Description:Methods of Producing Fungal Biomaterial and Post Processing
Field of invention
The present disclosure relates generally to isolated fungal strains and methods for growing/culturing said fungal strains, in particular, to produce fungal biomaterial or final bio leather. The fungal biomaterial is fungal bio-leather. The present invention also relates to a method of post processing of fungal biomaterial.
Background
Sustainable substitutes for biomaterial, like, bio-leather can be made from mushroom mycelium, which is an environmentally friendly alternative to animal and synthetic leather.
Traditional leather and its alternatives are obtained from animals and synthetic polymers. Leather production process is increasingly being considered to be ethically questionable and environmentally unfriendly, as it leads to deforestation for grazing, greenhouse gas emissions, use of hazardous substances, etc. The production of synthetic leather from plastics such as polyvinyl chloride (PVC) or polyurethane (PU) also depends on chemicals derived from fossil fuels. This is why leather-like materials from fungi, which are biodegradable, are important.
Mushroom based biomaterial, like bio-leather, is a relatively new technology that differs from animal leather production in several ways. Mushroom based biomaterial, bio-leather is made from mycelium which is a vegetative part of a mushroom. In contrast, animal-based leather comes from the skin of animals. Producing mushroom-based biomaterial, like bio-leather, has lower environmental impact, as it does not require the use of chemicals or large amount of water.
Mushroom based biomaterial, like bio-leather, is created by growing mycelium under controlled conditions, which allow customization in terms of thickness, texture, and durability. On the other hand, animal leather production involves a complex and resource-intensive process of skinning, cleaning, tanning, and dyeing.
Mushroom based biomaterial, like bio-leather, provide unique design aesthetic options due to its textural and natural look. Unlike animal leather, the growth process of mushroom-based bio-leather can form shapes which overcome the need of cutting. Overall mushroom based biomaterial, like bio-leather, is a promising alternative to animal leather that is more sustainable, customizable, and innovative.
At present, the market uses either animal-based leather or petroleum-based leather, both of which have disadvantages associated with them. Animal-based leather requires hazardous and toxic chemicals like chromium III, which is carcinogenic, for processing. The chemicals used for tanning give out toxic gases and effluents, which pose danger to workers in the leather industry. The modern tanning processes use considerable amount of energy, water, and chemicals. The processes pollute the land to such an extent that old tannery land cannot be used for cultivation. Similarly, petroleum-based leather is not biodegradable and leads to environmental pollution.
Mushroom based bio-leather is considered sustainable because it requires fewer resources for production as compared to animal leather. Conventional leather production involves a significant amount of water, energy, and chemicals, leading to pollution and other negative environmental impacts. In contrast, mushroom based bio-leather production as provided by the present invention, primarily uses agricultural waste or crop residues, such as rice straw, wheat straw, cotton wood, sawdust, wheat flour etc.
Along with being sustainable, mushroom based bio-leather is also biodegradable and compostable at the end of its life cycle. As a result, mushroom based bio-leather offers an environmentally friendly alternative to animal and synthetic leather, which has a long lifespan and takes many years to decompose.
Mushroom based bio-leather does not contain the allergens that are present in leather, making it a suitable choice for people with sensitive skin or allergies.
Also, mushroom based bio-leather can be produced to provide a high level of durability and strength, making it a long-lasting material that can withstand wear and tear.
Mushroom based bio-leather is a vegan alternative, making it an ethical choice for consumers who want to avoid products made from animal skin. Overall, mushroom based bio-leather offers a range of advantages that makes it a promising alternative to animal and synthetic leather materials.
Mushroom based bio-leather is, however, a relatively new technology that differs from current technology in several ways and companies and people working in the field are still developing ways to provide easy, simple, economical and sustainable ways and methods for producing biomaterial, like bio-leather, from fungi. Some companies are using genetically modified organisms. Other companies are using petroleum-based chemicals for strength and durability of the material. Yet others grow the mycelium in a highly customized growth chamber which leads to high costs.
The post-processing of mycelium-based bioleather is generally considered more environmentally friendly and offer a more sustainable alternative to traditional animal leather, with the potential to reduce the environmental footprint of the fashion and textile industries. The post-processing of animal or conventional leather typically involves a range of chemical treatments, such as tanning, dyeing, and finishing, which can have significant environmental impacts. These processes often involve the use of toxic chemicals, heavy metals, and large amounts of water, as well as generate harmful byproducts and waste that can pollute the environment. Additionally, challenges may arise in terms of meeting certain performance standards, such as water resistance and colorfastness, which are commonly associated with traditional leather. Overall, the field of mycelium-based bioleather is continuously evolving, and there is great potential for further innovation and improvement in post-processing techniques to unlock even more possibilities for this sustainable material.
Hence, there is a need for efficient and environmentally friendly methods for post processing of leather that can enhance the mechanical properties and texture of the biomaterial.
The invention described herein helps making biomaterial under controlled environmental conditions but does not require any specialized growth chamber which is a very costly alternative for production on a large scale. Furthermore, the post-processing of mycelium-based bioleather offers a more sustainable alternative to traditional animal leather.
OBJECTS OF THE INVENTION
An object of the present invention is to provide strains of fungi that can be used in various applications, particularly, in making biomaterials, like bio-leather, by a simple, easy, cost-effective method.
An object of the present invention is also to provide strains of fungi that produce mycelium in high density with good flexibility, good toughness and a complex mycelium network for production of biomaterial, like bio-leather.
An object of the present invention is to provide fungal strains and methods using which biomaterial, like bio-leather, can be made with ease. The bio-leather can be used for making leather garments, footwear, and accessories, such as jackets, coats, pants, shoes, bags, belts, etc.
An object of the present invention is to provide fungal strains using which biomaterial, like bio-leather, which, like animal leather has good strength, tensile strength, flexibility, stretching and colorization properties, can be obtained.
An object of the present invention is to provide a method using which biomaterial is made from agricultural/ organic waste and does not involve the use of animal hides, making it a sustainable and environmentally friendly alternative to animal leather.
An object of the present invention is to provide fungal strains and methods using which biomaterial, like bio leather, which is durable and very similar to animal leather, can be obtained.
An object of the present invention is to provide fungal strains and methods using which biomaterial, like bio-leather can be made which is water-resistant, and which makes it suitable for outdoor and sports applications.
An object of the present invention is to provide fungal strains and methods using which biomaterial, like bio-leather, can be made using a significantly low amount of energy and cost compared to animal leather and which lead to a very low carbon footprint.
An object of the present invention is to provide a method of making biomaterial, like bio-leather, using an easy, simple, and economical method.
An object of the present invention is to provide a method for culturing fungus or growing mycelium biomass using which a high density and good quality of mycelium biomass can be obtained on solid-state media comprising organic wastes, such as agricultural wastes, crop residues or forestry by-products.
An object of the present invention is to provide a method for culturing mycelium or growing mycelium biomass which can be performed under controlled environmental conditions, including temperature and humidity, without using any specialized growth chamber for production at any scale, including large scale.
An object of the present invention is to provide a method for making biomaterial, like bio-leather using an easy, simple, and economical method which can work by using easily available low-cost organic and/or agricultural waste.
An object of the present invention is to provide media and premix compositions for growth of mycelium biomass.
An object of the present invention is to provide a method of post processing of post harvested mycelium sheets.
An object of the present invention is to provide a method of post processing of post harvested fungal mycelium sheet using which biomaterial, like bio-leather, which, like animal leather has good mechanical strength and texture properties, can be obtained.

SUMMARY OF THE INVENTION
The present invention is directed to novel fungal strains of the Ganoderma and Dichomitus species.
The present invention is in particular directed to novel fungal strains selected from:-
Scientific name Applicant’s code MTCC accession number
Ganoderma multipileum A1 MTCC 25685
Ganoderma lucidium A2 MTCC 25683
Dichomitus sp. A3 MTCC 25684
Ganoderma carnosum A7 MTCC 25682

The present invention is further directed to a method of growing mycelium biomass for production of biomaterial, like bio-leather.
The present invention is directed to a method of growing mycelium biomass wherein, the fungal strain is inoculated in a solid-state culture media comprising organic waste, and wherein a premix is added to the mycelium layer at around the beginning of the stationary phase of the mycelium growth, wherein said solid-state media comprises cellulose in the range of from 30-60% (w/w), lignin in the range of from 20-40% (w/w) and hemicellulose in the range of from 10-30% (w/w).
In preferred embodiments of the method of growing mycelium biomass of the present invention, the solid-state media comprises a cellulose content of 50% (w/w), lignin content of 30% (w/w), and hemicellulose content of 20% (w/w).
The present invention is also directed to a method of preparing bio-leather from the fungal mycelium of the novel fungal strains of the present application. In preferred embodiments, the fungal mycelium of the novel fungal strains of the present application is obtained by the method of growing mycelium biomass described herein.
The present invention is further directed to solid-state media compositions and compositions of premix that enable excellent fungal mycelium growth.
The present invention is also directed to the method of post processing of harvested mycelium sheet comprising the steps denature or deproteination, deactylation, crosslinking, plasticization and coating.
In the present invention, the plasticizer comprises PEG400 5-10% by weight, Glycerol 10-15% by weight, Propylene glycol 5-10 % by weight, sorbitol 5-10 % by weight and water as solvent. The water is 65 to 80 % by weight in the plasticizer solution.
In the present invention, plasticizing is carried out by soaking the mycelium sheets in a plasticizer solution for 24-48 hours.
In the present invention, crosslinking solution comprises citric acid and glutaric acid in the range of 5 to 20%, preferably 5-10% by weight each in water. The water is 80 to 90 % by wight in the crosslinking solution.
In the present invention, crosslinking is carried out by soaking the mycelium sheets in a crosslinking solution for 24 hours to obtain 40-60 degree of crosslinking.
In the present invention, the tanning solution comprises tannic acid in the range of 5 to 15% by weight in water and maintained at pH 5.0. The water percentage in the tanning solution may be 85 to 95 %.
In the present invention, the tanning solution comprises tannic acid in the range of 5 to 15% by weight in water is mixed with 10 to 20% laccase powder and 10 to 20% ferric chloride. 85 to 95 % water is used for tannic acid solution preparation. Laccase and ferric chloride is added with respect to the quantity of tannic acid. For example, if 10g tannic acid is taken so 2g of laccase and 2g of ferric chloride will be taken.
In the present invention, the mycelium sheets are soaked in tanning solution and crosslinking solution individually or in combination.
In the present invention, coating comprises 8 to 13% by weight of polyvinyl alcohol and 2-3% by weight of plasticizer.
In the present invention, coating of 40 to 70 micron of polyvinyl alcohol is applied on mycelium sheets and dried at 50º C for 6 hours, followed by application of thin layer of bees wax on both sides of the sheets for hydrophobicity.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the isolated fungal strains. Figure 1A shows strain A1, that is, Ganoderma multipileum which has been deposited at Microbial Type Culture Collection and Gene bank, Chandigarh under deposit number MTCC 25685. Figure 1B shows strain A2, that is, Ganoderma lucidium which has been deposited at Microbial Type Culture Collection and Gene bank, Chandigarh under deposit number MTCC 25683. Figure 1C shows strain A3, that is, Dichomitus sp. which has been deposited at Microbial Type Culture Collection and Gene bank, Chandigarh under deposit number MTCC 25684. Figure 1D shows strain A7, that is, yes already mentionedGanoderma carnosum, which has been deposited at Microbial Type Culture Collection and Gene bank, Chandigarh under deposit number MTCC 25682.
Figure 2 shows the effect of the solid-state media composition on mycelium growth and demonstrates that the solid-state media with Cellulose- 50% w/w, Lignin- 30% w/w and Hemicellulose- 20% w/w (Figure 2B) gave better mycelium growth as compared to solid-state media with Cellulose- 30% w/w, Lignin- 40% w/w and Hemicellulose- 30% w/w (Figure 2A).
Figure 3 shows the growth of the fungal strain on different types of agricultural wastes. In figures 3A and 3B, column A represented wheat straw, column B represents saw dust, column C represents rice straw, column D represents corn cob, column E represents cotton wood and column F represents sugarcane bagasse. In figure 3B, row 1 represents the growth of strain A1 on different agricultural wastes, row 2 represents the growth of strain A2 on different agricultural wastes, row 3 represents the growth of strain A3 on different agricultural wastes and row 4 represents the growth of strain A7 on different agricultural wastes.
Figure 4 shows the effect of addition of premix of the desired particle size on growth of mycelium. Figure 4A shows the mycelium growth before addition of the premix while Figure 4B shows the mycelium growth after addition of the premix with average particle size 0.5 mm- 1 mm. The mycelium layer obtained was good in strength and showed high colour consistency, uniform growth and high smoothness.
Figure 5 shows the effect of addition of premix of particle size 2 mm to 2.5 mm on growth of mycelium. Figure 5A shows the mycelium growth before addition of the premix while Figure 5B shows the mycelium growth after addition of the premix with average particle size 2.0 mm-
2.5 mm. The obtained metabolite sheet was pale yellow and brownish colour, weak in strength and its smoothness was hindered.
Figure 6 shows the effect of the stage at which the premix is added on mycelium growth. Figure 6B shows that the culture in which the premix was added at the beginning of the stationary phase gave unexpected and surprising results in mycelial growth as compared to the culture in which the premix was added at a later stage (Figure 6A).
Figure 7 is a flow chart showing steps of post processing of harvested mycelium sheets.
Figure 8 is a flow chart showing post processing chemical treatments performed on harvested mycelium sheet.
Figure 9 shows different mycelium sheet obtained after each step of post processing chemical treatment
Figure 10 shows different types of processed mycelium sheets or bioleather material.

DETAILED DESCRIPTION
The present invention is directed to novel fungal strains of Ganoderma and Dichomitus species.
The present invention is in particular directed to a novel fungal strain selected from:-
Scientific name Applicant’s code MTCC accession number
Ganoderma multipileum A1 MTCC 25685
Ganoderma lucidium A2 MTCC 25683
Dichomitus sp A3 MTCC 25684
Ganoderma carnosum A7 MTCC 25682

The novel fungal strains of the present invention have one or more characteristics selected from the group consisting of: high growth rate, thick mycelium with good flexibility and thick mycelium with high toughness.
The present invention is also directed to a method of growing mycelium biomass for making biomaterial, like bio-leather and leather substitutes by up cycling organic wastes, such as, low-cost agricultural wastes, crop residue and forestry by-products. In an embodiment such organic wastes serve as feedstock for the growth of fungal mycelium, which comprises a mass of elongated tubular structures and represents the vegetative growth of filamentous fungi. Within a couple of weeks, the fungal biomass growing on organic wastes can be harvested and physically and chemically treated (e.g., by pressing, cross-linking, etc.) to obtain bio-leather.
The organic wastes used by the method of the present application are available at low cost and include agricultural wastes, crop residue and forestry by-products that contains cellulose, hemicellulose and lignin, which are easily digestible by fungi.
Leather substitute materials derived from fungi typically contain completely biodegradable chitin (which acts as a stabiliser in the material) and other polysaccharides, such as glucans.
The invention provides a unique fungi-based bio-leather product with good strength, flexibility, durability and close resemblance to animal leather. Biomaterial, like bio-leather of the present invention is preferably made by using novel and robust fungal strains identified and isolated through vigorous search and cultured in the laboratory environment.
Another aspect of the present invention relates to a method of growing mycelium biomass, comprising culturing a fungal strain on solid-state media comprising organic waste, and adding a premix to the solid-state media at around the beginning of the stationary phase of the mycelium growth.
In additional embodiments, the premix is added twice during mycelium growth- the first time, at around the beginning of the stationary phase and the second time, around the mid-stationary phase of mycelium growth. Preferably, the premix may be added 15-20 days after inoculation when the stationary phase starts, and then in mid-stationary phase, that is, 4-5 days after the first addition of the premix.
In preferred embodiments of the method of growing mycelium biomass, the organic waste comprises cellulose, lignin and hemicellulose. In more preferred embodiments, the solid-state media comprises cellulose in the range of from 30-60% (w/w), lignin in the range of from 20-40% (w/w), and hemicellulose in the range of from 10-30% (w/w). In particularly preferred embodiments, the solid-state media comprises a cellulose content of 50% (w/w), lignin content of 30% (w/w), and hemicellulose content of 20% (w/w).
In a preferred embodiment the fungal strain used in the method of growing mycelium biomassis either a Ganoderma species or a Dichomitus species.
In a further preferred embodiment, the fungal strain in the method of growing mycelium biomass is a strain selected from the group consisting of Ganoderma multipileum, Ganoderma lucidium, Dichomitus sp and Ganoderma carnosum.
In particularly preferred embodiments, the fungal strain is a strain selected from the following fungal strains:
Scientific name Applicant’s code MTCC accession number
Ganoderma multipileum A1 MTCC 25685
Ganoderma lucidium A2 MTCC 25683
Dichomitus sp A3 MTCC 25684
Ganoderma carnosum A7 MTCC 25682

In an embodiment of the present invention, the organic waste used in the method of growing fungal mycelium comprises agricultural waste, crop residues or forestry by-products, selected from rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran, corn cob, sugarcane bagasse or mixtures thereof. In an embodiment, the organic waste used in the method of growing fungal mycelium comprises agricultural waste, crop residues or forestry by¬products, selected from corn cob, rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran or mixtures thereof.
In an embodiment, the solid-state media for growing mycelium biomass in accordance with the present invention comprises cellulose in a content of from 30-60% (w/w), lignin in a content of from 20-40% (w/w) and hemicellulose in a content of from 10-30% (w/w). In preferred embodiments the solid-state media comprises a cellulose in a content of 50% (w/w), lignin in a content of 30% (w/w), and hemicellulose in a content of 20% (w/w).
In a preferred embodiment, the solid-state media has the following composition.
Material Content (% w/w)
Corn cob 15-30
Sawdust 20-30
Rice straw 15-30
Wheat straw 15-30
Cotton wood 10-30
Wheat bran 5-20
Wheat flour 5-20
Additives 2-10
The concentration of the above components or raw materials is adjusted, until a content of cellulose from 30-60% w/w, preferably 50 % w/w, lignin from 20-40% w/w, preferably 30% w/w, and hemicellulose from 10-30% w/w, preferably 20% w/w, is achieved.
The additives in the solid-state media can include one or more of calcium sulphate, calcium carbonate, dextrose and peptone.
The premix is a growth enhancer composition and is added to the culture at the beginning of the stationary phase to stimulate the growth of the fungal mycelium. In an embodiment, the premix is added 10-15 days after initiating culturing or inoculation of the fungal strain. The premix may also be added a second time around the mid-stationary phase. In an embodiment, the premix is added a second time 4-5 days after the first addition of the premix. The premix comprises cellulose, hemicellulose, lignin, and monosaccharides. The premix preferably comprises agricultural wastes and/or nutritional grains, such that the content of cellulose, hemicellulose, lignin, and monosaccharides in the premix is in the following ranges.
Component Content (% w/w)
Cellulose 40-50
Hemicellulose 15-35
Lignin 15-25
Monosaccharides 2-5

The premix may comprise agricultural wastes and/or nutritional grains selected from corn cob, sugarcane bagasse, corn starch, wheat flour, maize flour, wheat straw and mixtures thereof.
In one embodiment, the premix preferably comprises corncob, sugarcane bagasse, corn starch and wheat flour in the following ranges.
Component Content (% w/w) Preferred Content (% w/w)
Corn cob 30-45 40
Sugarcane bagasse 35-45 40
Corn starch 5-12 10
Wheat flour 5-15 10
The content of the above components is adjusted, till a cellulose content of 40-50% w/w, hemicellulose content of 15-35% w/w, lignin content of 15-25% w/w and a content of monosaccharides, in the range of 2-5% w/w, is achieved.
In another embodiment, the premix preferably comprises corn cob, maize flour, and wheat straw in the following ranges.
Component Content (% w/w) Preferred Content (% w/w)
Corn cob 55-65 60
Maize flour 25-32 30
Wheat straw 5-15 10

Again, the content of the above components is adjusted, till cellulose in the range of 40-50% w/w, hemicellulose in the range of 15-35% w/w, lignin in the range of 15-25% w/w and monosaccharides in the range of 5-10% w/w are achieved.
The monosaccharides in the premix composition are selected from the group consisting of glucose, dextrose, fructose and mixtures thereof.
It is also preferable that the premix is in the form of a powder that has a particle size in the range of from 0.5mm to 1.0mm. Said particle size can be achieved preferably by sieving.
In an embodiment, the solid-state media is prepared by a method comprising the following steps:-
a) hydrating organic waste comprising agricultural wastes, crop residues and/or forestry by-products with water;
b) preparing a mix by adding to the hydrated organic waste obtained in step a) above, additives selected from one or more of calcium salts and carbohydrate and nitrogen sources to obtain a mix, such that the mix comprises cellulose, lignin, and hemicellulose.
c) sterilizing the mix by heat and/or pressure to obtain a sterilized mix;
d) cooling the sterilized mix to ambient temperature.
In an embodiment, the agricultural waste, crop residue and forestry by-product in step a) of the method of preparing the solid-state media described above, is selected from rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran, corn cob, sugarcane bagasse or mixtures thereof. In the above method, the agricultural waste or crop residue may be mixed with water in a ratio of 1:2.
In an embodiment the carbohydrate source, in step b) of the method of preparing the solid-state media described above, is selected from dextrose, glucose and potato dextrose broth. In another embodiment the nitrogen source in step b) of the method above, is peptone. In yet another embodiment the calcium salts are calcium sulphate and calcium carbonate.
In an embodiment, in step b) of the method above, the mix is prepared by adding to the hydrated organic waste obtained in step a) of the method, additives selected from one or more calcium salts and carbohydrate and nitrogen sources to obtain a mix such that the mix comprises cellulose in the range of 30-60% w/w, preferably 50 % w/w, lignin in the range of 20-40% w/w, preferably 30% w/w, and hemicellulose in the range of 10-30% w/w, preferably 20% w/w.
In an embodiment, in step d) of the method of preparing the solid-state media described above, the sterilized mix is cooled to ambient temperature, preferably in the range of from 27ºC-30ºC to obtain the solid-state media.
In an embodiment, the solid-state media is prepared by the method comprising the following steps:
a) hydrating organic waste comprising agricultural waste or crop residues, selected from rice straw, wheat straw, cotton wood, sawdust, wheat flour or mixtures thereof with water in a ratio of 1:2;
b) preparing a mix by adding to the hydrated organic waste obtained in step a) above, additives selected from calcium salts and carbohydrate and nitrogen sources like, calcium carbonate, calcium sulphate, dextrose, glucose, peptone and potato dextrose broth (PDB), to obtain a mix, such that the mix comprises cellulose in the range of 30-60% w/w, preferably 50 % w/w, lignin in the range of 20-40% w/w, preferably 30% w/w, and hemicellulose in the range of 10-30% w/w, preferably 20% w/w.
c) sterilizing the mix by heat and/or pressure to obtain a sterilized mix;
d) cooling the sterilized mix to ambient temperature, preferably in the range of from 27ºC- 30ºC to obtain the solid-state media.
In an embodiment, sterilization is carried out at a temperature of 121°C and pressure of 15 psi.
In an embodiment, the solid-state media is filled in trays and the fungal strain is inoculated on said media for culturing.
In an embodiment, the premix is added to the mycelium layer at the beginning of the stationary phase. In an embodiment, the premix is added to the mycelium layer 15-20 days after inoculation of the fungal strain, when the stationary phase of mycelium growth starts.
In another embodiment, the method of growing mycelium comprises the additional step of adding premix to the solid-state media again in the mid-stationary phase of mycelium growth.
In an embodiment, the premix is preferably added 4-5 days after the first addition of the premix.
Another aspect of the present invention is directed to a method of making biomaterial, like bio¬leather. Said method comprises growing the mycelium biomass as described herein and further comprises the steps of:-
a) removing the thick mycelium sheet from the surface of the solid-state media;
b) cleaning the mycelium sheet to remove the remnant solid-state media from the mycelium sheet;
c) preserving the mycelium sheet in a preservative solution;
d) soaking the mycelium sheet in a plasticizer; and
e) drying the mycelium sheet.
In a preferred embodiment, the drying in step e) is carried out such that the mycelium sheet holds 15-20% moisture after drying.
In an embodiment, the additional step of soaking the mycelium in a preservation solution is carried out after step e) of drying the mycelium.
In an embodiment, the preservation solution comprises 30% glycerol or a solution of calcium chloride, methanol, and water in a ratio of 1:1:4.
In an embodiment, the method of making the biomaterial further comprises the following steps after step e) of drying the mycelium.
f) Deacetylation by soaking the mycelium sheet in ethanol and methanol to convert the chitin into chitosan and to deactivate the growth of the mycelium;
g) Crosslinking the chitosan by soaking the mycelium sheet in a crosslinking solution; and h) Plasticizing by soaking the mycelium sheet in a plasticizer.
In an embodiment, the method of making the biomaterial further comprises the following steps after step e) of drying the mycelium: -
f) Deacetylation by soaking the mycelium sheet in ethanol, sodium hydroxide, and a solution comprising methanol, calcium chloride and water in a 1:1:4 ratio, to convert the chitin into chitosan and to deactivate the growth of the mycelium;
g) Crosslinking the chitosan by soaking the mycelium sheet in a crosslinking solution;
h) Tanning into leather by using a combination of a crosslinking solution and tanning solution.
i) Colouring to produce natural black colour by using chemicals selected from tannic acid, acetic acid, laccase, ferric chloride in acetic buffer and combinations thereof;
j) Plasticizing by soaking the mycelium sheet in a plasticizer;
k) Coating with a biodegradable biopolymer selected from polyvinyl alcohol, ethylene glycol and glycerol, to increase the durability and smoothness of the material.
In an embodiment, any plasticizer can be used for mycelium sheet treatment. Preferably, the plasticizer is selected from glycerol, sorbitol, polyethylene glycol, ethylene glycol or mixtures thereof.
Tanning usually involves a method which permanently alters the structure of the material, making it more durable and less susceptible to decomposition. After the steps of deacetylation and crosslinking, the mycelium sheets are soaked in a solution comprising a combination of a tanning solution and crosslinking solution for 24-48 hours. The crosslinking solution is selected from the group consisting of glutaraldehyde, citric acid, adipic acid, polyphenol solution and combinations thereof while the tanning solution is selected from the group consisting of tannic acid, acetic acid, laccase, ferric chloride in acetic buffer solution and combinations thereof.
In an embodiment of the present invention, the plasticizer comprises PEG400 5-10% by weight, Glycerol 10-15% by weight, Propylene glycol 5-10 % by weight, sorbitol 5-10 % by weight and water as solvent.
In an embodiment the present invention, plasticizing is carried out by soaking the mycelium sheets in a plasticizer solution for 24-48 hours.
In an embodiment the present invention, crosslinking solution comprises citric acid and glutaric acid in the range of 5 to 20%, preferably 5-10% by weight each in water.
In an embodiment the present invention, crosslinking is carried out by soaking the mycelium sheets in a crosslinking solution for 24 hours to obtain 40-60 degree of crosslinking.
In an embodiment the present invention, the tanning solution comprises tannic acid in the range of 5 to 15% by weight in water and maintained at pH 5.0.
In an embodiment the present invention, the tanning solution comprises tannic acid in the range of 5 to 15% by weight in water is mixed with 10 to 20% laccase powder and 10 to 20% ferric chloride.
In an embodiment the present invention, the mycelium sheets are soaked in tanning solution and crosslinking solution individually or in combination.
In an embodiment the present invention, coating comprises 8 to 13% by weight of polyvinyl alcohol and 2-3% by weight of plasticizer.
In an embodiment the present invention, coating of 40 to 70 micron of polyvinyl alcohol is applied on mycelium sheets and dried at 50º C for 6 hours, followed by application of thin layer of bees wax on both sides of the sheets for hydrophobicity.
In an embodiment an embodiment of the present invention the post processing steps are described herein below:
Denature/Deproteinization: A fter harvesting of mycelium sheet, fungi are alive which need to be killed or denature before processing for further steps. Pure ethanol (100%) was used to denature any live organism in harvested mycelium sheets. These sheets were submerged in pure ethanol solution for 24h. Ethanol also deproteinate the mycelium sheets.Deacetylation: Deacetylation solution was prepared by the combination of methanol, calcium chloride and water. Molar solution (10M) of methanol and (10M) of calcium chloride were prepared in water, this solution is called ‘MAC solution’. Amount of water was optimized in such a way that the ratio of methanol, calcium chloride, and water remained (1:1:4). After the ethanol treatment, sheets were submerged in MAC solution for at least 24h. Deacetylation of mycelium fibres provides more reactive functional groups for crosslinking. The building material of mycelium fibre is known as chitin which is an acetylglucosamine carbohydrate. After the deacetylation process, acetyl amine groups in chitin are partially converted into acetyl amide.
Crosslinking: Crosslinking in mycelium sheet which originates from the presence of reactive functional groups i.e, acetyl amide. A combination of crosslinking agents are used in optimised composition in the range of 5 to 20 % by weight to give controlled degree of crosslinking. Organic acids with multifunctional carboxyl groups were chosen for this purpose. A combination of citric acid and glutaric acid were taken in the range 5- to 10 % by weight each in water. Mycelium sheets after deacetylation are submerged in a crosslinking solution for 24h at ambient temperature. It was found that increasing the temperature enhanced the crosslinking rate. Controlled crosslinking was optimised with 40 to 60% degree of crosslinking. Increasing crosslinking leads to the more rigid or brittle structure.
Tanning: A tanning process is formulated to further enhance the crosslinking of mycelium sheet. This tanning process resembles the process used for animal leather but this process is specifically designed to process fungal mycelium based products. This step is particularly used to provide crosslinking with tannic acid, which chemically contains polyphenolic groups in its backbone. Concentration of tannic acid used in the range of 5 to 15 % by weight in water. The solution was maintained at pH 5. In order to enhance the efficiency of tannic acid, a Laccase enzyme powder was added in tannic acid solution in the range of 10 to 20 % by weight of tannic acid used. Adding laccase enzyme converts phenolic groups into quinone functionality which is more reactive than phenol. Mycelium sheets were submerged in tannic acid solution for about 24 h. In some cases to further improve the tanning process Ferric chloride (FeCl3) was added in tannic acid solution, about 10-20 % by weight of FeCl3 with respect to tannic acid was added in the solution. Adding feCl3 provide dark purple colour to the mycelium sheets.
Plasticizer: A combination of plasticizers was used. Plasticizer solution was prepared by the combination of different types of chemicals with low molecular weight and polyhydroxyl functionality like polyethylene glycol (Mw 200, Mw 400), anhydrous Glycerol, Propylene glycol, and sorbitol. The primary function of plasticizer treatment is to develop more flexible mycelium sheets which give the feeling of natural animal leather. These polyhydroxy compounds extend the flexibility of mycelium sheets by providing more hydrogen bonding between plasticizer and mycelium fibres. The composition of these plasticizers is novel in this invention. In total the 30 to 40 % by weight amount of plasticizers was prepared in water. In this plasticizer solution about 5-10 % by weight was PEG400, 10-15 % by weight Glycerol, 5-10 % by weight Propylene glycol, and 5-10 % by weight sorbitol was mixed in water as solvent. Polyhydroxy based plasticizers provide durable flexibility because they blonde with mycelium backbone structure. After the crosslinking step mycelium sheets are washed with water till neutral pH and submerged in plasticizer solution for at least 24h or maximum 48 hr at ambient temperature.
Protective coating: After completing the chemical post processing of mycelium sheets, the sheets results in greater mechanical strength. The processed sheets then coated with polymer material to protect it from external exposure. This protective coating is biodegradable which gives excellent barrier properties against water, oils, chemicals, and solvents. A water soluble and biodegradable polymer polyvinyl alcohol (PVA) with specific properties such as molecular weight (Mw 100000 - 130000), viscosity (25 - 35 mPa.s), Hydrolysis (95 - 98%) was used for coating on top of post processed mycelium sheets. About 40 to 70 micron coating of PVA was applied on both sides of mycelium sheet. Coating of PVA also maintained the breathability and other aesthetic properties of mycelium sheets. Formulation of coating material contains about 8-13 % by weight of PVA and 2-3 % by weight of plasticizer. After coating, sheets were dried at 50 ? for 6 h and then heat pressed more obtained a uniform surface of coating material. At the end a very thin layer of beeswax was applied on both sides to confer hydrophobic properties.

EXAMPLES
Example 1- Selection of Strains
Various types of strains isolated from the different places from the wild were studied on various aspects and the best strains were selected based on robustness, toughness, flexibility, density of mycelium, hyphae interconnection and colour. The most critical aspects analysed were:-
1. Density of mycelium –higher density of mycelium provides the desired mechanical strength to the material.
2. Flexibility- higher flexibility provides smoothness and softness and higher tensile strength to the material.
3. Fruiting body toughness- fruiting body toughness provides high strength to the material. Tough fruiting body mycelium are thick, bright, and fast growing. In some strains the fruiting body is very soft, and the mycelium is very thin.
4. Complexity of mycelium network- higher interconnection between the hyphae provide higher strength and flexibility to the material.
Surprisingly, the strains of the invention as described below have excellent robustness of the mycelium, toughness, flexibility, density of mycelium, hyphae interconnection etc., and do not require any special equipment for growth of mycelium, and /or any specific media, culture conditions etc. Solid-state media made from agricultural and organic wastes can support good growth of the fungi, such that excellent robustness of mycelium, toughness, flexibility, density of mycelium, hyphae interconnection etc., can be achieved.

Table 1: Characteristics of Strains Selected
S. No Deposit details Image Selection based on morphology.
Colour, density of mycelium, flexibility, toughness, and complexity of mycelium network
1. A1
Ganoderma multipileum
(deposited at Microbial Type Culture Collection and Gene bank, Chandigarh* on 15th
September 2023 under
accession number MTCC
25685) Fig 1a Colour – white
Density of mycelium – medium
Flexibility – good
Toughness – high
Complexity of mycelium network – highly complex
2. A2
Ganoderma lucidium
(deposited at Microbial Type Culture Collection and Gene bank, Chandigarh* on 15th September 2023 under
accession number MTCC
25683) Fig 1b Colour – off white
Density of mycelium – high
Flexibility – good
Toughness – high
Complexity of mycelium network – highly complex
3. A3
Dichomitus sp
(deposited at Microbial Type Culture Collection and Gene bank, Chandigarh* on 15th September 2023 under
accession number MTCC
25684) Fig 1c Colour – white
Density of mycelium – high
Flexibility – highly flexible
Toughness – high
Complexity of mycelium network – highly complex
4. A7
Ganoderma carnosum
(deposited at Microbial Type Culture Collection and Gene bank, Chandigarh* on 15th
September 2023 under
accession number MTCC
25682) Fig. 1d Colour – white
Density of mycelium – low
Flexibility – average
Toughness – average
Complexity of mycelium network – low
*The international depository authority, Microbial Type Culture Collection and Gene bank is located at Institute of Microbial Technology, Shanti Path, 39A, Sector 39, Chandigarh, 160036, India.

Example 2- Selection of Solid-State Media
Various kinds of media were tested, on which the fungal strains can be cultured at lowest cost, and yet achieve unexpected growth and thickness of mycelium. Solid-state media was tested for the most optimal growth of mycelia. Different solid-state media were prepared by:
a) hydrating organic waste comprising agricultural waste, crop residues or forestry by¬products, selected from rice straw, wheat straw, cotton wood, sawdust, wheat flour, corn cob or mixtures thereof with water in the ratio of 1:2;
b) adding to the hydrated organic waste obtained in step a) above, additives selected from carbohydrate and nitrogen sources like, dextrose, glucose, peptone and potato dextrose broth (PDB) to obtain a mix, such that the mix comprises cellulose, lignin and hemicellulose;
c) sterilizing the mix by heat and/or pressure to obtain a sterilized mix;
d) cooling the sterilized mix to ambient temperature, preferably in the range of 27ºC-30ºC to obtain the solid-state media.
Different organic wastes were mixed in different ratio. The solid-state media were filled in trays and the individual fungal strains were inoculated on said media for culturing.
The following fungal strains were used individually for inoculating the media.
Ganoderma multipileum A1
Ganoderma lucidum A2
Dichomitus sp A3
Ganoderma carnosum, A7

It was found that the solid-state media with organic waste which comprises cellulose in the range of 30-60% w/w, preferably 50 % w/w, lignin in the range of 20-40% w/w, preferably 30% w/w, and hemicellulose in the range of 10-30% w/w, preferably 20% w/w, gave very good mycelium growth.
For instance, the following two media compositions comprising different amounts of cellulose, lignin and hemicellulose, were studied.
Media Composition A Media Composition B
Component Content (%w/w) Component Content (%w/w)
Cellulose 30 Cellulose 50
Lignin 40 Lignin 30
Hemicellulose 30 Hemicellulose 20

The results obtained, as provided in Figure 2, clearly show that out of media compositions A and B, media composition B gave excellent results.
The solid-state media composition A included corn cob, sawdust, rice straw, wheat straw, cotton
wood, wheat bran, wheat flour and additives.
Component Content (%w/w)
Corn cob 15-30
Sawdust 20-30
Rice straw 15-30
Wheat straw 15-30
Cotton wood 10-30
Wheat bran 5-20
Wheat flour 5-20
Additives 2-10
The additives added in this composition were calcium sulphate, calcium carbonate, dextrose and peptone.
The amount of the components was adjusted till the final desired levels of cellulose, lignin and hemicellulose was achieved.
Example 3- Determination of Composition of the Premix and the Effect of its Addition to the Mycelium Biomass
It was also surprisingly found that a premix of high nutritional value, if added in the solid-state fermentation of fungi in the beginning/start of the stationary phase, unexpectedly enhances the growth of the mycelium and a thick complex layer is obtained.
The premix preferably comprises one or more of corncob, sugarcane bagasse, and corn starch and wheat flour.
For experimental purposes, the following composition was used for the premix: -
Component Range (%w/w) Content (% w/w) used to achieve desired content of cellulose, hemicellulose, lignin and monosaccharides
Corn cob 30 to 45 40
Sugarcane bagasse 35 to 45 40
Corn starch 5 to 12 10
Wheat flour 5 to 15 10

Said premix was added at different phases of mycelium growth. The inventors of the present application found that the timing of addition of the premix to the culture is critical in determining growth of mycelium. As shown in Figure 6, the culture in which premix was added at the beginning of the stationary phase (Figure 6B) gave unexpected and surprising results in mycelium growth as compared to the culture in which the premix was added at a later stage (Figure 6A).
Another specific premix that was used successfully had the following composition:
Component Range (%w/w) Content (% w/w) used to achieve desired content of cellulose, hemicellulose, lignin and monosaccharides
Corn cob 55 to 65 60
Maize flour 25 to 32 30
Wheat straw 5 to 15 10

Fungal strains may preferably be grown for 40-50 days post inoculation for complete mycelium growth and to obtain a thick mycelium layer. Preferably, the stationary phase starts 15-20 days after inoculation, and it is preferable to add the premix after 15-20 days post inoculation.
Further, it may be preferable to add the premix twice, the first time, when the stationary phase of the mycelium growth starts and the second time, in mid-stationary phase. Preferably, the premix may be added 15-20 after inoculation when the stationary phase starts, and then in mid stationary phase, that is, 4-5 days after the first addition of the premix.
Further, the inventors of the present application surprisingly found that the growth of the mycelium is also dependent on the particle size of the premix. Premix of fine size (0.5 mm to 1.0 mm) is easily digestible, and the mycelium grows without producing metabolites. The mycelium digests the premix in 2-3 days and an increase in mycelium thickness is seen (0.5 m to 1 mm). This type of raw mycelium layer is good in strength, shows high colour consistency, uniform growth, and high smoothness in comparison to mycelium layer that has metabolites. On the other hand, when premix of larger size is used (2 mm – 2.5 mm), the premix is not easily digestible, and it takes a longer time for mycelium growth. In such a case, the mycelium has a chance to produce metabolites, can show mycelium pinning and produce pale yellow and brownish colour on the mycelium. These metabolites hinder the smoothness of the mycelium sheet and result in mycelium sheets which are weak in strength.
As shown in Figures 4 and 5, the growth of mycelium is much better when premix of particle size 0.5 mm-1.0 mm is used in comparison to premix of particle size of 2 mm-2.5 mm.
The method of growing the mycelium biomass according to the present invention comprises the following steps:
a) Preparing solid-state media comprising solid-state cellulose in the range of 30-60% w/w, preferably 50 % w/w, lignin in the range of 20-40% w/w, preferably 30% w/w, and hemicellulose in the range of 10-30% w/w, preferably 20% w/w;
b) Inoculating the solid-state media with a fungal strain selected from the group consisting of Ganoderma multipileum of accession number MTCC 25685, Ganoderma lucidium of accession number MTCC 25683, Dichomitus sp of accession number MTCC 25684 and Ganoderma carnosum of accession number 25682 and allowing the fungi to grow;
c) Adding a premix of fine particle size at the beginning of the stationary phase of the mycelium growth;
d) Optionally adding the premix again during mid-stationary phase;
e) Allowing the fungus to grow until a thick mycelium sheet is formed and harvesting the fungal biomass.
After growing and obtaining mycelial biomass, the biomaterial was formed by the following steps:
a) removing the thick mycelium sheet from the surface of the solid-state media;
b) cleaning the mycelium from the remnant solid-state media;
c) preserving the mycelium in a solution of CaCl2, methanol and water (1:1:4)
d) soaking the mycelium sheet in a plasticizer.
e) drying the mycelium sheet such that the sheet holds 15-20% moisture.

Example 4- Tensile strength testing of different bio leather products developed at lab level. The tensile strength testing of different types of leather was conducted at the lab level to compare their performance. The testing procedure and results are outlined in the table below.
After harvesting and before post processing the mycelium sheet is brittle, stiff and weak mechanical strength. After Going through post processing step mycelium sheet becomes flexible, smooth and improved mechanical strength.

For experimental purposes, the following parameters were assessed to evaluate the tensile strength:-

The present invention has several applications and advantages in a variety of sectors.
Applications of mycelium bioleather:
1. Fashion and Apparel: Mycelium bioleather can be used to create sustainable and cruelty-free fashion items such as shoes, handbags, wallets, belts, and clothing. Designers can leverage its versatility to craft unique textures, colors, and patterns that cater to eco-conscious consumers seeking ethical alternatives to traditional leather products.
2. Accessories and Home Goods: The innovative material can also be utilised in the production of accessories like watch straps, phone cases, and jewellery, as well as home goods such as upholstery, furniture, and interior decor items. Its durability and customization options make it a desirable choice for eco-friendly design projects.
3. Automotive: Mycelium bioleather's properties make it suitable for applications in the automotive industries. It can be used for interior components, seat covers, steering wheel wraps, and other parts where a durable and sustainable material is needed.
4. Medical and Textile Technology: The biodegradable nature of mycelium-based bioleather makes it a promising material for medical applications such as wound dressings, surgical implants, and biodegradable textiles. Its breathable and moisture-wicking properties can also benefit the textile industry in creating sustainable and comfortable fabrics.

Advnatages of mycelium bioleather
1. Sustainability: Mycelium-based bio leather is made by growing fungus on organic waste, which eliminates the need for intensive farming operations, hazardous tanning chemicals, and the enormous amounts of water generally needed in traditional leather manufacturing. This sustainable approach drastically minimizes the environmental effect of leather production.
2. Cruelty-Free: Unlike animal leather, mycelium bioleather is created without harming animals, making it a cruelty-free option that adheres to ethical and vegan standards.
3. Customizability: The culture process of mycelium bioleather allows for the customisation of texture, color, and thickness, giving designers and producers a versatile material that can be adjusted to individual requirements and tastes.
4. Biodegradability: Mycelium-based bio leather is biodegradable, providing a solution to the problem of synthetic leather waste that accumulates in landfills.
SOURCE AND ORIGIN OF BIOLOGICAL MATERIAL USED
All the fungal strains described in the present application were isolated from dead tree wood obtained from Kanpur and Greater Noida, Uttar Pradesh, India.
, Claims:We claim:

1. A method of making biomaterial comprising the following steps:
a. growing a mycelium biomass by culturing a fungal strain on solid ¬state media comprising organic waste;
b. removing a thick mycelium sheet from the surface of the solid-state media;
c. cleaning the mycelium sheet to remove the remnant solid-state media from the mycelium sheet;
d. preserving the mycelium sheet by soaking in a preservation solution;
e. deacetylation by soaking the mycelium sheet in ethanol and methanol to convert the chitin into chitosan and to deactivate the growth of the mycelium;
f. crosslinking the chitosan by soaking the mycelium sheet in a crosslinking solution;
g. Plasticizing by soaking the mycelium sheet in a plasticizer;
h. coating mycelium sheets by applying 40 to 70 micron of polyvinyl alcohol on mycelium sheets;
i. drying the mycelium sheets at 50º C for 6 hours;
j. applying thin layer of bees wax on both sides of the sheets for hydrophobicity.

2. The method as claimed in claim 1, wherein said solid-state media comprises cellulose in the range of from 30-60% (w/w), lignin in the range of from 20-40% (w/w), and hemicellulose in the range of from 10¬30% (w/w).

3. The method as claimed in claim 1, wherein the solid-state media comprises a cellulose content of 50% (w/w), lignin content of 30% (w/w), and hemicellulose content of 20% (w/w).

4. The method as claimed in claimed 1, wherein the fungal strain is selected from the group consisting of Ganoderma multipileum strain deposited under MTCC 25685, Dichomitus sp strain deposited under MTCC 25684, Ganoderma carnosum strain deposited under MTCC 25682, Ganoderma lucidium strain deposited under MTCC 25683.

5. The method of making biomaterial as claimed in claim 1, wherein drying the mycelium sheet is carried out such that the sheet holds 15-20% moisture after drying.

6. The method as claimed in claim 1, wherein the biomaterial is bio-leather.

7. The method as claimed in claim 1, wherein the additional step of soaking the mycelium in a preservation solution, is carried out after step f) of drying the mycelium.

8. The method as claimed in claim 1, wherein the preservation solution is selected from 30% glycerol or a solution comprising calcium chloride, methanol and water in a ratio of 1:1:4.

9. The method as claimed in claim 1, further comprising the steps of:
g. deacetylation by soaking the mycelium sheet in ethanol, sodium hydroxide and a solution comprising methanol, calcium chloride and water in a 1:1:4 ratio, to convert the chitin into chitosan and to deactivate the growth of the mycelium;
h. crosslinking the chitosan by soaking the mycelium sheet in a crosslinking solution;
i. tanning into leather by using a combination of a crosslinking solution and tanning solution;
j. colouring to produce natural black colour by using chemicals selected from tannic acid, acetic acid, laccase, ferric chloride in acetic buffer and combinations thereof;
k. plasticizing by soaking the mycelium sheet in a plasticizer;
l. coating with a biodegradable biopolymer selected from polyvinyl acetate, ethylene glycol and glycerol, to increase the durability and smoothness of the material.
10. The method as claimed in any one of claims 1 or 9, wherein the plasticizer is selected from the group consisting of glycerol, sorbitol, polyethylene glycol, ethylene glycol and mixtures thereof.

11. The method as claimed in any one of claims 1, 9 or 10, wherein the plasticizer comprises PEG400 5-10% by weight, Glycerol 10-15% by weight, Propylene glycol 5-10 % by weight, sorbitol 5-10 % by weight and water as solvent.

12. The method as claimed in any one of claims 1 or 9, wherein plasticizing is carried out by soaking the mycelium sheets in a plasticizer solution for 24-48 hours.

13. The method as claimed in any one of claims 1 or 9, wherein the crosslinking solution comprises citric acid and glutaric acid in the range of 5 to 20%, preferably 5-10% by weight each in water.

14. The method as claimed in any one of claims 1 or claim 9, wherein crosslinking is carried out by soaking the mycelium sheets in a crosslinking solution for 24 hours to obtain 40-60 degree of crosslinking.

15. The method as claimed in claim 9, wherein the tanning solution is selected from the group consisting of tannic acid, acetic acid, laccase, ferric chloride in acetic buffer solution and combinations thereof.
16. The method as claimed in any one of claims 9 or 15, wherein the tanning solution comprises tannic acid in the range of 5 to 15% by weight in water and maintained at pH 5.0.
17. The method as claimed in claim 15 or 16, wherein the tanning solution comprises tannic acid in the range of 5 to 15% by weight in water is mixed with 10 to 20% laccase powder and 10 to 20% ferric chloride.
18. The method as claimed in claim 9, wherein tanning is carried out by soaking the mycelium sheets in a combination of a crosslinking solution and tanning solution for 24-48 hours.

19. The method as claimed in claim 9, wherein coating comprises 8 to 13% by weight of polyvinyl alcohol and 2-3% by weight of plasticizer.