Method for producing bacterial cellulose by fermenting lignocellulose hydrolysate
By fermenting the production of bacterial cellulose by lignocellulose hydrolysate, the problems of medium and low yield and high cost in the prior art are solved, and the production of bacterial cellulose is achieved with high yield and economical production.
Patent Information
- Application Number
- CN202510010213.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-03
- Publication Date
- 2025-03-28
AI Technical Summary
In the prior art, the low yield and high production cost of Acetobacter xyloconate in the production of bacterial cellulose limit its wide application.
Bacterial cellulose is produced by fermenting the lignocellulose hydrolysate, including acid hydrolysis and enzymatic decomposition using corn stalks as raw material, fermentation is carried out after detoxification, and subsequent purification and decolorization of the bacterial cellulose membrane.
The high yield of bacterial cellulose is achieved comparable to that of commonly used HS liquid culture media, and reduces production costs, providing a new method for green biomanufacturing of bacterial cellulose and improving production economy.
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Figure CN119709910A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of bacterial cellulose preparation, and in particular to a method for producing bacterial cellulose by fermenting lignocellulose hydrolyzate. Background Art
[0002] Bacterial cellulose (BC) is an extracellular microbial product produced by fermentation. In 1886, British scientist Brown discovered that the type of cellulose produced by Acetobacter xylinum is a linear polysaccharide conjugated with β-d-glucose units linked by β-1,4 glycosidic bonds. Due to its excellent physical, chemical and mechanical properties, such as high crystallinity, high purity, good water retention, elasticity, formability, biodegradability and biocompatibility, bacterial cellulose can be widely used in industries such as food, cosmetics, drugs and biomedical products. However, the low yield of bacterial cellulose produced by Acetobacter xylinum and the high cost of production have greatly limited its wider application, so improving the yield of bacterial cellulose and reducing production costs have become top priorities.
[0003] Lignocellulose is a plant biomass composed of carbohydrate polymers, including cellulose (40%-60%), hemicellulose (20%-40%) and lignocellulose (10%-24%), and is the main component of plant cell walls. According to statistics, about 10 billion to 50 billion tons of lignocellulose are produced each year worldwide. It is a renewable resource available in large quantities on the earth, which can replace oil and reduce greenhouse gas emissions by producing carbon-neutral biofuels to reduce dependence on fossil fuels. Therefore, lignocellulosic biomass has broad development prospects and is a good choice for predictable, feasible and sustainable resources for biofuels and other value-added products.
[0004] Lignocellulose comes from various waste materials and is in large quantities, which determines that it can become the most promising resource for low-cost cultivation of bacterial cellulose. Since bacterial cellulose cannot directly metabolize lignocellulose and cannot make fuller use of cellulose resources, it is necessary to pretreat the lignocellulose in advance. Pretreatment can improve the material conversion rate and product yield of subsequent work. The production of bacterial cellulose from lignocellulose hydrolysate generally requires four processes: first, pretreat the lignocellulose, then perform enzymatic hydrolysis, then detoxify the inhibitors, and finally fermentation. Four processes are required to achieve the production of bacterial cellulose. Summary of the invention
[0005] To solve the above problems, the present invention provides a method for producing bacterial cellulose by fermenting lignocellulose hydrolyzate, which converts lignocellulose resources into high-value-added bacterial cellulose products, realizes green biomanufacturing of bacterial cellulose, and improves production economy.
[0006] To achieve the above object, the present invention provides a method for producing bacterial cellulose by fermenting lignocellulose hydrolysate, comprising the following steps:
[0007] S1, using corn straw as raw material, preparing lignocellulose hydrolyzate through acid hydrolysis and enzymatic hydrolysis;
[0008] S2, using the detoxified lignocellulose hydrolysate to ferment and produce bacterial cellulose;
[0009] S3. After the fermentation production is completed, the bacterial cellulose membrane is taken out for purification. The purification conditions are:
[0010] Soak the bacterial cellulose membrane in a 0.5M-1.5M NaOH solution in a 80°C water bath for 5-15 minutes;
[0011] S4. Decolorizing the purified bacterial cellulose membrane under the following decolorization conditions:
[0012] The purified bacterial cellulose membrane is immersed in a 0.1M-1M NaOH solution and decolorized in a 80°C water bath. During the decolorization process, the alkali solution is continuously replaced until the bacterial cellulose membrane becomes milky white and translucent.
[0013] The milky white translucent bacterial cellulose membrane was soaked in deionized water and de-alkalized in a water bath at 80°C, and the deionized water was continuously replaced until the pH value on the surface of the bacterial cellulose membrane was 7.
[0014] Preferably, the specific steps of step S1 include:
[0015] S11, cutting corn stalks into 2-3 cm small segments, drying naturally, and crushing the crushed corn stalks. The crushed corn stalk powder is passed through a 60-mesh sieve, dried at 60° C., and then sealed and stored;
[0016] S12, add 2% H2SO4 by mass to corn straw powder at a solid-liquid ratio of 1:15, mix gently, and treat at 120°C for 60 minutes;
[0017] S13. After the acid hydrolysis in step S12 is completed, the reaction solution is cooled to room temperature, a NaOH solution is added to adjust the pH of the reaction solution to 4.5, cellulase and hemicellulase are added in an amount of 150 U / g corn stalks and 200 U / g corn stalks, and enzymolysis is performed at 50° C. and 200 rpm on a shaker for 72 hours;
[0018] S14. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is filtered out using a Buchner funnel to obtain a corn straw hydrolyzate, i.e., a lignocellulose hydrolyzate.
[0019] Preferably, in step S2, the detoxification of the lignocellulose hydrolysate is performed by extracting the inhibitor in the lignocellulose hydrolysate with ethyl acetate.
[0020] Preferably, the volume ratio of the lignocellulose hydrolyzate to ethyl acetate during extraction is 1:5.
[0021] Preferably, in step S2, the strain used in the fermentation production is Gluconobacter xylinus CGMCC2955.
[0022] Preferably, in step S2, the fermentation medium configuration during fermentation production includes: adding 10 g / L peptone, 7.5 g / L yeast extract powder and 10 g / L disodium hydrogen phosphate to the detoxified lignocellulose hydrolysate, adjusting the pH to 4.5 with glacial acetic acid, and sterilizing at 115°C for 30 min for standby use.
[0023] Preferably, in step S3, the purification conditions are: immersing the bacterial cellulose membrane in a 1 M NaOH solution in a water bath at 80° C. for 10 min.
[0024] Preferably, in step S4, the decolorization conditions are as follows: immersing the purified bacterial cellulose membrane in a 0.5M NaOH solution, decolorizing in an 80°C water bath, and continuously replacing the alkali solution during the decolorization process until the bacterial cellulose membrane becomes milky white and translucent; immersing the milky white and translucent bacterial cellulose membrane in deionized water, de-alkaliizing in an 80°C water bath, and continuously replacing the deionized water until the pH on the surface of the bacterial cellulose membrane is 7.
[0025] The invention discloses a method for producing bacterial cellulose by fermenting lignocellulose hydrolysate. The yield of bacterial cellulose produced by fermenting lignocellulose hydrolysate is equivalent to that of a commonly used HS liquid culture medium, and the cost is lower than that of fermenting bacterial cellulose by a commonly used HS liquid culture medium. A new method for producing bacterial cellulose is provided, and lignocellulose resources are converted into high-value-added bacterial cellulose products, which is of great significance for realizing green biomanufacturing of bacterial cellulose and improving production economy.
[0026] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 The standard curves of glucose (Part A) and xylose (Part B) measured by the high performance liquid chromatography differential detector in Example 1;
[0028] Figure 2 The standard curves of furfural (Part A), phenol (Part B) and acetic acid (Part C) measured by the high performance liquid chromatography ultraviolet detector in Example 1 and Example 2;
[0029] Figure 3 This is a comparison chart of the yields of bacterial cellulose produced by fermentation of lignocellulose hydrolysate and bacterial cellulose produced by fermentation of HS liquid culture medium in Example 3. DETAILED DESCRIPTION
[0030] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention more clearly understood, the embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.
[0031] Embodiment 1
[0032] A method for preparing a lignocellulosic hydrolysate comprises:
[0033] S1. Preparation of lignocellulosic raw materials
[0034] The corn stalks were cut into 2-3 cm small segments, dried naturally in a fume hood, and then crushed with a micro-plant test grinder. The crushed stalk powder was sieved through a 60-mesh sieve, dried at 60°C, and stored in sealed bags.
[0035] S2. Weigh 100 g of crushed corn stalks, add 2% H2SO4 at a solid-liquid ratio (w / v) of 1:15, mix gently, and treat at 120°C for 60 min.
[0036] S3. After the reaction is completed, cool to room temperature, adjust pH to 4.5 with NaOH solution, add cellulase and hemicellulase at the amount of 150 U / g corn straw and 200 U / g corn straw, and shake on a shaker at 50°C and 200 rpm for 72 hours.
[0037] S4. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is filtered out using a Buchner funnel to obtain a corn straw hydrolyzate, i.e., a lignocellulose hydrolyzate.
[0038] S5. Determine the components of the lignocellulose hydrolysate using a high performance liquid chromatography differential detector and an ultraviolet detector.
[0039] HPLC differential detector analysis conditions: the chromatographic column is a Biorad Aminex HPX-87H chromatographic column, a guard column is connected before the chromatographic column, the injection volume is 10 μL, the mobile phase is 5 mM H2SO4, the flow rate is 0.6 mL / min, the column temperature is controlled at 65°C, the detector temperature is 50°C, and the analysis time for each sample is 15 min.
[0040] HPLC UV detector analysis conditions: the chromatographic column is Promosil C-18, the injection volume is 10 μL, the mobile phase is 20 mM disodium hydrogen phosphate (the pH is adjusted to 2.7 with phosphoric acid) and pure methanol, the aqueous phase ratio is 90% and the organic phase ratio is 10%, the flow rate is 0.8 mL / min, the column temperature is controlled at 30°C, the wavelength is 210 nm, and the analysis time for each sample is 40 min.
[0041] like Figure 1 Shown are standard curves of glucose (Part A) and xylose (Part B) measured by HPLC differential detection.
[0042] like Figure 2 Shown are the standard curves of furfural (part A), phenol (part B) and acetic acid (part C) using an HPLC UV detector.
[0043] The glucose concentration in the lignocellulose hydrolysate was detected to be 25.31 g / L, the xylose concentration was 9.19 g / L, the acetic acid concentration was 5.11 g / L, the furfural concentration was 1.45 g / L, and the phenol concentration was 0.065 g / L.
[0044] Embodiment 2
[0045] Detoxification of lignocellulosic hydrolysate, including:
[0046] S1. Measure the volume of the lignocellulose hydrolysate, add ethyl acetate at a liquid-to-liquid ratio (v / v) of 1:5, and shake to mix well so that the lignocellulose hydrolysate and ethyl acetate are fully combined.
[0047] S2. Allow to stand until the lignocellulose hydrolysate and ethyl acetate are separated and most of the ethyl acetate in the upper layer is discarded.
[0048] S3. Pour the remaining lignocellulose hydrolyzate and ethyl acetate mixture into the distillation flask of a rotary evaporator, set the temperature of the rotary evaporator to 65° C. for rotary evaporation, and take out the lignocellulose hydrolyzate after no residual ethyl acetate is left.
[0049] S4. Analyze the components again using high performance liquid chromatography (HPLC) with the same analysis conditions as in Example 1.
[0050] After measurement, the inhibitor concentration of the detoxified lignocellulose hydrolysate was 0g / L.
[0051] Embodiment 3
[0052] The fermentation of lignocellulose hydrolysate to produce bacterial cellulose comprises the following steps:
[0053] S1. Activated strain
[0054] (a) Prepare HS medium: glucose (25 g / L), peptone (10 g / L), yeast powder (7.5 g / L), disodium hydrogen phosphate (10 g / L), adjust the pH of the medium to 4.5 with glacial acetic acid, add 1%-2% agar to the solid medium before adjusting the pH, sterilize at 115°C for 30 min, and set aside.
[0055] (b) K. xylinus CGMCC2955 was cultured in HS medium. The glycerol tube of K. xylinus stored in a -80°C refrigerator was taken out, three-zoned on a HS solid plate, and the temperature of the static incubator was adjusted to 30°C. The plate was cultured for 1-2 days to obtain a single colony.
[0056] K. xylinus CGMCC2955 is a published strain.
[0057] (c) A single colony with good morphology was inoculated into HS liquid medium and cultured in a shaker at 30°C and 180 rpm for 18 h. The bacterial liquid was transferred to 30 mL HS liquid medium with cellulase added in advance at a 1% inoculum and cultured at 30°C and 180 rpm.
[0058] S2. Fermentation medium configuration
[0059] 10 g / L peptone, 7.5 g / L yeast extract powder and 10 g / L disodium hydrogen phosphate were added to the lignocellulose hydrolyzate in Example 2, and the pH was adjusted to 4.5 with glacial acetic acid. The solution was sterilized at 115° C. for 30 min for later use.
[0060] S3, the seed liquid prepared in step S1 is used to collect bacteria under sterile conditions, and the bacteria are centrifuged at 4°C and 4500rpm for 10min to retain the bacteria; the bacteria are resuspended in sterile HS liquid culture medium, and the bacteria are centrifuged again at 4°C and 4500rpm for 10min to retain the bacteria. The purpose of this step is to wash the bacteria; finally, the bacterial precipitate is resuspended in a small amount of HS culture medium, and the initial concentration of the bacterial liquid fermentation is adjusted to OD600=0.02, and an appropriate amount of seed liquid is inoculated into the fermentation medium and the same volume of HS liquid culture medium, and the culture is statically cultured in a constant temperature incubator at 30°C for 7 days, and three parallels are set for each fermentation group.
[0061] S4. Purification of BC
[0062] (a) After the static culture, the bacterial cellulose membrane was taken out and the surface of the bacterial cellulose membrane was rinsed with deionized water. In order to quickly remove the residual bacteria on the bacterial cellulose membrane, the membrane was immersed in NaOH solutions with concentrations of 0.5 M, 1 M and 1.5 M, respectively. The water bath temperature was set to 80 °C for 5 min, 10 min and 15 min, respectively, as shown in Table 1 below. The OD600 value was measured by sampling with a UV spectrophotometer, and finally 1 M NaOH solution in a water bath at 80 °C for 10 min was selected as the best condition for removing bacteria.
[0063] Table 1 Optimization results of bacterial removal conditions
[0064]
[0065] (b) Since the bacterial cellulose membrane produced by fermentation of lignocellulose hydrolysate has a dark color, the decolorization conditions of the bacterial cellulose membrane were optimized. The bacterial cellulose membrane with the bacteria removed was immersed in NaOH solutions with concentrations of 0.1M, 0.5M and 1M, respectively, and the water bath temperature was 80°C for decolorization. The alkali solution was continuously replaced until the BC membrane became milky white and translucent, and then immersed in deionized water and de-alkaliated in a water bath at 80°C. The deionized water was continuously replaced until the pH of the BC membrane surface was 7, as shown in Table 2 below. Based on the decolorization and de-alkali time as the standard, 0.5M MaOH solution was finally selected as the optimal decolorization and de-alkali conditions; the purification method of the bacterial cellulose membrane produced by fermentation of HS liquid culture medium is the same as that of the bacterial cellulose membrane produced by fermentation of lignocellulose hydrolysate.
[0066] Table 2 Optimization results of bacterial removal conditions
[0067] condition Decolorization and dealkali treatment time (h) 0.1MNaOH 168 0.5MNaOH 132 1MNaOH 144
[0068] S5. Determination of bacterial cellulose production
[0069] The yield of bacterial cellulose is expressed as dry weight, and the steps are as follows:
[0070] S1. The purified bacterial cellulose membrane is pressed into dry sheets using a sheet machine.
[0071] S2. Use a precision balance to weigh the mass of the bacterial cellulose membrane.
[0072] S3. The bacterial cellulose yield was calculated according to the formula: bacterial cellulose yield (g / L) = dry film weight (g) / initial volume of fermentation liquid (L), and the average value was calculated for the parallel groups.
[0073] Comparison of bacterial cellulose production by fermentation of lignocellulose hydrolysate and HS liquid medium Figure 3As shown, it can be seen that the yield of bacterial cellulose produced by fermentation of lignocellulose hydrolysate is comparable to that of the commonly used HS liquid culture medium.
[0074] Therefore, the present invention provides a method for producing bacterial cellulose by fermenting lignocellulose hydrolysate. The yield of bacterial cellulose produced by fermenting lignocellulose hydrolysate is comparable to that of the commonly used HS liquid culture medium, and the cost is lower than that of fermenting bacterial cellulose by the commonly used HS liquid culture medium. This provides a new method for the production of bacterial cellulose, converts lignocellulose resources into high-value-added bacterial cellulose products, and is of great significance for realizing green biomanufacturing of bacterial cellulose and improving production economy.
[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.
Claims
1. A method for producing bacterial cellulose by fermenting lignocellulose hydrolysate, characterized in that: The steps include: S1, using corn straw as raw material, preparing lignocellulose hydrolyzate through acid hydrolysis and enzymatic hydrolysis; S2, using the detoxified lignocellulose hydrolysate to ferment and produce bacterial cellulose; S3. After the fermentation production is completed, the bacterial cellulose membrane is taken out for purification. The purification conditions are: Soak the bacterial cellulose membrane in a 0.5M-1.5M NaOH solution in a 80°C water bath for 5-15 minutes; S4. Decolorizing the purified bacterial cellulose membrane under the following decolorization conditions: The purified bacterial cellulose membrane is immersed in a 0.1M-1M NaOH solution and decolorized in a 80°C water bath. During the decolorization process, the alkali solution is continuously replaced until the bacterial cellulose membrane becomes milky white and translucent. The milky white translucent bacterial cellulose membrane was soaked in deionized water and de-alkalized in a water bath at 80°C, and the deionized water was continuously replaced until the pH value on the surface of the bacterial cellulose membrane was 7.
2. The method for producing bacterial cellulose by fermenting lignocellulose hydrolysate according to claim 1, characterized in that: The specific steps of step S1 include: S11, cutting corn stalks into 2-3 cm small segments, drying naturally, and crushing the crushed corn stalks. The crushed corn stalk powder is passed through a 60-mesh sieve, dried at 60° C., and then sealed and stored; S12, add 2% H2SO4 by mass to corn straw powder at a solid-liquid ratio of 1:15, mix gently, and treat at 120°C for 60 minutes; S13. After the acid hydrolysis in step S12 is completed, the reaction solution is cooled to room temperature, a NaOH solution is added to adjust the pH of the reaction solution to 4.5, cellulase and hemicellulase are added in an amount of 150 U / g corn stalks and 200 U / g corn stalks, and enzymolysis is performed at 50° C. and 200 rpm on a shaker for 72 hours; S14. After the enzymatic hydrolysis is completed, the enzymatic hydrolyzate is filtered out using a Buchner funnel to obtain a corn straw hydrolyzate, i.e., a lignocellulose hydrolyzate.
3. The method for producing bacterial cellulose by fermenting lignocellulose hydrolysate according to claim 1, characterized in that: In step S2, the detoxification of the lignocellulose hydrolysate is performed by extracting the inhibitor in the lignocellulose hydrolysate with ethyl acetate.
4. The method for producing bacterial cellulose by fermentation of lignocellulose hydrolysate according to claim 3, characterized in that: The volume ratio of lignocellulose hydrolysate to ethyl acetate during extraction was 1:
5.
5. The method for producing bacterial cellulose by fermentation of lignocellulose hydrolysate according to claim 1, characterized in that: In step S2, the strain used in the fermentation production is Gluconacetobacter xylinus CGMCC2955.
6. The method for producing bacterial cellulose by fermenting lignocellulose hydrolysate according to claim 1, characterized in that: In step S2, the fermentation medium configuration during fermentation production includes: adding 10 g / L peptone, 7.5 g / L yeast extract powder and 10 g / L disodium hydrogen phosphate to the detoxified lignocellulose hydrolysate, adjusting the pH to 4.5 with glacial acetic acid, and sterilizing at 115°C for 30 minutes for standby use.
7. The method for producing bacterial cellulose by fermenting lignocellulose hydrolysate according to claim 1, characterized in that: In step S3, the purification conditions are as follows: the bacterial cellulose membrane is immersed in a 1 M NaOH solution in a water bath at 80° C. for 10 min.
8. The method for producing bacterial cellulose by fermenting lignocellulose hydrolysate according to claim 1, characterized in that: In step S4, the decolorization conditions are as follows: the purified bacterial cellulose membrane is immersed in a 0.5M NaOH solution, and decolorized in an 80°C water bath, and the alkali solution is continuously replaced during the decolorization process until the bacterial cellulose membrane becomes milky white and translucent; the milky white and translucent bacterial cellulose membrane is immersed in deionized water, and de-alkaliized in an 80°C water bath, and the deionized water is continuously replaced until the pH on the surface of the bacterial cellulose membrane is 7.