DESC:Method of production of fungal biomaterial, and fungal biomaterial produced thereof

Field of invention
The present invention is directed to a method for the production of fungal biomaterial. In particular, the present invention relates to a process of growing/culturing one or more strains of fungi (preferably mushroom) to produce fungal biomaterial, preferably, fungal bio-leather.

Background
Fungi, preferably mushroom mycelium offers a sustainable substitute to traditional biomaterials, like, bio-leather. By using mushroom mycelium, one can create bio-leather, an environmentally friendly substitute for animal and synthetic leather.

Traditional leather and its substitutes are obtained from animals and synthetic polymers. Leather production process is a growing concern about the ethical and environment implications, as it consumes a huge amount of water, which generates wastewater with high concentrations of pollutants (mainly, chromium (III), sodium sulphide, ammonium chloride, biocides, aldehydes, dyes, etc.), wherein chrome tanning is the most polluting operation during the production process. The agents such as chromium can be highly toxic and polluting depending on its existing form. Chrome tanning is very harmful to the environment and the finished product often retains chemical odor.

Synthetic leather substitutes made from polyvinyl chloride and polyurethane have captured a wide market and largely mitigate the environmental and social problems associated with leather production. These synthetic leather substitutes also require toxic chemicals in their production and are made from fossil fuels. As a result, they suffer from poor biodegradability and face the same limited end-of-life options as most plastics. This is why leather-like materials derived from fungi, which are biodegradable, are important.

Fungi (preferably mushroom) based biomaterial (Fungal biomaterial), like bio-leather (Fungal bio-leather), is a relatively new technology that differs from traditional animal leather production in various aspects. Mushroom is one of the fungi, which is used for producing biomaterial, and in a mushroom, biomaterial (preferably bio-leather) is made from mycelium which is a vegetative part of a mushroom. In contrast, animal-based leather derives from the skin of animals. Producing mushroom-based biomaterial, like mushroom based bio-leather, has lower environmental impact, as it eliminates the need of chemicals or large amount of water.

Mushroom based biomaterial 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 mushroom based bio-leather, provides unique design aesthetic options due to its textural and natural appearance. 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 inventive.

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.

Currently, the market employs, majorly, either animal-based or petroleum-based leather, both of which have drawbacks. To produce animal-based leather, dangerous and poisonous chemicals such as carcinogenic chromium III are required. Tanning chemicals emit toxic fumes and effluents, making the workplace hazardous to workers. Modern tanning procedures require a significant quantity of electricity, water, and chemicals. The operations degrade the land to the point where previous tannery land cannot be utilised for agriculture. Similarly, petroleum-based leather is non-biodegradable and pollutes the environment.

The approach to creating fungal biomaterial involves harnessing the natural growth capabilities of fungi to cultivate a network of fibres on organic waste materials. By optimising the cultivation process and fine-tuning the growth conditions, it produce a versatile and durable material that exhibits properties similar to animal leather and synthetic leather but without the negative environmental consequences.

There are certain limits to current procedures for creating fungal biomaterial or bio-leather. One problem is maintaining uniformity in the material's texture, thickness, and colour, as fluctuations in growing circumstances might affect the finished result. Furthermore, scaling up production to meet commercial demand while preserving quality and lowering prices is a barrier to broad adoption of fungal bio-leather. Further research and development is required to address problems such as the material's long-term durability, resilience to wear and tear, and compatibility with diverse production methods.

Fungal bio-leather is considered sustainable because it requires fewer resources to produce compared to animal leather. Traditional Leather production involves a significant amount of water, energy, and chemicals, leading to pollution and other negative environmental impacts. In contrast, fungal bio-leather production uses primarily agri-waste or crop residues, such as rice straw, wheat straw, cotton seed, sawdust, wheat flour etc.

There is a further need, however, to produces a robust and flexible biomaterial that may be utilised as a sustainable substitute for traditional leather. There is a further need, to provide a method which has better scalability, improved production and can help produce biomaterial that may be utilised as a sustainable substitute for traditional leather. There is also need for improved methods of production of fungal biomaterial [mushroom (mycelium based)] that reduces the production time to 30-35 days as compared to 60-70 days in case of existing mushroom (mycelium based) bio-leather production process.

There is also need for improved methods of production of fungal biomaterial and improved fungal biomaterial for new applications in the fashion, design, and manufacturing industries.

Summary of the invention
The present invention is directed to a process for the production of fungal biomaterial, and in particular, mushroom based biomaterial. The invention is also directed to fungal biomaterial, and in particular, mushroom biomaterial so produced.

In an embodiment of the present invention, the fungal biomaterial is fungal bio-leather.

In an aspect, the present invention is directed to a method of growing mycelium bio-mass, comprising:
a) inoculating one or more fungal strains on a solid substrate;
b) spraying a liquid feed onto the growing fungal colonies during initial log phase; and
c) covering the growing fungal colonies with a cellulose-based fabric during or around mid-log phase to obtain mycelium bio-mass.

In said aspect, the solid substrate comprises organic waste selected from the group comprising of agri-waste, crop residues and forestry by-products. In a preferred embodiment of said aspect, the solid substrate is an agri-waste based substrate.

In an aspect, the present invention is directed to a process to obtain fungal bio-material, preferably fungal bio-leather, comprising the steps of:-
a) inoculating one or more fungal strains on the solid substrate to allow culturing of the fungal strains;
b) incubating the culture;
c) after colonization of the upper surface of the solid substrate (preferably in the initial log phase, or after 5-7 days of inoculation), spraying a liquid media-based feed onto the fungal colonies growing on the upper surface of the solid substrate;
d) covering the growing fungal colonies with a cellulose based fabric and incubating the culture; and
e) optionally spraying a liquid media-based feed onto the fungal colonies growing on the upper surface of the solid substrate;
f) harvesting the mycelium biomass from the solid-substrate, air drying the harvested mycelium biomass at room temperature and/ or heat pressing at a temperature in the range of from 50 to 120°C for 20 to 60 seconds to obtain fungal bio-material sheet.

In the said aspect, the solid substrate comprises organic waste selected from the group comprising of agri -waste, crop residues and forestry by-products.

In the said aspect, the cellulose-based fabric is added to the growing culture to cover the fungal colonies, during around mid-log phase, after 1 - 5 days of spraying the liquid media in step c), preferably, after 2-3 days of spraying the liquid media in step c).

In the said aspect, after addition of the cellulose-based fabric, the culture is incubated and optionally to further improve the growth, sprayed with a liquid media-based feed after 3- 7 days of covering the culture with a cellulose based fabric, preferably 4-5 days after covering of the culture with a cellulose based fabric.

In the said aspect, in specific embodiment of the invention, the solid substrate comprising organic waste is obtained by:
a) hydrating the organic waste selected from the group comprising of agri -waste, crop residues and forestry by-products.;
b) mixing the hydrated organic waste and additives to obtain a mixture;
c) sterilizing the mixture obtained in step b) under heat and pressure to obtain a sterilized mixture; and
d) cooling down the sterilized mixture to a temperature of around 270C-300C to obtain the agri-waste based substrate.

The hydrating step a) as described above, is carried out by mixing water with the organic waste. The ratio of the organic waste and water is in the rage of 1:1 to 1:2, preferably, 1:1.6. The additives that may be added, include components that can provide carbon and nitrogen source, including but not limited dextrose, peptone and yeast extract. To sterilize the mixture obtained in step b) as described above under heat and pressure, a temperature of around 121 ? (Temperature), a pressure in the range of from 10-20psi, preferably 15 psi (Pressure) is applied for 60 min - 120 min (Time).

The solid substrate of the present invention is preferably filled into trays and inoculated with one or more fungal strains to allow culturing of the fungal strains. The preferable fungal strain to be used in respect of the present invention is Ganoderma multipileum, or other species in combination with Ganoderma multipileum, like, Ganoderma lucidium, Dichomitus sp, Ganoderma carnosum.

The organic waste to be used in the invention, comprises crop residue and forestry by-products that contains cellulose, hemicellulose and lignin, carbohydrates and additives.

In the provided method, a liquid media-based feed is sprayed onto the fungal colonies on the upper surface of the organic-waste based substrate after colonization of the upper surface of the organic-waste based substrate, preferably after 5-7 days of inoculation. Liquid media is formulated by using nitrogen and glucose sources. Liquid media composition contains dextrose, peptone, yeast extract, malt extract and potato dextrose broth.

The cellulose-based fabric comprises cotton, bio-polyester, or nylon and the same is spread over the growing fungal colonies.

In specific embodiment of the invention, the fungal bio-material sheet obtained is processed to obtain fungal biomaterial, and fungal bio-leather. Such processing comprises the steps:-
a) Denaturing the harvested mycelium sheet, after harvesting fungi are alive which need to be killed or denatured before processing further.
b) Deacetylation by soaking the mycelium sheet in ethanol and/or methanol to convert the chitin into chitosan and to deactivate the growth of the mycelium.
c) Crosslinking the chitosan by soaking the mycelium sheet in a crosslinking solution.
d) Tanning process is formulated to further enhance the crosslinking of mycelium sheet.
e) Plasticizing by soaking the mycelium sheet in a plasticizer.
f) Coating with a biodegradable biopolymer selected from polyvinyl alcohol, ethylene glycol and glycerol, to increase the durability and smoothness of the material.

Brief Description of the Drawings
The present invention is described by way of embodiments illustrated in the accompanying drawings wherein:
Figure 1 shows an image of agriculture waste used as substrate for fungal growth
Figure 2 shows an image of inoculated agriculture waste-based substrate used for fungal mycelium growth
Figure 3 shows an image of fungal mycelium growth after 2-3 days
Figure 4 shows an image of primary stage of mycelium growth
Figure 5 shows an image of cellulose based fabric insertion over the mycelium
Figure 6 shows an image of final growth of mycelium which ready for harvest
Figure 7 shows an image of the harvested mycelium sheet
Figure 8 shows a comparative mushroom (mycelium) growth at 30-35 days of present invention (Fig 8c) (as per example 1) as compared to mushroom (mycelium) growth for existing mushroom (mycelium based) bio-leather production method at 30-35 days (Fig 8a), and 60 days (Fig 8b)
Figure 9 shows (mycelium) growth at 30-35 days of present invention as per example 2.
Figure 10 (a to d) shows a comparative mushroom (mycelium) growth with cellulose fibric being added at various stages of culture growth
Figure 11 (a to d) shows a comparative mushroom (mycelium) growth with liquid media being added at various stages of culture growth

Detailed description of the Invention
The present invention is directed to a process for the production of fungal biomaterial. The present invention is also directed to a process of producing fungal biomaterial that is robust and flexible material that may be utilised as a sustainable substitute for traditional leather. In particular, the present invention combines biotechnology, material science, and sustainability concepts to create fungal biomaterial that is a cruelty-free and ecologically friendly alternative to animal leather.

In an embodiment, the fungal biomaterial is fungal bio-leather.

In an embodiment the present invention is relates to a method of growing mycelium bio-mass, comprising:
a) inoculating one or more fungal strains on a solid substrate;
b) spraying a liquid feed onto the growing fungal colonies during initial log phase; and
c) covering the growing fungal colonies with a cellulose-based fabric during or around mid-log phase to obtain mycelium bio-mass.

In the preferred embodiment, the solid substrate comprises organic waste selected from the group comprising of agri -waste, crop residues and forestry by-products.

In an embodiment, the solid substrate is an agri-waste based substrate.

In an embodiment, the solid substrate comprises organic waste selected from the group comprising of rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran, corn cob, sugarcane bagasse or mixtures thereof.

In an embodiment, the solid substrate comprises a cellulose content of 40-50% w/w, preferably 43-48% % w/w, hemicellulose content 5-15% w/w, preferably 7-14%w/w, lignin content 35-45% w/w, preferably 35-45% w/w, and additives 2-5%, preferably 3-5%w/w.

In an embodiment, the solid substrate comprises corn cob at 5 to 20% (w/w), sawdust at 20 to 35% (w/w), rice straw at 15 to 35% (w/w), wheat straw at 15 to 35% (w/w), cotton seed at 5 to 15% (w/w), wheat bran at 5 to 15 % (w/w), and additives at 2 to 5% (w/w).

In an embodiment, the liquid feed comprises dextrose in the range of 2-4 g/L, peptone in the range of 2-4 g/L, yeast extract in the range of 2-4 g/L, malt extract in the range of 2-4 g/L and potato dextrose broth in the range of 2-4 g/L.

In an embodiment, after step a) the culture is incubated at 25-28°C temperature and 60-95% humidity.

In an embodiment, the method comprises spraying the liquid feed in step b) after 5-7 days of inoculation.

In an embodiment, the cellulose-based fabric is placed over the growing fungal colonies in step c) after 1-5 days of spraying the liquid feed in step b).

In an embodiment, the cellulose-based fabric contains around at least 95% cellulose.

In an embodiment, the cellulose-based fabric comprises cotton, bio-polyester, or nylon or linen or flax or hemp containing natural cellulose fibres derived from plant sources or combination thereof. In an embodiment any cellulose fabric that is strong and durable and has the capability to colonise the cellulose based fabric and produce composite sheet can be used.

In an embodiment, in step c) of the method above, the culture is incubated for 4-7 days until a Mycelium layer/ chitin layer is formed above the solid substrate.

In an embodiment, the method comprises the additional step d) of spraying the liquid feed over the Mycelium layer/ chitin layer to promote further growth of mycelium biomass.

In an embodiment, the additives are selected from the group consisting of dextrose, peptone, yeast extract and combinations thereof.
In an embodiment, the present invention is also directed to a process to obtain bio-material, preferably bio-leather, as per the present invention comprises the steps of:-
a) inoculating one or more fungal strains on the solid substrate to allow culturing of the fungal strains;
b) incubating the culture;
c) after colonization of the upper surface of the solid substrate (preferably after 5-7 days of inoculation or during the initial log phase), spraying a liquid media-based feed onto the fungal colonies growing on the upper surface of the solid substrate;
d) covering the growing fungal colonies with a cellulose based fabric and incubating the culture; and
e) optionally spraying a liquid media-based feed onto the fungal colonies growing on the upper surface of the solid substrate;
f) harvesting the mycelium biomass from the solid-substrate, air drying the harvested mycelium biomass at room temperature and/ or heat pressing at a temperature in the range of from 50 to 120°C for 20 to 60 seconds to obtain fungal bio-material sheet.

In an embodiment, the fungal strains are inoculated on the solid substrate to allow culturing of the fungal strains, which is then incubated after culture growth initiation and until an upper surface colonisation of substrate is achieved. Once the upper surface of the solid substrate is fully colonized substrate (preferably after 5-7 days of inoculation or during the initial log phase), a liquid media-based feed is sprayed onto the fungal colonies on the upper surface of the solid substrate. After spraying the liquid media, the culture is then incubated for 1-5 days, preferably 2-3 days.

After 1- 5 days, preferably 2-3 days, the process of growing mycelium comprises an additional step of adding cellulose based fabric over the growing fungal colonies.

The cellulose based fabric is added to the growing culture to cover the fungal colonies, during the around mid-log phase, after 1 - 5 days of spraying the liquid media in step c), preferably after 2-3 days of spraying the liquid media in step c).

As an option, after covering with cellulose based fabric, the culture is incubated for 3- 7 days, preferably 4-5 days, and more liquid media-based feed sprayed onto the fungal colonies on the upper surface of the solid substrate. That is, after addition of the cellulose based fabric, the culture is incubated and optionally to further improve the growth, sprayed with a liquid media-based feed during the later part of the log phase, after 3- 7 days of covering the culture with a cellulose based fabric in step d), preferably 4-5 days after covering of the culture with a cellulose based fabric in step d).

Harvest the thick mycelium sheet from the surface of the solid-substrate, then air dry the harvested mycelium sheet at room temperature or heat pressing at a temperature in the range of from 60 °C to 120°C.

Fungal Strains
The various types of fungal strain used for the bio-leather production, such as Ganoderma multipileum or other species can be used for present invention.

The preferable fungal strain to be used in respect of the present invention is Ganoderma multipileum, or other species in combination with Ganoderma multipileum, like, Ganoderma lucidium, Dichomitus sp , Ganoderma carnosum.

The present invention in particular uses fungal strain selected from the following 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

The most preferred fungal strain of the present invention is Ganoderma multipileum which is a type of polypore fungus belonging to the Ganoderma genus. It is characterised by its reddish-brown, woody fruiting bodies with a shiny, varnished appearance. By growing the fungus on unique substrate composition such as agri - waste, the mycelium creates a dense network that can be collected / harvested and processed into a leather-like substance. Ganoderma multipileum helps to improve the strength, flexibility, and texture of bio-leather. Its inherent adhesive capabilities help bind the fibres together, resulting in a robust material that may be moulded and coloured as traditional leather.

In an embodiment, the one or more fungal strain are selected from the group consisting of Ganoderma multipileum strain deposited under MTCC 25685, or other species in combination with Ganoderma multipileum strain deposited under MTCC 25685, like, Ganoderma lucidium strain deposited under MTCC 25683, Dichomitus sp. strain deposited under MTCC 25684 and Ganoderma carnosum strain deposited under MTCC 25682, or any combination thereof.

In an embodiment, the fungal strains are selected from the group consisting of Dichomitus sp. strain deposited under MTCC 25684, or other species in combination with Dichomitus sp. strain deposited under MTCC 25684, like, Ganoderma lucidium strain deposited under MTCC 25683, Ganoderma multipileum strain deposited under MTCC 25685 and Ganoderma carnosum strain deposited under MTCC 25682.

In an embodiment, the selected fungal strains are Ganoderma multipilum and Dichomitus sp, and the same are present in a consortium in a ratio of 1:1.

Selecting the right agriculture substrate that supports the growth and development of the chosen fungal strain is essential for optimal bio-leather yield and quality.

Solid- substrate
The choice of solid substrate plays a crucial role in the production of mycelium-based bio-leather, as it directly influences the growth, quality, and characteristics of the final biomaterial.

The organic wastes used by the method of the present application are available at low cost and include agri-wastes, crop residue and forestry by-products that contains cellulose, hemicellulose and lignin, carbohydrates and additives. The solid substrate provides nutrients for the mycelium to develop and thrive. It offers cellulose, hemicellulose, lignin carbohydrates, proteins, and other important nutrients required by the fungi to construct a robust and interlinked network of mycelium fibres. The solid substrates can provide varied textures and structures of bio-leather material. For example, agri-waste, straw, or sawdust can all have an impact on the density, elasticity, and overall feel of the bio-leather.

The present invention also directed to prepare the appropriate combination of carbon/nitrogen ratio of cellulose, hemicellulose and lignin, and additives in such substrate. This yields better result compared and produces high mycelium:

Component Content (% w/w) Preferred content %
Cellulose 40-50 43-48
Hemicellulose 5-15 7-14
Lignin 35-45 36-42
Additives 2-5 3-5

The above ranges can be obtained by using various different wastes in various combinations, and preferably: -
Material Content (% w/w)
Corn cob 5-20
Sawdust 20-35
Rice straw 15-35
Wheat straw 15-35
Cotton seed 5-15
Wheat bran 5-15
Additives 2-5

In specific embodiment of the invention, the solid substrate comprising organic waste is obtained by:
a) hydrating organic waste comprising agri-waste, crop residues and/or forestry by-products with water, in the rage of 1:1 to 1:2, preferably in a ratio of 1:1.6;
b) preparing a mixture by adding one or more carbon and nitrogen sources to the hydrated organic waste obtained in step a) above;
c) sterilizing the mixture obtained in step b) under heat and pressure to obtain a sterilized mixture; and
d) cooling down the sterilized mixture to a temperature of around 27 0C-30 0C to obtain the agri-waste based substrate,
wherein the carbon and nitrogen sources are selected from the group comprising of dextrose, peptone and yeast extract.

In an embodiment, the solid substrate is selected from rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran, corn cob, sugarcane bagasse or mixtures thereof. The hydrating step a) as described above, is carried out by mixing water with organic waste selected from the group comprising of agri-waste, crop residues and/or forestry by-products. The ratio of the organic waste and water is in the range of 1:1 to 1:2, or lower than 1:2, preferably, 1:1.6.

The hydrated organic waste obtained in step a) is mixed with the additives. The additives that may be added, include components that can provide carbon and nitrogen source, including but not limited dextrose, peptone and yeast extract.

In an embodiment, the mixture in step b) comprises cellulose in the range of 40-50% w/w, lignin in the range of 35-45% w/w hemicellulose in the range of 5-15% w/w and additives in the range of 2-5% w/w.

In an embodiment, the mixture in step b) comprises cellulose in the range of 43-48% w/w, lignin in the range of 36-42% w/w hemicellulose in the range of 7-14% w/w and additives in the range of 3-5% w/w.

In an embodiment, the solid substrate is filled into trays and inoculated with one or more fungal strains to allow culturing of the fungal strains.

To sterilize the mixture obtained in step b) as described above under heat and pressure, a temperature of around 121 ? (Temperature), a pressure in the range of from 10-20 psi preferably 15 psi (Pressure) is applied for 90 min to 120 min (Time).

After sterilization, the next step is cooling down of the sterilized mixture to a temperature of around 27 0C-30 0C to obtain the agri-waste based substrate.

The choice of agricultural substrate is crucial in the production of mycelium-based bio-leather as it directly influences the growth, quality, and characteristics of the final material.
a) Nutritional Availability: The agricultural substrate provides nutrients for the mycelium to develop and thrive. It offers cellulose, hemicellulose, lignin carbohydrates, proteins, and other important nutrients required by the fungi to construct a robust and interlinked network of mycelium fibres.

b) Texture and structure: Different agricultural substrates can provide varied textures and structures of bio-leather material. For example, agricultural waste, straw, or sawdust can all have an impact on the density, elasticity, and overall feel of the bio leather.
c) Compatibility with Fungal Strains: In the basidiomycete’s family various types of fungal strain used for the bio-leather production Certain fungal strains, such as Ganoderma multipileum or other species used in bio-leather production, may have specific substrate preferences. Selecting the right agriculture substrate that supports the growth and development of the chosen fungal strain is essential for optimal bio-leather yield and quality.

Liquid media
In preferred embodiments liquid media with high nutritional value contains a combination of glucose and nitrogen sources. Liquid media sprayed during the log phase over the growing mycelium. Mycelium absorbs the nutrients completely and helps to increase the biomass of the mycelium and thickness of mycelium sheet.

In a further preferred embodiment, liquid media is formulated by using the nitrogen and glucose sources. It's a composition of dextrose, peptone, yeast extract, malt extract and potato dextrose broth. The absorption of liquid media by mycelium is higher than the solid media and mycelium consumes almost 100% liquid media in comparison to solid media. After the spray of liquid media over the mycelium it absorbs the nutrients from the media and increases the rate of mycelium growth and maintains the moisture content of the substrate.

In an embodiment liquid media composition of dextrose, peptone, yeast extract, malt extract and potato dextrose broth are in the following ranges.
Chemical g/l
Dextrose 2-4
Peptone 2-4
Yeast extract 2-4
Malt extract 2-4
Potato dextrose broth 2-4
Cellulose based fabric
To increase the quality of the mycelium sheet further apply the cellulose-based fabric during the incubation period over the mycelium growth. The composition of the fabric is 95% cellulose and 5 % other components. The compatibility of Polypore fungus mycelium with cellulose-based fabric is very high. It was noticed that the hyphal bonding with cellulose based fabric is very high which provides more strength to the material.

In a preferred embodiment the addition of cellulose based fabric sheet on mycelium layer provides complex material with enhanced properties. The cellulose-based fabric fibres reinforced structure with the mycelium which increases tensile strength and tear resistance of the material. This results in the material not only being durable but also flexible, making it suitable for a wide range of the applications. Composition of the cellulose-based fabric with mycelium allows for the integration of different texture and patterns offering the unique aesthetic appeal to the bio-leather. The natural look and feel of the cellulose-based fibre blended with mycelium create the material Stand out from the traditional alternative material.

Cellulose-based fabric has moisture absorption capacity which helps in providing the humid conditions so that mycelium grows very fast inside the fabric. The application of cellulose based fabric sheet on mycelium layer provides complex material with enhanced properties. The cellulose based fabric fibres reinforced structure with the mycelium which increases tensile strength and tear resistance of the material. This results in the material not only being durable but also flexible, making it suitable for a wide range of the applications. Composition of the cellulose based fabric with mycelium allows for the integration of different texture and patterns offering the unique aesthetic appeal to the bio leather.

The natural look and feel of the cellulose based fibre blended with mycelium create the material stand out from the traditional alternative material.

Another aspect of the present invention is directed to a method of making fungal biomaterial, like bio leather. Said method comprises growing the mycelium biomass as described herein and further comprises the steps of:-
a. harvesting the mycelium biomass from the surface of the solid substrate before the mycelium initiates pinning to develop fruiting bodies;
b. cleaning the mycelium biomass to remove the remnant solid substrate;
c. drying the mycelium bio-mass to obtain mycelium sheet; and
d. processing the mycelium sheet obtained in step c) to obtain fungal biomaterial

In an embodiment, the mycelium bio-mass is harvested in step b) when the solid substrate is fully utilized and mycelium growth ceases.

In an embodiment, drying the mycelium bio-mass in step c) is carried out at room temperature and/or heat pressing at a temperature in the range from 50 to 120ºC for 20 to 60 seconds to obtain mycelium sheet.

In an embodiment, processing the mycelium sheet comprises the steps of deacetylating, crosslinking, tanning, coloring, plasticizing and/or coating.

Post processing of Sheet
In specific embodiment of the invention, the sheet obtained is processed to obtain biomaterial, and bio-leather. Such processing comprises the steps : -
a) 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;
b) Crosslinking the chitosan by soaking the mycelium sheet in a crosslinking solution;
c) Tanning into leather by using a combination of a crosslinking solution and tanning solution.
d) Colouring to produce natural black colour by using chemicals selected from tannic acid, acetic acid, laccase, ferric chloride in acetic buffer and combinations thereof;
e) Plasticizing by soaking the mycelium sheet in a plasticizer; and
f) 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 utilized for treating mycelium sheet. Preferably, the plasticizer is selected from glycerol, sorbitol, polyethylene glycol, ethylene glycol or mixtures thereof.

Tanning typically involves a method that permanently changes the structure of the material, enhancing its durability and reducing its susceptibility 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.

The invention not only reduces culture time from 60 days (generally state of the art) to 30-35 days, but also has various other advantages:-
a) Texture and feel- cellulose-based fabric can improve the texture and feel of mycelium-based bio leather, resulting in a soft and comforting touch akin to typical leather. Fabric combined with mycelium can provide a novel material with attractive properties for a variety of uses.
b) Strength- cellulose-based fabrics are noted for their strength, flexibility and durability, which can help supplement the structural integrity of mycelium in bio leather manufacturing. Combining these elements can produce a durable and long-lasting material appropriate for a variety of goods.
c) Thickness- fabric helps to produce the desirable thickness of the mycelium sheet.

EXAMPLES
Example 1
Agri- waste based substrate is obtained as described. The following agri- waste material is taken:-
Material Content (% w/w)
Corn cob 10
Sawdust 20
Rice straw 25
Wheat straw 20
Cotton seed 13
Wheat bran 8
Additives comprising dextrose, peptone, yeast extract 4

The agri-waste hydrated by adding water to the agri-waste in the ratio of 1:1.6. The hydrated agri-waste was then mixed with additives to obtain a mixture. The mixture was sterilized under heat and pressure. The sterilized mixture was cooled to a temperature of 270C to obtain the agri-waste based substrate. Figure 1 provides an image for the agri-waste based substrate of the invention.

The agri-based substrate is put into trays and inoculated with Ganoderma multipileum (MTCC 25685). The source and location is Village: Surajpur Panchayat: Surajpur Block: Bisrakh District: Gautam Buddh Nagar State: Uttar Pradesh. Figure 2 provides an image of the agri-waste based substrate being inoculated by the strain.

The culture is incubated and after colonization of the upper surface of the agri-waste based substrate (as shown in Figure 3) a liquid media-based feed comprising the following constituents was sprayed onto the fungal colonies on the upper surface of the agri-waste based substrate:
Chemical g/l
Dextrose 3.5
Peptone 2.5
Yeast extract 3
Malt extract 2.5
Potato dextrose agar 3

After 3 days (See image in Figure 4), the growing fungal colonies were covered with a cotton fabric and incubated as provided in the image in Figure 5.

After 7 days, the liquid media-based feed was again sprayed onto the fungal colonies growing on the upper surface of the agri-waste based substrate (the spray was enough to soak the surface). After thick growth as shown in Figure 6, the culture was harvested, and the mycelium biomass of the culture was air dried at room temperature and then heat pressed at a temperature to obtain a sheet.

Figure 8c provides the image showing the growth in the present invention after 30-35 days of inoculation.

Example 2
Similar to Example 1, in Example 2 Agri- waste based substrate is obtained as described. The agri-waste hydrated by adding water to the agri-waste in the ratio of 1:1.6. The hydrated agri-waste is then mixed with additives to obtain a mixture. The mixture is then sterilized under heat and pressure- at 121°C and 15 psi for a duration around 100 minutes. The sterilized mixture is cooled to a temperature of 27 0C to obtain the agri-waste based substrate.

Following the sterilization process, the substrate was colonized with spawns to introduce the desired fungal culture. In this example fungal strains Ganoderma multipilum (MTCC 25685) and Dichomitus (MTCC 25684), were spread in a 1:1 ratio across the substrate. Once the substrate was inoculated, the culture was incubated at a temperature in the range of 25-28°C, maintaining a humidity level 60-95%. These environmental conditions help to healthy growth and development of the fungal mycelium.

To promote the healthy growth and development of the fungal mycelium, After the colonization of the upper surface of the agricultural waste-based substrate during the initial log phase, spray a liquid media-based feed onto the fungal colonies growing on the substrate's surface. The other steps were similar to those described in Example 1. The mycelium sheet so obtained is depicted in Figure 9 and has the following dimension and thickness: -

Material Liquid media consortium
Dimension 1*1.25 ft
Thickness 1-2 mm

COMPARITIVE EXAMPLE 1
In comparison when the culturing is done as per the method provided below the growth at day 30 and 60 is shown is figure 8a and 8b respectively: -
• hydrating organic waste comprising agricultural waste or crop residues, comprising rice straw, wheat straw, cotton wood, sawdust, wheat flour with water in a ratio of 1:2;
• preparing a mix by adding to the hydrated organic waste obtained above and additives to obtain a mix,
• sterilizing the mix by heat and/or pressure to obtain a sterilized mix;
• cooling the sterilized mix to ambient temperature to obtain a solid-state media;
• the solid-state media is filled in trays and the fungal strain (A1) is inoculated on said media for culturing;
• a premix (comprising - Corn cob 40 wt%, Sugarcane bagasse 40 wt%, Corn starch 10 wt%, Wheat flour 10wt%) is added to the mycelium layer at the beginning of the stationary phase;

The present invention achieves better growth, strength, flexibility, and thickness than Comparative example. The invention for instance reduces culture time from 60 days (generally state of the art) to 30-35 days, reason for the good growth of the mycelium sheet appears to be lower water content in the solid substrate and the use of liquid media in the initial log phase for feeding the substrate instead of a solid pre-mix.

COMPARITIVE EXAMPLE 2
The addition of cellulose-based fabric at a specific stage enhances the strength and durability of the bio leather material. The fabric provides structural reinforcement and appearance of the bio leather.

In this experiment addition of the liquid media is constant. But cellulose-based fabric is added at different stages of the culture growth curve.

- From the beginning of substrate incubation (lag phase) – By introducing the cellulose based fabric in lag phase, the culture starts grow from different inoculation point and takes time to colonize the rest part of the upper layer. Mycelium starts colonizing the fabric at initial point of growth of the culture and rest of fabric uncolonized. Due to uneven growth of mycelium, the thickness of mycelium varies. This process ensures that addition of the fabric at lag phase is not favourable for the mycelium sheet generation. See Figure 10 a.

- Starting of substrate colonization (Initial stage of log phase)- After the incubation of the inoculated substrate. culture start colonise substrate at different inoculation point and hyphae spread over the upper surface of substrate. Adding the cellulose based fabric at initial log phase stage, mycelium colonise fabric only the hyphae grown area, and the rest of the fabric takes time to colonise the fabric. Due to the irregular growth of the mycelium the thickness of the mycelium sheet varies. This process ensures that the fabric insertion at the initial stage of the lag phase is not beneficial for the generation of the mycelium sheet. See Figure 10b.

- After upper layer substrate colonization (log phase) - After colonization of the upper surface of the Agri-waste based substrate. spraying a liquid media-based feed onto the fully colonise upper surface of the agri-waste based substrate (preferably after 5-7 days of inoculation or during the initial log phase), after 2 days of the liquid media feed spray mycelium cover all the substrate growing fungal colonies. Adding cellulose based fabric over the colonised surface and incubate optimum temperature and humidity after 3-4 days mycelium colonise fabric evenly. In this process the thickness of the mycelium is same across the sheet. See Figure 10c.

- After the whole substrate colonization (stationary phase)- After the colonisation of the whole substrate (stationary phase), on adding the cellulose-based fabric to the colonised surface. At this stage Mycelium has a slow growth rate and produces fruiting bodies when exposed to oxygen. Due to the nutrient depletion in the substrate causes the production of the metabolite over the fabric. Application of cellulose based fabric at stationary phase does not for the generation of the mycelium sheet. See Figure 10d

COMPARITIVE EXAMPLE 3
The addition Liquid media feed at a specific stage enhances mycelium density. In this experiment addition of the liquid is done at various point in times to show the difference.

- From the beginning of substrate incubation (lag phase)- Adding the liquid media at a lag phase of the substrate. the moister content increase over the upper layer of substrate due to the high moisture content the growth of the mycelium hindered and the colonization rate automatically slow down. Due to improper growth of the mycelium substrate not ready for the addition of the fabric. See Figure 11a.

- Starting of substrate colonization (initial log phase)- After the incubation of the inoculated substrate. culture start colonise substrate at different inoculation point and hyphae starts spread over the upper surface of substrate. spraying the liquid media at this stage the hyphae grown area absorbs the nutrient from the liquid media and grow but uncolonized part of the substrate moister content increase and the air exchange between the substrate hampered. So, the growth of mycelium slow down. Growth of the mycelium is uneven over the upper layer of substrate. Due to uneven growth of the mycelium substrate not ready for the addition of the fabric. See Figure 11b.

- After upper layer substrate colonization (log phase)- After colonization of the upper surface of the Agri-waste based substrate. spraying a liquid media-based feed onto the fully colonise upper surface of the based (preferably after 5-7 days of inoculation or during the log phase), after 2 days of the liquid media feed spray mycelium cover all the substrate growing fungal colonies. At this phase Add cellulose based fabric over the mycelium and incubate. After few 2-4 days of incubation mycelium colonised the fabric and form chitin film over the fabric. The density of mycelium is high. See Figure 11c.

- After the whole substrate colonization (stationary phase)- After colonization of the upper surface of the Agri-waste based substrate. spraying a liquid media-based feed onto the fully colonise upper surface of the based (preferably after 5-7 days of inoculation or during the log phase), after 2 days of the liquid media feed spray mycelium cover all the substrate growing fungal colonies. At this phase Add cellulose based fabric over the mycelium and incubate. After few 2-4 days of incubation mycelium colonised the fabric and form chitin film over the fabric. The density of mycelium is high. After the colonisation of the whole substrate (stationary phase), Due to the nutrient depletion in the substrate causes the production of the metabolite over the colonized substrate spraying the liquid media on the colonised surface enhance the production of the metabolite over the surface. See Figure 11d.

OTHER EXAMPLES
The test of various parameters of strength, density, elongation etc. of fungal biomaterial or bioleather of the present invention was conducted to evaluate the quality and performance of bioleather. The tests were conducted at External Testing Laboratory- SLEEN Testing Lab, Agra, Uttar Pradesh

S. No Key parameters Bioeather (Sleen Lab Test)
1 Preparation technology Example 1
2 Weight in GSM approx 1050 gsm
3 Density 1.27 g/cm3
4 Thickness 1.5 mm
5 Breaking strength (N/cm) D1: 31.3 N/cm D2:31.1 N/cm
6 Elongation (%) D1: 42.1% D2: 37.1%
7 Tensile Strength D1: 5.38 MPa D2: 5.19 MPa
8 Bally Flex resistance Rank 1 (No damage) 100k cycles
9 Water resistance Water penetration = 0.41 g Water absorption = 20.04 %
10 Tear Resistance 54.2 N (Double hole stitched tear Method)
11 Colour fastness (rubbing method) Dry (10 cycle) - Class 4 (slightly changed) Wet (10 cycle) - Class 3 (Noticeably changed)
12 Moisture retention/Water uptake capacity 23.6 mg/cm2
(It is water vapour absorption, standard SATRA TM178)
13 Stretchability&Recoverability 30 s = 98% 30 min= 98% 1h = 97.5% 2h = 97.5%
14 Stitching capability Yes

The present invention not only reduces culture time from around 60 days (generally state of the art) to 30-35 days to achieve a high density, but also has various other advantages:-
• Texture and feel- cellulose-based fabric can improve the texture and feel of mycelium-based bioleather, resulting in a soft and comforting touch akin to typical leather. Fabric combined with mycelium can provide a novel material with attractive properties for a variety of uses.
• Strength- cellulose-based fabrics are noted for their strength, flexibility and durability, which can help supplement the structural integrity of mycelium in bioleather manufacturing. Combining these elements can produce a durable and long-lasting material appropriate for a variety of goods.
• Thickness- fabric helps to produce the desirable thickness of the mycelium sheet.

APPLICATION AND USES
This invention has several uses, applications, and advantages in a variety of sectors such as,
• Fashion and Apparel: Mycelium bio-leather 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, colours, and patterns that cater to eco-conscious consumers seeking ethical alternatives to traditional leather products.
• 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.
• Automotive: Mycelium bio-leather'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.
• Medical and Textile Technology: The biodegradable nature of mycelium-based bio-leather 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.

SOURCE AND GEOGRAPHICAL ORIGIN
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 growing mycelium bio-mass, comprising:
a) inoculating one or more fungal strains on a solid substrate;
b) spraying a liquid feed onto the growing fungal colonies during initial log phase; and
c) covering the growing fungal colonies with a cellulose-based fabric during or around mid-log phase to obtain mycelium bio-mass.

2. The method as claimed in claim 1, wherein the solid substrate comprises organic waste selected from the group comprising of agri -waste, crop residues and forestry by-products.

3. The method as claimed in claim 1 or 2, wherein the solid substrate comprises organic waste selected from the group comprising of rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran, corn cob, sugarcane bagasse or mixtures thereof.

4. The method as claimed in claim 1, wherein the solid substrate comprises a cellulose content of 40-50% w/w, hemicellulose content 5-15% w/w, lignin content 35-45% w/w, and additives 2-5%.

5. The method as claimed in claim 1, wherein the solid substrate comprises corn cob at 5 to 20% (w/w), sawdust at 20 to 35% (w/w), rice straw at 15 to 35% (w/w), wheat straw at 15 to 35% (w/w), cotton wood at 5 to 15% (w/w), wheat bran at 5 to 15 % (w/w), additives at 2 to 5% (w/w).

6. The method as claimed in claim 1, wherein the one or more fungal strain are selected from the group consisting of Ganoderma multipileum strain deposited under MTCC 25685, or other species in combination with Ganoderma multipileum strain deposited under MTCC 25685, like, Ganoderma lucidium strain deposited under MTCC 25683, Dichomitus sp. strain deposited under MTCC 25684 and Ganoderma carnosum strain deposited under MTCC 25682.

7. The method as claimed in claim 1, wherein the fungal strains are selected from the group consisting of Dichomitus sp. strain deposited under MTCC 25684, or other species in combination with Dichomitus sp. strain deposited under MTCC 25684, like, Ganoderma lucidium strain deposited under MTCC 25683, Ganoderma multipileum strain deposited under MTCC 25685 and Ganoderma carnosum strain deposited under MTCC 25682.

8. The method as claimed in claim 6 or claim 7, wherein the selected fungal strains are Ganoderma multipilum and Dichomitus sp, in a ratio of 1:1.

9. The method as claimed in claim 1, wherein the liquid feed comprises dextrose in the range of 2-4 g/L, peptone in the range of 2-4 g/L, yeast extract in the range of 2-4 g/L, malt extract in the range of 2-4 g/L and potato dextrose broth in the range of 2-4 g/L.

10. The method as claimed in claim 1, wherein after step a) the culture is incubated at 25-28°C temperature and 60-95% humidity.

11. The method as claimed in claim 1, wherein the method comprises spraying the liquid feed in step b) after 5-7 days of inoculation.

12. The method as claimed in claim 1, wherein the cellulose-based fabric is placed over the growing fungal colonies in step c) after 1-5 days of spraying the liquid feed in step b).

13. The method as claimed in claim 1, wherein the cellulose-based fabric contains 95% cellulose.

14. The method as claimed in claim 1 or claim 13, wherein the cellulose-based fabric comprises cotton, bio-polyester, or nylon or combination thereof.

15. The method as claimed in claim 1, wherein in step c), the culture is incubated for 4-7 days until a chitin layer is formed above the solid substrate.

16. The method as claimed in claim 1, wherein the method comprises the additional step d) of spraying the liquid feed over the chitin layer to promote further growth of mycelium bio-mass.
17. The method as claimed in claim 4 or claim 5, wherein the additives are selected from the group consisting of dextrose, peptone, yeast extract and combinations thereof.

18. The method as claimed in claim 1, wherein the solid substrate comprising organic waste is prepared by a method comprising the following steps:
a. hydrating organic waste comprising agri-waste, crop residues and/or forestry by-products with water, in the rage of 1:1 to 1:2, preferably in a ratio of 1:1.6;
b. preparing a mixture by adding one or more carbon and nitrogen sources to the hydrated organic waste obtained in step a) above;
c. sterilizing the mixture obtained in step b) under heat and/or pressure to obtain a sterilized mixture; and
d. cooling down the sterilized mixture to a temperature, preferably in the range of 27ºC-30ºC to obtain solid substrate,
wherein the carbon and nitrogen sources are selected from the group comprising of dextrose, peptone and yeast extract.

19. The method as claimed in claim 18, wherein the organic waste is selected from the group consisting of rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran, corn cob, sugarcane bagasse or mixtures thereof.

20. The method as claimed in claim 18, wherein the mixture in step b. comprises cellulose in the range of 40-50% w/w, lignin in the range of 35-45% w/w hemicellulose in the range of 5-15% w/w and additives in the range of 2-5% w/w.

21. The method as claimed in claim 18 or claim 20, wherein the mixture in step b. comprises a cellulose content of 40-50% w/w, hemicellulose content 5-15% w/w, lignin content 35-45% w/w, and additives 2-5%.

22. The method as claimed in claim 18, wherein sterilizing the mixture obtained in step b) is carried out at a temperature of 121°C and pressure in the range of 10-20 psi, preferably 15 psi for 90 to 120 min.

23. The method as claimed in claim 1 or claim 18, wherein the solid substrate is filled into trays and inoculated with one or more fungal strains to allow culturing of the fungal strains.

24. A method of making fungal biomaterial comprising the following steps:
a. growing the mycelium bio-mass as claimed in any of claims 1 to 23,
b. harvesting the mycelium bio-mass from the surface of the solid substrate before the mycelium initiates pinning to develop fruiting bodies;
c. cleaning the mycelium bio-mass to remove the remnant solid substrate;
d. drying the mycelium bio-mass to obtain mycelium sheet; and
e. processing the mycelium sheet obtained in step d) to obtain fungal biomaterial.

25. The method as claimed in claim 24, wherein the mycelium bio-mass is harvested in step b) when the solid substrate is fully utilized and mycelium growth ceases.

26. The method as claimed in claim 24, wherein drying the mycelium bio-mass obtained in step c) is carried out at room temperature and/or heat pressing at a temperature in the range from 50 to 120ºC for 20 to 60 seconds to obtain mycelium sheet.

27. The method as claimed in claim 24, wherein processing the mycelium sheet comprises the steps of deacetylating, crosslinking, tanning, coloring, plasticizing and/or coating.

28. The method as claimed in claim 24, wherein the fungal biomaterial is fungal bio-leather.

29. The method as claimed in claim 24 or claim 27, comprising the steps of:
a) deacetylation by soaking the mycelium sheet in ethanol, sodium hydroxide and a deacetylation 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;
b) crosslinking the chitosan by soaking the mycelium sheet in a crosslinking solution;
c) tanning into leather by using a combination of a crosslinking solution and tanning solution;
d) colouring to produce natural black colour by using chemicals selected from tannic acid, acetic acid, laccase, ferric chloride in acetic buffer and combinations thereof;
e) plasticizing by soaking the mycelium sheet in a plasticizer;
f) coating with a biodegradable biopolymer selected from polyvinyl acetate, ethylene glycol and glycerol, to increase the durability and smoothness of the material.

30. The method as claimed in claim 29, wherein the plasticizer is selected from the group consisting of glycerol, sorbitol, polyethylene glycol, ethylene glycol or mixtures thereof.

31. The method as claimed in claim 29, wherein the crosslinking solution is selected from the group consisting of glutaraldehyde, citric acid, adipic acid, polyphenol solution and combinations thereof.

32. The method as claimed in claim 29, 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.

33. The method as claimed in claim 29, wherein tanning is carried out by soaking the mycelium sheets in a combination of a crosslinking solution and tanning solution for 24-48 hours.

34. The method as claimed in any one of claims 1 to 33, wherein the method reduces culture time from 60 days to 30-35 days.