Method for modifying clay and use thereof
By immobilizing Trichoderma reesei with modified clay, the problem of excessive cell growth during Trichoderma reesei fermentation was solved, achieving efficient cellulase production and reducing fermentation costs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUHAN SUNHY BIOLOGICAL
- Filing Date
- 2024-06-25
- Publication Date
- 2026-05-12
AI Technical Summary
Excessive cell growth during Trichoderma reesei fermentation leads to a viscous fermentation broth, affecting mass transfer and enzyme activity, and increasing post-processing costs.
A clay modification method was adopted, in which clay was treated with long-chain fatty acids and inorganic acids to prepare modified clay as an immobilization material to immobilize Trichoderma reesei seed liquid, thereby achieving separation of the mycelium and fermentation broth, reducing mass transfer resistance and lowering post-treatment pressure.
It ensured the normal progress of the fermentation process, reduced fermentation costs, increased fermentation enzyme activity, simplified post-processing, and shortened the fermentation cycle.
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a clay modification method and its application. Background Technology
[0002] Cellulase is a complex enzyme system composed of exoglucanase, endoglucanase, and β-glucanase. The cellulase system produced by *Trichoderma reesei* is complete and its activity is stable. However, during the fermentation process of *Trichoderma reesei*, when the cell mass is too large, the fermentation broth becomes viscous, affecting the mass transfer process and preventing the fermentation enzyme activity from reaching a high level. Furthermore, after fermentation, the post-processing of the fermentation broth will also encounter significant resistance due to the cell mass, thus affecting the post-processing and increasing fermentation costs. Summary of the Invention
[0003] The purpose of this invention is to overcome the problems of excessive growth of Trichoderma reesei in the later stage of fermentation, which prevents the fermentation enzyme activity from reaching a high level and results in high post-processing costs in the existing technology.
[0004] Therefore, the present invention provides a clay modification method, comprising the following steps:
[0005] (1) Clay is crushed and sieved. Long-chain fatty acids are added to the sieved clay to react. After the reaction, the clay is filtered, washed and dried to obtain primary modified clay.
[0006] (2) Add inorganic acid and fatty amine to the primary modified clay to react. After the reaction, filter, wash and dry to obtain secondary modified clay.
[0007] (3) Add water to the secondary modified clay and bake it to obtain modified clay.
[0008] Specifically, in step (1) above, a long-chain fatty acid solution with a mass concentration of 3-5% is added to the sieved clay, and the solid-liquid ratio of the clay to the long-chain fatty acid solution is 1:5-7.
[0009] Specifically, in step (2) above, 0.02-0.05 mol / L of inorganic acid and 3-5% of fatty amine solution are added to the primary modified clay in a mass ratio of 1:3:3.
[0010] Specifically, in steps (1) and (2) above, the reaction temperature is 70-80℃ and the reaction time is 16-24h.
[0011] The present invention also provides a modified clay prepared by the above-described clay modification method.
[0012] The present invention also provides an immobilized Trichoderma reesei, comprising Trichoderma reesei and the above-mentioned modified clay.
[0013] Specifically, the aforementioned immobilized Trichoderma reesei includes modified clay with a solid-liquid ratio of 1:2 to 3.5 and Trichoderma reesei seed liquid.
[0014] This invention also provides a method for producing cellulase using immobilized Trichoderma reesei, which specifically includes the following steps:
[0015] (1) Prepare the fermentation medium;
[0016] (2) Immobilized Trichoderma reesei was added to the fermentation medium to produce cellulase.
[0017] Specifically, in step (2) above, the amount of immobilized Trichoderma reesei added is 3-5% based on the mass of the fermentation medium.
[0018] Specifically, during the fermentation process in step (2) above, when the dissolved oxygen level drops to its lowest point and rebounds to above 50%, feeding begins. During the feeding process, the dissolved oxygen level is controlled at 10-20%.
[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0020] This invention provides a clay modification method that modifies the wettability and other properties of clay. The resulting modified clay can be used as an immobilization material to fix Trichoderma reesei seed culture and is applied in Trichoderma reesei fermentation. Due to the unique characteristics of immobilized Trichoderma reesei, the fungus does not enter the fermentation broth, but its metabolic products gradually dissolve in the broth, thus achieving separation of the fungal cells from the fermentation broth. This reduces mass transfer resistance during fermentation, allowing the fermentation process to proceed normally. Simultaneously, it also reduces the pressure on post-processing, lowers the overall fermentation cost, and solves the problem of excessive Trichoderma reesei growth in the later stages of fermentation. After fermentation, the modified clay blocks are removed, yielding the cellulase fermentation broth. The modified clay blocks can also be reused for the immobilization of new batches of Trichoderma reesei strains. Detailed Implementation
[0021] The technical solutions of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Although representative embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.
[0022] This invention provides a method for modifying clay, comprising the following steps:
[0023] (1) After the clay is crushed and sieved, long-chain fatty acids are added to the sieved clay. A long-chain fatty acid solution with a mass concentration of 3-5% is preferred for the reaction. The reaction temperature is 70-80℃, and the reaction time is 16-24h. The solid-liquid ratio of clay to long-chain fatty acid solution is 1:5-7. The long-chain fatty acid is a fatty acid with more than 12 carbon atoms, preferably one or more of oleic acid, linoleic acid, stearic acid, linolenic acid, palmitic acid, and myristic acid.
[0024] After the reaction, the mixture is filtered and washed. The filter cake is then preferably dried at 65°C until all surface moisture is evaporated, followed by drying at 105°C to obtain primary modified clay. After drying, the wettability of the clay is improved.
[0025] (2) Add 0.02-0.05 mol / L inorganic acid and 3-5% fatty amine solution to the modified clay in a mass ratio of 1:3:3. The reaction temperature is 70-80℃ and the reaction time is 16-24h. After the reaction, filter and wash. After taking out the filter cake, it is preferable to dry it at 65℃ until the surface moisture is completely evaporated, and then dry it at 105℃ to obtain the secondary modified clay.
[0026] The inorganic acid is a strong inorganic acid, preferably one or more of hydrochloric acid, nitric acid, sulfuric acid, and perchloric acid; the fatty amine is preferably one or more of stearylamine, oleylamine, palmitamine, etc.
[0027] (3) Add water to the secondary modified clay at a solid-liquid ratio of 1:3 to 6, and place it in the corresponding mold according to actual needs. Generally, a spherical mold is selected. It is preferable to bake the mold at 105°C for 2 hours, then take it out and demold it. Bake it at 400°C in a muffle furnace for 2 hours to obtain a well-shaped modified clay model.
[0028] After high-temperature baking, the modified clay is placed in a sterile environment for later use to prevent contamination.
[0029] The present invention also provides an immobilized Trichoderma reesei, comprising Trichoderma reesei and modified clay prepared by the above method, preferably comprising modified clay with a solid-liquid ratio of 1:2 to 3.5 and Trichoderma reesei seed liquid.
[0030] In a more detailed embodiment, the immobilized Trichoderma reesei is prepared by the following steps: under sterile conditions, modified clay is mixed with Trichoderma reesei seed liquid cultured to the logarithmic growth phase, fixed for 1-2 hours, and then the surface moisture is wiped off with a sterile cloth. It is then placed in a sterile bag for later use to prevent contamination and facilitate subsequent fermentation operations.
[0031] This invention also provides a method for producing cellulase using immobilized Trichoderma reesei, which specifically includes the following steps:
[0032] (1) Prepare the fermentation medium and put it into the fermenter. By mass percentage, the fermentation medium includes 2.4-3.0% carbon source, 3-5% nitrogen source, 0.02-0.04% trace elements, 0.02% defoamer, and the balance is water.
[0033] The carbon source is selected from one or more types of sugar hydrolysate; the nitrogen source is selected from one or more types of soybean meal, corn steep liquor, peptone, soybean meal, ammonium sulfate, and ammonium chloride; and the trace elements are selected from one or more types of ferrous sulfate, cobalt chloride, manganese sulfate, zinc sulfate, copper chloride, and magnesium bromide.
[0034] Carbohydrate hydrolysate: Select one or more of lactose, sucrose, glucose, starch, glycerol, and microcrystalline cellulose, dissolve them in water at a solid-liquid ratio of 1:1, add 45% sulfuric acid at a ratio of 15 mL / kg, mix thoroughly, and react at 0.1 MPa for 40–50 min. Then add 20% NaOH solution to adjust the pH to 3.5–4.0 to obtain the corresponding carbohydrate hydrolysate.
[0035] (2) Based on the mass of the fermentation medium, 3-5% of the immobilized Trichoderma reesei was added to a fermenter containing the fermentation medium to produce cellulase.
[0036] The initial fermentation temperature was 32±2℃, the stirring speed was 30% of the maximum stirring speed of the fermenter, the initial aeration rate was 0.5vvm, and the initial fermentation pH was controlled at 4.50±0.20.
[0037] When the dissolved oxygen in the fermenter is below 50%, the air supply and rotation speed can be alternately increased, with each increase being 12% of the maximum speed. The initial ventilation ratio is 0.5vvm, and each increase is 0.5vvm, up to a maximum of 2.0vvm.
[0038] After the stirring speed and aeration rate are increased to the maximum, when the dissolved oxygen level in the fermenter drops to its lowest point and rebounds to above 50%, feeding begins. The initial feeding rate is 40 mL / h. The feeding rate is adjusted to keep the dissolved oxygen level between 10% and 20%, with the feeding rate increased by 15 mL / h each time, up to a maximum of 145 mL / h. If the dissolved oxygen level is above 20% when the maximum feeding rate is reached, the stirring speed and aeration rate can be appropriately reduced to keep the dissolved oxygen level below 20%.
[0039] By weight percentage, the fed culture medium includes 2.4-3.0% carbon source, 0.3-0.5% nitrogen source, 0.01-0.02% trace elements, 0.02% defoamer, and the balance is water.
[0040] The types of carbon sources, nitrogen sources, and trace elements can be selected from the types of carbon sources, nitrogen sources, and trace elements in the fermentation medium.
[0041] The effects of the clay modification method, modified clay, immobilized Trichoderma reesei, and method for producing cellulase from immobilized Trichoderma reesei of the present invention are studied through specific embodiments below.
[0042] Example 1:
[0043] This embodiment provides a modified clay, prepared using the following steps:
[0044] (1) After the clay is crushed and sieved, stearic acid with a mass concentration of 4% is added to the sieved clay at a solid-liquid ratio of 1:6. The reaction temperature is 75℃ and the reaction time is 24h. The filter cake is washed three times with distilled water. The filter cake is then dried at 65℃ for 1h until the surface moisture is completely evaporated, and then dried at 105℃ for 2h to obtain modified clay. After drying, the wettability of the clay will be improved.
[0045] (2) Add 0.02 mol / L hydrochloric acid and 4% stearamine solution to the modified clay in a mass ratio of 1:3:3 and react at 75°C for 24 hours. After the reaction, filter the mixture and wash the filter cake three times with distilled water. After removing the filter cake, preferably dry it at 65°C for 1 hour until the surface moisture is completely evaporated, and then dry it at 105°C for 2 hours to obtain the secondary modified clay.
[0046] (3) Add water to the secondary modified clay at a solid-liquid ratio of 1:4, place it in a mold (the mold is a sphere with a diameter of 5 cm), bake the mold at 105℃ for 2 hours, then remove it from the mold and bake it at 400℃ in a muffle furnace for 2 hours to obtain a well-shaped modified clay model.
[0047] After high-temperature baking, the modified clay is placed in a sterile environment to prevent bacterial contamination.
[0048] Example 2:
[0049] The present invention also provides an immobilized Trichoderma reesei, prepared by the following steps:
[0050] 1. Prepare the culture medium
[0051] PDA liquid culture medium: 1000 mL potato extract, 20 g glucose, sterilized at 121°C for 15 min.
[0052] PDA solid culture medium: 1000 mL potato extract, 20 g glucose, 15 g agar, sterilized at 121℃ for 15 min.
[0053] Potato extract: Take 200g of peeled potatoes, cut them into small pieces, add 1000mL of water, boil for 30 minutes, filter out the potato pieces, and add water to make up the filtrate to 1000mL.
[0054] 2. Trichoderma reesei seed culture
[0055] Take a loopful of Trichoderma reesei from the freeze-dried tube and inoculate it onto solid PDA medium. After incubating at 30°C for 24 hours, use a spatula to scrape off approximately 1 cm of the sample. 3 Small pieces are inoculated into sterilized liquid PDA medium and cultured at 24°C for 24 hours to obtain mature Trichoderma reesei seed culture.
[0056] 3. Immobilization
[0057] Under sterile conditions, the modified clay provided in Example 1 was mixed with Trichoderma reesei seed liquid cultured to the logarithmic growth phase at a solid-liquid ratio of 1:2.5. After fixation for 2 hours, the surface moisture was wiped off with a sterile cloth to obtain immobilized Trichoderma reesei. The immobilized Trichoderma reesei was placed in a sterile bag to prevent contamination and facilitate subsequent fermentation operations.
[0058] Example 3:
[0059] This embodiment provides a method for producing cellulase using immobilized Trichoderma reesei, including the following steps:
[0060] (1) Prepare the fermentation medium, put it into the fermenter, sterilize it at 115℃ for 30 minutes, and cool it down after sterilization, while keeping the temperature in the fermenter positive.
[0061] The fermentation medium, by mass percentage, comprises 2.5% glucose hydrolysate, 4% ammonium sulfate, 0.03% trace elements (manganese sulfate and magnesium bromide are selected here, with a mass ratio of manganese sulfate:magnesium bromide = 2:3), 0.02% defoamer, and the remainder is water.
[0062] (2) Based on the mass of the fermentation medium, 4% of the immobilized Trichoderma reesei was added to a fermenter containing the fermentation medium to produce cellulase.
[0063] The initial fermentation temperature was 32℃, the stirring speed was 30% of the maximum stirring speed of the fermenter, the initial aeration rate was 0.5vvm, and the initial fermentation pH was controlled at 4.50.
[0064] When the dissolved oxygen in the fermenter is below 50%, alternately increase the air volume and speed, each time increasing the speed by 12% of the maximum speed. The initial ventilation ratio is 0.5vvm, and it is increased by 0.5vvm each time, with a maximum of 2.0vvm.
[0065] After the stirring speed and aeration rate are increased to the maximum, when the dissolved oxygen level in the fermenter drops to its lowest point and rebounds to above 50%, feeding begins. The initial feeding rate is 40 mL / h. The feeding rate is adjusted to keep the dissolved oxygen level between 10% and 20%, with the feeding rate increased by 15 mL / h each time, up to a maximum of 145 mL / h. If the dissolved oxygen level is above 20% when the maximum feeding rate is reached, the stirring speed and aeration rate are appropriately reduced to keep the dissolved oxygen level below 20%.
[0066] Once the enzyme activity stopped increasing, the container was removed from the tank, and the cellulase activity was tested. The results are shown in Table 1.
[0067] Comparative Example 1:
[0068] This comparative example provides a method for producing cellulase from *Trichoderma reesei*, which differs from Example 3 in that the *Trichoderma reesei* seed culture was not solidified with modified clay and was directly inoculated into a fermenter for fermentation. When the enzyme activity stopped increasing, the fermenter was removed from the tank, and the cellulase activity was measured. The results are shown in Table 1.
[0069] Table 1. Enzyme activity detection results
[0070] Fermentation cycle Enzyme activity Example 3 130h 2514 U / mL Comparative Example 1 180h 2285U / mL
[0071] As shown in Table 1, after immobilizing Trichoderma reesei with the modified clay provided by this invention, the fermentation cycle was shortened by 27% and the enzyme activity increased by 10% compared with the non-immobilized Trichoderma reesei.
[0072] Example 4:
[0073] In this embodiment, the fermentation broth of Example 3 and Comparative Example 1 was post-treated to isolate cellulase.
[0074] 15 L of fermentation broth from Example 3 and Comparative Example 1 were each used in a plate and frame filtration experiment. Diatomaceous earth was used as the filter cake. Under the same filtration pressure, number of plates and frames, and amount of filter cake added, the required filtration time for the fermentation broths of Example 3 and Comparative Example 1 was compared. The results are as follows:
[0075] Solid content / % Filtering time / min Filtrate volume / L Example 3 7.8 77 13 Comparative Example 1 12.8 118 12
[0076] As can be seen from the table above, when filtering the same fermentation broth, Example 3, due to its lower solids content, allowed the liquid to more quickly fill the filtration plate and frame during the filtration process, resulting in an overall filtration time 26% faster than Comparative Example 1. Simultaneously, due to the lower solids content, the same volume of fermentation broth yielded 8% more filtrate. Therefore, using modified clay to immobilize Trichoderma reesei can alleviate post-processing pressure to some extent.
[0077] Example 5:
[0078] This embodiment recycles the modified clay from Example 3 after fermentation. The specific steps are as follows:
[0079] 1. Crushing
[0080] After fermentation, the modified clay blocks are dried at 45°C until all surface moisture is evaporated. Then, they are pulverized and sieved through a 40-mesh sieve. This pulverizing and sieving process is repeated until all clay powder can pass through the 40-mesh sieve, thereby removing insoluble solid particles that may have been embedded in the clay blocks during fermentation.
[0081] 2. Cleaning
[0082] Add 0.02–0.05 mol / L hydrochloric acid at a solid-liquid ratio of 1:2, based on the mass of clay powder, to remove soluble impurities adhering to the clay particles during fermentation. After shaking at room temperature for 30 minutes, filter out the liquid and rinse the clay particles with water for 30 minutes.
[0083] 3. Shaping
[0084] The clay is then placed in a mold (a spherical mold with a diameter of 5 cm), and baked at 105°C for 2 hours. Afterward, it is removed from the mold and baked in a muffle oven at 400°C for 2 hours to obtain a well-shaped modified clay model. If the modified clay is used too frequently or under extreme conditions (strong acid or alkali environments), it needs to be modified again after fermentation.
[0085] The above examples are merely illustrative of the present invention and do not constitute a limitation on the scope of protection of the present invention. All designs that are the same as or similar to the present invention are within the scope of protection of the present invention.
Claims
1. A method for modifying clay, characterized in that, Includes the following steps: (1) After the clay is crushed and sieved, long-chain fatty acids are added to the sieved clay to react. After the reaction, the clay is filtered, washed and dried to obtain primary modified clay. The long-chain fatty acids are one or more of oleic acid, linoleic acid, stearic acid, linolenic acid, palmitic acid and myristic acid. (2) Add an inorganic acid and a fatty amine to the primary modified clay to react. After the reaction, filter, wash and dry to obtain secondary modified clay. The inorganic acid is one or more of hydrochloric acid, nitric acid, sulfuric acid and perchloric acid. The fatty amine is one or more of stearylamine, oleylamine and palmitamine. (3) Add water to the secondary modified clay and bake it to obtain modified clay.
2. The clay modification method as described in claim 1, characterized in that: In step (1), a long-chain fatty acid solution with a mass concentration of 3-5% is added to the sieved clay, and the solid-liquid ratio of the clay to the long-chain fatty acid solution is 1:(5-7).
3. The clay modification method as described in claim 1, characterized in that: In step (2), 0.02-0.05 mol / L inorganic acid and 3-5% fatty amine solution are added to the primary modified clay at a mass ratio of 1:3:
3.
4. The clay modification method as described in claim 1, characterized in that: The reaction temperature in steps (1) and (2) is 70-80℃, and the reaction time is 16-24h.
5. A modified clay, characterized in that, The modified clay is prepared using the clay modification method described in any one of claims 1-4.
6. An immobilized Trichoderma reesei, characterized in that: Including Trichoderma reesei and the modified clay of claim 5.
7. The immobilized Trichoderma reesei as described in claim 6, characterized in that: It includes modified clay with a solid-liquid ratio of 1:2 to 3.5 and Trichoderma reesei seed liquid.
8. A method for producing cellulase by immobilizing Trichoderma reesei, characterized in that: The immobilized Trichoderma reesei as described in any one of claims 6-7 specifically includes the following steps: (1) Prepare the fermentation medium; (2) Immobilized Trichoderma reesei was added to the fermentation medium to produce cellulase.
9. The method for producing cellulase by immobilizing Trichoderma reesei as described in claim 8, characterized in that: In step (2), the amount of immobilized Trichoderma reesei added is 3-5% based on the mass of the fermentation medium.
10. The method for producing cellulase by immobilizing Trichoderma reesei as described in claim 8, characterized in that: When the dissolved oxygen level drops to its lowest point and rebounds to above 50% during the fermentation process in step (2), feeding begins, and the dissolved oxygen level is controlled at 10-20% during the feeding process.