Method for producing butanol system through fungus-bacterium co-culture fermentation alkali pretreatment of rice straw
Through fungus-bacterial co-culture fermentation technology, alkali-pretreated rice straw is used as substrate to solve the problem of excessive use of cellulase preparations during lignocellulose biomass fermentation in the prior art, and achieve efficient and low-cost butanol production.
Patent Information
- Application Number
- CN202510022147.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-03-14
AI Technical Summary
In the prior art, lignocellulosic biomass requires a large amount of cellulase preparations during butanol fermentation, which increases the cost of refining biobutanol and becomes a key obstacle to its commercialization.
The method of pretreatment of rice straw by fungus-bacterial co-culture fermentation base was used to pretreat butanol production. By introducing Trichoderma Reesei RUT-C30 as a cellulase-producing strain and co-culture with the butanol fermentation bacteria Clostridium beijerinckii NCIMB 8052, the alkali-pretreated rice straw was directly fermented as substrate.
It is realized that the production of butanol is directly fermented from alkali-pretreated rice straw without adding additional cellulase preparations, reducing production costs and overcoming the difficulties of differences in the growth conditions of the two strains.
Smart Images
Figure CN119614645A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of producing clean energy through microbial fermentation. Background Art
[0002] As a renewable raw material, lignocellulosic biomass mainly comes from residues and byproducts of agriculture and forestry. Among many biofuels, butanol has attracted much attention due to its high calorific value and safe transportation and handling characteristics. Biobutanol is mainly produced by butanol fermentation bacteria, among which the acetone-butanol-ethanol (ABE) fermentation process carried out by Clostridium beijerinckii NCIMB 8052 under anaerobic conditions is widely used due to its excellent fermentation efficiency. However, it lacks the ability to degrade lignocellulose and can only ferment lignocellulose hydrolysis products such as glucose and xylose. Commercial cellulase preparations must be used to hydrolyze and saccharify lignocellulose. However, the use of commercial cellulase preparations increases the refining cost of biobutanol, which is a key obstacle to its commercialization. Summary of the invention
[0003] The present invention aims to solve the problem that a large amount of cellulase preparations need to be used in the butanol fermentation process of existing lignocellulosic biomass, and further provides a method for a butanol production system of fungus-bacteria co-culture fermentation and alkali pretreatment of rice straw.
[0004] A method for producing butanol by fungus-bacteria co-culture fermentation and alkali pretreatment of rice straw, which is carried out according to the following steps:
[0005] 1. Pre-culturing Clostridium beijerinckii in a reinforced Clostridium medium, and collecting the suspension as Clostridium beijerinckii inoculum;
[0006] The enhanced Clostridium culture medium is composed of peptone, beef extract powder, yeast extract powder, glucose, NaCl, L-cysteine, sodium acetate and 0.1% w / v resazurin solution;
[0007] The Clostridium beijerinckii is Clostridium beijerinckii NCIMB 8052;
[0008] 2. Cultivate Trichoderma reesei in potato dextrose agar, then rinse with NaCl solution and collect spores, inoculate the spores into spore culture medium, and collect the suspension as Trichoderma reesei inoculum;
[0009] The Trichoderma Reesei is Trichoderma Reesei RUT-C30;
[0010] 3. Inoculate the Trichoderma reesei inoculum in a co-culture medium with straw as a carbon source at an inoculation amount of 10% to 40% v / v, culture for 72h to 216h at a rotation speed of 100rpm to 200rpm and a temperature of 25°C to 40°C, and then inoculate the Clostridium beijerinckii inoculum at an inoculation amount of 10% to 40% v / v, and culture at a temperature of 25°C to 40°C for 24h to 96h, thereby completing the method of the fungus-bacteria co-culture fermentation alkali pretreatment rice straw butanol production system;
[0011] The co-culture medium with straw as carbon source consists of alkali-pretreated rice straw and co-culture medium; the co-culture medium consists of KH2PO4, K2HPO4, CaCl2, (NH4)2SO4, MgSO4, peptone, yeast extract powder, Tween-80, FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O and CoCl2·6H2O.
[0012] The beneficial effects of the present invention are:
[0013] Trichoderma Reesei RUT-C30 is an aerobic fungus with efficient cellulase production capacity and biosafety. Introducing Trichoderma Reesei RUT-C30 as a cellulase-producing strain for co-cultivation with butanol fermentation bacteria can be an effective means to reduce the addition of cellulase preparations. However, the growth conditions of the two strains are quite different, and how to co-cultivate is a difficulty in ABE fermentation.
[0014] The present invention constructs a co-culture fermentation system consisting of Trichoderma Reesei RUT-C30 and Clostridium beijerinckiiNCIMB 8052, overcomes the difference in growth conditions between the two strains, and realizes direct fermentation production of butanol using alkali-pretreated rice straw as a substrate. The operation method is simple, no additional commercial cellulase preparation is required, and cost is saved. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 This is a comparison curve of reducing sugar in the co-culture system of Examples 1 to 4;
[0016] Figure 2 This is a comparative curve diagram of butanol production in the co-cultivation system of Examples 1 to 4;
[0017] Figure 3 This is a comparison curve of the ABE solvent yield of the co-culture system of Examples 1 to 4;
[0018] Figure 4 This is a comparison curve of acetic acid production in the co-culture system of Examples 1 to 4;
[0019] Figure 5 This is a comparative curve diagram of butyrate production in the co-cultivation system of Examples 1 to 4;
[0020] Figure 6 This is a scanning electron microscope image of the microorganisms on the surface of the straw after the fermentation in Example 1. DETAILED DESCRIPTION
[0021] Specific implementation method 1, this implementation method is a method for producing butanol by fungus-bacteria co-culture fermentation alkali pretreatment of rice straw, which is carried out according to the following steps:
[0022] 1. Pre-culturing Clostridium beijerinckii in a reinforced Clostridium medium, and collecting the suspension as Clostridium beijerinckii inoculum;
[0023] The enhanced Clostridium culture medium is composed of peptone, beef extract powder, yeast extract powder, glucose, NaCl, L-cysteine, sodium acetate and 0.1% w / v resazurin solution;
[0024] The Clostridium beijerinckii is Clostridium beijerinckii NCIMB 8052;
[0025] 2. Cultivate Trichoderma reesei in potato dextrose agar, then rinse with NaCl solution and collect spores, inoculate the spores into spore culture medium, and collect the suspension as Trichoderma reesei inoculum;
[0026] The Trichoderma Reesei is Trichoderma Reesei RUT-C30;
[0027] 3. Inoculate the Trichoderma reesei inoculum in a co-culture medium with straw as a carbon source at an inoculation amount of 10% to 40% v / v, culture for 72h to 216h at a rotation speed of 100rpm to 200rpm and a temperature of 25°C to 40°C, and then inoculate the Clostridium beijerinckii inoculum at an inoculation amount of 10% to 40% v / v, and culture at a temperature of 25°C to 40°C for 24h to 96h, thereby completing the method of the fungus-bacteria co-culture fermentation alkali pretreatment rice straw butanol production system;
[0028] The co-culture medium with straw as carbon source consists of alkali-pretreated rice straw and co-culture medium; the co-culture medium consists of KH2PO4, K2HPO4, CaCl2, (NH4)2SO4, MgSO4, peptone, yeast extract powder, Tween-80, FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O and CoCl2·6H2O.
[0029] The beneficial effects of this specific implementation are:
[0030] Trichoderma Reesei RUT-C30 is an aerobic fungus with efficient cellulase production capacity and biosafety. Introducing Trichoderma Reesei RUT-C30 as a cellulase-producing strain for co-cultivation with butanol fermentation bacteria can be an effective means to reduce the addition of cellulase preparations. However, the growth conditions of the two strains are quite different, and how to co-cultivate is a difficulty in ABE fermentation.
[0031] This specific embodiment constructs a co-culture fermentation system consisting of Trichoderma Reesei RUT-C30 and Clostridium beijerinckii NCIMB 8052, overcomes the difference in growth conditions between the two strains, and realizes direct fermentation production of butanol using alkali-pretreated rice straw as a substrate. The operation method is simple, no additional commercial cellulase preparation is required, and cost is saved.
[0032] Specific embodiment 2: This embodiment is different from specific embodiment 1 in that: the concentration of peptone in the fortified Clostridium culture medium described in step 1 is 5g / L-10g / L, the concentration of beef extract powder is 5g / L-10g / L, the concentration of yeast extract powder is 1g / L-3g / L, the concentration of glucose is 3g / L-10g / L, the concentration of NaCl is 3g / L-5g / L, the concentration of L-cysteine is 0.1g / L-0.5g / L, the concentration of sodium acetate is 1g / L-3g / L, and the concentration of 0.1% w / v resazurin solution is 0.1mL / L-0.5mL / L. The rest is the same as specific embodiment 1.
[0033] Specific embodiment 3: This embodiment is different from specific embodiment 1 or 2 in that the enhanced Clostridium culture medium described in step 1 is specifically prepared according to the following steps: deionized water is boiled, and then argon gas is introduced for 10 min to 20 min, and then the culture medium components are dissolved in water in sequence, and the pH value of the culture medium is adjusted to 6.5 to 7.0 using a KOH solution with a concentration of 0.5M to 1M, and finally sterilized. The rest is the same as specific embodiment 1 or 2.
[0034] Specific embodiment 4: This embodiment is different from specific embodiments 1 to 3 in that: in step 1, Clostridium beijerinckii is inoculated in a reinforced Clostridium culture medium at an inoculation amount of 3% to 5% v / v, and then cultured for 24h to 72h at a rotation speed of 100rpm to 200rpm and a temperature of 30°C to 40°C. Others are the same as specific embodiments 1 to 3.
[0035] Specific implementation method 5: This implementation method is different from the specific implementation methods 1 to 4 in that: in step 2, the inoculation amount is 10 5 ~107 spores, inoculate Trichoderma reesei into potato dextrose agar, culture at a temperature of 25°C to 35°C for 3 to 7 days, and then wash and collect the spores with a 0.5% to 0.9% by mass NaCl solution. The rest is the same as the first to fourth embodiments.
[0036] Specific embodiment 6: This embodiment is different from specific embodiments 1 to 5 in that: in step 2, the spores are inoculated into the spore culture medium at an inoculation amount of 0.1% to 0.5% v / v, and cultured for 24h to 72h at a rotation speed of 100rpm to 200rpm and a temperature of 25°C to 35°C. Others are the same as specific embodiments 1 to 5.
[0037] Specific embodiment 7: This embodiment is different from any one of specific embodiments 1 to 6 in that: the spore culture medium described in step 2 is composed of sodium carboxymethyl cellulose, peptone, Tween-80, MgSO4, CaCl2, FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O and CoCl2·6H2O; the concentration of sodium carboxymethyl cellulose in the spore culture medium is 5g / L-10g / L, the concentration of peptone is 3g / L-5g / L, the concentration of Tween-80 is The concentration of MgSO4 is 0.1g / L to 0.3g / L, the concentration of CaCl2 is 0.1g / L to 0.3g / L, the concentration of FeSO4·7H2O is 1mg / L to 5mg / L, the concentration of MnSO4·H2O is 0.5mg / L to 2mg / L, the concentration of ZnSO4·7H2O is 0.5mg / L to 2mg / L, and the concentration of CoCl2·6H2O is 0.5g / L to 2mg / L. The rest is the same as the first to sixth embodiments.
[0038] Specific embodiment eight: This embodiment differs from any one of specific embodiments one to seven in that: the amount of alkali pretreated rice straw added in step three is 3% to 10% w / v of the co-culture medium; the concentration of KH2PO4 in the co-culture medium in step three is 0.2g / L to 2g / L, the concentration of K2HPO4 is 0.1g / L to 0.5g / L, the concentration of CaCl2 is 0.1g / L to 0.3g / L, the concentration of (NH4)2SO4 is 0.4g / L to 1.4g / L, and the concentration of MgSO4 is 0.1g / L to 1.5g / L. L~0.3g / L, the concentration of peptone is 0.1g / L~0.5g / L, the concentration of yeast extract is 0.1g / L~0.5g / L, the concentration of Tween-80 is 0.05mL / L~0.1mL / L, the concentration of FeSO4·7H2O is 1mg / L~5mg / L, the concentration of MnSO4·H2O is 0.5mg / L~2mg / L, the concentration of ZnSO4·7H2O is 0.5mg / L~2mg / L, and the concentration of CoCl2·6H2O is 0.5mg / L~2mg / L. Others are the same as the first to seventh embodiments.
[0039] Specific embodiment 9: This embodiment is different from specific embodiments 1 to 8 in that: the alkali pretreated rice straw described in step 3 is specifically prepared according to the following steps: the rice straw is crushed, then added to the NaOH solution, and left to stand for 24h to 96h at a temperature of 40℃ to 60℃, and then washed and dried at a constant temperature; the mass ratio of the rice straw to the volume of the NaOH solution is 1g: (10-15)mL; the concentration of the NaOH solution is 0.2% to 1.0% w / v. The rest is the same as specific embodiments 1 to 8.
[0040] Specific embodiment 10: This embodiment differs from Specific embodiments 1 to 9 in that the pH value of the co-culture medium using straw as a carbon source in step 3 is 7.2. The rest is the same as Specific embodiments 1 to 9.
[0041] The following examples are used to verify the beneficial effects of the present invention:
[0042] Embodiment 1:
[0043] A method for producing butanol by fungus-bacteria co-culture fermentation and alkali pretreatment of rice straw, which is carried out according to the following steps:
[0044] 1. Inoculate Clostridium beijerinckii in a reinforced Clostridium medium at an inoculum volume of 3% v / v, then culture at a speed of 150 rpm and a temperature of 37°C for 48 hours, and collect the suspension as the Clostridium beijerinckii inoculum;
[0045] The Clostridium beijerinckii is Clostridium beijerinckii NCIMB 8052 (C. beijerinckii NCIMB 8052, stored at -20°C);
[0046] The reinforced Clostridium medium (RCM) is composed of peptone, beef extract powder, yeast extract powder, glucose, NaCl, L-cysteine, sodium acetate and 0.1% w / v resazurin solution; the concentration of peptone in the reinforced Clostridium medium is 10 g / L, the concentration of beef extract powder is 10 g / L, the concentration of yeast extract powder is 3 g / L, the concentration of glucose is 5 g / L, the concentration of NaCl is 3 g / L, the concentration of L-cysteine is 0.5 g / L, the concentration of sodium acetate is 3 g / L, and the concentration of 0.1% w / v resazurin solution is 0.5 mL / L;
[0047] The enhanced Clostridium culture medium is specifically prepared according to the following steps: boiling deionized water, then passing argon gas for 10 minutes, then dissolving the culture medium components in water in turn, adjusting the pH value of the culture medium to 6.8±0.1 using a 1M KOH solution, and finally sterilizing at a temperature of 121°C for 20 minutes;
[0048] 2. According to the inoculation volume of 10 7 spores, inoculating Trichoderma reesei into potato dextrose agar (PDA), culturing at 28°C for 7 days, then washing with 10 mL of 0.9% NaCl solution and collecting the spores, inoculating the spores into spore culture medium at an inoculum size of 0.3% v / v, culturing at a speed of 150 rpm and a temperature of 28°C for 72 h, and collecting the suspension as the Trichoderma reesei inoculum;
[0049] The Trichoderma Reesei is Trichoderma Reesei RUT-C30 (T. reesei RUT-C30, stored in spore form at -20°C);
[0050] The spore culture medium is composed of sodium carboxymethyl cellulose (CMC-Na), peptone, Tween-80, MgSO4, CaCl2, FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O and CoCl2·6H2O; the concentration of sodium carboxymethyl cellulose in the spore culture medium is 7.5 g / L, the concentration of peptone is 5.0 g / L, the concentration of Tween-80 is 2.0 mL / L, the concentration of MgSO4 is 0.3 g / L, the concentration of CaCl2 is 0.3 g / L, the concentration of FeSO4·7H2O is 5.0 mg / L, the concentration of MnSO4·H2O is 1.6 mg / L, the concentration of ZnSO4·7H2O is 1.4 mg / L, and the concentration of CoCl2·6H2O is 2.0 mg / L; the pH of the spore culture medium is a natural pH value;
[0051] 3. Inoculate the Trichoderma reesei inoculum in 100 mL of a co-culture medium with straw as a carbon source at an inoculation rate of 10% v / v, and culture at a rotation speed of 150 rpm and a temperature of 28° C. for 3 days to saccharify the rice straw and produce cellulase, and then inoculate the Clostridium beijerinckii inoculum at an inoculation rate of 10% v / v, and culture at a temperature of 37° C. for 4 days, thereby completing the method of producing butanol from rice straw by alkali pretreatment of rice straw through fungal-bacterial co-culture fermentation;
[0052] The co-culture medium with straw as carbon source is composed of alkali pretreated rice straw and co-culture medium; the co-culture medium is composed of KH2PO4, K2HPO4, CaCl2, (NH4)2SO4, MgSO4, peptone, yeast extract powder, Tween-80, FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O and CoCl2·6H2O; the amount of alkali pretreated rice straw added is 4.5% w / v of the co-culture medium; the concentration of KH2PO4 in the co-culture medium is 2.0 g / L, K2HPO4 The concentration of is 0.5g / L, the concentration of CaCl2 is 0.3g / L, the concentration of (NH4)2SO4 is 1.4g / L, the concentration of MgSO4 is 0.3g / L, the concentration of peptone is 0.5g / L, the concentration of yeast extract powder is 0.5g / L, the concentration of Tween-80 is 0.1mL / L, the concentration of FeSO4·7H2O is 5.0mg / L, the concentration of MnSO4·H2O is 1.6mg / L, the concentration of ZnSO4·7H2O is 1.4mg / L, and the concentration of CoCl2·6H2O is 2.0mg / L;
[0053] The alkali pretreated rice straw (RS) is prepared according to the following steps: the rice straw is crushed, then added into a NaOH solution, left to stand for 72 hours at a temperature of 50°C, then washed and dried at a constant temperature; the mass ratio of the rice straw to the volume of the NaOH solution is 1g:15mL; the concentration of the NaOH solution is 1% w / v;
[0054] The pH value of the co-culture medium using straw as a carbon source is 7.2.
[0055] Example 2: This example is different from Example 1 in that the amount of alkali pretreated rice straw added in step 3 is 6% w / v of the co-culture medium. Others are the same as Example 1.
[0056] Example 3: This example is different from Example 1 in that the amount of alkali pretreated rice straw added in step 3 is 3% w / v of the co-culture medium. Others are the same as Example 1.
[0057] Example 4: This example is different from Example 1 in that the amount of alkali-pretreated rice straw added in step 3 is 7.5% w / v of the co-culture medium. The rest is the same as Example 1.
[0058] Figure 1 This is a comparison curve of reducing sugar in the co-culture system of Examples 1 to 4;
[0059] Figure 2 This is a comparative curve diagram of butanol production in the co-cultivation system of Examples 1 to 4;
[0060] Figure 3 This is a comparison curve of the ABE solvent yield of the co-culture system of Examples 1 to 4;
[0061] Figure 4 This is a comparison curve of acetic acid production in the co-culture system of Examples 1 to 4;
[0062] Figure 5 This is a comparative curve diagram of butyrate production in the co-cultivation system of Examples 1 to 4;
[0063] Figure 6 This is a scanning electron microscope image of microorganisms on the surface of straw after fermentation in Example 1;
[0064] Example 1 When the addition amount of alkali pretreated rice straw was 4.5%, the accumulation of reducing sugars in T. reesei RUT-C30 after 3 days of culture was 1.32 g / L. It can be seen that the saccharification effect can be significantly improved by increasing the straw loading, providing a substrate for the subsequent co-culture of C. beijerinckii NCIMB 8052 to produce butanol. After inoculation of C. beijerinckii NCIMB 8052, the reducing sugars in the co-culture system were rapidly consumed 24 to 48 hours after inoculation, and only 0.36 g / L reducing sugars were present at the end of fermentation. During the fermentation process, the highest butanol production was 1496 ± 128 mg / L after 5 days of culture, and the ABE solvent production was 2231 ± 96 mg / L. Butyric acid is an important product in the acid production stage of ABE fermentation, which helps stimulate the formation of butanol in the solvent production stage. The butyric acid concentrations at the end of fermentation were 2376 ± 6 mg / L. Generally speaking, during the acid production stage, acetic acid and butyric acid will gradually accumulate, and then during the solvent production stage, C.beijerinckii NCIMB 8052 will reabsorb these volatile fatty acids and convert them into butanol under the reducing power provided by reduced coenzyme I (NADH). No significant decrease in butyric acid concentration was observed in this embodiment. It may be that during the solvent production stage, the concentration of reducing sugars in the system is low, resulting in the inability to provide sufficient NADH to convert butyric acid into butanol. Scanning electron microscope images after the fermentation ended showed that there were T.reesei RUT-C30 attached to the surface of the straw in filamentous form and C.beijerinckii NCIMB 8052 in rod-shaped form, indicating that the co-cultivation of the two strains was successfully achieved.
[0065] Example 2 When the addition amount of alkali pretreated rice straw was 6%, after 3 days of cultivation, the reducing sugar accumulation of T. reesei RUT-C30 was 1.88 g / L. After inoculation of C. beijerinckii NCIMB 8052, the reducing sugar concentration in the co-culture system was rapidly exhausted within 24 hours, and only 0.38 g / L reducing sugar was present at the end of fermentation. During the fermentation process, the maximum butanol production was 974 ± 31 mg / L, the ABE solvent production was 1542 ± 98 mg / L, and the butyric acid accumulation was 3180 ± 351 mg / L.
[0066] Comparative experiment 1 When the addition amount of alkali pretreated rice straw was 3%, after 3 days of cultivation, the reducing sugar accumulation of T. reesei RUT-C30 was 0.13 g / L, and the reducing sugar concentration was still at a very low state after inoculation with C. beijerinckii NCIMB 8052. At the end of fermentation, the main products in the system were ethanol (183 ± 53 mg / L) and acetic acid (470 ± 22 mg / L), which were mainly the products of T. reesei RUT-C30 to maintain its own metabolism under anaerobic conditions.
[0067] In comparative experiment 2, when the addition amount of alkali pretreated rice straw was 7.5%, the reducing sugar accumulation of T. reesei RUT-C30 was 3.54 g / L after 3 days of culture. After inoculation with C. beijerinckii NCIMB 8052, the reducing sugar in the co-culture system was rapidly consumed, but there was still 1.77 g / L reducing sugar at the end of fermentation. During the fermentation process, the maximum butanol production was 430 ± 197 mg / L, the ABE solvent production was 1109 ± 261 mg / L, and the butyric acid accumulation was 2805 ± 403 mg / L. It can be seen that increasing the substrate loading amount is conducive to the accumulation of reducing sugars in the system, but it will limit the mass transfer process of the co-culture system, resulting in low butanol production.
Claims
1. A method for producing butanol by fungus-bacteria co-culture fermentation alkali pretreatment of rice straw, characterized in that It is carried out in the following steps:
1. Pre-culturing Clostridium beijerinckii in a reinforced Clostridium medium, and collecting the suspension as Clostridium beijerinckii inoculum; The enhanced Clostridium culture medium is composed of peptone, beef extract powder, yeast extract powder, glucose, NaCl, L-cysteine, sodium acetate and 0.1% w / v resazurin solution; The Clostridium beijerinckii is Clostridium beijerinckii NCIMB 8052; 2. Cultivate Trichoderma reesei in potato dextrose agar, then rinse with NaCl solution and collect spores, inoculate the spores into spore culture medium, and collect the suspension as Trichoderma reesei inoculum; The Trichoderma Reesei is Trichoderma Reesei RUT-C30; 3. Inoculate the Trichoderma reesei inoculum in a co-culture medium with straw as a carbon source at an inoculation amount of 10% to 40% v / v, culture for 72h to 216h at a rotation speed of 100rpm to 200rpm and a temperature of 25°C to 40°C, and then inoculate the Clostridium beijerinckii inoculum at an inoculation amount of 10% to 40% v / v, and culture at a temperature of 25°C to 40°C for 24h to 96h, thereby completing the method of the fungus-bacteria co-culture fermentation alkali pretreatment rice straw butanol production system; The co-culture medium with straw as carbon source consists of alkali-pretreated rice straw and co-culture medium; the co-culture medium consists of KH2PO4, K2HPO4, CaCl2, (NH4)2SO4, MgSO4, peptone, yeast extract powder, Tween-80, FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O and CoCl2·6H2O.
2. The method of claim 1, wherein the method comprises the following steps: In the fortified Clostridium medium described in step 1, the concentration of peptone is 5g / L-10g / L, the concentration of beef extract powder is 5g / L-10g / L, the concentration of yeast extract powder is 1g / L-3g / L, the concentration of glucose is 3g / L-10g / L, the concentration of NaCl is 3g / L-5g / L, the concentration of L-cysteine is 0.1g / L-0.5g / L, the concentration of sodium acetate is 1g / L-3g / L, and the concentration of 0.1% w / v resazurin solution is 0.1mL / L-0.5mL / L.
3. The method of claim 1, wherein the method comprises the following steps: The enhanced Clostridium culture medium described in step 1 is specifically prepared according to the following steps: deionized water is boiled, and then argon gas is introduced for 10 minutes to 20 minutes, and then the culture medium components are dissolved in water in turn, and the pH value of the culture medium is adjusted to 6.5 to 7.0 using a 0.5M to 1M KOH solution, and finally sterilized.
4. The method of claim 1, wherein the method comprises the following steps: In step 1, Clostridium beijerinckii is inoculated into a reinforced Clostridium culture medium at an inoculation amount of 3% to 5% v / v, and then cultured for 24 hours to 72 hours at a rotation speed of 100 rpm to 200 rpm and a temperature of 30° C. to 40° C.
5. The method of claim 1, wherein the method comprises the following steps: In step 2, the inoculation volume is 10 5 ~10 7 spores, inoculating Trichoderma reesei into potato dextrose agar, culturing at a temperature of 25° C. to 35° C. for 3 to 7 days, and then washing with a NaCl solution with a mass percentage of 0.5% to 0.9% and collecting the spores.
6. The method of claim 1, wherein the method comprises the following steps: In step 2, the spores are inoculated into the spore culture medium at an inoculation amount of 0.1% to 0.5% v / v, and cultured for 24h to 72h at a rotation speed of 100rpm to 200rpm and a temperature of 25°C to 35°C.
7. The method of claim 1, wherein the method comprises the following steps: The spore culture medium described in step 2 is composed of sodium carboxymethyl cellulose, peptone, Tween-80, MgSO4, CaCl2, FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O and CoCl2·6H2O; the concentration of sodium carboxymethyl cellulose in the spore culture medium is 5g / L~10g / L, the concentration of peptone is 3g / L~5g / L, and the concentration of Tween-80 is 0.05mL / L~0.1m L / L, the concentration of MgSO4 is 0.1g / L~0.3g / L, the concentration of CaCl2 is 0.1g / L~0.3g / L, the concentration of FeSO4·7H2O is 1mg / L~5mg / L, the concentration of MnSO4·H2O is 0.5mg / L~2mg / L, the concentration of ZnSO4·7H2O is 0.5mg / L~2mg / L, and the concentration of CoCl2·6H2O is 0.5g / L~2mg / L.
8. The method of claim 1, wherein the method comprises the following steps: The amount of alkali pretreated rice straw added in step 3 is 3% to 10% w / v of the co-culture medium; the concentration of KH2PO4 in the co-culture medium in step 3 is 0.2g / L to 2g / L, the concentration of K2HPO4 is 0.1g / L to 0.5g / L, the concentration of CaCl2 is 0.1g / L to 0.3g / L, the concentration of (NH4)2SO4 is 0.4g / L to 1.4g / L, the concentration of MgSO4 is 0.1g / L to 0.3g / L, and the concentration of peptone is The concentration of yeast extract powder is 0.1g / L~0.5g / L, the concentration of Tween-80 is 0.05mL / L~0.1mL / L, the concentration of FeSO4·7H2O is 1mg / L~5mg / L, the concentration of MnSO4·H2O is 0.5mg / L~2mg / L, the concentration of ZnSO4·7H2O is 0.5mg / L~2mg / L, and the concentration of CoCl2·6H2O is 0.5mg / L~2mg / L.
9. The method of claim 1, wherein the method comprises the following steps: The alkali pretreated rice straw described in step three is specifically prepared according to the following steps: the rice straw is crushed, then added to a NaOH solution, left to stand for 24h to 96h at a temperature of 40°C to 60°C, then washed and dried at a constant temperature; the mass ratio of the rice straw to the volume of the NaOH solution is 1g: (10-15)mL; the concentration of the NaOH solution is 0.2% to 1.0% w / v.
10. The method of claim 1, wherein the method comprises the following steps: The pH value of the co-culture medium using straw as a carbon source described in step 3 is 7.2.