DESC:Fungal Strains, Consortium of Fungal Strains and Methods of Producing Fungal Biomaterial

FIELD OF INVENTION
The present disclosure relates generally to isolated fungal strains, consortium of such isolated fungal strains, method for growing/culturing said fungal strains or said consortium of fungal strains, in particular, to produce fungal biomaterial or final bio-leather. The fungal biomaterial is fungal bio-leather.

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 without 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 make it a dangerous place for the workers. 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 to produce compared to animal leather. 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 uses primarily 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 skins.

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, 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 cost. 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.

OBJECTS OF THE INVENTION
An object of the present invention is to provide strains of fungi and a consortium of fungal strains that can be used for various applications, particularly, in making biomaterials, like bio- leather, by a simple, easy, cost-effective process.

An object of the present invention is also to provide strains of fungi and a consortium of fungal strains 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 a consortium of fungal strains, and methods, using which biomaterial, like bio-leather can be made with ease, to develop 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 and a consortium of fungal strains and methods, 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 a consortium of 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 a consortium of 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 a consortium of 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 process of making biomaterial, like bio-leather, using an easy, simple, and economical method.

An object of the present invention is to provide a process for culturing mycelium which can be performed under controlled environmental conditions, like temperature and humidity.
However, said method does not require any specialized growth chamber for production at any scale, including large scale.

An object of the present invention is to provide a process 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.

SUMMARY OF THE INVENTION
The present invention is directed to fungal strains of the Ganoderma and Dichomitus species.

The present invention is also directed to a consortium of fungal strains of Ganoderma, Dichomitus species.

The present invention is in particulate directed to a consortium of fungal strains of Ganoderma, Dichomitus species, wherein the Ganoderma and Dichomitus species are present in the ratio of from 05:1 to 1: 0.5, preferably 1:1.

The present invention is in particular directed to fungal strains selected from:
Scientific name Applicant’s code MTCC accession number
Ganoderma multipileum A1 MTCC 25685
Ganoderma lucidum A2 MTCC 25683
Dichomitus sp A3 MTCC 25684
Ganoderma Carnosum A7 MTCC 25682

In another aspect, the present invention is directed to a consortium of one or more fungal strains selected from:
Scientific name Applicant’s code MTCC accession number
Ganoderma multipileum A1 MTCC 25685
Ganoderma lucidum A2 MTCC 25683
Dichomitus sp A3 MTCC 25684
Ganoderma Carnosum A7 MTCC 25682

Optionally, the consortium may include further fungal strains of one or more of Ganoderma, Dichomitus, Trametes, or Pluerotus species. In another preferred embodiment, the consortium may include further strains selected from the group consisting of Ganoderma carnosum, Trametes versicolor, Ganoderma lucidum, Pleurotus ostreatus.

In another embodiment, the invention is directed to a consortium of Ganoderma multipileum, Ganoderma Lucidum, Dichomitus sp, Ganoderma Carnosum species. Preferably in said embodiment Ganoderma and Dichomitus species are present in a ratio of from 05:1 to 1: 0.5, preferably 1:1.

The present invention is also directed to a method of growing mycelium biomass for production of biomaterial, like bio-leather. The present invention is also directed to a method of growing mycelium biomass wherein, one or more fungal strains are inoculated in a solid state culture media comprising organic waste, and wherein a premix is added to the culture at around the beginning of the stationary phase of the culture, wherein said organic waste comprises: cellulose in the range of from 30-60%, preferably 50 %, lignin in the range of from 20-40%, preferably 30%, and hemicellulose in the range of from 10-30%, preferably 20%.

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 lucidum which has been deposited at Microbial Type Culture Collection and Gene bank, Chandigarh under deposit number MTCC 8 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, Ganoderma 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 shows the effect premix on mycelium growth wherein the mycelium consortium is used. Figure 7(A) shows stage 1 of culture growth after 4-6 days incubation. Figure 7(B) shows stage 2 of culture growth after 12-15 days + premix. Figure 7(C) shows final culture growth.

DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to fungal strains of Ganoderma and Dichomitus species and to a consortium of fungal strains of Ganoderma and Dichomitus species.

The present invention is directed to fungal strains of Ganoderma and Dichomitus species and to a consortium of fungal strains of Ganoderma and Dichomitus species.

The present invention is directed to fungal strains of Ganoderma and Dichomitus species and to a consortium of fungal strains of Ganoderma and Dichomitus species, wherein Ganoderma and Dichomitus species are present in the ratio of from 05:1 to 1: 0.5, preferably 1:1.

The present invention is in particular directed to a fungal strain selected from:
Scientific name Applicant’s code MTCC accession number
Ganoderma multipileum A1 MTCC 25685
Ganoderma lucidum A2 MTCC 25683
Dichomitus sp A3 MTCC 25684
Ganoderma Carnosum A7 MTCC 25682

In another embodiment, the present invention is directed to a consortium of one or more fungal strains selected from:
Scientific name Applicant’s code MTCC accession number
Ganoderma multipileum A1 MTCC 25685
Ganoderma lucidum A2 MTCC 25683
Dichomitus sp A3 MTCC 25684
Ganoderma Carnosum A7 MTCC 25682

Optionally the consortium may include further fungal strains of one or more of Ganoderma, Dichomitus, Trametes, or Pluerotus species. In another preferred embodiment, of the present aspect, the consortium may include further strains selected from the group consisting of Ganoderma carnosum, Trametes versicolor, Ganoderma lucidum, Pluerotus oyster.

In another embodiment, the present invention is directed to a consortium of Ganoderma multipileum, Ganoderma lucidum, Dichomitus sp, Ganoderma carnosum species. Preferably in said embodiment the different species are present in in the ratio of from 05:1 to 1: 0.5, preferably 1:1. Preferably in said embodiment, Ganoderma and Dichomitus species are present in the ratio of from 05:1 to 1: 0.5, preferably 1:1

The fungal strains of the present invention have one or more of the characteristics selected from the group consisting of: high growth rate, thick mycelium with good flexibility and thick mycelium with high toughness. The present invention in preferred embodiments is also directed to a consortium of fungal strains that have excellent growth characteristics and can be used particularly for making the biomaterials, like bio-leather. The present invention is also directed to a process of growing mycelium biomass for making biomaterial, like bio-leather and leather substitutes by upcycling organic wastes, such as, low-cost agricultural and forestry by-products (e.g. sawdust). 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 can be harvested and physically and chemically treated (e.g., by pressing, cross-linking, etc.).

The organic waste used by the process of the present application is available at low cost, and includes agricultural waste, crop residue 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, or their consortiums, identified and isolated through vigorous search and cultured in the laboratory environment.

Another aspect of the present invention relates to a process of growing mycelium biomass, comprising culturing one or more fungal strains of the present invention on solid-state media comprising organic waste, and adding a pre-mix to the solid-state media in the beginning of the stationary phase of the culture.

In preferred embodiments the organic waste comprises cellulose in the range of from 30-60%, preferably 50 %, lignin in the range of from 20-40%, preferably 30%, and hemicellulose in the range of from 10-30%, preferably 20%.

In an embodiment of the present invention, the organic waste used in the process of growing fungal mycelium comprises agricultural waste or crop residues, selected from rice straw, wheat straw, cotton wood, sawdust, wheat flour, or mixtures thereof. In an embodiment, the organic waste used in the process of growing fungal mycelium comprises agricultural waste or crop residues, selected from rice straw, wheat straw, cotton wood, sawdust, wheat flour, or mixtures thereof in the following preferred ranges:-
Material Range (% 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 range of the above raw materials is adjusted, until a content of cellulose in the range of from 30-60% w/w, preferably 50 % w/w, lignin in the range of from 20-40% w/w, preferably 30% w/w, and hemicellulose in the range of 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 pre-mix is a growth enhancer and is added to the culture at the beginning of the stationary phase to stimulate the growth of the culture. In an embodiment, the premix is added 10-15 days after initiating culturing. The pre-mix preferably comprises agricultural wastes and nutritional grains, such that the content of cellulose, hemicellulose, lignin, and carbohydrate in the premix is in the following ranges.

Component Range (% w/w)
Cellulose 40-50
Hemicellulose 15-30
Lignin 15-25
Carbohydrate 5-10

In one embodiment, the pre-mix preferably comprises corncob, sugarcane bagasse, corn starch and wheat flour in the following ranges.
Component % w/w Preferred % w/w
Corn cob 30-45 40
Sugarcane bagasse 35-45 40
Corn starch 5-12 10
Wheat flour 5-15 10

The range of the above components is adjusted, till cellulose in the range of 40-50% w/w, hemicellulose in the range of 15-30% w/w, lignin in the range of 15-25% w/w and carbohydrates in the range of 5-10% w/w are achieved.

In another embodiment, the pre-mix preferably comprises corn cob, maize flour, and wheat straw in the following ranges.
Component % w/w Preferred % w/w
Corn cob 55-65 60
Maize flour 25-32 30
Wheat straw 5-15 10

Again, the range of the above components is adjusted, till cellulose in the range of 40-50% w/w, hemicellulose in the range of 15-30% w/w, lignin in the range of 15- 25% w/w and carbohydrates in the range of 5-10% w/w are achieved.

It is also preferable that the pre-mix 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 the process 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, corn cob or mixtures thereof with water in the ratio of 1:2;
b) preparing a mix by 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 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 temp, 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 or consortium of fungal strains are inoculated on said media for culturing.

In an embodiment, the premix is added to the solid-state media at the beginning of the stationary phase. In an embodiment, the premix is added to the solid-state media 15-20 days after inoculation when the stationary phase starts.

In another embodiment, the process of growing mycelium comprises the additional step of adding pre-mix to the solid-state media again around the mid-stationary phase of culturing. 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 process of making biomaterial, like bio-leather. Said process comprises growing the mycelium biomass as provided above 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 from the remnant solid-state media;
c) preserving the mycelium sheet;
d) adding organic moisturizer to the upper and lower part of the mycelium sheet; and
e) drying the mycelium sheet.

In an embodiment, the step of preserving the mycelium is carried out by soaking the mycelium in a preservation solution, like combination of methanol CaCl2 solution.

In an embodiment, any organic moisturizer or organic plasticizers can be used for mycelium sheet treatment. Preferably, the organic moisturizer is biobased and 100% biodegradable like polyethylene glycol, glycerol and/or ethylene glycol.

In an embodiment, the process of making the biomaterial further comprises the steps of :-
a. Deacetylation by soaking the mycelium sheet into ethanol and methanol to convert the chitin into chitosan and to deactivate the growth of the mycelium;
b. Crosslinking the chitosan by soaking the mycelium sheet into crosslinking solution; and
c. Plasticizing by soaking the mycelium sheet into the biobased plasticiser solution.

In an embodiment, the process of making the biomaterial further comprises the steps of: -
a. Deacetylation by soaking the mycelium sheet into ethanol, NaOH 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 into crosslinking solution, wherein the crosslinking solution is selected from the group comprising glutaraldehyde, citric acid, adipic acid, polyphenol solution and combinations thereof;
c. Tanning into leather by using a combination of a crosslinking solution and tannic acid, acetic acid, laccase, ferric chloride in acetic buffer solution.
d. Colouring 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 into the bio-based plasticiser solution;
f. Coating with a biodegradable biopolymer selected from polyvinyl alcohol, ethylene glycol and glycerol, to make the material water resistant.

Tanning usually involves a process 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 comprising 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.

EXAMPLES

SELECTION OF STRAINS
Various different 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 culturing, 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 lucidum

(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.

In addition to the above, various consortiums were also studied, and it was found that the consortium of the fungal strains also had synergistic potential., specifically Consortium of Ganoderma sp and Dichomitus sp had synergistic potential.

SELECTION OF 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 or crop residues, 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 or consortiums thereof were inoculated on said media for culturing.

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
Cellulose- 30% w/w Cellulose- 50% w/w
Lignin- 40% w/w Lignin- 30% w/w
Hemicellulose- 30% w/w Hemicellulose- 20% w/w

The results obtained, as provided in Figure 2 (For strain A1) , 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. The amount of the components was adjusted till the final desired levels of cellulose, lignin and hemicellulose was achieved.

The solid-state media composition A included
Component Content (%w/w)
Corn cob 10
Sawdust 25
Rice straw 22
Wheat straw 20
Cotton wood 7
Wheat bran 5
Wheat flour 5
Additives 6

The additives added in this composition were calcium sulphate, calcium carbonate, dextrose
and peptone.

The solid-state media composition/Media B included
Component Content (%w/w)
Corn cob 10
Sawdust 22
Rice straw 17
Wheat straw 20
Cotton wood 10
Wheat bran 10
Wheat flour 5
Additives 6

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, corn starch and wheat flour. For experimental purposes, the following composition was used for the premix: -
Component % w/w
Corn cob 40%
Sugarcane bagasse 40%
Corn starch 10%
Wheat flour 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 mycelial growth as compared to the culture in which the premix was added at a later stage (Figure 6A).

Fungal strains may preferably be grown for 40-50 days post inoculation for the 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 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. The inventors of the present application also 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 (0.5m to 1mm). This type of raw mycelium layer is good in strength, shows high colour consistency, uniform growth, and high smoothness in comparison to mycelium layer sheet 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. 23

Example – Consortium
Agri- waste based substrate is obtained as described. The following agri- waste is taken:-

Component Content (%w/w)

Corn cob 10
Sawdust 22
Rice straw 17

Wheat straw 20
Cotton wood 10
Wheat bran 10
Wheat flour 5

Additives 6

The agri-waste hydrated by adding water to the agri-waste in the ratio of 1:2. The hydrated agri-waste is then mixed with additives to obtain a mixture. The mixture was then sterilized under heat and pressure. The sterilized mixture is cooled to a temperature of 270 C to obtain the agri-waste based substrate.

The agri-based substrate is put into trays and inoculated with fungal strains Ganoderma multipilum (MTCC 25685) and Dichomitus (MTCC 25684) Consortium wherein the same are in a 1:1 ratio across the substrate. Figure 7A provides an image of the agri-waste based substrate being inoculated by the consortium.

For the Consortium as well, it was 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.

For experimental purposes, the following composition was used for the premix: -
Component % w/w
Corn cob 40%
Sugarcane bagasse 40%
Corn starch 10%
Wheat flour 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 7B, the culture in which premix was added at the beginning of the stationary phase (Figure 7B) gave unexpected and surprising results in mycelial.

Fungal strains in consortium are preferably grown for 40-50 days post inoculation for the complete mycelium growth and to obtain a thick mycelium layer. See Figure 7c. 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 starts and the second time, in mid-stationary phase.Premix of fine size (0.5 mm to 1.0 mm) was used.

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 consortium of one or more of :
a. isolated Ganoderma multipileum strain which is deposited under MTCC 25685.;
b. isolated Dichomitus sp strain or culture thereof which is deposited under MTCC 25684;
c. isolated Ganoderma carnosum strain which is deposited under MTCC 25682; or
d. isolated Ganoderma lucidum strain which is deposited under MTCC 25683.

2. A consortium as claimed in claim 1, wherein Ganoderma sp. and Dichomitus sp are present in the ratio of from 05:1 to 1: 0.5, preferably 1:1.

3. A method of growing mycelium biomass, comprising culturing a consortium of fungal strain as claimed in claim or 2 on solid-state media comprising organic waste, wherein a pre-mix is added to the mycelium layer at around the beginning of the stationary phase of the mycelium growth, and 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).

4. The method as claimed in claim 3, 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).

5. The method as claimed in claim 3, wherein the organic waste comprises agricultural wastes, crop residues or forestry by-products selected from the group consisting of rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran, corn cob, sugarcane bagasse and mixtures thereof.

6. The method as claimed in claim 3, wherein the solid-state media comprises corn cob at 15-30 % (w/w), sawdust at 20-30% (w/w), rice straw at 15-30% (w/w), wheat straw at 15-30% (w/w), cotton wood at 10-30% (w/w), wheat bran at 5-20% (w/w), wheat flour at 5-20% (w/w) and additives at 2-10% (w/w).

7. The method as claimed in claim 6, wherein the additives are selected from the group consisting of calcium sulphate, calcium carbonate, dextrose and peptone and combinations thereof.

8. The method as claimed in claim 6, wherein the concentration of the components 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.

9. The method as claimed in claim 3, wherein the method comprises an additional step of adding premix a second time to the mycelium biomass around the mid-stationary phase of mycelium growth.

10. The method as claimed in claim 3 or claim 9, wherein the premix is a solid powder with particle size in the range of from 0.5 mm to 1.0 mm.

11. The method as claimed in claim 3 or claim 9, wherein the premix comprises cellulose, hemicellulose, lignin, and monosaccharides.

12. The method as claimed in claim 3 or claim 9, wherein the premix comprises cellulose in a content from 40-50% (w/w), hemicellulose in a content from 15-35% (w/w), lignin in a content from 15-25% (w/w) and monosaccharides in the range from 2-5% (w/w).

13. The method as claimed in claim 3 or claim 9, wherein the premix comprises agricultural wastes and/or nutritional grains selected from corn cob, sugarcane bagasse, corn starch, wheat flour, maize flour, wheat straw and mixtures thereof.

14. The method as claimed in claim 3 or claim 9, wherein the premix comprises corn cob at 30 to 45% (w/w), sugarcane bagasse at 35 to 45% (w/w), corn starch at 5 to 12% (w/w) and wheat flour at 5 to 15% (w/w).

15. The method as claimed in claim 14 wherein the premix comprises corn cob at 40% (w/w), sugarcane bagasse at 40% (w/w), corn starch at 10% (w/w) and wheat flour at 10% (w/w). 19.

16. The method as claimed in claim 3 or claim 9, wherein the premix comprises corn cob at 55 to 65% (w/w), maize flour at 25 to 32% (w/w) and wheat straw at 5 to 15% (w/w).

17. The method as claimed in claim 16, wherein the premix comprises corn cob at 60% (w/w), maize flour at 30% (w/w) and wheat straw at 10% (w/w).

18. The method as claimed in claim 3, wherein 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, preferably 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 one or more 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, preferably in the range of 27ºC-30ºC.

19. The method as claimed in claim 18, wherein the agricultural waste, crop residues and/or forestry by-products, in step a. are selected from 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 carbohydrate source, in step b. is selected from dextrose, glucose and potato dextrose broth, the nitrogen source is peptone and the calcium salts are calcium sulphate and calcium carbonate.

21. The method as claimed in claim 18, wherein the mix is prepared by adding to the hydrated organic waste obtained in step a. 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, lignin in the range of 20-40% w/w and hemicellulose in the range of 10-30% w/w.

22. The method as claimed in claim 21, wherein the mix is prepared by adding to the hydrated organic waste obtained in step a. additives selected from one or more calcium salts and carbohydrate and nitrogen sources to obtain a mix such that the mix comprises a cellulose content of 50% w/w, lignin content of 30% w/w and hemicellulose content of 20% w/w.

23. The method as claimed in claim 3, wherein 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, preferably, 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 agar to obtain a mix, such that the mix comprises cellulose in the range of from 30-60% w/w, preferably 50 % w/w, lignin in the range of from 20-40% w/w, preferably 30% w/w, and hemicellulose in the range of from 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.

24. The method as claimed in claim 3, wherein the solid-state media is filled in trays and the fungal strain is inoculated on said media for growth of mycelium biomass.

25. The method as claimed in claim 3, wherein the premix is added at the beginning of the stationary phase, around 15-20 days after inoculation of the fungal strain.

26. The method as claimed in claim 9, wherein the premix is added in the mid-stationary phase, around 4-5 days after first addition of the premix.

27. A method of making biomaterial comprising the following steps:
a. growing the mycelium biomass through a method as claimed in any of claims 3 to 26,
b. removing the 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. soaking the mycelium sheet in a plasticizer.
f. drying the mycelium sheet, wherein drying the mycelium sheet is carried out such that the sheet holds 15-20% moisture after drying.

28. . The method as claimed in claim 27, wherein the biomaterial is bio-leather.

29. The method as claimed in claim 28, wherein the additional step of soaking the mycelium in a preservation solution, is carried out after step f) of drying the mycelium, and 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.

30. The method as claimed in claim 27, 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;
j. plasticizing by soaking the mycelium sheet in a plasticizer;
k. coating with a biodegradable biopolymer selected from polyvinyl acetate, ethylene glycol and glycerol, to increase the durability and smoothness of the material.

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

32. The method as claimed in claim 30, wherein the crosslinking solution is selected from the group consisting of glutaraldehyde, citric acid, adipic acid, polyphenol solution and combinations thereof.

33. The method as claimed in claim 30, 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.

34. The method as claimed in claim 30, wherein tanning is carried out by soaking the mycelium sheets in a combination of a crosslinking solution and tanning solution for 24-48 hours.