A method for the production of polyhydroxyalkanoates using textile biotransformation

Abstract

This invention relates to the field of biomaterial preparation technology, specifically to a method for preparing polyhydroxyalkanoates (PHA) using fabric biotransformation. The method includes: (1) treating cotton-containing fabrics with alkali, followed by enzymatic hydrolysis to fully destroy the crystalline structure of cotton fibers in the fabrics; (2) sterilizing the hydrolysate obtained in step (1) and using it as a carbon source to inoculate PHA synthesizing bacteria for fermentation, allowing the PHA synthesizing bacteria to accumulate PHA during secondary metabolism. This invention treats cotton-containing fabrics with alkali and performs enzymatic hydrolysis to reduce the crystallinity of cellulose and destroy its dense structure, thereby improving the accessibility of the enzyme to the substrate. The resulting material is then directly used for the biosynthesis of PHA, thus realizing the high-value transformation of waste textiles from low-value waste to biodegradable polymer materials.

Description

Technical Field

[0001] This invention belongs to the field of biomass resource utilization and biomaterial preparation technology, and in particular relates to a method for preparing polyhydroxy fatty acid esters by bioconversion of textiles. Background Technology

[0002] Cotton fiber, as an important component of blended textiles, is mainly composed of cellulose. However, due to the complex composition and dense structure of blended materials, it is difficult to achieve efficient separation and recycling through traditional methods. As a result, a large amount of waste textiles are landfilled or incinerated, which not only wastes resources but also causes serious environmental pollution.

[0003] Compared to traditional methods, biological methods offer advantages such as high selectivity, low energy consumption, and a smaller environmental impact. Utilizing biotechnology to hydrolyze cotton fibers into fermentable sugars can realize the resource utilization of waste textiles and increase the added value of waste resources. However, in existing research, cellulose hydrolysis products are mostly used to produce low-value-added products such as fuel ethanol or organic acids, limiting their overall economic value, and there is a lack of technological pathways for the high-value utilization of waste textiles.

[0004] Polyhydroxyalkanoates (PHAs) are a class of biodegradable polymers synthesized by microorganisms. Poly(3-hydroxybutyrate) (PHB) is the most typical type of PHA biodegradable material, exhibiting good biocompatibility and degradability, and showing broad application prospects in packaging materials, medical materials, and other fields. Currently, the industrial production of PHAs mainly uses primary raw materials such as glucose and vegetable oils as carbon sources, which is not only costly but also dependent on food resources.

[0005] Therefore, developing a method for utilizing waste textiles to produce fermentable sugars through hydrolysis and further biosynthesizing polyhydroxyalkanoates is of great theoretical and practical significance for improving the resource utilization efficiency of waste textiles, expanding the low-cost raw material sources for polyhydroxyalkanoates, and realizing the high-value conversion and utilization of waste biomass resources. Summary of the Invention

[0006] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a method for preparing polyhydroxyalkanoates (PHA) through fabric bioconversion. By using alkali treatment and enzymatic hydrolysis to fully destroy the crystalline structure of cotton fibers in oxygen-containing fabrics, the enzymatic accessibility of cellulose is improved, allowing it to be directly used in the biosynthesis of PHAs. This achieves high-value conversion and utilization of waste textiles, thereby solving the technical problem of low cellulose enzymatic hydrolysis efficiency and difficulty in directly using it for PHA synthesis in existing technologies.

[0007] To achieve the objective of this invention, a method for preparing polyhydroxyalkanoates using fabric bioconversion is provided, comprising the following steps: (1) After treating the cotton-containing fabric with alkali, a bio-enzymatic hydrolysis reaction is carried out to fully destroy the crystalline structure of the cotton fibers in the cotton-containing fabric. (2) After sterilizing the enzymatic hydrolysate obtained in step (1), use it as a carbon source to inoculate polyhydroxy fatty acid ester synthesizing bacteria for fermentation culture, so that the bacteria accumulate polyhydroxy fatty acid esters.

[0008] As a preferred embodiment of the present invention, in step (1), the alkaline solution used for the alkaline treatment is an aqueous solution of NaOH and KOH, the concentration of the alkaline solution is 1-15 wt%, the alkaline treatment temperature is -20-0℃, and the alkaline treatment time is 1-6 h; preferably, the concentration of the alkaline solution is 7-15 wt%, the alkaline treatment temperature is -20-0℃, and the alkaline treatment time is 1-6 h.

[0009] As a preferred embodiment of the present invention, in step (1), the enzymatic hydrolysis reaction specifically includes: washing the alkali-treated cotton fabric to neutrality with water, and then carrying out the enzymatic hydrolysis reaction with cellulase and β-glucosidase in a buffer system.

[0010] The alkali used in the alkali treatment is an aqueous solution of NaOH and KOH with a concentration of 1-15 wt%, the alkali treatment temperature is -20 to 0℃, and the alkali treatment time is 1-6 h.

[0011] As a preferred embodiment of the present invention, in step (1), the enzymatic hydrolysis specifically includes: washing the alkali-treated cotton fabric to neutral with water, and then carrying out the enzymatic hydrolysis reaction with cellulase and β-glucosidase in a buffer system.

[0012] As a preferred embodiment of the present invention, the cellulase has a concentration of 40-60 FPU / g, and the β-glucosidase has a concentration of 40-60 IU / g.

[0013] As a preferred embodiment of the present invention, in step (1), the conditions for the enzymatic hydrolysis reaction are: water bath at 45-55°C, processing speed at 150-200 r / min, and hydrolysis time at 24-168 h.

[0014] As a preferred embodiment of the present invention, in step (1), the cotton content in the cotton-containing fabric is ≥50%.

[0015] As a preferred embodiment of the present invention, in step (2), the sugar concentration of the enzymatic hydrolysate is 2~80 g / L, preferably 5~50 g / L.

[0016] As a preferred embodiment of the present invention, in step (2), the polyhydroxy fatty acid ester synthesizing strain is *Rhodotorula roximatei*. Ralstoniaeutropha H16 or Pseudomonas Pseudomonas .

[0017] As a preferred embodiment of the present invention, in step (2), the fermentation culture conditions are as follows: prepare the seed liquid of the polyhydroxy fatty acid ester synthesizing strain, inoculate and ferment, the initial OD600 of the fermentation medium after inoculation is 0.1-0.2, the fermentation temperature is 28-32℃, the pH is 6.5-7.5, the rotation speed is 150-250 r / min, and the fermentation time is 24-120 h.

[0018] As a preferred embodiment of the present invention, in step (2), the fermented cells are separated, dried, and extracted with an organic solvent to obtain the polyhydroxy fatty acid ester product. In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages: 1. This invention provides a method for preparing polyhydroxyalkali esters (PHA) through the bioconversion of textiles. Alkali treatment and enzymatic hydrolysis work synergistically to achieve deep saccharification of cotton fibers. Alkali treatment not only causes swelling but, more importantly, significantly reduces the crystallinity of cotton fibers. Due to the high natural crystallinity, dense structure, and stable hydrogen bond network of cotton fibers, direct enzymatic hydrolysis is inefficient, limiting subsequent bioconversion processes. Alkali pretreatment helps weaken intermolecular hydrogen bonds in cellulose molecules, reduces structural order, and improves enzyme accessibility to the substrate, thereby enhancing saccharification efficiency. Based on this, a cellulase complex system is subsequently used for enzymatic hydrolysis, completely hydrolyzing the cotton fibers into glucose monomers, significantly improving enzymatic hydrolysis efficiency and glucose yield. The hydrolysate obtained by this method can be directly used as the sole carbon source for subsequent PHA fermentation without any separation steps. Therefore, this application, for the first time, seamlessly connects the two key steps of "alkali treatment-enzymatic saccharification" and "PHA fermentation" in waste cotton textiles, constructing a direct bioconversion pathway from solid waste to biodegradable high-value polymers.

[0019] 2. The present invention preferably conducts the alkali treatment process under low-temperature conditions. The main purpose of choosing this low-temperature condition is to ensure that the alkali solution effectively swells the cellulose structure and breaks hydrogen bonds, while significantly inhibiting the excessive degradation of cellulose and the occurrence of side reactions by the alkali, reducing the loss of sugars, thereby improving the yield of sugars in subsequent enzymatic hydrolysis. At the same time, it can also avoid excessive damage to other components in the fabric, improve the integrity of the substrate structure, and facilitate the graded utilization and resource recycling of different components in the blended material.

[0020] 3. The cotton content in the cotton-containing fabric of this invention is preferably ≥50%, which ensures that the sugar concentration of the enzymatic hydrolysate meets the fermentation requirements and that the crystalline structure of the cotton fibers is fully destroyed. Furthermore, this invention has great adaptability to the range of cotton content and other components in cotton-containing fabrics, and therefore can be widely applied to the recycling and treatment of various waste fabrics.

[0021] In summary, this invention utilizes alkali treatment and enzymatic hydrolysis to fully destroy the crystalline structure of cotton fibers in cotton-containing fabrics, and then directly uses the hydrolysate for subsequent polyhydroxyalkanoate fermentation. This achieves continuous process integration from pretreatment and hydrolysis to biosynthesis of waste textiles, improves resource conversion efficiency, realizes the conversion of waste biomass into high-value-added materials, broadens the carbon source sources for polyhydroxyalkanoate production, and reduces dependence on grain sugar sources. Attached Figure Description

[0022] Figure 1 This is a comparison of the scanning electron microscope microstructure properties of waste textiles, pure cotton fibers, and pure polyester fibers after treatment with 12% NaOH at a low temperature of -20℃ for 2 hours in Example 1.

[0023] Figure 2 The results of thermogravimetric analysis (TGA) and derivative thermogravimetric analysis (DTG) of waste textiles used in Example 1 and Comparative Example 1 are shown in the figure.

[0024] Figure 3 The graph shows the change in crystallinity of waste textiles before and after enzymatic hydrolysis when treated with 12% NaOH at a low temperature of -20°C for 2 hours in Example 1.

[0025] Figure 4 The image shows the infrared chromatograms of the waste textiles before and after enzymatic hydrolysis when treated with 12% NaOH at a low temperature of -20°C for 2 hours in Example 1.

[0026] Figure 5 This is a graph showing the sugar yield of waste textiles under different loading amounts in Example 2.

[0027] Figure 6 The graph shows the sugar yield of waste textiles in Example 2 at different enzymatic hydrolysis reaction times.

[0028] Figure 7 Comparative Example 2: A comparison of the appearance of waste textiles after low-temperature alkali treatment and after acid-alkali high-temperature treatment. Figure 7 The left image shows the appearance of waste textiles after low-temperature alkali treatment, while the right image shows the appearance of waste textiles after acid-alkali high-temperature treatment.

[0029] Figure 8 This is a diagram of the synthesis of polyhydroxy fatty acid esters by shake-flask fermentation of waste textiles in Example 2.

[0030] Figure 9 Figure 3 shows the synthesis of polyhydroxyalkanoates from untreated textiles after enzymatic fermentation. Figure 10 This is a magnified image of a single batch of polyhydroxy fatty acid esters synthesized by fermentation in a small container of waste textiles in Example 3.

[0031] Figure 11 This is a diagram of the fermentation of waste textiles in a small container corresponding to Example 3. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0033] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing polyhydroxyalkanoates using fabric bioconversion includes the following steps: (1) After treating the cotton fabric with alkali, a biological enzymatic hydrolysis reaction is carried out to fully reduce the crystallinity of cotton cellulose in the cotton fabric and fully destroy its crystal structure, so as to obtain a glucose-containing enzymatic hydrolysate and solids corresponding to other components of the fabric. After alkali treatment and biological enzymatic hydrolysis, a fermentable glucose-containing enzymatic hydrolysate and other solid components of the fabric are obtained. The other components of the fabric are recovered, and the glucose-containing enzymatic hydrolysate is used as a fermentation culture medium for subsequent fermentation.

[0034] (2) After sterilizing the enzymatic hydrolysate obtained in step (1), use it as a carbon source to inoculate polyhydroxy fatty acid ester synthesizing bacteria for fermentation culture, so that the bacteria accumulate polyhydroxy fatty acid esters.

[0035] Preferably, the alkaline solution used for alkaline treatment is an aqueous solution of NaOH and KOH, with an alkaline solution concentration of 1-15 wt%, an alkaline treatment temperature of -20 to 0°C, and an alkaline treatment time of 1-6 h; more preferably, the alkaline solution concentration is 7-15 wt%, the alkaline treatment temperature is -20 to 0°C, and the alkaline treatment time is 1-6 h; more preferably, under the low-temperature alkaline treatment conditions of the present invention, when the NaOH concentration is 10%-12% and the treatment time is 1-4 h, the enzymatic hydrolysis sugar yield of waste textiles is >90%.

[0036] During alkali treatment, the high natural crystallinity, dense structure, and stable hydrogen bond network of cotton fibers typically result in low direct enzymatic hydrolysis efficiency, limiting subsequent biotransformation processes. Alkali pretreatment helps weaken intermolecular hydrogen bonds in cotton cellulose, reduces structural order, and improves enzyme accessibility to substrates, thereby enhancing saccharification efficiency. For cotton-containing blended systems, alkali treatment is a low-temperature reduction process that not only improves the sugar release efficiency of the cotton component but also avoids excessive damage to other components of the fabric, enabling the graded utilization of different components in the blended material. In particular, choosing low-temperature conditions of -20 to 0°C for alkali treatment aims to significantly inhibit excessive degradation and side reactions of cellulose by the alkali while ensuring effective swelling and hydrogen bond disruption of the cellulose structure, reducing sugar loss and thus increasing the yield of sugars from subsequent enzymatic hydrolysis. Low-temperature treatment also slows down the chemical damage of alkali to other fiber components, facilitating component separation and resource utilization in blended materials. In practice, the degree of alkali treatment needs to be controlled until the cotton-containing fabric structure is noticeably loosened and hydrogen bonds are effectively broken, but the fibers have not yet undergone severe dissolution or excessive degradation.

[0037] Specifically, the degree of treatment is preferably controlled by synergistic effects of the alkaline solution concentration (10-15 wt%), treatment time (1-6 h), and treatment temperature (-20-0℃). At lower temperatures, the treatment time can be appropriately extended or the concentration increased to maintain sufficient action; conversely, at temperatures close to 0℃, the time can be appropriately shortened or the concentration reduced. Preferably, after subsequent washing to neutrality, the enzymatic sugar yield of the treated cotton fabric should be no less than 90%, the accessibility of cellulase should be significantly improved, and the physical form should retain a partially solid state to facilitate solid-liquid separation.

[0038] Compared to existing technologies, without alkali treatment, the cotton fiber's crystalline structure remains intact, resulting in low enzyme accessibility and a significant decrease in enzymatic sugar yield. This, in turn, affects the carbon source supply and yield of polyhydroxyalkanoate fermentation and makes it difficult to separate and recover cotton fibers from other components in cotton-containing fabrics. Alternatively, combined acid-alkali treatment can lead to side reactions in the enzymatic hydrolysate and the production of furfural inhibitors, preventing microbial growth and inhibiting product synthesis. Furthermore, heat or acid treatment can reach the PET degradation threshold, carbonize glucose, produce numerous inhibitors and side reactions, hindering normal microbial growth.

[0039] Preferably, the enzymatic hydrolysis reaction specifically includes: washing the alkali-treated cotton fabric to neutral pH, followed by enzymatic hydrolysis with cellulase and β-glucosidase in a buffer system. Specifically, the enzymatic hydrolysis can also employ one or more combinations of the following systems: a polysaccharide monooxygenase system, a cellobiose phosphorylase system, a cellobiase and glucosidase synergistic system, or a crude enzyme solution system, to improve saccharification efficiency, reduce product inhibition, and promote the formation of cellobiose and glucose.

[0040] Preferably, the enzymatic hydrolysis involves 40-60 FPU cellulase and 40-60 IU β-glucosidase, more preferably 50 FPU cellulase and 60 IU β-glucosidase, respectively. The hydrolysis conditions are a water bath at 45-55°C, a rotation speed of 150-200 r / min, and a hydrolysis time of 24-168 h. An appropriate ratio of cellulase to β-glucosidase reduces cellobiose accumulation, thereby relieving product inhibition and increasing glucose yield. Further preference is given to 50 FPU cellulase and 60 IU β-glucosidase, which ensures high saccharification efficiency while avoiding increased costs due to excessive enzyme use. Regarding reaction temperature, the preferred temperature range corresponds to the optimal reaction temperature for most cellulases. This condition maintains enzyme activity, promotes effective substrate-enzyme binding, and avoids enzyme inactivation due to excessively high temperatures. Regarding oscillation speed, moderate oscillation can increase the contact frequency between the enzyme and the substrate, enhance mass transfer, and thus promote the enzymatic hydrolysis reaction; however, the speed should not be too high to prevent shear force from damaging the enzyme or substrate structure. In addition, because cotton fibers have a high degree of crystallinity and a relatively slow enzymatic hydrolysis rate, appropriately extending the hydrolysis time helps to ensure that hydrolysis is complete.

[0041] Preferably, the cotton content in the cotton-containing fabric is ≥50%. This serves two purposes: firstly, it ensures that the sugar concentration of the enzymatic hydrolysate meets the fermentation requirements. When the cotton content is ≥50%, a suitable concentration of glucose solution can be obtained after enzymatic hydrolysis, serving as a carbon source for polyhydroxyalkanoate synthesizing bacteria, supporting cell growth and product accumulation, and preventing a decrease in fermentation efficiency due to excessively low sugar concentration. Secondly, it ensures that the crystalline structure of the cotton fibers is fully destroyed. When the cotton content is high, the alkali treatment and enzymatic hydrolysis are concentrated on the cotton fibers, which is beneficial for the full destruction of the hydrogen bond network and crystalline regions of cellulose, improving enzyme accessibility and hydrolysis efficiency. If the cotton content is too low, the synthetic fiber components may encapsulate or isolate the cotton fibers, reducing the treatment effect.

[0042] Furthermore, other synthetic fiber components in cotton-containing fabrics include, but are not limited to, polyethylene terephthalate, polyamide, polyacrylonitrile, polyurethane, or polypropylene. Because this invention has broad applicability to the range of cotton content and other components in cotton-containing fabrics, it can be widely applied to the recycling and processing of various types of waste fabrics. Specifically, this invention provides a method for preparing polyhydroxyalkanoates using the bioconversion of waste fabrics.

[0043] Preferably, the concentration of the enzymatic hydrolysate is 2-80 g / L, more preferably 5-50 g / L. If the sugar concentration is too low, it will not meet the carbon source requirements for the growth and product accumulation of polyhydroxyalkanoate synthesizing bacteria, resulting in a prolonged fermentation cycle and reduced product yield. If the concentration is too high, it may cause substrate inhibition or an increase in osmotic pressure, inhibiting the metabolic activity of the bacteria and thus reducing the synthesis efficiency of polyhydroxyalkanoate. Therefore, a suitable sugar concentration can ensure normal bacterial growth while achieving efficient carbon source conversion and improving the yield and productivity of polyhydroxyalkanoate. The concentration of the enzymatic hydrolysate obtained by the reaction in step (1) of this invention is adjusted to between 2-80 g / L by adjusting the amount of substrate added and by concentration.

[0044] Preferably, the polyhydroxyalkanoate synthesizing strain is *Rhodotorula gravidarum*. Ralstoniaeutropha H16 Pseudomonas Pseudomonas When the polyhydroxyalkanoate synthesizing strain is *Ralstonia eutropha* H16, the resulting polyhydroxyalkanoate is poly(3-hydroxybutyric acid). For example, the polyhydroxyalkanoate synthesizing strain is *Ralstonia eutropha* H16. A seed culture of *Ralstonia eutropha* H16 is prepared and inoculated for fermentation. That is, through the corresponding synthesizing strain, a polyhydroxyalkanoate with a controllable structure can be obtained.

[0045] Preferably, the fermentation conditions are as follows: prepare seed culture of polyhydroxyalkanoate synthesizing strain, inoculate and ferment, the initial OD600 of the fermentation medium after inoculation is 0.1-0.2, the fermentation temperature is 28-32℃, the pH is 6.5-7.5, the rotation speed is 150-250 r / min, and the fermentation time is 24-120 h.

[0046] Preferably, the seed culture is LB medium.

[0047] Preferably, the fermented cells are separated, dried, and extracted with an organic solvent to obtain the polyhydroxyalkanoate product. The cells are collected by centrifugation at 5000-8000 r / min for 3-10 min, then frozen at -80℃ for 6-12 h, followed by vacuum freeze-drying for 12-24 h. The freeze-dried powder is then collected by grinding. The organic solvent is methanol, chloroform, or other mixed solutions.

[0048] A polyhydroxy fatty acid ester prepared by the above method is characterized by its uniqueness and the absence of inhibitors.

[0049] The present invention will be further described in detail below with reference to specific embodiments.

[0050] Example 1: The effect of low-temperature alkaline treatment on the enzymatic hydrolysis and sugar production of waste textiles This example illustrates the effect of low-temperature alkaline treatment on the enzymatic hydrolysis of waste textiles to produce sugars. The specific experimental procedure is described below: The waste textile raw materials used in this example are derived from industrial waste. Thermogravimetric analysis showed that the raw materials contain two components: cotton fibers and polyethylene terephthalate (PET). Figure 1 , Figure 2 Of the raw materials, cotton fiber accounted for 63.36 wt%.

[0051] In this experiment, 30 g / L of waste textiles were placed in NaOH alkaline solutions of different concentrations (1%, 3%, 5%, 7%, 10%, 12%, and 15%) and treated at -20°C for 1–6 h. The textiles were then removed, washed with water until neutral, dried to constant weight, and placed in an enzyme solution of 50 FPU cellulase and 60 IU β-glucosidase. The reaction was carried out in a water bath at 50°C for 72 h at a rotation speed of 150 r / min. After the reaction, the supernatant was centrifuged and filtered, and eluted using a high-performance liquid chromatography (HPLC) system equipped with a differential refractive index detector and an Aminex HPX-87H column (300 × 7.8 mm, 9 μm; Bio-Rad). Elution was performed isocratically with 0.005 M sulfuric acid (0.4 mL / min) at 60°C. The glucose content was determined by comparing the retention time / peak area with that of the standard, and the corresponding glucose yield was calculated. Each treatment was performed in triplicate.

[0052] The formula for calculating glucose yield is as follows: Glucose yield =

[0053] Table 1. Experimental results of low-temperature alkaline treatment on the enzymatic hydrolysis and sugar production of waste textiles.

[0054] The data in Table 1 are expressed as mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences between different treatment times at the same NaOH concentration (P < 0.05); data containing the same lowercase letter indicate no significant difference. The F-value and P-value are the results of one-way ANOVA of the effect of different treatment times on the enzymatic hydrolysis sugar production rate at the same NaOH concentration. P < 0.05 indicates that the treatment time has a significant effect on the enzymatic hydrolysis sugar production rate.

[0055] The experimental results of this embodiment are shown in Table 1. Under the conditions described in this invention, the NaOH concentration and treatment time both had a significant impact on the enzymatic sugar yield (P<0.05). When the NaOH concentration was 1%-3%, the sugar yield was generally low at each time point, with maximum values ​​of 34.81% and 48.79%, respectively. When the NaOH concentration was increased to 5%-7%, the sugar yield significantly improved. Specifically, the sugar yield reached 82.98% after 4 h of treatment with 7% NaOH and 87.12% after 5 h, showing a significant improvement in the lower concentration treatment group. When the NaOH concentration was 10%-12%, the sugar yield reached a relatively high level. The sugar yield was 97.04% after 2 h of treatment with 10% NaOH and reached 99.41% after 2 h of treatment with 12% NaOH, which was the highest value measured in this experiment. This indicates that within this concentration range, low-temperature alkaline treatment can significantly improve the efficiency of substrate enzymatic hydrolysis. When the NaOH concentration was further increased to 15%, the sugar yield did not continue to increase, and it showed a downward trend when the treatment time was extended to 5-6 h, indicating that excessively high alkali concentration or excessively long treatment time is not conducive to subsequent enzymatic hydrolysis reaction.

[0056] like Figure 3 and Figure 4 As shown, structural analysis of textiles treated with 12% NaOH for 2 h revealed that after alkali treatment and enzymatic hydrolysis, the characteristic signals of cellulose were significantly weakened, while the characteristic absorption peaks of ester groups in PET remained clearly visible. At the same time, the diffraction characteristics of the samples gradually shifted from cellulose-dominated to polyester-dominated, and the apparent crystallinity decreased significantly, indicating that the ordered crystalline regions of cellulose were significantly damaged.

[0057] The results of this embodiment show that, under the low-temperature alkaline treatment conditions described in this invention, alkaline treatment causes cellulose to swell, weaken the hydrogen bond network, and partially decrystallize, thereby improving its accessibility to enzymes and promoting rapid enzymatic hydrolysis. More preferably, when the NaOH concentration is 12% and the treatment time is 2 h, the enzymatic sugar yield reaches 99.41%.

[0058] Comparative Example 1: This comparative example illustrates the effect of using and not using low-temperature alkaline treatment on the yield of enzymatically hydrolyzed sugars from waste textiles. The specific experimental procedures are described below: To verify the effect of low-temperature alkali treatment on the enzymatic hydrolysis of waste textiles, this comparative example, based on the method in Example 1, used untreated waste textiles (CK) and waste textiles treated with 12% NaOH at low temperature for 2 h as substrates. Enzymatic hydrolysis experiments were conducted under the same conditions, and the glucose yield was measured under different substrate addition amounts. The substrate addition amounts were 10 g / L, 20 g / L, 30 g / L, 40 g / L, and 50 g / L. The experimental results are as follows: Figure 5 As shown.

[0059] The results showed that, under all substrate addition conditions, waste textiles treated with low-temperature alkali exhibited significantly higher glucose yields. Specifically, within the substrate concentration range of 10–30 g / L, the glucose yield of the untreated group was only about 33%–35%, while after treatment with 12% NaOH for 2 h, the glucose yield significantly increased to about 98%–99%. When the substrate concentration was increased to 40 g / L, the glucose yield of the untreated group decreased to about 24%, while the alkali-treated group still maintained a glucose yield of about 68%. Further increasing the substrate concentration to 50 g / L, the glucose yield of the untreated group was only about 16%–17%, while the alkali-treated group still reached about 56%–58%.

[0060] like Figure 6 As shown, the optimal alkaline treatment conditions (12% NaOH treatment for 2 hours) from Example 1 were selected for optimization of the enzymatic hydrolysis time. The results showed that optimizing the hydrolysis time with a substrate concentration of 30 g / L revealed that the sugar yield initially increased and then decreased with increasing hydrolysis time, reaching a peak of 99.5% on the third day, which was 65.73% higher than the control group. This indicates that after low-temperature alkaline treatment, the previously inaccessible cotton cellulose components in the substrate were effectively activated, allowing for rapid hydrolysis in the initial stages of enzymatic hydrolysis.

[0061] The results indicate that low-temperature alkaline treatment can significantly improve the enzymatic hydrolysis efficiency of cellulose in waste textiles. Compared with untreated samples, waste textiles treated with low-temperature alkaline treatment showed significantly increased glucose yields under various substrate concentration conditions, indicating that this pretreatment method can effectively improve the enzymatic hydrolysis performance of waste textiles, thereby increasing the efficiency of subsequent fermentation and utilization.

[0062] Comparative Example 2: This comparative study investigated the effects of different treatment methods on the enzymatic hydrolysis and saccharification efficiency of waste textiles and the subsequent fermentation process. Based on the method in Example 1, the following treatment groups were set up: Group CK (low-temperature alkali treatment): treated with 5 wt% NaOH solution at -20℃ for 2 h; Group A (acid-alkali high-temperature treatment): treated with a combination of 1 wt% H₂SO₄ solution and 5 wt% NaOH solution at 115℃ for 30 min. Experimental results are as follows: Figure 7 As shown.

[0063] like Figure 7As shown in the right figure, the results indicate that after acid-alkali high-temperature treatment, the appearance of waste textiles underwent significant color changes, and fermentation inhibitors were detected during the enzymatic hydrolysis and saccharification process. In contrast, the waste textiles treated with low-temperature alkali (CK group) maintained a good appearance, without significant color changes, and retained their shape, indicating milder treatment conditions. During acid-alkali high-temperature treatment, the combined action of strong acid and strong alkali under high-temperature conditions easily triggers excessive degradation of cellulose and side reactions, such as the dehydration of sugars to generate inhibitors like furfural and hydroxymethylfurfural. These inhibitors have significant toxic effects on subsequent microbial fermentation, severely affecting the growth and product accumulation of polyhydroxyalkali synthesizing bacteria. Furthermore, high-temperature treatment may also cause thermal oxidation or degradation of other components in the fabric, producing colored substances and affecting substrate quality.

[0064] In comparison, such as Figure 7 As shown in the left figure, the low-temperature alkali treatment (-20℃) used in this invention is mild. The alkali solution mainly affects the cellulose structure through swelling and hydrogen bond disruption, avoiding excessive degradation of cellulose and side reactions. Therefore, it does not produce fermentation inhibitors and does not cause significant changes in the appearance and shape of textiles. This allows the treated enzymatic hydrolysate to be directly used as a carbon source for PHA fermentation without additional detoxification steps, which simplifies the process, reduces production costs, and improves fermentation stability.

[0065] In summary, the low-temperature alkali treatment preferred in this invention has significant advantages over the acid-alkali high-temperature combination treatment, such as mild reaction conditions, good substrate structural integrity, no inhibitors generated, and high fermentation compatibility.

[0066] Example 2: Experimental results of the synthesis of polyhydroxyalkanoates by shake-flask fermentation of waste cotton fiber hydrolysate. This example presents the experimental results of synthesizing polyhydroxyalkanoates by shake-flask fermentation of waste cotton fiber hydrolysate. The specific experimental procedure is described below: The enzymatic hydrolysate from Example 1, treated with 12% NaOH at -20°C for 2 h, was placed in a 250 mL Erlenmeyer flask and adjusted to pH 6.5-7.5. It was then autoclaved at 115°C for 30 min. After cooling to room temperature, *Rhodotorula elegans* H16 was inoculated with an initial inoculum concentration of OD600 = 0.1-0.2. Simultaneously, 35 mL / L phosphate buffer, 1 mL / L Stock A, and 1 mL / L Trace element solution were added to the culture system. The culture was incubated with shaking at 30°C for 1-5 days. After fermentation, the cells were collected by centrifugation at 6000 r / min for 5 min. The cells were then frozen at -80°C for 6-12 h and freeze-dried under vacuum for 12-24 h to obtain lyophilized cell powder. 0.1 g of the lyophilized powder was weighed and placed in a heat-resistant sealed reaction tube. 2 mL of reaction solution (containing 1.7 mL methanol and 0.3 mL 98% sulfuric acid) was added, followed by 2 mL of chloroform. After sealing, the mixture was thoroughly vortexed for 30 s and shaken for 5-10 min. The reaction was then carried out at 100℃ for 140 min. After the reaction, the mixture was cooled to room temperature, 1 mL of ultrapure water was added, and the mixture was briefly vortexed and shaken for 5-10 min. The mixture was centrifuged at 3000 r / min for 5 min, and the lower organic phase was transferred to a GC sample vial. After drying to near dryness under nitrogen, 1 mL of n-hexane was added to dissolve the methyl hydroxybutyrate (MOH) to obtain the polyhydroxybutyrate. The MOH was filtered through a 0.22 μm PTFE membrane and transferred to a GC sample vial for analysis using gas chromatography-mass spectrometry (GC-MS). The content of polyhydroxybutyrate was calculated by detecting the peak area of ​​methyl 3-hydroxybutyrate.

[0067] To investigate the effects of different culture conditions on the synthesis of polyhydroxyalkanoates, shake-flask culture experiments were conducted under various fermentation conditions, including pH 6.0, 6.5, 7.0, 7.5, and 8.0; temperatures of 15℃, 20℃, 25℃, 30℃, and 35℃; shaker speeds of 50 r / min, 100 r / min, 150 r / min, 200 r / min, and 250 r / min; and fermentation times of 1 day, 2 days, 3 days, 4 days, and 5 days. Each experiment was conducted in triplicate.

[0068] The formulas for calculating the yield and production of polyhydroxyalkanoates are as follows: Yield of polyhydroxy fatty acid esters (%) =

[0069] Polyhydroxyalkanoate yield (g / L) = Cell dry weight × Polyhydroxyalkanoate yield The test results of this embodiment are as follows: Figure 8 As shown: (1) Effect of pH on the synthesis of polyhydroxy fatty acid esters Fermentation experiments were conducted within a pH range of 6.0–8.0. Results showed that as the culture medium pH increased from 6.0 to 7.0, cell dry weight (CDW) and polyhydroxyalkanoate (PHA) accumulation gradually increased, reaching a high level at pH 7.0, where the cell dry weight was approximately 2.3–2.5 g / L, the PHA content was approximately 1.8–2.0 g / L, and the PHA percentage of the cell dry weight was approximately 80 wt%. When the pH was further increased to 7.5 and 8.0, the PHA content and yield decreased slightly. Preferably, pH 7.0 was the most suitable culture condition.

[0070] (2) Effect of culture temperature on the synthesis of polyhydroxyalkanoates Culture experiments were conducted within a temperature range of 15-35℃. Results showed that as the culture temperature increased, cell dry weight and polyhydroxyalkanoate (PHA) accumulation gradually increased, reaching a high level at 30℃, with a cell dry weight of approximately 3.3 g / L, a PHA content of approximately 1.3-1.5 g / L, and a PHA yield of approximately 40-45 wt%. When the temperature continued to rise to 35℃, the PHA content decreased slightly. Preferably, 30℃ is the suitable culture temperature.

[0071] (3) Effect of shaking speed on the synthesis of polyhydroxy fatty acid esters Fermentation experiments were conducted at speeds of 50-250 r / min. Results showed that with increasing shaker speed, cell dry weight and polyhydroxyalkanoate (PHA) accumulation gradually increased, reaching a high level at 200 r / min, with cell dry weight approximately 3.1 g / L, PHA content approximately 1.0-1.1 g / L, and PHA yield approximately 35 wt%. When the shaker speed was further increased to 250 r / min, the PHA content decreased slightly. Preferably, 200 r / min was the optimal culture speed.

[0072] (4) Effect of fermentation time on the synthesis of polyhydroxy fatty acid esters Experiments were conducted within a culture time range of 1–5 days. Results showed that cell dry weight and polyhydroxyalkanoate (PHA) accumulation gradually increased with prolonged culture time, reaching a high level on day 4, with a cell dry weight of approximately 5.4 g / L, a PHA content of approximately 1.8–2.0 g / L, and a PHA yield of approximately 35 wt%. When the culture time was extended to day 5, the PHA content decreased slightly.

[0073] The results showed that when the culture conditions were pH approximately 7.0, temperature approximately 30℃, shaker speed approximately 200 r / min, and fermentation culture for 4 days, the cell growth and accumulation of polyhydroxy fatty acid esters reached a high level. This further indicates that the waste cotton fiber hydrolysate described in this invention can serve as an effective carbon source to support the synthesis of polyhydroxy fatty acid esters by Rochecholesterolus H16.

[0074] Comparative Example 3: This comparative study investigated the effect of untreated waste textiles as a carbon source on the fermentation synthesis of polyhydroxyalkali (PHA) under different enzymatic hydrolysis times. Using untreated waste textiles from Comparative Example 1 as the substrate, seven treatment groups were established with hydrolysis times of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, and 7 days. The hydrolysate was placed in 250 mL Erlenmeyer flasks, adjusted to pH 6.5-7.5, and autoclaved at 115 °C for 30 min. After cooling to room temperature, *Rhodotorula gravidarum* H16 was inoculated with an initial inoculum concentration of OD600 = 0.1-0.2. Simultaneously, 35 mL / L phosphate buffer, 1 mL / L Stock A, and 1 mL / L Trace element solution were added to the culture system. The culture was incubated at 30 °C with shaking for 1-5 days. After fermentation, the cells were collected by centrifugation at 6000 r / min for 5 min. Bacterial cells were frozen at -80℃ for 6-12 h and then freeze-dried under vacuum for 12-24 h to obtain lyophilized bacterial powder. 0.1 g of the lyophilized powder was weighed and placed in a heat-resistant sealed reaction tube. 2 mL of reaction solution (containing 1.7 mL methanol and 0.3 mL 98% sulfuric acid) was added, followed by 2 mL of chloroform. After sealing, the mixture was thoroughly vortexed for 30 s and shaken for 5-10 min. The mixture was then reacted at 100℃ for 140 min. After the reaction, the mixture was cooled to room temperature, and 1 mL of ultrapure water was added. The mixture was briefly vortexed and shaken for 5-10 min. The mixture was centrifuged at 3000 r / min for 5 min, and the lower organic phase was transferred to a GC sample vial. After drying to near-dryness under nitrogen, 1 mL of n-hexane was added to dissolve the hydroxy fatty acid methyl ester. The ester was filtered through a 0.22 μm PTFE membrane and transferred to a GC sample vial for analysis using gas chromatography-mass spectrometry (GC-MS). The results are shown below. Figure 9 As shown.

[0075] The PHA yield of untreated waste textiles was low at different enzymatic hydrolysis times (maximum only about 0.077 g / L). As the hydrolysis time increased from 1 day to 3 days, the PHA yield increased slightly, reaching a relative peak on day 3; thereafter, with further extension of the hydrolysis time (4–7 days), the PHA yield gradually decreased, reaching the lowest level on day 7 (about 0.034–0.036 g / L).

[0076] In untreated waste textiles, cotton fibers naturally exhibit high crystallinity, a dense structure, and a stable hydrogen bond network, making it difficult for cellulase to effectively access the substrate. This results in extremely low enzymatic hydrolysis and saccharification efficiency, and a significantly insufficient production of reducing sugars (glucose). Even with an extended hydrolysis time of 7 days, the lack of alkali treatment to disrupt the crystalline structure keeps the sugar concentration in the hydrolysate at a consistently low level, failing to meet the carbon source requirements for the growth of Roche's fungus H16 and PHA synthesis. Consequently, the overall PHA yield is low. This clearly demonstrates that the low-temperature alkali pretreatment employed in this invention is a crucial and indispensable step in achieving efficient saccharification of waste textiles and subsequent PHA fermentation synthesis.

[0077] Example 3: Experimental results of single-batch fermentation synthesis of polyhydroxyalkanoates from waste cotton fiber hydrolysate in small tanks. This example presents the experimental results of a single-batch fermentation synthesis of polyhydroxyalkanoates using waste cotton fiber hydrolysate in a small tank. Figure 11 As shown, the specific experimental procedure is described below: Based on the waste cotton fiber pretreatment and enzymatic hydrolysis methods described in Examples 1 and 2, this example uses the obtained enzymatic hydrolysate for a scale-up fermentation experiment in a small fermenter (5L).

[0078] The experimental group was set with a fermentation temperature of 30℃, an initial pH of 7.0, and a rotation speed of 300 r / min. During fermentation, samples were taken periodically to determine cell dry weight and polyhydroxyalkanoate (PHA) content. Fermentation samples were centrifuged at 6000 r / min for 5 min to collect the cells, which were then frozen at -80℃ and freeze-dried under vacuum to obtain cell powder. Subsequently, the PHA was converted to the corresponding hydroxyalkanoate methyl esters via acid methanol hydrolysis, and the powder was analyzed by GC-MS.

[0079] The test results of this embodiment are as follows: Figure 10 As shown: In the early stage of fermentation (1-2 days), the cells enter a rapid growth phase, with cell dry weight increasing from approximately 2 g / L to about 4 g / L, and polyhydroxyalkanoates (PHA) beginning to accumulate. As fermentation continues, cell dry weight and PHA accumulation reach high levels on days 3-4. On day 4, cell dry weight is approximately 5.5-5.8 g / L, and PHA content reaches approximately 5.0 g / L, with PHA accounting for the highest proportion of cell dry weight, approximately 90 wt%. When fermentation continues to days 5-7, as nutrients in the culture medium are gradually consumed, cell growth tends to stabilize, and the PHA content slightly decreases, with the proportion of PHA in cell dry weight decreasing to approximately 60-70 wt%.

[0080] The results show that using waste cotton fiber enzymatic hydrolysate as a carbon source, polyhydroxy fatty acid esters can be effectively synthesized under small-scale fermentation conditions. The bacterial cells can accumulate a high proportion of polyhydroxy fatty acid esters, indicating that the method of the present invention has good potential for fermentation scale-up applications.

[0081] In summary, this invention provides a method for preparing polyhydroxyalkanoates from waste textiles through high-value conversion. By subjecting waste textiles to alkaline treatment and enzymatic hydrolysis, the cotton fiber components are converted into fermentable sugar substrates, which are then further converted into biodegradable polyhydroxyalkanoates using microbial fermentation, thus realizing the resource utilization and high-value utilization of waste textiles.

[0082] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of protection of this invention and its equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for preparing polyhydroxyalkanoates using fabric bioconversion, characterized in that, Includes the following steps: (1) After treating the cotton-containing fabric with alkali, a bio-enzymatic hydrolysis reaction is carried out to fully destroy the crystalline structure of the cotton fibers in the cotton-containing fabric. (2) After sterilizing the enzymatic hydrolysate obtained in step (1), it is used as a carbon source to inoculate polyhydroxy fatty acid ester synthesizing bacteria for fermentation culture, so that the polyhydroxy fatty acid ester synthesizing bacteria accumulate polyhydroxy fatty acid esters during secondary metabolism.

2. The method for preparing polyhydroxyalkanoates using fabric bioconversion according to claim 1, characterized in that, In step (1), the alkaline solution used for the alkaline treatment is an aqueous solution of NaOH or KOH, the concentration of the alkaline solution is 1-15 wt%, the alkaline treatment temperature is -20-0℃, and the alkaline treatment time is 1-6 h; preferably, the concentration of the alkaline solution is 7-15 wt%, the alkaline treatment temperature is -20-0℃, and the alkaline treatment time is 1-6 h.

3. The method for preparing polyhydroxyalkanoates using fabric bioconversion according to claim 1, characterized in that, In step (1), the enzymatic hydrolysis reaction specifically includes: washing the alkali-treated cotton fabric to neutral with water, and then carrying out the enzymatic hydrolysis reaction with cellulase and β-glucosidase in a buffer system.

4. The method for preparing polyhydroxyalkanoates using fabric bioconversion according to claim 3, characterized in that, The cellulase content is 40-60 FPU / g, and the β-glucosidase content is 40-60 IU / g.

5. The method for preparing polyhydroxyalkanoates using fabric bioconversion according to claim 1, characterized in that, In step (1), the conditions for the biological enzymatic hydrolysis reaction are: water bath at 45-55℃, processing speed at 150-200 r / min, and enzymatic hydrolysis time at 24-168 h.

6. The method for preparing polyhydroxyalkanoates using fabric bioconversion according to claim 1, characterized in that, In step (1), the cotton content in the cotton-containing fabric is ≥50%.

7. The method for preparing polyhydroxyalkanoates using fabric bioconversion according to claim 1, characterized in that, In step (2), the sugar concentration of the enzymatic hydrolysate is 2~80 g / L, preferably 5~50 g / L.

8. The method for preparing polyhydroxyalkanoates using fabric bioconversion according to claim 1, characterized in that, In step (2), the polyhydroxy fatty acid ester synthesizing strain is *Rhodotorula roximatei*. Ralstoniaeutropha H16 or Pseudomonas Pseudomonas .

9. The method for preparing polyhydroxyalkanoates using fabric bioconversion according to claim 1, characterized in that, In step (2), the fermentation culture conditions are as follows: prepare the seed liquid of the polyhydroxy fatty acid ester synthesizing strain, inoculate and ferment, the initial OD600 of the fermentation medium after inoculation is 0.1-0.2, the fermentation temperature is 28-32℃, the pH is 6.5-7.5, the rotation speed is 150-250 r / min, and the fermentation time is 24-120 h.

10. The method for preparing polyhydroxyalkanoates using fabric bioconversion according to claim 1, characterized in that, In step (2), the fermented cells are separated, dried, and extracted with organic solvents to obtain polyhydroxy fatty acid ester products.

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