. Figure 17 The photo of species W is shown.

[0172] The carbonyl index (the change in intensity or area of ​​carbonyl groups in an FTIR spectrum) is used as a quantitative data point to measure the level of plastic decomposition (as an alternative to general decomposition). The presence of carbonyl groups indicates that the polymer has been decomposed and that further decomposition is favorable due to the instability of carbonyl groups. The carbonyl index can be calculated from the FTIR spectrum by analyzing peaks indicating the presence of carbonyl and methylene groups (see Equation 1 below). Due to oxidation during decomposition, methylene groups are reduced and eventually converted to carbonyl groups. The amount of decomposition can be assessed by comparing the carbonyl index (CI) of the decomposed sample with that of the undecomposed control sample.

[0173]

[0174] FTIR data confirmed that all species showed signs of polymer degradation after one month, with carbonyl index (CI>1) calculations indicating better degradation compared to the control. After two months, the carbonyl index increased significantly to an average of 6, while PP still maintained CI>1, but no significant increase was observed. Figure 18 As shown, all engineered species (species S and species W) exhibited higher decomposition efficiency than their respective unengineered controls.

[0175] Two months later, the samples were also analyzed by differential scanning calorimetry (DSC). These samples were composites of film film (PE) and film nonwoven fabric (PP), and the method used a heating cycle to calculate crystallinity. Both species W samples showed significantly reduced crystallinity, indicating substantial decomposition. For PE, species W3 exhibited 79.96% crystallinity, while species W4 showed 69.08%. For PP, species W3 showed 31.59% crystallinity, while species W4 showed 92.25%.

[0176] Table 5: Decreased crystallinity of fungal species W sample

[0177]

[0178] This paper also discloses an implementation scheme for a composite fungal community configured to enhance the decomposition of polymer waste products. The pre-selected fungi are not naturally occurring in a specific combination, allowing adaptation to biotic and abiotic factors. The community is designed to identify and utilize symbiotic relationships among its members, providing an effective and robust inoculum under diverse environmental conditions. The importance of this community lies in its ability to synergistically enhance the decomposition of recalcitrant polymers, ensuring the efficient decomposition of complex polymer mixtures commonly found in waste products.

[0179] Figure 19 The composition of the fungal community is described, comprising two or more pre-selected fungi. These fungi were selected based on their ability to generate a vibrant community in the treatment environment, synergistically enhancing the decomposition of recalcitrant polymeric materials. This community is capable of handling complex polymer products containing multiple types of polymers. The selectivity of the community members is highlighted, emphasizing their advantages in a given product mixture environment. These fungi were selected from pre-screened species, adapted to characteristics observed in nature, ensuring that the enzymatic and metabolic capabilities of each member are optimized for polymer decomposition.

[0180] Figure 20 A unique mixture of fungal communities is shown, demonstrating how it can be modulated to target specific polymer products. For example, products with a higher proportion of homogeneous chains require more oxidase-producing fungi compared to products primarily containing heterochain and natural polymers. Community members are mixed in proportion to ensure efficient colonization and decomposition. This encapsulated mixture is configured to emerge and colonize from all members (even at scaled-down inoculum ratios). This ratio is a function of factors such as doubling time and emergence lag time, highlighting the complexity of producing an efficient species mix at inoculum ratios less than 1:1 (and in some cases less than 0.1:1). The disclosed embodiments ensure efficient polymer decomposition communities even at reduced inoculum concentrations.

[0181] · Example 8 Species community inoculum mixture formulation

[0182] Example 8 illustrates a fungal species mixture formulation in which the fungal species effectively colonize and work synergistically without competing with each other. The lag time of emergence, growth rate, and compatibility of each species were considered and analyzed, affecting the proportion of each species in the inoculum mixture to ensure effectiveness. Four species S and two species W were mixed in various proportions according to Table 6 below. The total weight of the beads inoculated into each petri dish was 10 g.

[0183] Table 6: Species Ratio Mixture

[0184] Species Mixture 1 Mixture 2 Mixture 3 Mixture 4 <![CDATA[Species W1]]> 25％ 25％ 30％ 20％ <![CDATA[Species S1]]> 25％ 15％ 10％ 10％ <![CDATA[Species S2]]> 25％ 25％ 20％ 30％ <![CDATA[Species W2]]> 25％ 35％ 40％ 40％

[0185] The observations showed that mixtures 1 and 2 produced effective growth from species S1 and W1, while producing little or no growth from species S2 and W2. Mixture 3 showed effective growth from all species except S2. The results for mixture 4 were inconsistent; however, one replicate showed significant growth from all four species. Figure 21 Includes photographs showing the growth of each mixture in Table 6 after one week.

[0186] The mixture was optimized to ensure balanced growth of the three species W and species S. Figure 22 The images include photographs of fungal growth from these optimized mixtures, demonstrating balanced and efficient growth of all selected species.

[0187] Example 9A: Testing of Species Community Inoculum Mixtures

[0188] In Example 9, two species W and two species S were tested on contaminated diapers using 10% inoculum, mixed separately and with each other to form a mixture, with each treatment repeated 12 times. The diapers and their replicates were placed together in a bucket and buried in the surrounding soil. PE film samples were retrieved after one and two months for FTIR analysis. Figure 23 The carbonyl indexes for each species individually and collectively are shown. Box plots and whisker diagrams indicate that while some individual species do achieve carbonyl indices exceeding 1, species mixtures yield the most consistent and efficient results, demonstrating the synergistic effects achieved by the species community. This highlights the importance of species diversity in real-world environments where fungi must compete with biotic and abiotic stressors.

[0189] • Example 9B: Testing of Species Community Inoculum Mixtures

[0190] In Example 9B, the highest-performing single species from Example 9A was tested, separately, with the highest-performing three-species mixture and the highest-performing four-species mixture. Samples were collected three months later for DSC analysis and cut to obtain PP nonwoven fabric, PE film, and PET nonwoven fabric layers. Table 7 shows that species W3 and W4 demonstrated effective degradation of PE and PP, but failed to colonize and degrade the PET layer. The three-species mixture showed moderate effectiveness in degrading PE, was effective in degrading the PET layer, but did not colonize on the PP nonwoven fabric. The four-species mixture was effective for all three layers, most effective for PE and PET, but less effective for PP than W3 alone. Example 9B demonstrates that the inoculum implementation combined with multiple engineered fungal species in the community for degrading a group of polymers achieved synergistic results without inhibiting the degradation of other polymers. Species W was more effective for homogeneous polymers but less effective for heterogeneous polymers, while species S was effective for heterogeneous polymers. A mixture of four species was required to degrade all three polymers.

[0191] Table 7: Crystallinity decreased after 3 months

[0192]

[0193] In practice, inoculants containing fungal communities of the disclosed engineered fungal species have been shown to effectively inoculate contaminated diapers at initial concentrations of less than 1% fungal biomass:plastic ratio. These engineered fungal species have undergone the disclosed treatment, are more accepting of plastic materials as a metabolic carbon source, and are more tolerant to abiotic stresses. Preferably, the engineered fungal species and strains and / or communities of engineered fungal species and strains are engineered to inoculate at an initial mass ratio of fungal biomass:plastic no greater than 1:1. More preferably, the engineered fungal species and strains and / or communities of engineered fungal species and strains can be inoculated at an initial mass ratio of fungal biomass:plastic no greater than 1:10. Embodiments have shown that effective inoculation is achieved at initial mass ratios of fungal biomass:plastic from 1:10 to 1:200. Unlike known treatment methods that rely on applying enzymes to plastic materials, the disclosed implementation scheme utilizing communities of engineered fungal species and strains is self-sustaining, in which the fungal species can effectively continue to grow, thereby expressing more enzymes and effectively using the plastic material as a carbon source for further reproduction and growth.

[0194] Immobilized fungal inoculants: generation, control, and binding of polymer-based material decomposition

[0195] As described above, in another embodiment, the immobilized composition comprises an engineered fungal inoculant capable of targeting and degrading polymers present in polymer-based materials. These compositions have proven to be storage-resistant, and crucially, can be reactivated for use. In a particular embodiment, the composition comprises a number of fungal species, strains, or combinations thereof, which are kept quiescent by dehydration or lyophilization. This provides an extended shelf life without compromising the effectiveness of promoting the degradation of polymer-based materials upon reactivation from the immobilized state.

[0196] This composition can be taken as a standalone product configured as an additive or the like, or incorporated into a variety of polymer-based materials, such as disposable diapers, sanitary napkins, incontinence pads, wound dressings, wiping products, and other liquid or solid waste management products. It can be added in the form of encapsulated alginate beads or other substrates, allowing for precise delivery of units and protecting against fungal contamination.

[0197] This article describes a method for forming a storage-resistant fungal inoculum specifically designed for decomposition applications. The formulation involves encapsulating filamentous fungi in a stable form with an encapsulating material capable of gradual rehydration and activation in the presence of moisture. This method ensures that the fungi remain in a metabolically dormant state until environmental conditions are suitable for their activity, thereby facilitating their use in a variety of conditions. The fungal species used are primarily filamentous fungi, selected due to their robustness and broad adaptability to adequately moist environments.

[0198] The encapsulation techniques employed in this study are important for the stability and efficacy of the inoculum. Fungi can be encapsulated with or without a polymer matrix and can be fixed using only preservatives such as trehalose. A dehydrated state is achieved, where the fungi are preserved in an anhydrous state. Encapsulated particles prevent premature emergence and are formulated into beads using alginate (1-5%). Optimization factors (e.g., percentage of biomass, fragment size, and potential nutrients) ensure fungal viability and functionality. Crosslinking density is adjusted to influence bead strength and porosity, thereby allowing for proper growth and respiration. Genetic stability at room temperature is ensured. Delivery forms may include discrete beads 122, tablets 124, sheets 126 (similar to dried sheets deposited via fiber deposition), Velcro strips, capsules 128 (similar to laundry detergent capsules or pharmaceuticals), nonwoven bags, foam deposited articles 130, film deposited articles 132, and absorbent nonwoven components, generally illustrated in Figure 25.

[0199] Following rehydration and activation, the inoculum gradually absorbs moisture through the controlled microenvironment generated by the complex. The addition of hydrophilic abiotic components to the composition facilitates more aggressive absorption of liquid moisture, while the mixture of live mycelial fragments and hygroscopic components absorbs moisture from the environment, including water vapor. For delayed activation and optimal microenvironment, spores can be incorporated into the composition. The rehydration process allows for the safe rehydration of the fungal cell wall, re-initiating metabolic processes and enabling the mycelial tips to grow and detach from the encapsulating substrate. This prompts the fungi to search for food sources in their environment, ultimately leading to the efficient decomposition of waste material. Table 8 below details the various stages of preparation, storage, waste mixture, rehydration, and metabolic activation, highlighting the interactions of biotic and abiotic factors in this innovative inoculum delivery system.

[0200] Table 8

[0201]

[0202]

[0203] Example 10: Preparation and testing of fungal inoculum

[0204] The fungal inoculum of strain W, engineered according to the above method, was prepared by harvesting biomass from liquid fermentation and homogenizing it with a 2% sodium alginate mixture. Beads were formed by cross-linking the mixture by extruding it into a calcium chloride solution. The beads were then coated with trehalose and freeze-dried until moisture was removed. Different concentrations of fungal biomass, ranging from 2% to 20%, were tested in the mixture.

[0205] Figure 24 The percentage of beads appearing from each mixture after 7 and 10 days is shown, demonstrating an increase in appearance with increasing biomass content. This data illustrates the ability to control appearance through mixture composition. Quality assurance and quality control (QA / QC) were performed on the 2% and 4% fungal mixtures, as the lower limit represents the effectiveness of the minimum biomass dosage. These mixtures were allowed to appear after 14 days, and the results showed that over 90% of beads had successfully appeared after 14 days. This example demonstrates the effectiveness of the preparation method and the ability to control fungal appearance based on the composition of the inoculum mixture.

[0206] This wet formulation includes essential biological elements, such as fungal mycelial biomass or spores, mixed with preservatives (e.g., trehalose) to stabilize and protect the fungi. Depending on the application, other components, including conditioned media, polymer encapsulation matrices, processing aids, and secondary nutrients, are added to ensure optimal fungal growth and activity during decomposition. These components are described in more detail in Table 9 below.

[0207] Table 9: Components of the wet inoculum mixture

[0208]

[0209] Fixation techniques are used to dry wet mixtures, preserving the viability and functionality of fungal components. Methods such as dehydration, freeze-drying, lyophilization, and spray drying remove moisture from the mixture, producing a stable, dry product that is easy to store and use in a variety of applications. These techniques are described in more detail in Table 11 below.

[0210] Table 11: Fixing Technology

[0211]

[0212] The dried mixtures produced by the immobilization method can be used as is or further enhanced by mixing with other compounds. This includes the addition of other immobilized bio-elements, hydrophilic and hygroscopic complexes, fillers, disintegrants, and desiccants to form comprehensive compositions suitable for specific decomposition and / or degradation applications. Table 12 below describes the components of the dried inoculum in more detail.

[0213] Table 12: Components of dried inoculum

[0214]

[0215] Unlike any known technology, the dried inoculum according to the disclosed embodiments is storable and can be mixed with waste products at a low weight ratio. This method allows for effective decomposition and / or degradation in real-world conditions, whereas in clean laboratory conditions, the product typically consists of a mixture of synthetic and natural polymers contaminated with waste. By taking into account the complexity of these products, the method ensures effectiveness in practical, non-laboratory environments. For example, in the presence of moist contaminants, dried inoculum at an inoculum rate of less than 10% of the synthetic polymer weight, combined with contaminated polymer products, exhibits significant decomposition capabilities, such as... Figure 26 As shown.

[0216] Example 10: Encapsulated Polymer Matrix

[0217] In Example 10, a storage-resistant encapsulation of filamentous fungi was established, focusing on the use of a protective polymer matrix and a carefully formulated mixture of fungal hyphae. 1% of wet fungal biomass from an engineered basidiomycete of species W was used; however, in this embodiment, the concentration can reasonably range from 0.1% to 50%. The biomass was mixed with a 2% sodium alginate solution of medium viscosity to form a homogeneous mixture. To produce the encapsulation matrix, the alginate-fungus mixture was cross-linked with calcium chloride, resulting in the formation of uniform beads with a wet diameter of approximately 5 mm. These wet beads were then freeze-dried to improve their storage stability and stored with a desiccant to prevent moisture absorption during storage. Testing showed that the freeze-dried encapsulated fungi successfully emerged within 14 days when placed on petri dishes and in contaminated diaper environments. The specific percentages of the protective polymer matrix and the fungal hyphae mixture are important for maintaining the fungi in a stagnant state. This composition allows for slow, controlled rehydration, ensuring gradual reactivation of the fungal metabolism. This process prevents damage to the cells from rapid rehydration and protects the fungi from contamination by biological or abiotic moisture present in the treatment environment.

[0218] Repeat the above procedure using the same components and techniques, and place the dried encapsulated beads into the soiled diaper core. Figure 27 The photograph shows fungi emerging from these fixed encapsulated mycelium beads in an exemplary waste product environment.

[0219] Notably, storage-resistant encapsulated fungi were prepared using non-adapted basidiomycete species according to this method, further confirming the effectiveness of this method on basidiomycetes.

[0220] Example 11: Encapsulation with refrigerant

[0221] In Example 11, trehalose was used as a cryoprotectant to enhance the stability and viability of fungal biomass, encapsulating filamentous fungi without cross-linking. 1% of wet fungal biomass from an engineered basidiomycete of species W was used; however, concentrations from 0.1% to 50% can be reasonably used, as in Example 11. Instead of cross-linking, the fungal biomass was mixed with trehalose to create a cryoprotective environment. The mixture was then processed into a stable composition. Unlike bead formation, the fungal biomass bound to trehalose, combined with a hydrophilic filler component, ensured a consistent and protective matrix. The mixture was then freeze-dried to maintain the fungi in a storage-resistant state and stored with a desiccant to keep it dry. This embodiment was successfully rehydrated when exposed to moisture and combined with the hydrophilic filler component. The presence of trehalose as a cryoprotectant allowed for slow and appropriate rehydration of the fungal cells, avoiding damage caused by rapid moisture absorption. The hydrophilic filler played a crucial role in ensuring controlled rehydration, which is essential for the gradual reactivation of fungal metabolism.

[0222] Example 12: Encapsulated mycelium in a dry state for appearance and adhesion

[0223] In Example 12, the efficacy of trehalose and alginate as encapsulation methods for fungal inoculum was evaluated, with particular attention paid to the appearance and adhesion of the encapsulated inoculum on various nonwoven materials. This example demonstrates that encapsulated / fixed mycelia in a dried, stagnant state can be effectively reactivated and thus effectively used, proving its application potential in polymer-matrix encapsulation and trehalose encapsulation forms. Fungal inoculum of 96% alginate and 4% biomass (species S and species W) was mixed with trehalose and alginate powders, respectively, to produce an inoculum concentration of 20%. These mixtures were thoroughly mixed to ensure uniform distribution of the inoculum in the powder. The encapsulated inoculum powder was then spread in duplicate on agar plates and labeled accordingly. Different nonwoven materials (loops, films, baby wipes) were selected as substrates for adhesion experiments. Each material was divided into portions, labeled, and sprayed with an adhesive before application of the encapsulated inoculum powder. Control plates without adhesive were also prepared. After inoculation, the appearance of fungal colonies was detected and recorded. The results showed that both trehalose and alginate-encapsulated inoculum powders successfully adhered to the nonwoven material and exhibited significant fungal emergence. The trehalose and alginate encapsulation methods were comparable in their effectiveness in promoting fungal emergence. However, subtle differences in growth patterns and adhesion efficiency were noted on different substrates. Control plates confirmed that adhesive spraying enhanced the adhesion and subsequent emergence of the encapsulated inoculum.

[0224] Therefore, Example 12 demonstrates the feasibility of using encapsulated mycelium in a dry and storage-stable state to promote fungal emergence and adhesion on nonwoven substrates. The results indicate that trehalose and alginate are both effective encapsulation methods with potential applications in incorporating fungal inoculants into polymer-based products to enhance decomposition (see reference). Figure 28 (This shows the presence of nonwoven fabric-bonded and fixed fungal inoculum).

[0225] As described above, the dried inoculum described herein can be formed into various end-product shape factors to suit different applications, as shown in Figure 25. These include discrete beads or powder forms 122, tablets 124, dried sheets, Velcro strips, laundry detergent capsules 134, and nonwoven bags, as shown in Figure 25. Furthermore, the inoculum can be incorporated into foam / sponge components 130, absorbent nonwoven components 126, and Listerine strip-like nonwoven ultrasonic bonds 132. These different delivery forms enhance the versatility and scalability of the inoculum, enabling its effective use in a variety of waste decomposition environments. All of these shape factors demonstrate the efficacy of allowing the fungal inoculum to rehydrate and activate through gradual rehydration over time. In addition, the product contains nutrients that provide a controlled microenvironment conducive to the rehydration process. These may include hygroscopic trehalose for higher activity concentrations. The presence of hygroscopic components in the live mycelial fragments serves to absorb moisture from the surrounding environment. Furthermore, spores may be included to provide delayed activation. When combined with a desiccant, the disclosed composition products exhibit genetic stability at room temperature.

[0226] The fungal microcolonies used in the beads can be as small as 0.5 micrometers, representing the smallest viable hyphal fragments that cannot survive in nature without novel inoculum engineering. By isolating and engineering these microcolonies to survive and reproduce even at such a small size, the inoculum composition can be efficiently distributed over large areas with minimal material, reducing costs and increasing efficiency. This approach allows for a more controlled and consistent inoculation process, making it ideal for industrial and commercial applications involving polymer degradation.

[0227] In nature, fungal growth typically manifests as interconnected networks of hyphae or large, dispersed colonies, originating from spores that spread and colonize over a wide area. These natural colonies exhibit uniformity in their development and interactions with the environment. In contrast, the microcolonies in this inoculum composition are not derived from spores but are engineered and recovered from the biomass harvest of specifically engineered fungi. Each encapsulated microcolony is isolated, allowing it to develop unique properties and behaviors. This engineering process results in multiple microcolonies with different tendencies, allowing them to adapt to their specific microenvironments. This diversity within the microcolonies produces a more robust and adaptable community, enhancing the overall efficacy and function of the inoculum composition in decomposing polymer materials.

[0228] • Example 13A: The emergence of fungal inoculum compositions

[0229] In Example 13, nonwoven materials embedded with encapsulated mycelium and tablets containing encapsulated mycelium were prepared, each encapsulated with engineered species S. The absorbent pads were hydrated, but no additional liquid was added to the mixture. This assembly was then placed in a water tank. Figure 29 The photographs, including those showing the rehydration process and the appearance of the two inoculums after 5 and 14 days respectively, demonstrate the introduction of rehydrated water and the subsequent appearance of fungi.

[0230] • Example 13B: The emergence of fungal inoculum compositions

[0231] Example 13B tested tablets with a composition similar to that of Example 13A. The test environment was a soiled diaper placed in an outdoor bucket. The bucket was opened after 18 days. Figure 30 This includes photographs showing the appearance of fungal hyphae and their extensive colonization on diaper material.

[0232] Scaffold encapsulation for fungal fixation

[0233] This embodiment relates to a method for culturing filamentous fungi on a polymer scaffold, and then encapsulating them to produce a storage-resistant product, such as... Figure 31 As shown. This method and the resulting articles allow fungi to adapt to various polymer types, increasing their occurrence rate and viability. The encapsulated fungi can be applied to films, nonwovens, foams, and other polymer-based materials.

[0234] Scaffold types include nonwoven materials in direct contact with fibers or films, enhancing the absorbability of the encapsulated living organisms. The growth and encapsulation process involves growing filamentous fungi into the nonwoven scaffold, utilizing a new technology to provide a larger surface area for better adaptation. This process incorporates polymers to promote efficient fungal metabolism.

[0235] Scaffolds can take various forms, including foams, granules, and actual plastic resins, in which fungal spores are embedded. Both solid and liquid encapsulation methods are used to ensure fungal viability and effectiveness. This approach allows for a wide range of applications, including the treatment of films, nonwovens, foams, and other polymer-based materials. The process of growing filamentous fungi into nonwovens and encapsulating them (whether or not) provides a versatile and effective method for decomposition and / or degradation in a variety of environmental contexts.

[0236] Example 14: Solid-state fermentation combined with non-woven fabric

[0237] In Example 14, solid-state fermentation was used to colonize engineered filamentous fungi. The fungi were guided to produce aerial hyphae, which bonded to fibers in a nonwoven material placed on top of the fungal culture. This process ensured complete bonding between the fungal hyphae and the nonwoven fibers, forming a robust physical scaffold. The entire nonwoven sheet, now colonized with fungal hyphae, was then bonded to a sodium alginate mixture. This mixture provided additional structural support and protection. The bonded sheet was subsequently cross-linked in a calcium chloride solution, forming a robust, immobilized structure. This method provides the fungal hyphae with a physical scaffold for attachment, structural support for immobilization, and a directional microenvironment for controlled rehydration. Testing showed that this change successfully rehydrated, resulting in the emergence and effective colonization of the fungi on the material. As the metabolic processes of the fungi, which were dormant during immobilization, were reactivated, enzyme activity accelerated, promoting rapid degradation of the polymer scaffold. Figure 32 This includes a photograph showing the appearance of a fiber scaffold inoculum of the engineered species W after 7 days.

[0238] Example 15: Coating fibers with an alginate-mycelium mixture

[0239] In Example 15, instead of simply growing mycelia on the nonwoven material, a sodium alginate mixture with a specific viscosity and mycelial size was prepared. This preparation ensured that the alginate-mycelium mixture could completely coat the fibers of the nonwoven material. The alginate-mycelium mixture was then applied to the nonwoven material, allowing the fungal mycelia to bind to the fibers. This coating process provides a protective layer around the fibers. The entire composition was crosslinked using calcium chloride to produce a solid, immobilized structure. This method and the resulting article also ensured that the fungi had a physical scaffold for support and a directional microenvironment for controlled rehydration. This modification, as in Example 14, was tested to successfully rehydrate and allow the fungi to emerge and effectively colonize the material. The fungal metabolic processes were preserved during the immobilization process and reactivated upon rehydration, thereby accelerating the breakdown of the polymer scaffold through enzymatic activity. Figure 33 This includes a photograph showing the appearance of the engineered species W's fiber-coated inoculum after 7 days.

[0240] Example 16: Absorbable product with embedded, storage-resistant fungal inoculum

[0241] In Example 16, the diaper-like liquid absorbent product comprises multiple layers, including a liquid-impermeable backsheet, an absorbent core of short fiber pulp and superabsorbent polymer encapsulated in a nonwoven core wrapper, a collection and distribution layer, and a polypropylene topsheet, such as... Figure 34As shown. A control product was prepared using the same structure, but without any fixed living organisms, with 1g of superabsorbent polymer and 2g of film, nonwoven fabric, and adhesive added. Several test samples were also prepared to test the shape factors mentioned above: one sample containing 1g of inoculum and engineered fungal species S in the form of dried alginate beads added to the core mixture; one sample containing 3g of dried engineered mycelial species S embedded in the core envelope; one sample containing 1g of the same alginate beads in the core and 3g of the same mycelium in the core envelope; and one sample with a "receiving area" on the substrate, including a polymer film layer, 1g of encapsulated mycelium, and a nonwoven layer bonded to the substrate.

[0242] To confirm the effectiveness of these absorbent products as usable products, all samples underwent liquid retention and rewetting tests. For liquid retention, the product was immersed in water for one minute, hung to dry for two minutes, and the retained liquid was calculated by subtracting the dry weight from the wet weight. The rewetting test involved placing dry filter paper on the product, applying a 1 kg weight for 10 seconds, and comparing the wet and dry weights of the filter paper to measure moisture retention.

[0243] In addition, the presence of fungi was observed by placing the samples in an aquarium; the presence of fungi was defined as exceeding 1 cm. 2 Visible growth was observed. As expected, the control samples showed no fungal growth, while the samples with encapsulated engineered fungi showed different emergence times for different shape factors: 11 days for alginate beads, 5 days for embedded mycelia, and no growth in the outer attachment layer. The results are summarized in Table 13 below.

[0244] Table 13: Absorbance and emergence time of embedded fungal inoculum

[0245]

[0246] Liquid retention results showed that the control sample absorbed 43.79 g of liquid. Samples with fungal encapsulation in the polymer layer absorbed a similar amount of liquid, fluctuating within the 2% range. Samples with added encapsulated mycelial microbeads absorbed approximately 6 g more, indicating that encapsulated fungi within the nonwoven fabric inside and outside the absorbent product do not affect absorbency; however, the addition of alginate beads enhanced liquid retention at a 6:1 ratio. Rewetting tests showed that all samples retained similar amounts of moisture, ranging from 0.5 g to 0.56 g, indicating that the fungal inoculum does not affect the product's ability to remain dry. Regarding emergence, the control sample showed no fungal growth, while samples with encapsulated mycelium emerged within approximately 5 days, compared to 11 days for alginate beads, and no growth was observed in the chassis of the outer attachment layer. These results demonstrate the feasibility of encapsulating immobilized living organisms within absorbent products without compromising their absorbency, while simultaneously allowing for effective fungal growth. Figure 35 The photograph shows the emergence of a fungal inoculum of an engineered species S derived from an absorbent product.

[0247] Example 17: Liquid impermeable container with encapsulated biological agents for waste treatment

[0248] In Example 17, 1 g of unencapsulated, fixed, engineered species S mycelium was adhered to a plastic film bag. A No. 4 diaper without added mycelium was moistened with a mixture of 200 mL saline and urea. The moistened diaper was then placed in the mycelium embedding bag. After 7 days, fungal growth was observed on both the bag and the outer layer of the diaper. Figure 36 This includes a photograph showing the emergence of species S inoculum from a film container. The results of Example 17 demonstrate that fungi can be encapsulated in, for example, garbage bags or other packaging commonly used in feminine hygiene products, and achieve effective colonization and decomposition. By adding fungal inoculum to the post-use waste stream, the fungi utilize the contaminated product for rehydration and emergence, promoting decomposition in the treated environment.

[0249] In one embodiment, a method for controlling the rehydration and presence of immobilized fungal inoculants in absorbent products includes rehydrating the immobilized fungal inoculants using specific conditions. This method allows for the use of fungal inoculants where their rehydration can be strategically controlled. The immobilized fungal inoculants are typically kept stagnant and then configured to rehydrate in a manner that allows contamination of the absorbent product. This could be in an environment where a hydrogel swells in the absorbent core of a diaper, or in an environment where encapsulation draws liquid into a nonwoven substrate of encapsulating material.

[0250] Hydrogels are used as culture media for fungal inoculant immobilization due to their unique physical properties. As a three-dimensional cross-linked network of polymer chains, hydrogels have the ability to absorb and retain large amounts of water in the gaps between the polymer chains. These properties, along with their softness, hydrophilicity, superabsorbency, viscoelasticity, biodegradability, and biocompatibility, make hydrogels ideally suited for biological applications, including those disclosed in the embodiments.

[0251] It is noteworthy that the application of smart or stimulus-responsive hydrogels represents a significant shift. Smart hydrogels are sensitive to specific environmental changes and exhibit a response by altering their shape or volume when exposed to these conditions. This sensitivity to stimuli can be categorized as internal or external, depending on their source when applied to the hydrogel in vivo. External stimuli that can be used to control the rehydration and emergence of immobilized fungal inoculants include physical conditions such as temperature, pressure, light, electric fields, magnetic fields, and ultrasonic radiation. Alternatively, hydrogels can be configured to respond to chemical stimuli such as pH, ionic strength, and CO2.

[0252] pH-sensitive hydrogels can be designed to allow only the immobilized fungal inoculum to rehydrate and emerge at a specific pH value. For example, the hydrogel can be configured to not react in the presence of water, but only rehydrate and activate the fungal inoculum when exposed to urine. This approach is both safe and practical for the use of immobilized fungal inoculum in consumer absorbent products such as diapers, sanitary pads, and incontinence pads.

[0253] The hydrogel coating used in this paper ensures that the immobilized fungal inoculum is rehydrated only under specific conditions, enabling the safe and effective use of these fungi in consumer absorbable products. By providing precise control over the rehydration and re-emergence of the fungal inoculum, this product brings a higher level of complexity and specificity to the application of fungi in absorbable products.

[0254] A method for embedding immobilized fungal inoculants in a nonwoven or foam substrate is also provided. These substrates possess the ability to handle liquids due to their capillary action and permeability, properties that ensure efficient flow of liquid to the immobilized fungal inoculant. In this context, capillary action refers to the phenomenon where liquid rises within the substrate due to intermolecular forces overcoming gravity. Similarly, permeability defines the substrate's ability to allow liquid to pass through. Nonwoven or foam substrates with high capillary action and permeability effectively absorb liquid, ensuring that the liquid reaches the encapsulated fungal inoculant, triggering its rehydration and emergence. This strategic positioning within the substrate, and the subsequent hydration only upon exposure to specific liquid conditions (such as urine), provides a highly targeted and efficient mechanism for the activation of the fungal inoculant, further enhancing the safety and usability of this invention in consumer absorbable products.

[0255] Water absorption capacity (WAC) is determined by the following relationship:

[0256] ((osmotic pressure^2) + affinity) / rubber elasticity, where

[0257] • Osmotic pressure: In the formulations of this invention, the osmotic pressure is influenced by the ion concentrations in the hydrogel and the surrounding liquid. Sodium alginate is a polymer that forms a gel in the presence of calcium ions (from CaCl2). Sodium ions from sodium alginate and calcium ions from CaCl2 contribute to the ion concentration within the hydrogel. The ion concentration difference between the interior of the hydrogel and the surrounding liquid generates the osmotic pressure that drives water absorption.

[0258] • Affinity: The affinity in the formulation of this invention is determined by the hydrophilic groups present in the hydrogel. Sodium alginate is a hydrophilic polymer, meaning it has a high affinity for water. This high affinity contributes to the water absorption capacity of the hydrogel. Fungal hyphae may also contribute to the hydrophilicity of the hydrogel, depending on the specific species of fungus used.

[0259] • Rubber Elasticity: The rubber elasticity of the formulation of this invention is a function of the crosslinking density. Depending on the situation, the crosslinking is provided by calcium ions (from CaCl2), which crosslink sodium alginate to form a gel. The density of these crosslinks determines the rubber elasticity of the hydrogel. By adjusting the concentration of CaCl2, the crosslinking density can be controlled, thereby controlling the rubber elasticity of the hydrogel.

[0260] Osmotic pressure, generated by ion difference, is the primary driving force of swelling, increasing with the square of the ion concentration difference. Affinity associated with hydrophilic groups plays a minor role. The rubber's elasticity, determined by the degree of cross-linking, resists swelling. Particles will swell until osmotic pressure, affinity, and rubber elasticity reach equilibrium.

[0261] Synthetic absorbent hydrogel Synthetic polymers produced by monomer polymerization.

[0262] • The synthesized superabsorbent undergoes polymerization of monomers such as NaPA in multiple polymerization steps and crosslinks with the polymer.

[0263] Natural hydrogel Natural polymers that undergo ionic or chemical cross-linking.

[0264] • Natural polymers, such as alginate (or cellulose, chitosan, agar, pectin, starch, hyaluronic acid, carrageenan, guar gum, xanthan gum), are grafted or cross-linked (ionic or chemical cross-linking agents) to enhance water absorption capacity.

[0265] When sodium polyacrylate (NaPA) crosslinked with 5% polyethylene glycol (PEG) is mixed with sodium alginate crosslinked with CaCl2, ion exchange occurs. Na from NaPA... +Ions replaced Ca in the alginate network 2+ Ions. This exchange reduces the cross-linking density in alginate (because Ca... 2+ Na ion ratio + The ions produce stronger cross-linking, thus reducing its rubber elasticity and making it more swollen. Meanwhile, due to Ca... 2+ The influx of ions makes SAP more cross-linked, increasing its rubber elasticity and reducing its swelling capacity.

[0266] Changes in osmotic pressure (increasing with the square of the ion concentration gradient) significantly affect the overall absorbability of the system. While the SAP becomes more elastic and less absorbent, alginate becomes more swellable due to the loss of cross-linking. This ion exchange optimally utilizes the strengths of both the natural hydrogel (alginate) and the synthetic hydrogel (SAP). Alginate can absorb more liquid, making the system more efficient, while the increased elasticity of the SAP provides structural support. This balanced approach reduces the amount of SAP required and leverages the enhanced swelling capacity of the natural polymer hydrogel.

[0267] Example 18: Liquid retention of different amounts of dried SAP and alginate-encapsulated hydrogels

[0268] In Example 18, liquid retention was tested under conditions comprising different amounts of dried superabsorbent polymer (SAP) and dried alginate-encapsulated hydrogels and combinations thereof. Three different samples were prepared: Sample A containing only 1 g of alginate hydrogel; Sample B containing only 1 g of SAP; and Sample C containing a combination of 1 g of SAP and 1 g of alginate beads. Each mixture was placed in a tray, 100 g of 1% NaCl solution was added, and the tray was placed in a zip-lock bag. The samples were allowed to be exposed to liquid at three different time intervals: 2 minutes, 1 hour, and 4 hours. Each mixture was tested three times. After the specified exposure time, the mixture was filtered for 2 minutes, and the liquid retention was calculated as the wet weight after filtration minus the original dry weight.

[0269] · Sample A 5.8 grams of liquid were retained after 2 minutes, 4.6 grams after 1 hour, and 4.4 grams after 4 hours.

[0270] · Sample B It can continuously absorb and retain 40-45 grams of liquid, regardless of the contact time with water.

[0271] · Sample C After 2 minutes, 49.1 grams of liquid were retained; after 1 hour, 78.7 grams were retained; and after 4 hours, 98.7 grams (almost all) of liquid were retained.

[0272] The results showed that the mixture of SAP and alginate beads (sample C) exhibited an unexpected synergistic effect, significantly enhancing liquid retention over time. While SAP alone retained a consistent amount of liquid, the addition of alginate beads significantly increased total liquid retention, particularly over long periods. This suggests that combining synthetic hydrogels (such as SAP) with natural hydrogels (such as alginate-encapsulated hydrogels) can significantly improve the absorbency and liquid retention properties of absorbent products, making them more effective for long-term use. Figure 37 Includes photographs showing the results of Example 18.

[0273] Example 19: Field testing of alginate-encapsulated hydrogels in diapers

[0274] In Example 19, to test the effectiveness of the alginate-encapsulated hydrogel in a real-world environment, a size 4 diaper was used. The diaper, contaminated with 225 grams of urine, was cut open to access the absorbent core. One gram of dried sodium alginate beads (crosslinked with calcium chloride, with a diameter of 0.9-1.1 mm when dry) was added to the core.

[0275] After one month, the beads swelled to a diameter greater than 3 mm. In contrast, when the same beads were mixed with water or urine without contact with the superabsorbent polymer (SAP), they swelled to only 1.5–1.6 mm. This significant difference in swelling indicates that the presence of SAP in the contaminated diaper core promoted the rehydration and swelling of the alginate beads.

[0276] Through interaction with the swollen SAP, the beads rehydrate, allowing the natural polymer hydrogel to access the moisture within the SAP. This interaction causes the beads to swell beyond typical osmotic equilibrium due to ion exchange from the SAP. This field trial demonstrates that encapsulating alginate beads in post-soaked absorbent cores of diapers effectively utilizes the moisture retained in the SAP, maximizing the swelling and functionality of the hydrogel. Figure 38 This is a photograph showing the absorbency of the natural and synthetic hydrogel mixture of Example 16 in a diaper after 1 month. Figure 39 This unexpected synergistic effect of combining natural hydrogel 140 and synthetic hydrogel 142 is described.

[0277] Example 20:

[0278] In Example 20, an encapsulated, engineered fungal / alginate mixture was prepared. This mixture was designed to encapsulate fungi in a protective alginate matrix, thereby immobilizing them while maintaining their viability and the possibility of reappearance under specific conditions.

[0279] The encapsulated fungal / alginate mixture was then embedded in a cellulose-based nonwoven substrate. This substrate was chosen because of its liquid handling capabilities, particularly its permeability and capillary action, ensuring effective liquid absorption and transport to the encapsulated fungi.

[0280] Following encapsulation, the mixture-substrate composition was dehydrated, causing the fungi to enter a stagnant state, further enhancing their immobilization. This dehydration process effectively preserved the encapsulated fungi / alginate mixture, preparing it for future rehydration and emergence. Subsequently, the dehydrated mixture-substrate composition was rehydrated under controlled conditions. These conditions were specifically designed to simulate the types of liquid exposure the composition would encounter in absorbent product applications. Upon successful rehydration, the previously immobilized fungi were observed to emerge from their encapsulated state and begin colonizing on the provided agar plates. This colonization not only demonstrates the successful reemergence of the fungi from their encapsulated and immobilized state but also their continued viability and functionality after rehydration.

[0281] This embodiment demonstrates the controlled rehydration and emergence of fungi under specific conditions. This capability can be used to develop consumer absorbent products with advanced functionalities, leveraging the potential of fungi in material decomposition or degradation.

[0282] In another embodiment, a method for encapsulating and immobilizing preselected fungi is provided for the decomposition of recalcitrant long-chain carbon materials, particularly for commercial applications of plastics and other polymer-based wastes. This process is a natural progression following the scale-up of the preselected fungal species in a bioreactor.

[0283] Once the mycelial growth reaches its maximum expansion, the fungal hyphae undergo bioprocessing steps, typically including homogenization and, possibly, filtration, to produce homogeneous biomass. This homogenized biomass is then mixed with a polymer solution (e.g., alginate) to form a stable, manageable material for further processing.

[0284] Subsequently, the fungal hyphae-alginate mixture can be mixed with a solidification solution to form a hydrogel, which is then spray-dried. The spray-dried mixture can be directly deposited into a designated collection area for future use, or sprayed directly onto the substrate where the fungus will act. Alternatively, the fungal hyphae-alginate mixture can be extruded into a calcium carbonate solution to form beads, which are then recovered.

[0285] Beads or spray-dried materials containing immobilized fungi can now be further processed to improve shelf life and tolerance. This processing may include coating with trehalose or other similar compounds known to have protective properties during drying. This further processing step is optional and may be omitted depending on the product's end use and storage conditions.

[0286] The treated or untreated fungal products are then dehydrated by freeze-drying or heating in an oven, with the temperature maintained below the species' temperature threshold to prevent denaturation. This dehydration process significantly reduces the product weight by 10–75 times, improving handling convenience and lowering transportation costs.

[0287] Dehydrated fungal products are packaged for storage and sale. Encapsulated and immobilized fungi are storable and retain their ability to break down recalcitrant long-chain carbon materials, ready for commercial deployment in waste management applications.

[0288] This approach modifies and optimizes developed small-scale waste decomposition methods for large-scale industrial applications, starting with the collection and aggregation of waste products consisting of a mixture of polymer-based materials and organic waste, such as food waste, yard waste, and other organic matter. Waste streams can be collected from a variety of sources, including municipal waste management facilities, manufacturing plants, and other industries that generate polymer-based waste.

[0289] The collected waste material is then processed in a manner similar to that described in the previous invention, introducing a pre-selected fungal inoculant. Selected fungal species (adept at decomposing specific polymer-based materials) are introduced into the waste mixture. Industrial-scale applications, considering the large quantities of waste material, require substantial amounts of fungal inoculant.

[0290] Waste materials are placed in designated waste containers, which in this case can be industrial-scale bioreactors or similar large-scale waste treatment systems. Optimal environments for fungal growth and decomposition are created and maintained within the system by controlling factors such as temperature, humidity, pH, and ventilation.

[0291] In addition, processing aids, such as nutrient sources, biological enzymes, chemical acids or other additives, can be introduced to enhance the decomposition performance of rehydrated fungal inoculants.

[0292] The biomass produced can be harvested for further use, such as composting, soil improvement, or as a feedstock for renewable energy production, such as biofuel or biogas production.

[0293] Ultimately, this method provides an efficient, sustainable, and scalable solution for industrial waste management by leveraging the ability of preselected fungi to decompose recalcitrant long-chain carbon materials, thereby reducing the environmental footprint of polymer-based waste.

[0294] 1. Add before use

[0295] In this embodiment, during the manufacturing stage of the polymer product, the fungal inoculant is added to the polymer product itself in one or more of the following ways:

[0296] • Adding fungal inoculants to the core mixture of absorbent products: This method involves adding fungal inoculants directly to the absorbent core material.

[0297] • Fungal inoculants are included as a component in the mixture of product material components.

[0298] • Fungal inoculants are added to the substrate for offline treatment by mixing with the substrate material in a separate treatment stage.

[0299] Fungal inoculants exist in their non-dehydrated form.

[0300] • Add fungal inoculants to the exterior of the product that requires activation. Here, the inoculant is placed on the exterior of the product in the form of fragile beads, powder, or a similar structure or form, which can be activated by the consumer when needed, for example, by contact with a liquid.

[0301] 2. Add after use

[0302] In this alternative implementation, the consumer or waste container introduces a fungal inoculant into contact with the used polymer product during disposal. This can be achieved through a number of methods or actions, all of which are simple for everyday use.

[0303] Consumers can apply inoculants directly to used polymer products in one or more different forms, including but not limited to fragile beads, capsules, wipes, and powders. This proactive approach to the degradation process helps enable more personalized and controlled waste management methods.

[0304] Alternatively, consumers can place the fungal inoculant in a waste container or bag designed to hold one or more used products. This process involves placing the inoculant in the container, where it interacts with the used polymeric products to initiate the degradation process.

[0305] In another implementation, the waste container or bag itself has the capacity to disperse or contain inoculum. These containers can be pre-filled with fungal inoculum, allowing the degradation process to begin once the used product is introduced. This integrated approach minimizes the need for user interaction and maximizes convenience.

[0306] • In another implementation, the fungal inoculant can be added at any stage of the waste management flow. In these cases, the waste management facility or system can provide the necessary inoculant for the used product, further reducing consumer liability.

[0307] As described above, a superabsorbent hydrogel is also provided, formulated with the addition of a fungal inoculant, possessing unique characteristics that mimic the properties of superabsorbent polymers (SAPs). This hydrogel product exhibits high swelling capacity, rapid hydration rate, and excellent liquid retention, while also possessing a fungal inoculant for bioactive applications. The embodiments of the invention described herein also include novel biodegradable superabsorbent hydrogel formulations.

[0308] The superabsorbency of hydrogels stems from specialized formulations, including optimized crosslinking density, careful polymer selection, the incorporation of ionizable groups, and advanced processing techniques. The addition of fungal inoculants is crucial for efficacy. Fungal strains are selected based on the desired bioactivity. They are encapsulated within the hydrogel matrix, where they remain dormant until rehydration. The superabsorbency of the hydrogel ensures controlled and sustained rehydration of the fungal inoculants, promoting their bioactivity under specific conditions.

[0309] Furthermore, the size and dispersion of the discrete fungal hydrogel provide optimal available surface area to enhance the absorption rate and overall absorbency of the embedded hydrogel. This not only offers an efficient method for the rehydration of fungal inoculants but also provides another important source of absorbency for the performance of absorbent products such as diapers. In one embodiment, SAP can be completely replaced by this novel fungal hydrogel to achieve the performance characteristics of absorbent products.

[0310] The product of this invention combines the superabsorbent properties of SAP with the bioactivity of fungal inoculants, creating a versatile and environmentally friendly material. This superabsorbent hydrogel with fungal inoculants is suitable for a variety of applications, including but not limited to agriculture, bioremediation, wastewater treatment, and personal care products.

[0311] Other implementations involve methods for incorporating fungal inoculants into the manufacturing process of absorbent products. This innovative process is designed for seamless integration into existing production lines, particularly those utilizing core-molded components.

[0312] This method begins with the preparation of an immobilized fungal inoculum. The fungal inoculum is prepared in a form compatible with existing machinery, specifically, as particles with a size range similar to that of superabsorbent polymers (SAPs), typically 0.5 mm to 1.5 mm, with an average size of less than 1 mm. This size compatibility allows the fungal inoculum to be handled and processed in a manner similar to SAPs, facilitating its integration into the production process.

[0313] The immobilized fungal inoculum is then loaded into a hopper located upstream of the core-forming assembly. This hopper is specifically designed for the fungal inoculum, ensuring its accurate and controlled addition to the absorbent product.

[0314] The fungal inoculum is then mixed with the pulp fiber mixture. The ratio of fungal inoculum to pulp fiber is carefully calibrated to ensure sufficient fungal activity to break down the polymer product while maintaining the absorbency of the final product.

[0315] After being thoroughly mixed with pulp fibers, the combined mixture is transferred to the core forming assembly. Here, the mixture is placed in a rotating drum, where it forms the absorbent core of the product.

[0316] This method aims to ensure that the temperature experienced by most (>90%) fungal inoculum particles does not reach levels that kill stagnant fungi. This is important for maintaining the bioactivity of the fungal inoculum in the final product.

[0317] These implementations uniquely integrate bioactive fungal inoculants into the manufacturing process of absorbent products. When used alone or in combination with SAP, the fungal inoculants are specifically designed to be compatible with existing core-forming machines. This compatibility allows for the production of absorbent products with enhanced biodegradability without requiring significant changes to existing manufacturing processes.

[0318] The method of the present invention provides a practical and effective way to add fungal inoculants to absorbent products, which helps to develop more sustainable and environmentally friendly absorbent products.

[0319] In an alternative implementation, the fungal inoculant is embedded and immobilized within a carrier nonwoven substrate. This substrate can be applied between any layers of absorbent products, providing a universal method for incorporating fungal inoculants into the product.

[0320] This method begins with the preparation of inoculum patches. Fungal inoculum is embedded in a nonwoven substrate, forming a roll of material that can be cut into sheets of the desired size. These rolls are pre-prepared and can be stored until needed, providing a ready-made supply of inoculum patches for the manufacturing process.

[0321] When the inoculum needs to be added to the absorbent product, the roll is unrolled and the patch is cut to the appropriate size. This cutting method can be automated to ensure consistent patch size and simplify the manufacturing process.

[0322] Once the patches are cut, they are picked up by a vacuum rotary roller. This roller grips the patch and rotates it toward the partially assembled absorbent product. The use of a vacuum rotary roller ensures precise placement of the patches and minimizes the risk of misalignment or displacement during application.

[0323] The patch is then applied to the absorbent product. This application can be facilitated by using tackifiers or adhesives that help hold the patch in place. The choice of tackifier or adhesive can be tailored to the specific requirements of the absorbent product, taking into account factors such as the materials used in the product, desired product performance, and the environmental conditions under which the product will be used.

[0324] After the patch is applied, another layer (film or nonwoven fabric) is applied over the patch in the absorbent product. This layer is used to hold the patch in place and protect the fungal inoculum from damage or displacement during the remainder of the manufacturing process and subsequent use of the product.

[0325] Alternative embodiments of the present invention provide a practical and effective method for incorporating fungal inoculants into absorbent products. By embedding the inoculant in a nonwoven substrate and using it as a patch, the present invention allows for precise placement of the inoculant within the product, contributing to the development of more sustainable and environmentally friendly absorbent products.

[0326] Storage-resistant delivery of living organisms, discrete capsules and related shape factors

[0327] In another embodiment, a particle delivery system for storage-resistant living organisms is configured to be integrated into existing absorbent product manufacturing processes. This system utilizes hydrogel particles that can be seamlessly mixed with various species after drying / curing for targeted application in absorbent products. These embodiments offer flexibility in final packaging, similar to capsule-like dry detergent boxes, and provide a gel suspension option for post-use applications. This approach allows for the customization of disintegrating agents to suit specific consumer needs and environmental conditions.

[0328] This implementation utilizes a controlled-release system using hydrogels to deliver shelf-stable live organisms. These dehydrated hydrogels are preferably incorporated into absorbent products made of polymers, such as diapers or sanitary napkins, where they are positioned to come into contact with liquid after use. Upon contact with the liquid, the hydrogel rehydrates, triggering the emergence of encapsulated live fungal organisms. This controlled-release mechanism ensures that the activation of the degradative agent occurs only in the presence of moisture, thus aligning the system's activation with the product's functional lifecycle.

[0329] The encapsulation of these living organisms is achieved through various techniques, with alginate microparticles being particularly preferred. These microparticles provide a stable environment for the fungi, protecting them until rehydration. Furthermore, this system allows for the addition of extra nutrients and enzymes to the formulation, enhancing the potency of the fungal inoculum after activation. Once the hydrogel is rehydrated, this process facilitates a robust decomposition process. Additionally, in some embodiments, the formulation of these particles can be modified to include a delayed-release coating. This coating is designed to prevent premature fungal activation, ensuring they only become active after product use, thereby maximizing their decomposition potential.

[0330] A key feature of these implementations is their compatibility with a wide range of existing absorbent product designs. Discrete capsules formulated as alginate microparticles or similar encapsulated hydrogels can be seamlessly incorporated into the structure of these products. These capsules are carefully designed to remain inert and inconspicuous before and during product use. However, upon contact with moisture, they are activated, facilitating the breakdown of the product. This integration represents a significant advancement as it adds breakdown capabilities to a diverse range of products without altering the core design or user experience.

[0331] In another embodiment, storage-resistant living organisms are embedded in a mesh substrate, creating an advanced system for effective encapsulation and rehydration. In one particular embodiment, the fibrous material is impregnated with alginate to ensure adhesion and curing for effective encapsulation. In alternative embodiments, encapsulated beads are embedded in textiles, optionally with microencapsulation functionalization or adhesive bonding. This approach utilizes a fibrous scaffold to enhance efficiency and effectiveness in absorbent products and other applications, thereby providing living organisms for degradation.

[0332] Furthermore, storage-resistant live fungal organisms can be embedded in a nonwoven mesh substrate, which serves as an effective scaffold for encapsulation and rehydration. This embodiment utilizes advanced technologies such as screen printing, slot coating, and microgravure roll-to-roll printing. These methods precisely impregnate the fibrous nonwoven material with alginate (a biocompatible and biodegradable hydrogel), ensuring effective encapsulation of the fungal organisms within the fibrous substrate. This process not only provides a stable environment for the fungi but also ensures their direct contact with the nonwoven scaffold upon rehydration, which is crucial for accelerating their growth and decomposition activities.

[0333] In another embodiment, the nonwoven fiber material can be impregnated with alginate. This process is similar to screen printing, where alginate mixed with a fungal inoculant is uniformly distributed on the nonwoven fabric. This distribution allows for controlled and effective encapsulation of fungi, ensuring they are evenly dispersed throughout the material. After impregnation, the alginate undergoes a curing process, solidifying the encapsulation and placing the fungi in a dormant but viable state. This curing can be achieved using a variety of known techniques, including UV curing or heat treatment, thus providing flexibility in the manufacturing process.

[0334] In another implementation, slot coating or microgravure printing processes are utilized. These techniques provide precision in the application of alginate-fungus mixtures, ensuring consistent and effective encapsulation across various types of nonwoven materials. After rehydration, the fungi are in direct contact with the nonwoven scaffold. This proximity provides an ideal environment for rapid rehydration of the fungi and the initiation of their growth and decomposition processes. The scaffold not only physically supports the fungi but also facilitates the efficient transfer of moisture and nutrients, which is crucial for fungal activation and growth.

[0335] The system is designed to accelerate fungal growth during rehydration. The nonwoven scaffold, filled with encapsulated fungi, becomes an active site for decomposition upon contact with moisture. This immediate activation is particularly advantageous in absorbent products, where the presence of liquid triggers the emergence of fungi, thus initiating the decomposition process without delay. Once activated, the fungi utilize the nonwoven material as a growth medium, rapidly swelling and beginning to decompose the product.

[0336] It should be understood that the foregoing description is merely an explanation of various exemplary embodiments. Various alternatives and modifications can be devised by those skilled in the art without departing from this disclosure. Therefore, this disclosure is intended to include all such alternatives, modifications, and variations that fall within the scope of this disclosure.

Claims

1. An inoculum composition comprising: First fungal species or strain; A second fungal species or strain that is different from the first fungal species or strain. Among them, at least the first fungal species or strain is a saprophytic fungus. Each of the first fungal species or strain and the second fungal species or strain is present in the composition at a concentration relative to each other, such that the fungal biomass and polymer material are inoculated on one or more polymer materials at a mass ratio not greater than 1:

1.

2. The inoculum according to claim 1, wherein at least the first fungal species or strain is classified as a white-rot fungal species.

3. The inoculum composition according to claim 1, wherein each of the first fungal species or strain and the second fungal species or strain is present in the composition at a concentration relative to each other, such that the fungal biomass and polymer material are inoculated on one or more polymer materials at a mass ratio not greater than 0.5:

1.

4. The inoculum composition according to claim 1, wherein each of the first fungal species or strain and the second fungal species or strain is present in the composition at a concentration relative to each other, such that the fungal biomass and polymer material are inoculated on one or more polymer materials at a mass ratio not greater than 0.1:

1.

5. The inoculum composition according to claim 2, wherein the second fungal species or strain is classified as a brown rot fungus species or a soft rot fungus species.

6. The inoculum composition according to claim 2, wherein the second fungal species or strain is classified as a white-rot fungal species.

7. The inoculum composition according to claim 1, wherein... The first fungal species or strain is an engineered fungal species or strain that is engineered to recognize plastic products as a food source and to decompose recalcitrant polymers more effectively than their naturally occurring counterparts by initiating enzymatic degradation. The second fungal species or strain is an engineered fungal species or strain that is engineered to recognize plastic products as a food source and to decompose recalcitrant long-chain carbon more effectively than its naturally occurring counterparts by initiating enzymatic decomposition.

8. The inoculum composition of claim 6, wherein each of the first fungal species or strain and the second fungal species or strain is engineered by a process to produce improved tolerance to abiotic stress in the respective engineered fungal species or strain compared to the naturally occurring counterpart of the same fungal species or strain.

9. The inoculum composition of claim 1, wherein each of the first fungal species or strain and the second fungal species or strain is engineered by a process to produce improved tolerance to one or both of biotic and abiotic stresses in the respective engineered fungal species or strain compared to the naturally occurring counterpart of the same fungal species or strain.

10. The inoculum composition of claim 1, further comprising a fixation material, wherein each of the first fungal species or strain and the second fungal species or strain is fixed in the composition such that the first engineered fungal species or strain and the second fungal species or strain are in a metabolically dormant state.

11. The inoculum composition according to claim 7, wherein one of the first fungal species or strain and the second fungal species or strain originates from the Basidiomycota phylum.

12. The inoculum composition according to claim 11, wherein the first fungal species or strain and the second fungal species or strain are selected from the group consisting of *Bacillus buddingus*, *Microsporum simulans*, *Aspergillus* species, *Fusarium* species, *Aspergillus versicolor*, *Aspergillus fumigatus*, *Pleurotus ostreatus*, *Pleurotus erythropus*, *Pleurotus ostreatus*, *Pleurotus ostreatus*, *Pleurotus erythropus*, *Pterygodium japonicum*, *Pterygodium chrysosporum ... and *Pterygodium sclerotiorum*.

13. The inoculum composition according to claim 1, wherein one of the first fungal species or strain and the second fungal species or strain originates from the Basidiomycota phylum.

14. The inoculum composition according to claim 1, wherein the first fungal species or strain originates from a different fungal phylum than the second fungal species or strain.

15. The inoculum composition according to claim 1, wherein the first fungal species or strain and the second fungal species or strain are selected from the group consisting of *Bacillus buddingus*, *Microsporum simonii*, *Aspergillus* species, *Fusarium* species, *Aspergillus versicolor*, *Aspergillus fumigatus*, *Pleurotus ostreatus*, *Pleurotus erythropus*, *Pleurotus ostreatus*, *Polyporus scabra*, *Polyporus scabra*, *Procambarus chrysosporus*, *Pleurotus ostreatus ... and *Pleurotus ostreatus*.

16. The inoculum according to claim 15, wherein each of the first fungal species or strain, the second fungal species or strain, and the third fungal species or strain is selected from the group consisting of *Bacillus buddingus*, *Microsporum simulans*, *Aspergillus* species, *Fusarium* species, *Aspergillus versicolor*, *Aspergillus fumigatus*, *Pleurotus ostreatus*, *Pleurotus erythropus*, *Pleurotus ostreatus*, *Polyporus scabra*, *Polyporus scabra*, *Procambarus chrysosporus*, *Pleurotus ostreatus ... and *Pleurotus sclerotiorum*.

17. The inoculum composition according to claim 1, wherein one or both of the first fungal species or strain and the second fungal species or strain exist in the composition in the form of spores of the first fungal species or strain.

18. The inoculum composition according to claim 1, wherein one or both of the first fungal species or strain and the second fungal species or strain are filamentous fungi from any phylum of fungi that have a hyphal stage in their life cycle.

19. The inoculum composition according to claim 1, wherein The inoculum was dehydrated, causing the fungi to be in a stagnant state under initial conditions; and The inoculum is configured to rehydrate by exposure to moisture, thereby allowing the fungus to reappear from its initial conditions.

20. An inoculum composition comprising: The first engineered fungal species or strain is engineered to recognize plastic products as a food source and to decompose recalcitrant long-chain carbon more effectively than its naturally occurring counterparts. The first engineered fungal species or fungal strain is fixed within a fixation material, thereby placing the first engineered fungal species or fungal strain into a metabolic dormant state.

21. The inoculum composition according to claim 20, wherein the engineered fungal species or fungal strain is engineered through the following process: Fungal mycelium was introduced into a growth medium containing an initial concentration of simple carbon and plastic material, and The concentration of the simple carbon is reduced from the initial concentration over time, while the presence of the plastic material is maintained in the culture medium.

22. The inoculum composition of claim 21, wherein the first engineered fungal species or fungal strain is engineered to have improved tolerance to abiotic stress compared to the naturally occurring counterpart of the same fungal species or strain.

23. The inoculum composition of claim 20, wherein the first engineered fungal species or fungal strain is engineered to have improved tolerance to abiotic stress compared to the naturally occurring counterpart of the same fungal species or strain.

24. The inoculum composition of claim 20, wherein the immobilizing material is configured to absorb moisture, thereby inducing the movement of the first engineered fungal species or fungal species strain from its immobilized state.

25. The inoculum composition of claim 22, wherein the immobilizing material is configured to absorb moisture, thereby inducing the movement of the first engineered fungal species or fungal species strain from its immobilized state.

26. The inoculum composition of claim 21, wherein the immobilizing material is configured to absorb moisture, thereby inducing the movement of the first engineered fungal species or fungal species strain from its immobilized state.

27. The inoculum composition of claim 20, further comprising a second engineered fungal species or strain engineered to recognize plastic products as a food source and to decompose recalcitrant long-chain carbon more effectively than its naturally occurring counterpart, wherein... The second engineered fungal species or fungal strain is different from the first engineered fungal species or fungal strain.

28. The inoculum composition according to claim 20, wherein the first fungal species or species strain is derived from one of the phyla Basidiomycota and Ascomycota.

29. The inoculum composition of claim 20, wherein the first fungal species or species strain is derived from the Basidiomycota phylum.

30. The inoculum composition of claim 20, wherein the first fungal species or species strain is a filamentous fungus from any phylum of fungi that has a hyphal stage in its life cycle.

31. The inoculum composition of claim 20, wherein the first fungal species or strain exists in the composition in the form of spores of the first fungal species or strain.

32. A method for reducing large quantities of plastic waste, comprising: a) Forming an inoculum comprising one or more preselected fungal species or preselected fungal strains or combinations thereof, said inoculum being capable of consuming long-chain carbon molecules in a polymer material through metabolism; b) Provide a plastic substrate containing long-chain carbon molecules; c) Add the inoculum to the plastic substrate to form an inoculum / substrate complex; d) Decompose the plastic substrate in the composite by the one or more fungal species or strains or combinations thereof, thereby converting the long-chain carbon material therein.

33. The method of claim 32, wherein at least one of the one or more preselected fungal species or preselected fungal strains exists in the inoculum in the form of spores of the corresponding preselected fungal species or preselected fungal strain.

34. The method of claim 32, wherein prior to step (a) of forming the inoculum, the preselected fungal species or fungal strain is cloned and amplified by a hyphal propagation bioprocess.

35. The method of claim 32, wherein the plastic substrate is part of an absorbent consumer product.

36. The method of claim 35, wherein the absorbent consumer product is selected from diapers, sanitary products, and absorbent pads.

37. The method of claim 32, wherein The inoculum is formed by immobilizing one or more fungal species or strains or combinations thereof, and The inoculum is activated by contact with moisture in the inoculum / substrate complex, thereby resulting in the appearance of one or more fungal species or strains or combinations thereof.

38. The method of claim 32, wherein the inoculum / substrate complex is a combination of an inoculum and a substrate formed during the product manufacturing process.

39. The method of claim 32, wherein the preselected fungal species or preselected fungal strain or combination thereof comprises filamentous fungi from any phylum of fungi that have a hyphal stage in their life cycle.

40. The method of claim 32, wherein the inoculum comprises One or more fungal species classified as white-rot fungi, and One or more fungal species classified as soft-rot fungi or one or more fungal species classified as brown-rot fungi.

41. The method of claim 32, wherein the inoculum comprises One or more fungal species classified as soft-rot fungi, and One or more fungal species classified as white-rot fungi or brown-rot fungi.

42. The method of claim 32, wherein the inoculum comprises One or more fungal species classified as brown-rot fungi, and A fungal species classified as soft-rot fungi or a fungal species classified as white-rot fungi.

43. The method of claim 32, wherein the inoculum comprises one or more fungal species classified as white-rot fungi, one or more fungal species classified as soft-rot fungi, and one or more fungal species classified as brown-rot fungi.

44. The method according to claim 43, wherein the one or more preselected fungal species are selected from the group consisting of *Bacillus buddingus*, *Microsporum simulans*, *Aspergillus* species, *Fusarium* species, *Aspergillus versicolor*, *Aspergillus fumigatus*, *Pleurotus ostreatus*, *Pleurotus erythropus*, *Pleurotus ostreatus*, *Pleurotus erythropus*, *Pterocarpus scabra ...

45. The method according to claim 32, wherein the one or more preselected fungal species are selected from the group consisting of *Bacillus buddingus*, *Microsporum simulans*, *Aspergillus* species, *Fusarium* species, *Aspergillus versicolor*, *Aspergillus fumigatus*, *Pleurotus ostreatus*, *Pleurotus erythropus*, *Pleurotus ostreatus*, *Pleurotus erythropus*, *Pterocarpus scabra ...

46. ​​The method of claim 32, wherein the one or more preselected fungal species include at least one species of the Basidiomycota phylum.

47. The method of claim 46, wherein the preselected fungal species or preselected fungal strain or combination thereof comprises filamentous fungi.

48. The method of claim 32, wherein the inoculum is in a dehydrated form and is configured to be reactivated by contact with moisture.

49. The method of claim 48, wherein The inoculant is added to the substrate by embedding the encapsulated fungal inoculant into a substrate selected from the group consisting of natural fiber nonwoven materials, foam materials, and combinations thereof; The inoculum is dehydrated, wherein the fungi are in a stagnant state under initial conditions; and The inoculum is rehydrated by exposure to moisture, thereby allowing the fungus to reappear before step (d).

50. The method of claim 49, wherein the inoculum / substrate complex is incorporated into the absorbent product under initial conditions.

51. The method of claim 32, further comprising one or more of the following: The environmental variables of the inoculum / substrate complex are altered, and the environmental variables are selected from temperature, humidity, pH and oxygen content; Introducing one or more chemical or biological additives for the pretreatment of the plastic substrate; and The substrate is subjected to ultraviolet (UV) pretreatment.

52. The method of claim 51, wherein The chemical auxiliaries are selected from the group consisting of oxygen-releasing compounds, nitrates, pH buffers, and acid treatment agents. The bio-adjuvant is selected from groups consisting of enzyme treatments to generate microsites on the surface of the plastic substrate, and / or UV treatment sterilizes the plastic substrate and alters its surface properties to increase its susceptibility to fungal decomposition.

53. An inoculum composition comprising: Multiple different preselected fungal microcolonies were immobilized in multiple encapsulated fungal beads, among which Each bead comprises a single fungal species or strain, and Various engineered fungal species or strains have formed communities of species or strains not found in nature.

54. The inoculum composition of claim 53, wherein at least one of the plurality of preselected fungal species or strains thereof is engineered to recognize plastic products as food sources and to decompose recalcitrant long-chain carbon more effectively than naturally occurring counterparts of the same fungal species or strains thereof.

55. The inoculum composition of claim 54, wherein at least one of the plurality of preselected fungal species or strains thereof is engineered to have improved tolerance to abiotic stresses compared to naturally occurring counterparts of the same fungal species or strains thereof.

56. The inoculum composition of claim 53, wherein at least one of the plurality of preselected fungal species or strains thereof is engineered to have improved tolerance to abiotic stresses compared to naturally occurring counterparts of the same fungal species or strains thereof.

57. The inoculum composition of claim 32, wherein the preselected fungal species or strain thereof is selected from a plurality of different fungal phyla.

58. The inoculum composition according to claim 53, wherein the preselected fungal species or strain thereof is selected from the group consisting of *Bacillus buddingus*, *Microsporum simulans*, *Aspergillus* species, *Fusarium* species, *Aspergillus versicolor*, *Aspergillus fumigatus*, *Pleurotus ostreatus*, *Pleurotus erythropus*, *Pleurotus ostreatus*, *Pleurotus erythropus*, *Pterygodium japonicum*, *Pterygodium chrysosporum ...

59. The inoculum composition of claim 53, wherein the beads comprise alginate.

60. The inoculum composition of claim 53, wherein at least one of the plurality of first fungal species or strains thereof exists in the composition in the form of spores.

61. A fixed and storage-resistant fungal inoculant composition, comprising: A fungal inoculant selected from one or more fungal species or strains thereof, said fungal inoculant being engineered to recognize plastic products as a food source and to decompose recalcitrant long-chain carbon more effectively than naturally occurring counterparts of the same fungal species or strains thereof; and A fixation material is used to encapsulate the fungal inoculum, keeping it in a stagnant state and protecting it from contamination.

62. The composition of claim 32, wherein the fungal inoculum is preserved by dehydration, freezing, or lyophilization.

63. The composition according to claim 61, wherein the fixing material is alginate beads.

64. The composition of claim 61, further comprising a refrigerant.

65. The composition of claim 61, wherein the composition is capable of rehydration upon exposure to moisture, thereby allowing the formation of a fungal inoculum.

66. The composition of claim 61, wherein the composition is formed as beads, a nonwoven substrate, or a foam structure.

67. The composition of claim 61, wherein the immobilizing material encapsulates the fungal inoculum through a cross-linking process with calcium ions.

68. The composition of claim 67, wherein the composition is porous, thereby allowing oxygen to reach the fungal inoculum while remaining dehydrated until reactivated by contact with moisture.

69. The composition of claim 61, comprising a trehalose-containing coating for improving storage stability.

70. A method for manufacturing an inoculum composition, comprising: Select one or more fungal species or strains thereof; The selected one or more fungal species or strains thereof are fixed by dehydration or freeze-drying; and One or more fungal species or strains of the fixed fungus are encapsulated in a fixation material to form an encapsulated fungal inoculum.

71. The method of claim 70, wherein the one or more fungal species or strains thereof comprise at least one species of the Basidiomycota phylum.

72. The method of claim 70, wherein the one or more fungal species or strains thereof comprise filamentous fungi.

73. The method of claim 70, wherein the encapsulated fungal inoculum further comprises a conditioned medium having transcription factors such as cytokinins and hormones recovered from the fungal biomass during fermentation to aid fungal emergence.

74. The method of claim 70, wherein the encapsulated fungal inoculum further comprises a conditioned medium containing one or more of a recovered metabolite, a hydrophobic compound, a competitive compound, and a β-glucan, thereby enhancing fungal activity and stability.

75. A method for controlled biotransformation of a polymer-based product using a fungal inoculant, comprising the following steps: a. Add the fungal inoculant to the polymer-based product before or after use; b. To keep the fungal inoculum dormant until exposed to specific environmental stimuli or conditions; and c. Activate the dormant fungal inoculum to initiate the biotransformation of the polymer-based product at a controlled rate.

76. A hydrogel composite comprising: One or more natural hydrogels having a first liquid absorption capacity C1 and a first mass M1; and One or more synthetic hydrogels having a second liquid absorption capacity C2 and a second mass M2, wherein The total liquid absorption capacity C of the complex T It is equal to or greater than the liquid absorption capacity C2 of one or more synthetic hydrogels alone.

77. The hydrogel complex of claim 76, wherein each of the one or more natural hydrogels and the one or more synthetic hydrogels is a cross-linked polymer.

78. The hydrogel complex of claim 76, further comprising one or more fungal inoculants embedded within the complex.

79. The hydrogel complex according to claim 76, wherein the natural hydrogel is alginate and the synthetic hydrogel is SAP.

80. The hydrogel composite of claim 76, wherein the natural hydrogel and the synthetic hydrogel are combined to create a microenvironment in which ionic interactions occur in the presence of a liquid, the ionic interactions affecting one or both of the rubber elasticity and osmotic pressure of the hydrogel composite.

81. An absorbent article comprising at least one layer comprising the hydrogel composite of claim 76.

82. The absorbent article of claim 81 further comprises one or more fungal inoculants embedded in the hydrogel complex, wherein the one or more fungal inoculants are immobilized such that the fungi in the inoculants are in a metabolically dormant state.

83. The absorbent article of claim 82, wherein the one or more fungal inoculants comprise a fixation material configured to absorb moisture, thereby inducing the movement of fungi within the inoculant from their fixed state.

84. A method for manufacturing an inoculum product, comprising the following steps: a) Provide seed inoculants from preselected fungal species with the ability to degrade long-chain carbon compounds; b) Introducing the inoculum into a nutrient mixture, the nutrient mixture being formulated to provide the metabolic needs of the preselected fungal species; c) In a bioreactor, under controlled conditions, promote the mycelial expansion of a preselected fungal species until the maximum expansion level is reached to provide expanded mycelial biomass. d) The extended mycelial biomass is treated by homogenization and subsequently combined with a polymer solution to provide a mycelial polymer solution; e) Introducing a conditioned medium into a mycelial polymer solution to provide processed fungal biomass, said conditioned medium comprising one or more minor biotic elements to support fungal growth and activity, said minor biotic elements being selected from the group consisting of biomass, enzymes, polysaccharides, transcription factors, and antimicrobial agents; and f) Harvest, encapsulate, and fix the processed fungal biomass to produce a stable, storage-resistant inoculum product.

85. The method of claim 84, wherein the conditioned medium further comprises transcription factors such as cytokinins and hormones recovered from fungal biomass during fermentation to aid in fungal emergence.

86. The method of claim 84, wherein the conditioned medium comprises one or more of recovered metabolites, hydrophobic substances, competitive compounds, and β-glucan, thereby enhancing fungal activity and stability.

87. The method of claim 84, wherein the seed inoculum is a progeny or clone obtained directly from the preselected fungus, thereby facilitating the propagation of the desired trait.

88. The method of claim 84, wherein the nutrient mixture is formulated based on the specific metabolic needs of the preselected fungal species to improve growth and decomposition capabilities.

89. The method of claim 84, wherein conditions in the bioreactor are controlled, taking into account factors such as nutrients, temperature, pH and moisture, to achieve maximum mycelial expansion.

90. The method of claim 84, wherein the homogenization process includes a filtration step prior to combination with the polymer solution.

91. The method of claim 84, wherein the polymer solution is an alginate solution.

92. The method of claim 84, wherein the encapsulation and fixation procedures impart stability and shelf life to the fungal inoculum product.

93. The method of claim 84, wherein the fungal inoculant product is used to decompose recalcitrant long-chain carbon materials in waste management applications.

94. The method of claim 84, wherein the preselected fungal species belongs to a saprophytic fungal species.

95. The method of claim 84, wherein the recalcitrant long-chain carbon material comprises plastic or other polymer-based waste.

96. A method for preparing a storage-resistant fungal composition, comprising the following steps: a) Provide fungal inoculum from preselected filamentous fungi; b) Prepare polymer scaffolds selected from nonwoven materials, foams, granules and plastic resins; c) Introducing the fungal inoculum into the polymer scaffold via solid-state or liquid fermentation to provide a fungal scaffold complex; d) Encapsulate the fungal scaffold complex with an encapsulating preservative and a polymer matrix to provide an encapsulated scaffold; and e) Secure the encapsulated support to form a storage-resistant fungal composition.

97. The method of claim 96, wherein the fungal inoculum comprises fungal mycelial biomass or spores.

98. The method of claim 96, wherein the polymer scaffold is a nonwoven material that directly contacts the fiber or film to enhance absorbency.

99. The method of claim 96, wherein introducing the fungal inoculum into the polymer scaffold via solid-state fermentation comprises allowing filamentous fungi to grow into the nonwoven scaffold.

100. The method of claim 96, wherein introducing the fungal inoculum into the polymer scaffold via liquid fermentation comprises mixing a wet mycelial mixture with the polymer scaffold for fixation.

101. The method according to claim 96, wherein the encapsulating preservative is selected from the group consisting of trehalose, alginate, carrageenan, dextran, maltodextrin, sucrose and sorbitol.

102. The method of claim 96, wherein the polymer matrix is ​​sodium alginate crosslinked with calcium chloride.

103. The method of claim 96, wherein the encapsulated scaffold is crosslinked in a calcium chloride solution to form a fixed structure.

104. The method of claim 96, wherein the fungal inoculum is introduced into the polymer scaffold by coating the fibers of the nonwoven material with a sodium alginate mixture.

105. The method of claim 96, wherein the method allows the fungus to adapt to various polymer types, thereby enhancing decomposition efficiency and vigor.

106. The method of claim 96, wherein the fungal inoculum comprises a conditioned medium containing one or more minor biological elements selected from the group consisting of biomass, enzymes, polysaccharides, transcription factors, and antimicrobial agents.

107. The method of claim 96, wherein the conditioned medium further comprises transcription factors such as cytokinins and hormones recovered from fungal biomass during fermentation to enable the fungi to emerge more rapidly and vigorously.

108. The method of claim 96, wherein the scaffold is selected from foam, granules and plastic resin, wherein fungal spores are embedded in the scaffold.

109. The method of claim 96, wherein the encapsulated fungus is suitable for films, nonwovens, foams and other polymer-based materials for decomposition.

110. The method of claim 96, wherein The metabolic processes of the fungus are preserved during fixation and have the ability to be reactivated upon rehydration, thereby leading to accelerated decomposition of the polymer scaffold through enzymatic activity.

111. The method of claim 96, wherein the encapsulated scaffold is used in the product to induce the decomposition of recalcitrant long-chain carbon materials.

112. A composition comprising: a) Products selected from absorbent products or waste containers; b) Storage-resistant fungal inoculum incorporated into the product in, During product use, the storage-resistant fungal inoculum remains in a metabolic dormant state. The storage-resistant fungal inoculum is activated when exposed to moisture after use.

113. The composition of claim 112, wherein the fungal inoculum is added to one or more portions of the absorbent product, the absorbent product being selected from a hydrogel in an absorbent layer, laminated on a membrane of an impermeable layer without altering breathability, added to a nonwoven permeable layer without altering permeability or rewetting, added to a cuff, and added to a receiving area.

114. The composition of claim 112, wherein the fungal inoculum is encapsulated to ensure dormancy until activated by moisture.

115. The composition of claim 113, wherein the hydrogel containing the fungal inoculum is incorporated into the absorbent core mixture of the product.

116. The composition of claim 113, wherein the fungal inoculum is laminated onto a film to create an impermeable layer for use in layers such as film, packaging, or wrapping paper.

117. The composition of claim 113, wherein the fungal inoculum is incorporated into one or more nonwoven permeable layers selected from the top sheet, distribution layer, back sheet nonwoven fabric, and cuff.

118. The composition of claim 113, wherein the fungal inoculum is incorporated into the receiving area, elastic ear, or other structural component of the absorbent product.

119. The composition of claim 113, wherein the fungal inoculum is incorporated into the membrane to produce a substantially impermeable waste container for waste treatment applications.

120. The composition of claim 112, wherein the encapsulation structure of the fungal inoculum is configured to delay its appearance during product use.

121. The composition of claim 112, wherein the fungal inoculum is applied in combination with other biological agents for enhancing waste treatment.

122. The composition of claim 112, wherein the product is an impermeable waste container and the fungal inoculum remains in a metabolically dormant state until it is activated after treatment.

123. The composition of claim 112, wherein the fungal inoculum enhances the post-use degradation process of the product.

124. A method for manufacturing an absorbent product, comprising: a) Prepare storage-resistant fungal inoculums in which their biological components are preserved; and b) Add the fungal inoculum to the absorbent product, embed the fungal inoculum in a mesh substrate, or add the fungal inoculum as discrete beads into the absorbent core.

125. The method of claim 124, wherein the fungal inoculum is added in the form of discrete beads, the discrete beads comprising fixed fungal inoculum particles having absorption properties.

126. The method of claim 124, wherein the fungal inoculum is embedded in a mesh substrate and then incorporated into the absorbent product by preparing a roll of the mesh substrate and cutting the roll into pieces of the desired size.

127. The method of claim 124, wherein the fungal inoculum is added as discrete beads, then loaded into a hopper located upstream of the core forming assembly, mixed with the pulp fiber mixture, and transferred to the core forming assembly to form an absorbent core.

128. The method of claim 124, wherein the fungal inoculum is embedded in a mesh substrate applied to the absorbent product using a vacuum rotary roller, secured in place with an adhesive or tackifier, and covered with an additional protective layer.

129. The method of claim 124, wherein the discrete beads or mesh substrate is encapsulated to remain dormant until activated by moisture after product use.

130. The method of claim 124, wherein the fungal inoculum comprises a conditioned medium containing one or more minor biological elements selected from the group consisting of enzymes, polysaccharides, and transcription factors to support fungal growth and activity upon activation.

131. The method of claim 124, wherein the encapsulation structure of the fungal inoculum is configured to delay its appearance during use of the product and to ensure activation only upon exposure to moisture.

132. The method of claim 124, wherein the fungal inoculum enhances the biodegradation process of the absorbent product after use.

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