Method for enhancing cellulose degradation and sugar fermentation by using Aspergillus niger HKS11 solid fermentation broth in cooperation with cellulase

Abstract

The invention relates to a method for enhancing cellulose degradation and sugar fermentation by using Aspergillus niger HKS11 solid fermentation liquor in cooperation with cellulase. The method comprises the following steps: preparing fermentation liquor containing various enzymes through a specific solid fermentation process; the problems that in the prior art, cellulase is low in catalytic efficiency, high in production cost, limited in single enzyme system, inhibited in activity and poor in adaptability can be effectively solved, and according to the technical scheme, the method comprises the following steps that 1, aspergillus niger HKS11 is cultured and fermented; (2) degrading cellulose by the aspergillus niger HKS11 solid fermentation liquor in cooperation with cellulase; according to the method, through the synergistic effect of the aspergillus niger HKS11 solid fermentation liquor and the cellulase, the diversity and coordination of an enzyme system are effectively improved through multiple enzymes in the fermentation liquor, the defect that components of a single enzyme system are incomplete is overcome, and the solid fermentation process is simple and easy to implement, low in production cost, environmentally friendly and suitable for large-scale application.

Description

1. Technical Field

[0001] The present invention relates to the field of microbial fermentation, and particularly to a method for enhancing cellulose degradation and fermentable sugar by using the solid fermentation broth of Aspergillus niger HKS11 in combination with cellulase. 2. Background Art

[0002] Cellulose is the most abundant renewable biological resource on earth, and its efficient degradation is of great significance in the fields of biomass energy development, food industry, and feed processing. However, there are still multiple deficiencies in the existing technology for cellulose degradation: the catalytic efficiency of the traditional cellulase system is limited, with insufficient enzyme activity, incomplete enzyme components, or uncoordinated ratios, resulting in hydrolysis effects that are difficult to meet the requirements of industrial production; the industrial production of cellulase mainly relies on liquid submerged fermentation, although the enzyme production is relatively high, but the process conditions are difficult to control and the cost is relatively high; the limitations of single-strain fermentation also restrict the efficient degradation of cellulose, and even in the case of multi-strain mixed fermentation, there are problems such as unstable strain synergy and complex processes. In addition, the accumulation of intermediate products such as cellobiose during hydrolysis will inhibit the activity of cellulase, further reducing the hydrolysis efficiency. The problems existing in the existing technology specifically include:

[0003] (1) Low catalytic efficiency of cellulase: Due to insufficient enzyme activity and incomplete enzyme components in the existing cellulase system, the degradation efficiency of cellulose is low and cannot meet the requirements of industrial production.

[0004] (2) High production cost of cellulase: The liquid submerged fermentation process is complex and costly, not suitable for large-scale applications, which restricts the wide application of cellulase in the fields of biomass degradation and so on.

[0005] (3) Limitations of a single enzyme system: The enzyme production efficiency of single-strain fermentation is limited, and the enzyme system is incomplete, making it difficult to achieve efficient hydrolysis of cellulose; while multi-strain fermentation has problems such as complex processes and unstable strain synergy.

[0006] (4) Inhibition of cellulase activity: During the hydrolysis process, the accumulation of intermediate products (such as cellobiose) has an inhibitory effect on cellulase, further reducing the degradation efficiency of cellulose.

[0007] (5) Poor adaptability to substrate diversity: The existing cellulase system has insufficient adaptability to cellulose substrates from different sources (such as wood powder, straw, etc.), restricting its application in various industrial scenarios.

[0008] Therefore, inventing a method to enhance cellulose degradation and fermentable sugar is a technical problem that urgently needs to be solved at present. 3. Summary of the Invention

[0009] In view of the above situation, to solve the defects of the prior art, the purpose of the present invention is to provide a method for enhancing cellulose degradation and fermentable sugar by using the solid fermentation broth of Aspergillus niger HKS11 in cooperation with cellulase. By a specific solid fermentation process, a fermentation broth containing various enzymes is prepared, which can effectively solve the problems of low catalytic efficiency of cellulase in the prior art, high production cost, limitations of a single enzyme system, inhibited activity and poor adaptability.

[0010] In view of the deficiencies of the prior art, the present invention provides a method for enhancing cellulose degradation and fermentable sugar by using the solid fermentation broth of Aspergillus niger HKS11 in cooperation with cellulase, comprising the following steps:

[0011] 1) Cultivation and fermentation of Aspergillus niger HKS11

[0012] Inoculate the spore suspension of Aspergillus niger HKS11 on a PDA plate medium, and incubate it in an inverted position at 30 °C until the spores of Aspergillus niger HKS11 are formed. Prepare a solid fermentation medium, sterilize it at 121 °C for 20 min, evenly inoculate the spores of Aspergillus niger HKS11 on the surface of the sterilized solid medium, control the fermentation conditions to incubate at 28 °C for 24 h, and then dry it to a constant weight in an environment of 40 °C to obtain a fermentation product. Extract the fermentation product with distilled water for 1 h, filter to remove the solid residue, and obtain a solid fermentation broth;

[0013] 2) Degradation of cellulose by the solid fermentation broth of Aspergillus niger HKS11 in cooperation with cellulase

[0014] Mix the cellulose substrate, ascorbic acid solution, CuCl 2 solution, cellulase, the solid fermentation broth obtained in step 1) and PBS buffer, and react at 35 °C and 240 rpm for 20 min. After the reaction, determine the content of reducing sugar produced in the reaction solution by the DNS method to evaluate the degradation efficiency of cellulose.

[0015] In the above step 1), the composition of the PDA plate medium is: 200 g of potatoes, 20 g of reducing sugar, 15 g of agar powder, and 1 L of distilled water.

[0016] In the above step 1), the composition of the solid fermentation medium is: 90 g of bran, 10 g of soybean meal, and 100 ml of distilled water.

[0017] In the above step 1), the solid fermentation medium is placed in a 500 mL Erlenmeyer flask, and the medium is evenly spread at the bottom of the Erlenmeyer flask to facilitate uniform inoculation and fermentation of the bacterial strain.

[0018] In the above step 2), the dosage of the cellulose substrate is 20 mg, the dosage of the ascorbic acid solution is 100 mM, 100 μL, CuCl 2The dosage of the solution is 500 mM, 2 μL, the dosage of cellulase is 70 μg pro. and the dosage of the solid fermentation broth is 20 μg pro. , and the above components are made up to 1 mL with 100 mM, pH 7.5 PBS buffer solution.

[0019] In the said step 2), the content of reducing sugar produced in the reaction solution determined by DNS method is as follows: The reaction system is centrifuged at 8000 rpm for 5 min, 50 μL of the supernatant of the reaction system is taken into a new 1.5 mL centrifuge tube, 50 μL of DNS solution is added and boiled in a water bath for 10 min. The reaction system after boiling treatment is centrifuged at 12000 rpm for 1 min to remove impurities. After dilution with 900 μL of deionized water, 200 μL of the supernatant diluted 10 times is taken, and the absorbance value at OD540 is measured. The amount of reducing sugar produced is calculated according to the drawn standard curve of reducing sugar.

[0020] In the said step 2), the cellulose substrate is one of microcrystalline cellulose, wheat bran, wheat straw, corn straw, corn cob powder, poplar powder, rice straw, among which wheat bran, corn cob powder, poplar powder, and rice straw all pass through a 100-mesh sieve, and wheat straw and corn straw all pass through an 80-mesh sieve.

[0021] The present invention provides a method for enhancing cellulose degradation and fermentable sugar production by using the solid fermentation broth of Aspergillus niger HKS11 in cooperation with cellulase. By optimizing the solid fermentation process and constructing a multi-enzyme synergistic system, the cellulose degradation efficiency and the yield of reducing sugar are significantly improved. The beneficial technical effects are as follows:

[0022] 1. Improvement in yield and quality

[0023] (1) Significantly improve the degradation efficiency and the yield of reducing sugar: The solid fermentation broth of Aspergillus niger HKS11 contains rich multi-enzyme components (such as LPMOs, β-glucosidase, hemicellulase, etc.). Acting synergistically with cellulase, it can improve the efficiency of cellulose hydrolysis, and the conversion rate of cellulose is increased by 513.0±1.3% compared with using cellulase alone.

[0024] (2) Reduce the accumulation of by-products: A variety of enzymes in the fermentation broth can effectively decompose intermediate products (such as cellobiose), reduce its inhibition on the activity of cellulase, and ensure the continuous and efficient progress of the degradation reaction.

[0025] 2. Improvement in process efficiency and precision

[0026] (1) Increase the reaction rate: By optimizing the reaction conditions and the synergistic system, the present invention can achieve rapid degradation under mild conditions, effectively shortening the reaction time.

[0027] (2) Enhanced coordination of enzyme system functions: The synergistic effect among multiple enzyme components in the fermentation broth is superior to that of the traditional single-enzyme system, improving the enzymatic efficiency and the utilization rate of substrates.

[0028] 3. Energy consumption and raw material savings

[0029] (1) Low-energy-consuming reaction: The degradation reaction can be carried out at a relatively low temperature (about 35°C) and in a neutral buffer system, without the need for high temperature or special solvents, significantly reducing energy consumption.

[0030] (2) High substrate utilization rate: The fermentation broth can process a variety of cellulose substrates (such as corn straw, wheat straw, rice straw, wood powder, etc.), reducing raw material waste and making full use of biomass resources.

[0031] 4. Simplification of processes and convenience of operation

[0032] (1) Simple and efficient solid fermentation process: The solid fermentation process of Aspergillus niger HKS11 does not require complex equipment and conditions, and the production process is stable and easy to control, suitable for large-scale industrial applications.

[0033] (2) Direct use of fermentation broth: The solid fermentation broth of Aspergillus niger HKS11 can be directly compounded with cellulase without purification, greatly simplifying the process steps and significantly improving the operation convenience.

[0034] 5. Environmentally friendly

[0035] (1) Green and environmentally friendly: The fermentation process uses agricultural wastes (such as wheat bran and soybean meal) as the culture medium raw materials, which not only reduces costs but also avoids the use of chemical reagents and is harmless to the environment.

[0036] (2) Reduction of industrial pollution: This method hardly produces waste liquid and harmful by-products during the cellulose degradation process, meeting the sustainable development requirements of green bio-manufacturing.

[0037] 6. Great potential for comprehensive application

[0038] (1) Wide substrate adaptability: This method has good adaptability to cellulose substrates from different sources (such as microcrystalline cellulose, wood powder, straw, etc.), significantly broadening its industrial application scope.

[0039] (2) Meeting the needs of multiple industries: This technology can be applied to multiple fields such as biomass energy development, preparation of cellulose-based materials, feed processing, and food industry, providing low-cost and high-efficiency technical solutions for related industries. IV. Description of the drawings

[0040] Figure 1 This is the amount of reducing sugar produced in the present invention at different reaction times.

[0041] Figure 2 The amount of reducing sugar produced under different temperatures in the present invention.

[0042] Figure 3 The amount of reducing sugar produced under different pH values in the present invention.

[0043] Figure 4 The amount of reducing sugar produced under different ratios of the solid fermentation broth of Aspergillus niger HKS11 and cellulase in the present invention.

[0044] Figure 5 The production of reducing sugar in the reaction with different substrates involved in the present invention.

[0045] Figure 6 The test results of the temperature stability of the solid fermentation broth of Aspergillus niger HKS11 in the present invention.

[0046] Figure 7 The test results of the storage stability of the solid fermentation broth of Aspergillus niger HKS11 in the present invention. V. Specific Embodiments

[0047] The following further elaborates on the specific embodiments of the present invention in conjunction with the accompanying drawings and examples.

[0048] Example 1

[0049] A method for enhancing cellulose degradation by synergistically using the solid fermentation broth of Aspergillus niger HKS11 and cellulase, comprising the following steps:

[0050] 1) Cultivation and fermentation of Aspergillus niger HKS11

[0051] Inoculate the spore suspension of Aspergillus niger HKS11 on the PDA plate medium, and incubate it in an inverted manner at 30 °C until the spores of Aspergillus niger HKS11 are formed. Prepare the solid fermentation medium, sterilize it at 121 °C for 20 min, evenly inoculate the spores of Aspergillus niger HKS11 on the surface of the sterilized solid medium, control the fermentation conditions to incubate at 28 °C in an electrothermal constant temperature incubator for 24 h, and then dry it to a constant weight in an environment of 40 °C to obtain the fermentation product. Extract the fermentation product with distilled water for 1 h, and use a pressure filtration pump and filter paper to filter out the solid residues to obtain the solid fermentation broth;

[0052] 2) Degrading cellulose by the solid fermentation broth of Aspergillus niger HKS11 synergistically with cellulase

[0053] Add 20 mg of cellulose substrate, 100 mM of ascorbic acid solution, 100 μL, 500 mM of CuCl 2 solution, 2 μL, 70 μg of cellulase pro. , and 20 μg of the solid fermentation broth obtained in step 1) pro., after the above components were made up to 1 mL with 100 mM PBS buffer at pH 7.5 and mixed in a 5 mL glass reaction flask, the reaction was carried out at 35 °C and 240 rpm in a constant temperature culture oscillator for 20 min. After the reaction, the content of reducing sugar produced in the reaction solution was determined by the DNS method to evaluate the degradation efficiency of cellulose.

[0054] Example 2

[0055] A method for enhancing cellulose degradation by synergistically using the solid fermentation broth of Aspergillus niger HKS11 and cellulase, comprising the following steps:

[0056] 1) Cultivation and fermentation of Aspergillus niger HKS11

[0057] Inoculate the spore suspension of Aspergillus niger HKS11 on a PDA plate medium and incubate it upside down at 30 °C until the spores of Aspergillus niger HKS11 are formed. Prepare a solid fermentation medium and sterilize it at 121 °C for 20 min. Uniformly inoculate the spores of Aspergillus niger HKS11 on the surface of the sterilized solid medium. Control the fermentation conditions to incubate at 28 °C in an electrothermal constant temperature incubator for 24 h, and then dry it to a constant weight at 40 °C to obtain a fermentation product. Extract the fermentation product with distilled water for 1 h, and use a pressure filtration pump and filter paper to filter out the solid residue to obtain a solid fermentation broth;

[0058] 2) Degradation of cellulose by the solid fermentation broth of Aspergillus niger HKS11 synergistically with cellulase

[0059] Add 20 mg of microcrystalline cellulose, 100 μL of 100 mM ascorbic acid solution, 2 μL of 500 mM CuCl 2 solution, 70 μg of cellulase pro. , 20 μg of the solid fermentation broth obtained in step 1) pro. , after the above components were made up to 1 mL with 100 mM PBS buffer at pH 7.5 and mixed in a 5 mL glass reaction flask, the reaction was carried out at 35 °C and 240 rpm in a constant temperature culture oscillator for 20 min. After the reaction, the content of reducing sugar produced in the reaction solution was determined by the DNS method to evaluate the degradation efficiency of cellulose.

[0060] The present invention provides a new and efficient cellulose degradation solution with low cost by using the solid fermentation broth of Aspergillus niger HKS11 in cooperation with cellulase. Aspergillus niger HKS11 (preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms with the preservation number of CGMCC No. 6027) is used to prepare a multi-enzyme complex fermentation broth through a solid fermentation process, which makes up for the shortcoming of insufficient enzyme components in a single enzyme system and significantly improves the cellulose degradation efficiency. Through the synergistic effect of the fermentation broth and cellulase (Solarbio Science & Technology Co., Ltd., Cat: C8271), the accumulation of cellobiose during the degradation of cellulose substrate is effectively reduced, the enzyme activity inhibition effect is alleviated, and the substrate utilization rate is improved. By using a low-cost and environmentally friendly solid fermentation process, the enzyme production cost is reduced, which is suitable for large-scale industrial production. This method is applicable to cellulose substrates from various sources and has good effects on substrates such as microcrystalline cellulose, corn straw, wheat straw, bran, corn cob powder, wood powder, and rice straw, broadening its industrial application scope, meeting diverse production needs, and having broad industrial application potential. Significant beneficial effects have been obtained through experiments, and the relevant experimental data are as follows:

[0061] 1. Optimization of reaction conditions for the reaction involving the solid fermentation broth of Aspergillus niger HKS11 and cellulase

[0062] Weigh 20 mg of microcrystalline cellulose into a reaction flask and perform ultrasonic treatment to evenly disperse the reaction substrate. Then add 100 mM ascorbic acid (100 μL), 500 mM CuCl 2 solution (2 μL), cellulase (70 μg pro. ), and the solid fermentation broth of Aspergillus niger HKS11, and make up to 1 mL of the reaction system with PBS buffer (100 mM). Place the reaction flask in a constant temperature oscillator at a certain temperature and continuously stir at 240 rpm.

[0063] The conditions that need to be optimized during the construction of the reaction include: reaction time (10 - 120 min), reaction temperature (20 - 50 °C), pH of the PBS buffer (pH 6.0 - 9.0), and mass ratio of cellulase to the solid fermentation broth of Aspergillus niger HKS11 (μg pro. :μg pro. , 70:6 - 70:20). After the reaction ends, determine the content of reducing sugar produced in the reaction solution by the DNS method.

[0064] 1.1 Optimization of reaction time

[0065] As one of the important conditions for enzymatic reactions, the length of the reaction time is related to the production cost. From Figure 1It can be clearly seen that the amount of reducing sugar produced increases with the increase of the catalytic reaction time. When the reaction time reaches 20 min, the most reducing sugar is produced, and the concentration of reducing sugar obtained by hydrolysis at this time is 11.02 mM ± 0.04 mM. Subsequently, when the reaction time is further extended, the reducing sugar yield decreases and then levels off. Therefore, 20 min is selected as the final enzymatic reaction time.

[0066] 1.2 Optimization of reaction temperature

[0067] After obtaining the optimal temperature of the reaction, the reaction temperature was optimized. As Figure 2 shown, during the process of the temperature rising from 20 °C to 35 °C, the amount of reducing sugar produced increased slowly and reached a peak value of 11.68 mM ± 0.22 mM at 35 °C. With the further increase of temperature, the amount of reducing sugar produced decreased significantly, probably because high temperature caused partial denaturation and inactivation of some enzymes, resulting in a decrease in the reaction rate. Finally, 35 °C was selected as the optimal temperature for this reaction.

[0068] 1.3 pH optimization

[0069] As one of the important conditions for enzymatic reactions, changes in the pH environment can change the process of enzymatic reactions. In extreme cases, it may even cause irreversible damage to enzymes. As Figure 3 shown, as the pH environment changes from acidic to alkaline, the yield of reducing sugar produced by the catalytic reaction of the solid fermentation broth of Aspergillus niger HKS11 and cellulase increases from low to high and reaches a peak value of 11.65 mM ± 0.61 mM at pH 7.5. Subsequently, when the pH environment further tends to be alkaline, the reducing sugar yield decreases. Therefore, pH 7.5 was selected as the optimal pH condition for the enzymatic reaction involving the solid fermentation broth of Aspergillus niger HKS11 and cellulase.

[0070] 1.4 Optimization of the input ratio of the solid fermentation broth of Aspergillus niger HKS11 and cellulase

[0071] The rate of enzymatic reactions is closely related to the concentration of enzyme molecules. When the enzyme concentration is low, the reaction rate is proportional to the enzyme concentration; when the enzyme concentration is high enough, the reaction rate levels off, and substrate inhibition may even occur. To explore the optimal enzyme addition amount for this synergistic reaction, first, the addition amount of our cellulase was set to 70 μg pro. , and the amount of the solid fermentation broth of Aspergillus niger HKS11 was gradually increased. The results showed (as Figure 4 shown) that the amount of reducing sugar produced increased continuously with the increase of the solid fermentation broth of Aspergillus niger HKS11. When the input amount of the solid fermentation broth of Aspergillus niger HKS11 reached 20 μg pro.When the time is reached, the reducing sugar yield is 12.68 ± 0.14 mM. Since the fermented broth was not concentrated after filtration and the protein concentration in the fermented broth was low, although the reducing sugar yield still showed an upward trend with the increase of the fermented broth, the maximum value in this experiment was only taken up to 20 μg pro. According to the above results and analysis, the optimal input ratio of cellulase to the solid fermented broth of Aspergillus niger HKS11 was determined to be 70:20.

[0072] 2. Substrate expansion

[0073] Under the optimal reaction conditions, 7 different substrates such as microcrystalline cellulose (20 mg), wheat bran (100 mesh, 20 mg), wheat straw (80 mesh, 20 mg), corn straw (80 mesh, 20 mg), corn cob powder (100 mesh, 20 mg), poplar wood powder (100 mesh, 20 mg), and rice straw (100 mesh, 20 mg) were used to participate in the reaction, and the production of reducing sugar was detected.

[0074] Control group A: No solid fermented broth of Aspergillus niger HKS11 was added; Control group B: No cellulase was added, and the other conditions remained unchanged.

[0075] In order to further study the substrate applicability of the solid fermented broth of Aspergillus niger HKS11 and cellulase in the synergistic reaction, their hydrolysis effects on different cellulose substrates were studied. As Figure 5 can be seen, the hydrolysis effect of the solid fermented broth of Aspergillus niger HKS11 on cellulose substrates itself is not obvious, but its introduction significantly improves the hydrolysis effect of cellulase on cellulose substrates. Taking microcrystalline cellulose as an example, in the reaction of the solid fermented broth of Aspergillus niger HKS11 synergistic with cellulase, the production of reducing sugar increased by 513.0 ± 1.3% compared with the use of cellulase alone, and increased by 313.9 ± 0.8% compared with the use of the solid fermented broth of Aspergillus niger HKS11 alone. At the same time, the effect of this synergistic reaction system on different cellulose substrates is good. When using microcrystalline cellulose, wheat bran, wheat straw, corn straw, corn cob powder, poplar wood powder, and rice straw as substrates, the production of reducing sugar in the reaction system reached 12.91 ± 0.03 mM, 16.61 ± 0.38 mM, 15.74 ± 0.32 mM, 16.62 ± 0.56 mM, 16.28 ± 0.63 mM, 13.87 ± 0.31, and 16.65 ± 0.50 mM respectively, showing a certain preference for different substrates, which may be related to the crystallinity, degree of polymerization, etc. of different cellulose substrates.

[0076] 3. Stability evaluation of the solid fermented broth

[0077] 3.1 Temperature stability

[0078] The solid fermentation broth of Aspergillus niger HKS11 was incubated at the optimal reaction temperature (35 °C) for 0 h - 12 h respectively, and then the activity was measured. Taking the activity of the fermentation broth at 0 h as 100%, the ratio of the detection result under other conditions to the initial activity was called relative activity.

[0079] Using microcrystalline cellulose as the model substrate, the effect of temperature on the activity of the solid fermentation broth of Aspergillus niger HKS11 was investigated. As Figure 6 shown, although the relative activity of the solid fermentation broth of Aspergillus niger HKS11 decreased to a certain extent with the prolongation of the incubation time, more than 90% of the initial activity was still retained after being placed at 35 °C for 12 h, and the relative activity remained at about 90.60%. Temperature change can cause the peptide chain on the enzyme to be damaged, thus changing the protein conformation and affecting the enzyme activity. Therefore, it is normal for the enzyme activity to decrease after incubation at a temperature higher than 4 °C for a period of time. Without any protection, the fermentation broth only lost about 9.40% of the initial activity after incubation at 35 °C for 12 h, indicating that it has good temperature stability.

[0080] 3.2 Storage stability

[0081] The solid fermentation broth of Aspergillus niger HKS11 was stored at 4 °C for 7 days, and samples were taken every day to test the activity. The activity shown by the fermentation broth of Aspergillus niger HKS11 before storage (0 d) was defined as 100%, and the ratio of the detection results at other storage days to the initial activity was called relative activity.

[0082] The storage stability of the enzyme determines its service life to a certain extent. Figure 7 The storage stability of the solid fermentation broth of Aspergillus niger HKS11 at 4 °C was described. After 3 days of storage, the relative activity decreased significantly, reaching 91.10 ± 5.41%. The fermentation broth contains impurities and lacks a protective agent, and is vulnerable to protease degradation, oxidative damage and environmental changes at 4 °C. However, after 7 days of storage, the relative activity of the solid fermentation broth of Aspergillus niger HKS11 was 85.37 ± 2.12%, with good stability and possibly good industrial properties.

[0083] Through the synergistic effect of the solid fermentation broth of Aspergillus niger HKS11 and cellulase, various enzymes in the fermentation broth effectively improved the diversity and coordination of the enzyme system, made up for the shortcomings of the incomplete components of a single enzyme system. The solid fermentation process is simple and easy to operate, with low production cost and environmental friendliness, suitable for large-scale application. The various enzymes in the fermentation broth can also reduce the accumulation of cellobiose, relieve the inhibitory effect on cellulase, and improve the hydrolysis rate. It solves multiple bottleneck problems in traditional cellulose degradation methods from the technical path and process flow, and has the advantages of high yield, high efficiency, low cost, strong environmental protection and wide applicability. It provides important technical support for the development of biomass energy and the high-value utilization of cellulose resources, and has broad prospects for industrial promotion.

[0084] It should be noted that the above are only the preferred embodiments of the present invention, and do not impose any formal restrictions on the present invention. Any person skilled in the relevant art can, without departing from the scope of the technical solution of the present invention, make modifications or variations to equivalent embodiments using the technical content disclosed above, and all fall within the protection scope of the present invention.

Claims

1. A method for enhancing cellulose degradation and sugar fermentation by using Aspergillus niger HKS11 solid fermentation broth in conjunction with cellulase, characterized in that: The following steps are involved: 1) Cultivation and fermentation of Aspergillus niger HKS11 The Aspergillus niger HKS11 spore liquid was inoculated into a PDA plate medium, and inverted and cultured at 30°C until the Aspergillus niger HKS11 spores were formed, a solid fermentation medium was prepared, and the medium was sterilized at 121°C for 20 min. The Aspergillus niger HKS11 spores were evenly inoculated on the surface of the sterilized solid medium, and the fermentation conditions were controlled to be cultured at 28°C for 24 h, and then dried to constant weight at 40°C to obtain a fermentation product, and the fermentation product was extracted with distilled water for 1 h, and the solid residue was removed by filtration to obtain a solid fermentation liquid; 2) Degradation of cellulose by solid fermentation broth of Aspergillus niger HKS11 and cellulase The cellulose substrate, ascorbic acid solution, CuCl2 solution, cellulase, the solid fermentation broth obtained in step 1) and PBS buffer were mixed and reacted at 35°C and 240 rpm for 20 min. After the reaction, the reducing sugar content produced in the reaction solution was determined by the DNS method to evaluate the cellulose degradation efficiency.

2. The method for enhancing cellulose degradation and sugar fermentation by using Aspergillus niger HKS11 solid fermentation broth in coordination with cellulase according to claim 1, characterized in that: In the step 1), the PDA plate culture medium is composed of: 200 g potatoes, 20 g reducing sugar, 15 g agar powder, and 1 L distilled water.

3. The method for enhancing cellulose degradation and sugar fermentation by using Aspergillus niger HKS11 solid fermentation broth in coordination with cellulase according to claim 1, characterized in that: In the step 1), the solid fermentation medium consists of: 90 g bran, 10 g soybean meal, and 100 ml distilled water.

4. The method for enhancing cellulose degradation and sugar fermentation by using Aspergillus niger HKS11 solid fermentation broth in coordination with cellulase according to claim 1, characterized in that: In the step 1), the solid fermentation medium is placed in a 500 mL Erlenmeyer flask, and the medium is evenly spread on the bottom of the Erlenmeyer flask to allow the bacteria to be evenly inoculated and fermented.

5. The method for enhancing cellulose degradation and sugar fermentation by using Aspergillus niger HKS11 solid fermentation broth in coordination with cellulase according to claim 1, characterized in that: In the step 2), the amount of cellulose substrate is 20 mg, the amount of ascorbic acid solution is 100 mM, 100 μL, the amount of CuCl2 solution is 500 mM, 2 μL, and the amount of cellulase is 70 μg. pro. , the dosage of solid fermentation liquid is 20 μg pro. The above components were made up to 1 mL with 100 mM PBS buffer, pH 7.

5.

6. The method of utilizing Aspergillus niger HKS11 solid fermentation broth in coordination with cellulase to enhance cellulose degradation and sugar fermentation according to claim 1, characterized in that: In the step 2), the reducing sugar content produced in the reaction solution is determined by the DNS method as follows: the reaction system is centrifuged at 8000 rpm for 5 min, 50 μL of the supernatant of the reaction system is taken into a new 1.5 mL centrifuge tube, 50 μL of DNS solution is added to a boiling water bath for 10 min, the reaction system after boiling is centrifuged at 12000 rpm for 1 min to remove impurities, 900 μL of deionized water is added for dilution, 200 μL of the supernatant diluted 10 times is taken, the absorbance value at OD540 is measured, and the amount of reducing sugar generated is calculated according to the drawn reducing sugar standard curve.

7. The method of utilizing Aspergillus niger HKS11 solid fermentation liquid in coordination with cellulase to enhance cellulose degradation and sugar fermentation according to claim 1, characterized in that: In the step 2), the cellulose substrate is one of microcrystalline cellulose, bran, wheat straw, corn straw, corn cob powder, poplar powder, and rice straw, wherein the bran, corn cob powder, poplar powder, and rice straw are all sieved through a 100-mesh sieve, and the wheat straw and corn straw are all sieved through an 80-mesh sieve.

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