Method for producing bacterial cellulose through large-scale fermentation

Abstract

The invention relates to a fermentation method of bacterial cellulose, in particular to a method for producing bacterial cellulose through large-scale fermentation. On the basis of integration of low-cost culture medium design and cooperation of intelligent fermentation control, carbon and nitrogen sources in a fermentation culture medium, corn steep liquor pretreatment and fermentation process conditions are optimized, and static fermentation culture is performed in combination with a layered standing reaction tank, so that the technical bottlenecks of low fermentation yield, poor economy and small scale of bacterial cellulose are broken through; compared with dynamic fermentation, the method has the advantages that the energy consumption is obviously reduced, meanwhile, the problems of low fermentation yield and difficulty in decoloration of the bacterial cellulose in the static fermentation process are effectively solved, and a solid foundation is laid for commercial application of the bacterial cellulose.

Description

Technical Field

[0001] This invention relates to a fermentation method for bacterial cellulose, and more specifically to a method for large-scale fermentation to produce bacterial cellulose. Background Technology

[0002] Bacterial cellulose (BC) is a high-purity nanocellulose synthesized by microorganisms such as Komagataeibacter spp. It possesses a unique three-dimensional network structure, high crystallinity, excellent mechanical strength, and biocompatibility, showing broad application prospects in medical dressings, food packaging, tissue engineering, and flexible electronic devices. However, existing production technologies still face key bottlenecks such as low yield (traditional static culture yields are typically below 8 g / L), high cost (carbon source content reaches 30%-50%), and complex processes (requiring precise control of pH and dissolved oxygen, and high energy consumption during purification), limiting its large-scale industrial application. Although studies have improved the process through strain mutagenesis (such as ARTP mutation), gene editing (such as enhancing the bcsABCD cellulose synthesis gene), or culture medium optimization (such as using agricultural waste to replace traditional carbon and nitrogen sources), none of these methods have simultaneously solved the problems of high yield, low cost, and process stability. For example, dynamic culture is prone to strain mutation, while low-cost culture media may introduce impurities that affect product uniformity.

[0003] Currently, the efficient production of bacterial cellulose still faces multiple challenges. At the strain level, long-term subculturing can lead to degradation of synthetic capacity and insufficient genetic stability. Regarding the culture system, traditional carbon sources (such as fructose and mannitol) are expensive, while inexpensive alternative raw materials (such as bagasse and corn steep liquor) often result in the accumulation of byproducts, affecting fermentation efficiency. In terms of process optimization, although airlift reactors combined with pulse feeding strategies can increase the yield to over 10 g / L, the significant increase in equipment investment and energy consumption makes it less economical, thus hindering the commercial application of bacterial cellulose.

[0004] To address the aforementioned issues, there is an urgent need to develop a collaborative innovation technology that integrates low-cost culture medium design and intelligent fermentation control. This patent aims to provide a high-yield, economical, and scalable method for preparing bacterial cellulose, thereby overcoming existing technological bottlenecks and promoting its widespread application in fields such as biomedicine and environmental materials. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for large-scale fermentation of bacterial cellulose, which addresses the shortcomings of existing technologies such as low yield, poor economic efficiency, and small scale.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for large-scale fermentation to produce bacterial cellulose involves mixing the fermentation medium with the seed liquid of Acetobacter xylinum in a stirred tank and then transferring the mixture to a reaction tank for multiple batch static fermentation culture or single batch static fermentation culture.

[0008] The multiple batches of static fermentation culture consist of two or more batches. After the first batch of static fermentation culture is completed, fresh fermentation culture medium is sent into the reaction vessel for the second batch of static fermentation culture, and so on.

[0009] In an embodiment of the present invention, the multiple batches of static fermentation culture are 2 to 10 batches.

[0010] In an embodiment of the present invention, the multiple batches of static fermentation culture are 2 to 5 batches.

[0011] In an embodiment of the present invention, the multiple batches of static fermentation culture are two batches.

[0012] In an embodiment of the present invention, the time for feeding each batch of fermentation medium is after the static fermentation culture of the previous batch of fermentation medium has ended.

[0013] In order to save equipment costs and simplify equipment design, in the embodiments of the present invention, the fresh fermentation medium that is fed in after the first batch of static fermentation culture is also fed in through a stirred tank.

[0014] In an embodiment of the present invention, the feed is pumped in by a pump.

[0015] The fermentation medium contains corn steep liquor pretreated with alkali and liquefying enzyme.

[0016] The scale mentioned here refers to a scale of 120L or more.

[0017] In an embodiment of the present invention, the Acetobacter xylinus is Komagataeibacter xylinus CICC 10529.

[0018] The seed culture of Acetobacter xylinum is obtained by shaking flask seed culture of Acetobacter xylinum strain, followed by secondary seed culture expansion of the seed culture.

[0019] Specifically, the seed culture medium for shake flask culture and secondary seed expansion culture has the following formula: 10-30 g / L glucose, 10-20 g / L yeast extract, 10-20 g / L peptone, with water as the solvent and natural pH.

[0020] In an embodiment of the present invention, the seed culture medium for shake flask culture and secondary seed expansion culture has the following formula: 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, with water as the solvent and natural pH.

[0021] In an embodiment of the present invention, the shake flask culture is carried out under the following conditions: 28-32°C and 150-200 r / min until flocculent matter appears.

[0022] In an embodiment of the present invention, the secondary seed expansion culture is carried out under the following conditions: 28-32℃ and 150-200r / min for 12-24 hours.

[0023] In an embodiment of the present invention, the mixing time is 10 to 30 minutes.

[0024] In an embodiment of the present invention, the mixing time is 10 minutes.

[0025] The corn steep liquor pretreated with alkali and liquefying enzyme is prepared by the following method: the pH of the corn steep liquor is adjusted to 5-6.5, the temperature is raised to 50-70℃, liquefying enzyme with 2-10 U / g dry matter is added and treated for 1-4 hours, and then the pH is adjusted to 7.5-8.0. The liquid part is obtained by solid-liquid separation.

[0026] In an embodiment of the present invention, the solid-liquid separation liquid portion is obtained by centrifuging at 5000-10000 r / min for 10-20 minutes, and then passing the supernatant through a microfiltration membrane to obtain the permeate.

[0027] The fermentation medium has the following formula: 20-40 g / L glucose, 20-80 mL / L corn steep liquor pretreated with alkali and liquefying enzyme, 2-3 g / L disodium hydrogen phosphate, 4-10 g / L succinic acid, 5-6 g / L magnesium sulfate, 1.5-3 g / L ammonium sulfate, and pH 5.0-6.5.

[0028] In an embodiment of the present invention, the fermentation medium has the following formulation: 33.22 g / L glucose, 72.0 mL / L corn steep liquor pretreated with alkali and liquefying enzyme, 2.1 g / L disodium hydrogen phosphate dodecahydrate, 8.25 g / L succinic acid, 5.7 g / L anhydrous magnesium sulfate, 2 g / L ammonium sulfate, and pH 5.0.

[0029] In an embodiment of the present invention, by using a corn steep liquor fermentation medium that has been optimized by response surface methodology and pretreated, the bacterial cellulose yield is increased, while the problem of difficult bacterial cellulose decolorization is solved by alkali solution and liquefying enzyme treatment.

[0030] The reaction vessel is a stratified static reaction vessel.

[0031] In embodiments of the present invention, the layered settling reaction vessel may be the layered settling reaction vessel described in Chinese Patent CN108441416, or it may be an improved layered settling reaction vessel based on the layered settling reaction vessel described in the patent.

[0032] In an embodiment of the present invention, the improved layered static reaction tank is provided with a plurality of trays, and a tray hole is formed by punching a hole on one side of each tray. A corresponding reaction tank hole is formed by punching a hole on the tank wall near the hole of each tray. The tray hole is connected to its corresponding reaction tank hole. The reaction tank hole is connected to a main pipeline through parallel branch pipes. The main pipeline is connected to a stirring tank. A steam pipeline and a venting pipeline are provided on the main pipeline.

[0033] In an embodiment of the present invention, the plurality of trays is 20 to 40 trays.

[0034] In an embodiment of the present invention, the plurality of trays is 20 trays.

[0035] In an embodiment of the present invention, the diameter of the tray is 32-36cm, the height is 2.5-5cm, and the gap between the trays is 0.5-2cm.

[0036] In an embodiment of the present invention, the tray has a diameter of 34cm, a height of 3.5cm, and a gap of 0.5 to 2cm between the trays.

[0037] In an embodiment of the present invention, the liquid level in the tray is 1 to 3 cm.

[0038] In an embodiment of the present invention, the liquid level in the tray is 2 cm.

[0039] The seed culture of Acetobacter xylinum accounts for 2-3% of the volume of the reaction vessel; when performing multiple batches of static fermentation culture, the total volume of the added fermentation medium is 25-30% of the volume of the reaction vessel; when performing a single batch of static fermentation culture, the volume of the fermentation medium is 25-30% of the volume of the reaction vessel.

[0040] In an embodiment of the present invention, the seed culture of Acetobacter xylinum accounts for 2.5% of the volume of the reaction vessel; when performing multiple batches of static fermentation culture, the total volume of the added fermentation medium is 27.5% of the volume of the reaction vessel; when performing a single batch of static fermentation culture, the volume of the fermentation medium is 27.5% of the volume of the reaction vessel.

[0041] When conducting multiple batches of static fermentation culture, the first batch of static fermentation culture is cultured under the following conditions: temperature 28-32℃, aeration rate 0.04-0.05 vvm, tank pressure 0.01-0.08 MPa for 2-3 days; subsequent batches of static fermentation culture are cultured under the following conditions: temperature 28-32℃, aeration rate 0.08-0.11 vvm, tank pressure 0.01-0.08 MPa for 2-3 days.

[0042] In this embodiment of the invention, the first batch of static fermentation culture is cultured under the following conditions: temperature 30℃, aeration rate 0.05vvm, tank pressure 0.01Mpa for 3 days; the subsequent static fermentation cultures are cultured under the following conditions: temperature 30℃, aeration rate 0.11vvm, tank pressure 0.01Mpa for 3 days.

[0043] When conducting single-batch static fermentation culture, the fermentation culture conditions are: temperature 28-32℃, aeration rate 0.04-0.11 vvm, and tank pressure 0.01-0.08 MPa, and culture for 8-10 days.

[0044] In this embodiment of the invention, when performing a single-batch static fermentation culture, the fermentation culture conditions are: a temperature of 30°C, an aeration rate of 0.05 to 0.11 vvm, and a tank pressure of 0.01 to 0.075 MPa, under which static fermentation culture is carried out for 8 days.

[0045] In the static fermentation culture, aeration is performed for 0.5 to 2 minutes every 3 to 6 hours.

[0046] In this embodiment of the invention, the static fermentation culture is carried out by aeration from the bottom of the tray inside the reactor for 1 minute every 4 hours during the static fermentation culture.

[0047] Beneficial effects:

[0048] (1) Based on the integration of low-cost culture medium design and intelligent fermentation control, this invention optimizes the carbon and nitrogen sources, corn steep liquor pretreatment and fermentation process conditions in the fermentation culture medium, and combines static fermentation culture with static fermentation device to overcome the technical bottlenecks of low bacterial cellulose fermentation yield, poor economy and small scale. It can quickly and significantly increase the yield of bacterial cellulose and expand the production capacity of bacterial cellulose to 120L.

[0049] (2) The bacterial cellulose product obtained by fermentation using the pretreated fermentation medium formula of this invention is easier to purify, reducing the raw material and power costs of the separation process. Compared with dynamic fermentation, energy consumption is significantly reduced. At the same time, it effectively solves the problem of difficult decolorization of bacterial cellulose during fermentation, laying a solid foundation for the commercial application of bacterial cellulose. Attached Figure Description

[0050] The present invention will be further described in detail below with reference to the accompanying drawings, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0051] Figure 1 Images show the bacterial cellulose fermentation process after completion and purification. In the images, A and C represent the untreated corn steep liquor group after bacterial cellulose fermentation and purification, respectively; B and D represent the pretreated corn steep liquor group after bacterial cellulose fermentation and purification, respectively.

[0052] Figure 2 This is a schematic diagram of the overall structure of a static fermentation device.

[0053] Figure 3 The fermentation of bacterial cellulose is shown under an aeration rate of 0.11 vvm and a tank pressure of 0.01 MPa.

[0054] Figure 4 This is a schematic diagram of the improved 120L static fermentation device. Detailed Implementation

[0055] The present invention will be further described in detail below with reference to specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0056] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0057] In the following examples, the *Komagataeibacter xylinus* CICC 10529 is commercially available from the China Industrial Microbial Culture Collection Center; the HS medium is a commercially available culture medium for culturing *Acetobacter xylinus* and other related microorganisms.

[0058] Example 1: Single-factor optimization experiment of fermentation medium

[0059] 1. Seed culture

[0060] Add 150 mL of seed culture medium YPD (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) to a clean 500 mL Erlenmeyer flask, sterilize at 121 °C for 15 minutes, cool, and place in a clean bench. Inoculate one loop of Komagataeibacter xylinus CICC 10529 slant culture into the shake flask seed culture medium, shake well, and incubate at 150 rpm and 30 °C for about 2 days until obvious flocculent matter appears to obtain the seed solution.

[0061] 2. Fermentation culture

[0062] The seed culture was inoculated at a rate of 10% v / v into 500 mL Erlenmeyer flasks containing 150 mL of different fermentation media, with three replicates for each experiment. The flasks were incubated at 30℃ for 8 days. The effects of different glucose, corn steep liquor, and succinic acid concentrations in the fermentation media on the production of bacterial cellulose by *Xylostella spp.* were investigated.

[0063] Basic fermentation medium: glucose 20 g / L, corn steep liquor 20 mL / L, disodium hydrogen phosphate dodecahydrate 2.1 g / L, succinic acid 1 g / L, anhydrous magnesium sulfate 5.7 g / L, ammonium sulfate 2 g / L, pH adjusted to 5.0 with sodium hydroxide.

[0064] (1) Glucose concentration optimization experiment: 0 g / L, 20 g / L, 40 g / L, 60, 80 g / L, 100 g / L, with the other components remaining unchanged.

[0065] (2) Optimization test of corn steep liquor concentration: 20 mL / L, 40 mL / L, 60 mL / L, 80 mL / L, with the other components remaining unchanged.

[0066] (3) Succinic acid concentration optimization test: 0 g / L, 2 g / L, 4 g / L, 6 g / L, 8 g / L, 10 g / L, with the other components remaining unchanged.

[0067] 3. Analysis and testing

[0068] After fermentation, the reducing sugar concentration was measured, and the bacterial cellulose was purified. The bacterial cellulose membrane was soaked in a 0.1 mol / L sodium hydroxide solution (50℃) for 12 hours, then the solution was changed and the membrane was soaked again until it turned milky white. Finally, it was repeatedly soaked and washed with pure water at 50-80℃ to obtain a pH-neutral transparent gel membrane, which was then dried and weighed.

[0069] Table 1. Effects of different concentrations of glucose, corn steep liquor, and succinic acid on bacterial cellulose production by *Xylose* fermentation.

[0070]

[0071] The results are shown in Table 1. From the table, it can be seen that (1) when the glucose concentration in the fermentation medium is 20-40 g / L, the bacterial cellulose yield is at a high level. Afterwards, as the glucose concentration in the fermentation medium continues to increase, the bacterial cellulose content gradually decreases. (2) The bacterial cellulose yield increases significantly with the increase of corn steep liquor concentration, showing an upward trend in the range of 20-80 mL / L. Moreover, the growth momentum gradually increases with the increase of concentration, which indicates that nitrogen source is the key factor limiting bacterial cellulose synthesis. (3) After adding succinic acid to the basic fermentation medium, it was found that the bacterial cellulose yield first increases and then decreases. When the amount of succinic acid added is 4-8 g / L, the bacterial cellulose yield is at a high level overall.

[0072] Example 2: Response surface methodology optimization of fermentation medium

[0073] Response surface methodology was employed using Design-Expert software to study the interactions among three key factors: glucose concentration (A), corn steep liquor concentration (B), and succinic acid concentration (C). The optimal combination of these three factors for bacterial cellulose production was determined to optimize the fermentation medium formulation. Three levels were selected for each factor, as shown in Table 2.

[0074] Table 2 Factor Level Table

[0075]

[0076] Based on the response surface methodology in Table 3, 17 groups of fermentation media with different component concentrations were precisely prepared. The optimized fermentation medium formulation for *Xylostella xylostella* was evaluated using bacterial cellulose yield as the standard. The seed culture, fermentation culture, and analysis of bacterial cellulose production during fermentation were conducted as described in Example 1, and the response surface methodology results were recorded.

[0077] Table 3 Response Surface Experiment Scheme and Results

[0078] serial number Glucose (g / L) Corn steep liquor (mL / L) Succinic acid (g / L) BC membrane dry weight g / L 1 35 45 6 9.71 2 10 45 1 3.28 3 35 45 6 9.99 4 35 80 1 9.54 5 60 45 1 3.49 6 10 80 6 4.47 7 35 10 10 7.85 8 60 45 10 9.69 9 10 10 6 3.34 10 35 45 6 8.66 11 35 45 6 9.63 12 60 80 6 11.85 13 35 80 10 13.2 14 10 45 10 5.69 15 35 10 1 1.74 16 35 45 6 11.23 17 60 10 6 3.11

[0079] Response surface methodology was performed on the experimental data in Table 3 using glucose, corn steep liquor, and succinic acid as independent variables and bacterial cellulose (BC) membrane dry weight as the response value. The results are shown in Table 4. The p-value of the model was 0.0001 < 0.01, indicating that the quadratic regression model was highly significant; the p-value of the lack-of-fit term was 0.4796 > 0.05, which was not significant, indicating that the lack-of-fit term had a small effect; the correlation coefficient R... 2A value of 0.9713 indicates a high degree of fit in the regression equation; AP (adeq precision), the signal-to-noise ratio, reflects the influence of external interference on the experimental model, with an AP value of 14.0593 (>4), indicating a low degree of interference on the model; the coefficient of variation (CV) is 12.41%, indicating low model dispersion. Using Design Expert to fit the experimental data, a quadratic polynomial regression equation was generated between BC yield (Y) and the components of the fermentation medium: Y = 9.84 + 1.43A + 2.29B + 2.88C + 0.97AB + 1.90AC - 0.0.61BC - 3.36A. 2 -0.97B 2 -0.79C 2 Where A, B, and C represent glucose concentration, corn steep liquor concentration, and succinic acid concentration, respectively.

[0080] Table 4 Analysis of Response Surface Experiment Results

[0081]

[0082] Response surface methodology analysis revealed significant interactions between glucose, corn steep liquor, and succinic acid. The predicted values ​​for the three factors in bacterial cellulose production by *Xylostella xylostella* fermentation were: glucose concentration 33.22 g / L, corn steep liquor concentration 72.80 mL / L, and succinic acid concentration 8.25 g / L. The optimal bacterial cellulose yield was predicted to be 12.10 g / L.

[0083] Under these conditions, shake-flask fermentation was performed based on the optimal fermentation medium formulation. The bacterial cellulose yield was measured to be 12.67 g / L, which was largely consistent with the simulated prediction and showed no significant difference, fully demonstrating the accuracy and practicality of the model. Compared with HS medium specifically used for culturing *Acetobacter xylinum* and other related microorganisms (bacterial cellulose fermentation yield 3.12 g / L), the yield increased by more than four times, indicating that the optimized fermentation medium significantly improved the efficiency of bacterial cellulose synthesis.

[0084] Example 3: Effects of Corn Stew Pretreatment

[0085] Corn steep liquor pretreatment method: Adjust the pH of corn steep liquor to 5.5 with calcium hydroxide, heat to 50℃, add 5U / g (dry matter) of liquefying enzyme, that is, 5 units of liquefying enzyme activity per gram (g) of dry matter, treat for 2 hours, continue to add calcium hydroxide to adjust the pH to 8.0, then centrifuge at 5000r / min for 10 minutes and take the supernatant, and finally take the permeate through a microfiltration membrane.

[0086] Fermentation medium: glucose 33.22 g / L, pretreated corn steep liquor 72.80 mL / L, disodium hydrogen phosphate dodecahydrate 2.1 g / L, succinic acid 8.25 g / L, anhydrous magnesium sulfate 5.7 g / L, ammonium sulfate 2 g / L, pH adjusted to 5.0 with sodium hydroxide.

[0087] The bacterial cellulose produced during fermentation was analyzed and detected using the seed culture, fermentation culture, and analysis process described in Example 1. The experimental results are as follows: Figure 1 and Figure 2 As shown, the color of the untreated corn steep liquor group after bacterial cellulose fermentation and purification was darker than that of the pretreated group, even without alkali or liquefying enzyme treatment. Further testing showed that the bacterial cellulose yield of the pretreated group after drying reached 13.72 g / L, which was about 8% higher than that of the untreated corn steep liquor group.

[0088] Example 4: Large-scale fermentation using a 120L static fermentation unit

[0089] 1. Shake flask seed culture

[0090] Add 150 mL of seed culture medium YPD (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) to a clean 500 mL Erlenmeyer flask, sterilize at 121 °C for 15 minutes, cool, and place in a clean bench. Inoculate one loop of Komagataeibacter xylinus CICC 10529 slant culture into the shake flask seed culture medium, shake well, and incubate at 150 rpm and 30 °C for about 2 days until obvious flocculent matter appears, thus obtaining the shake flask seed solution.

[0091] 2. Secondary seed expansion culture

[0092] The shake flask seed culture was inoculated into a 10L seed tank containing 6L of seed culture medium at an inoculation rate of 10% v / v, and cultured at 30℃ and 150r / min for 24 hours to obtain a secondary seed culture.

[0093] 3. Large-scale fermentation using a 120L static fermentation unit

[0094] Large-scale fermentation is carried out using the static fermentation device in Chinese patent CN108441416. In this embodiment, the stirred fermentation tank in the patent is not used for fermentation, but is only used as a stirring tank. Figure 2 This is a schematic diagram of the overall structure of the static fermentation device in Chinese patent CN108441416.

[0095] Prepare 33L of fermentation medium (33.22g / L glucose, 72.80mL / L pretreated corn steep liquor, 2.1g / L disodium hydrogen phosphate dodecahydrate, 8.25g / L succinic acid, 5.7g / L anhydrous magnesium sulfate, 2g / L ammonium sulfate, pH adjusted to 5.0 with sodium hydroxide) in a 50L stirred tank 1, sterilize at 121℃ for 20 minutes, and then cool to 30℃.

[0096] 3L of mature secondary seed culture was inoculated into a 50L stirred tank 1 containing 33L of fermentation medium. After mixing for 10 minutes, it was pumped into a layered static reaction tank 2 (in this embodiment, the layered static reaction tank 2 has 20 trays, each tray is 34cm in diameter and 3.5cm in height, with a liquid level of about 2cm). The drainage pipe 71 was set at a certain height. After each tray was filled, the liquid overflowed into the next tray through the drainage pipe 71 until all trays were filled. Finally, the tank was aerated and pressurized for fermentation. Static fermentation was carried out for 8 days under the conditions of 30℃, aeration rate of 0.05~0.11vvm, and tank pressure of 0.01~0.075Mpa.

[0097] After fermentation, the bacterial cellulose was purified: the bacterial cellulose membrane was soaked in a 0.1 mol / L sodium hydroxide solution (50℃) for 12 hours, then soaked again in a fresh sodium hydroxide solution until it turned milky white. Finally, it was soaked several times in 50℃ hot water to obtain a pH-neutral gel membrane, which was then dried and weighed.

[0098] The results are shown in Table 5. In the stratified static reaction tank 2, by controlling factors such as aeration rate and tank pressure, it was found that under the same aeration conditions, higher tank pressure resulted in a denser and more resilient bacterial cellulose membrane with a higher degree of polymerization. However, excessive density led to insufficient dissolved oxygen in the lower layer, resulting in a thinner bacterial cellulose membrane and lower yield. Under the same tank pressure, a higher aeration rate promoted bacterial growth, thereby promoting bacterial cellulose synthesis. Figure 3 The fermentation of bacterial cellulose was demonstrated at an aeration rate of 0.11 vvm and a tank pressure of 0.01 MPa, with a yield reaching nearly 13 g / L. This shows that by adjusting the fermentation conditions, the yield and degree of polymerization of bacterial cellulose can be controlled to meet application requirements.

[0099] Table 5. Effects of different aeration rates and tank pressures on bacterial cellulose yield and degree of polymerization.

[0100] serial number Ventilation rate and tank pressure conditions Bacterial cellulose yield (g / L) Degree of aggregation 1 Ventilation rate: 0.05 vvm; Tank pressure: 0.01 MPa 9.82 689 2 Ventilation rate: 0.05 vvm / tank pressure: 0.03 MPa 7.27 780 3 Ventilation rate: 0.05 vvm / tank pressure: 0.05 MPa 6.16 895 4 Ventilation rate: 0.05 vvm / tank pressure: 0.075 MPa 4.98 1023 5 Ventilation rate: 0.08 vvm / tank pressure: 0.01 MPa 11.27 678 6 Ventilation rate: 0.11 vvm / tank pressure: 0.01 MPa 12.85 671

[0101] Example 5: Fermentation using a modified 120L static fermentation apparatus

[0102] A hole with a diameter of 0.4 cm is drilled on one side of the tray of the layered static fermentation tank 2, and a first short pipe with a length of 0.8 cm is welded into the hole. Corresponding holes with the same diameter of 0.4 cm are drilled on the wall of the layered static fermentation tank 2 on the same side of the tray, and a second short pipe with a length of 0.8 cm is welded into each hole. The first and second short pipes are connected using silicone tubing. Simultaneously, 20 independent branches are formed by welding stainless steel pipes to the outside of the holes in the wall of the layered static fermentation tank 2. Each branch is connected to the main pipeline via a separate one-way valve. A steam pipe, a vent pipe, and the stirring tank 1 are welded onto the main pipeline. A schematic diagram of the improved 120L static fermentation device is shown below. Figure 4 As shown.

[0103] 1. Shake flask seed culture

[0104] Add 150 mL of seed culture medium YPD (20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone) to a clean 500 mL Erlenmeyer flask, sterilize at 121 °C for 15 minutes, cool, and place in a clean bench. Inoculate one loop of Komagataeibacter xylinus CICC 10529 slant culture into the shake flask seed culture medium, shake well, and incubate at 150 rpm and 30 °C for about 2 days until obvious flocculent matter appears, thus obtaining the shake flask seed solution.

[0105] 2. Secondary seed expansion culture

[0106] The shake flask seed culture was inoculated into a 10L seed tank containing 6L of seed culture medium at an inoculation rate of 10% v / v, and cultured at 30℃ and 150r / min for 24 hours to obtain a secondary seed culture.

[0107] 3. Large-scale fermentation using the improved 120L static fermentation unit

[0108] In the first stage, 16.5L of fermentation medium (33.22g / L glucose, 72.80mL / L pretreated corn steep liquor, 2.1g / L disodium hydrogen phosphate dodecahydrate, 8.25g / L succinic acid, 5.7g / L anhydrous magnesium sulfate, 2g / L ammonium sulfate, pH adjusted to 5.0 with sodium hydroxide) was prepared in a 50L stirred tank 1. The medium was then sterilized at 121℃ for 20 minutes and then cooled to 30℃.

[0109] 3L of mature secondary seed culture was inoculated into a 50L stirred tank 1 containing 16.5L of fermentation medium. After mixing for 10 minutes, the one-way valves of 20 branches and valve 15 on the main pipeline of the stirred tank were opened, and the mixed material (metered by a peristaltic pump) was added to each tray of the stratified static reaction tank 2 (pre-emptively sterilized, including all side pipelines). Static fermentation was carried out for 3 days at a temperature of 30℃, an aeration rate of 0.05 vvm, and a tank pressure of 0.01 MPa. Then, in the second stage, 16.5L of fermentation medium was prepared again in the 50L stirred tank 1 and added to each tray of the stratified static reaction tank 2 in the same manner. The aeration rate was adjusted to 0.11 vvm, and static fermentation was continued for another 3 days. The bacterial cellulose yield was measured to be 12.18 g / L, and the fermentation time was shortened by 2 days.

[0110] Example 6: Large-scale fermentation using a modified 120L static fermentation apparatus

[0111] Seed culture and scale-up culture were performed in the same manner as in Example 5.

[0112] Large-scale fermentation using the improved 120L static fermentation unit: In the first stage, 16.5L of fermentation medium (33.22g / L glucose, 72.80mL / L pretreated corn steep liquor, 2.1g / L disodium hydrogen phosphate dodecahydrate, 8.25g / L succinic acid, 5.7g / L anhydrous magnesium sulfate, 2g / L ammonium sulfate, pH adjusted to 5.0 with sodium hydroxide) was prepared in a 50L stirred tank 1, sterilized at 121℃ for 20 minutes, and then cooled to 30℃.

[0113] 3L of mature secondary seed culture was inoculated into a 50L stirred tank 1 containing 16.5L of fermentation medium. After mixing for 10 minutes, the one-way valve on the branch line and valve 15 on the main pipeline of the stirred tank were opened, and the mixed material (metered by a peristaltic pump) was added to each tray of the stratified static reaction tank 2 (pre-emptively emptied, including all side pipelines). Static fermentation was carried out for 3 days at a temperature of 30℃, an aeration rate of 0.05 vvm, and a tank pressure of 0.01 MPa. Then, in the second stage, 16.5L of fermentation medium was prepared again in the 50L stirred tank 1 and added to each tray in the same manner. The aeration rate was adjusted to 0.11 vvm, and static fermentation was continued for another 3 days, for a total of 6 days of fermentation. An aeration device was installed at the bottom of the trays of the stratified static reaction tank 2, and aeration was carried out from the bottom of the trays for 1 minute every 4 hours during the fermentation period. The bacterial cellulose yield was further increased to 13.84 g / L.

[0114] This invention provides a method and approach for large-scale fermentation to produce bacterial cellulose. 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. A method for large-scale fermentation to produce bacterial cellulose, characterized in that, The fermentation medium and the seed liquid of Acetobacter xylinum are mixed in a stirred tank and then transferred to a reaction tank for multiple batch static fermentation culture or single batch static fermentation culture. The multiple batches of static fermentation culture consist of two or more batches. After the first batch of static fermentation culture is completed, fresh fermentation culture medium is sent into the reaction vessel for the second batch of static fermentation culture, and so on. The fermentation medium contains corn steep liquor pretreated with alkali and liquefying enzymes. The scale is defined as 120L or more.

2. The method according to claim 1, characterized in that, The Acetobacter xylinus mentioned is Komagataeibacter xylinus CICC 10529.

3. The method according to claim 1, characterized in that, The corn steep liquor pretreated with alkali and liquefying enzyme is prepared as follows: the pH of the corn steep liquor is adjusted to 5-6.5, the temperature is raised to 50-70℃, liquefying enzyme at 2-10 U / g dry matter is added and treated for 1-4 hours, then the pH is further adjusted to 7.5-8.0, and the liquid portion is obtained by solid-liquid separation.

4. The method according to claim 1, characterized in that, The fermentation medium has the following formula: 20-40 g / L glucose, 20-80 mL / L corn steep liquor pretreated with alkali and liquefying enzyme, 2-3 g / L disodium hydrogen phosphate, 4-10 g / L succinic acid, 5-6 g / L magnesium sulfate, 1.5-3 g / L ammonium sulfate, and pH 5.0-6.

5.

5. The method according to claim 1, characterized in that, The reaction vessel is a stratified static reaction vessel.

6. The method according to claim 5, characterized in that, The layered static reaction tank is equipped with several trays. A tray hole is drilled on one side of each tray. A corresponding reaction tank hole is drilled on the tank wall near the tray hole. The tray hole is connected to its corresponding reaction tank hole. The reaction tank hole is connected to the main pipeline through parallel branch pipes. The main pipeline is connected to the stirring tank. A steam pipeline and a venting pipeline are provided on the main pipeline.

7. The method according to claim 1, characterized in that, The seed culture of Acetobacter xylinum accounts for 2-3% of the volume of the reaction vessel; when performing multiple batches of static fermentation culture, the total volume of the added fermentation medium is 25-30% of the volume of the reaction vessel; when performing a single batch of static fermentation culture, the volume of the fermentation medium is 25-30% of the volume of the reaction vessel.

8. The method according to claim 1, characterized in that, When conducting multiple batches of static fermentation culture, the first batch of static fermentation culture shall be cultured under the following conditions: temperature 28-32℃, aeration rate 0.04-0.05 vvm, tank pressure 0.01-0.08 MPa for 2-3 days; subsequent batches of static fermentation culture shall be cultured under the following conditions: temperature 28-32℃, aeration rate 0.08-0.11 vvm, tank pressure 0.01-0.08 MPa for 2-3 days.

9. The method according to claim 1, characterized in that, When conducting single-batch static fermentation culture, the fermentation culture conditions are: temperature 28-32℃, aeration rate 0.04-0.11 vvm, tank pressure 0.01-0.08 MPa, and culture for 8-10 days.

10. The method according to claim 1, 8, or 9, characterized in that, During the static fermentation culture, aeration is performed for 0.5 to 2 minutes every 3 to 6 hours.

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