Culture medium for regulating acetyl coenzyme A derivative and use method thereof

Abstract

The invention relates to the technical field of synthesis gas fermentation, in particular to a culture medium for regulating an acetyl coenzyme A derivative and a use method of the culture medium. The culture medium is used for regulating and controlling the yield of ethanol and the yield of acetic acid generated by an acetyl coenzyme A derivative in the ethanol-producing clostridium; the culture medium comprises ferrous sulfate, the weight content of the ferrous sulfate is in negative correlation with the yield of ethanol, and the weight content of the ferrous sulfate is in positive correlation with the yield of acetic acid. According to the culture medium, along with the weight content change of the ferrous sulfate and the content change of the ethanol and the acetic acid generated by the ethanol-producing clostridium, the yield of the ethanol and the acetic acid in a synthetic product of the ethanol-producing clostridium is adjusted by adjusting the content change of the ferrous sulfate; therefore, the controllability of the synthetic product of the clostridium ethanoliforme can be comprehensively improved.

Description

Technical Field

[0001] The present application relates to the field of synthesis gas fermentation technology, and in particular to a culture medium for regulating acetyl-CoA derivatives and a method for using the culture medium. Background Art

[0002] Acetogenic bacteria can utilize CO2 to grow autotrophically and synthesize short-chain organic acids, alcohols and other bulk chemicals, and have low requirements for the ratio of gas components in the feed gas (CO / CO2 / H2). They are an ideal biocatalyst for synthesizing biofuels and chemicals using one-carbon gas. Currently, the most studied acetogenic bacteria include Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Acetobacterium woodii, Moorella thermoacetica, Eubacterium limosum, etc. Among them, Clostridium ethanologenum has achieved industrial production of ethanol synthesized from tail gas of the steel industry. Ethanolic Clostridium mainly fixes CO2 through the Wood-Ljungdahl Pathway (WLP); among all known carbon fixation pathways, WLP is considered to be the most efficient and the only one that couples CO2 fixation with energy metabolism to maintain cell growth. WLP is divided into methyl branch and carbonyl branch. In the methyl branch, CO2 is catalyzed by a series of metalloenzymes with tetrahydrofolate as a cofactor: formate dehydrogenase (Fdh), formyltetrahydrofolate synthetase (Fhs), methyltetrahydrofolate cyclase / dehydrogenase (Fch / Mdh), methylenetetrahydrofolate reductase (Mthfr) and methyltransferase (MT) to produce methyl-corrinoid iron-sulfur protein (Methyl-CoFeS) as a methyl donor, which is then mediated by carbon monoxide dehydrogenase / acetyl-CoA synthetase (CODH / ACS) to condense with coenzyme A and a molecule of CO from the carbonyl branch to produce acetyl-CoA. In this methyl branch, CO can come from the external environment or be produced by reducing CO2 through CODH / ACS. It can be seen from this that acetyl-CoA is the central metabolite of autoethanologenic Clostridium, which can further generate acetic acid under the action of phosphate acetyltransferase (Pta) and acetate kinase (Ack), accompanied by the synthesis of a molecule of ATP. Acetic acid will be synthesized into acetaldehyde under the action of aldehyde ferredoxin oxidoreductase (Aor), and finally synthesized into ethanol under the action of alcohol dehydrogenase (indirect ethanol synthesis pathway); in addition, acetyl-CoA can also be converted into acetaldehyde and ethanol in turn under the action of aldehyde dehydrogenase and alcohol dehydrogenase (direct ethanol synthesis pathway).

[0003] Current studies have shown that ethanol production by autoethanogenic Clostridium is mainly achieved through an indirect synthesis pathway when using CO2 for autotrophic fermentation, which may be related to the fact that this pathway can provide additional ATP for strain metabolism. In addition, studies have found that the autotrophic metabolism of acetogenic bacteria generally operates at the edge of the thermodynamic limit, and the reason why autotrophic metabolism can maintain its normal growth and synthesize various acids, alcohols and other chemicals is that there is a third energy conservation method in acetogenic bacteria - electron bifurcation. In addition, unlike other microorganisms that regulate metabolism at the transcription or translation level, the energy metabolism of acetogenic bacteria is affected by the redox level and thermodynamic control, so the redox balance is the key factor that determines the direction of carbon flow in the product synthesis process and the robustness of cell metabolism. However, different growth conditions will lead to changes in the ratio of reduced products to oxidized products, which is more controlled by the concentration of metabolites in the cell and the laws of thermodynamics, rather than being regulated at the transcription or translation level. For example, under fermentation conditions with an oversupply of CO, if the growth of the strain is limited by nutrition, the energy flow and carbon flow will shift from supplying bacterial growth to ethanol production, thereby achieving redox balance. Therefore, in the commercial operation of ethanol production from CO2-containing industrial gas fermentation, the supply of raw gas is a key factor affecting the synthesis rate of acetic acid and ethanol. However, in actual high-load operation, the increase in gas volume is limited, or simply increasing the intake volume cannot achieve effective control of acetic acid and thus enhance ethanol synthesis. Therefore, it is necessary to actively explore other regulation processes.

[0004] At present, the methods for regulating the distribution of metabolites of acetogenic bacteria include strain modification and fermentation process optimization. For example, under autotrophic conditions, knocking out aor2 of autoethanologenic Clostridium can increase the ethanol synthesis yield by 170%, while knocking out adhE can increase the ethanol synthesis yield under autotrophic conditions by 180%, but the corresponding strain lag period will increase and the biomass will decrease. However, these engineered strains have only been verified by batch fermentation in the laboratory, and the continuous fermentation conditions under commercial operation have not been simulated, so they cannot be directly used for commercial production. In addition, trace elements in the fermentation medium are often key cofactors for the catalytic activity of enzymes. For example, selenium is a cofactor of formate dehydrogenase (FDH), and tungsten is a cofactor of FDH and aldehyde ferredoxin oxidoreductase (AOR). FDH is responsible for fixing CO2 into formic acid, and AOR catalyzes acetic acid to synthesize acetaldehyde. These parameters indicate that selenium has a great influence on the growth and metabolism of the strain, and the increase in the amount of tungsten added to the culture medium will increase the ethanol / acetic acid ratio and increase the ethanol yield by at least 2.18 times. Therefore, adjusting the content of trace elements in the fermentation medium and then adjusting the catalytic performance of enzymes in the metabolic pathway may play a role in regulating carbon metabolism distribution. However, at present, the adjustment of trace elements in the culture medium is only targeted at specific enzyme systems, and it is impossible to comprehensively improve the controllability of the synthetic products of autoethanologenic Clostridium. Summary of the invention

[0005] The present application provides a culture medium for regulating acetyl-CoA derivatives and a method for using the same to solve the following technical problem: how to comprehensively improve the controllability of the synthetic products of autoethanologenic Clostridium.

[0006] In a first aspect, the present application provides a culture medium for regulating acetyl-CoA derivatives, wherein the culture medium is used to regulate the production of ethanol and acetic acid generated by acetyl-CoA derivatives in ethanologenic Clostridium; the culture medium comprises ferrous sulfate, the weight content of the ferrous sulfate is negatively correlated with the production of ethanol, and the weight content of the ferrous sulfate is positively correlated with the production of acetic acid.

[0007] Optionally, the mass concentration of ferrous sulfate is ≥0.0125 g / L.

[0008] Optionally, the culture medium also includes trace element solution and vitamin solution.

[0009] In a second aspect, the present application provides a method for using a culture medium for regulating acetyl-CoA derivatives, wherein the culture medium described in the first aspect is used to regulate the process of acetyl-CoA derivatives producing ethanol and acetic acid, and the method comprises:

[0010] Using a CaGM growth medium containing a first preset ferrous sulfate content and a preset gas source to perform primary fermentation culture on the autoethanologenic Clostridium to obtain a preliminary fermentation broth;

[0011] detecting the ethanol content of the preliminary fermentation broth and the acetic acid content of the preliminary fermentation broth;

[0012] determining a second preset ferrous sulfate content of the CaGM growth medium according to the ethanol content of the preliminary fermentation broth and the acetic acid content of the preliminary fermentation broth;

[0013] The preliminary fermentation broth is subjected to continuous fermentation culture according to the CaGM growth medium containing the second preset ferrous sulfate content and the preset gas source to adjust the ethanol yield and the acetic acid yield in the preliminary fermentation broth to obtain an ethanol product and an acetic acid product, respectively.

[0014] Optionally, the second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 satisfy the relationship: W1:W2≥25:100.

[0015] Optionally, the second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 satisfy the relationship: W1:W2=25:100; and / or

[0016] The second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 satisfy the relationship: W1:W2=50:100.

[0017] Optionally, the acetic acid content W3 of the preliminary fermentation liquid and the ethanol content W4 of the preliminary fermentation liquid satisfy the relationship: W3:W4≥2:1.

[0018] Optionally, the weight m3 of the acetic acid product and the weight m4 of the ethanol product satisfy the relationship: m3:m4≤1.1:1.

[0019] Optionally, the preset gas source includes a carbon-containing gas and an inert gas, and the volume V3 of the carbon-containing gas and the volume V4 of the inert gas satisfy the relationship: V3:V4≥7:3.

[0020] The above technical solution provided by the embodiment of the present application has the following advantages compared with the prior art:

[0021] The embodiment of the present application provides a culture medium for regulating acetyl-CoA derivatives. Since iron ions are widely present in various enzymes in the metabolic pathway of autoethanologenic Clostridium, such as carbon monoxide dehydrogenase, hydrogenase, formate dehydrogenase, aldehyde ferredoxin oxidoreductase, etc., the content of iron ions in autoethanologenic Clostridium can be adjusted by regulating the content of ferrous sulfate in the culture medium, so that the biological metabolic synthesis pathway in the autoethanologenic Clostridium can be adjusted; in addition, the weight content of ferrous sulfate is negatively correlated with the output of ethanol, and the weight content of ferrous sulfate is positively correlated with the output of acetic acid. It can be clearly seen that as the weight content of ferrous sulfate changes with the content of ethanol and acetic acid produced by autoethanologenic Clostridium, the output of ethanol and acetic acid in the synthetic products of autoethanologenic Clostridium can be adjusted by adjusting the change in the content of ferrous sulfate, thereby comprehensively improving the controllability of the synthetic products of autoethanologenic Clostridium. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0023] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0024] Figure 1 A schematic flow chart of a method for using a culture medium for regulating acetyl-CoA derivatives provided in an embodiment of the present application;

[0025] Figure 2This is a graph showing the changing trends of biomass, ethanol weight content and acetic acid weight content during the continuous fermentation of the lyophilized powder of Clostridium ethanolum DSM10061 provided in the examples of the present application as the amount of ferrous sulfate added changes. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0027] Various embodiments of the present application may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the present application; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range; for example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which applies regardless of the range; in addition, whenever a numerical range is indicated in this document, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0028] In this document, the terms including "comprising" and the like mean "including but not limited to". Relational terms such as "first" and "second" are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone; wherein A and B can be singular or plural. Unless otherwise specified, various raw materials, reagents, instruments and equipment used in this application can be purchased on the market or prepared by existing methods.

[0029] An embodiment of the present application provides a culture medium for regulating acetyl-CoA derivatives, wherein the culture medium is used to regulate the production of ethanol and acetic acid generated by acetyl-CoA derivatives in ethanologenic Clostridium; the culture medium includes ferrous sulfate, the weight content of the ferrous sulfate is negatively correlated with the production of ethanol, and the weight content of the ferrous sulfate is positively correlated with the production of acetic acid.

[0030] It should be noted that, in addition to ferrous sulfate, the culture medium also includes other substances that can promote the normal growth of Clostridium ethanolicum, such as trace element solution TSE and Wolfe's vitamin solution.

[0031] In some optional embodiments, the mass concentration of ferrous sulfate is ≥0.0125 g / L;

[0032] In these embodiments, the mass concentration of ferrous sulfate can be ≥0.0125 g / L, so that there is a sufficient mass concentration of ferrous sulfate in the culture medium, so as to adjust the content of ferrous sulfate to adjust the production of ethanol and acetic acid in the synthetic products of Clostridium autoethanogenum, thereby comprehensively improving the controllability of the synthetic products of Clostridium autoethanogenum.

[0033] In some optional embodiments, the culture medium further comprises a trace element solution and a vitamin solution;

[0034] In these embodiments, the culture medium may also include trace element solution and vitamin solution, which can ensure that there is a sufficient amount of trace element solution and vitamin solution in the culture medium, thereby conveniently adjusting the content of ferrous sulfate to adjust the production of ethanol and acetic acid in the synthetic products of Clostridium autoethanologenum, so as to comprehensively improve the controllability of the synthetic products of Clostridium autoethanologenum.

[0035] Figure 1 The schematic diagram of the method for using a culture medium for regulating acetyl-CoA derivatives provided in an embodiment of the present application is exemplarily shown;

[0036] Based on a general inventive concept, such as Figure 1 As shown, the embodiment of the present application provides a method for using a culture medium for regulating acetyl-CoA derivatives, wherein the culture medium is used to regulate the process of acetyl-CoA derivatives generating ethanol and acetic acid, and the method comprises:

[0037] S1. Using a CaGM growth medium containing a first preset ferrous sulfate content and a preset gas source, the autoethanologenic Clostridium was subjected to primary fermentation culture to obtain a preliminary fermentation broth;

[0038] S2. detecting the ethanol content of the preliminary fermentation broth and the acetic acid content of the preliminary fermentation broth;

[0039] S3. Determine a second preset ferrous sulfate content of the CaGM growth medium according to the ethanol content of the preliminary fermentation broth and the acetic acid content of the preliminary fermentation broth;

[0040] S4. The preliminary fermentation broth is subjected to continuous fermentation culture according to the CaGM growth medium containing the second preset ferrous sulfate content and the preset gas source to adjust the ethanol yield and the acetic acid yield in the preliminary fermentation broth to obtain an ethanol product and an acetic acid product, respectively.

[0041] The method of use is implemented based on the above-mentioned culture medium. The specific composition of the culture medium can refer to the above-mentioned embodiment. Since the method of use adopts part or all of the technical solutions of the above-mentioned embodiment, it has at least all the beneficial effects brought by the technical solutions of the above-mentioned embodiment, which will not be described one by one here.

[0042] It should be noted that, based on the ethanol content and acetic acid content of the preliminary fermentation broth, it is possible to accurately determine whether the amount of ferrous sulfate added to the CaGM growth medium needs to be adjusted, and determine the content of ferrous sulfate that needs to be added to the CaGM growth medium. The content of ferrous sulfate can be conveniently adjusted to adjust the production of ethanol and acetic acid in the synthetic products of the autoethanologenic Clostridium, thereby comprehensively improving the controllability of the synthetic products of the autoethanologenic Clostridium.

[0043] In some optional embodiments, the second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 satisfy the relationship: W1:W2≥25:100;

[0044] In these embodiments, the second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 may satisfy the relationship: W1:W2≥25:100, and the yield of ethanol and acetic acid synthesized products in the preliminary fermentation broth may be adjusted by reducing the ferrous sulfate content in the CaGM growth medium.

[0045] In some optional embodiments, the second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 satisfy the relationship: W1:W2=25:100; and / or

[0046] The second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 satisfy the relationship: W1:W2=50:100;

[0047] In these embodiments, the second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 can satisfy the relationship: W1:W2=25:100, or the second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 can satisfy the relationship: W1:W2=50:100, and CaGM growth medium with different ferrous sulfate contents can be selectively added according to the ethanol content and acetic acid content in the preliminary fermentation broth to comprehensively improve the controllability of the synthesis products of the autoethanol-producing Clostridium, thereby promoting the production of ethanol products mainly in the fermentation broth.

[0048] In some optional embodiments, the acetic acid content W3 of the preliminary fermentation broth and the ethanol content W4 of the preliminary fermentation broth satisfy the relationship: W3:W4≥2:1;

[0049] In these embodiments, the acetic acid content W3 of the preliminary fermentation broth and the ethanol content W4 of the preliminary fermentation broth can satisfy the relationship: W3:W4≥2:1, which can make it clear that the acetic acid content in the preliminary fermentation broth accounts for too large a proportion, so as to clarify the timing of adjusting the ferrous sulfate content in the CaGM growth medium, thereby facilitating the subsequent adjustment of the ferrous sulfate content in the CaGM growth medium to comprehensively improve the controllability of the synthetic products of the autoethanologenic Clostridium.

[0050] In some optional embodiments, the weight m3 of the acetic acid product and the weight m4 of the ethanol product satisfy the relationship: m3:m4≤1.1:1;

[0051] In these embodiments, the weight m3 of the acetic acid product and the weight m4 of the ethanol product can satisfy the relationship: m3:m4≤1.1:1. Compared with the contents of acetic acid and ethanol in the preliminary fermentation broth, the synthetic products of the fermentation broth can be converted into ethanol products by adjusting the content of ferrous sulfate in the CaGM growth medium. Therefore, the controllability of the synthetic products of the autoethanologenic Clostridium can be comprehensively improved by adjusting the content of ferrous sulfate in the CaGM growth medium.

[0052] In some optional embodiments, the preset gas source includes a carbon-containing gas and an inert gas, and the volume V3 of the carbon-containing gas and the volume V4 of the inert gas satisfy the relationship: V3:V4≥7:3;

[0053] In these embodiments, the preset gas source may include a carbon-containing gas and an inert gas, and the volume V3 of the carbon-containing gas and the volume V4 of the inert gas may satisfy the relationship: V3:V4≥7:3, which can ensure that there is a sufficient amount of carbon-containing gas in the preset gas source to promote the stable synthesis of ethanol and acetic acid by Clostridium ethanolum.

[0054] The present application is further described below in conjunction with specific examples. The experimental methods in the following examples that do not specify specific conditions are usually measured according to industry standards; if there are no corresponding industry standards, they are carried out according to common international standards, conventional conditions, or conditions recommended by the manufacturer.

[0055] Example 1

[0056] 1. Continuous fermentation of CO gas with self-producing ethanol Clostridium:

[0057] The autoethanogenic Clostridium DSM10061 was used as the fermentation strain. The ethanologenic Clostridium strain was purchased from the German strain collection DSMZ. The strain gas fermentation medium was a CaGM growth medium component (see Liew F, Henstra AM, Winzer K, M,Simpson SD,Minton NP.Insights into CO2 Fixation Pathway ofClostridium autoethanogenum by Targeted Mutagenesis.mBio.2016May 24;7(3):e00427-16.doi:10.1128 / mBio.00427-16.PMID:27222467; PMCID:PMC4895105.) as shown in Table 1.

[0058] Table 1. Components of CaGM growth medium

[0059]

[0060]

[0061] In Table 1, the specific components of TSE are shown in Table 2, and the specific components of Wolfe's vitamin solution are shown in Table 3.

[0062] Table 2 Components of trace element solution

[0063] TSE solution components Component content nitrilotriacetic acid 2g / L <![CDATA[MnSO4·H2O]]> 1g / L <![CDATA[Fe(SO4)2(NH4)2·6H2O]]> 0.8g / L <![CDATA[CoCl2·6H2O]]> 0.2g / L <![CDATA[ZnSO4·7H2O]]> 0.2mg / L <![CDATA[CuCl2·2H2O]]> 0.02g / L <![CDATA[NaMoO4·2H2O]]> 0.02g / L <![CDATA[Na2SeO3]]> 0.02g / L <![CDATA[NiCl2·6H2O]]> 0.02g / L <![CDATA[Na2WO4·2H2O]]> 0.02g / L

[0064] Table 3 Composition of Wolfe's vitamin solution

[0065] Wolfe's vitamin solution ingredients Component content Biotin (Vitamin H) 2mg / L folic acid (vitamin B) 2mg / L Pyridoxine hydrochloride(Vitamin B6) 10mg / L Thiamine HCl (Vitamin B1) 5mg / L Riboflavin (Vitamin B2) 5mg / L Nicotinic acid (vitamin B3) 5mg / L calcium pantothenate (vitamin B5) 5mg / L Vitamin B12 0.1mg / L p-aminobenzoic acid (Vitamin Bx) 5mg / L Thioctic acid 5mg / L

[0066] Then a 3L dual bioreactor (Dibier) was used as a fermentation equipment. A 1g / L acetic acid aqueous solution was prepared in the reactor according to a working volume of 2L, and then the acetic acid solution was fixed to 1.9L and sterilized at 121°C for 20min. After the sterilization was completed, 100% N2 was introduced into the reactor at a gas flow rate of 1L / min for 4h replacement to remove the oxygen in the fermentation equipment. Subsequently, 100mL of 20-fold concentrated CaGM growth medium was added to the fermentation equipment, and the gas source was switched to a preset gas source (50% CO by volume, 20% CO2 by volume, and 30% N2 by volume) for standby, and the ventilation volume of the preset gas source was set to 0.1L / min, and the parameters of the fermentation equipment were set according to the conditions of an initial stirring speed of 150rpm and a fermentation temperature of 37°C. Within the first half hour of inoculating the strain, 1 g of cysteine ​​was dissolved in 50 mL of CaGM growth medium to obtain a mixed culture solution, and then the prepared culture solution was added to the fermentation equipment. Ammonia water was then used to adjust the initial pH of the culture solution to 5.0. When the ORP of the culture solution dropped to -150 mV, 3 g of ethanol Clostridium DSM10061 freeze-dried powder strain was inoculated.

[0067] Figure 2 The variation trend graph of biomass, ethanol weight content and acetic acid weight content during the continuous fermentation of the lyophilized powder of Clostridium ethanolicum DSM10061 provided in the examples of the present application as the amount of ferrous sulfate added changes is shown by way of example;

[0068] After 2 days of inoculation of lyophilized powder of Clostridium ethanolicum DSM10061, Figure 2 As shown in the figure, lactic acid synthesis was detected in the fermentation broth. At this time, the pH and ORP of the fermentation broth showed a downward trend. The strain of ethanol Clostridium DSM10061 began to recover. Ammonia water was manually added to make the pH value of the fermentation broth not less than 4.8. After 4 days of fermentation, the accumulation of lactic acid reached a maximum value of 0.51 g / L. Subsequently, the lactic acid in the fermentation broth began to be consumed, while the biomass of acetic acid and the fermentation broth began to accumulate slowly. At this time, the pH of the fermentation broth was controlled at 5.0 by automatically adding ammonia water. After 7 days of fermentation, the lactic acid was consumed. At this time, the biomass of the fermentation broth reached 0.38 g / L, and the weight content of acetic acid in the fermentation broth was 2.15 g / L. In the subsequent fermentation process, the stirring speed and the ventilation volume of the preset gas source were gradually increased according to the metabolism of the strain to ensure the synchronous growth of the biomass, acetic acid and ethanol of the fermentation broth. At the same time, a 1% NaHS solution was added to keep the H2S content in the tail gas of the fermentation equipment at 200 ppmv. When the fermentation liquid reaches the 12.7th day, the biomass of the fermentation liquid can reach 1.66g / L, the weight content of ethanol is 2.7g / L, and the weight content of acetic acid is 9.78g / L. The continuous fermentation mode is started according to the following formula:

[0069] D=Dw=0.8×Day -1 ;

[0070] Wherein, D is the dilution rate calculated by the flow rate of the culture medium, and Dw is the dilution rate calculated by the discharge rate of the fermentation liquid). At this time, the culture medium added is the CaGM growth medium with normal ferrous sulfate content, and the weight content of ferrous sulfate in the culture medium is 0.05g / L, which is recorded as 100%. When the continuous fermentation runs to the 17.8th day, the biomass in the primary fermentation liquid is basically stable at 2.1g / L, the weight content of ethanol is basically stable at 5.9g / L, and the weight content of acetic acid increases to 13.31g / L. At this time, the ethanol content and acetic acid content in the subsequent fermentation liquid are regulated.

[0071] Example 2

[0072] 2. Switching culture medium with different ferrous sulfate content to regulate the synthesis of acetic acid and ethanol during continuous fermentation:

[0073] When the fermentation broth in Example 1 is continuously fermented to the 17.8th day, the weight content of ferrous sulfate in the CaGM growth medium is reduced to 25% of the flow-added CaGM growth medium, and the weight content of ferrous sulfate is 0.0125g / L at this time. Subsequent experiments found that the biomass of the strain in the fermentation broth remained essentially unchanged, but the weight content of acetic acid in the primary fermentation broth decreased rapidly, and the weight content of ethanol increased, but the weight content of acetic acid in the primary fermentation broth decreased by more than the weight content of ethanol. When the fermentation was continued for the 22.8th day, the weight content of ethanol in the primary fermentation broth increased to 8g / L, and the weight content of acetic acid decreased to 8.5g / L. At this time, the weight content of ferrous sulfate in the CaGM growth medium was increased to 50% of the ferrous sulfate content in the above-mentioned CaGM growth medium, and the weight content of ferrous sulfate was 0.025g / L at this time. Acetic acid began to accumulate in the primary fermentation broth, and the weight content of ethanol decreased. Subsequently, the same CaGM growth medium was repeated for 2 rounds according to the above-mentioned ferrous sulfate adjustment method, and the weight content of acetic acid and ethanol showed the same trend during the experiment.

[0074] Comparative Example 1

[0075] Based on the contents disclosed in Example 2, the following modifications are further made:

[0076] The weight content of ferrous sulfate was adjusted to 10% of the CaGM growth medium, at which time the weight content of ferrous sulfate was 0.005 g / L, and the rest remained unchanged. After 1 day of continuous culture, it was found that the biomass in the primary fermentation broth continued to decrease to 1.3 g / L, and the mass concentrations of the metabolites acetic acid and ethanol in the primary fermentation broth also decreased, with the weight content of acetic acid decreasing to 2.8 g / L and the weight content of ethanol decreasing to 6.3 g / L.

[0077] It can be seen from Example 2 and Comparative Example 1 that when the weight content of ferrous sulfate is insufficient, it will not only lead to a decrease in the biomass in the fermentation broth, but also affect the final weight content of acetic acid and ethanol, resulting in insufficient output of the final ethanol product and acetic acid product.

[0078] In summary, the embodiment of the present application provides a culture medium for regulating acetyl-CoA derivatives, which clearly defines the changes in the weight content of ferrous sulfate and the changes in the content of ethanol and acetic acid produced by Clostridium autoethanogenum. Therefore, by adjusting the change in the content of ferrous sulfate to adjust the yield of ethanol and acetic acid in the synthetic products of Clostridium autoethanogenum, the controllability of the synthetic products of Clostridium autoethanogenum can be comprehensively improved.

[0079] In addition, a culture medium for regulating acetyl-CoA derivatives provided in an embodiment of the present application provides a new regulation process for controlling the production of acetic acid produced in the commercial operation of fermenting CO2-containing industrial gas to produce ethanol. For example, when the weight content of acetic acid produced by fermentation is too high and it is impossible to promote the conversion of acetic acid into ethanol by increasing the gas volume of raw gas, the acetic acid synthesis can be reduced by reducing the weight content of ferrous sulfate in the culture medium, while strengthening the ethanol synthesis pathway in the fermentation broth, so as to ultimately comprehensively improve the controllability of the synthesis products of the autoethanologenic Clostridium.

[0080] The above is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. It will be apparent to those skilled in the art that various modifications to these embodiments are possible, and the general principles defined in the present application can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown in the present application, but will conform to the widest scope consistent with the principles and novel features applied for by the present application.

Claims

1. A culture medium for regulating acetyl-CoA derivatives, characterized in that: The culture medium is used to regulate the production of ethanol and acetic acid generated by acetyl-CoA derivatives in ethanol-producing Clostridium; the culture medium includes ferrous sulfate, the weight content of which is negatively correlated with the production of ethanol, and the weight content of which is positively correlated with the production of acetic acid.

2. The culture medium according to claim 1, characterized in that The mass concentration of the ferrous sulfate is ≥0.0125 g / L.

3. The culture medium according to claim 1, characterized in that The culture medium also includes trace element solution and vitamin solution.

4. A method for using a culture medium for regulating acetyl-CoA derivatives, characterized in that: The method of use is to use the culture medium according to any one of claims 1 to 3 for regulating the process of acetyl-CoA derivatives producing ethanol and acetic acid, and the method of use comprises: Using a CaGM growth medium containing a first preset ferrous sulfate content and a preset gas source to perform primary fermentation culture on the autoethanologenic Clostridium to obtain a preliminary fermentation broth; detecting the ethanol content of the preliminary fermentation broth and the acetic acid content of the preliminary fermentation broth; determining a second preset ferrous sulfate content of the CaGM growth medium according to the ethanol content of the preliminary fermentation broth and the acetic acid content of the preliminary fermentation broth; The preliminary fermentation broth is subjected to continuous fermentation culture according to the CaGM growth medium containing the second preset ferrous sulfate content and the preset gas source to adjust the ethanol yield and the acetic acid yield in the preliminary fermentation broth to obtain an ethanol product and an acetic acid product, respectively.

5. The method of use according to claim 4, characterized in that: The second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 satisfy the relationship: W1:W2≥25:

100.

6. The method of use according to claim 4 or 5, characterized in that: The second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 satisfy the relationship: W1:W2=25:100; and / or The second preset ferrous sulfate content W1 and the first preset ferrous sulfate content W2 satisfy the relationship: W1:W2=50:

100.

7. The method of use according to claim 4, characterized in that: The acetic acid content W3 of the preliminary fermentation liquid and the ethanol content W4 of the preliminary fermentation liquid satisfy the relationship: W3:W4≥2:

1.

8. The method of use according to claim 4, characterized in that: The weight m3 of the acetic acid product and the weight m4 of the ethanol product satisfy the relationship: m3:m4≤1.1:

1.

9. The method of use according to claim 4, characterized in that: The preset gas source includes a carbon-containing gas and an inert gas, and the volume V3 of the carbon-containing gas and the volume V4 of the inert gas satisfy the relationship: V3:V4≥7:3.

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