Method for biomanufacturing bacterial cellulose filaments and bioreactor according to the method

Abstract

The invention relates to a method for biomanufacturing bacterial cellulose filaments wherein the culture medium (5) is poured into a bioreactor having a spiral structure (2), the gap (A) of which structure corresponds to the thickness of the desired filament (6). In the bioreactor, the culture medium (5) undergoes static fermentation that creates a bacterial cellulose matrix (4) as a secondary product that is deposited on the surface of the culture medium (5), forming a solid film that adapts to the spiral structure (2) of the bioreactor such that when it is extracted from same, it forms a bacterial cellulose filament (6) having a length identical to that of the spiral and a diameter identical to that of the gap (A).

Description

[0001]

[0002] PROCEDURE FOR THE BIOFABRICATION OF BACTERIAL CELLULOSE FILAMENTS AND BIOREACTOR ACCORDING TO THE PROCEDURE

[0003] TECHNICAL SECTOR

[0004] The present invention relates to processes for producing bacterial cellulose and, in particular, bacterial cellulose filaments from a kombucha tea substrate and to a bioreactor for carrying out said process.

[0005] BACKGROUND OF THE INVENTION

[0006] Bacterial cellulose is a biopolymer that has attracted significant scientific and technological interest in recent decades due to its exceptional properties, including high purity, excellent mechanical strength, high hydration capacity, and biocompatibility.

[0007] Bacterial cellulose is produced primarily by acetic acid bacteria of the genus Gluconacetobacter, particularly Gluconacetobacter xylinus. This bacterium ferments culture media containing carbon sources and produces bacterial cellulose in the form of a floating hydrogel at the air-culture medium interface.

[0008] In recent years, the state of the art has revealed several procedures for the production of bacterial cellulose, which generally involve the fermentation of glucose-based culture media by acetic acid bacteria under controlled conditions.

[0009] The conventional process for producing bacterial cellulose involves the static fermentation of bacteria in a culture medium containing a sugar source, nutrients, and water. During the fermentation process, the bacteria secrete cellulose, which is deposited on the surface of the culture medium to form a film or hydrogel. After fermentation, the film is collected and processed to remove impurities and water, leaving a pure cellulose film.

[0010] One of the preferred uses of bacterial cellulose is in filaments, which have a variety of potential applications in different fields thanks to their unique properties, such as high mechanical strength, biocompatibility, and ability to form porous three-dimensional structures. Examples of these applications include:

[0011] Composite Materials: Bacterial cellulose filaments can be used as reinforcement in composite materials, improving their mechanical strength and other properties. This could be relevant in fields such as automotive manufacturing, construction, and materials engineering.

[0012] Textiles: Bacterial cellulose filaments are used to make fabrics and textile fibers. These can have a variety of applications, from fashion to medical and technical textiles.

[0013] Biomedical Applications: Due to their biocompatibility, bacterial cellulose filaments can be used in a variety of biomedical applications, including tissue engineering, bandages, sutures, and drug delivery vehicles.

[0014] Electronics: Bacterial cellulose filaments have potential for use in flexible and portable electronics, thanks to their flexibility and strength.

[0015] Food: In the food industry, bacterial cellulose filaments can be used as thickening, stabilizing or emulsifying agents.

[0016] Paper and Packaging: Bacterial cellulose filaments can also be used in the manufacture of paper and packaging materials, providing a sustainable and biodegradable alternative to traditional packaging materials.

[0017] The transition from a film or hydrogel of pure bacterial cellulose to a bacterial cellulose filament is the subject of further processing or an intermediate step in the process of producing bacterial cellulose.

[0018] The main known methods for producing bacterial cellulose filaments are:

[0019] Extrusion and Drying Method: This is the most commonly used method. First, a bacterial cellulose biomaterial is produced by fermenting specific bacteria in a culture medium. This biomaterial is then processed to form a paste, which is extruded through a nozzle to form filaments. The filaments then undergo a drying process to remove water and harden the filaments. Another variant of this method is wet-spinning, which uses a chemical dissolution and coagulation process using acetone or methanol.

[0020] These methods have proven to have limitations, as the resulting filaments often have unsatisfactory mechanical strength and a lack of uniformity in terms of thickness and length.

[0021] Modified Fermentation Method: In this approach, fermentation conditions are altered to promote the in situ formation of bacterial cellulose filaments. This may involve agitation of the growth medium during fermentation, which induces the bacteria to form filaments rather than cellulose films or hydrogels. Once fermentation is complete, the filaments are collected and processed for use. This method can provide greater control over filament properties, but has disadvantages, as agitation can interfere with bacterial growth and cellulose production, and precise control of fermentation conditions is required to obtain filaments with the desired properties.

[0022] Electrospinning Method: This method involves using an electric field to produce high-quality bacterial cellulose filaments. In this process, a solution of bacterial cellulose is placed in a syringe, and a high voltage is applied between the syringe and a collector. The solution spreads toward the collector due to the force of the electric field, forming extremely fine, high-quality filaments as it dries. This method can produce filaments with a high degree of uniformity and superior mechanical properties, but it can be more complicated and expensive to implement than other methods.

[0023] In conclusion, although bacterial cellulose production is a well-developed field, the targeted production of bacterial cellulose filaments with consistent mechanical properties remains a challenge in the current state of the art, necessitating the development of new procedures that can directly produce bacterial cellulose filaments with the desired characteristics, avoiding the problems associated with current production methods.

[0024] EXPLANATION OF THE INVENTION

[0025] The present invention consists of a process for the biofabrication of bacterial cellulose filaments by which cellulose filaments are obtained with a mechanical strength and simplicity of production equivalent to that of a cellulose culture by static fermentation and with a uniformity in terms of thickness and length equivalent to the filaments obtained by electrospinning.

[0026] This procedure is essentially applicable to the production of cellulose filaments from any appropriate substrate, but it is optimized to produce cellulose filaments as a by-product of the Kombucha Tea production process, specifically by fermentation of kombucha, since it has been demonstrated that the cellulose obtained from this substrate has great mechanical and thermal capacities, a high index of crystallinity and biocompatibility.

[0027] Kombucha tea is a fermented beverage made from sugar-sweetened tea and fermented with a symbiotic colony of bacteria and yeast, known as a SCOBY (Symbiotic Culture of Bacteria and Yeast). The top of the bacterial colony develops into a solid film of bacterial cellulose, which can be harvested and used for various applications.

[0028] Specifically, the new procedure involves the following phases:

[0029] 1.- Preparing the culture medium: The first step is to create a nutrient-rich culture medium that supports bacterial growth and cellulose production. This can be done with a kombucha tea base containing black, green, or white tea, depending on the desired color and texture, or with a pure culture (K. xylinus). The culture medium is sterilized to kill any undesirable organisms that might compete with the bacteria of interest. It is then allowed to cool to room temperature, followed by the addition of sugar.

[0030] 2.- Bioreactor loading: Once the culture medium has cooled, it is poured into a spiral-structured bioreactor with a wall perpendicular to the bottom plane and with a pitch equivalent to the desired filament thickness. The wall height is at least twice the spiral pitch. Alternatively, the bioreactor may consist of a tank with a spiral-structured scaffold at the bottom, with the characteristics described above. The culture medium is poured uniformly until it reaches a level approximately half the wall height of the spiral bioreactor. 3.- Inoculation: After loading the culture medium into the bioreactor to the appropriate level, it is inoculated with the appropriate bacteria, such as a K. xylinus colony or SCOBY, depositing a gelatinous and somewhat viscous matrix on the surface of the culture medium containing the mixture of bacteria and yeasts that will statically ferment the culture medium.

[0031] 4.- Cultivation: Immediately after inoculating the culture medium with the bacterial colony, the spiral bioreactor is closed with a cover that allows some air circulation but prevents other particles from entering. In this way, the inoculated medium is incubated at a suitable temperature (around 25-30 degrees Celsius) for a controlled time, during which the bacteria and yeasts statically metabolize the sugars in the culture, producing acetic acid, gas, and a cellulose matrix as a by-product that is deposited on the surface of the culture medium, forming a solid film that will adapt to the spiral shape of the bioreactor.

[0032] 5.- Filament extraction: Once static fermentation is complete, a thick matrix of bacterial cellulose has formed on the surface of the culture. This cellulose matrix has adapted to the spiral structure of the bioreactor, so that when extracted, it forms a bacterial cellulose filament with a length equal to that of the spiral and a diameter equal to its pitch.

[0033] 6.- Purification and treatment: The bacterial cellulose filament extracted from the spiral bioreactor is purified to eliminate metabolic byproducts, culture medium residues, and dead bacteria. The filament may then undergo a treatment that modifies its mechanical characteristics, since in its pure form, bacterial cellulose is rigid and fragile.

[0034] 7.- Drying: After purification and treatment, the bacterial cellulose filament is completely or partially dried at room temperature or in a low-temperature oven. The drying method can affect the final properties of the cellulose, so it should be chosen based on the final application.

[0035] In this way, a bacterial cellulose filament is obtained with a mechanical strength equivalent to that of a cellulose culture by static fermentation and with a uniformity in terms of thickness and length equivalent to the filaments obtained by electrospinning. Another aspect of the invention relates to a bioreactor for the biofabrication of bacterial cellulose filaments, which is comprised of a spiral structure with a wall perpendicular to the plane of the bottom and with a pitch equivalent to the thickness of the desired filament, inside which the bacterial cellulose filament is generated.

[0036] The wall height of the spiral bioreactor is at least twice the spiral pitch and is sealed with a cover that allows air recirculation to facilitate static fermentation.

[0037] Alternatively, the bioreactor may consist of a tank with a spiral-structured scaffold at the bottom with the characteristics described above.

[0038] This bioreactor allows for simplified biofabrication of bacterial cellulose filaments by static fermentation, avoiding the need for agitation or the use of electric fields during fermentation, which modified the final mechanical characteristics of the cellulose filament obtained.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] To complement the description being made and in order to help better understand the characteristics of the invention, a set of drawings is attached as an integral part of said description, in which the following has been represented for illustrative and non-limiting purposes:

[0041] Figure 1.- Shows a view of a bioreactor according to the invention consisting of a tank at the bottom of which a scaffold with a spiral structure is provided.

[0042] Figure 2.- Shows the method of extraction of the bacterial cellulose filament from the interior of the bioreactor.

[0043] Figure 3.- Shows a detailed view of the culture medium in the bioreactor with the bacterial cellulose filament before being extracted.

[0044] PREFERRED EMBODIMENT OF THE INVENTION

[0045] Example 1 Mixed Culture Kombucha Tea (SCOBY)

[0046] 1.- Preparation of the culture medium: In this case, the culture medium consists of an infusion of 2 g of Kombucha green tea in 180 ml of distilled water to which 5 g of panela sugar is added.

[0047] 2.- Loading the bioreactor: The sweetened Kombucha green tea, once cooled to room temperature, is poured into a spiral bioreactor which in the illustrated example is made up of a bucket (1) inside which there is a spiral structure scaffold (2) of 100 mm diameter, 3000 mm length, 15 mm wall height (P) and 2 mm pitch (A).

[0048] 3.- Inoculation: After pouring the culture medium into the bioreactor, it is inoculated with 20 ml of SCOBY culture that is deposited on the surface of the culture medium.

[0049] 4.- Cultivation: After inoculating the culture medium with the SCOBY colony, the spiral bioreactor is closed with a cover (3) and incubated at a temperature of 28 °C for 21 days.

[0050] 5.- Extraction of the filament: After 21 days of static fermentation, a matrix of bacterial cellulose (4) has been created on the surface of the culture medium (5) which, thanks to the structure of the spiral bioreactor, has the structure of a filament (6) of 3000 mm long and 2 mm in diameter, which is extracted by traction from the spiral bioreactor.

[0051] 6.- Purification and treatment: The bacterial cellulose filament extracted from the spiral bioreactor is placed in a shaken incubator at 19.5°C and 50 rpm containing a 20% sodium hydroxide (NaOH) solution for 24 hours to purify and separate any remaining culture medium residues that may have adhered. It is then placed in a shaken incubator at 19.5°C and 50 rpm containing a 10% glycerol solution for 24 hours to make the filament more flexible and improve its mechanical properties, since pure bacterial cellulose is rigid and fragile. At the same time, bacterial cellulose filaments treated with glycerol show greater stability against environmental factors such as humidity and temperature changes. Glycerol acts as a plasticizing agent, reducing the rigidity of the cellulose and helping to prevent its decomposition or degradation. This prolongs its useful life and improves its resistance to moisture.

[0052] 7.- Drying: After treatment with glycerol, the bacterial cellulose filament with optimal flexibility and stability characteristics against environmental factors is completely dried on waxed paper at room temperature, to obtain a flexible and durable bacterial cellulose filament, or it is partially dried and wet-wound to be able to weave with the wet filament.

[0053] Example 2 Pure culture (K.xylinus)

[0054] 1.- Preparation of the culture medium: In this case, the culture medium consists of a mixture of 0.4 g of Chitosan and 4 g of Peptone diluted in 200 ml of distilled water to which 4 g of d-glucose is added.

[0055] 2.- Loading the bioreactor: This culture medium at room temperature is poured into a spiral structure bioreactor with a diameter of 100 mm, a length of 3000 mm, a wall height (P) of 15 mm and a passage (A) of 5 mm.

[0056] 3.- Inoculation: After pouring the culture medium into the bioreactor, it is inoculated with 2 g of yeast and a colony of Komagataeibacter xylinus that is deposited on the surface of the culture medium.

[0057] 4.- Cultivation: After inoculating the culture medium with Komagataeibacter xylinus, the spiral bioreactor is closed with a cover and incubated at a temperature of 28°C for 6 days.

[0058] 5.- Extraction of the filament: After 6 days of static fermentation, a matrix of bacterial cellulose (4) has been created on the surface of the culture medium (5) which, thanks to the structure of the spiral bioreactor, has the structure of a filament (6) of 3000 mm long and 5 mm in diameter, which is extracted from the bioreactor.

[0059] 6.- Purification and treatment: The bacterial cellulose filament (6) extracted from the spiral bioreactor is introduced into a shaken incubator at 19.5 °C and 50 rpm containing a 20% sodium hydroxide (NaOH) solution for 24 hours to purify and separate any remains of the culture medium that may have adhered. It is then introduced into a shaken incubator at 19.5 °C and 50 rpm containing a 10% glycerol solution for 24 hours to make the filament more flexible and improve its mechanical properties, since bacterial cellulose in its pure form is rigid and fragile. At the same time, bacterial cellulose filaments treated with glycerol show greater stability against environmental factors, such as humidity and temperature changes. Glycerol acts as a plasticizing agent, reducing the rigidity of the cellulose and helping to prevent its decomposition or degradation. This prolongs its useful life and improves its resistance to humidity.

[0060] 7.- Drying: After treatment with glycerol, the bacterial cellulose filament with optimal flexibility and stability characteristics against environmental factors is completely dried on waxed paper at room temperature to obtain a flexible and durable bacterial cellulose filament, or it is partially dried and wet-wound to be able to weave with the wet filament.

Claims

CLAIMS 1.- Procedure for the biofabrication of bacterial cellulose filaments characterized in that the culture medium (5) is poured into a spiral structure bioreactor (2) closed by a cover (3) that allows a certain amount of air circulation, the passage (A) of which is equivalent to the thickness of the desired filament (6), and the wall of which is perpendicular to the plane of the bottom and has a height (P) at least twice the passage of the spiral (A), in which, after the culture medium (5) is inoculated, the bacteria and yeasts statically metabolize the sugars in the culture, producing acetic acid, gas and a bacterial cellulose matrix (4) as a secondary product that is deposited on the surface of the culture medium (5), forming a solid film that has adapted to the spiral structure (2) of the bioreactor, such that when it is extracted from it, it forms a bacterial cellulose filament (6) with a length equal to that of the spiral and a diameter equal to the passage (A). 2.- Bioreactor for the biofabrication of bacterial cellulose filaments according to the process of the invention, characterized in that it has a spiral structure (2) closed by a cover (3) that allows a certain amount of air circulation, the passage (A) of which corresponds to the thickness of the desired filament (6), and the wall of which is perpendicular to the plane of the bottom and has a height (P) at least twice as high as the passage of the spiral (A). 3.- Bioreactor for the biofabrication of bacterial cellulose filaments according to the second claim, characterized in that it is made up of a container (1) closed with a cover (3) that allows a certain amount of air circulation, at the bottom of which a scaffold with a spiral structure (2) has been provided, the pitch (A) of which corresponds to the thickness of the desired filament (6) and the wall of which is perpendicular to the plane of the bottom and has a height (P) at least twice as high as the pitch of the spiral (A).

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