A fermentation process for improving the solvent production of Clostridium beijerinckii

Abstract

The present invention relates to a fermentation process for improving the solvent production of Clostridium beijerinckii. The present invention improves the solvent production of Clostridium beijerinckii by controlling the production of byproduct glycoproteins. The specific fermentation process is that in the process of fermenting and producing solvents with Clostridium beijerinckii as a fermentation bacterium, a feed containing a nitrogen source is added to the fermentation liquid during the logarithmic growth phase of Clostridium beijerinckii, and the nitrogen source is ammonium acetate, so that the final concentration of ammonium acetate in the fermentation liquid at the end of the addition is 3.3-12.2 g / L; the fermentation produces solvents and suppresses the large-scale production of byproduct glycoproteins. The present invention adds an ammonium acetate solution during the logarithmic growth phase of Clostridium beijerinckii fermentation without controlling the pH of the fermentation liquid, so that Clostridium beijerinckii can supplement the nitrogen source during the fermentation process without causing excessive nitrogen source, can improve the utilization rate of substrate glucose, can improve the total solvent production, and can also suppress the large-scale production of byproduct glycoproteins.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a fermentation process for improving the solvent yield of Clostridium beijerinckii. Background Art

[0002] Glucose and starch can be fermented by Clostridium beijerinckii to produce acetone, butanol and ethanol (collectively referred to as solvents), a process known as ABE ( a cetone, b utanol and e The fermentation of acetone and butanol by biological fermentation has unique advantages over chemical synthesis. However, the fermentation process also faces some problems. First, the metabolic regulation mechanism of acetone and butanol is unclear. Knocking out the metabolic pathway of acetone does not increase the production of butanol, and it will cause a decrease in the sugar utilization of Clostridium beijerinckii, indicating that the synthesis of acetone and butanol is not in a competitive relationship, and the specific metabolic regulation mechanism has not been clarified. Secondly, when calculating the solvent yield, the yield in the ABE fermentation process is about 0.33, that is, 3 tons of glucose (or starch) can produce 1 ton of solvent. (ABE), but from the analysis of the metabolic pathways currently elucidated, the theoretical yield should be around 0.66, and the actual yield is twice as much as the theoretical yield, indicating that the metabolic pathway of Clostridium beijerinckii for synthesizing solvents is incomplete and needs further analysis; finally, butanol is the main product in the ABE fermentation process. It takes 5 moles of NADH to produce 1 mole of butanol, while 1 mole of glucose is metabolized to acetyl-CoA, which can only produce 2 moles of NADH. This shows that under the condition of carbon source balance, there is no scientific explanation for the energy metabolism balance problem, resulting in the energy metabolism balance problem of Clostridium beijerinckii not being thoroughly analyzed. The existence of the above problems has plagued the large-scale fermentation process of ABE fermentation, and thus the technical route of producing butanol by biological fermentation is at a disadvantage compared with the technical route of synthesizing butanol by chemical method. Therefore, it is urgent to overcome the above problems so that the fermentation of biological solvent generation can be mass-produced and generate economic benefits.

[0003] Chinese patent document CN108690853A (application number 201710218605.1) discloses a method for producing butanol by fermentation, comprising: (1) preparing a fermentation bacteria seed solution, wherein the fermentation bacteria is Clostridium beijerinckii XH0906; (2) preparing a fermentation medium, adding calcium carbonate to the fermentation medium, and inoculating the seed solution prepared in step (1) for fermentation culture to produce butanol. The inventive method produces butanol by fermentation under anaerobic conditions, and has the characteristics of high butanol yield and low organic acid byproducts. Chinese patent document CN101429527A (application number 200810225373.3) discloses a process for producing butanol by intermittent oxygen supply and feeding fermentation of microorganisms. During the exponential growth period of bacterial fermentation for 10-20 hours, 0.1-0.5 vvm of air is introduced, and during the late exponential growth period of 18-0 hours, carbon source and nitrogen source are supplemented at a carbon-nitrogen ratio of 20-100:1, which can significantly increase the butanol concentration. At the end of fermentation, butanol is separated and purified by distillation and vacuum distillation steps to prepare the product. Although there are reports on optimizing the fermentation medium to increase the solvent yield in the prior art, its mechanism and principle have not been explained. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a fermentation process for improving the solvent production of Clostridium beijerinckii. The fermentation process of the present invention improves the utilization of glucose and the production of solvent during the fermentation process of Clostridium beijerinckii, solving the problem of low solvent production during the fermentation process of Clostridium beijerinckii; on the other hand, the present invention also finds another synthetic product of glucose metabolism in addition to the solvent produced by Clostridium beijerinckii, and a method for controlling the product, so as to solve the problem of low solvent yield.

[0005] The technical solution adopted by the invention is as follows:

[0006] Application of a pathway to control the production of by-product glycoproteins in increasing the yield of solvents produced by Clostridium beijerinckii fermentation.

[0007] Preferably according to the present invention, the sugar chain content of the glycoprotein is 20-30%, the protein content is 45-55%, and the two monosaccharide components in the sugar chain are glucose and ethyl glucoside.

[0008] The byproduct glycoprotein of the present invention is preliminarily determined to be a new type of glycoprotein, and its specific structure and properties need to be further studied in detail.

[0009] Preferably, according to the present invention, the fermentation process for increasing the yield of solvent produced by Clostridium beijerinckii fermentation is, in the process of fermenting solvent with Clostridium beijerinckii as the fermentation bacteria, adding a feed containing a nitrogen source to the fermentation broth during the logarithmic growth phase of Clostridium beijerinckii, wherein the nitrogen source is ammonium acetate, so that the final concentration of ammonium acetate in the fermentation broth at the end of the addition is 3.3-12.2 g / L; fermentation produces solvent and inhibits the massive production of by-product glycoprotein.

[0010] More preferably, the final concentration of ammonium acetate in the fermentation broth is 3.3-7.2 g / L; most preferably, 4.4-5.5 g / L.

[0011] In a preferred technical solution of the present invention, the nitrogen source in the feeding feed is ammonium acetate. Other commonly used nitrogen sources in the technical field are also within the protection scope of the present invention.

[0012] In a preferred technical solution of the present invention, the logarithmic growth phase of the Clostridium beijerinckii is 8 to 24 hours after the start of fermentation.

[0013] The solvents produced in the present invention are mainly butanol, acetone and ethanol.

[0014] Further preferably, during the process of fermenting and producing the solvent using Clostridium beijerinckii as the fermentation bacterium, the pH of the fermentation liquid is not controlled and the pH is natural.

[0015] In the present invention, the production of solvent by fermentation using Clostridium beijerinckii as the fermentation bacteria can be carried out according to conventional operations in the art. In a preferred technical solution of the present invention, the steps are as follows:

[0016] (1) Cultivation of Clostridium beijerinckii seed solution: Spread Clostridium beijerinckii on a P2 medium plate and culture it anaerobically at 37° C. for 1 day to obtain an activated strain; inoculate the activated single strain into an anaerobic tube containing P2 medium and culture it anaerobically at 37° C. to obtain a primary seed solution; inoculate the primary seed solution into an anaerobic bottle containing P2 medium at an inoculation amount of 5% and culture it anaerobically at 37° C. to obtain a secondary seed solution;

[0017] (2) Fermentation: Prepare P2 culture medium and place it in a fermenter. During the fermentation process, the pH of the fermentation liquid is natural. The secondary seed liquid of step (1) is inoculated into the fermenter at an inoculation amount of 1-10%. The fermentation temperature is maintained at 36° C. and the rotation speed is maintained at 250 rpm. The initial glucose fermentation concentration is 40 g / L. Prepare and feed glucose solution so that the glucose concentration during the fermentation process is not less than 20 g / L, and ferment to produce solvent.

[0018] Further preferably, the components of the P2 medium are as follows:

[0019] Potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, ammonium acetate 2.2g / L, glucose 40g / L, yeast powder 1g / L, ferrous sulfate heptahydrate 0.01g / L, magnesium sulfate heptahydrate 0.2g / L, manganese sulfate monohydrate 0.01g / L, sodium chloride 0.01g / L, p-aminobenzoic acid 1mg / L, vitamin B1 1mg / L, biotin 0.1g / L, solvent water, natural pH is 6.5.

[0020] In a preferred technical solution of the present invention, the fermentation process for increasing the solvent production of Clostridium beijerinckii comprises the following specific steps:

[0021] (1) Cultivation of Clostridium beijerinckii seed solution: Spread and culture Clostridium beijerinckii on a P2 medium plate, and culture it anaerobically at 37° C. for 1 day to obtain an activated strain; inoculate the activated single strain into an anaerobic tube containing P2 medium, and culture it anaerobically at 37° C. to obtain a primary seed solution; inoculate the primary seed solution into an anaerobic bottle containing P2 medium at an inoculation amount of 5%, and culture it anaerobically at 37° C. to obtain a secondary seed solution;

[0022] (2) Fermentation: Prepare P2 culture medium and place it in a fermenter. During the fermentation process, the pH of the fermentation liquid is natural. The secondary seed liquid of step (1) is inoculated into the fermenter at an inoculation amount of 1-10%. The fermentation temperature is maintained at 36° C. and the rotation speed is maintained at 250 rpm. The initial glucose fermentation concentration is 40 g / L. Prepare 400 g / L glucose solution and feed the glucose solution so that the glucose concentration during the fermentation process is not less than 20 g / L. At the same time, prepare ammonium acetate solution and feed the ammonium acetate solution into the fermenter at a uniform rate during the logarithmic growth phase of Clostridium beijerinckii fermentation so that the final concentration of ammonium acetate in the fermentation system is 3.3-12.2 g / L at the end of the feed. Fermentation produces solvent and inhibits the massive production of by-product glycoprotein.

[0023] Technical features and beneficial effects of the present invention:

[0024] The present invention changes the fermentation conditions of the fermentation broth and finds that the actual sugar consumption of Clostridium beijerinckii differs greatly from the theoretical sugar consumption. It is preliminarily determined that a new substance is produced in the fermentation process. Preliminary verification shows that the new substance is a glycoprotein, the sugar chain content of the glycoprotein is 20-30%, the protein content is 45-55%, and the two monosaccharide components in the sugar chain are glucose and ethyl glucoside; and the production of the glycoprotein is closely related to the pH value and nitrogen source content of the fermentation broth, and is in a substrate competition relationship with the production of the solvent.

[0025] The present invention found that by changing the pH and nitrogen source of the fermentation broth of Clostridium beijerinckii, the metabolic process of the fermentation strain can be changed, resulting in changes in sugar utilization and product composition and concentration. On this basis, the inventor optimized the fermentation process of Clostridium beijerinckii, and the pH of the fermentation broth was natural. During the logarithmic growth period of Clostridium beijerinckii fermentation, an ammonium acetate solution was added at a uniform rate so that the final concentration of ammonium acetate in the fermentation broth was 3.3 to 12.2 g / L at the end of the addition. This allows Clostridium beijerinckii to supplement the nitrogen source without causing an excess of nitrogen source during the fermentation process, improve the utilization rate of the substrate glucose, increase the total solvent production, and inhibit the large-scale production of byproduct glycoproteins. When the concentration of the added ammonium acetate was 2.2 g / L, that is, the final concentration of ammonium acetate in the fermentation broth was 4.4 g / L, the maximum biomass OD of Clostridium beijerinckii was 2.2 g / L. 600 It reached 23, which was 35% higher than that of non-nitrogen supplementation culture. The stable growth period of Clostridium beijerinckii was extended, and the total solvent production reached 28g / L, which was 51% higher than that of non-nitrogen supplementation culture. The sugar utilization reached 82g / L, which was 71% higher than that of non-nitrogen supplementation culture. The production of by-product glycoprotein was only 0.34g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a diagram of the central metabolism of Clostridium beijerinckii using glucose as raw material.

[0027] Figure 2 This is a photo of the glycoprotein precipitate obtained after adding two volumes of ethanol to the fermentation supernatant.

[0028] Figure 3 The monosaccharide composition of the sugar chain in glycoprotein. M1, glucose; M2, sucrose; 1, acid-hydrolyzed heteropolysaccharide sample (sulfuric acid staining); 2, acid-hydrolyzed heteropolysaccharide sample (iodine staining).

[0029] Figure 4 This is the mass spectrum of ethyl glucoside.

[0030] Figure 5 This is the growth curve of Clostridium beijerinckii. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with the accompanying drawings and examples, but the protection scope of the present invention is not limited thereto. The reagents and raw materials involved in the examples, unless otherwise specified, are all common commercially available products; the operating steps and methods involved in the examples, unless otherwise specified, are all conventional operations in the art.

[0032] Fermentation mode of product changes of Clostridium beijerinckii: pH value and nitrogen source can change the fermentation metabolism process of Clostridium beijerinckii, resulting in changes in sugar utilization and product composition and concentration. In the fermentation tank, glucose concentration in the tank was not less than 20g / L by adding glucose, and pH value was kept constant at 6 and 7 by 4M potassium hydroxide, respectively, and fermentation was carried out. In addition, conventional batch feeding fermentation was carried out without controlling pH value. After the fermentation, conventional traditional product generation under three fermentation conditions was measured, including acetic acid, butyric acid, lactic acid, ethanol, acetone and butanol. On this basis, under three pH control strategies, 4M potassium hydroxide was replaced with 50% ammonia water, and the initial ammonium acetate concentration was adjusted from 2.2g / L to 4.4g / L. Fermentation was carried out under the condition of supplementing nitrogen source. After the fermentation, conventional traditional product generation under three fermentation conditions was measured, including acetic acid, butyric acid, lactic acid, ethanol, acetone and butanol.

[0033] Calculate the relationship between sugar consumption of Clostridium beijerinckii and glucose required for product production: During the fermentation of Clostridium beijerinckii, the glucose consumption can be calculated by measuring the glucose content before and after fermentation, and the product metabolic pathway ( Figure 1 ), calculate the amount of glucose required to produce traditional products, and preliminarily determine the distribution of glucose metabolism through the difference between the glucose consumption and the amount of glucose required to produce traditional products under different fermentation conditions. The corresponding relationship between traditional products and glucose is shown in Table 1:

[0034] Table 1. Molar correspondence between glucose and traditional products

[0035] Glucose (mol) Product (mol) 1. Glucose 1Butyric acid 1. Glucose 1-Butanol 1. Glucose 2 lactic acid 1. Glucose 2. Ethanol 1. Glucose 2-Acetic acid 1. Glucose 1. Acetone

[0036] Discovery and analysis of new glycoproteins: By calculating the relationship between glucose consumption and traditional product synthesis, it was found that Clostridium beijerinckii synthesized substances other than traditional products during the fermentation process with nitrogen supplementation and neutral pH. These substances were present in the fermentation broth. After the fermentation, the cells were removed by centrifugation, and the supernatant was transferred to a beaker. When 2 times the volume of anhydrous ethanol was added to the beaker, a large amount of precipitation was found ( Figure 2 ), and it was preliminarily determined that the substance was a glycoprotein. The supernatant was removed by centrifugation, and the precipitate was freeze-dried to measure the dry weight. The protein content in the glycoprotein was measured by the BCA method, and the sugar content was determined by the phenol-sulfuric acid method. The glycoprotein was hydrolyzed with 0.3M HCl at 100°C for 12 hours. After concentration, TLC analysis was performed, and it was found that the sugar in the glycoprotein contained two components. The molecular weight of the product was analyzed by liquid chromatography-mass spectrometry, and it was speculated that the two components were glucose and ethyl glucose ( Figure 3 ).

[0037] Improving glucose utilization and solvent production of Clostridium beijerinckii: Through fermentation condition setting and product analysis, it was found that Clostridium beijerinckii would utilize a large amount of glucose when the nitrogen source was supplemented and the pH was neutral, but most of the glucose was used to synthesize a glycoprotein of unknown function, and the solvent production was low. The results of batch fed fermentation without controlling the pH value showed that Clostridium beijerinckii had a high growth rate and solvent yield, but the glucose consumption was low and Clostridium beijerinckii quickly autolyzed when the growth reached the maximum. Therefore, an ammonium acetate solution was prepared and added during the logarithmic growth phase after the start of fermentation, starting at 8 hours. The flow rate was designed so that the solution was added within a period of 8 to 24 hours, so that the concentration of ammonium acetate added to the fermentation medium was 2.2 g / L. By controlling the method of adding nitrogen source, Clostridium beijerinckii can supplement nitrogen source during the fermentation process without causing excessive nitrogen source, thereby increasing the OD of the maximum biomass of Clostridium beijerinckii. 600 It reached 23, which was 35% higher than that of non-nitrogen supplementation culture, and the total solvent production reached 28g / L, which was 51% higher than that of non-nitrogen supplementation culture, and the sugar utilization reached 82g / L, which was 71% higher than that of non-nitrogen supplementation culture.

[0038] The Clostridium beijerinckii used in the embodiment is Clostridium beijerinckii NCIMB 8052, which is an internationally used Clostridium beijerinckii strain, which can be purchased from the National Center for Industry, Marine and Food Preservation (NCIMB) of the United Kingdom. Clostridium beijerinckii from other sources is also suitable for the present invention.

[0039] The components of P2 medium are as follows:

[0040] Potassium dihydrogen phosphate 0.5g / L, dipotassium hydrogen phosphate 0.5g / L, ammonium acetate 2.2g / L, glucose 40g / L, yeast powder 1g / L, ferrous sulfate heptahydrate 0.01g / L, magnesium sulfate heptahydrate 0.2g / L, manganese sulfate monohydrate 0.01g / L, sodium chloride 0.01g / L, p-aminobenzoic acid 1mg / L, vitamin B1 1mg / L, biotin 0.1g / L, solvent water, initial pH is 6.5.

[0041] Example 1: Analysis of glucose consumption and traditional product production of Clostridium beijerinckii under different fermentation conditions:

[0042] (1) Fermentation of Clostridium beijerinckii without nitrogen supplementation

[0043] 1) Spread Clostridium beijerinckii on a P2 medium plate, and culture it anaerobically at 37° C. for 1 day to obtain an activated strain; inoculate the activated single strain into an anaerobic tube containing P2 medium, and culture it anaerobically at 37° C. to obtain a primary seed solution; inoculate the primary seed solution into an anaerobic bottle containing P2 medium at an inoculation amount of 5%, and culture it anaerobically at 37° C. to obtain a secondary seed solution;

[0044] 2) preparing P2 culture medium and placing it in a fermenter, setting different fermentation conditions in the fermenter, controlling the pH value of the fermentation liquid to 7 or 6, or not controlling the pH value of the fermentation liquid, wherein the pH value of the fermentation liquid is controlled by a 4M potassium hydroxide solution; inoculating the secondary seed liquid of step 1) in the fermenter, the inoculation amount is 5%, the fermentation temperature is maintained at 36° C., the rotation speed is maintained at 250 rpm, the initial glucose fermentation concentration is 40 g / L, the glucose concentration is measured every six hours, and a 400 g / L glucose solution is prepared, and the glucose concentration during the fermentation process is not less than 20 g / L by adding it through a peristaltic pump; and the fermentation is terminated after 72 hours.

[0045] The various parameters were determined as follows:

[0046] Glucose concentration: The glucose concentration during the fermentation process was detected using an SBA-40D biosensor analyzer;

[0047] Bacterial growth: The absorbance value (OD) was measured at 600 nm using a spectrophotometer. 600 ) reflects the growth status of bacteria. Deionized water is used as a blank control. 1 mL of fermentation liquid is taken into a cuvette for determination. If the measured value is greater than 0.5, the fermentation liquid is diluted to a credible value range (0.2-0.5) and then measured again. The measured value is multiplied by the dilution multiple and recorded;

[0048] Acetic acid, butyric acid and lactic acid in the fermentation broth: high performance liquid chromatography (Agilent 1260) was used with a parallax refractometer. The fermentation broth was centrifuged at 12000 rpm for 2 min, and the supernatant was taken and diluted to an appropriate concentration. The supernatant was filtered into a sample injection bottle using a 0.22 μm filter membrane. The chromatographic column was a HiPlex H column, with a mobile phase of 0.5 mM H2SO4, a flow rate of 0.6 mL / min, and a column temperature of 55 °C.

[0049] Butanol, acetone and ethanol in the fermentation broth were determined by gas chromatography (Agilent 7890B). The fermentation broth was appropriately diluted and extracted with three volumes of ethyl acetate. The upper extract was transferred to a special gas phase bottle. The chromatographic column was HP-INNOWAX, the carrier gas was N2, and isobutanol was used as the internal standard.

[0050] The concentrations of traditional products in the fermentation broth at the end of fermentation under different fermentation conditions are shown in Table 2.

[0051] Table 2. Concentrations of traditional products at the end of fermentation under different fermentation conditions

[0052]

[0053]

[0054] (2) Fermentation of Clostridium beijerinckii under the condition of supplementing nitrogen source:

[0055] 1) Spread Clostridium beijerinckii on a P2 medium plate, and culture it anaerobically at 37° C. for 1 day to obtain an activated strain; inoculate the activated single strain into an anaerobic tube containing P2 medium, and culture it anaerobically at 37° C. to obtain a primary seed solution; inoculate the primary seed solution into an anaerobic bottle containing P2 medium at an inoculation amount of 5%, and culture it anaerobically at 37° C. to obtain a secondary seed solution;

[0056] 2) Prepare P2 medium, and directly supplement nitrogen source in P2 medium so that ammonium acetate concentration is adjusted from 2.2g / L to 4.4g / L, place in a fermenter, set different fermentation conditions in the fermenter, control the pH value of the fermentation liquid to 7 or 6, or do not control the pH value of the fermentation liquid, wherein the pH value of the fermentation liquid is controlled by 50% ammonia water (a mixture of Chinese medicine ammonium hydroxide and deionized water in a ratio of 1:1 (v / v), which not only adjusts the pH but also further supplements the nitrogen source); inoculate the secondary seed liquid of step 1) in the fermenter, the inoculation amount is 5%, the fermentation temperature is maintained at 36°C, the rotation speed is maintained at 250 rpm, the initial glucose fermentation concentration is 40g / L, the glucose concentration is measured every six hours, a 400g / L glucose solution is prepared, and the glucose concentration during the fermentation process is not less than 20g / L by adding it through a peristaltic pump; fermentation ends after 72 hours.

[0057] The above method was used to determine the concentration of traditional products in the fermentation broth at the end of fermentation. The results are shown in Table 3.

[0058] Table 3. Concentration of traditional products at the end of fermentation under different fermentation conditions

[0059]

[0060] It can be seen from the data in Tables 2 and 3 that changes in pH value and nitrogen source will lead to changes in the fermentation metabolism process of Clostridium beijerinckii, resulting in changes in the production concentration of traditional products, indicating that both pH value and nitrogen source can affect product composition and product concentration.

[0061] 3) Result analysis

[0062] According to the central metabolic pathway of Clostridium beijerinckii ( Figure 1 ) and the molar correspondence between glucose and traditional products in the metabolic pathway of Clostridium beijerinckii (Table 1), combined with the fermentation conditions under different fermentation conditions, the glucose consumption and the amount of glucose required for the synthesis of traditional products were calculated, and the results are shown in Table 4.

[0063] Table 4. Glucose consumption under different fermentation conditions and the amount of glucose required for the synthesis of traditional products

[0064]

[0065] It can be seen from the data in Table 4 that changes in pH and nitrogen source will lead to changes in the fermentation metabolism process of Clostridium beijerinckii, resulting in changes in sugar utilization and product concentration. Among them, under the fermentation conditions without supplementing nitrogen source, changing the pH of the fermentation broth, although the glucose consumption in the fermentation broth will increase, the proportion of glucose used to generate traditional products will be significantly reduced, that is, the solvent yield is reduced, and the solvent production is also reduced. Under the fermentation conditions of supplementing nitrogen source, compared with the fermentation results without supplementing nitrogen source, the glucose consumption increased, but the solvent yield decreased, especially under the conditions of supplementing nitrogen source in the fermentation broth and pH7, the difference between the glucose consumption and the amount of glucose required to produce traditional products was the largest; and when the nitrogen source was supplemented under natural pH conditions to produce solvents, the solvent yield was most significantly reduced compared with the fermentation results without supplementing nitrogen source, indicating that the addition of nitrogen source may also affect the production of solvents. A sudden increase in nitrogen source is not conducive to the production and accumulation of solvents. In summary, it is preliminarily judged that a new product may be produced in this fermentation process.

[0066] In addition, the growth of bacteria under different fermentation conditions is shown in Table 5.

[0067] Table 5. Maximum biomass (OD) under different fermentation conditions 600 Value)

[0068] Fermentation conditions <![CDATA[Maximum biomass (OD 600 )]]> Fermentation time to reach maximum biomass (h) pH 7, nitrogen supplement 6.2 16 pH7 7.0 18 pH 6, supplemented with nitrogen source 2.5 9 pH6 13 12 Natural pH, supplemented nitrogen source 16 18 Natural pH 17 16

[0069] It can be seen from the data in Table 5 that changes in pH and nitrogen source will also affect the growth of Clostridium beijerinckii, especially when the pH value is controlled during fermentation, the biomass is significantly lower than that of fermentation without pH control. However, when the biomass reaches the maximum under fermentation conditions without nitrogen source supplementation and natural pH, Clostridium beijerinckii quickly autolyzes.

[0070] Example 2: Preliminary determination of new products

[0071] 1) Quality measurement

[0072] Take 1L of fermentation liquid obtained under the conditions of nitrogen supplementation and pH 7, centrifuge at 5500rpm for 30min, discard the precipitate, take the supernatant, add 2 times the volume of anhydrous ethanol, stir evenly and let it stand, a large amount of precipitate will be obtained ( Figure 2 ), and it was preliminarily determined that the precipitate was a glycoprotein. Take a 50mL centrifuge tube, weigh it, record the weight, collect the glycoprotein precipitate and transfer it to a 50mL centrifuge tube, place it in a vacuum freeze dryer overnight, remove water and ethanol, weigh it, and subtract the weight of the empty tube to obtain the dry weight of the precipitate, which is the mass of glycoprotein produced in 1L fermentation broth. According to this method, the glycoprotein yield under different fermentation conditions was measured (Table 6).

[0073] Table 6. Glycoprotein yield under different fermentation conditions

[0074]

[0075]

[0076] 2) Determination of total sugar content in glycoprotein by phenol-sulfuric acid method: With glucose as the standard, accurately prepare 0.4546g / L glucose solution, accurately pipette 40, 60, 100, 120, 160, 200μL into a glass tube, then add the corresponding volume of deionized water to 2mL, shake well to obtain glucose solutions of different concentrations, take 2mL deionized water sample as blank control, add 1mL 5% phenol solution to each tube, shake well and immediately add 5mL concentrated sulfuric acid, shake well again and let stand at room temperature for 30min; measure the absorbance of each solution at 485nm; dilute the glycoprotein sample after 0.3M hydrochloric acid hydrolysis into samples of different concentrations (10×, 20×, 30×, 40×) for preliminary experiment, still use deionized water as blank control. Appropriate dilution makes the absorbance measurement result between 0.2-0.8, and the preliminary experiment results show that the reading result is between 0.2-0.8 after the sample is diluted 20 times. Pipette 2mL of sample diluted to appropriate concentration into a dry glass tube, add 1mL of 5% phenol solution, shake well and immediately add 5mL of concentrated sulfuric acid, shake well again and let stand at room temperature for 30min. Three parallel samples are made at the same time, and 2mL of deionized water sample is still used as blank control. The absorbance of each solution is measured at 485nm; the sugar chain content in glycoprotein is calculated to be 20-30%.

[0077] 3) Determination of protein content in fermentation broth by BCA method: The protein in the fermentation broth was first precipitated and separated, and then dissolved for determination. 15 mL of fermentation broth and 15 mL of P2 medium were taken as controls, 30 mL of ice ethanol pre-cooled in a -20 ° C refrigerator was added, mixed and placed in a -20 ° C refrigerator overnight; centrifuged at 4 ° C, 5000 rpm for 30 min, the supernatant was discarded, the residual ethanol was evaporated, and 5 mL of deionized water was added to dissolve the protein precipitate. Using P2 medium as a control, the protein concentration was determined using the BCA protein quantification kit. 200 μL of A solution, 4 μL of B solution and 25 μL of fermentation broth / P2 medium were mixed, incubated at 37 ° C for 30 min, and 200 μL was taken to measure the light absorption intensity at 562 nm of the microplate reader. It was measured that protein accounted for 45-55% of the total glycoprotein mass.

[0078] 4) Determination of sugar chain monosaccharide components in glycoproteins by TLC and high-resolution mass spectrometry: Weigh an appropriate amount of freeze-dried glycoprotein precipitate into an anaerobic glass tube, add 10 mL of 0.3 M hydrochloric acid, seal with a rubber stopper, place in a 100°C incubator for acid hydrolysis, take out after 2 days, and the precipitate is completely acid-hydrolyzed. At the same time, prepare 10 g / L glucose and 10 g / L sucrose as controls. Take a TLC glass plate of appropriate size, separate it from the middle with a pencil line, run two controls and one sample on the left, and run three samples on the right, load 0.5μL of the control and 6μL of the sample, put the glass plate with the sample into the developing tank with the developing agent, and when the developing agent runs to 90% of the glass plate, take out the glass plate and blow it dry, cut the glass plate along the line drawn by the pencil, dye the glass plate on the left with sulfuric acid, pour the color developer evenly on the dried glass plate, blow it dry again, and place it in a 100℃ oven for 5-8min to develop the color; put the glass plate on the right directly into the specimen tank and dye it with iodine, and place it in a 100℃ oven for 5-8min and observe it directly. The results are as follows: Figure 3 .

[0079] Using the colored glass plate on the left as a position reference, scrape off the two points separated by chromatography on the right glass plate, collect them in a centrifuge tube, and add deionized water to dissolve; after dissolving overnight, separate the precipitate from the supernatant, and the monosaccharide components are present in the supernatant. Filter the supernatant using a 0.22μm filter membrane, concentrate the supernatant with a vacuum concentrator, and run TLC again to verify whether the two monosaccharide components are successfully separated.

[0080] After successful separation, the sample was sent for high-resolution mass spectrometry analysis. The sample was analyzed by mass spectrometry using a high-resolution mass spectrometer (Bruke Maxis UHRTOF). Mass spectrometry conditions: point spray ion (ESI) source; negative ion mode; capillary voltage: 2200V; drying heater temperature: 180℃; drying gas flow rate: 6L / min. Figure 4 ) to determine the molecular weight, and combined with the verification results of the TLC plate, it was inferred that the two monosaccharide components in the sugar chain were glucose and ethyl glucoside.

[0081] Example 3: Fermentation process for increasing solvent production of Clostridium beijerinckii

[0082] The analysis results of Example 1 show that when the fermentation broth is not supplemented with nitrogen source and the pH is natural, the solvent yield is the highest, but the glucose consumption is the lowest, resulting in the lowest utilization rate of the substrate glucose; while when the fermentation broth is supplemented with nitrogen source and the pH is 7, the glucose consumption is the highest, which improves the utilization rate of the substrate glucose, but the solvent yield is the lowest. On this basis, the present invention optimizes a fermentation process for improving the solvent production of Clostridium beijerinckii, which can not only improve the utilization rate of glucose, but also increase the solvent production.

[0083] 1) Optimization of supplementary nitrogen source concentration

[0084] The nitrogen source was supplemented by feeding: a P2 culture medium was prepared and placed in a fermenter. During the fermentation process, the pH of the fermentation liquid was natural. The secondary seed liquid of Clostridium beijerinckii was inoculated in the fermenter with an inoculation amount of 5%. The preparation method of the secondary seed liquid was as described in Example 1. The fermentation temperature was maintained at 36°C, the rotation speed was maintained at 250 rpm, the initial glucose fermentation concentration was 40 g / L, the glucose concentration was measured every six hours, a 400 g / L glucose solution was prepared, and the glucose concentration during the fermentation process was not less than 20 g / L by adding a peristaltic pump; an ammonium acetate solution was prepared. During the logarithmic growth phase of the fermentation of Clostridium beijerinckii (time period: 8-24 h), the flow rate of the peristaltic pump was controlled, and the ammonium acetate solution was uniformly added to the fermenter, so that the increased ammonium acetate concentrations in the fermentation system were 1.1 g / L, 2.2 g / L, 3.3 g / L, 5 g / L, and 10 g / L, respectively.

[0085] Table 7. Fermentation results of different concentrations of ammonium acetate

[0086]

[0087] From the data in Table 7, it can be seen that as the concentration of added ammonium acetate increases, the consumption of glucose in the fermentation broth by Clostridium beijerinckii increases, and the total solvent production is also higher than that of the control group without nitrogen source addition. However, when the concentration of added ammonium acetate is too high, the content of its byproduct glycoprotein will also be too high, which is not conducive to the flow of substrate glucose to the direction of solvent synthesis. Therefore, when the increased ammonium acetate concentration is 1.1-10 g / L, it can not only improve the utilization rate of substrate glucose, but also improve the total solvent production, and can also inhibit the large-scale production of byproduct glycoprotein, especially when the increased ammonium acetate concentration is 2.2 g / L, the total solvent production is the highest.

[0088] 2) Optimization of the feeding time period

[0089] A P2 culture medium is prepared and placed in a fermenter. During the fermentation process, the pH of the fermentation liquid is natural. A secondary seed liquid of Clostridium beijerinckii is inoculated in the fermenter at an inoculation amount of 5%. The preparation method of the secondary seed liquid is referred to Example 1. The fermentation temperature is maintained at 36° C., the rotation speed is maintained at 250 rpm, the initial glucose fermentation concentration is 40 g / L, the glucose concentration is measured every six hours, a 400 g / L glucose solution is prepared, and the glucose concentration during the fermentation process is not less than 20 g / L by adding it through a peristaltic pump; an ammonium acetate solution is prepared, and the flow rate of the peristaltic pump is controlled at the initial stage (0-16 h) of the fermentation of Clostridium beijerinckii, the logarithmic growth phase (time period: 8-24 h), or the growth stationary phase, and the ammonium acetate solution is uniformly added to the fermenter, so that the increased ammonium acetate concentration in the fermentation system at the end of the flow addition is 2.2 g / L, and the final concentration of ammonium acetate in the fermentation system is 4.4 g / L.

[0090] Table 8. Fermentation results at different feeding time periods

[0091]

[0092] It can be seen from the data in Table 8 that only by adding ammonium acetate at a uniform rate during the logarithmic growth phase can the total solvent output be increased while significantly increasing the substrate glucose consumption and inhibiting the production of a large amount of by-product glycoproteins.

[0093] Under the fermentation conditions in which ammonium acetate was added during the logarithmic growth phase to increase the concentration of ammonium acetate in the fermentation system to 2.2 g / L, the change in biomass in the fermentation system was measured. The results are as follows: Figure 5 The results showed that under the condition of flowing ammonium acetate, the biomass in the fermentation system increased significantly, and OD 600 The OD value was 23, which was significantly higher than that of the control group without ammonium acetate addition. 600 =17), and significantly prolonged the stable growth period of Clostridium beijerinckii, which is beneficial to the production of solvents by microbial fermentation.

Claims

1. Application of a method for controlling the production of byproduct glycoprotein in increasing the yield of solvent produced by fermentation of Clostridium beijerinckii. The fermentation process for increasing the yield of solvent produced by fermentation of Clostridium beijerinckii comprises the following steps: in the process of fermentation of solvent produced by Clostridium beijerinckii as a fermentation bacterium, a feed containing a nitrogen source is added to the fermentation broth during the logarithmic growth phase of Clostridium beijerinckii, wherein the nitrogen source is ammonium acetate, so that the final concentration of ammonium acetate in the fermentation broth is 3.3-12.2 g / L at the end of the addition; the fermentation produces solvent and inhibits the massive production of byproduct glycoprotein; the solvent is mainly butanol, acetone and ethanol; the sugar chain content of the glycoprotein is 20-30%, the protein content is 45-55%, and the two monosaccharide components in the sugar chain are glucose and ethyl glucoside; the pH of the fermentation broth is not controlled during the process of fermentation of solvent produced by Clostridium beijerinckii as a fermentation bacterium, and the pH is natural.

2. The use according to claim 1, characterized in that The final concentration of ammonium acetate in the fermentation broth is 3.3-7.2 g / L.

3. The use according to claim 1, characterized in that The final concentration of ammonium acetate in the fermentation broth is 4.4-5.5 g / L.

4. The use according to claim 1, characterized in that The logarithmic growth phase of the Clostridium beijerinckii is 8 to 24 hours after the start of fermentation.

5. The use according to claim 1, characterized in that The steps of fermenting and producing the solvent using Clostridium beijerinckii as the fermentation bacteria are as follows: (1) Cultivation of Clostridium beijerinckii seed solution: Spread Clostridium beijerinckii on a P2 medium plate and culture it anaerobically at 37°C for 1 day to obtain an activated strain; inoculate the activated single strain into an anaerobic tube containing P2 medium and culture it anaerobically at 37°C to obtain a first-level seed solution; inoculate the first-level seed solution into an anaerobic bottle containing P2 medium at an inoculation rate of 5% and culture it anaerobically at 37°C to obtain a second-level seed solution; (2) Fermentation: Prepare P2 culture medium and place it in a fermenter. During the fermentation process, the pH of the fermentation liquid is natural. The secondary seed liquid of step (1) is inoculated into the fermenter at an inoculation amount of 1-10%. The fermentation temperature is maintained at 36°C and the rotation speed is maintained at 250 rpm. The initial glucose fermentation concentration is 40 g / L. Prepare and feed glucose solution so that the glucose concentration during the fermentation process is not less than 20 g / L, and ferment to produce solvent.

6. The use according to claim 5, characterized in that The components of the P2 medium are as follows: Potassium dihydrogen phosphate 0.5 g / L, potassium dihydrogen phosphate 0.5 g / L, ammonium acetate 2.2 g / L, glucose 40 g / L, yeast powder 1 g / L, ferrous sulfate heptahydrate 0.01 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate monohydrate 0.01 g / L, sodium chloride 0.01g / L, p-aminobenzoic acid 1 mg / L, vitamin B1 1 mg / L, biotin 0.1 g / L, solvent water, pH natural, 6.5.

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