Method for improving biobutanol conversion efficiency

Abstract

The present invention relates to the field of microbial engineering technology, and in particular to a method for improving the conversion efficiency of biobutanol. There are four butanol dehydrogenases in Clostridium, namely BDH1, BDH2, BDH3 and BDH4. Studies have found that BDH1 and BDH4 each have two Fe 3+ binding sites, therefore, by adding Fe 3+ More butanol can be produced. Experiments show that exogenous addition of Fe 3+ The optimized system not only promotes the growth of fermentation strains and their utilization efficiency of substrates such as glucose or galactose, but also improves the butanol conversion rate while shortening the fermentation time to 48 hours, thereby increasing the butanol production rate and significantly saving the fermentation cost. This technology solves the problems of incomplete carbon source utilization, low butanol conversion rate and long fermentation time in the traditional fermentation process. It is simple to operate, does not require special instrument conditions, and has good application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of microbial engineering, and in particular to a method for improving biobutanol conversion efficiency. Background Art

[0002] At present, the development and utilization of new biofuels can not only alleviate the energy crisis caused by policy reasons, but also avoid environmental pollution problems caused by the mining process of fossil energy, which meets the national "carbon neutral" energy demand and has huge market development potential. As an important member of new fuels, biobutanol has an energy density (29.2MJ / L) significantly higher than ethanol (19.6MJ / L) and is similar to gasoline (32.0MJ / L). As a blending component of gasoline, butanol can be miscible with gasoline in any ratio, and can release 36065.84KJ / kg of energy after full combustion, which is equivalent to the energy value of gasoline combustion, but much higher than ethanol (29639.68KJ / kg). In addition, butanol also has the advantages of low corrosiveness, low volatility, low hygroscopicity, and low corrosiveness. It can be used directly without modifying existing engines and is widely regarded as an ideal substitute for ethanol and biodiesel. In addition, compared with some gaseous fuels, liquid butanol also has advantages in terms of economy and safety in storage and transportation.

[0003] The traditional microbial fermentation method for producing biobutanol mainly uses solvent-producing Clostridium as the fermentation strain, and converts substrates such as glucose into product butanol through the acetone-butanol-ethanol (ABE) pathway under anaerobic conditions. However, some common problems in the traditional fermentation process, such as low butanol yield and conversion rate caused by the toxic effect of butanol, low butanol ratio in the total solvent (<60%), and long fermentation time, all increase the production and purification costs of the fermentation process and constitute the reason for hindering the industrialization process. Therefore, constructing an engineered strain by metabolic engineering transformation can reduce the accumulation of by-products while further improving the conversion efficiency, which is one of the ways to improve the quality of strains at this stage. However, due to the complexity of genetic manipulation of Clostridium strains, it is a very economical and simple fermentation engineering method to promote strain growth activity and increase product synthesis rate by analyzing the regulatory factors of key enzymes in the metabolic pathway of the strain and optimizing the culture medium system. In recent years, some research reports on the interaction between metal ions and microbial proteases have shown that by optimizing the concentration of metal cofactors of key enzymes in the fermentation medium of solvent-producing Clostridium, or by adding substances such as amino acids to increase the activity of key enzymes in the metabolic pathway, the strain's tolerance to butanol can be improved, and ultimately the production of biobutanol can be increased.

[0004] Related studies have reported that the effect of adding zinc ions on improving fructose / xylose utilization and butanol production during acetone-butanol-ethanol fermentation showed that adding a certain amount of ZnSO 4 7H 2 O can not only promote cell growth and glucose utilization, shorten the fermentation cycle, but also increase the production and yield of butanol. 3+ Related reports on improving the efficiency of biobutanol conversion by solventogenic Clostridium. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for improving the conversion efficiency of biobutanol.

[0006] The present invention provides Fe 3+ Application in improving the butanol conversion efficiency of Clostridium.

[0007] In the application of the present invention, the Clostridium comprises butanol dehydrogenase; the BDH1 butanol dehydrogenase has an amino acid sequence as shown in SEQ ID NO.1; and the BDH4 butanol dehydrogenase has an amino acid sequence as shown in SEQ ID NO.2.

[0008] The strain of the present invention is known to have four butanol dehydrogenases, namely BDH1, BDH2, BDH3 and BDH4. Studies have shown that BDH1 and BDH4 each have two Fe 3+ The binding site of Fe 3+ to promote butanol production.

[0009] In the application of the present invention, the Clostridium includes the strain with the deposit number of GDMCC61493.

[0010] The strains described in the present invention include solvent-producing clostridium, which is Clostridium sp. WK (patent application number: 202110179660.0), which is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a collection number of GDMCC61493.

[0011] The present invention also provides a composition comprising Fe 3+ and a carbon source; the Fe 3+ The molar ratio of the carbon source is 1:10 to 70; the carbon source is glucose and / or galactose.

[0012] In some embodiments, in the composition of the present invention, Fe 3+ The molar ratio of Fe to glucose is 1:10~70; 3+ The molar ratio of galactose to 1:9-60.

[0013] In the composition of the present invention, the Fe 3+ Including FeCl 3 and / or Fe 2 (SO 4 ) 3 .

[0014] The present invention proves through comparative experiments that the reducing agent converts part of Fe 3+ Fe 2+ It is not a factor that accelerates the utilization of glucose and increases butanol production by the strain, but it is indeed through Fe 3+ The presence of improves the utilization of glucose and the efficiency of converting butanol.

[0015] In some specific embodiments, Fe 3+ The metal ion compound solution is FeCl 3 or Fe 2 (SO 4 ) 3 One of them was prepared into a 50g / L mother solution and filtered for sterilization.

[0016] The present invention also provides a culture medium, which comprises a basic culture medium and the composition.

[0017] In the culture medium of the present invention, Fe 3+ The concentration of is 5mM~20mM, and the concentration of carbon source is 50g / L~60g / L.

[0018] In the culture medium of the present invention, the basic culture medium includes anaerobic culture medium.

[0019] In some embodiments, the anaerobic culture medium described in the present invention includes ACM anaerobic culture medium.

[0020] The ACM anaerobic culture medium of the present invention is an ACM anaerobic culture medium prepared with pure water, and the formula is:

[0021] Yeast extract 10g / L; NaHCO 3 2.52g / L; 2-(N-morpholino)ethanesulfonic acid (MES) 2.132g / L; 100× salt solution 10mL / L; 1000× trace element solution 1mL / L; Resazurin anaerobic indicator 0.001g / L; Dithiothreitol 0.077g / L; L-cysteine ​​0.0242g / L; Na 2 S 0.016 / L.

[0022] The 100× salt solution contains: NaCl, 1.0 g / L; MgCl 2 6H 2 O, 0.5 g / L; KH2 PO 4 , 0.2g / L; NH 4 Cl, 0.3g / L; KCl, 0.3g / L; CaCl 2 ·2H 2 O, 0.015g / L.

[0023] The 1000× trace element solution contains: FeCl 2 ·4H 2 O, 1.5 g / L; CoCl 2 6H 2 O, 0.19 g / L; MnCl 2 ·4H 2 O, 0.1 g / L; ZnCl 2 , 0.07g / L; H 3 BO 3 , 0.006g / L; Na 2 MoO 4 ·2H 2 O, 0.036 g / L; NiCl 2 6H 2 O, 0.024 g / L; CuCl 2 ·2H 2 O, 0.002g / L.

[0024] In some embodiments, the culture medium of the present invention uses 4M NaOH to adjust the initial pH of the culture medium to 6.7±0.3.

[0025] The invention provides a method for improving the butanol conversion efficiency of clostridium, which comprises culturing clostridium by using the composition or the culture medium.

[0026] In the method of the present invention, the Clostridium includes the Clostridium with the deposit number of GDMCC61493.

[0027] The clostridium described in the present invention is a solvent-producing clostridium, which is named Clostridium sp. WK (patent application number: 202110179660.0), and is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a collection number of GDMCC61493.

[0028] In some embodiments, the culturing comprises inoculating the Clostridium with the deposit number GDMCC61493 into a culture medium containing Fe 3+ and carbon source culture medium.

[0029] The inoculation amount is 3% to 8% of the total volume of the culture medium.

[0030] The Fe 3+and a carbon source culture medium, which includes a basic culture medium and the Fe 3+ and carbon source.

[0031] In some embodiments, the Fe 3+ The initial concentration of Fe is 5mM to 30mM. 3+ The starting concentration is 10mM to 25mM; in some other specific embodiments, the Fe 3+ The starting concentration was 20 mM.

[0032] In some embodiments, the initial concentration of the carbon source of the present invention is 40 g / L to 60 g / L. In some specific embodiments, the initial concentration of the carbon source is 60 g / L, and in other specific embodiments, the initial concentration of the carbon source is 50 g / L.

[0033] In the method of the present invention, the conditions for culturing Clostridium include 48 to 96 hours, pH 6 to 7, and an anaerobic environment.

[0034] In some specific embodiments, the culture is to inoculate 4% of the total volume of the culture medium with a seed solution containing Clostridium with a deposit number of GDMCC61493, and the anaerobic fermentation temperature is 30° C. and the fermentation is carried out at 150 rpm for 48 to 96 hours. In some specific embodiments, the fermentation time is 96 hours.

[0035] During the fermentation culture of the present invention, sampling is performed every 12 or 24 hours, and a UVmini-1240 ultraviolet / visible spectrophotometer is used to measure the growth curve of the strain at a wavelength of 600 nm; a 3,5-dinitrosalicylic acid (DNS) method is used to measure the consumption of the carbon source substrate; and a gas chromatography-FID method (GC-FID) is used to detect the fermentation products: acetone, butanol, ethanol, butyric acid, acetic acid, etc., to monitor the changes in the products under different fermentation conditions.

[0036] The present invention not only promotes the utilization of substrates by the strain, but also increases the consumption rate of carbon sources by the strain, and at the same time, the production rate of butanol and the biomass of the strain during the fermentation process are also significantly improved. 3 + ) 3+ The fermentation results of the low concentration Fe 3+ It cannot significantly improve the utilization of carbon sources and the production of butanol by the strain. 3+ Only in this way can the effect of improving the strain's utilization of carbon sources and butanol production be achieved.

[0037] There are four butanol dehydrogenases in Clostridium, namely BDH1, BDH2, BDH3 and BDH4. Studies have found that BDH1 and BDH4 each have two Fe3+ binding sites, therefore, by adding Fe 3+ More butanol can be produced. Experiments show that the exogenous addition of Fe established by the present invention 3+ The optimized system not only promotes the growth of fermentation strains and their utilization efficiency of substrates such as glucose or galactose, but also improves the butanol conversion rate while shortening the overall fermentation time to 48 hours, further improving the butanol production rate and significantly saving the fermentation cost. It solves the common problems that affect the industrialization process in the acetone-butanol-ethanol fermentation process of traditional solvent-producing Clostridium, such as incomplete utilization of carbon sources by strains, low butanol conversion rate and long fermentation time. 3+ The method for improving the conversion efficiency of biobutanol is simple to operate, does not require special instrument conditions, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific implementation of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation or the prior art description are briefly introduced below. Obviously, the drawings in the following description are some implementations of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative work:

[0039] Figure 1 The growth curve of the strain, glucose consumption and fermentation product production during the fermentation process of Example 1 of the present invention are shown;

[0040] Figure 2 The growth curve of the strain, glucose consumption and fermentation product production during the fermentation process of Example 2 of the present invention are shown;

[0041] Figure 3 The growth curve of the strain, the consumption of galactose and the production of fermentation products during the fermentation process of Example 3 of the present invention are shown;

[0042] Figure 4 The growth curve of the strain, the consumption of galactose and the production of fermentation products during the fermentation process of Example 4 of the present invention are shown;

[0043] Figure 5 The growth curve of the strain, glucose consumption and fermentation product production during the fermentation process of Example 5 of the present invention are shown;

[0044] Figure 6 The growth curve of the strain, the consumption of galactose and the production of fermentation products during the fermentation process of Example 6 of the present invention are shown;

[0045] Figure 7The growth curve of the strain, the glucose consumption and the production of the fermentation product during the fermentation process of Comparative Example 1 of the present invention are shown;

[0046] Figure 8 The growth curve of the strain, the glucose consumption and the production of the fermentation product during the fermentation process of Comparative Example 2 of the present invention are shown;

[0047] Fig. 9 The growth curve of the strain, the glucose consumption and the production of the fermentation product during the fermentation process of Comparative Example 3 of the present invention are shown;

[0048] Fig.10 The growth curve of the strain, the glucose consumption and the production of the fermentation product during the fermentation process of Comparative Example 4 of the present invention are shown;

[0049] Fig.11 The structural model diagram of two butanol dehydrogenases BDH1 and BDH4 in Clostridium with the deposit number GDMCC61493 is shown, wherein Fig.11 A in the figure shows the schematic diagram of the structure of BDH1 (ID1860), Fig.11 B in the figure shows a schematic diagram of the structure of BDH4 (ID5100). DETAILED DESCRIPTION

[0050] The present invention provides a method for improving the biobutanol conversion efficiency. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0051] The invention of the present invention is completely different from the invention concept of the disclosed technology. The laboratory modeled the key enzyme for butanol production, butanol dehydrogenase (BDH), through the SWISS-MODEL website and found that BDH1 and BDH4 of the four butanol dehydrogenases of strain WK each have one Fe 3+ Binding sites (such as Fig.11 Therefore, theoretically, a certain concentration of Fe can be added to the fermentation medium. 3+ To regulate the activity of butanol dehydrogenase, thereby increasing the production of butanol. 3+ The compound is mainly Fe 2 (SO4) 3 and FeCl 3 , which is why the present invention selects these two compounds.

[0052] Technical solution of the present invention

[0053] 1. Anaerobic culture medium (ACM), carbon source and Fe 3+ Preparation of metal ion solutions

[0054] (1) ACM anaerobic medium was prepared with pure water. The formula was (g / L): yeast extract, 10; NaHCO 3 , 2.52; 2-(N-morpholinyl)ethanesulfonic acid (MES), 2.132; 100× salt solution mother liquor, 10 mL / L (wherein the mother liquor contains NaCl, 1.0 g / L; MgCl 2 6H 2 O, 0.5 g / L; KH 2 PO 4 , 0.2g / L; NH 4 Cl, 0.3g / L; KCl, 0.3g / L; CaCl 2 ·2H 2 O, 0.015 g / L); 1000×trace element solution mother liquor, 1 mL / L (where the mother liquor contains FeCl 2 ·4H 2 O, 1.5 g / L; CoCl 2 6H 2 O, 0.19 g / L; MnCl 2 ·4H 2 O, 0.1 g / L; ZnCl 2 , 0.07g / L; H 3 BO 3 , 0.006g / L; Na 2 MoO 4 ·2H 2 O, 0.036 g / L; NiCl 2 6H 2 O, 0.024 g / L; CuCl 2 ·2H 2 O, 0.002 g / L); then add 0.001 g / L of resazurin anaerobic indicator; finally add deoxygenation reducing agent (dithiothreitol, 0.077 g / L; L-cysteine, 0.0242 g / L; Na 2 S, 0.016 g / L). Prepare the above solution and pass N 2 After deoxygenation, dispense into serum bottles, immediately seal with butyl rubber stoppers and aluminum caps, adjust pH to 6.0, sterilize at 121°C for 20 min, and cool for later use.

[0055] (2) The carbon source is either glucose or galactose, both of which are prepared into 50% mother liquor and filtered for sterilization.

[0056] (3) Fe content3+ The metal ion compound solution is FeCl 3 or Fe 2 (SO 4 ) 3 One of them was prepared into a 50g / L mother solution and filtered for sterilization.

[0057] (4) Carbon source and Fe 3+ The metal ion compound solution is added to ACM in a certain proportion and mixed thoroughly to obtain a fermentation medium for later use.

[0058] 2. Strain activation and seed liquid acquisition

[0059] The solvent-producing clostridium involved in the present invention is Clostridium sp. WK (patent application number: 202110179660.0), which is deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC61493. After the low-temperature preserved strain WK was placed in an 80°C water bath for heat shock for 10 minutes, it was inoculated into the ACM medium at 4% of the total volume of the culture medium, and anaerobically cultured for 12 hours at 37°C and 150rpm for activation (OD600=0.8). Take the activated strain culture solution, and the same 4% inoculation amount is inoculated into the seed culture medium (same as ACM), and anaerobically cultured for 12 hours at 37°C and 150rpm to obtain a seed solution for inoculation of the fermentation medium (OD600=0.8).

[0060] 3. Add Fe 3+ Optimization of fermentation conditions

[0061] (1) Add glucose or galactose to the new ACM as a carbon source for fermentation, and control the initial concentration of the carbon source to 40 g / L to 60 g / L.

[0062] (2) Then add FeCl 3 or Fe 2 (SO 4 ) 3 Mother liquor, added Fe 3+ The starting concentration is 5mM~30mM.

[0063] (3) The initial pH of the culture medium was adjusted to 6.7±0.3 using anaerobic 4 M NaOH.

[0064] (4) Inoculate 4% of the total volume of the culture medium with the seed solution of strain WK, and ferment at 30° C. and 150 rpm for 48 to 96 hours for anaerobic fermentation.

[0065] (5) During the fermentation period, samples were taken every 12 or 24 hours, and the strain growth curve was measured at a wavelength of 600 nm using a UVmini-1240 ultraviolet / visible spectrophotometer; the consumption of the carbon source substrate was determined using the 3,5-dinitrosalicylic acid (DNS) method; and the fermentation products (acetone, butanol, ethanol, butyric acid, acetic acid, etc.) were detected using gas chromatography (GC-FID) to monitor the changes in the products under different fermentation conditions.

[0066] The solvent-producing clostridium involved in the present invention is Clostridium sp. WK (patent application number: 202110179660.0), which is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC61493.

[0067] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0068] Example 1: 60 g / L glucose was used as the carbon source, and 3.24 g / L FeCl was added 3 (20 mM Fe 3+ ) biobutanol fermentation process

[0069] 1. Preparation of anaerobic culture medium

[0070] Prepare ACM medium, adjust the pH to 6.0, and continuously introduce N into the medium. 2 Deoxygenate, then dispense into anaerobic serum bottles, seal with butyl rubber stoppers and aluminum caps, sterilize at 121°C for 20 min for later use; add a certain amount of 50% glucose mother solution to the sterilized ACM medium to make its initial concentration 60 g / L; at the same time add a certain amount of 50 g / L FeCl 3 solution, making Fe 3+ The starting concentration of was 20 mM, and the medium was adjusted to an initial pH of 6.7 ± 0.3 with anaerobic 4 M NaOH.

[0071] 2. Seed Activation and Preparation

[0072] The solvent-producing clostridium involved in the present invention is Clostridium sp. WK (patent application number: 202110179660.0), which is preserved in the Guangdong Provincial Microbiological Culture Collection Center with a preservation number of GDMCC61493. The low-temperature preserved strain WK was placed in an 80°C water bath for heat shock for 10 minutes, then inoculated into ACM culture medium at 4% of the total volume of the culture medium, and anaerobically cultured at 37°C and 150rpm for 12 hours for activation (OD600 = 0.8). After the strain WK was activated, 4% of the total volume of the culture medium was inoculated with glucose but no Fe 3+ The culture medium was anaerobically cultured at 37°C and 150 rpm for 12 hours to obtain a seed solution (OD600 = 0.8).

[0073] 3. Monitoring of the fermentation process

[0074] According to step 1, 4% of the total volume of the culture medium was inoculated with the WK seed liquid activated in step 2, and anaerobically fermented at 30°C and 150rpm for 96 hours; during the entire fermentation process, samples were taken every 12 or 24 hours, and during the fermentation period, samples were taken every 12 or 24 hours, and the strain growth curve was measured at a wavelength of 600nm using a UVmini-1240 UV / visible spectrophotometer; the consumption of carbon source substrates was measured using the 3,5-dinitrosalicylic acid (DNS) method; the fermentation products (acetone, butanol, ethanol, butyric acid, acetic acid, etc.) were detected using gas chromatography (GC-FID) to monitor the changes in products under different fermentation conditions. The strain growth curve, glucose consumption and fermentation product production were measured respectively, and the results are shown in Tables 1 and Figure 1 shown.

[0075] Example 2: 60 g / L glucose as carbon source, 4 g / L Fe 2 (SO 4 ) 3 (20 mM Fe 3+ )

[0076] The production process and operation mode of this embodiment are the same as those of Example 1, except that Fe 2 (SO 4 ) 3 Replace FeCl 3 , also ensure that Fe 3+ The initial concentration of 20 mM was then activated and expanded, and then the strain was inoculated into the above medium for 96 hours. Samples were collected at the same intervals to measure the growth curve of the strain, glucose consumption, and fermentation product production. The results are shown in Tables 1 and Figure 2 Example 3: 50 g / L galactose was used as the carbon source and 3.24 g / L FeCl was added.3 (20 mM Fe 3+ ) biobutanol fermentation process

[0077] The production process and operation method of this embodiment are the same as those of Example 1, except that galactose is used instead of glucose as the carbon source for fermentation. The initial concentration of galactose is adjusted to 50 g / L. 3+ The initial concentration of 20 mM was then activated and expanded, and then the strain was inoculated into the above medium for 96 hours. Samples were collected at the same intervals to measure the growth curve of the strain, galactose consumption, and fermentation product production. The results are shown in Tables 1 and Figure 3 shown.

[0078] Example 4: Using 50 g / L galactose as the carbon source and adding 4 g / L Fe 2 (SO 4 ) 3 (20 mM Fe 3+ ) biobutanol fermentation process

[0079] The production process and operation mode of this embodiment are the same as those of embodiment 3, except that Fe 2 (SO 4 ) 3 Replace FeCl 3 , also ensure that Fe 3+ The initial concentration of 20 mM was then activated and expanded, and then the strain was inoculated into the above medium for 96 hours. Samples were collected at the same intervals to measure the growth curve of the strain, galactose consumption, and fermentation product production. The results are shown in Tables 1 and Figure 4 shown.

[0080] Example 5 Biobutanol fermentation process using 60 g / L glucose as carbon source without adding iron ions

[0081] The production process and operation method of this embodiment are the same as those of Example 1, except that no Fe is added to the culture medium. 3+ ions, and then the strain was activated and expanded and inoculated into the above medium for 96 hours. Samples were collected at the same intervals to measure the growth curve of the strain, glucose consumption and fermentation product production. The results are shown in Tables 1 and Figure 5 shown.

[0082] Example 6 Biobutanol fermentation process using 50 g / L galactose as carbon source without adding iron ions

[0083] The production process and operation method of this embodiment are the same as those of Example 3, except that no Fe is added to the culture medium. 3+ions. The strain was then activated and expanded and inoculated into the above medium for 96 hours of fermentation. Samples were collected at the same intervals to measure the strain growth curve, glucose consumption and fermentation product production. The results are shown in Tables 1 and Figure 6 shown.

[0084] Comparative Example 1: 60 g / L glucose was used as the carbon source and 0.81 g / L FeCl was added 3 (5mM Fe 3+ ) biobutanol fermentation process

[0085] The production process and operation mode of this comparative example are the same as those of Example 1, except that Fe 3+ The initial concentration was adjusted to 5 mM (i.e. 0.81 g / L FeCl 3 ), and then the strain was activated and expanded and inoculated into the above medium for 96 hours. Samples were collected at the same intervals to measure the growth curve of the strain, glucose consumption and fermentation product production. The specific results are shown in Tables 1 and Figure 7 shown.

[0086] Comparative Example 2: 60 g / L glucose was used as the carbon source and 1 g / L Fe was added 2 (SO 4 ) 3 (5mM Fe 3+ ) biobutanol fermentation process

[0087] The production process and operation mode of this comparative example are the same as those of Example 2, except that Fe 3+ The initial concentration was adjusted to 5 mM (i.e. 1 g / L Fe 2 (SO 4 ) 3 ), and then the strain was activated and expanded and inoculated into the above medium for 96 hours. Samples were collected at the same intervals to measure the growth curve of the strain, glucose consumption and fermentation product production. The specific results are shown in Tables 1 and Figure 8 shown.

[0088] Comparative Example 3: 60 g / L glucose was used as the carbon source and 3.96 g / L FeCl was added 2 ·4H 2 O(20 mM Fe 2+ ) biobutanol fermentation process

[0089] The production process and operation mode of this comparative example are the same as those of Example 1, except that FeCl 2 ·4H 2 O replaces FeCl 3 , to ensure Fe 2+The initial concentration of 20 mM was then activated and expanded, and then the strain was inoculated into the above medium for 96 hours. Samples were collected at the same intervals to measure the growth curve of the strain, glucose consumption, and fermentation product production. The specific results are shown in Tables 1 and Fig. 9 shown.

[0090] Comparative Example 4: 60 g / L glucose was used as the carbon source and 5.56 g / L FeSO was added. 4 7H 2 O(20 mM Fe 2+ )

[0091] The production process and operation mode of this comparative example are the same as those of Comparative Example 3, except that FeSO 4 7H 2 O replaces FeCl 2 ·4H 2 O, also ensure Fe 2+ The initial concentration of 20 mM was then activated and expanded, and then the strain was inoculated into the above medium for 96 hours. Samples were collected at the same intervals to measure the growth curve of the strain, glucose consumption, and fermentation product production. The specific results are shown in Tables 1 and Fig.10 shown.

[0092] Table 1 Comparison of fermentation conditions of different embodiments and comparative examples

[0093]

[0094] As shown in Table 1, in Examples 1 and 2, 3.24 g / L FeCl was added respectively under the condition that glucose was used as the carbon source. 3 and 4 g / L Fe 2 (SO 4 ) 3 , fermented for 72h, compared with Example 5, the sugar consumption increased by 31.76% and 23.07%, and the biobutanol production increased by 27.71% and 5.81%, respectively. In Examples 3 and 4, galactose was used as the carbon source and 3.24g / L FeCl was added. 3 and 4 g / L Fe 2 (SO 4 ) 3 , fermented for 72h, compared with Example 6, the sugar consumption increased by 37.74% and 24.9%, and the butanol production increased by 56.74% and 47.2%. 3+ The fermentation results of the compound (0.81g / L FeCl 3 and 1g / L Fe 2 (SO4 ) 3 ), compared with Example 5, there was no significant change in sugar consumption and butanol production, indicating that a small amount of ferric sulfate or ferric chloride cannot significantly improve the utilization of carbon sources and butanol production of the strain. Comparative Examples 3 and 4 were added with 3.96 g / LFeCl respectively under the condition of glucose as the carbon source. 2 ·4H 2 O and 5.56g / L FeSO 4 7H 2 O fermentation results, indicating that Fe 2+ It is not the reason for promoting strain WK to utilize glucose and increase butanol production.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Sequence Listing <110> Guangzhou Qianxiang Biotechnology Co., Ltd. <120> Method for improving biobutanol conversion efficiency <130> MP22008207 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 387 <212> PRT <213> Artificial Sequence <400> 1 Met Glu Asn Phe Asn Tyr Ser Ile Pro Thr Lys Val Tyr Phe Gly Lys 1 5 10 15 Gly Gln Ile Lys Asn Leu Ala Ala Ile Ile Lys Glu Tyr Gly Asn Lys 20 25 30 Ile Leu Ile Ala Tyr Gly Gly Gly Ser Ile Lys Lys Ile Gly Leu Tyr 35 40 45 Asp Glu Met Ile Lys Ile Leu Asn Asp Asn Ser Ile Ser Tyr Val Glu 50 55 60 Leu Ser Gly Ile Glu Pro Asn Pro Arg Ile Glu Thr Val Arg Lys Gly 65 70 75 80 Ile Lys Ile Cys Lys Glu Asn Asn Val Glu Val Val Leu Ala Val Gly 85 90 95 Gly Gly Ser Thr Ile Asp Cys Ala Lys Val Ile Ala Ala Gly Val Lys 100 105 110 Tyr Glu Gly Asp Pro Trp Asp Leu Val Thr Ser Pro Gln Lys Ile Asn 115 120 125 Glu Val Leu Pro Ile Val Thr Ile Leu Thr Leu Ser Ala Thr Gly Ser 130 135 140 Glu Met Asp Pro His Ala Val Ile Ser Asp Met Thr Thr Asn Gln Lys 145 150 155 160 Leu Gly Thr Gly His Glu Asn Met Lys Pro Lys Ala Ser Ile Leu Asp 165 170 175 Pro Glu Tyr Thr Tyr Ser Val Pro Lys Asn Gln Thr Ala Ala Gly Thr 180 185 190 Ala Asp Ile Met Ser His Ile Phe Glu Thr Tyr Phe Asn His Thr Lys 195 200 205 Gly Val Asp Ile Gln Asp Ser Thr Ala Glu Gly Leu Leu Arg Ala Cys 210 215 220 Ile Lys Tyr Gly Lys Ile Ala Ile Glu Asn Pro Lys Asp Tyr Asp Ala 225 230 235 240 Arg Ala Asn Leu Met Trp Ala Ser Ser Trp Ala Ile Asn Gly Leu Ile 245 250 255 Ser Tyr Gly Thr Asn Ser Pro Trp Val Val His Pro Met Glu His Glu 260 265 270 Leu Ser Ala Phe Tyr Asp Ile Thr His Gly Val Gly Leu Ala Ile Leu 275 280 285 Thr Pro His Trp Met Lys Tyr Ser Leu Asp Asp Thr Thr Val Phe Lys 290 295 300 Phe Ala Gln Tyr Gly Ile Asn Val Trp Gly Ile Asp Lys Asn Leu Asp 305 310 315 320 Lys Phe Glu Ile Ala Asn Lys Ala Ile Glu Lys Thr Ser Glu Phe Phe 325 330 335 Lys Glu Leu Gly Ile Pro Ser Thr Leu Arg Glu Val Gly Ile Glu Glu 340 345 350 Asp Lys Leu Glu Leu Met Ala Lys Lys Ala Met Asn Pro Tyr Phe Lys 355 360 365 Tyr Ala Phe Lys Pro Leu Asp Glu Asn Asp Ile Leu Lys Ile Phe Lys 370 375 380 Ala Ala Leu 385 <210> 2 <211> 392 <212> PRT <213> Artificial Sequence <400> 2 Met Lys Asn Phe Glu Phe Tyr Ala Pro Thr Arg Val Ile Phe Gly Lys 1 5 10 15 Asp Ser Glu Lys Gln Ile Gly Thr Ile Ile Lys Asn Gln Asn Cys Lys 20 25 30 Lys Val Leu Val His Phe Gly Gly Ser Ser Ala Lys Lys Ser Gly Leu 35 40 45 Leu Asp Lys Ile Phe Glu Ser Leu Lys Glu Ala Glu Ile Asp Tyr Val 50 55 60 Ser Leu Gly Gly Val Val Pro Asn Pro Arg Leu Ser Lys Val Tyr Glu 65 70 75 80 Gly Ile Asn Leu Cys Lys Lys Glu Lys Val Asp Phe Ile Leu Ala Val 85 90 95 Gly Gly Gly Ser Val Ile Asp Ser Ala Lys Ala Ile Gly Tyr Gly Ile 100 105 110 Ser Asn Glu Cys Asp Val Trp Asp Ile Tyr Ser Lys Lys Val Ile Pro 115 120 125 Thr Gly Cys Leu Pro Val Gly Ala Val Leu Thr Ile Ala Ala Ala Gly 130 135 140 Ser Glu Met Ser Asn Ser Ser Val Ile Thr Asn Glu Glu Gly Trp Leu 145 150 155 160 Lys Arg Gly Cys Asn Ser Glu Tyr Ala Arg Cys Lys Phe Ala Ile Met 165 170 175 Asn Pro Glu Leu Thr Tyr Thr Leu Pro Lys Tyr Gln Thr Ala Ser Gly 180 185 190 Ala Thr Asp Ile Leu Met His Thr Met Glu Arg Tyr Phe Thr Lys Glu 195 200 205 Gln Ser Met Glu Ile Thr Asp Arg Ile Ser Glu Gly Leu Met Arg Thr 210 215 220 Val Ile His Asn Val Lys Ile Leu Met Lys Asn Pro Lys Asp Tyr Asn 225 230 235 240 Ala Arg Ala Glu Val Met Trp Ala Gly Ser Leu Ser His Asn Asp Leu 245 250 255 Thr Gly Cys Gly Ser Val Gly Asp Trp Ser Cys His Gln Leu Glu His 260 265 270 Glu Leu Gly Gly Met Phe Asp Val Ala His Gly Ala Gly Leu Ala Ala 275 280 285 Val Trp Gly Ser Trp Ala Arg Tyr Val Tyr Lys Ser Asn Ile Ser Arg 290 295 300 Phe Val Gln Phe Ala Val Asn Val Met Gly Ile Thr Asn Asp Phe Tyr 305 310 315 320 Asn Pro Glu Lys Val Ala Leu Glu Gly Ile Glu Ala Met Glu Ser Phe 325 330 335 Tyr His Ser Ile Asp Met Pro Ile Ser Ile Lys Glu Leu Gly Val Asn 340 345 350 Leu Thr Asp Asp Gln Ile Ala Asp Leu Ala Tyr Lys Cys Ser Phe Lys 355 360 365 Asp Thr Arg Thr Ile Gly Glu Phe Gln Lys Leu Asn Met Glu Asp Met 370 375 380 Lys Lys Ile Tyr Ile Met Ala Arg 385 390

Claims

1. 20 mM Fe 3+ The invention is used for improving the butanol conversion efficiency of Clostridium, wherein the Clostridium is a strain with a preservation number of GDMCC61493.

2. Methods for improving the conversion efficiency of Clostridium butanol, It is characterized in that Use 20 mM Fe 3+ and a medium containing a carbon source to culture the Clostridium with a deposit number of GDMCC61493; The Fe 3+ From FeCl 3 and / or Fe 2 (SO 4 ) 3 ; the Fe 3+ The molar ratio of the carbon source is 1:(9-70); the carbon source is glucose and / or galactose; The conditions for culturing Clostridium include 48 to 96 hours, pH 6 to 7, and an anaerobic environment.

3. The method according to claim 2, It is characterized in that 20 mM Fe 3+ and a carbon source in the culture medium, wherein the basic culture medium is an ACM anaerobic culture medium; the ACM anaerobic culture medium includes 10 g / L yeast extract; NaHCO 3 2.52 g / L; 2-(N-morpholino)ethanesulfonic acid (MES) 2.132 g / L; 100× salt solution 10 mL / L; 1000× trace element solution 1 mL / L; Resazurin anaerobic indicator 0.001 g / L; Dithiothreitol 0.077 g / L; L-cysteine ​​0.0242 g / L; Na 2 S0.016 / L.

4. The method according to claim 3, It is characterized in that The 100× salt solution contains: NaCl, 1.0 g / L; MgCl 2 6H 2 O, 0.5 g / L; KH 2 PO 4 , 0.2 g / L; NH 4 Cl, 0.3 g / L; KCl, 0.3 g / L; CaCl 2 ·2H 2 O, 0.015 g / L; The 1000× trace element solution contains: FeCl 2 ·4H 2 O, 1.5 g / L; CoCl 2 6H 2 O, 0.19 g / L; MnCl 2 ·4H 2 O, 0.1 g / L; ZnCl 2 , 0.07 g / L; H 3 BO 3 , 0.006 g / L; Na 2 MoO 4 ·2H 2 O, 0.036 g / L; NiCl 2 6H 2 O, 0.024 g / L; CuCl 2 ·2H 2 O, 0.002 g / L.

5. The method according to any one of claims 2 to 4, It is characterized in that The culture medium contained 60 g / L glucose and 3.24 g / L FeCl 3 .

6. The method according to any one of claims 2 to 4, It is characterized in that The culture medium contained 60 g / L glucose and 4 g / L Fe 2 (SO 4 ) 3 .

7. The method according to any one of claims 2 to 4, It is characterized in that The culture medium contained 50 g / L galactose and 3.24 g / L FeCl 3 .

8. The method according to any one of claims 2 to 4, It is characterized in that The culture medium contained 50 g / L galactose and 4 g / L Fe 2 (SO 4 ) 3 .

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