Bacterial cellulose and method for its production

Abstract

The present disclosure relates to a method of producing bacterial cellulose, comprising culturing a starting culture of about 20 L to about 50 L together with at least about 200 L of a production medium to form a culture for forming the bacterial cellulose; wherein the starting culture comprises at least one Komagataeibacter strain selected from the group consisting of Komagataeibacter cohnii ( Komagataeibacter nataicola ), Komagataeibacter rhaeticus ( Komagataeibacter rhaeticus ), Komagataeibacter xylinus ( Komagataeibacter xylinus ), and Komagataeibacter intermedius ( Komagataeibacter intermedius ); wherein the production medium comprises a waste yeast broth that is not heat treated or chemically treated. The present disclosure also relates to the bacterial cellulose produced thereby; wherein the step of culturing the culture is performed in a static condition in a fermentation tray of 1 L to 2 L.

Description

Technical Field

[0001] This invention relates in general to bacterial cellulose and its preparation method. Background Technology

[0002] Biopolymers are natural polymers produced by the cells of living organisms. Like other polymers, biopolymers consist of monomer units linked by covalent bonds to form chains that create larger molecules. Based on the monomers used and the structure of the resulting biopolymer, there are three main classes of biopolymers: polynucleotides, polypeptides, and polysaccharides. Polynucleotides, such as RNA and DNA, are long-chain polymers composed of nucleotides. Polypeptides include proteins and shorter amino acid polymers; some major examples include collagen, actin, and fibrous proteins. Polysaccharides are linear or branched carbohydrates; examples include starch, cellulose, and alginate. Other examples of biopolymers include natural rubber (isoprene polymers), suberin and lignin (complex polyphenol polymers), cutin and cutan (complex polymers of long-chain fatty acids), melanin, and polyhydroxyalkanoates (PHAs).

[0003] Bacterial cellulose (BC) is a biopolymer with a wide range of applications across various industries due to its unique properties, including high purity, mechanical strength, biocompatibility, and ability to retain large amounts of water. Its primary applications are in the food industry, medical fields, and materials science. For example, BC can be used to manufacture edible films and coatings for fruits, vegetables, and other foods, extending their shelf life and maintaining freshness by providing the necessary oxygen and moisture barrier properties. In medical applications, BC can be used in wound care products, including dressings and bandages, thanks to its excellent water retention, biocompatibility, and transparency, facilitating wound monitoring. As a material, BC fibers can be used to produce sustainable textiles and clothing with unique properties such as moisture wicking, breathability, and biodegradability. Furthermore, BC can be combined with other biomaterials to create nanocomposites with even superior properties for a variety of applications.

[0004] Certain bacterial strains can produce bacterial cellulose (BC), including Gram-negative bacteria such as *Komagataeibacter* (Gluconacetobacter), *Agrobacterium*, *Achromobacter*, *Aerobacter*, *Alcaligenes*, *Rhizobium*, *Pseudomonas*, and *Salmonella*, as well as Gram-positive bacteria such as *Sarcina* and *Leifsonia*. Commercial production of BC involves culturing specific bacterial strains during a controlled fermentation process. Specific details of BC production vary depending on the strains used, the intended use of the BC, and the available equipment and facilities.

[0005] Despite the numerous advantages of bacterial cellulose in many applications, its production cost remains relatively high, primarily due to the high cost of culture media and the low productivity of known strains. Therefore, low-cost raw materials and biowaste are needed to reduce the production cost of bacterial cellulose. These raw materials can include agricultural residues (molasses and pretreated wheat straw), food industry byproducts (fruit peels), and municipal organic waste and paper mill sludge. Furthermore, it is recognized that breweries and bioethanol production generate substantial amounts of waste grains and yeast (approximately 36.4 million tons annually), posing significant challenges to waste management. This situation is both costly and environmentally problematic. These byproducts can serve as suitable carbon sources for certain bacterial cellulose (BC)-producing bacteria. For example, it has been reported that bacterial cellulose can be produced using only waste brewer's yeast as a nutrient source from *Gluconobacterium hannella* CGMCC 3917. However, in the aforementioned laboratory studies, the waste brewer's yeast had to undergo harsh chemical and thermal treatments, increasing operating costs and limiting the commercial application of this process.

[0006] Overcoming or improving at least one of the above problems would be beneficial. Summary of the Invention

[0007] This disclosure relates to a method for producing bacterial cellulose, comprising: Approximately 20 L to approximately 50 L of the starting culture was cultured together with at least approximately 200 L of the production medium to form a culture for the formation of bacterial cellulose. The starting culture contains *Coprinus cocovenenans* (cocovenenans) Komagataeibacter nataicola ), Reuteria paniculata ( Komagataeibacter rhaeticus ), Komatobacterium xylinum ( Komagataeibacter xylinus ) and Intercalozoa ( Komagataeibacter intermediusAt least one strain of Komagataeibacter; The production culture medium contains waste yeast liquid that has not been heat-treated or chemically treated; The step of culturing the culture is carried out under static conditions in a 1 L to 2 L fermentation tray.

[0008] In some embodiments, the starting culture further comprises a strain of *Glucosobacterium* selected from the following strains: *Glucosobacterium sucrofermentans*, *Glucosobacterium hansenii*, *Glucosobacterium europaeus*, *Glucosobacterium entanii*, *Glucosobacterium saccharivorans*, *Glucosobacterium swingsii*, *Glucosobacterium oboediens*, *Glucosobacterium liquefaciens*, *Glucosobacterium sacchari*, *Glucosobacterium diazotrophicus*, *Glucosobacterium johannae*, *Glucosobacterium azotocaptans*, *Glucosobacterium medellinensis*, or a combination thereof.

[0009] In some implementations, the waste yeast liquid originates from breweries and / or bioethanol fermentation processes.

[0010] In some implementations, the production culture medium comprises: a) Sugars of approximately 1% w / v to approximately 18% w / v relative to the production medium; b) Waste yeast broth, approximately 2% v / v to approximately 12% v / v relative to the production culture medium; c) Sodium hydrogen phosphate (Na₂HPO₄) of approximately 0.1% w / v to approximately 1% w / v relative to the production medium; and e) Ethanol, approximately 0.5% v / v to approximately 2.5% v / v relative to the production medium.

[0011] In some embodiments, the production medium also includes waste grain filtrate at 5% v / v to 50% v / v relative to the production medium.

[0012] In some embodiments, the waste grain filtrate is formed by suspending the waste grain in an aqueous medium at a ratio of about 1:3 to form sludge, and then filtering the sludge.

[0013] In some implementations, the waste grains are derived from breweries and / or bioethanol fermentation processes.

[0014] In some embodiments, the production medium also contains citric acid at about 0.05% w / v to about 1% w / v relative to the production medium.

[0015] In some embodiments, the production culture medium is characterized by a pH of about 4 to about 6, or a Brix value of about 3 to about 6.

[0016] In some embodiments, the culture culturing step is performed at about 20°C to about 40°C. In some embodiments, the culture culturing step is performed at about 30°C.

[0017] In some embodiments, the step of culturing the culture is performed without stirring the starting culture.

[0018] In some embodiments, the culture culturing step is performed for approximately 2 days to approximately 20 days. In some embodiments, the culture culturing step is performed for approximately 4 days to approximately 7 days.

[0019] In some implementations, the starting culture is added to the production medium at a ratio of about 5% v / v to about 40% v / v relative to the medium.

[0020] In some embodiments, the method further includes the step of culturing the seed inoculum in a propagation medium and / or an activation medium to form a starting culture. In some embodiments, the seed inoculum comprises a biofilm formed by at least one strain of *Coprinus coccinea*.

[0021] In some embodiments, the seed inoculum is added to the propagation and / or activation media at a ratio of about 10% v / v to about 30% v / v relative to the starting culture. In some embodiments, the seed inoculum is added to the propagation and / or activation media at a ratio of about 20% v / v relative to the starting culture.

[0022] In some embodiments, the step of forming the starter culture is performed at a temperature of about 20°C to about 40°C. In some embodiments, the step of forming the starter culture is performed at about 30°C.

[0023] In some embodiments, the step of forming the starter culture is performed under static conditions. In some embodiments, the step of forming the starter culture is performed without stirring the starter culture.

[0024] In some embodiments, the step of forming the starter culture takes about 2 days to about 20 days. In some embodiments, the step of forming the starter culture takes about 4 days to about 7 days.

[0025] In some embodiments, the culture medium comprises: a) Sugars of approximately 3% w / v to approximately 20% w / v relative to the propagation medium; and b) Waste yeast culture, approximately 2% v / v to approximately 12% v / v relative to the propagation medium.

[0026] In some embodiments, the propagation medium also contains citric acid at about 0.05% w / v to about 1% w / v relative to the propagation medium.

[0027] In some embodiments, the propagation medium also contains ethanol at a concentration of 0.5% v / v to 2.5% v / v relative to the propagation medium.

[0028] In some embodiments, the propagation medium is characterized by a pH of about 4 to about 6.

[0029] In some embodiments, the step of forming the seed inoculum is performed at about 20°C to about 40°C. In some embodiments, the step of forming the seed inoculum is performed at about 30°C.

[0030] In some implementations, the step of forming the seed inoculum is carried out under static conditions.

[0031] In some embodiments, the seed inoculum formation step is carried out for about 2 to about 10 days. In some embodiments, the seed inoculum formation step is carried out for about 4 to about 7 days.

[0032] In some embodiments, the method further includes the step of activating at least one strain of *Streptococcus foetida* in an activation medium to form a seed inoculum.

[0033] In some embodiments, the activation culture medium comprises: a) Sugars of approximately 1% w / v to approximately 20% w / v relative to the activating medium; and b) Yeast extract powder, approximately 0.1% w / v to approximately 2% w / v relative to the activated medium.

[0034] In some embodiments, the activating medium also contains citric acid at about 0.05% w / v to about 1% w / v relative to the activating medium.

[0035] In some embodiments, the activating medium also contains ethanol at a concentration of 0.5% v / v to 2.5% v / v relative to the activating medium.

[0036] In some embodiments, the activation medium is characterized by a pH of about 4 to about 6.

[0037] In some embodiments, the step of culturing the culture includes forming a bacterial cellulose membrane to form bacterial cellulose.

[0038] In some embodiments, the method further includes the step of harvesting the bacterial cellulose membrane when the pH of the culture is about 2 to about 3.

[0039] In some embodiments, the method further includes the steps of washing the bacterial cellulose membrane and compressing the bacterial cellulose membrane.

[0040] In some implementations, after harvesting bacterial cellulose, the residual culture medium from the method is recycled to produce another batch of bacterial cellulose.

[0041] In some implementations, the bacterial cellulose yield of this method is greater than about 80%.

[0042] This disclosure relates to bacterial cellulose formed using the methods disclosed herein.

[0043] In some embodiments, the bacterial cellulose is characterized by a thickness of about 1 cm to about 3 cm.

[0044] In some embodiments, bacterial cellulose is characterized by a tensile strength of about 100 MPa to about 200 MPa.

[0045] In some embodiments, bacterial cellulose is characterized by a breaking elongation of about 20% to about 50%.

[0046] In some embodiments, bacterial cellulose can be formed into foam, hydrogel, aerogel, cardboard sheet, biofilm, film, sheet, or membrane. Attached Figure Description

[0047] Embodiments of the present invention will now be described by way of non-limiting example with reference to the accompanying drawings, wherein: Figure 1 A flowchart is shown for producing bacterial cellulose using waste grains and waste yeast.

[0048] Figure 2 A bioreactor system for continuous operation of bacterial cellulose production is shown.

[0049] Figure 3 A stacked tray is shown for static fermentation.

[0050] Figure 4 The production of bacterial cellulose (opaque membrane) is shown when grown in Buyo2 production medium containing waste grain filtrate and waste yeast broth. Detailed Implementation

[0051] Brewery and bioethanol production processes generate substantial amounts of waste grains and yeast, posing significant challenges to waste management due to both high costs and environmental problems. It is estimated that brewery waste grains alone generate approximately 36.4 million tons annually. Without effective strategies to reuse these byproducts, the valuable resources contained within waste grains and yeast, including nutrients, carbon sources, and potential energy, will be wasted. For example, brewery waste grains are rich in hemicellulose, cellulose, protein, lignin, oils, and phenolic compounds, while brewery yeast is a source of protein, minerals, vitamins, and sugars. Traditional treatment methods, such as using byproducts as animal feed, have many limitations. Exploring more diverse and sustainable applications for these byproducts requires innovative solutions and technologies.

[0052] Furthermore, bacterial cellulose is a high-value biomaterial with a wide range of applications, but its large-scale industrial production remains challenging due to the need for expensive, carbon-rich virgin feedstocks and the lack of optimized, efficient, and large-scale production processes. At least one of these challenges can be addressed by using specific bacterial strains and combinations to provide more efficient and sustainable methods for utilizing brewery and bioethanol byproducts on an industrial scale.

[0053] This disclosure is based on the understanding that, in laboratory-scale testing, the aforementioned brewery waste must undergo chemical or thermal pretreatment via a two-step or one-step process to form bacterial cellulose. Furthermore, bacterial cellulose production was conducted only in small volumes (50 mL to 110 mL), with only one strain used in each study, and no wastewater recycling was reported. This indicates that these laboratory-scale processes cannot be linearly scaled up, and optimizing the process for industrial-scale production is extremely challenging.

[0054] This disclosure relates to a method for efficiently utilizing brewery or bioethanol production byproducts (including waste grains and waste yeast) using specific bacteria or mixtures of bacteria. This disclosure also relates to applying these specific microorganisms to the large-scale conversion of the aforementioned byproducts into bacterial cellulose. For example, the method includes steps such as starter culture preparation, byproduct addition, culture medium concentration and composition, and other fermentation parameters and techniques. This integrated method is designed to improve the yield of industrial-scale bacterial cellulose production, thereby contributing to a more sustainable and efficient utilization of brewery and bioethanol byproducts.

[0055] Biopolymers consist of monomeric units formed from monomers. Biopolymers typically have a well-defined structure, but this is not their defining characteristic (e.g., lignocellulose). For proteins, the exact chemical composition and sequence of these units is called the primary structure. Many biopolymers spontaneously fold into characteristic compact shapes (secondary and tertiary structures) that determine their biological functions and have complex dependencies on their primary structure. In contrast, synthetic polymers have much simpler and more random (or random) structures. This results in a molecular weight distribution absent in biopolymers. In fact, because the synthesis of biopolymers in most in vivo systems is controlled by template-directed processes, biopolymers of the same type (e.g., a specific protein) are similar: they all contain similar monomer sequences and quantities, and therefore have similar mass. This monodispersity contrasts sharply with the polydispersity common in synthetic polymers. Therefore, the dispersion of biopolymers is typically 1.

[0056] "Biopolymers" are also distinct from "bio-based polymers." Bio-based polymers are polymers synthesized (wholly or partially) from biomass monomers through chemical or biological methods, such as polyesters (e.g., polyhydroxyalkanoates (PHAs) and polylactic acid (PLA)). In this regard, the only polymers that can be considered both biopolymers and bio-based polymers are those biosynthesized (through microorganisms) from biomass carbon sources (e.g., carbohydrates and lipids), examples of which include PHAs, bacterial cellulose, gellan gum, xanthan gum, and curdlan.

[0057] Polysaccharides (sugar polymers) can be linear or branched, typically linked by glycosidic bonds. The specific location of the bonds can vary, and the orientation of the linking functional groups is also important, resulting in α- and β-glycosidic bonds, with the position of the linking carbon atom within the ring determined by numbering. Furthermore, many sugar units can undergo various chemical modifications, such as amination, and can even form part of other molecules, such as glycoproteins.

[0058] Cellulose is composed of stacked chain-like structures, which gives it stability and strength. This strength and stability stem from the linear shape of cellulose, which is formed by glucose monomers linked by glycosidic bonds. This linear shape allows the molecules to align closely. Cellulose is widely used in the form of nanofibers (called nanofibers). Low concentrations of nanocellulose can form transparent gel materials. This material can be used to prepare biodegradable, homogeneous, and dense films that are highly valuable in the biomedical field.

[0059] Therefore, this disclosure relates to a method for producing bacterial cellulose, comprising: The starting culture was cultured together with the production medium to form a culture for the formation of bacterial cellulose; The starting culture contains *Coprinus cocovenenans* (cocovenenans) Komagataeibacter nataicola ), Reuteria paniculata ( Komagataeibacter rhaeticus ), Komatobacterium xylinum ( Komagataeibacter xylinus ) and Intercalozoa ( Komagataeibacter intermedius At least one strain of *Strombyx mori*; and The production culture medium contains waste yeast liquid that has not been heat-treated or chemically treated.

[0060] In some embodiments, the method for producing bacterial cellulose includes: Approximately 20 L to approximately 50 L of the starting culture was cultured together with at least approximately 200 L of the production medium to form a culture for the formation of bacterial cellulose. The starting culture contains *Coprinus cocovenenans* (cocovenenans) Komagataeibacter nataicola ), Reuteria paniculata ( Komagataeibacter rhaeticus ), Komatobacterium xylinum ( Komagataeibacter xylinus ) and Intercalozoa ( Komagataeibacter intermedius At least one strain of *Strombyx mori*; and The production culture medium contains waste yeast liquid that has not been heat-treated or chemically treated.

[0061] Pretreatment of spent grains and yeast can maximize their nutritional value. For example, it is recommended to use sulfuric acid or phosphoric acid for pretreatment to break down proteins and lignocellulose, thus enabling microorganisms to utilize them effectively. As another example, Mitri, S., Salameh et al., in Fermentation, 2022, 8(2), 50 (Valorization of Brewers' spent grains: pretreatments and fermentation, areview), highlight the crucial role of pretreatment of spent grains before fermentation.

[0062] The inventors discovered that when using certain bacteria for fermentation, such pretreatment steps are unnecessary and potentially more advantageous. Eliminating the need for chemical or thermal pretreatment improves cost-effectiveness, reduces environmental impact, and simplifies the process. In particular, omitting these chemical and thermal pretreatment steps lowers operating costs, making the entire utilization process more economically viable.

[0063] Research has found that *Cocovenenans* (… Komagataeibacter nataicola ), Reuteria paniculata ( Komagataeibacter rhaeticus), Komatobacterium xylinum ( Komagataeibacter xylinus ) and Intercalozoa ( Komagataeibacter intermedius It exhibits high adaptability to culture media containing waste yeast culture and waste grain filtrate. In some embodiments, the starting culture contains *Reticulitermes repens* (…). Komagataeibacter rhaeticus ) and Cladosporium ( Komagataeibacter xylinus In some embodiments, the starting culture contains *Coprinus cocovenenans* (…). Komagataeibacter nataicola ), Reuteria paniculata ( Komagataeibacter rhaeticus ) and Cladosporium ( Komagataeibacter xylinus In some embodiments, the starting culture contains *Rhizoctonia solani* (…). Komagataeibacter rhaeticus ), Komatobacterium xylinum ( Komagataeibacter xylinus ) and Intercalozoa ( Komagataeibacter intermedius In some embodiments, the starting culture contains *Codonopsis Cocos* (cocovenenans). Komagataeibacter nataicola ), Reuteria paniculata ( Komagataeibacter rhaeticus ), Komatobacterium xylinum ( Komagataeibacter xylinus ) and Intercalozoa ( Komagataeibacter intermedius ).

[0064] Furthermore, the presence of other *Coprinus* strains in combination with at least one of the above four strains will not negatively affect the method. In some embodiments, the starting culture contains *Coprinus cocovenenans* (… Komagataeibacter nataicola ), Reuteria paniculata ( Komagataeibacter rhaeticus ), Komatobacterium xylinum ( Komagataeibacter xylinus ), Intermediate Columbium ( Komagataeibacter intermedius ), *Glucosibirica* strains (e.g., *Glucosibirica sacchariformis*). G. sucrofermentans ), Staphylococcus hannii ( G. hansenii ), European Staphylococcus aureus ( G. europaeus ), Staphylococcus entanediol ( G. entanii ), Staphylococcus sucraniosus ( G. saccharivorans ), Staphylococcus aureus ( G. swingsii ), docile glucosamine ( G. oboediens ), liquefied glucosamine ( G. liquefaciens ), glucosidobacterium sugarcane ( G. sacchari ), nitrogen-fixing glucosamine ( G. diazotrophicus ), Johannesburg glucosamine ( G. johannae ), nitrogen-trapping glucosamine ( G. azotocaptans ), Medellin Staphylococcus aureus ( G. medellinensisBacteria (or combinations thereof). In some embodiments, the starting culture also comprises a strain of *Glucosobacterium* selected from *Glucosobacterium sugar-fermenting*. G. sucrofermentans ), Staphylococcus hannii ( G. hansenii ), European Staphylococcus aureus ( G. europaeus ), Staphylococcus entanediol ( G. entanii ), Staphylococcus sucraniosus ( G. saccharivorans ), Staphylococcus aureus ( G. swingsii ), docile glucosamine ( G. oboediens ), liquefied glucosamine ( G. liquefaciens ), glucosidobacterium sugarcane ( G. sacchari ), nitrogen-fixing glucosamine ( G. diazotrophicus ), Johannesburg glucosamine ( G. johannae ), nitrogen-trapping glucosamine ( G. azotocaptans ), Medellin Staphylococcus aureus ( G. medellinensis (or a combination thereof).

[0065] Combinations of multiple bacterial strains enable bacterial communities to effectively utilize a wider range of nutrients, particularly those from complex substrates such as spent grains and yeast. Using bacterial mixtures also improves robustness and adaptability to changes in fermentation conditions.

[0066] In some implementations, the production culture medium comprises: a) Sugars of approximately 1% w / v to approximately 18% w / v relative to the production medium; b) Waste yeast broth, approximately 2% v / v to approximately 12% v / v relative to the production culture medium; and c) Sodium hydrogen phosphate (Na2HPO4) at approximately 0.1% w / v to approximately 1% w / v relative to the culture medium.

[0067] "Carbohydrates" or "sugars" include monosaccharides, disaccharides, oligosaccharides, polysaccharides, and their isomers. Monosaccharides, also known as simple sugars, are the simplest forms of sugar and the most basic building blocks (monomers) of all carbohydrates. Examples of monosaccharides include glucose (dextrose), fructose (levose), and galactose. Monosaccharides are the building blocks of disaccharides (such as sucrose and lactose) and polysaccharides (such as cellulose and starch). Disaccharides are sugars composed of two monosaccharides linked by glycosidic bonds. Like monosaccharides, disaccharides are simple sugars that are soluble in water. Sucrose, lactose, and maltose are three common examples. Oligosaccharides are carbohydrate polymers containing a small number (typically 3 to 10) of monosaccharides. Polysaccharides are long-chain polymers of carbohydrates composed of monosaccharide units linked by glycosidic bonds. These carbohydrates can be reacted with water (hydrolyzed) using enzymes (such as amylase, cellulase, chitinase, etc.) as catalysts to produce constituent sugars (monosaccharides or oligosaccharides). Their structures range from linear to highly branched. Examples include storage polysaccharides such as starch, glycogen, and galactosogen, as well as structural polysaccharides such as cellulose and chitin.

[0068] In some embodiments, the sugar is selected from glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, sorbitol, mannitol, maltodextrin, raffinose, stachyose, fructooligosaccharides, amylose, amylopectin, modified starch, glycogen, dextran, chitosan, glycosaminoglycans, alginate, ulvan, gum arabic, gellan gum, cellulose, hemicellulose, ethylcellulose, methylcellulose, pectin, hydrocolloids, and combinations thereof. In some embodiments, the sugar is sucrose.

[0069] In some embodiments, the sugar is present relative to the production medium at about 1% w / v to about 18% w / v, about 1% w / v to about 16% w / v, about 1% w / v to about 14% w / v, about 1% w / v to about 12% w / v, about 1% w / v to about 10% w / v, about 1% w / v to about 9% w / v, about 1% w / v to about 8% w / v, about 1% w / v to about 7% w / v, about 1% w / v to about 6% w / v, about 1% w / v to about 5% w / v, about 1% w / v to about 4% w / v, about 2% w / v to about 4% w / v, or about 3% w / v to about 4% w / v.

[0070] Waste yeast liquid refers to the supernatant of the waste yeast mixture after yeast sedimentation. While this liquid can also be obtained by centrifuging the waste yeast, it is not essential. In some embodiments, the waste yeast liquid originates from brewery and / or bioethanol fermentation processes. The waste yeast liquid can be directly utilized after being obtained from brewery and / or bioethanol fermentation processes, i.e., without modification. Waste yeast liquid may contain proteins, minerals, vitamins, nitrogen, and enzymes.

[0071] In some embodiments, the waste yeast broth has the following characteristics: total organic carbon of about 8% w / v to about 15% w / v, total nitrogen of about 0.5% w / v to about 1.5% w / v, total protein of about 4% w / v to about 10% w / v, carbohydrate of about 3% w / v to about 5% w / v, total fat of about 0.1% w / v to about 0.5% w / v, and ash of about 1% w / v to about 2% w / v. In some embodiments, the waste yeast broth has the following characteristics: total organic carbon of about 13.8% w / v, total nitrogen of about 1.09% w / v, total protein of about 6.8% w / v, carbohydrate of about 4.35% w / v, total fat of about 0.39% w / v, and ash of about 1.26% w / v.

[0072] In some embodiments, the waste yeast broth has a Brix value of about 7 to about 14, a potential alcohol content of about 4 to about 7.0, a water content of about 65% to about 90%, and a pH of about 4.5 to 6.5. In some embodiments, the waste yeast broth has a Brix value of 8.7, a potential alcohol content of about 4.8, a water content of about 86.6%, and a pH of 5.9.

[0073] In some implementations, the waste yeast liquid is not subjected to heat treatment or chemical pretreatment. This means that the waste yeast liquid is not heat-treated and no chemicals are added.

[0074] In some embodiments, the waste yeast broth is expressed relative to the production culture medium at about 2% w / v to about 12% w / v, about 2% w / v to about 10% w / v, about 2% w / v to about 9.5% w / v, about 2% w / v to about 9% w / v, about 2% w / v to about 8.5% w / v, about 2% w / v to about 8% w / v, about 2% w / v to about 7.5% w / v, about 2% w / v to about 7% w / v, about 2% w / v to about 6.5% w / v, about 2% w / v to about 6% w / v, about 2% w / v to about 5.5% w / v, about 2% w / v to about 5% w / v, about 2.5% w / v to about 5% w / v, about 3% w / v to about 5% w / v, about 3.5% w / v to about 5% w / v, or about 4% w / v to about 5% w / v.

[0075] In some embodiments, the waste yeast broth is expressed relative to the production culture medium at about 2% v / v to about 12% v / v, about 2% v / v to about 10% v / v, about 2% v / v to about 9.5% v / v, about 2% v / v to about 9% v / v, about 2% v / v to about 8.5% v / v, about 2% v / v to about 8% v / v, about 2% v / v to about 7.5% v / v, about 2% v / v to about 7% v / v, about 2% v / v to about 6.5% v / v, about 2% v / v to about 6% v / v, about 2% v / v to about 5.5% v / v, about 2% v / v to about 5% v / v, about 2.5% v / v to about 5% v / v, about 3% v / v to about 5% v / v, about 3.5% v / v to about 5% v / v, or about 4% v / v to about 5% v / v.

[0076] In some embodiments, disodium hydrogen phosphate (Na2HPO4) is present at a concentration of about 0.1% w / v to about 1% w / v relative to the production medium. In other embodiments, the concentration is about 0.1% w / v to about 0.9% w / v, about 0.1% w / v to about 0.8% w / v, about 0.1% w / v to about 0.7% w / v, about 0.1% w / v to about 0.6% w / v, about 0.1% w / v to about 0.5% w / v, about 0.2% w / v to about 0.5% w / v, about 0.3% w / v to about 0.5% w / v, or about 0.4% w / v to about 0.5% w / v.

[0077] Grain filtrate is the filtrate obtained from grain waste. Grain filtrate may contain organic carbon, nitrogen, protein, carbohydrates, and other components.

[0078] In some embodiments, the waste grain filtrate has the following characteristics: total organic carbon of about 10% w / v to about 30% w / v, total nitrogen of about 0.5% w / v to about 1.5% w / v, total protein of about 4% w / v to about 10% w / v, carbohydrate of about 5% w / v to about 15% w / v, total fat of about 0.1% w / v to about 1% w / v, and ash of about 0.5% w / v to about 2% w / v. In some embodiments, the waste grain has the following characteristics: total organic carbon of about 20.6% w / v, total nitrogen of about 1.1% w / v, total protein of about 6.7% w / v, carbohydrate of about 10% w / v, total fat of about 0.6% w / v, and ash of about 0.7% w / v.

[0079] In some embodiments, the production medium also comprises waste grain filtrate of about 5% w / v to about 50% w / v relative to the production medium. In some embodiments, the waste grain filtrate is about 5% w / v to about 45% w / v, about 5% w / v to about 40% w / v, about 5% w / v to about 35% w / v, about 5% w / v to about 30% w / v, about 10% w / v to about 30% w / v, about 10% w / v to about 28% w / v, about 10% w / v to about 26% w / v, about 10% w / v to about 24% w / v, about 10% w / v to about 22% w / v, about 10% w / v to about 20% w / v, about 10% w / v to about 18% w / v, about 10% w / v to about 16% w / v, about 12% w / v to about 16% w / v, or about 14% w / v to about 16% w / v.

[0080] In some embodiments, the production medium also comprises waste grain filtrate at a concentration of about 5% v / v to about 50% v / v relative to the production medium. In some embodiments, the waste grain filtrate is about 5% v / v to about 45% v / v, about 5% v / v to about 40% v / v, about 5% v / v to about 35% v / v, about 5% v / v to about 30% v / v, about 10% v / v to about 30% v / v, about 10% v / v to about 28% v / v, about 10% v / v to about 26% v / v, about 10% v / v to about 24% v / v, about 10% v / v to about 22% v / v, about 10% v / v to about 20% v / v, about 10% v / v to about 18% v / v, about 10% v / v to about 16% v / v, about 12% v / v to about 16% v / v, or about 14% v / v to about 16% v / v.

[0081] In some implementations, the waste grain filtrate is not subjected to heat treatment or chemical pretreatment. This means that the waste grain filtrate is not heat-treated and no other chemicals are added.

[0082] In some embodiments, the waste grain filtrate is formed by suspending waste grains in an aqueous medium at a ratio of about 1:3 to form sludge, and then filtering the sludge. The resulting filtrate is the waste grain filtrate. In some embodiments, the waste grains are suspended in an aqueous medium such that the average moisture content of the waste grains is about 70% to about 90%, about 70% to about 85%, about 70% to about 80%, or about 70% to about 75%.

[0083] As used herein, the term "aqueous medium" refers to a water-based solvent or solvent system whose primary component is water. Such solvents can be polar or nonpolar, and / or protic or aprotic. A solvent system refers to a combination of solvents that ultimately form a single phase. Both "solvent" and "solvent system" can include, but are not limited to, pentane, cyclopentane, hexane, cyclohexane, benzene, toluene, dioxane, chloroform, diethyl ether, dichloromethane, tetrahydrofuran, ethyl acetate, acetone, dimethylformamide, acetonitrile, dimethyl sulfoxide, nitromethane, propylene carbonate, formic acid, butanol, isopropanol, propanol, ethanol, methanol, acetic acid, ethylene glycol, diethylene glycol, or water. Water-based solvents or solvent systems may also contain dissolved ions, salts, and molecules such as amino acids, proteins, carbohydrates, and phospholipids. This category of salts includes, but is not limited to, sodium chloride, potassium chloride, ammonium acetate, magnesium acetate, magnesium chloride, magnesium sulfate, potassium acetate, potassium chloride, sodium acetate, sodium citrate, zinc chloride, sodium HEPES, calcium chloride, ferric nitrate, sodium bicarbonate, potassium phosphate, and sodium phosphate. Therefore, biological fluids, physiological solutions, and culture media also fall within this definition.

[0084] In some implementations, the waste grains are derived from breweries and / or bioethanol fermentation processes.

[0085] In some embodiments, the production medium further comprises citric acid at a concentration of about 0.05% w / v to about 1% w / v relative to the production medium. In other embodiments, the concentration is about 0.05% w / v to about 0.9% w / v, about 0.05% w / v to about 0.8% w / v, about 0.05% w / v to about 0.7% w / v, about 0.05% w / v to about 0.6% w / v, about 0.05% w / v to about 0.5% w / v, about 0.05% w / v to about 0.4% w / v, about 0.05% w / v to about 0.3% w / v, about 0.05% w / v to about 0.2% w / v, or about 0.05% w / v to about 0.1% w / v.

[0086] In some implementations, the production culture medium is characterized by a pH of about 4 to about 6.

[0087] In some embodiments, the production medium is characterized in that the ethanol concentration is from about 0.5% v / v to about 2.5% v / v relative to the production medium. In some embodiments, the concentration is from about 0.5% v / v to about 2% v / v, from about 0.5% v / v to about 1.5% v / v, or from about 0.5% v / v to about 1% v / v.

[0088] Studies have found that the addition of ethanol is optional. This contrasts with bacterial cellulose production conducted in 50 mL working volume Erlenmeyer flasks with continuous oscillation at 200 rpm (in which case the addition of ethanol was found to be necessary). However, the addition of ethanol may lead to unfavorable transformation of the bacteria, generating mutants that do not produce cellulose, thus significantly reducing yield. This is described in Sani et al., J. Chem. Technol. Biotechnol.; 2010; 85, 151–164 (Improvements in the production of bacterial synthesized biocellulose nanofibrils using different culture methods). The processing conditions and requirements of this disclosure differ accordingly, and therefore ethanol may or may not be used.

[0089] In some embodiments, the production medium is characterized by a Brix value of about 3 to about 6. In some embodiments, the production medium is characterized by a Brix value of 4.5.

[0090] The method was able to stably produce bacterial cellulose when tested using at least three different waste grain and waste yeast samples from different breweries, as well as different batches of waste grain and waste yeast from the same brewery.

[0091] In some embodiments, the step of culturing the culture includes: culturing the starting culture with a first production medium to form an intermediate culture, and then culturing the intermediate culture with a second production medium to form a production culture. This allows the cells to adapt to the new medium, thereby providing a higher yield. Preferably, the first and second media are the same. For example, if the first media is Buyo2, the second media is preferably Buyo2.

[0092] In some embodiments, the step of culturing the culture is performed at about 20°C to about 40°C. In some embodiments, the temperature is about 20°C to about 38°C, about 20°C to about 36°C, about 20°C to about 34°C, about 20°C to about 32°C, about 22°C to about 32°C, about 24°C to about 32°C, about 26°C to about 32°C, or about 28°C to about 32°C. In some embodiments, the step of culturing the culture is performed at about 30°C.

[0093] In some embodiments, the culturing step is performed under static conditions. This means that the starting culture is allowed to be cultured without external disturbance. In some embodiments, the culturing step is performed without stirring the starting culture.

[0094] In some embodiments, the culture culturing step is performed for about 2 days to about 20 days. In some embodiments, the duration is about 2 days to about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days to about 12 days, about 2 days to about 10 days, or about 2 days to about 8 days. In some embodiments, the culture culturing step is performed for about 4 days to about 7 days.

[0095] In some embodiments, the starting culture is mixed with the production medium before being transferred to or cultured in the fermentation tray. Alternatively, in some embodiments, the starting culture is added to the fermentation tray containing the production medium. In this method, instead of mixing the medium and culture before pouring into the fermentation tray, fresh, sterilized, and relatively hot medium is poured into the fermentation tray (while simultaneously sterilizing the tray), and once the medium has cooled to below approximately 30°C, the starting culture is added directly to the fermentation tray and mixed.

[0096] In some embodiments, about 10 L to about 50 L of starting culture is mixed with at least about 200 L of production medium. In some embodiments, about 20 L to about 50 L of starting culture is mixed with about 400 L to about 1600 L of production medium. In some embodiments, about 40 L of starting culture is mixed with at least about 1500 L of production medium.

[0097] In some embodiments, about 20 L to about 50 L of a starting culture is mixed with at least about 200 L of a first production medium to prepare an intermediate culture. In some embodiments, about 100 L to about 300 L of a cultured intermediate culture is mixed with about 800 L to about 1000 L of a second production medium. In some embodiments, about 40 L of a starting culture is mixed with at least about 200 L of the first production medium. In some embodiments, about 200 L of an intermediate culture is mixed with at least about 800 L of the second production medium.

[0098] In some embodiments, the culture culturing step is carried out in a fermentation tray with a volume of about 1 L to about 2 L. Preferably, the volume can be about 1 L to about 1.5 L. Studies have found that if the volume is too large, the bottom cells have difficulty producing cellulose because cellulose formation occurs at the air / cellulose membrane interface, rather than the culture medium / cellulose interface. Large fermentation volumes maximize the use of production facilities, resulting in lower unit product production costs. The culture medium and starting culture are prepared and mixed in an autoclaved bioreactor and then transferred to stackable fermentation trays, facilitating the scaling up of the entire production process. Furthermore, when carried out under static conditions in the fermentation tray, the growth of non-cellulose-producing bacterial mutant cells is minimized, and the increased surface area and improved air exchange of the fermentation tray enable high oxygen transfer rates for aerobic bacterial reactions, resulting in higher bacterial cellulose yields.

[0099] In some embodiments, the starting culture is added to the production medium in an amount from about 5% v / v to about 40% v / v relative to the medium. In some embodiments, the concentration is from about 5% v / v to about 35% v / v, from about 5% v / v to about 30% v / v, from about 5% v / v to about 28% v / v, from about 5% v / v to about 26% v / v, from about 5% v / v to about 24% v / v, from about 5% v / v to about 22% v / v, from about 5% v / v to about 20% v / v, from about 6% v / v to about 20% v / v, from about 8% v / v to about 20% v / v, from about 10% v / v to about 20% v / v, from about 12% v / v to about 20% v / v, from about 14% v / v to about 20% v / v, or from about 18% v / v to about 20% v / v. In some implementations, the starting culture is added to the production medium at an amount of about 20% v / v relative to the medium.

[0100] In some embodiments, the method further includes the step of culturing the seed inoculum in a propagation medium and / or an activation medium to form a starting culture. In some embodiments, the method further includes the step of culturing the seed inoculum in a propagation medium to form a starting culture. In some embodiments, the seed inoculum comprises a biofilm formed by at least one strain of *Coprinus coccinea*.

[0101] Therefore, methods for producing bacterial cellulose include: a) Culturing a seed inoculum in a propagation medium and / or activation medium to form a starting culture, said seed inoculum comprising a biofilm formed by at least one strain of *Streptococcus*; and b) The starting culture is cultured together with the production medium to form a culture for the formation of bacterial cellulose; The production culture medium contains waste yeast liquid that has not been heat-treated or chemically treated; Wherein, at least one strain of *Coprinus cocovenenans* is selected from *Coprinus cocovenenans* (… Komagataeibacter nataicola ), Reuteria paniculata ( Komagataeibacter rhaeticus ), Komatobacterium xylinum ( Komagataeibacter xylinus ), Intermediate Columbium ( Komagataeibacter intermedius (or a combination thereof).

[0102] Seed inoculum refers to the material used for inoculation, that is, introducing microorganisms into a culture. Inoculation is carried out to allow the microorganisms to grow in the culture under specific growth conditions.

[0103] In some embodiments, the seed inoculum is added to the propagation medium and / or activation medium at an amount of about 10% v / v to about 30% v / v relative to the starting culture. In some embodiments, the concentration is about 10% v / v to about 28% v / v, about 10% v / v to about 26% v / v, about 10% v / v to about 24% v / v, about 10% v / v to about 22% v / v, about 10% v / v to about 20% v / v, about 12% v / v to about 20% v / v, about 14% v / v to about 20% v / v, or about 16% v / v to about 20% v / v. In some embodiments, the seed inoculum is added to the propagation medium and / or activation medium at an amount of about 20% v / v relative to the starting culture.

[0104] The study found that revived bacterial strains are more stable after being cultured into a biofilm and can establish a starter culture after several cycles. The biofilm contains highly active bacteria, resulting in faster growth and thus shorter culture times. The study found that biofilms can be used to establish starter cultures or revive strains because transferring biofilms during the production stage can be challenging.

[0105] In some embodiments, the step of forming the starter culture is performed at about 20°C to about 40°C. In some embodiments, the temperature is about 20°C to about 38°C, about 20°C to about 36°C, about 20°C to about 34°C, about 20°C to about 32°C, about 22°C to about 32°C, about 24°C to about 32°C, about 26°C to about 32°C, or about 28°C to about 32°C. In some embodiments, the step of forming the starter culture is performed at about 30°C.

[0106] In some embodiments, the step of forming the starter culture is performed under static conditions. This means that the starter culture is cultured without external disturbance. In some embodiments, the step of forming the starter culture is performed without stirring the starter culture.

[0107] In some embodiments, the step of forming the starter culture is performed for about 2 days to about 20 days. In some embodiments, the duration is about 2 days to about 18 days, about 2 days to about 16 days, about 2 days to about 14 days, about 2 days to about 12 days, about 2 days to about 10 days, or about 2 days to about 8 days. In some embodiments, the step of forming the starter culture is performed for about 4 days to about 7 days.

[0108] In some implementations, the step of forming the starter culture includes: i) Conditioning the seed inoculum in a first propagation medium and / or activation medium to form a conditioned seed inoculum; and ii) The regulated seed inoculum is cultured in a second propagation medium to form a starting culture.

[0109] Modifying the seed inoculum can prevent cells from being "shocked," a process that can lead to cell death. Studies have found that this can increase bacterial cellulose production.

[0110] The first culture medium may be the same as the second culture medium. Alternatively, the first culture medium may contain the same components as the second culture medium, but at least one component may be present at a lower concentration.

[0111] In some embodiments, the culture medium comprises: a) Sugars of approximately 3% w / v to approximately 20% w / v relative to the propagation medium; and b) Waste yeast culture, approximately 2% v / v to approximately 12% v / v relative to the propagation medium.

[0112] In some embodiments, the propagation medium comprises waste yeast liquid that has not been heat-treated or chemically treated.

[0113] In some embodiments, the sugar content relative to the propagation medium is about 3% w / v to about 20% w / v, about 3% w / v to about 18% w / v, about 3% w / v to about 16% w / v, about 3% w / v to about 14% w / v, about 3% w / v to about 12% w / v, about 6% w / v to about 12% w / v, or about 8% w / v to about 12% w / v. In some embodiments, the sugar content relative to the propagation medium is about 10% w / v.

[0114] In some embodiments, the waste yeast broth is expressed relative to the propagation medium at approximately 2% w / v to approximately 12% w / v, approximately 2% w / v to approximately 11% w / v, approximately 2% w / v to approximately 10% w / v, approximately 2% w / v to approximately 9.5% w / v, approximately 2% w / v to approximately 9% w / v, approximately 2% w / v to approximately 8.5% w / v, approximately 2% w / v to approximately 8% w / v, approximately 2% w / v to approximately 7.5% w / v, approximately 2% w / v to approximately 7% w / v, approximately 2% w / v to approximately 6.5% w / v, approximately 2% w / v to approximately 6% w / v, approximately 2% w / v to approximately 5.5% w / v, approximately 2% w / v to approximately 5% w / v, approximately 2.5% w / v to approximately 5% w / v, approximately 3% w / v to approximately 5% w / v, approximately 3.5% w / v to approximately 5% w / v, or approximately 4% w / v to approximately 5%. w / v.

[0115] In some embodiments, the waste yeast broth is expressed relative to the propagation medium at approximately 2% v / v to approximately 12% v / v, approximately 2% v / v to approximately 11% v / v, approximately 2% v / v to approximately 10% v / v, approximately 2% v / v to approximately 9.5% v / v, approximately 2% v / v to approximately 9% v / v, approximately 2% v / v to approximately 8.5% v / v, approximately 2% v / v to approximately 8% v / v, approximately 2% v / v to approximately 7.5% v / v, approximately 2% v / v to approximately 7% v / v, approximately 2% v / v to approximately 6.5% v / v, approximately 2% v / v to approximately 6% v / v, approximately 2% v / v to approximately 5.5% v / v, approximately 2% v / v to approximately 5% v / v, approximately 2.5% v / v to approximately 5% v / v, approximately 3% v / v to approximately 5% v / v, approximately 3.5% v / v to approximately 5% v / v, or approximately 4% v / v to approximately 5% v / v. v / v.

[0116] In some embodiments, the propagation medium further comprises citric acid at a concentration of about 0.05% w / v to about 1% w / v relative to the propagation medium. In other embodiments, the concentration is about 0.05% w / v to about 0.9% w / v, about 0.05% w / v to about 0.8% w / v, about 0.05% w / v to about 0.7% w / v, about 0.05% w / v to about 0.6% w / v, about 0.05% w / v to about 0.5% w / v, about 0.05% w / v to about 0.4% w / v, about 0.05% w / v to about 0.3% w / v, about 0.05% w / v to about 0.2% w / v, or about 0.05% w / v to about 0.1% w / v.

[0117] In some embodiments, the propagation medium further comprises ethanol at a concentration of 0.5% v / v to 2.5% v / v relative to the propagation medium. In some embodiments, the concentration is about 0.5% v / v to about 2% v / v, about 0.5% v / v to about 1.5% v / v, or about 0.5% v / v to about 1% v / v.

[0118] In some embodiments, the propagation medium is characterized by a pH of about 4 to about 6.

[0119] In some embodiments, the method further includes the step of activating at least one strain of *Streptococcus foetida* in an activation medium to form a seed inoculum.

[0120] In some embodiments, the step of forming the seed inoculum is performed at a temperature of about 20°C to about 40°C. In some embodiments, the temperature is about 20°C to about 38°C, about 20°C to about 36°C, about 20°C to about 34°C, about 20°C to about 32°C, about 22°C to about 32°C, about 24°C to about 32°C, about 26°C to about 32°C, or about 28°C to about 32°C. In some embodiments, the step of forming the seed inoculum is performed at about 30°C.

[0121] In some implementations, the seed inoculum formation step is carried out under static conditions. This means that the seed inoculum is cultured without external disturbance or stirring.

[0122] In some embodiments, the seed inoculum formation step is performed for about 2 to about 10 days. In some embodiments, the duration is about 2 to about 18 days, about 2 to about 16 days, about 2 to about 14 days, about 2 to about 12 days, about 2 to about 10 days, or about 2 to about 8 days. In some embodiments, the seed inoculum formation step is performed for about 4 to about 7 days.

[0123] In some embodiments, the activation culture medium comprises: a) Sugars of approximately 1% w / v to approximately 20% w / v relative to the activating medium; and b) Yeast extract powder, approximately 0.1% w / v to approximately 2% w / v relative to the activated medium.

[0124] In some embodiments, the sugar is present at about 1% w / v to about 20% w / v, about 1% w / v to about 18% w / v, about 1% w / v to about 16% w / v, about 1% w / v to about 14% w / v, about 1% w / v to about 12% w / v, about 1% w / v to about 10% w / v, about 1% w / v to about 8% w / v, or about 1% w / v to about 6% w / v relative to the activation medium. In some embodiments, the sugar is present at about 5% w / v relative to the activation medium.

[0125] Yeast extract powder is a mixture of amino acids, peptides, vitamins, and carbohydrates. In some embodiments, the yeast extract powder is present at a concentration relative to the activating medium of about 0.1% w / v to about 2% w / v, about 0.1% w / v to about 1.8% w / v, about 0.1% w / v to about 1.6% w / v, about 0.1% w / v to about 1.4% w / v, about 0.1% w / v to about 1.2% w / v, about 0.1% w / v to about 1% w / v, about 0.1% w / v to about 0.8% w / v, about 0.1% w / v to about 0.6% w / v, about 0.2% w / v to about 0.6% w / v, or about 0.4% w / v to about 0.6% w / v. In some embodiments, the yeast extract is present at a concentration relative to the activating medium of about 0.5% w / v.

[0126] In some embodiments, the activating medium further comprises citric acid at a concentration of about 0.05% w / v to about 1% w / v relative to the activating medium. In other embodiments, the concentration is about 0.05% w / v to about 0.9% w / v, about 0.05% w / v to about 0.8% w / v, about 0.05% w / v to about 0.7% w / v, about 0.05% w / v to about 0.6% w / v, about 0.05% w / v to about 0.5% w / v, about 0.05% w / v to about 0.4% w / v, about 0.05% w / v to about 0.3% w / v, about 0.05% w / v to about 0.2% w / v, or about 0.05% w / v to about 0.1% w / v.

[0127] In some embodiments, the activating medium further comprises ethanol at a concentration of 0.5% v / v to 2.5% v / v relative to the activating medium. In some embodiments, the concentration is about 0.5% v / v to about 2% v / v, about 0.5% v / v to about 1.5% v / v, or about 0.5% v / v to about 1% v / v.

[0128] In some embodiments, the activation medium is characterized by a pH of about 4 to about 6.

[0129] In some embodiments, the activated medium is characterized by a Brix value of about 3 to about 6. In some embodiments, the activated medium is characterized by a Brix value of 4.5.

[0130] In some embodiments, the step of culturing the culture medium includes forming a bacterial cellulose membrane to form bacterial cellulose. The bacterial cellulose membrane may be a biofilm. The bacterial cellulose membrane may be formed on the surface of the culture medium.

[0131] In some embodiments, the method further includes the step of harvesting the bacterial cellulose membrane when the pH of the culture medium is about 2 to about 3.

[0132] In some embodiments, the method further includes the step of wiping the bacterial cellulose membrane. In some embodiments, the method further includes the steps of washing and compressing the bacterial cellulose membrane. The bacterial cellulose membrane may be hydraulically compressed to remove solvent or water.

[0133] After the cleaning process, the biofilm mainly consists of water and cellulose, along with some trace bacteria. If necessary, the bacteria can be removed by treatment with NaOH and / or H2O2.

[0134] In some embodiments, the residual culture medium is recycled. In some embodiments, after harvesting the first batch of bacterial cellulose, the residual culture medium from the first method is recycled into a second production or method to form a second batch of bacterial cellulose. The residual culture medium can be recycled into activation medium, propagation medium, and / or production medium. Since the production yield exceeds 80%, less than 20% of the culture medium remains after production, and the residual culture medium can be reused. This reduces wastewater treatment costs and minimizes the environmental impact of the production process, thus aligning with sustainable development goals.

[0135] Ethanol can be added to all culture media, such as activation media, propagation media, and production media. The concentration can be from about 0.5% v / v to about 2.5% v / v relative to the medium. In other embodiments, the concentration is from about 0.5% v / v to about 2% v / v, from about 0.5% v / v to about 1.5% v / v, or from about 0.5% v / v to about 1% v / v.

[0136] In some embodiments, the method is characterized by a bacterial cellulose yield exceeding about 80%. In other embodiments, the bacterial cellulose yield is higher than about 82%, about 84%, about 86%, about 88%, or about 90%.

[0137] This disclosure relates to bacterial cellulose prepared using the methods disclosed herein.

[0138] This disclosure relates to a bacterial cellulose comprising less than about 1% w / w of *Coprinus comatus* strain residue. In other embodiments, the *Coprinus comatus* strain residue is less than about 0.9% w / w, about 0.8% w / w, about 0.7% w / w, about 0.6% w / w, about 0.5% w / w, about 0.4% w / w, about 0.3% w / w, about 0.2% w / w, or about 0.1% w / w.

[0139] In some embodiments, the bacterial cellulose is characterized by a thickness of about 1 cm to about 3 cm. In other embodiments, the thickness is about 1 cm to about 2.8 cm, about 1 cm to about 2.6 cm, about 1 cm to about 2.4 cm, about 1 cm to about 2.2 cm, about 1 cm to about 2 cm, or about 1 cm to about 1.8 cm.

[0140] In some embodiments, the bacterial cellulose is characterized by a tensile strength of about 100 MPa to about 200 MPa. In other embodiments, the tensile strength is about 110 MPa to about 200 MPa, about 110 MPa to about 190 MPa, about 110 MPa to about 180 MPa, about 110 MPa to about 170 MPa, about 110 MPa to about 160 MPa, about 110 MPa to about 150 MPa, or about 110 MPa to about 140 MPa.

[0141] In some embodiments, the bacterial cellulose is characterized by an elongation at break of about 20% to about 50%. In other embodiments, the elongation at break is about 25% to about 50%, about 30% to about 50%, about 30% to about 45%, or about 30% to about 40%.

[0142] In some embodiments, bacterial cellulose can be formed into foams, hydrogels, aerogels, cardboard, biofilms, films, sheets, or membranes.

[0143] Example Figure 1 The entire process of producing bacterial cellulose using waste grains and waste yeast was demonstrated.

[0144] Culture medium preparation Four culture media were prepared, including activation medium GY (Table 1), propagation medium BuyoF1 (Table 2), production medium Buyo2 (Table 3), and production medium Buyo4 (Table 4). Medium GY contained 50 g / L sucrose and 5 g / L yeast extract powder. Medium BuyoF1 contained 100 g / L sucrose and 45 mL / L spent yeast culture. Medium Buyo2 contained 150 mL / L spent grain filtrate, 40 mL / L spent yeast culture, 30 g / L sucrose, and 5 g / L disodium hydrogen phosphate (Na₂HPO₄). Medium Buyo4 contained 40 mL / L spent yeast culture, 30 g / L sucrose, and 5 g / L disodium hydrogen phosphate (Na₂HPO₄). All media were supplemented with 1 g / L citric acid and 5 mL / L ethanol, and the pH was adjusted to 4.0 with acetic acid.

[0145] The culture medium was sterilized by autoclaving (121°C, 15 minutes) or by a continuous sterilization system at the same temperature and flow rate of 1000 L / h. The medium was then cooled to 28–30°C before use. For a 1000 L scale fermentation with 20% culture volume, 800 L of medium was prepared.

[0146] Table 1. Composition of activation medium GY

[0147] Table 2. Composition of BuyoF1 propagation medium used for starter cultures

[0148] Table 3. Composition of Buyo2 culture medium used for bacterial cellulose production

[0149] Table 4. Composition of Buyo4 culture medium for bacterial cellulose production

[0150] Preparation of waste grain filtrate For the use of waste grains, no thermal or chemical pretreatment is required. Wet waste grains from breweries or ethanol producers are directly resuspended in water at a ratio of 1:3 (i.e., 50 kg of wet waste grains mixed with 150 L of water); the average moisture content of the wet waste grains is approximately 75%. The sludge is then filtered using a centrifuge (1000 rpm) equipped with a 200-mesh filter cloth, and the filtrate is collected and added to the Buyo2 culture medium.

[0151] Preparation of waste yeast liquid For the use of waste yeast broth collected from breweries or bioethanol fermentation processes, no thermal or chemical pretreatment is required. The waste yeast broth can be used directly or filtered using a centrifuge (1000 rpm) equipped with a 200-mesh filter cloth, and then added to culture media BuyoF1, Buyo2, and Buyo4.

[0152] Preparation of seed inoculum and starting culture on an industrial scale Bacillus cocovenenans (cocovenenans) has been found to be among the bacteria identified. Komagataeibacter nataicola ), Reuteria paniculata ( Komagataeibacter rhaeticus ), Komatobacterium xylinum ( Komagataeibacter xylinus ) and Intercalozoa ( Komagataeibacter intermedius The *Coprinus cocovenenans* strains adapt well to culture media containing waste grains and waste yeast, and are stored at -80°C for long-term preservation in the form of single strains or mixtures. Strain mixtures can contain all four strains (*Coprinus cocovenenans*, * ... K. nataicola ), Reuteria paniculata ( K. rhaeticus ), Komatobacterium xylinum ( K. xylinus ) and Intercalozoa ( K. intermedius It can also contain a combination of three strains (e.g., *Coprinus cocovenenans*). K. nataicola ), Reuteria paniculata ( K. rhaeticus ) and Cladosporium ( K. xylinus (), or a combination of two strains (e.g., *Rhizoctonia solani*). K. rhaeticus ) and Cladosporium ( K. xylinus )).

[0153] To prepare the seed inoculum, cryopreserved single or mixed strains were activated in sterilized propagation or activation medium GY cooled to 28–30°C. The seed inoculum was grown in glass petri dishes containing 30 mL of medium GY. For a 1000 L fermentation scale, cells were grown in 40 glass petri dishes under static conditions at 28–30°C for 4 days until a biofilm formed. These biofilms were then transferred to medium BuyoF1 and cultured under static conditions at 28–30°C for 4–7 days to acclimate the cells to the presence of spent yeast broth, thus forming a 40 L starting culture. Biofilm transfer is preferred to reactivate the strain and establish the starting culture after multiple solution transfer cycles.

[0154] The starting culture can then be transferred to the production medium (Buyo2 or Buyo4) for the production of bacterial cellulose.

[0155] Industrial-scale production of bacterial cellulose under static conditions Bacterial cellulose (BC) was produced using either Buyo2 medium containing both waste grain filtrate and waste yeast broth, or Buyo4 medium containing waste yeast broth but not waste grain filtrate. The medium (up to 800 L) was sterilized in a bioreactor at 121°C for 15 minutes, or via a continuous sterilization system at a flow rate of 1000-1200 L / h and a temperature of 121°C. The medium was then cooled to 28-30°C using a cold water heat exchange method. The exchanged water was collected for the preparation of various media, including activation media, propagation media, and production media. A series of bioreactors were used ( Figure 2 This enables the continuous production of bacterial cellulose.

[0156] In a 500L bioreactor, the starting culture (40L) was added to the culture medium (Buyo2 or Buyo4) at a ratio of 20% v / v, and the cells were grown under static conditions at 28-30℃ for 4-7 days to acclimate them to the production medium. After biofilm formation, the culture (20%, v / v) was transferred to the corresponding culture medium (Buyo2 or Buyo4) in a 1500L bioreactor, and the solutions were mixed. The mixture was then placed in each plastic fermentation tray (internal dimensions 32cm × 26cm × 4.5cm, maximum volume 3744 cm³). 3 Pump 1L into the container. Then stack the fermentation trays. Figure 3 Each stack contains a maximum of 40 pallets, with the top pallet empty and used as a cover for the pallets below.

[0157] To produce BC, keep the fermentation trays and stacks static and undisturbed for up to 7 days at 28-30°C. Bacterial cellulose forms a biofilm floating on the culture medium. Figure 4 Harvest the BC membrane (also known as the microbial film) when the culture medium is exhausted or after a maximum of 7 days of incubation. The pH of the culture medium at harvest is approximately 2.0-3.0. Briefly wipe the harvested BC membrane to remove the culture medium, then wash it with water; finally, use a hydraulic press to remove the absorbed water from the BC membrane. Collect the remaining culture medium, along with the water from the washing and compression steps, for recycling.

[0158] Coconut bacteria ( K. nataicolaThe BC yield of this strain was 85% to 89% (wet weight) (Table 5), and the strain grew and consumed almost all the liquid in the fermentation tray. The produced bacterial cellulose formed strong, flexible, and opaque films that were easy to handle and not easily broken or torn. It was also noted that when fermentation trays were not used (i.e., 24 mL and 100 mL flasks were used), the bacterial cellulose yield dropped to below 60%. It was also found that no cellulose films formed in the BC production medium in the absence of waste grain filtrate and / or waste yeast broth. Moreover, in the absence of Na2HPO4, the cellulose films were prone to rupture and tearing.

[0159] Table 5. *Coconutella cocovenenans* ( K. nataicola BC yield in 1 L Buyo2 or Buyo4 medium

[0160] Recycling residual culture medium and wastewater The residual culture medium was filtered using a centrifuge (1000 rpm) equipped with a 200-mesh filter cloth to remove any potential precipitates. The filtrate was then mixed with water used for washing the BCs and water used to press the BCs during harvest. If necessary, the pH of the collected water was adjusted to 4.0 using NaOH. The collected solution was then used to dissolve the chemical components of the BC production media (i.e., media Buyo2 and Buyo4).

[0161] Bacterium colata strains Under the same temperature and static conditions, *Codonopsis pilosula* was screened on a small scale (24 mL petri dishes). K. nataicola ) 、 Remyxobolus repens ( K. rhaeticus ) 、 Bacterium colata ( K. xylinus ) and Intercalozoa ( K. intermedius They found that these yeasts could efficiently produce cellulose in culture media containing untreated or chemically treated waste yeast broth (Table 6).

[0162] Table 6. BC yield of different strains of *Odontotrichum coccinea*

[0163] in conclusion Producing bacterial cellulose (BC) from waste or low-cost raw materials not only reduces production costs but also contributes to sustainable development by reusing waste that would otherwise be discarded. In this paper, we screened specific bacteria and mixtures thereof and established a fermentation process for BC production directly from waste grains or yeast without any chemical or thermal pretreatment. Industrial-scale BC production was achieved using bioreactors for inoculation and culture preparation, followed by static fermentation. Specifically, seed inoculum and starting cultures were prepared in 500L and 1500L bioreactors, with fermentation carried out on stackable fermentation trays. When using alcoholic fermentation byproducts, optimizing fermentation conditions is essential to maximizing BC yield and quality. We optimized the byproduct addition amount, culture medium composition, and fermentation conditions to achieve high yields of bacterial cellulose, with a maximum wet yield exceeding 85%. Furthermore, we recycled residual culture medium and wastewater to minimize production costs and environmental impact.

[0164] It should be understood that numerous further modifications and arrangements are possible to various aspects of the described embodiments. Therefore, the described aspects are intended to cover all such changes, modifications, and variations that fall within the spirit and scope of the appended claims.

[0165] In this specification and its appended claims, unless the context otherwise requires, the word “comprising” and its variations (e.g., “including” and “containing”) should be understood to imply inclusion of the said integer, step or set of integers or steps, but not to exclude any other integer, step or set of integers or steps.

[0166] In this specification and its appended claims, unless the context otherwise requires, the phrase "consistent with..." and variations thereof (e.g., "composed of...") should be understood to indicate that the listed elements are essential, i.e., necessary elements of the invention. This phrase allows for the presence of other unlisted elements that do not substantially affect the features of the invention, but excludes other unspecified elements that would affect the essential and novel features of the defined method.

[0167] Any prior publications (or information derived therefrom) or any known matters mentioned in this specification shall not constitute, nor should be construed as, an acknowledgment or endorsement, or any implication in any way that such prior publications (or information derived therefrom) or known matters constitute part of the general knowledge in the field to which this specification pertains.

Claims

1. A method for producing bacterial cellulose, comprising: Approximately 20 L to approximately 50 L of the starting culture was cultured together with at least approximately 200 L of the production medium to form a culture for the formation of bacterial cellulose. The starting culture contains *Coprinus cocovenenans* (cocovenenans) Komagataeibacter nataicola ), Reuteria paniculata ( Komagataeibacter rhaeticus ), Komatobacterium xylinum ( Komagataeibacter xylinus ) and Intercalozoa ( Komagataeibacter intermedius At least one strain of Komagataeibacter; The production culture medium contains waste yeast liquid that has not been heat-treated or chemically treated; The step of culturing the culture is carried out under static conditions in a 1 L to 2 L fermentation tray.

2. The method according to claim 1, wherein, The starting culture also contains *Glucosibirica* strains selected from the following: *Glucosibirica sacchariformis* (glucosibirica fermentans) G. sucrofermentans ), Staphylococcus aureus ( G. hansenii ), European Staphylococcus aureus ( G. europaeus ), Staphylococcus entanediol ( G. entanii ), Staphylococcus sucraniosus ( G. saccharivorans ), Staphylococcus aureus ( G. swingsii ), docile glucosamine ( G. oboediens ), liquefied glucosamine ( G. liquefaciens ), glucosidobacterium sugarcane ( G. sacchari ), nitrogen-fixing glucosamine ( G. diazotrophicus ), Johannesburg glucosamine ( G. johannae ), nitrogen-trapping glucosamine ( G. azotocaptans ), Medellin Staphylococcus aureus ( G. medellinensis (or a combination thereof).

3. The method according to claim 1 or 2, wherein, The production culture medium comprises: a) Sugars of about 1% w / v to about 18% w / v relative to the production medium; b) Waste yeast broth, approximately 2% v / v to approximately 12% v / v, relative to the production culture medium; c) Disodium hydrogen phosphate (Na2HPO4) of about 0.1% w / v to about 1% w / v relative to the production medium; d) Citric acid, approximately 0.05% w / v to approximately 1% w / v, relative to the production medium; and e) Ethanol of about 0.5% v / v to about 2.5% v / v relative to the production medium; The waste yeast liquid originates from a brewery and / or the bioethanol fermentation process; The production culture medium is characterized in that it has a pH of about 4 to about 6 and a Brix value of about 3 to about 6.

4. The method according to any one of claims 1-3, wherein, The production medium also contains waste grain filtrate of about 5% v / v to about 50% v / v relative to the production medium; The method for forming the waste grain filtrate involves suspending waste grains in an aqueous medium at a ratio of approximately 1:3 to form sludge, and then filtering the sludge; and The waste grains are derived from breweries and / or bioethanol fermentation processes.

5. The method according to any one of claims 1-4, wherein, The culture is cultivated at about 20°C to about 40°C for about 2 days to about 20 days.

6. The method according to any one of claims 1 to 5, wherein, The starting culture is added to the production culture medium at a ratio of about 5% v / v to about 40% v / v relative to the production culture.

7. The method according to any one of claims 1 to 6, wherein, The method further includes the step of culturing a seed inoculum in a propagation medium and / or an activation medium to form the starting culture, wherein the seed inoculum comprises a bacterial film formed by at least one strain of *Streptococcus foetida*. The seed inoculum is added to the propagation medium and / or activation medium at a ratio of about 10% v / v to about 30% v / v relative to the starting culture.

8. The method according to claim 7, wherein, The process of forming the starting culture is carried out at about 20°C to about 40°C under static conditions for about 2 to about 20 days.

9. The method according to claim 7 or 8, wherein, The culture medium comprises: a) Sugars of approximately 3% w / v to approximately 20% w / v relative to the aforementioned culture medium; b) Waste yeast culture, approximately 2% v / v to approximately 12% v / v, relative to the aforementioned culture medium; c) Citric acid, approximately 0.05% w / v to approximately 1% w / v, relative to the propagation medium; and d) Ethanol, 0.5% v / v to 2.5% v / v relative to the propagation medium; The propagation culture medium is characterized in that the pH is from about 4 to about 6.

10. The method according to any one of claims 7 to 9, wherein, The method further includes the step of activating the at least one strain of *Streptococcus* in an activation culture medium to form the seed inoculum.

11. The method according to claim 10, wherein, The process of forming the seed inoculum is carried out at about 20°C to about 40°C under static conditions for about 2 to about 20 days.

12. The method according to any one of claims 7 to 11, wherein, The activation culture medium comprises: a) Sugars of approximately 1% w / v to approximately 20% w / v relative to the activated medium; b) Yeast extract powder, approximately 0.1% w / v to approximately 2% w / v, relative to the activated culture medium; c) Citric acid, approximately 0.05% w / v to approximately 1% w / v, relative to the activated medium; and d) Ethanol, 0.5% v / v to 2.5% v / v, relative to the activated medium; The activated culture medium is characterized in that its pH is from about 4 to about 6.

13. The method according to any one of claims 1 to 12, wherein, The step of culturing the culture includes forming a bacterial cellulose membrane and harvesting the bacterial cellulose membrane when the pH of the culture is about 2 to about 3.

14. The method according to any one of claims 1 to 13, wherein, The method further includes the steps of cleaning the bacterial cellulose membrane and compressing the bacterial cellulose membrane.

15. The method according to any one of claims 1 to 14, wherein, After harvesting the bacterial cellulose, the residual culture medium from the method is recycled to form another batch of bacterial cellulose.

16. The method according to any one of claims 1 to 15, characterized in that, The method is characterized in that the bacterial cellulose yield is greater than about 80%.

17. A bacterial cellulose prepared by the method according to any one of claims 1 to 16.

18. The bacterial cellulose according to claim 17, wherein, The bacterial cellulose is characterized by at least one of the following features: a) The thickness is approximately 1 cm to approximately 3 cm; b) Tensile strength is from about 100 MPa to about 200 MPa; c) The elongation at break is approximately 20% to approximately 50%.

19. The bacterial cellulose according to claim 17 or 18, wherein, The bacterial cellulose can be formed into foam, hydrogel, aerogel, cardboard, biofilm, film, sheet or membrane.

Images