Fungal strains, consortium of fungal strains and methods of producing fungal biomaterial

Abstract

Fungal Strains, Consortium of Fungal Strains and Methods of Producing Fungal Biomaterial The present disclosure relates to novel fungal strains and consortium of the fungal strains and methods for growing / culturing said fungal strains, in particular, to produce fungal biomaterial or final bio - leather. The fungal biomaterial is fungal bio-leather. The present invention also provides novel methods and novel compositions for improving the growth of fungal mycelium.

Description

[0001] Field of invention The present disclosure relates generally to isolated fungal strains, consortium of such isolated fungal strains, method of growing / culturing said fungal strains or said consortium of fungal strains, in particular, to produce fungal biomaterial or final bio-leather. Background Sustainable substitutes for biomaterial, like, bio-leather can be made from mushroommycelium, 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-basedbio-leather can form shapes which overcome the need of cutting. Overall mushroom basedbiomaterial, like bio-leather, is a promising alternative to animal leather that is more sustainable, customizable, and innovative. At present, the market uses either animal-based leather or petroleum-based leather, both of which have disadvantages associated with them. Animal-based leather requires hazardous and toxic chemicals like chromium III, which is carcinogenic, for processing. The chemicals used for tanning give out toxic gases and effluents, which pose danger to workers in the leather industry. The modern tanning processes use considerable amount of energy, water, and chemicals. The processes pollute the land to such an extent that old tannery land cannot be used for cultivation. Similarly, petroleum-based leather is not biodegradable and leads to environmental pollution. Mushroom based bio-leather is considered sustainable because it requires fewer resources for production as compared to animal leather. Conventional leather production involves a significant amount of water, energy, and chemicals, leading to pollution and other negative environmental impacts. In contrast, mushroom based bio-leather production as provided by the present invention, primarily uses agricultural waste or crop residues, such as rice straw, wheat straw, cotton wood, sawdust, wheat flour etc. Along with being sustainable, mushroom based bio-leather is also biodegradable and compostable at the end of its life cycle. As a result, mushroom based bio-leather offers an environmentally friendly alternative to animal and synthetic leather, which has a long lifespan and takes many years to decompose. Mushroom based bio-leather does not contain the allergens that are present in leather, making it a suitable choice for people with sensitive skin or allergies. Also, mushroom based bio-leather can be produced to provide a high level of durability and strength, making it a long-lasting material that can withstand wear and tear. Mushroom based bio-leather is a vegan alternative, making it an ethical choice for consumers who want to avoid products made from animal skin. Overall, mushroom based bio-leather offers a range of advantages that makes it a promising alternative to animal and synthetic leather materials. Mushroom based bio-leather is, however, a relatively new technology that differs from current technology in several ways and companies and people working in the field are still developing ways to provide easy,simple, economical and sustainable ways and methods for producing biomaterial, like bio -leather, from fungi. Some companies are using genetically modified organisms. Other companies are using petroleum-based chemicals for strength and durability of the material. Yet others grow the mycelium in a highly customized growth chamber which leads to high costs. The 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 can be made, and which, like animal leather has good strength, tensile strength, flexibility, stretching and colorization properties. 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 asustainable 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 leads to a very low carbon footprint.An object of the present invention is to provide a method of making biomaterial, like bio -leather, using an easy, simple, and economical method. An object of the present invention is to provide a method for culturing mycelium or growing mycelium biomass which can be performed under controlled environmental conditions, including temperature and humidity, without using any specialized growth chamber for production at any scale, including large scale. An object of the present invention is to provide a method for 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 also to provide media and premix compositions for growing, culturing of mycelium biomass and / or production of fungal biomaterial. Summary of the Invention The present invention is directed to novel fungal strains of the Ganoderma and Dichomitus species. The present invention is in particular directed to novel fungal strains selected from: The present invention is also directed to a consortium of fungal strains of Ganoderma, Dichomitus species. In particular, the present invention is directed to a consortium of one or more fungal strains selected from: The present invention is also 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. 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. 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. The present invention is further directed to a method of growing mycelium biomass for production of biomaterial, like bio-leather. The present invention is directed to a method ofgrowing mycelium biomass wherein, the fungal strain is inoculated in a solid -state culturemedia comprising organic waste, and wherein a premix is added to the mycelium layer ataround the beginning of the stationary phase of the mycelium growth, wherein said solid -state media comprises cellulose in the range of from 30-60% (w / w), lignin in the range of from 20-40% (w / w) and hemicellulose in the range of from 10-30% (w / w). The present invention is also directed to a method of preparing bio-leather from the fungal mycelium of the novel fungal strains or a consortium of fungal strains of the present application. In preferred embodiments, the fungal mycelium of the novel fungal strains or a consortium of fungal strains of the present application is obtained by the method of growing mycelium biomass described herein. The present invention is further directed to solid-state media compositions and compositions of premix that enable excellent fungal mycelium growth. 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 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 wastesand 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 tothe 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 novel fungal strains of Ganoderma and Dichomitus species. The present invention is in particular directed to a novel fungal strain selected from: carnosum The present invention is also directed to a consortium of fungal strains of Ganoderma, Dichomitus species. In another aspect, the present invention is directed to a consortium of one or more fungal strains selected from: The present invention is also 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. 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. 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 method 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 method 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 method of growing mycelium biomass,comprising culturing one or more fungal strains of the present invention on solid -statemedia / substrate comprising organic waste, and adding a premix to the solid-state media ataround the beginning of the stationary phase of the mycelium growth.In additional embodiments, the premix is added twice during mycelium growth - the firsttime, at around the beginning of the stationary phase and the second time, around the mid -stationary phase of mycelium growth. Preferably, the premix may be added 15-20 days after inoculation when the stationary phase starts, and then in mid-stationary phase, that is, 4-5 days after the first addition of the premix. In preferred embodiments 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 a preferred embodiment the fungal strain used in the method of growing mycelium biomass is either a Ganoderma species or a Dichomitus species. In a further preferred embodiment, the fungal strain in the method of growing mycelium biomass is a strain selected from the group consisting of Ganoderma multipileum, Ganoderma lucidum, Dichomitus sp and Ganoderma carnosum: In an embodiment of the present invention, the organic waste used in the method of growing fungal mycelium comprises agricultural waste, crop residues or forestry by-products selected from rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran, corn cob, sugarcane bagasse or mixtures thereof. In an embodiment, the organic waste used in the method of growing fungal mycelium comprises agricultural waste, crop residues or forestry byproducts, selected from corn cob, rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran or mixtures thereof. In an embodiment, the solid-state media for growing mycelium biomass in accordance with the present invention comprises cellulose in a content of from 30-60% (w / w), lignin in a content of from 20-40% (w / w) and hemicellulose in a content of from 10-30% (w / w). In preferred embodiments the solid-state media comprises a cellulose in a content of 50% (w / w), lignin in a content of 30% (w / w), and hemicellulose in a content of 20% (w / w).In a preferred embodiment, the solid-state media has the following composition. The range of the above components or raw materials is adjusted, until a content of cellulose from 30-60% w / w, preferably 50 % w / w, lignin from 20-40% w / w, preferably 30% w / w, and hemicellulose from 10-30% w / w, preferably 20% w / w, is achieved. The additives in the solid-state media can include one or more of calcium sulphate, calcium carbonate, dextrose and peptone. The premix is a growth enhancer composition and is added to the culture at the beginning of the stationary phase to stimulate the growth of the fungal mycelium. In an embodiment, the premix is added 10-15 days after initiating culturing or inoculation of the fungal strain. The premix may also be added a second time around the mid-stationary phase. In an embodiment, the premix is added a second time 4-5 days after the first addition of the premix. The premix comprises cellulose, hemicellulose, lignin, and monosaccharides. The premix preferably comprises agricultural wastes and / or nutritional grains, such that the content of cellulose, hemicellulose, lignin, and monosaccharides in the premix is in the following ranges. The premix may comprise agricultural wastes and / or nutritional grains selected from corn cob, sugarcane bagasse, corn starch, wheat flour, maize flour, wheat straw and mixtures thereof. In one embodiment, the premix preferably comprises corncob, sugarcane bagasse, corn starch and wheat flour in the following ranges. The range of the above components is adjusted, till a cellulose content of 40-50% w / w, hemicellulose content of 15-35% w / w, lignin content of 15-25% w / w and a content of monosaccharides, in the range of 2-5% w / w, is achieved. In another embodiment, the premix preferably comprises corn cob, maize flour, and wheat straw in the following ranges. Again, the content of the above components is adjusted, till cellulose in the range of 40-50% w / w, hemicellulose in the range of 15-35% w / w, lignin in the range of 15-25% w / w and monosaccharides in the range of 5-10% w / w are achieved. The monosaccharides in the premix composition are selected from the group consisting of glucose, dextrose, fructose and mixtures thereof. It is also preferable that the premix is in the form of a powder that has a particle size in the range of from 0.5mm to 1.0mm. Said particle size can be achieved preferably by sieving.In an embodiment, the solid-state media is prepared by a method comprising the following steps:- a) hydrating organic waste comprising agricultural wastes, crop residues and / or forestry by-products with water; b) preparing a mix by adding to the hydrated organic waste obtained in step a) above, additives selected from one or more of calcium salts and carbohydrate and nitrogen sources to obtain a mix, such that the mix comprises cellulose, lignin, and hemicellulose. c) sterilizing the mix by heat and / or pressure to obtain a sterilized mix; d) cooling the sterilized mix to ambient temperature. In an embodiment, the agricultural waste, crop residue and forestry by-product in step a) of the method of preparing the solid-state media described above, is selected from rice straw, wheat straw, cotton wood, sawdust, wheat flour, wheat bran, corn cob, sugarcane bagasse or mixtures thereof. In the above method, the agricultural waste or crop residue may be mixed with water in a ratio of around 1:2.In an embodiment the carbohydrate source, in step b) of the method of preparing the solid -state media described above, is selected from dextrose, glucose and potato dextrose broth. In another embodiment the nitrogen source in step b) of the method above, is peptone. In yetanother embodiment the calcium salts are calcium sulphate and calcium carbonate.In an embodiment, in step b) of the method above, the mix is prepared by adding to the hydrated organic waste obtained in step a) of the method, additives selected from one or more calcium salts and carbohydrate and nitrogen sources to obtain a mix such that the mix comprises cellulose in the range of 30-60% w / w, preferably 50 % w / w, lignin in the range of 20-40% w / w, preferably 30% w / w, and hemicellulose in the range of 10-30% w / w, preferably 20% w / w.In an embodiment, in step d) of the method of preparing the solid -state media describedabove, the sterilized mix is cooled to ambient temperature, preferably in the range of from 27ºC-30ºC to obtain the solid-state media. In an embodiment, the solid-state media is prepared by the method comprising the following steps: a) hydrating organic waste comprising agricultural waste or crop residues, selected from rice straw, wheat straw, cotton wood, sawdust, wheat flour or mixtures thereof with water in a ratio of 1:2; b) preparing a mix by adding to the hydrated organic waste obtained in step a) above, additives selected from calcium salts and carbohydrate and nitrogen sources like, calcium carbonate, calcium sulphate, dextrose, glucose, peptone and potato dextrose broth (PDB), to obtain a mix, such that the mix comprises cellulose in the range of 30-60% w / w, preferably 50 % w / w, lignin in the range of 20-40% w / w, preferably 30% w / w, and hemicellulose in the range of 10-30% w / w, preferably 20% w / w. c) sterilizing the mix by heat and / or pressure to obtain a sterilized mix; d) cooling the sterilized mix to ambient temperature, preferably in the range of from 27ºC- 30ºC to obtain the solid-state media. In an embodiment, sterilization is carried out at a temperature of 121°C and pressure of 15 psi. In an embodiment, the solid-state media / substrate is filled in trays and the fungal strain is inoculated on said media for culturing. In an embodiment, the premix is added to the mycelium layer at the beginning of the stationary phase. In an embodiment, the premix is added to the mycelium layer 15-20 days after inoculation of the fungal strain, when the stationary phase of mycelium growth starts. In another embodiment, the method of growing mycelium comprises the additional step of adding premix to the solid-state media again in the mid-stationary phase of mycelium growth. In an embodiment, the premix is preferably added 4-5 days after the first addition of the premix. Another aspect of the present invention is directed to a method of making biomaterial, like bioleather. Said method comprises growing the mycelium biomass as described herein and further comprises the steps of: a) removing the thick mycelium sheet from the surface of the solid-state media; b) cleaning the mycelium sheet to remove the remnant solid-state media from the mycelium sheet; c) preserving the mycelium sheet in a preservative solution; d) soaking the mycelium sheet in a plasticizer; and e) drying the mycelium sheet. In a preferred embodiment, the drying in step e) is carried out such that the mycelium sheet holds 15-20% moisture after drying. In an embodiment, the additional step of soaking the mycelium in a preservation solution is carried out after step e) of drying the mycelium.In an embodiment, the preservation solution comprises 30% glycerol or a solution of calcium chloride, methanol, and water in a ratio of 1:1:4. In an embodiment, the method of making the biomaterial further comprises the following steps after step e) of drying the mycelium.f) Deacetylation by soaking the mycelium sheet in ethanol and methanol to convert the chitin into chitosan and to deactivate the growth of the mycelium; g) Crosslinking the chitosan by soaking the mycelium sheet in a crosslinking solution; and h) Plasticizing by soaking the mycelium sheet in a plasticizer. In an embodiment, the method of making the biomaterial further comprises the following steps after step e) of drying the mycelium: - f) Deacetylation by soaking the mycelium sheet in ethanol, sodium hydroxide, and a solution comprising methanol, calcium chloride and water in a 1:1:4 ratio, to convert the chitin into chitosan and to deactivate the growth of the mycelium; g) Crosslinking the chitosan by soaking the mycelium sheet in a crosslinking solution; h) Tanning into leather by using a combination of a crosslinking solution and tanning solution. i) Colouring to produce natural black colour by using chemicals selected from tannic acid, acetic acid, laccase, ferric chloride in acetic buffer and combinations thereof; j) Plasticizing by soaking the mycelium sheet in a plasticizer; k) Coating with a biodegradable biopolymer selected from polyvinyl alcohol, ethylene glycol and glycerol, to increase the durability and smoothness of the material.In an embodiment, any plasticizer can be used for mycelium sheet treatment. Preferably, the plasticizer is selected from glycerol, sorbitol, polyethylene glycol, ethylene glycol or mixtures thereof. Tanning usually involves a method which permanently alters the structure of the material, making it more durable and less susceptible to decomposition. After the steps of deacetylation and crosslinking, the mycelium sheets are soaked in a solution comprising a combination of a tanning solution and crosslinking solution for 24-48 hours. The crosslinking solution is selected from the group consisting of glutaraldehyde, citric acid, adipic acid, polyphenol solution and combinations thereof while the tanning solution is selected from the group consisting of tannic acid, acetic acid, laccase, ferric chloride in acetic buffer solution and combinations thereof. EXAMPLES Example 1- Selection of Strains Various types of strains isolated from the different places from the wild were studied on various aspects and the best strains were selected based on robustness, toughness, flexibility, density of mycelium, hyphae interconnection and colour. The most critical aspects analysed were:- 1. Density of mycelium –higher density of mycelium provides the desired mechanical strength to the material. 2. Flexibility- higher flexibility provides smoothness and softness and higher tensile strength to the material. 3. Fruiting body toughness- fruiting body toughness provides high strength to the material. Tough fruiting body mycelium are thick, bright, and fast growing. In some strains the fruiting body is very soft, and the mycelium is very thin.4. Complexity of mycelium network- higher interconnection between the hyphae provide higher strength and flexibility to the material. Surprisingly, the strains of the invention as described below have excellent robustness of the mycelium, toughness, flexibility, density of mycelium, hyphae interconnection etc., and do not require any special equipment for growth of mycelium, and / or any specific media, culture conditions etc. Solid-state media made from agricultural and organic wastes can support good growth of the fungi, such that excellent robustness of mycelium, toughness,flexibility, density of mycelium, hyphae interconnection etc., can be achieved.

[0002] Table 1: Characteristics of Strains Selected *The international depository authority, Microbial Type Culture Collection and Gene bank is located at Institute of Microbial Technology, Shanti Path, 39A, Sector 39, Chandigarh, 160036, India. Example 2- Selection of Solid-State Media Various kinds of media were tested, on which the fungal strains can be cultured at lowest cost, and yet achieve unexpected growth and thickness of mycelium. Solid-state media was tested for the most optimal growth of mycelia. Different solid-state media were prepared by: a) hydrating organic waste comprising agricultural waste, crop residues or forestry byproducts, 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 andthe individual fungal strains were inoculated on said media for culturing.The following fungal strains were used individually or in consortium for inoculating the media. 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. The results obtained, as provided in Figure 2vfor starin 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 additives added in this composition were calcium sulphate, calcium carbonate, dextrose and peptone. The solid-state media composition B included: The amount of the components was adjusted till the final desired levels of cellulose, lignin and hemicellulose was achieved. Example 3- Determination of Composition of the Premix and the Effect of its Addition to the Mycelium BiomassIt was also surprisingly found that a premix of high nutritional value, if added in the solid -statefermentation 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: - Said premix was added at different phases of mycelium growth. The inventors of the present application found that the timing of addition of the premix to the culture is critical in determining growth of mycelium. As shown in Figure 6, the culture in which premix was added at the beginning of the stationary phase (Figure 6B) gave unexpected and surprising results in mycelium growth ascompared to the culture in which the premix was added at a later stage (Figure 6A).Another specific premix that was used successfully had the following composition: Fungal strains may preferably be grown for 40-50 days post inoculation for complete mycelium growth and to obtain a thick mycelium layer. Preferably, the stationary phase starts 15-20 days after inoculation, and it is preferable to add the premix after 15-20 days post inoculation. Further, it may be preferable to add the premix twice, the first time, when the stationary phase of the mycelium growth starts and the second time, in mid-stationary phase. Preferably, the premix may be added 15-20 after inoculation when the stationary phase starts, and then in mid stationary phase, that is, 4-5 days after the first addition of the premix. Further, the inventors of the present application surprisingly found that the growth of the mycelium is also dependent on the particle size of the premix. Premix of fine size (0.5 mm to 1.0 mm) is easily digestible, and the mycelium grows without producing metabolites. The mycelium digests the premix in 2-3 days and an increase in mycelium thickness is seen (0.5 m to 1 mm). This type of raw mycelium layer is good in strength, shows high colour consistency, uniform growth, and high smoothness in comparison to mycelium layer that has metabolites. On the other hand, when premix of larger size is used (2 mm – 2.5 mm), the premix is not easily digestible, and it takes a longer time for mycelium growth. In such a case, the mycelium has a chance to produce metabolites, can showmycelium 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. Example 4- Consortium Agri- waste based substrate is obtained as described. The following agri- waste is taken: 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 (Calcium sulphate, calcium carbonate, dextrose and peptone) to obtain a mixture. The mixture was then sterilized under heat and pressure. The sterilized mixture is cooled to a temperature of 270C 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: - 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. The method of growing the mycelium biomass according to the present invention comprises the following steps: a) Preparing solid-state media comprising solid-state cellulose in the range of 30-60% w / w, preferably 50 % w / w, lignin in the range of 20-40% w / w, preferably 30% w / w, and hemicellulose in the range of 10-30% w / w, preferably 20% w / w; b) Inoculating the solid-state media with a fungal strain selected from the group consisting of Ganoderma multipileum of accession number MTCC 25685, Ganoderma lucidum of accession number MTCC 25683, Dichomitus sp of accession number MTCC 25684 and Ganoderma carnosum of accession number 25682 and allowing the fungi to grow; c) Adding a premix of fine particle size at the beginning of the stationary phase of the mycelium growth; d) Optionally adding the premix again during mid-stationary phase; e) Allowing the fungus to grow until a thick mycelium sheet is formed and harvesting the fungal biomass. After growing and obtaining mycelial biomass, the biomaterial was formed by the following steps: a) removing the thick mycelium sheet from the surface of the solid-state media; b) cleaning the mycelium from the remnant solid-state media; c) preserving the mycelium in a solution of CaCl2, methanol and water (1:1:4) d) soaking the mycelium sheet in a plasticizer. e) drying the mycelium sheet such that the sheet holds 15-20% moisture. 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. An isolated Ganoderma multipileum strain or culture thereof which is deposited under MTCC 25685.

2. An isolated Dichomitus sp strain or culture thereof which is deposited under MTCC 25684.

3. An isolated Ganoderma carnosum strain or culture thereof which is deposited under MTCC 25682.

4. An isolated Ganoderma lucidum strain or culture thereof which is deposited under MTCC 25683.

5. A consortium of one or more of : • isolated Ganoderma multipileum strain which is deposited under MTCC 25685; • isolated Dichomitus sp strain or culture thereof which is deposited under MTCC 25684; •isolated Ganoderma carnosum strain which is deposited under MTCC 25682; or• isolated Ganoderma lucidum strain which is deposited under MTCC 25683.

6. The consortium as claimed in claim 5, wherein Ganoderma sp. and Dichomitus sp are present in the ratio of from 05:1 to 1: 0.5, preferably 1:

1.

7. A method of growing mycelium biomass, comprising culturing a fungal strain or a consortium of fungal strains 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).

8. The method as claimed in claim 7, 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).

9. The method as claimed in claim 7, wherein the fungal strain or consortium of fungal strains is selected from the group consisting of Ganoderma multipileum strain deposited under MTCC 25685, Dichomitus sp strain deposited under MTCC 25684, Ganoderma carnosum strain deposited under MTCC 25682, Ganoderma lucidum strain deposited under MTCC 25683.

110. The method as claimed in claim 7, 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.

11. The method as claimed in claim 7, 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).

12. The method as claimed in claim 11, wherein the additives are selected from the group consisting of calcium sulphate, calcium carbonate, dextrose and peptone and combinations thereof.

13. The method as claimed in claim 11, wherein the range 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.

14. The method as claimed in claim 7, 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.

15. The method as claimed in claim 7 or claim 14, wherein the premix is a solid powder with particle size in the range of from 0.5 mm to 1.0 mm.

16. The method as claimed in claim 7 or claim 14, wherein the premix comprises cellulose, hemicellulose, lignin, and monosaccharides.

17. The method as claimed in claim 7 or claim 14, wherein the premix comprises cellulose in the range of from 40-50% (w / w), hemicellulose in the range of from 15-35% (w / w), lignin in the range of from 15-25% (w / w) and monosaccharides in the range of from 2-5% (w / w).

18. The method as claimed in claim 7 or claim 14, 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.

219. The method as claimed in claim 7 or claim 14, 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).

20. The method as claimed in claim 19, 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).

21. The method as claimed in claim 7 or claim 14, 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).

22. The method as claimed in claim 21, wherein the premix comprises corn cob at 60% (w / w), maize flour at 30% (w / w) and wheat straw at 10% (w / w).

23. The method as claimed in claim 19 or claim 21, wherein the range of the components is adjusted till a cellulose content from 40-50% (w / w), hemicellulose content from 1535% (w / w), lignin content from 15-25% (w / w) and monosaccharide content from 2-5% (w / w), is achieved.

24. The method as claimed in claim 7, 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.

25. The method as claimed in claim 24, 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.

26. The method as claimed in claim 24, wherein the carbohydrate source, in step b. is selected 3from dextrose, glucose and potato dextrose broth, the nitrogen source is peptone and the calcium salts are calcium sulphate and calcium carbonate.

27. The method as claimed in claim 24, 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.

28. The method as claimed in claim 27, 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.

29. The method as claimed in claim 7, 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 nitrogensources 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.

30. The method as claimed in claim 7, wherein the solid-state media is filled in trays and the fungal strain is inoculated on said media for growth of mycelium biomass.

31. The method as claimed in claim 7, wherein the premix is added at the beginning of the stationary phase, around 15-20 days after inoculation of the fungal strain.

432. The method as claimed in claim 14, wherein the premix is added in the mid-stationary phase, around 4-5 days after first addition of the premix.

33. The method as claimed in claim 24 or claim 29 wherein sterilizing is carried out at a temperature of 121°C and pressure of 15 psi.

34. A method of making biomaterial comprising the following steps: a. growing the mycelium biomass through a method as claimed in any of claims 5 to 31, 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; and f. drying the mycelium sheet, wherein drying the mycelium sheet is carried out such that the sheet holds 15-20% moisture after drying.

35. The method as claimed in claim 34, wherein the biomaterial is bio-leather.

36. The method as claimed in claim 34, wherein the additional step of soaking the mycelium in apreservation 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.

37. The method as claimed in claim 34, further comprising the steps of: g. deacetylation by soaking the mycelium sheet in ethanol, sodium hydroxide and a solution comprising methanol, calcium chloride and water in a 1:1:4 ratio, to convert the chitin into chitosan and to deactivate the growth of the mycelium; h. crosslinking the chitosan by soaking the mycelium sheet in a crosslinking solution; i. tanning into leather by using a combination of a crosslinking solution and tanning solution; j. colouring to produce natural black colour by using chemicals selected from tannic acid, acetic acid, laccase, ferric chloride in acetic buffer and combinations thereof; k. plasticizing by soaking the mycelium sheet in a plasticizer; l. coating with a biodegradable biopolymer selected from polyvinyl acetate, ethylene glycol and glycerol, to increase the durability and smoothness of the material.

538. The method as claimed in any one of claims 37, wherein the plasticizer is selected from the group consisting of glycerol, sorbitol, polyethylene glycol, ethylene glycol and mixtures thereof.

39. The method as claimed in claim 37, wherein the crosslinking solution is selected from the group consisting of glutaraldehyde, citric acid, adipic acid, polyphenol solution and combinations thereof.

40. The method as claimed in claim 37, 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.

41. The method as claimed in claim 37, wherein tanning is carried out by soaking the mycelium sheets in a combination of a crosslinking solution and tanning solution for 24 -48 hours.6

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