Application of Rhizopus oryzae nanofiber living gel in the treatment of lignocellulose biorefining wastewater

Abstract

This invention discloses the application of *Rhizopus oryzae* nanofiber living gel in the treatment of lignocellulose biorefining wastewater. The *Rhizopus oryzae* nanofiber living gel is prepared by fermentation of *Rhizopus oryzae*, with additional nanofibers added to the fermentation medium during the middle and late stages of the fermentation process. The *Rhizopus oryzae* nanofiber living gel is added to the lignocellulose biorefining wastewater for biological treatment. During the treatment process, the living gel adsorbs lignin from the wastewater. This lignin-adsorbed living gel possesses photothermal conversion capabilities and can be reused as a photothermal material. This invention applies *Rhizopus oryzae* nanofiber living gel to the treatment of lignocellulose biorefining wastewater, achieving effective treatment of the wastewater. Furthermore, the wastewater treatment method is highly operable, avoids secondary pollution, and reduces the economic cost of wastewater treatment.

Description

Technical Field

[0001] This invention belongs to the field of wastewater biological treatment technology, specifically relating to the application of Rhizopus oryzae nanofiber living gel in the treatment of wastewater from lignocellulose biorefining. Background Technology

[0002] Rhizopus oryzae, an important mold in Chinese medicine and brewing yeast, can saccharify starch, convert sucrose, and produce lactic acid, organic acids, and trace amounts of alcohol. Nanocellulose, as a novel biomass nanomaterial, not only possesses the renewable and biodegradable properties of natural cellulose, but also boasts advantages such as large specific surface area, high hydrophilicity, high transparency, high strength, high Young's modulus, and low coefficient of thermal expansion. This makes it possible to form various functional composite materials and has proven to be a promising self-assembled hydrogel or bio-based nanofiller.

[0003] Hydrogels are a class of polymer materials with a three-dimensional network structure made of synthetic and natural polymers. They can absorb large amounts of liquid in a short time and retain the liquid without dissolving. Due to their high water content and high elasticity, hydrogels are widely used in tissue fillers, drug carriers, cartilage regeneration, wound dressings, and environmental absorbents.

[0004] Living hydrogels are materials in which living cells (such as microorganisms, enzymes, or functional cells) are embedded in a three-dimensional polymer network structure, retaining the structural function of the gel while endowing it with bioactivity. Commonly used matrix materials include natural polymers (such as sodium alginate, chitosan, and gelatin) or synthetic polymers (such as polyethylene glycol diacrylate). Functionalized materials (such as nanocellulose and graphene oxide) improve the mechanical properties and biocompatibility of the gel. Combining these with biomaterials, and utilizing the extracellular polysaccharides or proteins secreted by microorganisms (such as fungi and bacteria) as the gel matrix, forms a "self-assembled" living gel. Preparation methods include physical cross-linking, chemical cross-linking, and 3D printing technology. These can be applied to pollutant degradation, catalysis and energy conversion, and the biomedical field.

[0005] Wastewater generated during the biorefining of lignocellulose is characterized by high concentrations of COD (Chemical Oxygen Demand), BOD (Biochemical Oxygen Demand), and complex components. Treatment challenges include: 1. High-molecular-weight organic matter (lignin and cellulose decomposition products); 2. Recalcitrant components: lignin derivatives (such as phenol and methoxybenzene) exhibit strong resistance to degradation; 3. High biotoxicity: certain intermediate products (such as aldehydes and organic acids) may be toxic to microorganisms. Traditional activated sludge processes have limited effectiveness in treating recalcitrant pollutants such as lignin derivatives. Highly toxic and concentrated pollutants can inhibit the activity of conventional microorganisms. Furthermore, the organic wastewater byproducts generated during the treatment process are not fully utilized, resulting in resource waste and high economic and environmental costs. Traditional treatment methods (such as chemical oxidation and physical adsorption) require high energy consumption or expensive chemical reagents, hindering large-scale implementation. Additionally, traditional lignocellulose biorefining wastewater treatment processes generate large amounts of solid waste, causing secondary pollution and negatively impacting environmental protection.

[0006] Lignin photothermal conversion technology is a technique that utilizes light energy to accelerate the decomposition of lignin and generate useful products, with broad application prospects, particularly in energy production and environmental protection. The basic principle of photothermal conversion is to irradiate lignin materials with a specific light source (such as sunlight, laser, or visible light). Through the absorption of light energy, the material's temperature rises, leading to the pyrolysis or chemical reaction of lignin molecules. The synergistic effect of photothermal and photocatalysis can improve lignin conversion efficiency, promoting the efficient utilization of biomass resources. Although lignin itself has certain light absorption characteristics, its photothermal conversion efficiency is usually low.

[0007] Therefore, in response to the problems of high COD and BOD wastewater treatment in the field of lignocellulose biorefining, there is an urgent need to develop a wastewater treatment technology for lignocellulose biorefining that has excellent treatment effect, low cost, and is environmentally friendly. Summary of the Invention

[0008] The purpose of this invention is to address the problem that existing technologies cannot effectively and economically treat lignocellulose biorefining wastewater, and that this causes secondary pollution to the environment, by providing a lignocellulose biorefining wastewater treatment technology that has excellent wastewater treatment effect, low cost, and is environmentally friendly.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] An application of Rhizopus oryzae nanofiber live gel in the treatment of lignocellulose biorefining wastewater, wherein the Rhizopus oryzae nanofiber live gel is prepared by fermentation of Rhizopus oryzae, and nanofibers are added to the fermentation culture medium in the middle and late stages of the fermentation process.

[0011] The *Rhizopus oryzae* nanofiber live gel is prepared by the following method: *Rhizopus oryzae* seed culture is inoculated into a fermentation medium at a volume ratio of 1% to 10%, and fermentation is carried out at a temperature of 30 to 40°C and a certain rotation speed; before the nanofibers are added to the fermentation medium, the rotation speed is 120 to 300 rpm; after the nanofibers are added to the fermentation medium, the rotation speed is 40 to 70 rpm.

[0012] Preferably, the *Rhizopus oryzae* nanofiber live gel is prepared by the following method: *Rhizopus oryzae* seed culture is inoculated into a fermentation medium at a volume ratio of 5%, and fermentation is carried out at a temperature of 30-40°C and a certain rotation speed; before the nanofibers are added to the fermentation medium, the rotation speed is 120-300 rpm; after the nanofibers are added to the fermentation medium, the rotation speed is 40-70 rpm.

[0013] The addition of nanofibers to the fermentation medium in the middle and late stages of the fermentation process refers to adding nanofibers to the fermentation medium after 10 to 72 hours of fermentation and continuing fermentation for 2 to 20 hours; preferably, nanofibers are added to the fermentation medium after 60 hours of fermentation and fermentation continues for 12 hours.

[0014] The nanofibers are any one of cellulose nanofibers, cellulose nanocrystals, and chitin nanofibers, preferably cellulose nanofibers. The carboxyl content of the cellulose nanofibers, cellulose nanocrystals, and chitin nanofibers must be 0.8–1.5 mmol / g. The cellulose nanofibers can be prepared by the method described in the literature (Tang X, Liu L, Wang Z, et al. Ahoneycomb-like hydrogel in-situ constructed by Streptococcus zooepidemicus and TOCN for the proliferation of bacteria[J]. Carbohydrate Polymers, 2022, 281:119099). The prepared cellulose nanofibers have a negatively charged surface and dimensions of: L (length): 200–500 nm, D (diameter): 10–20 nm; carboxyl content: 1.17 mmol / g; Zeta potential: -49 ± 1 mV. They can also be purchased commercially. The cellulose nanocrystals and chitin nanofibers are also commercially available.

[0015] The nanofibers are added to the fermentation culture medium in the form of a nanofiber dispersion; the nanofiber dispersion is a dispersion of the nanofibers in water; the mass fraction of the nanofibers in the nanofiber dispersion is 0.1% to 2%, preferably 0.5% to 2%; the nanofiber dispersion is added to the fermentation culture medium to obtain a mixed system, wherein the mass fraction of the nanofibers in the mixed system is 0.1% to 1%, preferably 0.5%.

[0016] Among them, Rhizopus oryzae includes, but is not limited to, Rhizopus oryzae ATCC20344.

[0017] The fermentation medium comprises: 10–40 g / L glucose, 0–1 g / L (NH4)2SO4, 0–1 g / L KH2PO4, 0–0.1 g / L FeSO4, 0.1–1 g / L MgSO4, 0–0.1 g / L ZnSO4, 10–40 g / L CaCO3, and 1–7% v / v citrate buffer; preferably, the fermentation medium comprises: 40 g / L glucose, 0.71 g / L (NH4)2SO4, 0.6 g / L KH2PO4, 0.004 g / L FeSO4·7H2O, 0.5 g / L MgSO4·7H2O, 0.01 g / L ZnSO4·7H2O, 30 g / L CaCO3, and 5% v / v citrate buffer.

[0018] The seed culture medium comprises: 10–40 g / L glucose, 2–5 g / L (NH4)2SO4, 0.2–1 g / L KH2PO4, 0–0.01 g / L FeSO4·7H2O, 0–1 g / L MgSO4·7H2O, and 0–0.2 g / L ZnSO4·7H2O; preferably, the seed culture medium comprises: 40 g / L glucose, 4.4 g / L (NH4)2SO4, 0.6 g / L KH2PO4, 0.005 g / L FeSO4·7H2O, 0.5 g / L MgSO4·7H2O, and 0.018 g / L ZnSO4·7H2O.

[0019] This invention uses Rhizopus oryzae as the main component, which is fermented in the fermentation medium to produce the organic acid fumaric acid. This fumaric acid, along with exogenously added nanofibers (the organic acid provides hydrogen bonds for gel formation), forms a living gel. The living gel is then added to the wastewater from the lignocellulose biorefining process for wastewater treatment.

[0020] The lignocellulose is lignocellulose from the Poaceae family and / or lignocellulose from the Woody family, preferably lignocellulose from the Poaceae family.

[0021] The method for applying the *Rhizopus oryzae* nanofiber live gel to the treatment of lignocellulose biorefining wastewater is as follows: the *Rhizopus oryzae* nanofiber live gel is added to the lignocellulose biorefining wastewater and treated for 1 to 10 days at a temperature of 30–40°C and a stirring speed of 50–80 rpm.

[0022] Preferably, the method for applying the *Rhizopus oryzae* nanofiber live gel to the treatment of lignocellulose biorefining wastewater is as follows: the *Rhizopus oryzae* nanofiber live gel is added to the lignocellulose biorefining wastewater and treated for 6 days at a temperature of 35°C and a stirring speed of 70 rpm.

[0023] In the process of treating the lignocellulose biorefining wastewater using the aforementioned *Rhizopus oryzae* nanofiber live gel, there is no special limitation on the amount of live gel added, with the standard being that the lignocellulose biorefining wastewater completely submerges the live gel; preferably, for every 100 mL of lignocellulose biorefining wastewater, 3 to 4 cylindrical live gels with a diameter of about 2 cm and a height of 2 to 3 cm are added.

[0024] In this process, the wastewater from the lignocellulose biorefining process is treated with the living gel of Rhizopus oryzae nanofibers to obtain gel solid waste, which is then reused as a photothermal material.

[0025] The gel solid waste is treated with laccase and then reused as a photothermal material.

[0026] Nanocellulose materials (such as TEMPO oxidized cellulose) serve as hydrogel matrices, providing excellent mechanical strength and porous structure to support the growth and activity of Rhizopus oryzae. Immobilization techniques can enhance the stability and recycling capacity of Rhizopus oryzae extracellular enzymes, thereby strengthening enzyme activity. Furthermore, nanofiber-based gels can not only degrade organic matter through Rhizopus oryzae but also adsorb heavy metals or toxic small molecules through the surface functional groups of cellulose.

[0027] Rhizopus oryzae nanofiber living gel has the following characteristics in the treatment of lignocellulose biorefining wastewater: 1. Highly efficient degradation of organic matter: Rhizopus oryzae can secrete a variety of extracellular enzymes (such as xylanase, lipase, and peroxidase), which can efficiently decompose lignin derivatives and other organic pollutants; 2. Reduced biotoxicity of pollutants: The nanofiber matrix provides a protective barrier for Rhizopus oryzae, reducing the toxic effects of high concentrations of pollutants on Rhizopus oryzae; 3. Improved resource utilization: Lignin degradation products (such as phenolic compounds and short-chain organic acids) can be further metabolized by microorganisms to generate valuable chemicals.

[0028] Rhizopus oryzae live gel can treat wastewater in two ways: 1. Biodegradation: The lignin-degrading enzymes and cellulases secreted by Rhizopus oryzae can break the chemical bonds (such as benzene rings and ether bonds) in complex macromolecular organic matter, converting them into simple small molecules (such as alcohols and phenols). 2. Physical adsorption: The nanofiber matrix has a high specific surface area and abundant functional groups (such as carboxyl and hydroxyl groups), which adsorb oxygen-containing organic matter and heavy metals in wastewater through electrostatic interactions and hydrogen bonding.

[0029] Lignin itself possesses certain light absorption properties, but its photothermal conversion efficiency is typically low. Adding laccase to lignin induces oxidation under its catalysis, forming more active oxidation products (such as quinones and free radicals). These oxidation products exhibit stronger light absorption and a wider light absorption wavelength range, thereby enhancing the gel's light absorption capacity and thus improving its photothermal conversion efficiency. The role of nanofibers in enhancing the photothermal conversion of living gels can be summarized as follows: improving light absorption and scattering capabilities, increasing thermal conductivity, providing more surface functional groups, promoting composite material formation, and exhibiting synergistic effects with photothermal materials. These characteristics make nanocellulose an ideal reinforcing agent, helping to improve the efficiency of living gels in photothermal conversion and expanding its application potential in environmental protection, energy conversion, and other fields. When lignin is adsorbed onto the surface of nanofibers and treated with laccase, the oxidation products of lignin interact with the nanofibers, forming a more compact composite structure. This structure is more conducive to light absorption and heat accumulation, improving the photothermal conversion efficiency of the gel. Meanwhile, laccase not only promotes the oxidation of lignin, but may also undergo cross-linking reactions with certain functional groups (such as phenolic hydroxyl groups and amino groups) in Rhizopus oryzae nanofiber gel, further enhancing the stability and structural integrity of the material. This cross-linking effect leads to a denser gel structure, thereby improving its heat accumulation effect under light conditions.

[0030] Rhizopus oryzae hyphae possess a strong adsorption capacity for organic pollutants such as lignin, and the composite material formed after Rhizopus oryzae nanofiber gel adsorbs lignin exhibits superior photothermal performance compared to nanofibers alone. After adsorbing lignin, the hyphae promote the distribution of lignin oxidation products, making them more uniformly distributed within the gel structure, thereby enhancing the overall photothermal conversion efficiency of the gel. Furthermore, through the catalytic action of laccase, lignin and its oxidation products can absorb light more efficiently and convert it into heat energy. Because these products are uniformly distributed in the gel and interact with the nanofibers, this localized photothermal effect is more concentrated, thus improving the overall photothermal effect of the gel. The Rhizopus oryzae hyphae themselves are also a biocatalyst, and in some cases, they can synergistically work with the lignin oxidation process, helping lignin to be oxidized by laccase more quickly and efficiently. This synergistic effect further enhances the photothermal conversion efficiency of lignin.

[0031] Rhizopus oryzae nanofiber-based living gel combines the high degradation capacity of Rhizopus oryzae with the structural advantages of nanofibers, providing a green, efficient, and sustainable solution for treating high COD and BOD wastewater generated in the field of lignocellulose biorefining. It not only significantly improves treatment efficiency and reduces environmental impact, but also enables the recycling of resources from wastewater and the secondary use of solid waste, providing strong support for the sustainable development of biorefining processes.

[0032] Beneficial effects:

[0033] 1. This invention applies Rhizopus oryzae nanofiber living gel to the treatment of lignocellulose biorefining wastewater. The living gel degrades and removes organic pollutants in the wastewater through biodegradation and physical adsorption, effectively reducing the COD and BOD content in the wastewater, thus achieving effective treatment of lignocellulose biorefining wastewater. Moreover, the wastewater treatment method is highly operable.

[0034] 2. This invention applies Rhizopus oryzae nanofiber live gel to the treatment of lignocellulose biorefining wastewater. After use, the live gel adsorbs lignocellulose, giving it photothermal conversion capabilities. It can be reused as a photothermal material, which not only avoids the generation of secondary pollution during wastewater treatment but also reduces the economic cost of wastewater treatment. Attached Figure Description

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0036] Figure 1 The images show the Rhizopus oryzae nanocellulose living gel from Example 3 and the gel solid waste formed after its use in wastewater treatment; where, image a is a photo of the Rhizopus oryzae nanocellulose living gel; and image b is a photo of the gel solid waste.

[0037] Figure 2 The images show SEM images of the *Rhizopus oryzae* nanocellulose live gel from Example 3 and the gel solid waste formed after its use in wastewater treatment; where, image a is the SEM image of the *Rhizopus oryzae* nanocellulose live gel; and image b is the SEM image of the gel solid waste.

[0038] Figure 3 This is a statistical chart showing the COD and BOD5 content in the gramineous lignocellulose cooking wastewater before and after treatment with Rhizopus oryzae nanocellulose live gel in Example 3.

[0039] Figure 4 These are experimental photos verifying the photothermal conversion effect of *Rhizopus oryzae* nanocellulose live gel, gel solid waste, and laccase-treated gel solid waste in Example 4.

[0040] Figure 5 The following figures illustrate the evaporation effect of laccase-treated gel solid waste as a solar evaporator in Example 5. Figure a shows the temperature change of the laccase-treated gel solid waste during sunlight irradiation; Figure b shows the mass change rate of water during sunlight irradiation; and Figure c shows a photograph of the temperature measurement experiment of the laccase-treated gel solid waste during sunlight irradiation. Detailed Implementation

[0041] The present invention will be further described below with reference to the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0042] For any specific techniques or conditions not specified in the examples, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0043] The Rhizopus oryzae used in the following examples is Rhizopus oryzae ATCC20344.

[0044] The seed culture medium used in the following examples has the following formulation: glucose 40 g / L, (NH4)2SO4 4.4 g / L, KH2PO4 0.6 g / L, FeSO4·7H2O 0.005 g / L, MgSO4·7H2O 0.5 g / L, ZnSO4·7H2O 0.018 g / L. It was pre-sterilized.

[0045] The fermentation medium used in the following examples was formulated as follows: glucose 40 g / L, (NH4)2SO4 0.71 g / L, KH2PO4 0.6 g / L, FeSO4·7H2O 0.004 g / L, MgSO4·7H2O 0.5 g / L, ZnSO4·7H2O 0.01 g / L, CaCO3 30 g / L, and citrate buffer 5% v / v. The medium was pre-sterilized.

[0046] The lignocellulose biorefining wastewater treated in the following examples is gramineous lignocellulose (corn stalk) cooking wastewater, which is wastewater generated from the hydrothermal pretreatment of corn stalks. Hydrothermal pretreatment of corn stalk wastewater: Using 170°C as the pretreatment temperature, 50g of oven-dried 20-80 mesh corn stalks and 500mL of water were added to an oil bath tank. The oil bath temperature was first raised to 60°C and held for 30 minutes, ensuring thorough mixing of the solid-liquid mixture. Then, the temperature was increased to 170°C at a rate of 1°C / min. The time to reach the target temperature was recorded, followed by holding at 170°C for 40 minutes. After the hydrothermal pretreatment was completed and the tank cooled, solid-liquid separation was performed using a bag filter or glass frosted funnel to obtain the gramineous lignocellulose (corn stalk) cooking wastewater.

[0047] In the following embodiments, the nanofibers have a particle size of 200–500 nm.

[0048] In the following embodiments, unless otherwise specified, "%" refers to mass percentage content.

[0049] In the following examples, the solvent for the nanocellulose dispersion is distilled water.

[0050] Example 1: Preparation of Rhizopus oryzae nanocellulose living gel

[0051] (1) Inoculate the slant culture of Rhizopus oryzae stored at 4℃ into seed culture medium and culture at 35℃ and 200rpm for 24 hours to obtain Rhizopus oryzae seed liquid.

[0052] (2) The obtained Rhizopus oryzae seed culture was inoculated into a shake flask containing 100 mL of fermentation medium at a volume of 5% v / v. The culture was carried out on a shaker under the following conditions: 130 rpm, 30 °C, and 10 h.

[0053] (3) The 1% nanocellulose dispersion was directly poured into the fermentation medium in step (2) which had been fermented for 10 hours, so that the final concentration of nanocellulose in the mixture was 0.1%. The rotation speed was reduced to 70 rpm and the mixture was cultured for another 2 hours. Finally, a light white gel-like substance was obtained, which is the living gel of Rhizopus oryzae nanocellulose. However, the strength of this gel-like substance was too low and it could not maintain its shape.

[0054] Example 2: Preparation of Rhizopus oryzae nanocellulose living gel and its application in the treatment of wastewater from lignocellulose biorefining.

[0055] Preparation of Rhizopus oryzae nanocellulose living gel:

[0056] (1) Inoculate the slant culture of Rhizopus oryzae stored at 4℃ into seed culture medium and culture at 35℃ and 200rpm for 24 hours to obtain Rhizopus oryzae seed liquid.

[0057] (2) The obtained Rhizopus oryzae seed culture was inoculated into a shake flask containing 100 mL of fermentation medium at a volume of 5% v / v. The culture was carried out on a shaker under the following conditions: 130 rpm, 40 °C, and 72 h.

[0058] (3) The 1% nanocellulose dispersion was directly poured into the fermentation medium that had been fermented for 72 hours in step (2) to make the final concentration of nanocellulose in the mixture 0.2%. The rotation speed was reduced to 70 rpm and the culture was continued for 20 hours. Finally, a light white gel-like substance was obtained, which is the living gel of Rhizopus oryzae nanocellulose.

[0059] The live gel obtained through the above steps was placed in the cooking waste liquid of lignocellulose from the Gramineae family and treated for 6 days. The temperature during the treatment was 40℃ and the rotation speed was 70rpm. After the wastewater treatment was completed, a dark brown live gel was finally obtained.

[0060] Example 3: Preparation of Rhizopus oryzae nanocellulose living gel and its application in the treatment of wastewater from lignocellulose biorefining.

[0061] Preparation of Rhizopus oryzae nanocellulose living gel:

[0062] (1) Inoculate the slant culture of Rhizopus oryzae stored at 4℃ into seed culture medium and culture at 35℃ and 200rpm for 24 hours to obtain Rhizopus oryzae seed liquid.

[0063] (2) The obtained Rhizopus oryzae seed culture was inoculated into a shake flask containing 100 mL of fermentation medium at a volume of 5% v / v. The culture was carried out on a shaker under the following conditions: 150 rpm, 35 °C, and 60 h.

[0064] (3) The 1% nanocellulose dispersion was directly poured into the fermentation medium that had been fermented for 60 hours in step (2) to make the final concentration of nanocellulose in the mixture 0.5%. The rotation speed was reduced to 70 rpm and the mixture was cultured for another 12 hours. Finally, a light white gel-like substance was obtained, which is the living gel of Rhizopus oryzae nanocellulose.

[0065] The live gel prepared through the above steps was added to gramineous lignocellulose cooking wastewater (3-4 cylindrical live gels approximately 2 cm in diameter and 2-3 cm in height were added per 100 mL of wastewater), and reacted for 6 days at a temperature of 35°C and a rotation speed of 70 rpm. After wastewater treatment, a live gel adsorbing lignin from the wastewater was obtained, i.e., gel solid waste. The morphology and microstructure of the Rhizopus oryzae nanocellulose live gel prepared in this example and the gel solid waste formed after wastewater treatment were characterized. Figure 1 The images show the Rhizopus oryzae nanocellulose live gel and the gel solid waste formed after its use in wastewater treatment. It is clear from the images that the initial live gel is milky white and transparent, containing many small white particles. Figure 1 a) is a hyphae of Rhizopus oryzae; after using the live gel for wastewater treatment, a live gel adsorbing lignin can be obtained. Figure 1 b) indicates that the living gel can treat wastewater. Figure 2 This is a SEM image of *Rhizopus oryzae* nanocellulose living gel and the gel solid waste formed after its use in wastewater treatment. The image shows that the *Rhizopus oryzae* nanocellulose living gel (… Figure 2a) The surface only had scattered Rhizopus oryzae spores. After using it for wastewater treatment for 6 days, lignin began to accumulate inside the gel. Figure 2 b).

[0066] The COD and BOD contents of the wastewater before and after treatment with the live gel were determined, and the results are as follows: Figure 3 As shown, the live gel exhibited significant wastewater treatment capabilities. The initial COD concentration of the wastewater was 26388 mg / L, which decreased to 9432 mg / L after treatment with the live gel, achieving a COD removal rate of 64.3%. Simultaneously, the BOD5 concentration in the wastewater also decreased from 10602 mg / L before treatment to 4780 mg / L after treatment, achieving a BOD5 removal rate of 54.9%.

[0067] Example 4: Characterization of the photothermal conversion capacity of gel solid waste

[0068] The photothermal conversion capacity of the lignin-adsorbed gel solid waste of Example 3 was characterized.

[0069] 20 μL of laccase (purchased from Aladdin, catalog number L419514-50 ml) was added to 100 mL of treated wastewater containing 3-4 pieces of gel solid waste (obtained in Example 3), and reacted at 35 °C and 70 rpm for 12 h to obtain laccase-treated gel solid waste.

[0070] The *Rhizopus oryzae* nanocellulose live gel, gel waste, and the laccase-treated gel waste obtained in Example 3 were subjected to vacuum freeze-drying at -80°C for 24 hours. Then, the three gels were irradiated with near-infrared light at a wavelength of 808 nm, and their temperatures were monitored using a HIKVISION handheld infrared thermal imager H10. Figure 4 These are experimental photos verifying the photothermal conversion effect of *Rhizopus oryzae* nanocellulose live gel, gel solid waste, and laccase-treated gel solid waste. The results show that the temperature of the *Rhizopus oryzae* nanocellulose live gel without lignin adsorption only increased to 18.7℃ under continuous irradiation with 808nm near-infrared light; the temperature of the gel solid waste with lignin adsorption could rise to 101℃ within a short time (10s) under irradiation with 808nm near-infrared light; and after adding laccase, the temperature of the gel solid waste could rise to 190℃ within a short time (less than 10s) under near-infrared light irradiation, an increase of 90% compared to the gel solid waste without laccase treatment. These experimental results indicate that the photothermal conversion effect of the lignin-adsorbed gel solid waste after wastewater treatment according to this invention is significantly improved compared to the live gel without lignin adsorption, and the photothermal conversion performance of the gel solid waste treated with laccase is even better.

[0071] Example 5: Characterization of the solar evaporation performance of gel solid waste

[0072] The solar evaporation performance of the laccase-treated lignin-adsorbed gel waste from Example 4 was characterized. The laccase-treated gel waste (a cylinder with a base radius of 1.2 cm and a height of 2 cm) was subjected to vacuum freeze-drying at -80℃ for 24 h. A beaker containing 50 mL of water was placed on an electronic balance (Mettler). One laccase-treated gel waste pellet after freeze-drying was placed in the beaker. The gel waste in the beaker was irradiated with standard sunlight (fiber optic xenon lamp light source, purchased from Shanghai Hefan Instrument Co., Ltd.). The temperature of the gel in the water was monitored using a HIKVISION handheld infrared thermal imager H10, and the balance reading was recorded every minute. The rate of change in the mass of water in the beaker was calculated to characterize the solar evaporation performance of the laccase-treated gel waste.

[0073]

[0074] Among them, M t The mass value displayed on the balance after the gel solid waste has been exposed to sunlight for a duration of t, in kg;

[0075] M0 is the mass value displayed on the balance when the gel solid waste is not exposed to sunlight, in kg;

[0076] S is the cross-sectional area of ​​the portion of the cylindrical gel solid waste exposed to sunlight. In this embodiment, S = 4.52 × 10⁻⁶. -4 Unit: m 2 .

[0077] Figure 5 The image shows the evaporation effect of laccase-treated gel waste as a solar evaporator on water. The experiment found that the temperature of the gel waste in water could rise to 36℃ within one hour under sunlight, achieving an evaporation rate of 0.7 kg / m³. 2 .

[0078] This invention provides an application concept and method for Rhizopus oryzae nanofiber living gel in the treatment of lignocellulose biorefining wastewater. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. Application of Rhizopus oryzae nanofiber living gel in the treatment of lignocellulose biorefining wastewater. in, The Rhizopus oryzae nanofiber live gel is prepared by fermentation of Rhizopus oryzae, with additional nanofibers added to the fermentation medium in the middle and late stages of the fermentation process.

2. The application according to claim 1, characterized in that, The *Rhizopus oryzae* nanofiber live gel was prepared by the following method: *Rhizopus oryzae* seed culture was inoculated into a fermentation medium at a volume ratio of 1% to 10%, and fermentation was carried out at a temperature of 30 to 40°C and a certain rotation speed; before the nanofibers were added to the fermentation medium, the rotation speed was 120 to 300 rpm; after the nanofibers were added to the fermentation medium, the rotation speed was 40 to 70 rpm.

3. The application according to claim 1, characterized in that, The additional addition of nanofibers to the fermentation medium in the middle and late stages of the fermentation process involves adding nanofibers to the fermentation medium after 10–72 hours of fermentation, and continuing fermentation for another 2–20 hours.

4. The application according to claim 1, characterized in that, The nanofibers are any one of cellulose nanofibers, cellulose nanocrystals, and chitin nanofibers.

5. The application according to claim 1, characterized in that, The nanofibers are added to the fermentation medium in the form of a nanofiber dispersion; the mass fraction of nanofibers in the nanofiber dispersion is 0.1% to 2%; the nanofiber dispersion is added to the fermentation medium to obtain a mixed system, and the mass fraction of nanofibers in the mixed system is controlled to be 0.1% to 1%.

6. The application according to claim 1, characterized in that, The fermentation medium comprises: 10–40 g / L glucose, 0–1 g / L (NH4)2SO4, 0–1 g / L KH2PO4, 0–0.1 g / L FeSO4, 0.1–1 g / L MgSO4, 0–0.1 g / L ZnSO4, 10–40 g / L CaCO3, and 1–7% v / v citrate buffer.

7. The application according to claim 1, characterized in that, The lignocellulose is lignocellulose from the Poaceae family and / or lignocellulose from the Oleaceae family.

8. The application according to claim 1, characterized in that, The method for applying the *Rhizopus oryzae* nanofiber live gel to the treatment of lignocellulose biorefining wastewater is as follows: the *Rhizopus oryzae* nanofiber live gel is added to the lignocellulose biorefining wastewater and treated for 1 to 10 days at a temperature of 30–40°C and a stirring speed of 50–80 rpm.

9. The application according to claim 1, characterized in that, After treating the lignocellulose biorefining wastewater with the aforementioned Rhizopus oryzae nanofiber live gel, gel solid waste is obtained, which is then reused as a photothermal material.

10. The application according to claim 9, characterized in that, The gel solid waste is treated with laccase and then reused as a photothermal material.

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