Biodegradable leather and preparation method thereof

Abstract

Described are methods of producing a bio-leather. A biomass having a plurality of cells and a mat of microbial cellulose fibers is obtained. The biomass is blended to obtain a suspension having a rati

Description

FIELD OF INVENTION [1] The present disclosure relates to a biodegradable leather. More particularly, the present disclosure relates to a biodegradable leather prepared from microbial cellulose. BACKGROUND OF INVENTION [2] Leather has been valued throughout history and continues to be sought because of its durability (i.e., due to its strength and longevity, it can withstand wear and tear), versatility (i.e., it can be treated and processed to make a wide range of textures and firmness), and / or aesthetic appeal. Specifically, leather is a popular choice in fashion and furniture industries because of its ability to develop patina over time. [3] The increase in consumption of leather-related products in recent decades has raised sustainability challenges. Making traditional leather from animal hides is a complex and resourceintensive process involving use of large amount of water, energy and harmful chemicals that have detrimental effects on the environment. Further, conventional leather is almost inert as it can take decades to decompose in a landfill due to the preservatives used in its production. Thus, it is difficult to manage leather-related waste in a sustainable manner. [4] Conventionally available bio-leather, made of synthetic polymers, are named either for their biological origin or biodegradability. They are widely used as they are often easily shaped, have superior physicochemical properties, and are economical. [5] Although few conventional bio-leathers are derived from microbial and / or plant sources, they include organic additives (for example, malic anhydride or similar, used as chemical crosslinkers) that render them non-biodegradable following the formation of covalent bonds. Even if they are made of organic components, the components themselves are not biodegradable in natural land-fill conditions. They often require industrially elevated conditions for their biodegradation. For example, leather made of petroleum-based plastics and bio-based synthetic polymer (like poly-lactic acid) are only degraded at industrial composting facilities at elevated temperatures of 58°C or more. Further, PLA emits approximately 1.3 kg CO2 equivalents / kg of synthesized plastic. [6] Sustainability of PLA can be enhanced by blending it with natural materials such as coconut coir fibers. However, such practices limit processability and / or scalability of the biodegradable polymers. Further, producing and refining PLA depends on unsustainable practices requiring the use of arable land, water, pesticides and fertilizers. [7] Other substrates for making leather include Poly(butylene adipate-co-terephthalate) (PBAT), Polybutylene succinate (PBS), petroleum-based plastics, etc. Although these substrates are naturally bio-degradable are sourced through non-sustainable means. For example, they have detrimental impact on the environment such as, emission of high amounts of greenhouse gases (GHGs), negative impact on wildlife, etc. Further, they may impart several health complications related to microplastic (PLA and other industrially compostable materials still generate microplastics if disposed of in the natural environment) and / or nano plastics causing issues relating to the leakage of additives within petroleum derived plastics. [8] Further, the end properties of the biodegradable polymers-based leather do not always meet market needs which leads to petroleum-based bioplastics overperforming such biodegradable polymers. The scalability of using biodegradable polymers over petroleum bioplastics may not be economically feasible. [9] To produce bio-leather, home compostable coatings have also been explored by many. Conventional home compostable coatings include algal derived hydrocolloid coatings, protein derived coatings and similar. However, such coatings do not exhibit adequate water barrier properties for leather-like applications. In addition to the home compostable coatings, few natural coatings like beeswax are also being explored as an alternative. However, beeswax and similar crystalize, and form an undesirable white patina.

[10] Thus, there arises a need for an alternative that overcomes the challenges associated with the conventional solutions. SUMMARY OF INVENTION

[11] Particular embodiments of the present disclosure are described herein below with reference to the accompanying drawings; however, it is to be understood that the disclosed embodiments are mere examples of the disclosure, which may be embodied in various forms. Well-known functions or constructions are not described in detail to avoid obscuring the present disclosure in unnecessary detail. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present disclosure in virtually any appropriately detailed structure.

[12] The present disclosure relates to a method to produce a bio-leather. The method commences by obtaining a biomass having a plurality of cells and a mat of microbial cellulose fibers. The biomass is blended to obtain a suspension having a ratio of microbial cellulose fibers to water between 10:90 to 1:99. The plurality of cells in the suspension is lysed to obtain a slurry. The slurry at least includes ruptured cells and the microbial cellulose fibers. One or more additives are added to the slurry to obtain a mixture. The mixture is then heated at a second predefined temperature. The second pre-defined temperature is lower than the first pre-defined temperature. The mixture is cooled to solidify the mixture. BRIEF DESCRIPTION OF DRAWINGS

[13] The summary above, as well as the following detailed description of illustrative embodiments, is better understood when read in conjunction with the apportioned drawings. For the purpose of illustrating the present disclosure, exemplary constructions of the disclosure are shown in the drawings. However, the disclosure is not limited to specific methods and instrumentality disclosed herein. Moreover, those in the art will understand that the drawings are not to scale.

[14] Fig. 1 depicts a method 100 for preparing a biodegradable leather, according to an embodiment of the present disclosure.

[15] Fig. 2 depicts a method 200 for preparing the biodegradable leather, according to an embodiment of the present disclosure. DETAILED DESCRIPTION OF THE DRAWINGS

[16] Prior to describing the disclosure in detail, definitions of certain words or phrases used throughout this patent document will be defined: the terms "include" and "comprise", as well as derivatives thereof, mean inclusion without limitation; the term "or" is inclusive, meaning and / or; the phrases "coupled with" and "associated therewith", as well as derivatives thereof, may mean to include, be included within, interconnect with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have a property of, or the like. Definitions of certain words and phrases are provided throughout this patent document, and those of ordinary skill in the art will understand that such definitions apply in many, if not most, instances to prior as well as future uses of such defined words and phrases.

[17] Reference throughout this specification to "one embodiment," "an embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment, but mean "one or more but not all embodiments" unless expressly specified otherwise. The terms "including," "comprising," "having," and variations thereof mean "including but not limited to" unless expressly specified otherwise. An enumerated listing of items does not imply that any or all of the items are mutually exclusive and / or mutually inclusive, unless expressly specified otherwise. The terms "a," "an," and "the" also refer to "one or more" unless expressly specified otherwise.

[18] Although the operations of exemplary embodiments of the disclosed method may be described in a particular, sequential order for convenient presentation, it should be understood that the disclosed embodiments can encompass an order of operations other than the particular, sequential order disclosed. For example, operations described sequentially may in some cases be rearranged or performed concurrently. Further, descriptions and disclosures provided in association with one particular embodiment are not limited to that embodiment, and may be applied to any embodiment disclosed herein. Moreover, for the sake of simplicity, the attached figures may not show the various ways in which the disclosed system, method, and apparatus can be used in combination with other systems, methods, and apparatuses.

[19] Furthermore, the described features, advantages, and characteristics of the embodiments may be combined in any suitable manner. One skilled in the relevant art will recognize that the embodiments may be practiced without one or more of the specific features or advantages of a particular embodiment. In other instances, additional features and advantages may be recognized in certain embodiments that may not be present in all embodiments. These features and advantages of the embodiments will become more fully apparent from the following description and apportioned claims, or may be learned by the practice of embodiments as set forth hereinafter.

[20] The term 'leather' in the below description corresponds to leather and leather-like material, at least partially made of inorganic constituents and is not naturally bio-degradable.

[21] The term 'bio-leather' in the below description corresponds to a material that has leatherlike properties and is entirely made of organic constituents. It also corresponds to being a theoretically carbon neutral material which is 100% naturally biodegradable (or compostable in natural environments).

[22] The term 'microbial cellulose' or 'microbial cellulose fibers' in the below description corresponds to cellulosic material produced naturally from microorganisms (both prokaryotic - bacterial and eukaryotic - fungi).

[23] The term 'symbiotic culture of bacteria and yeast' (or SCOBY) corresponds to a gelatinous, cellulose-based biofilm or microbial mat formed at the surface (air-liquid interface) of kombucha fermentation. In an exemplary embodiment, the microbial mat includes microbial cellulose, gram-negative acetic acid bacterial (AAB) species and gram-positive lactic acid bacterial (LAB) species. In an embodiment, the AABs include Acetobacter, Gluconobacter and Komagataeibacter, etc. In an embodiment, the LABs include Lactobacillus, as well as various yeasts such as Saccharomyces and Zygosaccharomyces.

[24] The present disclosure relates to a biodegradable leather (or bio-leather) and a method of preparation thereof. The bio-leather is derived from a microbial origin having organic additives, thus, serving as a sustainable and vegan alternative to traditional leather. The bioleather of the present disclosure is eco-friendly as it can easily be composted at home (i.e., home compostable).

[25] The method of preparing the bio-leather of the present disclosure involves preparing microbial cellulose and processing it with various sustainable techniques. Microbial cellulose is free of impurities like hemicellulose, lignin, etc. commonly found in cellulose sourced from plant origin. Hence, no chemical purification is required to process microbial cellulose. Microbial cellulose provides significantly higher strength thereby allowing manufacture of products having potentially similar mechanical properties as that of traditional leathers. Further, microbial cellulose can be easily scaled for large scale production as it is quickly produced in reactor vessels (unlike plant-based cellulose). Microbial cellulose requires 100-10,000 times less water for its processing compared to conventional processes.

[26] One or more organic additives are added during processing of the microbial cellulose for enhancing mechanical strength of the bio-leather without compromising its flexibility. Further, as the composition of the bio-leather formed from the method of the present disclosure is 100% organic, it is completely home compostable and eco-friendly. The bioleather of the present disclosure may easily be adopted by for example, the fashion and furniture industries. For example, the bio-leather of the present disclosure may be used to produce clothing, bags, beer mats, hats, etc.

[27] Now referring to the figures, Fig. 1 depicts an exemplary method 100 to prepare a bioleather from a microbial origin according to an embodiment of the present disclosure. The method 100 provides scalable and efficient purification and formulation of microbial cellulose (or microbial cellulose fibers) into bio-leather. The method 100 of the present disclosure is completely carbon neutral and does not involve leakage or emission of any pollutants in the environment. In an embodiment, the method 100 commences at step 101, by preparing an inoculum including at least one microorganism (or microbe). At least one of the microbes, including a plurality of cells, in the inoculum produces microbial cellulose. The microbe(s) includes at least one of Acetobacter xylinum, Gluconacetobacter xylinium, Saccharomyces cerevisiae, etc. or variants thereof. In an exemplary embodiment, the microbial cellulose is produced using a bacterium from genus Komagataeibacter. Using a microbial cellulose instead of plant-based cellulose is economically cheaper because microbes are easy to grow and the microbial cellulose obtained does not have any impurities like hemicellulose, lignin, etc. Hence, no purification step is required to separate cellulose from other impurities.

[28] The pre-defined number of cells of the at least one microbe (described above) is added and cultivated in a first pre-defined media to obtain an inoculated media via a first cultivation. The inoculated media acts as an inoculum for a second cultivation of the microbes in the subsequent steps. The first pre-defined media includes, without limitation, a carbon source, a nitrogen source, essential salts, organic fractions / biomasses, etc. Exemplary first predefined media is at least one of the described media below:

[29] Media 1: In an embodiment, Hestrin-Schramm media (also known as HS-media) includes 2% (w / v) glucose, 0.5% (w / v) yeast extract, 0.5% (w / v) peptone, 0.27% (w / v) NazHPCU, 0.15% (w / v) citric acid. HS-media is an exemplary simple media.

[30] Media 2: In an embodiment, Tryptic Soy Broth (also known as TSB) includes 17 g pancreatic digest of casein, 3 g peptic digest of soybean, 5 g sodium chloride, 2.5g dipotassium phosphate (K2HPO4), and 2.5g glucose per liter. TSB is an exemplary simple media.

[31] In a preferred embodiment, Symbiotic Culture of Bacteria and Yeast (SCOBY) is used as the inoculum instead of the inoculated media (described above).

[32] In an alternate embodiment, 250mL of the inoculated media having cells of an improved non-genetically modified strain of Gluconacetobacter xylinus (also known as acetobacter xylinium) is agitated at 200-350 rpm in a 500mL Erlenmeyer flasks (maintained at a temperature ranging from 15 °C to 42 °C) for 24-48 hours to produce spherical microbial cellulose particles having diameter in the range of 0.1 cm to 4 cm.

[33] At step 103, the inoculated media and / or SCOBY is used as the inoculum to produce a biomass via the second cultivation of a second pre-defined media. The biomass including at least a mat of microbial cellulose having a pre-defined thickness and the plurality of cells. The pre-defined thickness of the biomass is more than 1 cm.

[34] The inoculated media and / or SCOBY, as the inoculum, is added to the second pre-defined media such that the cells in the second predefined media ranges between 106 to 109 colony forming unit (CFU) / mL of the second pre-defined media.

[35] The second pre-defined media includes, without limitation, waste organic fractions, glucose rich waste streams and / or partial waste algae. Exemplary media is described as follows:

[36] Media 3: In an embodiment, the second pre-defined media (an exemplary complex media) includes 10% (w / v) spent grain, 10% (w / v) spent yeast and 15% (w / v) filtered brewery beer. The complex media is economically very cheap thereby helps reducing the cost of the bio-leather formed. The second pre-defined media may be used as is or may be pre-treated via physical, chemical and / or biological techniques depending upon a source of the second pre-defined media. Exemplary pre-treatment techniques include, without limitation, mechanical pre-treatment (physical grinding, high shear mixing or the like), mechanicalphysical pretreatment (autoclave sterilization or the like), chemical pre-treatment (dissolving in acid or the like), biochemical pre-treatment (enzymatic digestion or the like).

[37] In an embodiment, the second pre-defined media includes at least lOg / L carbohydrates (or the like), and at least 5g / L nitrogen (or the like). The pH of the second pre-defined media ranges between 3 and 6.5.

[38] In an alternate embodiment, either of Media 1 and Media 2 is used as the second predefined media.

[39] The second pre-defined media may either be sterilized or unsterilized before inoculation (i.e., adding of the inoculated media). The second pre-defined media after sterilization may be termed as a sterile media. The second pre-defined media, if used as is without sterilization, may be termed as an unsterile media. In an embodiment, after adding the inoculated media and / or SCOBY to the second pre-defined media, the number of cells in the sterile media ranges between 106 to 108CFU / mLand number of cells in the non-sterile media ranges from 106 to 109 CFU / mL. In an embodiment, the inoculated media and / or SCOBY added to the second pre-defined media ranges from 1 wt% to 25 wt%.

[40] The second-predefined media is subjected to the second cultivation to obtain a fermented media. The second cultivation increases the number of cells to form the biomass. While second cultivating the second-pre-defined media, one or more cultivation parameters are controlled to increase the yield of the second cultivation. The cultivation parameters include, without limitation, temperature, pH, incubation time, etc. In an embodiment, the temperature is maintained between 15°C to 42°C. In another embodiment, the temperature is maintained over 30 °C. In an embodiment, the pH is maintained between 3 and 6.5. In an embodiment, the incubation time ranges from 7 to 10 days (for example, in stagnant condition). In an embodiment, the incubation time ranges from 14 to 20 days in a batch (or fed-batch) reactor. In an embodiment, the second-predefined media is subjected to the second cultivation in the presence of a nanobubbler system to reduce the incubation time from 14-20 days to 7-10 days. The cultivation parameters may further depend upon the concentration of the inoculum and the cultivation scale, i.e., the amount of media being cultivated at a time and / or the volume of the reactor used.

[41] In an embodiment, the second cultivation is carried out in 12L water-baths (such as Grant JB Academy 12L vessels). In another embodiment, customized larger vessels are used for the second cultivation, for example, fermentation vats used in the brewery industry holding up to 240L or Edelstahl-Gartank 240L or OFV-1000A Opened fermentation vat holding up to 1000L.

[42] At step 105, post-second cultivation, the biomass is harvested from the fermented media using a pre-defined technique. The pre-defined technique includes, without limitation, filtration, centrifugation, sedimentation, or the like. In an embodiment, the fermented media is kept over a mesh filter having a porosity of 500 microns for 10-20 mins or until the water stops dripping. The biomass is retained over the mesh filter and the media (or spent media) is drained. After step 105, the microbial cellulose has 90-99% water content.

[43] In an embodiment, the media drained at step 105 may be used as the inoculum for setting up subsequent second cultivation. Recycling media drained at step 105 to set up subsequent second cultivations make the cells (or microbes) more efficient and adapted to the second pre-defined media.

[44] In a preferred embodiment, SCOBY obtained from kombucha fermentation is used as the biomass.

[45] At step 107, the biomass (i.e., the mat of microbial cellulose) obtained at step 105 is subjected to blending, to obtain a suspension. The blending process reduces particle size of the biomass. The biomass is subjected to blending by processing the biomass through, without limitation, high-shear mixing, high-pressure homogenization, twin screw compounding, hydrodynamic cavitation, etc. In an exemplary embodiment, the biomass is blended using a high-shear mixing technique. The reduced particle size, after blending the biomass, helps to increase the surface area of the biomass and helps to increase chain-to-chain interaction of the biomass and ultimately mechanical properties for the subsequent steps of the method 100.

[46] Post blending, in an embodiment, the particle size of the biomass (after it is completely dried) ranges between 200 microns to 1000 microns. The particle size of the biomass in an aqueous medium ranges between 500 microns to 2000 microns. The said particle size of the biomass helps in optimal interaction with additives in the subsequent steps of the method (100).

[47] Post blending, in an embodiment, the ratio of microbial cellulose to water is between 10:90 to 1:99.

[48] Additionally or optionally, water may be added depending on the additives (and their quantity) to be added in the subsequent steps of the method (100). For example, water is required additionally if gums and / or hydrocolloids are added as additives.

[49] At step 109, the cells present in the suspension obtained at step 107 are lysed to obtain a slurry. Lysing the cells prevents any unpleasant odor from the bio-leather and ensures that the bio-leather will not degrade over time while in use. Further, lysing the cells leads to release of protein fractions of the cells which significantly improves the strength and / or flexibility of the bio-leather.

[50] The cells may be lysed using a pre-defined technique including, but not limited to, mechanical disruption (for example, stirring, high shear mixing, sonication, bead-beating, mortar pestle, etc.), subjecting the solution to heating, enzymatic treatment, chemical treatment or a combination thereof. In an embodiment, after step 107, the slurry includes fibers of microbial cellulose (as insoluble cellulose fibers), ruptured cells, and a small amount of residual fermented media.

[51] In an embodiment, the cells in the suspension are lysed by subjecting the suspension at a first pre-defined temperature for a pre-defined time period ranging from 2 to 15 hours.

[52] In an embodiment, the suspension is heated between 90°Cto 100°C. The pre-defined time period depends upon a pre-defined thickness of the biomass and the ratio between biomass and water (10:90 or 1:99). In an embodiment, the suspension is heated for 3 hours, if the thickness of the biomass is 0.5 cm or less. In another embodiment, the suspension is heated for 5 hours, if the thickness of the biomass is in between 0.5 cm to 1 cm. In an embodiment, the suspension is heated for 7.5 hours, if the thickness of the biomass is more than 1 cm.

[53] In an embodiment, the suspension is heated in a water bath. In another embodiment, the suspension is washed cold in a 2% sodium hydroxide (NaOH) bath.

[54] In another embodiment, the solution was simultaneously heated and subjected to stirring and / or high shear mixing.

[55] At step 111, the slurry obtained post step 109 is cooled down to a pre-defined temperature. In an embodiment, the slurry is cooled by keeping the slurry under ambient room conditions. Alternatively, a cooling jacket may be used to cool the slurry. The slurry may be cooled to a temperature in the range of 30°C to 60°C depending upon additives that are to be added in the subsequent step. For example, temperature sensitive additives such as proteins and other small organic compounds require lower temperatures, whereas minerals such as clays, carbonates can tolerate high temperatures.

[56] At step 113, one or more additives (or organic additives) are added to the slurry obtained at step 111 in a pre-defined concentration to obtain a mixture. The one or more additives stabilize the physical cross-linking of the microbial cellulose, improve flexibility and / or softness of the bio-leather by reducing brittleness of the bio-leather, thereby allowing the bio-leather to be easily shaped during processing as desired by the user. The one or more additives improve durability of the bio-leather during its use. The one or more additives may be selected from a group of gums (added in the range of 1-15 wt%), hydrocolloid (added in the range of 1-15 wt%), protein (added in the range of 1-15 wt%), one or more salt (added in the range of 1-5 wt%), one or more organic plasticizer (added in the range of 5-35 wt%), etc. The one or more additives may further be selected from a group of one or more fillers (added in the range of 1-25 wt%), at least one coloring agent (added in the range of 0.01-5 wt%), at least one natural odor component (added in the range of 0.01-5 wt%), at least one filler (added in the range of 5-35 wt%), etc.

[57] In an embodiment, the gum, hydrocolloid and the protein act as a structural interpenetrating network component.

[58] In an embodiment, the salts provide physical interaction of the interpenetrating network. In an embodiment, the gums are one of guargum, xanthan gum, orthe like.

[59] In an embodiment, the hydrocolloid is one of alginate, carrageenan, agar, or the like. In an embodiment, the protein is one of hydrophobins, zein, pea protein, whey, or the like.

[60] In an embodiment, the salt is one of sodium chloride, magnesium sulfate, calcium chloride, or the like.

[61] In an embodiment, the organic plasticizer is one of glycerol, sorbitol, xylose, mannose, betaine, urea, polyglycerols, maltodextrin, orthe like.

[62] In an embodiment, the coloring agent is one of astaxanthin, beta carotene, beetroot extract, turmeric extract or other natural pigments.

[63] In an embodiment, the natural odor component is one of oregano, lavender, cedarwood, sage, eucalyptus or other essential oils.

[64] In an embodiment, the filler is one of carbonates, algal polysaccharides, water soluble arabinose xylans, clays, biomass including spend brewery waste, microalgae, macroalgae, yeast fungi, shellfish, antioxidants, flame retardants, or the like. The filler improves strength and water resistance of the bio-leather.

[65] Although the present disclosure is described with the examples of organic additives, naturally derived inorganic minerals (like calcium carbonate) are within the scope of the teachings of the present disclosure. In an exemplary embodiment, addition of 10 g / L of inorganic carbonate (at step 103) simulates (i.e., increases the speed) the growth of the microbe and improves the final quality and properties (like strength and consistency) of the bio-leather.

[66] At step 115, the mixture obtained in step 111 is subjected to heating at a second predefined temperature for a pre-defined time period. The second pre-defined temperature ranges between 75°C to 85°C. The first pre-defined temperature is higher than the second pre-defined temperature. Compared to the heating in step 109, the heating at step 115 is relatively gentle to avoid degradation of additives, i.e., setting the second pre-defined temperature lower than 85 °C protects the additives from thermal degradation. pH of the mixture may be maintained between 5 and 7. The pre-defined time period ranges from 20 to 120 minutes. In an embodiment, the mixture is simultaneously stirred while the mixture is subjected to the heat. Heating the mixture improves the mixing and physical interaction between the microbial cellulose and the additive(s). The purity / homogeneity of the mixture is increased, interpenetrating network is formed within the matrix-like network (present inside the mixture) and correct physical cross-linking of the components are ensured.

[67] In an embodiment, while the mixture is heated, the additives are slowly dissolved thereby reducing the optical density of the mixture. For example, the optical density may reduce from above 0.4 to below 0.2.

[68] In an alternate embodiment, while the mixture is heated, significant increase in viscosity of the mixture is an indication of complete dissolution of the additives.

[69] In an exemplary embodiment, dissolution of the additives leads to uniform physical interaction of the additives with the microbial cellulose fibers which ensures that a matrixlike network is formed. The additives are homogenously dispersed within the matrix-like network formed.

[70] At step 117, post-heating the mixture at step 115, the mixture is solidified to form the bio-leather. In an embodiment, the mixture is spread over a surface (for example, without limitation, silicone, glass, plastic, etc.). After spreading the mixture, the mixture is dried using, without limitations, a vacuum oven, sun drying, a food dehydrator, an oven, etc. In an embodiment, the mixture is dried with the help of lyophilization to obtain a foam. Other functionally equivalent desiccation methods to dry / solidify the mixture are within the scope of the teachings of the present disclosure. In some examples, solidifying comprises gelling.

[71] In an embodiment, the mixture is solidified inside a food dehydrator set at 40°C for 6-12 hours depending on the thickness 0.5 cm to 1 cm (of the spread mixture). In another embodiment, the mixture is solidified using a normal oven operated at 40°C for 12 to 16 hours depending on the thickness 0.5 cm to 1 cm (of the spread mixture).

[72] The solidified product obtained at step 117 is the bio-leather. The bio-leather offers superior properties in terms of mechanical performance, hydrophobicity, tear resistance, etc. The bio-leather may easily be adopted by for example, the fashion and furniture industries. For example, the bio-leather of the present disclosure maybe used to produce clothing, bags, beer mats, hats, etc.

[73] In an embodiment, the bio-leather includes the microbial cellulose fibers in the range of 5 wt% to 65 wt% for structural performance, a gum / hydrocolloid (in the range of 1 wt% to 15 wt%) as a structural interpenetrating network component, a salt (in the range of lwt% to 5 wt%) to enhance physical interaction of the interpenetrating network, a plasticiser (in the range of 5 wt% to 35 wt%), a colouring agent (in the range of 0.01 wt% to 5 wt%) which can be either natural or synthetic (but naturally degradable), a natural odour component (in the range of 0.01 wt% to 5 wt%), and / or natural fillers (in the range of 5wt% to 25wt%) to improve strength and water resistance as well as other properties including but not limited to antioxidants, carbonates, processing aids such as flame retardants. Advantageously, the fillers can act as inter-polymeric chain spacers to improve flowability, processability, and flexibility of the bio-leather.

[74] Additionally or optionally, at step 119, the solidified mixture (i.e., the bio-leather) is dried to remove moisture. The bio-leather may either be dried naturally at ambient temperature or be dried at an elevated temperature. In an embodiment, the bio-leather is dried at room temperature. In another embodiment, the bio-leather is dried in a sun bath. In yet another embodiment, the bio-leather is dried at an elevated temperature ranging from 30 °C to 80 °C.

[75] Additionally or optionally, at step 121, at least one layer of coating is provided over an outer surface of the bio-leather obtained at either step 117 or step 119. The coating provides finishing to the bio-leather. The coating may be at least one of natural waxes, hydrophobic proteins &oils, or a combination thereof dissolved in a solvent and / or an ionic liquid. In an exemplary embodiment, a coating of Polyhydroxyalkanoates (PHAs) dissolved in chloroform is uniformly applied over the outer surface of the bio-leather. In another exemplary embodiment, a coating of PHAs powder is thermally applied over the outer surface of the bio-leather.

[76] The method 100 to produce the bio-leather can be easily scaled to provide for the market needs without significantly increasing economical overhead. The method 100 does not produce any polluting discharge(s) or emission(s).

[77] Fig. 2 depicts another exemplary method 200 to prepare the bio-leather from a microbial origin according to an embodiment of the present disclosure. The method 200 provides scalable and efficient purification and formulation of microbial cellulose (or microbial cellulose fibers) into bio-leather. The method 200 of the present disclosure can be carbon neutral and the method 200 does not entail leakage or emission of any pollutants in the environment. In an embodiment, the method 200 commences at step 201, by obtaining SCOBY from kombucha fermentation and using it as the biomass. SCOBY is a gelatinous, cellulose-based biofilm or microbial mat formed at the surface (air-liquid interface) of kombucha fermentation. In an exemplary embodiment, the microbial mat includes microbial cellulose, gram-negative acetic acid bacterial (AAB) species and gram-positive lactic acid bacterial (LAB) species. In an embodiment, the AABs include Acetobacter, Gluconobacter and Komagataeibacter, etc. In an embodiment, the LABs include Lactobacillus, as well as various yeasts such as Saccharomyces and Zygosaccharomyces.

[78] At step 203, the biomass is blended to obtain the suspension as described in step 107 of method 100.

[79] At step 205, the cells are lysed to obtain the slurry as described in step 109 of method 100.

[80] At step 207, the one or more additives are added to the slurry to obtain the mixture as described in step 113 of the method 100.

[81] At step 209, the mixture is heated as described in step 115 of the method 100.

[82] At step 211, the mixture is spread and cooled to solidify as described in step 117 of the method 100. The solidified mixture yields the bio-leather of the present disclosure.

[83] The present disclosure will be described with the help of the following examples. As will be evident from the following examples, the variation in water content, and the additives produces bio-leather with different properties suitable for different applications.

[84] Example 1: Selective breeding of a microbe for producing microbial cellulose fibers. An improved non-genetically modified strain of Gluconacetobacter xylinus (aka acetobacter xylinium) was grown over a 6-months period in 500 mL Erlenmeyer flasks. The G. xylinus culture was grown in batches of strong growth, by removing 5 mL samples and using them to start new cultures after every 2-week. The strain of G. xylinus was grown at a temperature ranging from 15 °C to 42 °C, preferably above 20 °C. As a result, a fast growing strain of G. xylinus was obtained that produced thick biomass (1 cm or more) containing microbial cellulose fibers.

[85] Example 2: Preparing a biomass containing microbial cellulose fibers of the present disclosure in a simple media. Two liters of media was prepared by dissolving 40g glucose, 10g Yeast extract, 10g Peptone, 6g Salt, 3g citric acid in distilled water. The pH of the first predefined media was adjusted between 4 and 6.5. The microbe obtained from Example 1 was used as an inoculum by adding 106 to 108 CFU / mL of media.

[86] A fed batch system was preferred in which after 14-20 days, the biomass containing microbial cellulose was removed, and the media was replenished with approximately 70% of the initial sugars (between 35-85% of sugars). When replenished an identification plate was carried out to assess whether the replenished media had a sufficient number of cells (the same amount as those present in the initial inoculum). It was also observed whether contamination of other microorganisms took place. If not, another inoculum presenting 107 CFU / ml of media was added to the cultivation vessel. A thick biomass (1cm or more) having microbial cellulose fibers was obtained.

[87] Example 3: Preparing a biomass containing microbial cellulose fibers of the present disclosure in a complex media. Two liters of media was prepared by mixing 10% (w / v) spent grain, 10% (w / v) spent yeast and 15% (w / v) filtered brewery beer (discarded by breweries as waste). A little amount (1-5% (w / v)) of waste algal component was added to the media. The microbe obtained from Example 1 was used as an inoculum by adding 109 CFU / mL of media.

[88] A fed batch system was preferred in which after 14-20 days, the biomass containing microbial cellulose was removed, and the media was replenished with approximately 70% of the initial sugars (between 35-85% of sugars). When replenished an identification plate was carried out to assess whether the replenished media had sufficient number of cells (the same amount as those present in the initial inoculum). It was also observed whether contamination of other microorganisms took place. If not, another inoculum presenting 107 CFU / mL of media was added to the cultivation vessel. A thick biomass (1cm or more) having microbial cellulose fibers was obtained.

[89] A yield of the biomass, both qualitatively as well as quantitatively, obtained from Example 2 and Example 3 was observed to be same.

[90] Example 4: Preparing a tough variant of the bio-leather of the present disclosure.

[91] 500g of biomass, as was obtained from Example 2, was blended using a high-shear mixing technique at 1000-5000 rpm to obtain a suspension. The suspension had microbial cellulose and water in a ratio of 2:98 and particle size of 500 microns. The suspension was heated between 90°C to 100°C for 2 hours to 15 hours to obtain a slurry. The slurry was cooled between 30 °C to 60°C. 15g of vegetable triglycerides (Spiganord Grade SG-3), 15g Xanthan gum, 3g of hydroxyapatite, 3g of beetroot extract, 4 drops of rosewood essential oil was added to the slurry to form a mixture. The mixture was heated between 75°C to 85°C. The mixture was then cooled to obtain the bio-leather. The bio-leather has significantly increased fiber size that achieved 40-80% increase in tensile properties.

[92] In an embodiment, the tough variant of bio-leather obtained from Example 4 was used for clothing.

[93] Example 5: Preparing a rigid variant of the bio-leather of the present disclosure.

[94] 500g of SCOBY, as was obtained from Kombucha fermentation, was blended using a high- shear mixing technique at 1000-5000 rpm to obtain a suspension. The suspension had microbial cellulose and water in a ratio of 5:95. The suspension was heated between 90°C to 100°C for 2 hours to 15 hours to obtain a slurry. The slurry was cooled between 30 °C to 60°C. 10g of vegetable glycerol, 105g of wood particles (200-500 microns particle size), 50g of ground pecan or egg shells (below 300 microns particle size) and 3g of natural color was added to the slurry to form a mixture. The mixture was heated between 75°C to 85°C. The mixture was then cooled to obtain the bio-leather. The bio-leather has significantly increased fiber size that achieved 80-100% increase in tensile properties.

[95] In an embodiment, the rigid variant of bio-leather obtained from Example 5 was used for patches.

[96] Example 6: Preparing a flexible variant of the bio-leather of the present disclosure.

[97] 500g of SCOBY, as was obtained from Kombucha fermentation, was blended using a high- shear mixing technique at 1000-5000 rpm to obtain a suspension. The suspension had microbial cellulose and water in a ratio of 5:95. The suspension was heated between 90°C to 100°Cfor 2 hours to 15 hours to obtain a slurry. The slurry was cooled between 30 °C to 60°C. 10g of iota carrageenan, 0.25g potassium sorbate, 200g of higher degree polyglycerol (e.g., Nona-polyglycerol), 2.5g of titanium dioxide 10mL natural color (e.g., cabbage extract for purple coloration) was added to the slurry to form a mixture. The mixture was heated between 75°C to 85°C. The mixture was then cooled to obtain the bio-leather. The bio-leather had increased elongation from 10% to 50-120%.

[98] The foregoing description of preferred embodiments of the present disclosure provides illustration and description, but is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Modifications and variations are possible in light of the above teachings or may be acquired from practice of the disclosure.

[99] No element, act, or instruction used in the description of the present disclosure should be construed as critical or essential to the invention unless explicitly described as such. Also, as used herein, the article "a" is intended to include one or more items. Where only one item is intended, the term "one" or similar language is used.

Claims

1. A method (100) to produce a bio-leather, the method (100) comprising:I. obtaining a biomass having a plurality of cells and a mat of microbial cellulose fibers;II. blending the biomass to obtain a suspension having a ratio of microbial cellulose fibers to water between 10:90 to 1:99;III. lysing the plurality of cells in the suspension obtained at step II to obtain a slurry, the slurry at least including ruptured cells and the microbial cellulose fibers;IV. adding one or more additives to the slurry obtained at step III to obtain a mixture;V. heating the mixture obtained at step IV at a second pre-defined temperature; andVI. cooling the mixture obtained at step V to solidify the mixture.

2. The method (100) as claimed in claim 1, wherein the step of lysing the plurality of cells in the suspension includes subjecting the suspension to at least one of mechanical disruption, heating, enzymatic treatment, and chemical treatment.

3. The method (100) as claimed in claims 1 or 2, wherein the step of lysing the plurality of cells in the suspension includes:I. heating the suspension at a first predefined temperature to obtain the slurry, the first pre-defined temperature is higher than the second pre-defined temperature; andII. cooling the slurry.

4. The method (100) as claimed in any of the claims 1 to 3, wherein the step of obtaining the biomass includes:I. preparing an inoculated media using at least one microbe in a first pre-defined media, the at least one microbe including the plurality of cells producing the microbial cellulose fibers; orII. obtaining a symbiotic culture of bacteria and yeast (SCOBY) from kombucha fermentation.

5. The method (100) as claimed in claim 4, wherein the stepof obtaining the biomass further includes:I. adding at least one of:i. the inoculated media in a second pre-defined media such that the plurality of cells in the second pre-defined media ranges from 106 to 109 colony forming unit (CFU) / mL, orii. the SCOBY in the second pre-defined media, the SCOBY added to the second pre-defined media ranges from 1 wt% to 25 wt%; andII. second cultivating the second pre-defined media to obtain a fermented media including the biomass.

6. The method (100) as claimed in any of the claims 4 to 5, wherein the microbes include at least one of Acetobacter xylinum, Gluconacetobacter xylinium, or Saccharomyces cerevisiae.

7. The method (100) as claimed in any of the claims 4 to 6, wherein the first pre-defined media includes:I. 2% (w / v) glucose, 0.5% (w / v) yeast extract, 0.5% (w / v) peptone, 0.27% (w / v) disodium phosphate Na2HP04, 0.15% (w / v) citric acid; orII. 17 g / L pancreatic digest of casein, 3 g / L peptic digest of soybean, 5 g / L sodium chloride, 2.5 g / L dipotassium phosphate (K2HPO4), and 2.5 g / Lglucose.

8. The method (100) as claimed in any of the claims 5 to 7, wherein the second pre-defined media includes:I. 10% (w / v) spent grain, 10% (w / v) spent yeast and 15% (w / v) filtered brewery beer; orII. the first pre-defined media as claimed in claim 6.

9. The method (100) as claimed in any of the claims 4 to 8, wherein the step of preparing the inoculated media includes agitating 250mL of the inoculated media having cells of an improved non-genetically modified strain of Gluconacetobacter xylinus in a 500mL Erlenmeyer flasks maintained at a temperature ranging from 15 °C to 42 °C for 24 hours to 48 hours.

10. The method (100) as claimed in any of the claims 5 to 9, wherein the step of second cultivating the second pre-defined media includes:I. maintaining the temperature of the second pre-defined media between 15°C to 42°C;II. maintaining the pH of the second pre-defined media between 3 and 6.5; andIII. incubating the second pre-defined media fori. 7 days to 10 days in stagnant conditions, orii. 14 days to 20 days in a batch or fed-batch reactor.

11. The method (100) as claimed in any of the claim 5 to 10, wherein after the step of second cultivating the second pre-defined media, harvesting the biomass from the fermented media by keeping the fermented media over a mesh filter having a porosity of 500 microns till a water content of the microbial cellulose is in the range of 90% to 99%.

12. The method (100) as claimed in any of the claims 1 to 11, wherein the step of blending the biomass includes reducing a particle size of the biomass to a range of 200 microns to 1000 microns.

13. The method (100) as claimed in any of the claims 3 to 12, wherein the step of heating the suspension includes heating the suspension at the first pre-defined temperature ranging between 90°C and 100°C for 2 hours to 15 hours in one of:I. a water bath, orII. a 2% sodium hydroxide (NaOH) bath.

14. The method (100) as claimed in any of the claims 2 to 13, wherein the step of heating the suspension includes simultaneously subjecting the suspension to at least one of stirring or high shear mixing.

15. The method (100) as claimed in any of the claims 2 to 14, wherein the step of heating the suspension includes:I. heating the suspension for 3 hours, if thickness of the biomass is 0.5 cm or less; orII. heating the suspension for 5 hours, if the thickness of the biomass is in between0.5 cm to 1 cm, orIII. heating the suspension is heated for 7.5 hours, if the thickness of the biomass is more than 1 cm.

16. The method (100) as claimed in any of the claims 3 to 15, wherein the step of cooling the slurry includes cooling the slurry to a temperature ranging from 30 °C to 60 °C.

17. The method (100) as claimed in any of the claims 1 to 16, wherein adding one or more additives to the slurry includes adding at least one of 1-15 wt% gums, 1-15 wt% hydrocolloid, 1-15 wt% protein, 1-5 wt% one or more salt, 5-35 wt% one or more organic plasticizer, 1-25 wt% one or more fillers, 0.01-5 wt% at least one coloring agent, or 0.01-5 wt% at least one natural odor component.

18. The method (100) as claimed in any of the claims 1 to 17, wherein the step of heating the mixture includes heating the mixture at the second pre-defined temperature ranging between 75°C and 85°C.

19. The method (100) as claimed in any of the claims 1 to 18, wherein the step of heating the mixture includes simultaneously subjecting the mixture to at least one of stirring or high shear mixing.

20. The method (100) as claimed in as claimed in any of the claims 1 to 19, wherein the step of cooling the mixture includes solidifying the mixture inside one of:I. a food dehydrator set at 40 °C for 6 hours to 12 hours, orII. a normal oven operated at 40 °C for 12 hours to 16 hours.

21. The method (100) as claimed in any of the claims 1 to 20, wherein the method (100) further includes drying the bio-leather to remove moisture.

22. The method (100) as claimed in any of the claims 1 to 21, wherein the method (100) further includes providing at least one layer of coating over an outer surface of the bioleather.

23. The method (100) as claimed in claim 22, wherein the at least one layer of coating is prepared by dissolving at least one of natural waxes, hydrophobic proteins or oils, in at least one of a solvent or an ionic liquid.

24. The method (100) as claimed in any of the claims 22 to 23, wherein providing the at least one layer of coating includes uniformly applying Polyhydroxyalkanoates (PHAs) over the outer surface of the bio-leather.

25. A bio-leather obtained by the method (100) as claimed in any of the preceding claims.

26. The bio-leather as claimed in claim 25, wherein the bio-leather includes the microbial cellulose fibers in the range of 5 wt% to 65 wt%.

27. The bio-leather as claimed in claims 25 or 26, wherein the bio-leather further include at least one of 1-15 wt% gums, 1-15 wt% hydrocolloid, 1-5 wt% one or more salt, 5-35 wt% one or more organic plasticizer, 1-25 wt% one or more fillers, 0.01-5 wt% at least one coloring agent, or 0.01-5 wt% at least one natural odor component.

28. The bio-leather as claimed in any of the claims 25-27, wherein an outer surface of the bioleather includes at least one layer of coating selected form natural waxes, hydrophobic proteins or oils dissolved in at least one of a solvent or an ionic liquid.21

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