Process for producing microbial cellulose from waste textiles

Abstract

The present invention relates to a process for producing microbial cellulose from a waste textile material, such as 100% cotton or a mixed cotton / polyester / elastic fiber waste textile, said waste textile material comprising cellulose, the process comprising the steps of: providing an amount of said waste textile material; pretreating the waste textile material with an aqueous solution comprising NaOH and urea to obtain a pretreated textile wherein the step of treating the waste textile with an aqueous solution comprising NaOH and urea is carried out at a temperature in the range of-25 DEG C to + 30 DEG C; enzymatically treating the pretreated fabric with a cellulase to convert the cellulose to glucose, thereby obtaining a mixture comprising glucose; preparing a culture medium comprising the mixture comprising glucose; inoculating the medium with a microorganism producing microbial cellulose; and incubating the microorganism producing microbial cellulose to obtain microbial cellulose.

Description

TECHNICAL FIELD

[0001] The present invention relates to a process for producing microbial cellulose from waste textile material. More specifically, the present invention relates to a process for producing microbial cellulose starting from waste textile material comprising cellulose fibers and optionally also synthetic fibers. BACKGROUND

[0002] Microbial cellulose is one of the most well-known and studied bacterial biopolymers. Microbial cellulose is an organic compound (same as plant cellulose) of formula (C6H 10 O5) n produced by certain types of microorganisms (e.g. bacteria) as an extracellular polymer. Although microbial cellulose (e.g. bacterial cellulose) has the same molecular formula as plant cellulose, its macromolecular properties are different from those of plant cellulose. In fact, microbial cellulose is generally more chemically pure (i.e. free from hemicellulose or lignin) than plant cellulose and has a higher water holding capacity, greater tensile strength, higher degree of polymerization and higher crystallinity. Due to these unique properties, microbial cellulose has been applied in several technical fields such as the food industry, the medical field (e.g. as wound dressing and for blood vessel regeneration) and paper restoration, among others. The use of microbial cellulose (e.g. bacterial cellulose) in the textile field is also known.

[0003] The recycling of old textiles (i.e. "waste textiles") has become a primary goal of the textile industry; new and efficient recycling processes are actively being researched. One of the goals is to recycle the materials contained in waste textiles (e.g. the fibers contained in waste textiles), for example, by converting the fibers of the waste textiles into a raw material suitable for different processes (e.g. processes for producing microbial cellulose).

[0004] Processes for obtaining bacterial cellulose starting from waste textile material are known in the art. In these processes it is known to pretreat fabrics of yarns containing cotton fibers with organic salts (e.g. N-methylmorpholine-N-oxide (NMMO) or 1-alkyl-3-methylimidazolium ionic liquids) prior to enzymatic hydrolysis.

[0005] For example, CN102080114 discloses a method for preparing bacterial cellulose from waste cotton fabric. The method according to CN102080114 comprises the step of pretreating the cotton fabric in an ionic liquid at a temperature of 90°C-130°C for a time of 15 minutes to 10 hours. Then, a cellulase enzyme is added to perform enzymatic hydrolysis to obtain an enzymatic hydrolysate. The enzymatic hydrolysate is used to prepare a culture medium for bacteria producing bacterial cellulose.

[0006] CN111269953 discloses a method for preparing bacterial cellulose from waste polyester fabric, which includes adding barium hydroxide solution and treating the fabric at a temperature of 120°C to 126°C for 90-100 minutes. After hydrolysis, sulfuric acid is added until no precipitate is formed, the pH of the solution is adjusted to neutral, and centrifugation is performed to obtain the supernatant, which is the polyester hydrolysis product. The polyester hydrolysis product is used as a carbon source in the culture medium for preparing bacteria that produce bacterial cellulose.

[0007] Currently available processes for obtaining microbial cellulose from waste textile materials have several drawbacks.

[0008] For example, currently feasible processes typically result in reduced efficiency of enzymatic conversion to glucose; the applicant has recognized that this reduction is due to the presence of dissolved cellulose in the liquid phase obtained after pretreatment, and / or due to at least partial degradation of the cellulose fibers before their conversion to glucose. For example, it is known from “Enzymatic saccharification of dissolution-pretreated waste cellulosic fabrics for bacterial cellulose production by Gluconacetobacter xylinus” CHIA-HUNG KUO ET AL, JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY, vol. 85, no. 10, pages 1346-1352, that treating fabrics with ionic liquids leads to cellulose dissolution. When textiles containing cellulose are treated with ionic liquids (such as NMMO or 1-butyl-3-methylimidazolium chloride ([BMIM]Cl)) or strong acids (such as phosphoric acid and its solutions), most of the cellulose dissolves, thus requiring a regeneration step to obtain cellulose in solid form before enzymatic treatment with cellulase. Therefore, cellulose solutions are unsuitable as starting materials for the enzymatic conversion of cellulase into glucose.

[0009] In addition to the issues mentioned above, currently feasible processes for waste textiles containing both cellulosic fibers (such as cotton, lyocell, and viscose) and non-cellulosic fibers (such as synthetic fibers, like polyester) result in at least partial degradation of the non-cellulosic materials (such as polyester fibers). This degradation of polyester fibers through polymer hydrolysis may impair the possibility of reusing polyester fibers by melting them to extrude new polyester fibers with the desired mechanical properties.

[0010] On the other hand, it is necessary to provide methods to increase the rate at which fabric cellulose fibers are enzymatically converted into glucose, and to reduce the overall cost of a cyclic recycling process that starts with cellulose fibers and ends with (recycled) cellulose fibers.

[0011] Furthermore, given the conditions of the process, currently feasible processes require a large amount of energy. Additionally, the reagents used in currently feasible processes (e.g., ionic liquids) require expensive chemical processes to recover and potentially reuse. Summary of the Invention

[0012] The purpose of this invention is to solve the above-mentioned problems and to provide a process for producing microbial cellulose from waste textiles in an efficient manner. In particular, the purpose of this invention is to provide a cellulose saccharification process that is efficient and easy to scale up for industrial application.

[0013] Another object of the present invention is to provide a process for producing microbial cellulose from waste textiles, suitable for use on waste textiles containing cellulose fibers and non-cellulose, synthetic fibers (particularly polyester fibers); the process provides a composition comprising or substantially free of cotton and cellulose fibers (e.g., polyester fibers). The process also reduces or helps prevent the depolymerization of polyester or other synthetic fibers present in textiles treated according to the present invention.

[0014] Another objective of this invention is to provide a process for producing microbial cellulose from waste textiles, which can reduce energy consumption.

[0015] These and other objectives are achieved by the present invention, which relates to the process according to claim 1, the enzymatic saccharification process of cellulosic fabrics according to claim 2, and the recycling process of blended textiles according to claim 19. Preferred embodiments of the invention are based on the dependent claims. Attached Figure Description

[0016] Figure 1 An embodiment of the process of the present invention is illustrated schematically.

[0017] Figure 2 SEM images of exemplary waste textile materials before and after pretreatment according to the process of the present invention are shown.

[0018] Figure 3A and Figure 3B Microscopic images of exemplary bacterial cellulose obtained according to the process of the present invention are shown at two magnifications.

[0019] Figure 4A and Figure 4BSEM images of 100% polyester fabric are shown, pretreated at room temperature using an aqueous solution comprising NaOH and urea according to the present invention. Figure 4A ), and after pretreatment at 50°C with an aqueous solution including NaOH and urea ( Figure 4B ).

[0020] Figure 5A , Figure 5B and Figure 5C The following are illustrations showing the pretreatment and enzymatic treatment according to the present invention (…). Figure 5A ) and afterwards ( Figure 5B and Figure 5C SEM images of exemplary waste textiles. Specifically, Figure 5B An exemplary waste textile material after pretreatment at 0°C and treatment with cellulase is shown as an embodiment of the process according to the present invention; Figure 5C An exemplary waste textile material, after pretreatment at room temperature and treatment with cellulase, is shown as an embodiment of the process according to the present invention. Detailed Implementation

[0021] like Figure 1 As shown, according to one aspect, the present invention relates to a process for producing microbial cellulose from waste textile materials, said waste textile materials comprising cellulose in the form of cellulose fibers, the process comprising the following steps:

[0022] a) Provide waste textile materials;

[0023] b) Pretreating the waste textile material with an aqueous solution containing an alkali (preferably NaOH) and an amide (preferably urea) to obtain a pretreated textile material, wherein the step of pretreating the waste textile material is carried out at a temperature in the range of -25°C to +30°C;

[0024] c) Treat the pretreated textile material with cellulase to convert the cellulose into glucose, thereby obtaining a mixture comprising a solid phase and a liquid phase;

[0025] d) Separate the liquid phase from the solid phase to obtain a liquid phase mixture containing glucose;

[0026] e) Using the liquid-phase mixture containing glucose to prepare a microbial culture, wherein the microbial culture comprises microorganisms that produce microbial cellulose;

[0027] f) Incubate the microbial culture to obtain microbial cellulose.

[0028] Advantageously, the present invention enables the efficient extraction of microbial cellulose from waste textile materials. Furthermore, the present invention enables the extraction of microbial cellulose from waste textile materials in an energy-efficient and cost-effective manner.

[0029] In a possible embodiment, the microbial cellulose obtained by the process of the present invention is used as a starting cellulose material in a viscose-like process that transforms the microbial cellulose into regenerated cellulose fibers.

[0030] In addition to cost-effective and affordable cellulose pretreatment solutions, the presence of NaOH (or other alkalis) and urea (or other amides, such as thiourea) offers advantages such as improved cellulose properties, which enhance subsequent steps in the microbial cellulose production process. More specifically, it has been found that the use of the aforementioned reagents at specific temperatures, particularly at room temperature, facilitates enzymatic degradation and increases the yield of glucose available after enzymatic saccharification. It has also been unexpectedly found that the conversion yield of cellulose to glucose is higher when the textile to be treated (i.e., the fabric) is shredded (or it typically undergoes a size reduction process) after the NaOH / urea pretreatment step; preferably, the textile is dried before shredding.

[0031] The first believed reason is that the amount of cellulose dissolved during the NaOH and urea pretreatment is small enough not to impair or significantly reduce the enzymatic activity of cellulase in subsequent steps. Furthermore, since only a very small amount of cellulose dissolves during the NaOH and urea pretreatment of the fabric, especially when the treated fabric area is large, no regeneration step is required before shredding; that is, the cellulose is precipitated as a solid by adding water to the cellulose solution after dissolution. A further believed reason is that the addition of urea captures free water and prevents hydrogen bonding interactions between cellulose chains, and the treatment reduces the crystallinity of cellulose while the change in the degree of polymerization is negligible.

[0032] Therefore, the present invention also relates to a process for enzymatic saccharification of waste textile materials, wherein the waste textile materials include cellulose fibers, and the process includes the following steps:

[0033] a) Provide waste textile materials;

[0034] b) Pretreating the waste textile material with an aqueous solution comprising NaOH and urea to obtain pretreated textile material, wherein the step of pretreating the waste textile material with an aqueous solution comprising NaOH and urea is carried out at a temperature in the range of -25°C to +30°C;

[0035] c) Treat the pretreated textile material with cellulase to convert the cellulose into glucose, thereby obtaining a mixture comprising a solid and a liquid phase.

[0036] Prior to step c), the pretreated textile obtained in step b) is shredded into multiple pieces of a size suitable for enzymatic saccharification.

[0037] In fact, the pretreatment used in this invention reduces the amount of enzyme required for the subsequent enzymatic conversion of cellulose fibers into glucose. That is, the enzyme amount is low enough that it is economically feasible to avoid recycling the enzyme after the enzymatic reaction. This effect allows the use of a glucose-containing solution as a basis for the production of microbial cellulose without the removal of cellulase: in fact, the cellulase is inactivated before the cellulose-producing microorganisms are added to the glucose + enzyme solution, providing both nitrogen and carbon sources for the production of biocellulose.

[0038] In the examples, the base is selected from sodium hydroxide (NaOH), potassium hydroxide (KOH), calcium hydroxide Ca(OH)2, lithium hydroxide (LiOH), and sodium carbonate (Na2CO3).

[0039] In the examples, the amide is selected from urea and thiourea. In a preferred embodiment of the pretreatment solution, the weight ratio of alkali / amide (i.e., NaOH / urea) is in the range of 1.0 / 1.0 to 1.0 / 2.0, preferably 1.0 / 1.5 to 1.0 / 2.0, more preferably 1.0 / 1.5 to 1.0 / 1.8; the preferred ratio is about 1.0 / 1.7.

[0040] The energy savings are achieved by using an aqueous solution comprising an alkali (preferably NaOH) and an amide (preferably urea) at a temperature ranging from -25°C to +30°C. Suitable temperatures are between -25°C and +25°C, preferably between -10°C and +25°C, more preferably between +10°C and +25°C, and most preferably at room temperature (i.e., within the range of 20°C ± 5°C). In practice, using an aqueous solution comprising NaOH and urea at the aforementioned temperatures (preferably within the range of +15°C to +25°C) yields a pretreated fabric in which the yarns swell (fibers do not dissolve) and voids are created between the cellulose fibers, making it easier for cellulase to be used. Simultaneously, the degradation and dissolution levels of the cellulose fibers are minimized. Another advantage of the process of this invention is that, in blended textiles (e.g., blended fabrics), the process minimizes potential damage to the polyester portion of the blended textile, i.e., minimizes the degradation of polyester filaments or fibers, so as to recycle and reuse the polyester material.

[0041] In this process, NaOH and urea solutions alter the structure of crystalline cellulose by converting cellulose I to cellulose II, much like the hydrolysis of amorphous regions of cellulose during mercerizing. Advantageously, it has been observed that cellulase can more efficiently convert cellulose to glucose when it has structure II compared to cellulose with structure I. Natural cotton cellulose was found to be predominantly cellulose I. After NaOH / urea pretreatment, the cellulose was converted to a predominantly cellulose II structure. As mentioned above, cellulose I is more difficult for cellulase to convert to glucose compared to cellulose II. In fact, cellulose I is more resistant to enzymatic hydrolysis. Cellulose I has a highly ordered structure, making it more difficult for cellulase to access and hydrolyze the cellulose chains. On the other hand, cellulose II has a more disordered structure, making it easier for cellulase to access and hydrolyze. Without being bound by specific scientific explanations, it has been observed that pretreatment with an aqueous solution of NaOH (or other alkali) and urea (or other amide) can convert cellulose I into cellulose II, thereby enhancing the accessibility of cellulase to cellulose and improving the efficiency of cellulose hydrolysis, while keeping the amount of dissolved cellulose to a minimum, similar to what happens during mercerizing.

[0042] Furthermore, treatment with NaOH and urea promotes the swelling and dissolution of the amorphous regions of cellulose, which allows for the extraction of impurities and increases the surface area and reactivity of the fibers, preparing them for enzymatic pretreatment. In other words, the pretreatment step of this invention increases the surface area of ​​cellulose fibers in the treated textiles (particularly in the yarn of the fabric), making them available for contact with cellulase, thereby significantly improving the efficiency of the enzymatic conversion of cellulose to glucose by treating the pretreated yarn or fabric with cellulase. Moreover, using an aqueous solution with a temperature range of -25°C to +30°C (preferably 0°C to room temperature), especially when the process is carried out at room temperature, can reduce energy consumption and overall cost.

[0043] In other words, the process according to the invention enables the extraction of microbial cellulose from waste textile materials at a lower cost than known processes without compromising the effectiveness of the process. Advantageously, the process of the invention significantly reduces the degradation and / or dissolution of cellulose fibers during the pretreatment step, thereby achieving efficient conversion of cellulose to glucose. Advantageously, the process of the invention does not include (i.e., does not require) a step of regenerating solid cellulose after pretreatment with an aqueous solution comprising an alkali (preferably NaOH) and an amide (preferably urea).

[0044] Furthermore, the process of the present invention is particularly suitable when waste textiles contain both natural fibers (e.g., cotton) and synthetic fibers (e.g., polyester). In fact, using an aqueous solution comprising an alkali (preferably NaOH) and an amide (preferably urea) at a temperature ranging from -25°C to +30°C, preferably at room temperature (20°C ± 5°C), it is possible to obtain a pretreated fabric with less polyester (or other synthetic) fiber degradation, which can be recycled and reused. Advantageously, the process of the present invention can efficiently and potentially completely convert cellulose into glucose, and high yields of microbial cellulose production are also achieved when the starting textile material is waste textile material containing both cellulose and non-cellulose materials.

[0045] According to an embodiment, waste textile materials include cellulose in the form of cellulose fibers and non-cellulose fibers.

[0046] In this embodiment, the cellulose fiber is a virgin fiber or recycled fiber selected from plant fibers, man-made cellulose fibers, recycled plant fibers, recycled man-made cellulose fibers, and mixtures thereof. According to this embodiment, the plant fiber may be selected from cotton fiber, hemp fiber, flax fiber, jute fiber, coconut fiber, sisal fiber, abaca (Manila hemp) fiber, kapok fiber, ramie fiber, kenaf fiber, agave fiber, henna fiber, lotus fiber, nettle fiber, bagasse fiber, pineapple leaf fiber, banana tree trunk fiber, rice straw fiber, and mixtures thereof. According to this embodiment, the man-made cellulose fiber may be selected from viscose fiber, lyocell fiber, modal fiber, acetate fiber, cuprammonium fiber, urethane fiber, and mixtures thereof. In this embodiment, the cellulose fiber is selected from cotton fiber, viscose fiber, and mixtures thereof.

[0047] In the embodiments, the non-cellulose fibers are selected from synthetic fibers, biosynthetic fibers, recycled synthetic fibers, recycled biosynthetic fibers, and mixtures thereof.

[0048] According to embodiments, the synthetic fibers are virgin fibers or recycled fibers selected from polyester fibers, polyamide fibers, elastic fibers, polypropylene (PP) fibers, acrylic fibers, polyurethane fibers, CO2-based fibers, and mixtures thereof. For example, polyester fibers may be polyethylene terephthalate (PET) fibers, polypropylene terephthalate (PTT) fibers, polybutylene terephthalate (PBT) fibers, polybutylene succinate (PBS) fibers, polybutylene adipate terephthalate (PBAT) fibers, polylactic acid (PLA) fibers, or polyhydroxyalkanoate (PHA) fibers. Recycled fibers can also be obtained by making fibers from recycled polymers; for example, recycled polyester fibers can be obtained from recycled polyester polymers obtained from waste textiles (e.g., from cotton-polyester fabrics).

[0049] Biosynthetic fibers can be selected from fossil-based fibers, bio-based fibers, and mixtures thereof. Fossil-based biosynthetic fibers can be selected from polylactic acid (PLA) fibers, polyhydroxyalkanoate (PHA) fibers, polyurethane (PU) fibers, polypropylene terephthalate (PTT) fibers, polybutylene succinate (PBS) fibers, polybutylene adipate (PBAT) fibers, and mixtures thereof. According to examples, bio-based biosynthetic fibers can be selected from bio-based polylactic acid (Bio-PLA) fibers, bio-based polyhydroxyalkanoate (Bio-PHA) fibers, bio-based polyurethane (Bio-PU) fibers, bio-based polypropylene terephthalate (Bio-PTT) fibers, bio-based polybutylene succinate (Bio-PBS) fibers, bio-based polybutylene adipate (Bio-PBAT) fibers, and mixtures thereof. According to the embodiments, the recycled synthetic fibers may be selected from recycled polyethylene terephthalate (rPET) fibers, recycled polyamide (nylon) fibers, recycled elastic fibers, recycled polypropylene (PP) fibers, recycled acrylic fibers, recycled polyurethane (PU) fibers, recycled polypropylene terephthalate (PTT) fibers, polybutylene succinate (PBS) fibers, and mixtures thereof.

[0050] According to embodiments, the waste textile material is selected from clothing, fabrics, or yarns, and the process is typically carried out on fabrics and clothing. According to embodiments, the waste textile material is clothing. For example, the fabric or clothing may be cotton fabric or clothing (i.e., 100% cotton fabric or clothing), or may include a combination of cotton and / or viscose fibers, possibly also including polyester and / or elastic fibers. In embodiments, the waste textile is waste textile containing viscose fibers (or cotton, or viscose and cotton), polyester, and elastic fibers. According to embodiments, the waste textile material is a mixed textile material containing polyester fibers or silk and cellulose fibers.

[0051] According to an embodiment, the waste textile material can be washed before the step of pretreating the waste textile material with an aqueous solution including NaOH and urea.

[0052] In an embodiment, prior to the step of pretreating the waste textile material with an aqueous solution comprising NaOH and urea, or optionally after washing, non-textile elements optionally present in the waste textile material, such as labels (e.g., made of leather or polyurethane), buttons, and zippers (e.g., made of aluminum, brass, or plastic), may be removed. The removal of non-textile elements may be performed manually and / or automatically using techniques and / or machines known per se in the art.

[0053] According to embodiments, the process may further include a step of sorting the waste textile materials before or after the step of pretreating the waste textile materials with an aqueous solution comprising NaOH and urea. Preferably, the process further includes a step of sorting the waste textile materials before pretreating them with an aqueous solution comprising NaOH and urea. In embodiments, the sorting is based on the color and type of dyes present in the waste textile materials (e.g., indigo, sulfur dyes, or black reactive dyes), the textile (e.g., fabric) composition, the weight percentage of cellulose, non-cellulose components, or combinations thereof. For example, near-infrared (NIR) sorting of textiles (e.g., fabrics and / or clothing) based on color and / or textile (e.g., fabrics and / or clothing) composition is known in the art.

[0054] According to an embodiment, the waste textile material contains dyes. When dyes are present, the process of the present invention preferably includes a step of removing at least a portion of the dyes from the waste textile material before the step of treating the textile with cellulase, and preferably before the step of pretreating the waste textile with the aqueous solution comprising NaOH and urea. Advantageously, when the dyes are removed from the waste textile material before the enzymatic step of using cellulase, a particularly efficient conversion of cellulose to glucose can be obtained.

[0055] According to embodiments, dyes can be removed using techniques known in the art. According to embodiments, dyes can be removed from waste textile materials by methods selected from ozone washing, washing with a reducing solution, dye extraction (e.g., using a Soxhlet extractor), and combinations thereof.

[0056] According to embodiments, dyes can be removed from waste textile materials by treating them with a reducing solution containing NaOH and sodium dithionite (Na₂S₂O₄) preferably at a temperature of 60°C to 80°C (e.g., 70°C), preferably in a closed washing machine. According to embodiments, the concentration of NaOH in the reducing solution can be from 1 g / L to 60 g / L, preferably from 10 g / L to 30 g / L, more preferably from 15 g / L to 22.5 g / L. The concentration of sodium dithionite (Na₂S₂O₄) in the reducing solution can be from 1 g / L to 30 g / L, preferably from 5 g / L to 15 g / L, more preferably from 7.5 g / L to 10 g / L.

[0057] According to an embodiment, the process further includes a step of reducing the size of the waste textile material, preferably performed after the step of pretreating the waste textile material with the aqueous solution comprising NaOH and urea. For example, the pretreated textile material can be shredded or torn, optionally after at least some of the dye has been removed, and before the step of treating the pretreated textile material with cellulase. In this case, it is advantageous to increase the contact area between the pretreated textile material and the cellulase. In an embodiment, before step c), the pretreated textile material as in step b) is torn into multiple pieces of a size suitable for enzymatic saccharification. For example, before step c), the textile material obtained in step b) is torn into pieces with an average size of 0.5 mm. 2 (0.005 cm) 2 ) to 2500 mm 2 (25 cm) 2 (, preferably 1 mm) 2 (0.01 cm) 2 Up to 625 mm 2 (6.25cm) 2 ), more preferably 10 mm 2 (0.1 cm) 2 ) to 400 mm 2 (4 cm) 2 ), for example, 100 mm 2 Multiple films.

[0058] Furthermore, pre-treating waste textile materials before shredding reduces the amount of cellulose dissolved in the pre-treatment step, i.e., reduces the amount of cellulose dissolved during pre-treatment with an aqueous solution including NaOH and urea, without impairing the enzymatic conversion of cellulose to glucose. Additionally, when waste textile materials contain non-cellulose fibers (e.g., polyester fibers), shredding or tearing the textile materials after the pre-treatment step reduces the amount of non-cellulose material (e.g., polyester) that may be lost during shredding or tearing. Moreover, when waste textile materials contain non-cellulose fibers (e.g., polyester fibers), shredding or tearing the textile materials after the pre-treatment step reduces or avoids the potentially harmful effects of NaOH / urea solution on non-cellulose materials (e.g., polyester). Advantageously, shredding or tearing the textile materials after the pre-treatment step allows for the recycling and reuse of a larger amount of non-cellulose material (e.g., polyester fibers) compared to shredding or tearing the textile materials before the pre-treatment step with an aqueous solution including NaOH and urea, because some polyester particles will be lost with the NaOH / urea solution.

[0059] As is known in the art, shredding can be carried out mechanically. In an embodiment, waste textiles are transferred to an insulated container and liquid nitrogen is poured onto them. The resulting frozen waste textiles are then ground until small pieces between 1 mm and 5 mm are obtained. Preferably, the average size is 1 mm. The reduced-size particles are then treated according to the process steps of the invention (e.g., cellulase treatment). Advantageously, the fabric to be pretreated can be quite large, for example, a piece of fabric about 180 cm wide and 4-6 meters long. Similarly, garments from which non-woven elements have been removed can be pretreated only after a NaOH / urea pretreatment.

[0060] As previously stated, the step of pretreating waste textile materials with an aqueous solution comprising NaOH and urea results in improved time and efficiency for the enzymatic conversion of fabric cellulase to glucose. Without being bound by specific scientific explanations, it has been observed that pretreating waste textile materials with an aqueous solution comprising NaOH and urea at temperatures ranging from -25°C to +30°C, preferably from +10°C to +25°C, and more preferably from room temperature to +15°C to +25°C, causes the textile materials to swell, thereby enabling the efficient conversion of cellulose to glucose by cellulase. In other words, it has been observed that the pretreatment of the present invention causes the textile fibers to separate (e.g., the distance between fibers increases), an effect also known as swelling, allowing cellulase to effectively reach the cellulose chains, thereby achieving efficient conversion of cellulose to glucose.

[0061] According to embodiments, the aqueous solution comprising NaOH and urea includes NaOH in a content ranging from 1% to 15% w / v, preferably from 2% to 10% w / v, and more preferably from 3% to 9% w / v. For example, the aqueous solution comprising NaOH and urea may include NaOH in a content ranging from 6% to 8% w / v, such as 7% w / v.

[0062] According to embodiments, the aqueous solution comprising NaOH and urea includes a urea content ranging from 1% to 25% w / v, preferably from 3% to 20% w / v, and more preferably from 5% to 15% w / v. For example, the aqueous solution comprising NaOH and urea may include a urea content ranging from 11% w / v to 13% w / v, such as 12% w / v.

[0063] According to an embodiment, in the step of pretreating the waste textile material using the aqueous solution comprising NaOH and urea, the content of the waste textile material is in the range of 1% w / v to 20% w / v, preferably 2% w / v to 15% w / v, and more preferably 2.5% w / v to 10% w / v, relative to the volume of the aqueous solution comprising NaOH and urea. For example, the content of the waste textile material may be in the range of 3% w / v to 7% w / v, preferably 3.5% w / v to 6.5% w / v, and more preferably 4% w / v to 6% w / v, for example, 5% w / v, relative to the volume of the aqueous solution comprising NaOH and urea.

[0064] According to an embodiment, the step of pretreating the waste textile material with the aqueous solution comprising NaOH and urea is carried out in a temperature range of -15°C to +25°C, preferably at room temperature (+15°C to +25°C).

[0065] According to an embodiment, the duration of the step of pretreating the waste textile material with the aqueous solution comprising NaOH and urea is from 0.5 hours to 10 hours, preferably from 1 hour to 8 hours, and more preferably from 2 hours to 7 hours. For example, the duration of the step of pretreating the waste textile material with the aqueous solution comprising NaOH and urea can be from 4 hours to 6 hours (e.g., 5 hours).

[0066] For example, the pretreatment step of the waste textile material can be carried out using an aqueous solution comprising NaOH and urea at a temperature ranging from -15°C to +25°C, preferably at room temperature, for 1 to 8 hours, more preferably 2 to 7 hours. This aqueous solution comprises 3% to 9% w / v of NaOH, preferably 6% to 8% w / v, and 5% to 15% w / v of urea, preferably 11% to 13% w / v. In an embodiment, a pretreatment solution containing approximately 7% w / v of NaOH and 12% w / v of urea is used at room temperature for 2 hours to provide the desired fiber separation for cotton / polyester blended fabrics (5% w / v), for example, by fiber swelling.

[0067] According to an embodiment, the pretreated textiles obtained after a pretreatment step with an aqueous solution comprising NaOH and urea are washed. Washing can be performed, for example, with a weak acid solution (e.g., acetic acid solution) or with water. The water can be, for example, tap water, distilled water, reverse osmosis water, or mixtures thereof. Washing is preferably performed at a temperature in the range of 10°C to 30°C, preferably 15°C to 28°C (e.g., 25°C). According to an embodiment, after the washing step, the pH of the aqueous extract of the pretreated textiles is in the range of 6 to 8, preferably 6.5 to 7.5 (e.g., 7). The pH of the aqueous extract of the pretreated textiles is measured according to ISO 3071:2020 standard.

[0068] According to an embodiment, the textiles obtained after a pretreatment step with an aqueous solution comprising NaOH and urea, and optionally after washing, are dried. For example, drying is preferably carried out in an industrial dryer (e.g., a drum dryer or a conveyor belt dryer) known in the art itself, at a temperature ranging from 50°C to 100°C, preferably from 70°C to 90°C (e.g., 90°C). According to an embodiment, the textiles obtained after a pretreatment step with an aqueous solution comprising NaOH and urea, optionally after washing, and optionally after drying, are shredded or torn (e.g., cut into small pieces or fragments) to reduce the size of the pretreated textile material. For example, the textiles obtained after a pretreatment step with an aqueous solution comprising NaOH and urea are shredded or torn into pieces with an average size (area) of 0.5 mm. 2 (0.005 cm) 2 ) to 2500 mm 2 (25 cm) 2 (, preferably 1 mm) 2 (0.01 cm) 2 Up to 625 mm 2 (6.25 cm) 2 ), more preferably 10 mm 2 (0.1 cm) 2) to 400 mm 2 (4 cm) 2 ), for example, 100mm 2 (1 cm) 2 For example, multiple pieces (e.g., 1x1 cm).

[0069] According to embodiments, after the step of pretreating textiles with an aqueous solution comprising NaOH (or other alkali) and urea (or other amide), the aqueous solution comprising NaOH (or other alkali) and urea (or other amide) can be reused. According to embodiments, the aqueous solution comprising NaOH (or other alkali) and urea (or other amide) can be reused 1 to 20 times, preferably 5 to 15 times, more preferably 8 to 12 times (e.g., 10 times). In embodiments, if necessary, the concentrations of NaOH (or other alkali) and urea (or other amide) can be adjusted before reuse. For example, if necessary, the NaOH concentration can be adjusted to a range of 1% to 15% w / v, preferably 2% to 10% w / v, more preferably 3% to 9% w / v. For example, if necessary, the urea concentration can be adjusted to a range of 1% to 25% w / v, preferably 3% to 20% w / v, more preferably 5% to 15% w / v.

[0070] According to an embodiment, the step of pretreating the waste textile material with the aqueous solution comprising NaOH and urea is carried out in a reaction chamber; wherein the aqueous solution comprising NaOH and urea is provided to the waste textile material in the reaction chamber and circulated through the waste textile material. Preferably, the waste textile material is in a static state, and the solution comprising NaOH and urea is circulated through it to provide the pretreated textile material. When the aqueous solution comprising NaOH and urea is circulated through the waste textile material, the duration of pretreatment can be particularly short.

[0071] The term "circulation" refers to an aqueous solution comprising NaOH (or other alkali) and urea (or other amide) intended to define a solution subjected to a forced flow through the textile and through a reaction chamber holding the textile. This flow is preferably generated by a pump that supplies the solution into the reaction chamber, through the textile, and through the chamber. The solution leaves the reaction chamber and is subsequently supplied back to it for further circulation through the textile and through the chamber. According to an embodiment, when washing pretreated textiles obtained after a pretreatment step with an aqueous solution comprising NaOH and urea, the washing solution may also be circulated through the textile several times, similar to the solution comprising NaOH and urea. In an embodiment, the facility for performing the step of pretreating the waste textile material with the aqueous solution comprising NaOH and urea includes a fluid loop that typically includes an inlet and an outlet to the reaction chamber. Preferably, in this embodiment, the bath ratio (i.e., the ratio of fabric weight to solution volume) is 5% w / v relative to the volume of the aqueous solution comprising NaOH and urea.

[0072] The terms "stationary" or "static" state are intended to define a state where textiles are contained within a reaction chamber and held there as an aqueous solution (or washing solution) comprising NaOH and urea is supplied through the textiles. The textiles fill the chamber so that they do not move with the solution (i.e., they do not undergo displacement under the influence of the solution flow). In embodiments, the textiles are compressed within the chamber.

[0073] According to one aspect, the process of the present invention further includes the step of treating the textile material with cellulase to convert the cellulose present in the textile material into glucose, the textile material being obtained by pretreatment with an aqueous solution comprising NaOH and urea.

[0074] According to an embodiment, the pretreated textile material is treated with a solution including cellulase (i.e., cellulase enzyme).

[0075] According to an embodiment, the amount of pretreated textile material, by weight, relative to the volume of the solution including cellulase, is in the range of 1% w / v to 20% w / v, preferably 2% w / v to 15% w / v, and more preferably 2.5% w / v to 10% w / v. For example, 5 gr of pretreated waste textiles can be treated with 100 ml of a solution including cellulase: in this case, the amount of pretreated textile material is 5% w / v relative to the volume of the solution including cellulase.

[0076] According to embodiments, the amount of cellulase results in an enzyme activity of 1 FPU (filter paper unit) to 50 FPU per gram of cellulose, preferably 10 FPU to 30 FPU per gram of cellulose, and more preferably 15 FPU to 25 FPU per gram of cellulose. The enzyme activity of the cellulase can be measured according to methods known per se in the art. For example, the enzyme activity of the cellulase can be measured according to Adney B. and Baker J., "Measurement of cellulase activity", National Renewable Energy Laboratory, 1996; or according to Selig M., Weiss N., and Ji Y., "Enzymatic saccharification of lignocellulosic biomass", National Renewable Energy Laboratory, 2008. The cellulase suitable for use in this invention is known per se in the art. For example, suitable currently available products can be used: Novozymes Cellic® CTec3 HS, AB Enzyme – Flashzyme Plus 200, Novozymes Cellic® CTec2, and Novozymes Cellusoft® AB Conc.

[0077] According to embodiments, the enzymatic step of treating the pretreated textile material with cellulase is carried out at a temperature ranging from 30°C to 70°C, preferably from 40°C to 60°C, and more preferably from 45°C to 55°C. For example, the step of treating the pretreated textile material with cellulase can be carried out at a temperature of 50°C.

[0078] According to the embodiments, the time range for treating the pretreated textile material with cellulase is from 10 minutes to 144 hours, preferably from 30 minutes to 96 hours, more preferably from 12 hours to 72 hours, even more preferably from 18 hours to 48 hours, and even more preferably from 20 hours to 36 hours. For example, the step of treating the pretreated textile material with cellulase can be carried out for 24 hours.

[0079] According to an embodiment, the step of treating the pretreated textile material with cellulase can be carried out under stirring, with the stirring speed ranging from 5 rpm to 200 rpm, for example, 50 rpm to 100 rpm. Preferably, the enzymatic step of treating the pretreated textile material with cellulase is carried out without stirring (i.e., without agitation).

[0080] According to an embodiment, the step of treating the pretreated textile material with cellulase is carried out at a pH range of 3.0 to 6.0, preferably 4.0 to 5.0. For example, the step of treating the pretreated textile material with cellulase can be carried out at a pH of 5.0.

[0081] According to an embodiment, the pretreated textile material contains dye, and at least a portion of the dye is removed from the pretreated textile material. In other words, in an embodiment, at least a portion of the dye can be removed after the step of pretreating the waste textile material with an aqueous solution comprising NaOH and urea and before the step of treating the pretreated textile material with cellulase.

[0082] In a preferred embodiment, the pretreated textile material is not dyed. In other words, in this embodiment, the step of treating the pretreated textile material with cellulase is performed on the undyed pretreated textile material.

[0083] For example, the step of treating pretreated textile materials with cellulase can be carried out without stirring using an aqueous solution containing cellulase at a temperature ranging from 30°C to 70°C, preferably from 40°C to 60°C, more preferably from 45°C to 55°C, with the amount of cellulase being from 1 FPU (filter paper unit) to 50 FPU per gram of cellulose, preferably from 10 FPU to 30 FPU per gram of cellulose, more preferably from 15 FPU to 25 FPU per gram of cellulose, for a duration of 10 min to 144 hours, preferably from 30 min to 96 hours, more preferably from 12 hours to 72 hours, even more preferably from 18 hours to 48 hours, more preferably from 20 hours to 36 hours.

[0084] According to an embodiment, at the end of the step of treating the pretreated textile material with cellulase, a mixture comprising a solid phase and a liquid phase is obtained. For example, the solid phase may comprise non-cellulose fibers (e.g., polyester fibers) and optionally insoluble dyes (e.g., indigo dyes and sulfur dyes, if present) contained in the waste textile material, as well as optionally undigested cellulose fibers and material. For example, the liquid phase may comprise a cellulase solution for treating the pretreated textile material, glucose, and optionally soluble dyes (e.g., reactive black dyes, if present).

[0085] According to one aspect, the process of the present invention includes the step of separating the liquid phase from the solid phase to obtain a liquid mixture containing glucose.

[0086] According to an embodiment, when waste textile materials contain non-cellulose fibers, for example after the separation step, the non-cellulose fibers are recovered and reused from the solid phase of the mixture comprising a solid phase and a liquid phase.

[0087] According to embodiments, the separation of the liquid and solid phases can be carried out by gravity, preferably by waiting for the heavy material (polyester, elastic fiber) to settle to the bottom of the reactor, and then discharging the supernatant liquid phase. Alternatively, the separation of the liquid and solid phases can include filtration or multi-stage filtration (i.e., sequential cascade filtration), preferably through a sieve with a pore size in the range of 0.3 mm to 0.5 cm, by centrifugation, preferably at a speed in the range of 5000 rpm to 50000 rpm, or a combination of both. For example, in an embodiment, a sieve with a pore size preferably of 0.3 mm can be used to filter a mixture containing both solid and liquid phases. In an embodiment, the mixture containing both solid and liquid phases, or the liquid phase itself, can be centrifuged, for example, at a speed of 9000 rpm for 30 minutes. The supernatant obtained after centrifugation can be recycled and reused as a liquid mixture containing glucose. In other words, the liquid phase obtained after the separation step, such as after separating the liquid phase from the solid phase (e.g., by filtration or multi-stage filtration, or by centrifugation, or a combination thereof), can be recycled and reused as a liquid mixture containing glucose. A suitable separator is a clarifier, particularly for food components.

[0088] According to embodiments, cellulase can be recovered and reused from the liquid phase, and preferably can be reused repeatedly. Cellulase can be recovered and reused using techniques known in the art itself, such as dialysis or ultrafiltration. According to embodiments, the recovered and reused cellulase can be reused 1 to 10 times, preferably 2 to 8 times, more preferably 2 to 5 times (e.g., 3 times). In embodiments, the amount of cellulase can be adjusted if necessary. For example, if necessary, the amount of cellulase can be adjusted for reuse such that the enzyme activity is from 1 FPU (filter paper unit) to 50 FPU per gram of cellulose, preferably from 10 FPU to 30 FPU per gram of cellulose, more preferably from 15 FPU to 25 FPU per gram of cellulose.

[0089] According to an embodiment, cellulase can be recycled and reused to treat the solid phase obtained after the separation step and / or to treat one or more additional pretreated waste textile materials.

[0090] According to embodiments, the cellulase can be recycled and reused, for example, 1 to 10 times, preferably 2 to 8 times, more preferably 2 to 5 times, for example 3 times, to treat the solid phase obtained after the separation step. Reusing the cellulase to treat the solid phase after the separation step enables the digestion of cellulose fibers and materials that were not digested in previous treatments or in treatment with the cellulase, thereby increasing the yield of cellulose to glucose. As described above, enzymatic treatment of the solid phase yields an additional amount of a mixture comprising both solid and liquid phases.

[0091] According to embodiments, cellulase can be recycled and reused, for example, 1 to 10 times, preferably 2 to 8 times, more preferably 2 to 5 times (e.g., 3 times), to treat one or more additional pretreated waste textile materials.

[0092] According to an embodiment, preferably after the step of separating the liquid phase from the solid phase, at least a portion of the dye can be removed and / or recycled from the solid phase or from the liquid phase of the mixture comprising the solid and liquid phases. For example, if the dye is to be removed and / or recycled from the liquid phase, it can be removed from the liquid phase by passing the liquid phase through an activated carbon column.

[0093] According to one aspect, the process of the present invention includes the step of preparing a microbial culture using a mixture containing glucose, preferably obtained after a separation step, wherein the microbial culture comprises microorganisms that produce microbial cellulose.

[0094] According to an embodiment, the step of preparing a microbial culture using a glucose-containing mixture may optionally include a step of concentrating the glucose-containing mixture to increase the glucose concentration. Advantageously, by concentrating the glucose-containing mixture, the volume of the mixture is reduced, thereby making the handling and optionally storage of the mixture easier. According to an embodiment, the concentration step may be carried out using a heat exchanger, a rotary evaporator, a vacuum evaporator, or a combination thereof. For example, the glucose concentration in the glucose-containing mixture may be increased by treating the glucose-containing mixture in a heat exchanger (e.g., at 100°C for 1 to 12 hours, optionally, depending on the volume of the glucose-containing mixture to be concentrated and / or the final glucose concentration to be obtained).

[0095] According to an example, the glucose content in a mixture containing glucose can be measured using the 3,5-dinitrosalicylic acid (DNS) method. The DNS method is a method known per se in the art. The DNS method is disclosed, for example, in Ghose, TK, 1987, “Measurement of Cellulase Activities”, Pure & Applied Chemistry 59:257-268; and Miller GL, 1959, “Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar”, Analytical Chemistry 31:426-428.

[0096] In this embodiment, the percentage of cellulose converted to glucose was determined. Preferably, the percentage of cellulose converted to glucose was calculated using the following formula:

[0097]

[0098] Wherein: "Glucose released from enzyme solution" is determined using the DNS method; "Reaction volume" is the volume of cellulase-containing solution used to treat pretreated textiles; "Base weight" is the initial weight of waste textile material; "Cellulose content" is the cellulose content in waste textile material, expressed as a percentage of the initial weight of waste textile material, and "0.9" is a correction factor for water molecules added during cellulose hydrolysis.

[0099] According to an embodiment, the step of preparing a microbial culture using the glucose-containing mixture includes adjusting the glucose concentration in the glucose-containing mixture to a concentration of 10 g / L to 30 g / L, preferably 15 g / L to 25 g / L. According to an embodiment, the step of adjusting the glucose concentration in the glucose-containing mixture can be performed by concentrating the glucose-containing mixture, by diluting the glucose-containing mixture, or a combination of both. For example, the glucose-containing mixture can be concentrated, and after measuring the amount of glucose in the concentrated glucose-containing mixture, it can be diluted to obtain the desired glucose concentration, for example, 10 g / L to 30 g / L, preferably 15 g / L to 25 g / L. In an embodiment, the unconcentrated glucose-containing mixture can be diluted to obtain the desired concentration. In an embodiment, if cellulase is removed from the liquid phase obtained after the separation step by means of dialysis, a reduced glucose concentration in the glucose-containing mixture is obtained; in this case, the glucose concentration in the mixture can be adjusted to obtain the desired glucose concentration, for example, 10 g / L to 30 g / L, preferably 15 g / L to 25 g / L.

[0100] According to an embodiment, the mixture containing glucose is preferably sterilized in an autoclave. Advantageously, any enzymes present in the mixture are inactivated and can be used as an amino acid source for microorganisms producing microbial cellulose. In an embodiment, cellulase is not removed from the liquid phase obtained after the separation step. In an embodiment, the concentration of amino acids in the mixture can be adjusted if necessary. For example, the concentration of amino acids in the mixture can be adjusted by adding peptone to the mixture. For example, if the enzyme is recycled and reused to treat additional pretreated textiles, additional nitrogen and carbon sources (e.g., peptone) can be added. In practice, enzymatic treatment of each additional textile results in more glucose in the mixture, leading to insufficient amounts of amino acids in the glucose-containing mixture. In an embodiment, if the enzyme is recycled and reused to treat other pretreated textiles, cellulase is not removed from the liquid phase obtained from the separation step performed after the enzymatic treatment of the last textile. In embodiments, if the cellulase is recycled and reused to treat the solid phase obtained after the separation step (i.e., a second enzymatic treatment on the solid phase obtained from a single piece of starting waste textile material), it may be unnecessary to add additional nitrogen and carbon sources (e.g., peptone). In embodiments, if the cellulase is recycled and reused to treat the solid phase obtained after the separation step (i.e., a second enzymatic treatment on the solid phase obtained from a single piece of starting waste textile material), the cellulase is not removed from the liquid phase obtained in the final separation step following the final enzymatic treatment.

[0101] According to one aspect, a mixture containing glucose, preferably with a glucose concentration of 10 g / L to 30 g / L, preferably 15 g / L to 25 g / L (e.g., 20 g / L), can be used as a “waste textile glucose” (WTG) medium for preparing microbial cultures.

[0102] According to an embodiment, the step of preparing a microbial culture using a mixture containing glucose includes mixing the mixture containing glucose with a starter microbial culture containing microorganisms that produce microbial cellulose.

[0103] According to an embodiment, the amount of the mixture containing glucose in the microbial culture is 99% to 80% by weight, preferably 95% to 85% by weight, of the weight of the microbial culture.

[0104] According to an embodiment, the amount of the microbial culture comprising microorganisms that produce microbial cellulose in the microbial culture is 1% to 20% by weight, preferably 5% to 15% by weight, of the weight of the microbial culture.

[0105] As used herein, the term "starter culture" refers to a microbial culture typically used in small quantities to inoculate a batch of culture medium for fermentation. A starter culture is usually the microbial culture that actually undergoes fermentation, which is then added to the culture medium to initiate the fermentation process.

[0106] According to an embodiment, the step of preparing a microbial culture using the mixture containing glucose includes adding at least one additive to the mixture containing glucose, wherein the additive is preferably selected from titanium dioxybis(ammonium lactate)(IV) (TiBALDH), glycerol, ascorbic acid, ethephon (2-chloroethylphosphonic acid), and mixtures thereof.

[0107] Advantageously, when one or more additives are used, the amount of microbial cellulose (e.g., bacterial cellulose) obtained is increased relative to the amount obtained without the use of additives.

[0108] According to the embodiments, the additive is titanium dihydroxybis(ammonium lactate) (IV) (TiBALDH), preferably in an amount of 0.0005% to 1% (w / v), more preferably 0.0025% to 0.4% (w / v), even more preferably 0.05% to 0.3% (w / v), for example 0.25% (w / v).

[0109] According to the embodiments, the additive is glycerol, preferably in an amount of 0.1% to 10% (w / v), more preferably 0.5% to 2% (w / v), even more preferably 0.75% to 1.5% (w / v), for example 1% (w / v).

[0110] According to the embodiments, the additive is ascorbic acid, preferably in an amount of 0.05% to 10% (w / v), more preferably 0.25% to 2% (w / v), even more preferably 0.3% to 1% (w / v), for example 0.5% (w / v).

[0111] According to the examples, the additive is ethephon (2-chloroethylphosphoric acid), preferably in an amount of 0.001 mM to 10 mM, more preferably 0.005 mM to 2 mM, and even more preferably 0.01 mM to 1 mM, for example 0.01 mM.

[0112] According to an embodiment, the additive is a mixture of glycerol and titanium di(lactic acid)bis(lactic acid)IV (TiBALDH), wherein the glycerol is preferably added to the mixture containing glucose in an amount of 0.1% to 10% (w / v), more preferably between 0.5% and 2% (w / v), and even more preferably between 0.5% and 2% (w / v), and wherein the titanium di(lactic acid)IV (TiBALDH) is preferably added to the mixture containing glucose in an amount of 0.0005% to 1% (w / v), more preferably between 0.0025% and 0.4% (w / v), and even more preferably between 0.25% (w / v).

[0113] As used herein, the term "microbe" refers to organisms too small to be seen with the naked eye. As used herein, the term "microbe" encompasses both ungenetically modified (i.e., wild-type) and genetically modified microbes.

[0114] According to embodiments, the microorganisms producing microbial cellulose are selected from bacteria, algae, yeast, and mixtures thereof. According to embodiments, the microorganisms producing microbial cellulose may be genetically modified microorganisms. In a preferred embodiment, the microorganisms producing microbial cellulose are bacteria.

[0115] In the embodiments, the microbial cellulose may be selected from microbial cellulose produced by bacteria (i.e., bacterial cellulose), microbial cellulose produced by algae, microbial cellulose produced by yeast, and mixtures thereof. According to the embodiments, the microbial cellulose may be selected from microbial cellulose produced by bacteria (i.e., bacterial cellulose), microbial cellulose produced by algae, and mixtures thereof. Preferably, the microbial cellulose is bacterial cellulose.

[0116] According to the embodiments, the bacteria used to produce microbial cellulose are selected from *Glucosibibrio*, transgenic *Glucosibrio*, *Columella*, transgenic *Columella*, *Aerobiculbium*, transgenic *Aerobiculbium*, *Acetobacter*, transgenic *Acetobacter*, *Achromobacter*, transgenic *Achromobacter*, *Agrobacterium*, transgenic *Agrobacterium*, *Azotobacter*, transgenic *Azotobacter*, *Dinospermia*, transgenic *Dinospermia*, *Salmonella*, transgenic *Salmonella*, *Alcaligenes*, transgenic *Alcaligenes*, *Pseudomonas*, transgenic *Pseudomonas*, *Rhizobium*, transgenic *Rhizobium*, *Dinospermia*, transgenic *Dinospermia*, transgenic *Enterobacter*, *Escherichia*, transgenic *Escherichia*, *Bacillus*, transgenic *Bacillus*, *Klebsiella*, transgenic *Klebsiella*, and mixtures thereof. According to a preferred embodiment, the bacteria producing microbial cellulose are selected from *Gluconacetobacter hansenii*, *Gluconacetobacter xylinus*, *Komagateibacter xylinus*, and mixtures thereof. According to a preferred embodiment, the bacteria producing bacterial cellulose are selected from *Gluconacetobacter hansenii* ATCC 53582, *Gluconacetobacter xylinus* ATCC 23770, *Komagateibacter xylinus* DSM46604, and mixtures thereof. For example, the bacteria producing bacterial cellulose may be *Gluconacetobacter hansenii* ATCC 53582.

[0117] According to embodiments, the algae used for producing microbial cellulose are selected from the following phyla: Phaeophyta, Chlorella, Rhodophyta, Ochrophyta, Ascopyllum nodosum, Chlorella vulgaris, Nannochloropsis gaditana, Ulva prolifera, Ulva pertusa, Cladophora glomerata, Valonia ventricosa, and mixtures thereof. According to embodiments, the algae used for producing microbial cellulose are selected from the following phyla: Ascopyllum nodosum, Chlorella vulgaris, Nannochloropsis gaditana, Ulva prolifera, Ulva pertusa, Cladophora glomerata, Valonia ventricosa, and mixtures thereof. It is known in the art that heterotrophic and fascicial algae require glucose as a carbon source under dark conditions.

[0118] Yeasts that produce cellulose are known, for example, from Jasme, Nurshafqah et al. (2022) First report of biocellulose production by an indigenous yeast, Pichia kudriavzevii USM-YBP2. Green Processing and Synthesis. Vol 11 issue 1.

[0119] According to one aspect, the process of the present invention includes the step of incubating a microbial culture to obtain microbial cellulose. In an embodiment, a bacterial culture is incubated to obtain bacterial cellulose.

[0120] According to an embodiment, the incubation step is carried out at a temperature ranging from 15°C to 35°C, preferably from 20°C to 30°C. For example, the incubation step may be carried out at a temperature of 25°C to 30°C (e.g., 28°C).

[0121] According to an embodiment, the duration of the incubation step can be in the range of 1 to 30 days, preferably 5 to 20 days, and more preferably 10 to 16 days. For example, the duration of the incubation step can be 13 to 15 days (e.g., 14 days).

[0122] In this embodiment, the starter culture medium may be Hestrin Schramm (HS) medium, and may have, for example, the following components:

[0123] 20.0 gr / L glucose; 5.0 gr / L soybean peptone, enzyme digest; 5.0 gr / L yeast extract; 3.4 gr / L disodium hydrogen phosphate dihydrate; 1.5 gr / L citric acid monohydrate; 1.0 L distilled water.

[0124] In this embodiment, the mixture containing glucose may have, for example, the following components before the addition of the starting culture:

[0125] 20.0 gr / L glucose, derived from the enzymatic degradation of cellulose in waste textile materials; 2.85 mL / L peptone, derived from the degrading cellulase; 10.5 gr / L citric acid, derived from the citrate buffer used in the cellulase treatment; 1.0 L used water, derived from the mixture used in the cellulase treatment.

[0126] According to an embodiment, a mixture containing glucose and starter culture can be mixed to form a mixture containing glucose:starter culture in a volume ratio of 99:1 to 80:20, preferably 95:5 to 85:15 (e.g., 90:10).

[0127] Cultures of microorganisms, including those producing microbial cellulose, can be incubated, for example, in one or more sterile containers. The containers may have a lid or no lid. Preferably, the containers have a lid. For example, the containers may have a lid that serves as a physical barrier (i.e., an airtight barrier, such as a plastic lid) or a lid that serves as a partial physical barrier (e.g., a spunbond nonwoven layer with high air permeability, or a meltblown nonwoven layer with low air permeability).

[0128] Incubation can be carried out at a temperature ranging from 15°C to 35°C, preferably from 20°C to 30°C, for 1 to 30 days, preferably 5 to 20 days, and more preferably 10 to 16 days. For example, the incubation step can be carried out at a temperature of 25°C to 30°C (e.g., 28°C) for 13 to 15 days (e.g., 14 days).

[0129] According to an embodiment, the obtained microbial cellulose (e.g., bacterial cellulose) is washed and optionally dried.

[0130] According to an embodiment, washing is performed using an aqueous solution containing a bleach. In an embodiment, the bleach may be selected from NaOCl, NaOH, H2O2, or mixtures thereof. In a preferred embodiment, the bleach is NaOCl. According to an embodiment, washing is performed using an aqueous solution containing an amount ranging from 0.01% to 10% (w / v), preferably from 0.1% to 0.05% (w / v) of NaOCl.

[0131] According to the embodiments, the liquid ratio of the microbial cellulose layer to the washing solution by weight is 1 / 10, preferably 1 / 4.

[0132] According to an embodiment, washing is performed using an aqueous solution containing an amount of NaOH ranging from 0.01% to 10% (w / v), preferably from 1% to 5% (w / v).

[0133] According to an embodiment, washing is performed using an aqueous solution containing an amount of H2O2 ranging from 0.01% to 10% (w / v), preferably from 1% to 5% (w / v).

[0134] According to an embodiment, the step of washing microbial cellulose (e.g., bacterial cellulose) is carried out at a temperature ranging from 20°C to 80°C, preferably from 25°C to 50°C, and more preferably from 25°C to 35°C. The step of washing bacterial cellulose is carried out at a temperature of 30 ± 2°C. According to an embodiment, the step of washing microbial cellulose is performed on microbial cellulose in a laundry bag. For example, the laundry bag may be made of a fabric having a mesh size between 50 micrometers and 100 micrometers, preferably 75 micrometers.

[0135] According to an embodiment, the step of washing microbial cellulose (e.g., bacterial cellulose) can be carried out with agitation at 5 rpm to 250 rpm, preferably 50 rpm to 75 rpm. For example, the step of washing microbial cellulose (e.g., bacterial cellulose) can be carried out with agitation at 100 rpm.

[0136] Advantageously, the process of the present invention enables the production of microbial cellulose, particularly bacterial cellulose, with a low content of inorganic compounds. Advantageously, the content of inorganic material in the microbial cellulose of the present invention, as measured according to ISO 1762:2019 (determination of ash content in residue on ignition at 525°C), is less than 1% by weight.

[0137] As described above, one advantage of the process of the present invention is that, in blended textiles (e.g., blended fabrics), the process minimizes potential damage to the polyester portion of the blended textile, i.e., the degradation of polyester filaments or fibers is kept to a minimum so that the polyester material can be recycled and reused for recycling.

[0138] Therefore, the present invention also relates to a process for recycling a blend of textiles comprising polyester fibers or filaments and cellulose fibers, the process comprising the following steps:

[0139] g) Provide waste textile materials;

[0140] h) Pretreating the waste textile material with an aqueous solution comprising NaOH and urea to obtain pretreated textile material, wherein the step of pretreating the waste textile material with an aqueous solution comprising NaOH and urea is carried out at a temperature in the range of -25°C to +30°C;

[0141] i) Treat the pretreated textile material with cellulase to convert the cellulose into glucose, thereby obtaining a mixture comprising a solid phase and a liquid phase, the solid phase comprising polyester fibers or filaments;

[0142] j) Separating the liquid phase from the solid phase to obtain a liquid mixture containing glucose;

[0143] k) Recycle and reuse the polyester fibers or filaments from the solid phase.

[0144] All the features discussed in this article regarding the production of microbial cellulose from waste textile materials and the enzymatic saccharification process of waste textile materials also apply to the recycling of the aforementioned mixed textiles containing polyester or filament and cellulose fibers.

[0145] Figure 1 Exemplary embodiments of the process of the present invention are schematically summarized below.

[0146] Collect waste textile materials, and optionally wash them for cleaning purposes. Figure 1 (See box 1 in the text). Waste textile materials include cellulose, such as natural cellulose fibers (e.g., cotton fibers) and / or regenerated cellulose fibers (e.g., viscose fibers), and may optionally include non-cellulose fibers (e.g., polyester and / or elastic fibers). For example, waste textile materials may be selected from yarns, fabrics, garments, and combinations thereof.

[0147] After collecting and washing waste textile materials, non-textile elements, such as labels (e.g., made of leather or polyurethane), buttons, and zippers (e.g., typically made of aluminum, brass, or plastic), are removed from the waste textile materials, manually or automatically (using machines and equipment known in the art itself), if necessary. Figure 1 (Box 2 in the middle).

[0148] Then, waste textile materials are classified, for example, based on the color and type of dyes present in the waste textile materials (e.g., indigo, sulfur dyes, or black reactive dyes) or the composition of the textiles (e.g., fabrics) (e.g., 100% cotton textiles can be separated from textiles containing both cotton and polyester fibers). For example, near-infrared (NIR) classification of textiles (e.g., fabrics and / or clothing) based on color and / or the composition of the textiles (e.g., fabrics and / or clothing) is known in the art. Figure 1 (Box 3 in the middle).

[0149] If the waste textile material contains one or more dyes, the dyes are preferably removed by treating the fabric with a reducing solution of, for example, NaOH and sodium dithionite (Na2S2O4). Figure 1 (Box 4).

[0150] Waste textile materials are pretreated with an aqueous solution comprising NaOH and urea at a temperature of -25°C to +30°C (box 5) to swell the yarn and fibrillate the cellulose fibers. The pretreatment step can be carried out for varying durations depending on the temperature and concentration of the pretreatment solution. Typical treatment times are 1 to 8 hours, more preferably 2 to 5 hours. The treated textile materials can then be washed with, for example, a weak acid solution (e.g., acetic acid solution) or water. Figure 1(Box 5 in the text). The pretreated textile material can preferably be dried after washing. Then, the pretreated waste textile is preferably shredded to reduce the size of the waste textile material and increase the surface area that can contact the solution including cellulase. Figure 1 (See box 5a in the text). The aqueous solution comprising NaOH and urea can optionally be reused up to 20 times (e.g., for pretreatment of other waste textile materials); if necessary, the concentration of NaOH and / or urea can be adjusted for reuse.

[0151] Following the pretreatment and washing steps, the preferably dried and shredded textile material obtained therefrom is contacted with a solution containing cellulase to convert the cellulose fibers of the textile material into glucose (box 6). Preferably, the step of treating the pretreated textile material with cellulase is carried out without agitation at a pH in the range of 4.0 to 5.0. At the end of the enzymatic conversion step of cellulose fibers to glucose, a mixture containing a solid phase and a liquid phase is obtained ( Figure 1 (Box 6 in the middle).

[0152] The solid phase will contain non-cellulose fibers (e.g., polyester and / or elastic fibers), undigested cellulose fibers, and (if dyes are still present) insoluble dyes (e.g., indigo and sulfur dyes). The liquid phase typically contains a cellulase solution for treating the pretreated textile material, glucose, and optionally soluble dyes (e.g., reactive black dyes, if present).

[0153] After the step of treating the pretreated textile material with cellulase, the resulting mixture is processed and the liquid phase is separated from the solid phase. Figure 1 (See box 7 in the diagram). At the end of the separation step, a liquid mixture containing glucose is obtained ( Figure 1 (Box 9 in the text); the liquid mixture contains cellulase added in the previous step. In embodiments, the cellulase can be recovered and optionally reused to treat the solid phase obtained after the separation step up to 10 times. Figure 1 (Not shown in the image). In embodiments, cellulase can be recycled and reused to treat one or more additional pretreated waste textile materials (…). Figure 1 (Not shown in the image). Cellulase can be recovered and reused, for example, by means of dialysis. Advantageously, the enzyme is not removed from the mixture at the end of the enzymatic treatment. Materials such as non-cellulosic fibers (e.g., polyester fibers and / or elastic fibers) and insoluble dyes (e.g., indigo dyes and sulfur dyes, if present) can be recovered and reused from the solid phase. Figure 1(Box 8). The polyester fibers or other synthetic fibers thus recycled can be used as starting materials to prepare filaments and fibers for recycling yarns and fabrics (Box 8a).

[0154] The resulting liquid mixture containing glucose and enzymes was then used to prepare microbial cultures. Figure 1 Box 12 in the diagram is used to produce bio-cellulose (i.e., bacterial cellulose). For this purpose, the mixture is heat-treated to inactivate any enzymes present therein. Preferably, the mixture containing glucose is sterilized, for example, by heating to, for example, 121°C in a reactor suitable for producing microbial cellulose. Optionally, additives may be added to the mixture containing glucose (…). Figure 1 (See box 10 in the text) and sterilize. Optionally, the concentration of glucose in the mixture can be adjusted.

[0155] The microorganisms that produce microbial cellulose are then added to a liquid mixture containing glucose as a starter culture. Figure 1 (See box 11 in the text). The degrading enzymes are no longer active, but they provide a source of nitrogen and carbon as nutrients for the microorganisms (e.g., in the form of amino acids or amino acid fractions). Optionally, if necessary, the concentration of amino acids in the mixture can be adjusted. For example, the concentration of amino acids in the mixture can be adjusted by adding peptone to the mixture.

[0156] The resulting culture, which includes microorganisms that produce microbial cellulose, is then incubated to grow microbial cellulose. Figure 1 (See box 13 in the diagram). At the end of the incubation, microbial cellulose is obtained as a layer that is typically used as microbial cellulose.

[0157] The process involves recovering and reusing microbial cellulose (e.g., a layer of microbial cellulose) from one or more containers, washing, and optionally drying. Figure 2 (Box 14 in the text). Washing can be performed using an aqueous solution containing bleach. The washed microbial cellulose is then optionally dried. In an embodiment, the microbial cellulose is preferably partially dried after washing. For example, partial drying of the microbial cellulose results in a water content of 99% to 80% by weight, preferably 90% to 80% by weight, and more preferably 85% by weight.

[0158] The microbial cellulose obtained in box 14 can be advantageously used as a starting cellulose material in the process of preparing regenerated cellulose fibers. Figure 4A The suitable process for step 15 in box 15 is the process used for viscose production. Then, the regenerated cellulose fibers can be used to manufacture yarns and fabrics in the entire cycle (box 15a).

[0159] The invention will now be further described with reference to the following non-limiting embodiments.

[0160] Example 1 – Producing bacterial cellulose from waste textile materials

[0161] The process of this invention is to produce bacterial cellulose from waste textile materials.

[0162] Waste textile materials

[0163] Undyed mixed waste fabrics (fabric composition: 76% viscose, 23% polyester, 1% elastane) were used as waste textile materials.

[0164] Pretreatment with an aqueous solution including NaOH and urea

[0165] Dissolve 7% (w / v) NaOH and 12% (w / v) urea in 40 L of distilled water and cool to 0°C in a climate-controlled chamber to obtain an aqueous solution of NaOH and urea. Immerse 2 kg of fabric in the cold aqueous solution of NaOH and urea and pretreat at 0°C for 5 hours. After 5 hours, neutralize the fabric with tap water at 20°C until the pH reaches 7. Alternatively, the fabric can be neutralized by washing with a weak acid (e.g., acetic acid). The pretreated fabric is then dried at 120°C in a tenter frame.

[0166] Treatment with cellulase

[0167] The pretreated fabric was torn into small pieces (approximately 1 cm x 1 cm) and soaked in 40 L of citrate buffer (0.05 M, pH 5.0) and heated at 50°C in a bioreactor. The 40 L citrate buffer (0.05 M, pH 5.0) was prepared as follows: 420 g of citrate monohydrate was weighed and dissolved in 35 L of distilled water. The mixture was stirred until the citrate monohydrate was completely dissolved. The pH was adjusted to 5.0 with NaOH. Distilled water was added to a final volume of 40 L. 114.28 mL of Novozyme Cellic® CTec3 HS cellulase (equivalent to 20 FPU per gram of cellulose) was added to the solution. The enzyme was then added to the solution and stirred at 100 rpm for 1 min to ensure uniform dissolution. The fabric was then incubated undisturbed at 50°C for 24 hours in the solution containing the cellulase. At the end of the process, a mixture is obtained comprising a solid phase (primarily non-cellulose materials, such as, but not limited to, polyester and elastic fibers, and partially degraded cellulose materials, such as, partially degraded viscose) and a liquid phase containing glucose.

[0168] Separation of liquid and solid phases

[0169] After incubation, the solid phase of the mixture was separated from the liquid phase by filtration through a 0.3 mm sieve. The filtered liquid phase was centrifuged at 9000 RPM for 30 minutes. After centrifugation, the supernatant was collected as a mixture containing glucose (e.g., glucose-rich syrup).

[0170] Preparation of microbial cultures

[0171] Optionally, the mixture containing glucose can be concentrated (e.g., via a rotary evaporator). For example, a 40 L mixture can be concentrated to a final volume of 10 L.

[0172] The yield of the enzymatic reaction was determined using a mixture containing glucose, without the need for a concentration step.

[0173] In this example, 38.8 gr / L of glucose was released from 2 kg of a mixed fabric containing 1.52 kg of viscose according to the DNS method (measured on the above 40 L mixture containing glucose).

[0174] If the mixture is concentrated to a final volume of 10 L, the glucose concentration in the mixture will increase to 155.2 gr / L.

[0175] In this example, the yield is 91.9% according to the NREL method and is calculated as follows:

[0176]

[0177]

[0178] The value of 0.9 is a correction factor for the water molecules added during cellulose hydrolysis.

[0179] 40 L of the mixture containing glucose (38.8 g / L) was mixed with 37.6 L of distilled water to obtain a diluted mixture containing glucose at a concentration of 20 g / L. 77.6 L of the diluted glucose mixture was then autoclaved at 121°C for 15 min.

[0180] The resulting mixture containing glucose can also be called "Waste Textile Glucose" (WTG) medium.

[0181] The resulting mixture containing glucose can have the following components:

[0182] 20.0 gr / L glucose, derived from the enzymatic conversion of cellulose in waste textile materials into glucose;

[0183] 2.85 ml / L peptone, derived from cellulase degradation enzymes;

[0184] 10.5 gr / L citric acid, from citrate buffer;

[0185] Water is derived from the mixture obtained after the step of treatment with cellulase.

[0186] Preparation of starter bacteria cultures for bacterial cellulose production

[0187] Produce starter bacteria cultures according to the following procedure. Prepare 8 L of Hestrin & Schramm (HS) medium by mixing and autoclaving the following ingredients:

[0188] 160 gr glucose

[0189] 40g soybean peptone, enzyme digest

[0190] 40g yeast extract

[0191] 27.2 gr Disodium hydrogen phosphate dihydrate

[0192] 12g citric acid monohydrate

[0193] 8 L of water

[0194] ATCC 53582 of *Glucosium henneruis* was inoculated into 0.8 L of autoclaved HS medium and incubated at 28°C for 2 days.

[0195] Mix 0.8 L of freshly prepared bacterial culture with 7.2 L of autoclaved HS medium. Then, stir the mixture on a shaker at 150 rpm for 2 days.

[0196] Obtain 8L of freshly prepared starter culture for later use.

[0197] Mix the glucose-containing mixture (WTG medium) with the Spore bacteria culture.

[0198] 77.6 L of diluted glucose-containing mixture (20 gr glucose / L) (i.e., waste textile glucose medium) was aseptically mixed with 8 L of freshly prepared starter culture. Then, 4 L of the newly obtained bacterial mixture was dispensed into 21 plastic containers (730 mm wide x 460 mm long x 193 mm high) and capped.

[0199] incubation of bacterial cultures

[0200] Bacterial cellulose was obtained by incubating covered plastic containers at 28°C for 14 days in a climate-controlled room.

[0201] Washing and drying of bacterial cellulose

[0202] An average of 17 ± 0.35 kg of wet and turbid bacterial cellulose layer was collected from 85.6 L of bacterial culture. This bacterial cellulose layer was removed from the container and collected into the reactor. The bacterial cellulose layer was then washed with 100 L of hot water (80°C) containing 1% bleach (5% w / v sodium hypochlorite) while stirring at 100 rpm for 3 hours. Afterward, 100 L of wastewater was drained, and another 100 L of clean hot water (80°C) was added, followed by stirring at 100 rpm for 3 hours.

[0203] An average of 16 ± 0.32 kg of wet and clean bacterial cellulose layer can be obtained from 17 ± 0.35 kg of wet and dirty bacterial cellulose layer.

[0204] After drying these bacterial cellulose layers at 28°C for 12 hours, dry bacterial cellulose was obtained. An average of 470 ± 10 gr of dry bacterial cellulose could be obtained from 16 ± 0.32 kg of wet and clean bacterial cellulose.

[0205] Example 2 – Exemplary room temperature chemical pretreatment and enzymatic treatment of undyed mixed waste fabrics

[0206] Undyed mixed waste fabrics (fabric composition: 76% viscose, 23% polyester, 1% elastane) were used as waste textile materials.

[0207] Dissolve 7% (w / v) NaOH and 12% (w / v) urea in 1 L of distilled water.

[0208] 50 kg of fabric was immersed in an aqueous solution (1 L) of NaOH and urea and pretreated for 2 hours at room temperature (between 20 and 25°C). After 2 hours, the fabric was neutralized with tap water at 20°C until the pH reached 7. Alternatively, the fabric could be neutralized by washing with a weak acid (e.g., acetic acid). The pretreated fabric was then dried in an oven at 80°C.

[0209] The pretreated fabric was shredded into small pieces (e.g., 1 cm x 1 cm), soaked in 1 L of citrate buffer (0.05 M, pH 5.0), and heated at 50°C in a bioreactor. 2.85 mL of NovozymeCellic CTec3 HS cellulase (equivalent to 20 FPU per gram of cellulose) was added to the solution. Following this, the enzyme was added to the solution, and the solution was stirred at 100 rpm for 1 minute to ensure uniform dissolution. The fabric was incubated undisturbed at 50°C for 24 hours in the cellulase-containing solution. At the end of the treatment, a mixture was obtained comprising a solid phase (primarily non-cellulose materials, such as polyester and elastic fibers, and partially degraded cellulose materials, such as partially degraded viscose) and a liquid phase containing glucose.

[0210] After incubation, the solid phase of the mixture was separated from the liquid phase by filtration through a 0.3 mm sieve. The filtered liquid phase was centrifuged at 9000 RPM for 30 minutes. After centrifugation, the supernatant was collected as a mixture containing glucose (e.g., glucose-rich syrup).

[0211] The yield of the enzyme-catalyzed reaction was determined.

[0212] According to the DNS method, 36.2 gr / L of glucose was released from 50 g of a mixed fabric containing 38 g of viscose.

[0213] According to the NREL method, the yield is 85.7% and is calculated as follows:

[0214]

[0215] The value of 0.9 is a correction factor for the water molecules added during cellulose hydrolysis.

[0216] A mixture containing glucose (36.2 gr / L) can be mixed with distilled water to obtain a diluted mixture containing glucose at a concentration of 20 g / L, i.e., to obtain waste textile glucose (WTG) medium.

[0217] Example 3 - Exemplary chemical pretreatment and enzymatic treatment of undyed 100% cotton waste fabric

[0218] Undyed waste fabric (fabric composition: 100% cotton) is used as waste textile material.

[0219] Dissolve 7% (w / v) NaOH and 12% (w / v) urea in 1 L of distilled water and cool to 0°C in a refrigerator to obtain an aqueous solution of NaOH and urea. Soak 50 g of fabric in the aqueous solution of NaOH and urea (1 L) and pretreat at 0°C for 5 hours. After 5 hours, neutralize the fabric with tap water at 20°C until the pH reaches 7. Alternatively, the fabric can be neutralized by washing with a weak acid (e.g., acetic acid). The pretreated fabric is then dried in an oven at 80°C.

[0220] The pretreated fabric was shredded into small pieces (e.g., 1 cm x 1 cm), soaked in 1 L of citrate buffer (0.05 M, pH 5.0), and heated at 50°C in a bioreactor. 3.76 mL of NovozymeCellic® CTec3 HS cellulase (equivalent to 20 FPU per gram of cellulose) was added to the solution. Following this, the enzyme was added to the solution, and the solution was stirred at 100 rpm for 1 min to ensure uniform dissolution. The fabric was then incubated undisturbed at 50°C for 96 hours in the solution containing the cellulase. At the end of the treatment, a mixture containing a solid phase (primarily partially degraded cellulose material, e.g., partially degraded cotton) and a liquid phase containing glucose was obtained.

[0221] Centrifuge the mixture containing the solid and liquid phases at 9000 RPM for 30 minutes. After centrifugation, collect the supernatant as a mixture containing glucose (e.g., glucose-rich syrup).

[0222] The yield of the enzyme-catalyzed reaction was determined.

[0223] According to the DNS method, 51.3 gr / L of glucose is released from 50 g of 100% cotton fabric.

[0224] According to the NREL method, the yield is 92.34% and is calculated as follows:

[0225]

[0226] The value of 0.9 is a correction factor for the water molecules added during cellulose hydrolysis.

[0227] A mixture containing glucose (51.3 gr / L) can be mixed with distilled water to obtain a diluted mixture containing glucose at a concentration of 20 g / L, i.e., to obtain waste textile glucose (WTG) medium.

[0228] Example 4 – Exemplary chemical pretreatment and enzymatic treatment of undyed mixed waste fabrics

[0229] Undyed mixed waste fabrics (fabric composition: 76% viscose, 23% polyester, 1% elastane) were used as waste textile materials.

[0230] Dissolve 7% (w / v) NaOH and 12% (w / v) urea in 1 L of distilled water and cool to 0°C in a refrigerator to obtain an aqueous solution of NaOH and urea. Soak 50 g of fabric in the cold aqueous solution of NaOH and urea (1 L) and pretreat at 0°C for 5 hours. After 5 hours, neutralize the fabric with tap water at 20°C until the pH reaches 7. Alternatively, the fabric can be neutralized by washing with a weak acid (e.g., acetic acid). The pretreated fabric is then dried in an oven at 80°C.

[0231] The pretreated fabric was shredded into small pieces (e.g., 1 cm x 1 cm), soaked in 1 L of citrate buffer (0.05 M, pH 5.0), and heated at 50°C in a bioreactor. 2.85 mL of NovozymeCellic® CTec3 HS cellulase (equivalent to 20 FPU per gram of cellulose) was added to the solution. Following this, the enzyme was added to the solution and stirred at 100 rpm for 1 min to ensure uniform dissolution. The fabric was then incubated undisturbed at 50°C for 24 hours in the solution containing the cellulase. At the end of the treatment, a mixture was obtained comprising a solid phase (primarily non-cellulose materials, such as polyester and elastomers, and partially degraded cellulose materials, such as partially degraded viscose) and a liquid phase containing glucose.

[0232] After incubation, the solid phase of the mixture was separated from the liquid phase by filtration through a 0.3 mm sieve. The filtered liquid phase was centrifuged at 9000 RPM for 30 minutes. After centrifugation, the supernatant was collected as a mixture containing glucose (e.g., glucose-rich syrup).

[0233] The yield of the enzyme-catalyzed reaction was determined.

[0234] According to the DNS method, 38.4 gr / L of glucose was released from 50 g of a mixed fabric containing 38 g of viscose.

[0235] According to the NREL method, the yield is 90.9% and is calculated as follows:

[0236]

[0237] The value of 0.9 is a correction factor for the water molecules added during cellulose hydrolysis.

[0238] A mixture containing glucose (38.4 gr / L) can be mixed with distilled water to obtain a diluted mixture containing glucose at a concentration of 20 g / L, i.e., to obtain waste textile glucose (WTG) medium.

[0239] Example 5 - Exemplary chemical pretreatment and enzymatic treatment of mixed waste fabrics dyed with indigo.

[0240] Indigo-dyed mixed waste fabrics (fabric composition: 99% cotton, 1% elastic fiber; indigo dyed) are used as waste textile materials.

[0241] Dissolve 7% (w / v) NaOH and 12% (w / v) urea in 1.2 L of distilled water and cool to 0°C in a refrigerator to obtain an aqueous solution of NaOH and urea. Immerse a 60 g sheet of fabric in the cold aqueous solution of NaOH and urea (1.2 L) and pretreat at 0°C for 5 hours. After 5 hours, neutralize the fabric with tap water at 20°C until the pH reaches 7. Alternatively, the fabric can be neutralized by washing with a weak acid (e.g., acetic acid). The pretreated fabric is then dried in an oven at 80°C.

[0242] The pretreated fabric was shredded into small pieces (e.g., 1 cm x 1 cm), soaked in 1.2 L of citrate buffer (0.05 M, pH 5.0), and heated at 50°C in a bioreactor. 4.47 mL of NovozymeCellic® CTec3 HS cellulase (equivalent to 20 FPU per gram of cellulose) was added to the solution. Following this, the enzyme was added to the solution, and the solution was stirred at 100 rpm for 1 min to ensure uniform dissolution. The fabric was then incubated undisturbed at 50°C for 24 hours in the solution containing the cellulase. At the end of the treatment, a mixture containing a solid phase (primarily partially degraded cellulose material, such as cotton, as well as non-cellulose elastic fibers) and a liquid phase containing glucose was obtained.

[0243] After incubation, the solid phase of the mixture was separated from the liquid phase by filtration through a 0.3 mm sieve. The filtered liquid phase was centrifuged at 9000 RPM for 30 minutes. After centrifugation, the supernatant was taken as a mixture containing glucose (e.g., a glucose-rich syrup), and the precipitate containing indigo dye was collected.

[0244] A mixture containing glucose (i.e., a glucose solution from waste textiles, i.e., a glucose-rich syrup) can be mixed with distilled water to obtain a diluted mixture containing glucose at a concentration of 20 g / L, i.e., to obtain a glucose (WTG) culture medium from waste textiles.

[0245] Example 6 – Exemplary chemical pretreatment and enzymatic treatment of mixed waste fabrics dyed with sulfur dyes

[0246] Mixed waste textiles dyed with sulfur dyes (textile composition: 92.5% cotton, 5% polyester, 2.5% elastic fiber; sulfur dyes) were used as waste textile materials.

[0247] Dissolve 7% (w / v) NaOH and 10.5% (w / v) urea in 2 L of distilled water and cool to 0°C in a refrigerator to obtain an aqueous solution of NaOH and urea. Soak 100 g of fabric in the cold aqueous solution of NaOH and urea (2 L) and pretreat at 0°C for 5 hours. After 5 hours, neutralize the fabric with tap water at 20°C until the pH reaches 7. Alternatively, the fabric can be neutralized by washing with a weak acid (e.g., acetic acid). The pretreated fabric is then dried in an oven at 80°C.

[0248] The pretreated fabric was shredded into small pieces (e.g., 1 cm x 1 cm), soaked in 2 L of citrate buffer (0.05 M, pH 5.0), and heated at 50°C in a bioreactor. 6.95 mL of NovozymeCellic® CTec3 HS cellulase (equivalent to 20 FPU per gram of cellulose) was added to the solution. Following this, the enzyme was added to the solution and stirred at 100 rpm for 1 min to ensure uniform dissolution. The fabric was then incubated undisturbed at 50°C for 24 hours in the solution containing the cellulase. At the end of the treatment, a mixture was obtained consisting of a solid phase (primarily, but not limited to, non-cellulose materials containing polyester and elastic fibers, and partially degraded cellulose materials such as cotton) and a liquid phase containing glucose.

[0249] After incubation, the solid phase of the mixture was separated from the liquid phase by filtration through a 0.3 mm sieve. The filtered liquid phase was centrifuged at 9000 RPM for 30 minutes. After centrifugation, the supernatant was taken as a mixture containing glucose (e.g., a glucose-rich syrup), and the precipitate containing the sulfur dye was collected.

[0250] A mixture containing glucose can be mixed with distilled water to obtain a diluted mixture containing glucose at a concentration of 20 g / L, i.e., to obtain waste textile glucose (WTG) medium.

[0251] Example 7 – Exemplary chemical pretreatment and enzymatic treatment of mixed waste fabrics dyed with black reactive dyes

[0252] Mixed waste textiles dyed with black reactive dyes (textile composition: 90% cotton, 6% polyester, 4% elastic fiber; black reactive dye) were used as waste textile materials.

[0253] Dissolve 7% (w / v) NaOH and 14.0% (w / v) urea in 2 L of distilled water and cool to 0°C in a refrigerator to obtain an aqueous solution of NaOH and urea. Immerse 100 kg of fabric in the cold aqueous solution of NaOH and urea (2 L) and pretreat at 0°C for 5 hours. After 5 hours, neutralize the fabric with tap water at 20°C until the pH reaches 7. Alternatively, the fabric can be neutralized by washing with a weak acid (e.g., acetic acid). The pretreated fabric is then dried in an oven at 80°C.

[0254] The pretreated fabric was shredded into small pieces (e.g., 1 cm x 1 cm), soaked in 2 L of citrate buffer (0.05 M, pH 5.0), and heated at 50°C in a bioreactor. 6.77 mL of NovozymeCellic® CTec3 HS cellulase (equivalent to 20 FPU per gram of cellulose) was added to the solution. Following this, the enzyme was added to the solution and stirred at 100 rpm for 1 min to ensure uniform dissolution. The fabric was then incubated undisturbed at 50°C for 24 hours in the solution containing the cellulase. At the end of the treatment, a mixture was obtained consisting of a solid phase (primarily, but not limited to, non-cellulose materials containing polyester and elastic fibers, and partially degraded cellulose materials, such as cotton) and a liquid phase containing glucose.

[0255] After incubation, the solid phase of the mixture was separated from the liquid phase by filtration through a 0.3 mm sieve. The filtered liquid phase was centrifuged at 9000 RPM for 30 minutes. After centrifugation, the supernatant was collected as a mixture containing glucose (e.g., a glucose-rich syrup) and also containing the black reactive dye.

[0256] Dyes were removed from liquid mixtures by activated carbon column chromatography. Specifically, to remove the dyes, a glucose-rich syrup was poured into an activated carbon column at a flow rate of 2 mL / min. The 5 cm x 50 cm column contained 1 kg of activated carbon beads with a diameter of 2.88 mm ± 0.26 mm.

[0257] The mixture obtained after removing the dye can be mixed with distilled water to obtain a diluted mixture containing glucose at a glucose concentration of 20 g / L, i.e., to obtain waste textile glucose (WTG) medium.

[0258] Example 8 - Comparison of Hestrin-Schramm (HS) medium and Waste Textile Glucose (WTG) medium

[0259] In a closed plastic container, *Glucosidobacterium henneruis* strain ATCC 53582 was incubated for 14 days at 28°C–30°C in 500 mL of waste textile glucose (WTG) medium or Hestrin-Schramm (HS) medium.

[0260] After incubation, the obtained wet bacterial cellulose was squeezed or pressed using mechanical equipment to remove excess culture medium. The amount of wet bacterial cellulose obtained was then measured.

[0261]

[0262] It was observed that using waste textile glucose medium increased the yield of wet bacterial cellulose by approximately 20% compared to HS medium.

[0263] Example 9 - Comparison of Additives (TiBALDH, Glycerin, Ascorbic Acid)

[0264] In a closed plastic box, *Glucosium henneruis* strain ATCC 53582 was incubated for 14 days at 28°C–30°C in 500 mL of glucose medium from waste textiles.

[0265] Different experiments were conducted to test the effects of adding different concentrations of different additives (TiBALDH, glycerol, ascorbic acid) on the production of bacterial cellulose.

[0266] At the end of incubation, the amount of wet bacterial cellulose obtained was measured. The comparison of the changes (%) in the amount of wet bacteria in Tables 2, 3, 4 and 5 is based on the data in Table 1.

[0267] TiBALDH

[0268] The first additive tested was a solution of dihydroxy bis(ammonium lactate)titanium(IV) solution (TiBALDH), CAS number 65104-06-5.

[0269]

[0270] Based on the results obtained, adding 0.25% TiBALDH to the glucose medium of waste textiles increased the amount of wet BC obtained by the most (+55.30%).

[0271] glycerin

[0272] The second additive tested was glycerin, CAS number: 56-81-5.

[0273]

[0274] Based on the results obtained, adding 1% glycerol to the glucose medium containing waste textiles increased the amount of wet BC obtained by the most (+19.74%).

[0275] Ascorbic acid (vitamin C)

[0276] The third additive tested was ascorbic acid (vitamin C), CAS number: 50-81-7.

[0277]

[0278] Based on the results obtained, the addition of 0.5% ascorbic acid to the glucose medium of waste textiles resulted in the greatest increase in the amount of wet BC obtained (+9.25%).

[0279] Combination of additives

[0280] Different combinations of three additives—TiBALDH, glycerol, and ascorbic acid—were tested.

[0281]

[0282] Based on the results obtained, the highest amount of wet BC (58.04%) was achieved by adding 1% glycerol and 0.25% TiBALDH.

[0283] Example 10 - Comparison of waste textile materials before and after pretreatment with aqueous solutions including NaOH and urea.

[0284] Mixed waste fabric (fabric composition: 76% viscose, 23% polyester, 1% elastane) was treated with a solution of 7% (w / v) NaOH and 12% (w / v) urea at 0°C for 5 hours. Images of the fabric were taken and analyzed using a scanning electron microscope (SEM) before and after treatment. Figure 4B It can be seen that the treated fabric expands, that is, the fibers in the fabric separate relative to the fibers in the fabric before treatment.

[0285] Example 11 – Effect of pretreatment with an aqueous solution containing NaOH and urea on polyester at room temperature and 50°C

[0286] Two 100% virgin polyester fabric samples were treated with 7% NaOH (w / v) and 12% urea (w / v) solutions at room temperature and 50°C, respectively, for 5 hours. After treatment, images of the samples were acquired and analyzed using a scanning electron microscope (SEM). Figure 5A It can be seen that pretreatment at room temperature does not damage polyester fibers. Conversely, pretreatment at 50°C damages polyester fibers. Figure 5B ).

[0287] Example 12 - Comparison of waste textile materials before and after pretreatment with an aqueous solution containing NaOH and urea (0°C and room temperature) and enzymatic treatment.

[0288] Mixed waste fabric samples (fabric composition: 76% viscose, 23% polyester, 1% elastane) were pretreated with a solution of 7% (w / v) NaOH and 12% (w / v) urea at 0°C or room temperature for 5 hours, followed by enzymatic treatment with cellulase. Images of the samples were captured and analyzed using scanning electron microscopy (SEM) before and after treatment. Figure 5C SEM images of the samples before pretreatment and enzymatic treatment are shown. Figure 3A The sample material after pretreatment at 0°C and treatment with cellulase is shown. Figure 3B The sample material after pretreatment at room temperature and treatment with cellulase is shown. It can be seen that the process of the present invention does not damage the polyester fibers.

[0289] Example 13 – Characterization of glucose obtained after treatment with cellulase

[0290] According to the process of the present invention, mixed waste fabrics (fabric composition: 76% viscose, 23% polyester, 1% elastic fiber) and 100% cotton waste fabrics are pretreated with NaOH and urea solution, and then treated with cellulase.

[0291] Glucose obtained after treatment with cellulase was analyzed by nuclear magnetic resonance (NMR). The following results were obtained.

[0292] Blended fabric: glucose in D2O 1 ¹H NMR; 40.7% α-glucose, 59.3% β-glucose 5.21 ppm (bs, H₂) 1-α ), 4.63 ppm (d, J=7.6 Hz, H 1-β ), 3.87 ppm (d, J=12.0 Hz, H 6-β ), 3.81 ppm (d, J=10.9 Hz, H 5-α & H 6-α ), 3.77-3.64 ppm (m, H), 6-α & H 3-α & H 6-β ), 3.52 ppm (d, J=8.0 Hz, H 2-α ), 3.46 ppm (dd, J=18.2, 9.1 Hz, H 3-β & H 5-β ), 3.38 ppm (dd, J=15.3, 8.6 Hz, H 4-α & H 4-β), 3.23 ppm (t, J=8.1 Hz, H 2-β )

[0293] 100% cotton fabric: glucose in D2O 1 ¹H NMR; 43.3% α-glucose, 56.7% β-glucose 5.23 ppm (d, J = 3.7 Hz, H2). 1-α ), 4.65 ppm (d, J=7.9 Hz, H 1-β ), 3.90 ppm (dd, J=12.3, 1.9 Hz, H 6-β ), 3.87-3.81 ppm (m, H), 5-α or H 6-α ), 3.76 ppm (dd, J=12.5, 5.5 Hz, H 6-α ), 3.75-3.69 ppm (m, H), 3-α & H 6-β ), 3.53 ppm (dd, J=9.8, 3.7 Hz, H 2-α ), 3.50-3.45 ppm (m, H3-β & H5-β), 3.41 ppm (dt, J=9.4, 6.1 Hz, H 4-α and H 4-β ), 3.24 ppm (dd, J=9.1, 8.1 Hz, H 2-β )

[0294] Example 14 - Exemplary bacterial cellulose structure obtained according to the process of the present invention

[0295] Exemplary bacterial cellulose samples obtained according to the process of the present invention were analyzed and characterized.

[0296] The following results were obtained.

[0297] Viscosity: 980 ml / g (measured according to ISO 5351:2010)

[0298] Molecular weight distribution: Mn = 88047 g / mol, Mw = 510577 g / mol, DI = 5.8 (SEC analysis in 0.5% (w / v) LiCl / DMAc mobile phase)

[0299] Bacterial cellulose contains >99% cellulose.

[0300] The bacterial cellulose contained less than 1% hemicellulose; 0.08 g / kg arabinose, 0.03 g / kg galactose, 0.05 g / kg xylose, 0.4 g / kg mannose, and 882 g / kg glucose (SCAN-CM 71:09).

[0301] Ash content % < 0.5% (according to ISO 1762:2019)

[0302] Figure 3A and Figure 3B The image shows bacterial cellulose structures at two magnifications as observed using an optical microscope. Figure 3A The middle is 150X and Figure 3B The value is 75X.

[0303] from ​ and ​ It can be observed that the structure of bacterial cellulose is covered with stripes of a network of fine and coarse fibers.

[0304] Example 15 - Washing of bacterial cellulose

[0305] After bacterial cellulose (BC) fermentation is complete, the BC layer is removed from the fermentation vessel and placed in a laundry bag sealed with resealable Velcro. The laundry bag is made of fabric with a mesh size between 50 and 100 micrometers (preferably 75 micrometers). The laundry bag is squeezed using a wringer to remove excess bacterial solution. After this, the laundry bag is placed in an industrial washing machine to inactivate and remove bacteria and whiten the BC layer. The washing process includes the following:

[0306] The liquid-to-water ratio (by weight) of the bacterial cellulose layer is 1 / 10, preferably 1 / 4.

[0307] Whitening agent: 1% bleach

[0308] Temperature: 30°C

[0309] Duration: 30 min

[0310] Example 16 – Exemplary room temperature chemical pretreatment and enzymatic treatment of undyed 100% cotton waste fabric

[0311] Undyed waste fabric (fabric composition: 100% cotton) is used as waste textile material.

[0312] Dissolve 7% (w / v) NaOH and 12% (w / v) urea in 1 L of distilled water.

[0313] 50 g of fabric was soaked in an aqueous solution (1 L) of NaOH and urea and pretreated for 2 hours at room temperature (between 20 and 25°C). After 2 hours, the fabric was neutralized with tap water at 20°C until the pH reached 7. Alternatively, the fabric could be neutralized by washing with a weak acid (e.g., acetic acid). The pretreated fabric was then dried in an oven at 80°C.

[0314] The pretreated fabric was shredded into small pieces (e.g., 1 cm x 1 cm), soaked in 1 L of citrate buffer (0.05 M, pH 5.0), and heated at 50°C in a bioreactor. 3.76 mL of NovozymeCellic CTec3 HS cellulase (equivalent to 20 FPU per gram of cellulose) was added to the solution. The enzyme was then added to the solution, and the solution was stirred at 100 rpm for 1 minute to ensure uniform dissolution. The fabric was incubated undisturbed at 50°C for 24 hours in the cellulase-containing solution. At the end of the treatment, a mixture containing a solid phase (partially degraded cellulose material, e.g., partially degraded cotton) and a liquid phase containing glucose was obtained.

[0315] After incubation, the solid phase of the mixture was separated from the liquid phase by filtration through a 0.3 mm sieve. The filtered liquid phase was centrifuged at 9000 RPM for 30 minutes. After centrifugation, the supernatant was collected as a mixture containing glucose (e.g., a glucose-rich syrup). The yield of the enzymatic reaction was determined.

[0316] According to the DNS method, 45.62 gr / L of glucose was released from 50 gr 100% cotton fabric. According to the NREL method, the yield was 82.12% and calculated as follows:

[0317]

[0318] The value of 0.9 is a correction factor for the water molecules added during cellulose hydrolysis.

[0319] A mixture containing glucose (45.62 gr / L) can be mixed with distilled water to obtain a diluted mixture containing glucose at a concentration of 20 g / L, i.e., to obtain waste textile glucose (WTG) medium.

[0320] Example 17 – Comparison of enzyme activities at different chemical pretreatment temperatures: -20°C, 0°C, and room temperature

[0321] (As in Example 1 and Example 2) Use undyed waste textile fabric (fabric composition: 76% viscose, 23% polyester, 1% elastane).

[0322] The cells were pretreated with NaOH and urea at -20°C for 24 hours. Then, the same procedure as in Example 1 was performed. The cellulose conversion yield obtained after enzymatic treatment was 89.6%.

[0323] According to Example 1, pretreatment with NaOH and urea was carried out at 0°C for 5 hours. The cellulose conversion yield obtained after enzymatic treatment was 91.9% (see also Example 1).

[0324] According to Example 2, the NaOH and urea pretreatment was carried out at room temperature (20 to 25°C) for 2 hours. The cellulose conversion yield obtained after enzymatic treatment was 87.5% (see also Example 2).

[0325] Results: Pretreatment with NaOH and urea at room temperature can achieve a high conversion yield of cellulose to glucose, while significantly shortening the pretreatment time.

[0326] Example 18 - Exemplary chemical pretreatment and enzymatic treatment of undyed 100% cotton waste fabric

[0327] Undyed waste textile fabrics (fabric composition: 100% cotton) are used as waste textile materials.

[0328] Perform the same process as in Example 3, using Enzyme AB – Flashzyme Plus 200 instead of Novozymes Cellic® CTec3 HS.

[0329]

[0330] The cellulose conversion rate was 93.51% after 144 hours of enzyme treatment.

[0331] Example 19 - Cellulase Recycling

[0332] Undyed waste textile fabrics (fabric composition: 100% cotton) are used as waste textile materials.

[0333] Dissolve 7% NaOH (w / v) and 12% urea (w / v) in 1 L of distilled water and cool to 0°C in a refrigerator to obtain an aqueous solution of NaOH and urea. Soak 50 g of fabric in the NaOH and urea aqueous solution (1 L) and pretreat at 0°C for 5 hours. After 5 hours, neutralize the fabric with tap water at 20°C until the pH reaches 7. Alternatively, the fabric can be washed with a weak acid (e.g., acetic acid) to neutralize it. Then, dry the pretreated fabric in an oven at 80°C.

[0334] The pretreated fabric was shredded into small pieces (approximately 1 cm x 1 cm), soaked in 1 L of citrate buffer (0.05 M, pH 5.0), and heated at 50°C in a bioreactor. 3.76 mL of Novozyme Cellic® CTec3 HS cellulase (equivalent to 20 FPU per gram of cellulose) was added to the solution. Following this, the enzyme was added to the solution, and the solution was stirred at 100 rpm for 1 min to ensure uniform dissolution. The fabric was then incubated undisturbed at 50°C for 24 hours in the solution containing the cellulase. At the end of the treatment, a mixture containing a solid phase (primarily partially degraded cellulose material, e.g., partially degraded cotton) and a liquid phase containing glucose was obtained.

[0335] Centrifuge the mixture containing the solid and liquid phases at 9000 RPM for 30 minutes. After centrifugation, collect the supernatant as a mixture containing glucose (e.g., glucose-rich syrup).

[0336] After centrifugation, the enzyme was removed from the supernatant (i.e., the mixture containing glucose) by dialysis (dialysis cartridge: Thermo Scientific - Slide-A-Lyzer cassette (10 K MWCO)), as follows:

[0337] Prepare 1 L of 0.05 M citrate (pH 5) buffer and place it on ice.

[0338] Place the dialysis chamber in the buffer solution and stir with a magnetic stirrer for 3 hours to remove glucose from the dialysis chamber resulting from cellulose degradation via enzyme cycling from the mixture.

[0339] Dialysis is performed at low temperatures to prevent enzyme degradation. A 10 kDa dialysis cartridge is selected based on the approximate molecular weight of the cellulase to prevent the enzyme from passing through the cartridge.

[0340] Three hours later, the solution in the dialysis chamber was used to contact the solid phase obtained above (mainly partially degraded cellulose material, such as partially degraded cotton) for continuous enzymatic degradation.

[0341] After incubation at 50°C without stirring for 24 hours (total enzymatic treatment time: 48 hours), the resulting mixture containing a solid phase and a liquid phase containing glucose was centrifuged; after centrifugation, the enzyme was removed from the supernatant and reused to contact the solid phase obtained after centrifugation.

[0342] The same enzymatic treatment steps were then performed twice more (72 hours and 96 hours): separating the liquid phase from the solid phase, recovering and reusing the enzyme by dialysis, and reusing the enzyme.

[0343] After the second application of the enzyme solution (i.e., after a total of 48 hours of enzymatic treatment), the conversion of cellulose to glucose (% yield) was 79.77%.

[0344] After the fourth application of the enzyme solution (i.e., after a total of 96 hours of enzymatic treatment), the conversion of cellulose to glucose (% yield) was 99.18%.

[0345] Example 20 - Effect of shredding on the amount of dissolved cellulose

[0346] Undyed waste textile fabrics (fabric composition: 76% viscose, 23% polyester, 1% elastane) were used as waste textile materials.

[0347] A 2 g fabric sample was pretreated by immersing it in an aqueous solution of NaOH and urea. After pretreatment, the fabric was neutralized with tap water at 20°C until the pH reached 7. The pretreated fabric was then dried in an oven at 80°C. The weight of the pretreated fabric was 1.86 g.

[0348] A 2 g fabric sample was shredded into small pieces. The shredded material was pretreated by soaking it in an aqueous solution of NaOH and urea. After pretreatment, the fabric was neutralized with tap water at 20°C until the pH reached 7. The pretreated fabric was then dried in an oven at 80°C. The weight of the pretreated shredded fabric obtained was 1.48 g.

[0349] It can be seen that if the fabric is not shredded before treatment, 0.14% of cotton will be lost during the pretreatment process, which is equivalent to a 7% loss of cellulose. Conversely, if the fabric is shredded before treatment, 0.52% of cotton will be lost during the pretreatment process, which is equivalent to a 26% loss of cellulose.

[0350] Shredding waste textile materials after pretreatment provides more cellulose for cellulase, which in turn produces a higher amount of glucose at the end of the cellulase-catalyzed conversion to glucose.

[0351] Example 21 - An exemplary comparison of chemical pretreatment versus no pretreatment of undyed mixed waste fabrics

[0352] Undyed mixed waste fabrics (fabric composition: 76% viscose, 23% polyester, 1% elastane) were used as waste textile materials.

[0353] Chemical pretreatment

[0354] Dissolve 7% (w / v) NaOH and 12% (w / v) urea in 100 mL of distilled water and cool to 0°C in a refrigerator to obtain an aqueous solution of NaOH and urea. Soak 5 g of fabric in the cold NaOH and urea aqueous solution and pretreat at 0°C for 5 hours. After 5 hours, neutralize the fabric with tap water at 20°C until the pH reaches 7. Alternatively, the fabric can be neutralized by washing with a weak acid (e.g., acetic acid). Then, dry the pretreated fabric in an oven at 80°C. Tear the pretreated fabric into small pieces (e.g., 1 cm x 1 cm).

[0355] Non-preprocessing

[0356] Tear 5 g of fabric into small pieces (e.g., 1 cm x 1 cm).

[0357] Treatment with cellulase

[0358] Pretreated and untreated fabrics were immersed in 100 mL of citrate buffer (0.05 M, pH 5.0) and heated at 50°C in a track shaker. 0.28 mL of Novozyme Cellic® CTec3 HS cellulase (equivalent to 20 FPU per gram of cellulose) was added to the solution. Following this, the enzyme was added to the solution and stirred at 100 rpm for 1 min to ensure uniform dissolution. The fabrics were then incubated undisturbed at 50°C for 24 hours in the cellulase-containing solution. At the end of the treatment, a mixture of two phases was obtained: a solid phase (primarily non-cellulose materials, such as polyester and elastomers, and partially degraded cellulose materials, such as partially degraded viscose) and a liquid phase containing glucose.

[0359] After incubation, the solid phase of the mixture was separated from the liquid phase by filtration through a 0.3 mm sieve. The filtered liquid phase was centrifuged at 9000 RPM for 30 minutes. After centrifugation, the supernatant was collected as a mixture containing glucose (e.g., glucose-rich syrup).

[0360] Enzyme reaction yield:

[0361] - Chemical pretreatment

[0362] According to the DNS method, 38.4 gr / L of glucose was released from 5 g of a mixed fabric containing 38 g of viscose.

[0363] According to the NREL method, the yield is 90.9% and is calculated as follows:

[0364]

[0365] The value of 0.9 is a correction factor for the water molecules added during cellulose hydrolysis.

[0366] - No preprocessing

[0367] According to the DNS method, 12.7 gr / L of glucose was released from 5 g of a mixed fabric containing 3.8 g of viscose.

[0368] According to the NREL method, the yield is 90.9% and is calculated as follows:

[0369]

[0370] The value of 0.9 is a correction factor for the water molecules added during cellulose hydrolysis.

[0371] It can be seen that, by pretreating waste textile materials according to the present invention, the conversion yield of cellulose to glucose can be increased from 30.1% to 89.3%.

[0372] The above examples demonstrate that the process of the present invention provides a method for recycling cotton and cellulose fibers, as well as polyester or other synthetic fibers, from waste textiles (i.e., fabrics). Cellulose fibers are converted into glucose for the production of microbial cellulose, which is then dissolved and transformed into viscose or similar cellulose fibers, thereby achieving a complete recycling cycle of the initial cellulose fibers.

[0373] Furthermore, in the temperature range of the claimed protection, particularly at room temperature (20°C ± 5°C), the degradation of polyester fibers in the pretreatment step with the claimed solution containing NaOH and urea is significantly reduced compared to prior art treatments. Therefore, polyester recovered and reused after enzymatic conversion of cellulose fibers is suitable for recycling and re-spinning into recycled polyester filaments.

[0374] Furthermore, this process allows the enzymes used in the enzymatic conversion of cellulose to consist of cellulase. The process also offers the advantage that enzymes present in glucose and glucose-containing solutions can be heat-inactivated, allowing the glucose-containing solution to be directly used in subsequent steps of culturing cellulosic microorganisms. Here, the inactivated enzymes serve as a nutrient source for the microorganisms.

[0375] This process is particularly advantageous in the recycling of blended yarns of synthetic and cellulose fibers, where the cellulose fibers are difficult to recycle directly (e.g., by mechanical processes that retain the fibers in their fibrous form). As an example, this process is useful when the cellulose fibers are derived from recycled fabrics and yarns and are therefore short in average length, resulting in recycled fibers that are too short to be used in yarn.

Claims

1. A process for producing microbial cellulose from waste textile materials, said waste textile materials comprising cellulose fibers, said process comprising the following steps: a) Provide waste textile materials; b) Pretreating the waste textile material with an aqueous solution comprising NaOH and urea to obtain pretreated textile material, wherein the step of pretreating the waste textile material with an aqueous solution comprising NaOH and urea is carried out at a temperature in the range of -25°C to +30°C; c) Treat the pretreated textile material with cellulase to convert the cellulose into glucose, thereby obtaining a mixture comprising a solid phase and a liquid phase; d) Separate the liquid phase from the solid phase to obtain a liquid mixture containing glucose; e) Using the mixture containing glucose to prepare a microbial culture, wherein the microbial culture comprises microorganisms that produce microbial cellulose; f) Incubate the microbial culture to obtain microbial cellulose.

2. An enzymatic saccharification process for waste textile materials, wherein the waste textile materials comprise cellulose fibers containing cellulose, the process comprising the following steps: a) Provide waste textile materials; b) Pretreating the waste textile material with an aqueous solution comprising NaOH and urea to obtain pretreated textile material, wherein the step of pretreating the waste textile material with an aqueous solution comprising NaOH and urea is carried out at a temperature in the range of -25°C to +30°C; c) Treat the pretreated textile material with cellulase to convert the cellulose into glucose, thereby obtaining a mixture comprising a solid and a liquid phase. Prior to step c), the pretreated textile obtained in step b) is shredded into multiple pieces of a size suitable for enzymatic saccharification.

3. The process according to claim 1 or 2, wherein the waste textile material comprises cellulose fibers and non-cellulose fibers, and the waste textile material is selected from yarns, fabrics, and articles containing said fabrics.

4. The process according to any one of claims 2 or 3, wherein the cellulose fiber is selected from cotton fiber, hemp fiber, flax fiber, jute fiber, viscose fiber, lyocell fiber, modal fiber, acetate fiber, cuprammonium fiber and mixtures thereof, preferably, the fiber is a recycled fiber, and wherein the non-cellulose fiber is selected from polyester fiber, polyamide fiber, elastic fiber, polypropylene (PP) fiber, acrylic fiber and mixtures thereof, preferably, the fiber is a recycled fiber.

5. The process according to any one of the preceding claims, comprising the aqueous solution of NaOH and urea, wherein the amount of NaOH is in the range of 1% to 15% w / v, preferably 2% to 10% w / v, more preferably 3% to 9% w / v, and the amount of urea in the aqueous solution is in the range of 1% to 25% w / v, preferably 3% to 20% w / v, more preferably 5% to 15% w / v.

6. The process according to any one of the preceding claims, wherein the weight ratio of NaOH to urea (NaOH / urea) is in the range of 1.0 / 1.0 to 1.0 / 2.0, preferably 1.0 / 1.5 to 1.0 / 2.0, and more preferably 1.0 / 1.5 to 1.0 / 1.

8.

7. The process according to any one of the preceding claims, wherein, In the step of pretreating the waste textile material with the aqueous solution comprising NaOH and urea, the amount of waste textile material by weight relative to the volume of the aqueous solution comprising NaOH and urea is in the range of 1% w / v to 20% w / v, preferably 2% w / v to 15% w / v, more preferably 2.5% w / v to 10% w / v, and most preferably 5% w / v.

8. The process according to any one of the preceding claims, wherein the step of pretreating the waste textile material with the aqueous solution comprising NaOH and urea is carried out at a temperature in the range of -25°C to +30°C, preferably in the range of -15°C to +25°C, more preferably in the range of 4°C to +25°C, and most preferably in the range of +15°C to +25°C.

9. The process according to any one of the preceding claims, wherein the pretreatment step is performed for a period of 0.5 hours to 10 hours, preferably 1 hour to 8 hours, and more preferably 2 hours to 7 hours.

10. The process according to any one of the preceding claims, wherein the cellulase has an enzyme activity of 1 FPU to 50 FPU per gram of cellulose, preferably 10 FPU to 30 FPU per gram of cellulose, more preferably 15 FPU to 25 FPU per gram of cellulose, and optionally wherein the cellulase is in the form of a solution comprising cellulase.

11. The process according to any one of the preceding claims, wherein the waste textile material contains dye, and at least a portion of the dye is removed from the waste textile material before step c), preferably before step b), or after step d), it is removed from the solid phase or the liquid phase of the mixture comprising a solid and a liquid phase.

12. The process according to any one of the preceding claims, wherein the non-cellulose fibers are recovered and reused from the solid phase of the mixture comprising a solid phase and a liquid phase.

13. The process according to any one of the preceding claims, wherein prior to its use in step e), the mixture comprising glucose obtained from step d) is heat-treated to inactivate the cellulase, thereby providing a source of nutrients for the microorganism.

14. The process according to claim 1 or any one of claims 3 to 13, wherein the concentration of glucose in the mixture comprising glucose is in the range of 10 g / L to 30 g / L, preferably in the range of 15 g / L to 25 g / L, and the amount of the mixture comprising glucose is 99% to 80% by weight, preferably 95% to 85% by weight of the microbial culture.

15. The process according to claim 1 or any one of claims 3 to 14, wherein the mixture containing glucose is mixed with a starter microbial culture comprising microorganisms producing microbial cellulose, wherein the amount of the starter microbial culture comprising microorganisms producing microbial cellulose is 1% to 20% by weight, preferably 5% to 15% by weight, of the microbial culture.

16. The process according to claim 1 or any one of claims 3 to 15, wherein the step of preparing the microbial culture using the mixture containing glucose comprises adding at least one additive to the mixture containing glucose, wherein the additive is preferably selected from dihydroxybis(ammonium lactate)titanium(IV) (TiBALDH), glycerol, ascorbic acid, ethephon (2-chloroethyl phosphate), and mixtures thereof.

17. The process according to claim 1 or any one of claims 3 to 16, wherein the microbial cellulose is washed and optionally dried, wherein the washing is performed using an aqueous solution containing a bleach, wherein the bleach is preferably NaOCl in an amount ranging from 0.01% to 10% (w / v), preferably from 0.1% to 0.05% (w / v).

18. The process according to claim 1 or any one of claims 3 to 17, wherein the microbial cellulose is used to produce cellulose fibers.

19. A process for recycling a blend of textiles comprising polyester fibers or filaments and cellulose fibers, comprising the following steps: g) Provide waste textile materials; h) Pretreating the waste textile material with an aqueous solution comprising NaOH and urea to obtain pretreated textile material, wherein the step of pretreating the waste textile material with an aqueous solution comprising NaOH and urea is carried out at a temperature in the range of -25°C to +30°C; i) Treat the pretreated textile material with cellulase to convert the cellulose into glucose, thereby obtaining a mixture comprising a solid phase and a liquid phase, the solid phase comprising polyester fibers or filaments; j) Separating the liquid phase from the solid phase to obtain a liquid mixture containing glucose; k) Recycle and reuse the polyester fibers or filaments from the solid phase.

20. The process according to claim 19, further characterized in that... According to any one of claims 3 to 12.

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