CN118222647A - Method for improving lysine fermentation yield and reducing by-product acetic acid - Google Patents
Method for improving lysine fermentation yield and reducing by-product acetic acid Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 177
- 230000004151 fermentation Effects 0.000 title claims abstract description 177
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 title claims abstract description 126
- 238000000034 method Methods 0.000 title claims abstract description 34
- 239000004472 Lysine Substances 0.000 title claims abstract description 33
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 239000006227 byproduct Substances 0.000 title claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 37
- 238000004519 manufacturing process Methods 0.000 claims abstract description 37
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 26
- 235000015097 nutrients Nutrition 0.000 claims abstract description 20
- 241000588724 Escherichia coli Species 0.000 claims abstract description 19
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 13
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- 229910052799 carbon Inorganic materials 0.000 claims abstract description 12
- 239000000126 substance Substances 0.000 claims abstract description 10
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 33
- 239000007788 liquid Substances 0.000 claims description 29
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 26
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- 239000008103 glucose Substances 0.000 claims description 23
- 239000002609 medium Substances 0.000 claims description 19
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 17
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 17
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 15
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 13
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 13
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 13
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 13
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 12
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- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 12
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- 235000005822 corn Nutrition 0.000 claims description 12
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- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 9
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- 239000000413 hydrolysate Substances 0.000 claims description 7
- 238000009423 ventilation Methods 0.000 claims description 7
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
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- 239000002253 acid Substances 0.000 abstract description 25
- 239000000463 material Substances 0.000 abstract description 11
- 230000003204 osmotic effect Effects 0.000 abstract description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 32
- 235000018977 lysine Nutrition 0.000 description 24
- 238000002474 experimental method Methods 0.000 description 18
- 230000012010 growth Effects 0.000 description 16
- 239000000758 substrate Substances 0.000 description 14
- 241001052560 Thallis Species 0.000 description 12
- 230000000694 effects Effects 0.000 description 10
- 235000016709 nutrition Nutrition 0.000 description 10
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- 229910021529 ammonia Inorganic materials 0.000 description 8
- 230000008569 process Effects 0.000 description 7
- 235000019764 Soybean Meal Nutrition 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 150000001413 amino acids Chemical class 0.000 description 6
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- 235000019766 L-Lysine Nutrition 0.000 description 4
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- 125000001477 organic nitrogen group Chemical group 0.000 description 2
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- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
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- 238000010923 batch production Methods 0.000 description 1
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention provides a method for improving lysine fermentation yield and reducing byproduct acetic acid. The method utilizes escherichia coli to ferment and produce lysine, on the basis of examining the adding concentration and adding time of a culture medium, 15% -45% of nutrient substances (except carbon sources and nitrogen sources) of a fermentation base material are transferred to fed-batch in the middle of fermentation, acetic acid under the strategy is reduced to 0.1g/L from 1.0g/L, and the acid production by fermentation reaches 200g/L, which is 107% of the original fermentation process. By selecting proper initial concentration and reasonable feeding time, the osmotic pressure of a fermentation system in the earlier stage of fermentation can be reduced, and high yield, high yield and high production strength are realized.
Description
Technical Field
The invention relates to the field of fermentation, in particular to a method for improving the fermentation yield of lysine and reducing the byproduct acetic acid.
Background
Coli is widely used as one of the most important industrial production strains for the production of various biological products, such as proteins and amino acids. In order to increase the yield or concentration of the desired product, high-density cell culture is often performed using glucose as a main carbon source. However, E.coli accumulates a large amount of by-product acetic acid even when fermentation is performed under aerobic conditions. As a byproduct of aerobic fermentation of E.coli, acetic acid exists in the fermentation broth as acetate under neutral pH conditions. Accumulation of acetic acid not only limits the growth of cells, but also inhibits the synthesis of the product, and the formation of acetic acid also causes loss of carbon source and thus decreases the substrate conversion rate. It is reported that acetic acid content exceeding 1g/L in fermentation broth can produce stronger inhibition effect on the growth of colibacillus thallus and the synthesis of target products, reduce the yield of the target products, and acetic acid mainly begins to accumulate in the logarithmic phase.
One of the key problems of the fermentation culture of E.coli is how to reduce the production of organic acids such as acetic acid caused by high concentration of matrix, which are caused by saturation of TCA cycle and electron transfer chain, and these organic acids penetrate into E.coli cell membrane, decrease intracellular pH, affect the action of enzyme system, interfere with energy metabolism process, thereby hindering cell growth and product synthesis of E.coli. Therefore, in the fermentation process of the escherichia coli, the nutrient source is rich in the early stage of the culture process, and the method is suitable for the growth of thalli; in the middle and later stages of culture, along with the continuous accumulation of metabolic byproducts, the inhibition effect of the byproducts on the growth of the thalli is gradually dominant, the concentration of the thalli is prevented from being increased, and the acid production is influenced. The fed-batch culture technology is adopted, the feeding rate of nutrient substances is controlled by a certain strategy, and the substrate concentration in the culture medium is maintained at a lower level, so that the mass accumulation of metabolites of escherichia coli is facilitated.
In the lysine fermentation stage, the thallus absorbs the nutrients first, and the substrate consumption reaches a small peak at about 8 hours. This is in preparation for the propagation of the cells themselves. The bacterial growth enters the vigorous period about 12 hours and reaches about 18 hours, the bacterial growth is basically completed, but the substrate consumption enters a new peak, which is prepared for acid production, the acid production reaches the fastest in the stable period, the substrate consumption rate is the peak period about 18-24 hours, and nutrient substances should be fed from 18-24 hours in a fed-batch mode, so that the consumed substrate is supplemented. The lysine fermentation process has intermediate discharging, and nutrition is supplemented in a process by feeding nutrition, so that the activity of the thallus in the middle and later stages is maintained. Because the fed-batch fermentation has the catabolite repression effect of reducing the substrate inhibition and relieving the substrate, the formation of inhibitory byproducts is reduced, and partial nutrient components of the bottom materials are fed-batch fed, the nutrition utilization is effectively improved, and the generation of the inhibitory byproducts, namely acetic acid, is effectively reduced.
Disclosure of Invention
The invention aims to provide a method for improving the fermentation yield of lysine and reducing the byproduct acetic acid.
The invention is characterized in that: the method utilizes escherichia coli to ferment and produce lysine, and on the basis of examining the adding concentration and adding time of a culture medium, 15% -45% (preferably 30%) of nutrient substances (except a carbon source and a nitrogen source) of a fermentation base material are transferred to the fed-batch process in the middle of fermentation, acetic acid under the strategy is reduced to 0.1g/L from 1.0g/L, and the acid production by fermentation reaches 200g/L, which is 107% of the original fermentation process. By selecting proper initial concentration and reasonable feeding time, the osmotic pressure of a fermentation system in the earlier stage of fermentation can be reduced, and high yield, high yield and high production strength are realized.
In order to achieve the object of the present invention, the present invention provides a method for improving lysine fermentation yield and reducing acetic acid as a byproduct, the method comprising: in the initial fermentation culture medium, other nutrient substances in the fermentation culture medium except a carbon source and a nitrogen source use C 0 concentration, feeding is started in the middle period of fermentation, and other nutrient substances in the fermentation culture medium with the concentration of (100% -C 0) are fed into the fermentation system.
Further, C 0 is 55%, 70% or 85%, preferably 70%.
Specifically, the method comprises the steps of:
(1) Preparing primary seed liquid;
(2) Preparing secondary seed liquid;
(3) Fermentation culture: when the OD 600 value reaches 0.8, inoculating the secondary seed liquid into a fermentation tank for fermentation, wherein an initial fermentation medium is filled in the fermentation tank, the liquid loading amount of the fermentation tank is 15L/50L, and inoculating according to the volume ratio of 18-22% (preferably 20 percent);
After the secondary seed liquid is inoculated into an initial fermentation culture medium, fermenting and culturing for 18-24 hours under the conditions of 36-38 ℃ and 0.07-0.09MPa of fermentation tank pressure, 0.8-1.2vvm of ventilation rate and 6.7-7.1 of pH, then pumping feed supplement liquid into a fermentation system, simultaneously pumping a feeding culture medium (namely feeding is started when the residual sugar content in the fermentation liquid is 0.1-0.2 g/L), controlling the flow rate to be 90-110mL/h (preferably 100 mL/h), controlling the feeding quantity of the feeding culture medium to be 600mL at the temperature of 36-38 ℃, and continuously fermenting under the conditions of 0.06-0.08MPa of fermentation tank pressure, 300-700rpm of ventilation rate to be 0.5-0.8vvm, 18-22% of dissolved oxygen and 7.0-7.2 of pH, wherein the residual sugar content is controlled to be 0.8-1.2g/L, the ammonia nitrogen concentration is controlled to be 0.8-1.2g/L, and the pH is controlled to be 600mL; the total fermentation period is 35-37h (preferably 36 h). The fermentation tank is placed when the volume of the fermentation liquid is 65-75% (preferably 70%) of the volume of the fermentation tank.
Wherein, the initial fermentation culture medium comprises the following components: 19-21g/L of glucose, 9-11g/L of molasses, 58-62g/L of corn steep liquor, 29-31g/L,H3PO4(1.2-1.7)×C0 g/L,KCl(0.4-0.6)×C0g/L,MgSO4(0.6-0.8)×C0 g/L,MnSO4(0.0018-0.0022)×C0 g/L,FeSO4(0.0018-0.0022)×C0 g/L,VB1(40-80)×C0μg/L of soybean meal hydrolysate and 190-210 of biotin (multiplied by C 0 mu g/L).
The ingredients of the feed supplement liquid are as follows: 550-650g/L of glucose solution, 450-550g/L of ammonium sulfate solution, 3-5g/L of threonine solution and 25% -28% of ammonia water; wherein the mass ratio of threonine fed-batch amount to glucose fed-batch amount is (0.0014-0.0016): 1.
The components of the fed-batch culture medium are ：H3PO4(1.2-1.7)×(100％-C0)g/L,KCl(0.4-0.6)×(100％-C0)g/L,MgSO4(0.6-0.8)×(100％-C0)g/L,MnSO4(0.0018-0.0022)×(100％-C0)g/L,FeSO4(0.0018-0.0022)×(100％-C0)g/L,VB1(40-80)×(100％-C0)μg/L and biotin (190-210) x (100% -C 0) mug/L.
Preferably, the seed culture medium used for preparing the primary seed liquid and the secondary seed liquid is as follows: 39-41g/L of glucose, 1.4-1.6g/L of KH 2PO4 1.4-1.6g/L,MgSO4, 11-13g/L of molasses, 24-26g/L of corn steep liquor, 11-13g/L of ammonium sulfate, 0.0015-0.0025g/L of MnSO 4 and 0.0015-0.0025g/L of FeSO 4.
In the invention, ammonia water is used for regulating and controlling pH.
Preferably, the escherichia coli is escherichia coli (ESCHERICHIA COLI) MHZ-0914 with the preservation number of CGMCC No. 22648. Strain MHZ-0914 can be found in CN114875090a.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
The invention provides a novel lysine fermentation production control scheme, which avoids the inhibition of eutrophic to the activity of thalli by reducing the concentration of a culture medium of a fermentation substrate, reduces the generation of acetic acid, relieves the influence of acetic acid on the growth and metabolism of thalli, improves the activity of thalli while not influencing the growth and the quantity of thalli, successfully solves the substrate inhibition and the byproduct feedback inhibition in the fermentation process, and greatly improves the yield of lysine. The material cost is not increased, so that the method has important industrial application value, and has certain guiding significance for fermentation production of other amino acids and related compounds thereof.
Drawings
FIGS. 1 and 2 show the fermentation acid production and process acetic acid conditions of the control and experimental groups, respectively, in example 1 of the present invention. Wherein, the control group, the experimental group 1 and the experimental group 2 and the experimental group 3 respectively represent the nutrition proportion of the bottom materials of 100%,85%, 70% and 55% respectively.
FIGS. 3 and 4 show the fermentation acid production and process acetic acid in the control and experimental groups, respectively, of example 2 according to the present invention. Wherein, experiment 2 in example 1 is used as a control group, experiment group 4, experiment group 5 and experiment group 6 respectively show that the ratio of fed-batch nutrient solution is 15%, 30% and 45%.
Detailed Description
The invention provides a method for reducing lysine fermentation byproduct acetic acid and improving fermentation acid production (improving lysine fermentation yield).
The invention adopts the following technical scheme:
The invention provides a method for reducing the by-product of fermentation lysine, which can effectively reduce the generation of inhibitory by-product acetic acid by dividing a fermentation formula into a bottom material and feeding in the middle period of fermentation and maintaining the concentration of a substrate in a culture medium at a lower level.
On the one hand, the nutrition of the fermentation is reduced in a gradient way for 0h, and the growth of the bacteria and the maximum OD (OD) are not influenced at the stage without influencing the growth of the bacteria, and the optimal concentration is preferred.
On the other hand, in the fermentation acid production stage, a certain concentration of nutrient substances are fed, preferably, the acid production is fastest, the mixed acid is lowest, and the optimal addition concentration is determined, so that the fermentation acid production and conversion rate are improved.
It was found that if the organic nitrogen source is subtracted from the fermentation start, the cell growth becomes slow, the sugar consumption rate decreases, and the overall fermentation strength is greatly affected. In the case of a long cell, a sufficient organic nitrogen source is required. Therefore, only the components other than the carbon source and the nitrogen source are considered at the start of the gradient-reduced nutrition.
In the specific research and development, the gradient experiments of 100%,85%,70% and 55% are carried out on other components except carbon sources and nitrogen sources in the culture medium of the fermentation base material, so that the growth of the earlier-stage thalli and the acid production by fermentation are verified. As a result, when the initial concentration of the culture medium is 100%,85%,70% and 55%, the OD value of 0-18h is normal, the acetic acid level in the middle fermentation period corresponding to 85%,70% and 55% is obviously reduced compared with that in the control group, and the acetic acid level is respectively 0.3g/L,0.2g/L and 0.4g/L, and the corresponding fermentation results are as follows: 188g/L,190g/L and 181g/L of acid. Therefore, the 70% effect is optimal, the concentration of the culture medium for 0h of fermentation is reduced, the growth of thalli is not affected, and the concentration of the substrate in the culture medium is maintained at a lower level, so that the mass accumulation of metabolites of escherichia coli is facilitated, and the fermentation acid production in the middle stage of fermentation can be improved.
In addition, in practical development, the method is characterized in that 15%,30% and 45% of an initial culture medium (except a carbon source and a nitrogen source) are prepared into 600mL, the adding time is 18-24h, the flow acceleration is 100mL/h, and the specifically adopted scheme is a test for verifying the influence on the fermentation result on the basis of a 70% experimental group. As a result, it was found that 15% of the initial medium corresponded to a fermentation index of 192g/L of acidogenesis and 0.4g/L of acetic acid. 30% of the corresponding fermentation indexes are 200g/L of acidogenesis and 0.2g/L of acetic acid, and 45% of the corresponding fermentation indexes are 193g/L of acidogenesis and 0.2g/L of acetic acid. Therefore, the bacterial cells grow rapidly into the logarithmic phase in the earlier stage of fermentation, the growth is not affected, nutrition starts from 18 to 24 hours, the acid production and conversion rate of 30% fed-batch fermentation are higher than those of a control, and the acetic acid is reduced to the lowest value, so that the effect is optimal.
Preferably, the concentration of the fermentation initial medium (except the carbon source and the nitrogen source) is 70%, the concentration of the feeding medium (except the carbon source and the nitrogen source) is 30%, the feeding time is 18-24h, and the feeding flow rate is 100mL/h.
In the invention, the carbon source of the fermentation medium comprises glucose or sucrose, and the nitrogen source comprises molasses, corn steep liquor, soybean meal hydrolysate, ammonium sulfate or ammonia water.
In the present invention, when the amino acid is lysine, the fermentation medium comprises: 19-21g/L of glucose, 9-11g/L of molasses, 58-62g/L of corn steep liquor and 133-147 mu g/L of bean pulp hydrolysate 29-31g/L,H3PO4 0.84-1.19g/L,KCl0.28-0.42g/L,MgSO4 0.42-0.56g/L,MnSO4 0.00126-0.00154g/L,FeSO40.00126-0.00154g/L,VB1 28-56μg/L, biotin; the concentration of the glucose solution used in the feeding is 550-650g/L, the concentration of the ammonium sulfate solution is 450-550g/L, the concentration of the ammonia water is 25-28%, the concentration of the threonine solution is 3-5g/L, and the concentration of the nutrient solution (namely other nutrient substances in the fermentation medium) is ：H3PO40.36-0.51g/L,KCl 0.12-0.18g/L,MgSO4 0.18-0.24g/L,MnSO4 0.00054-0.00066g/L,FeSO4 0.00054-0.00066g/L,VB1 12-24μg/L, biotin 57-63 mug/L.
In the invention, the fermentation bacteria are escherichia coli.
In the present invention, when the amino acid is lysine, the seed medium comprises: 39-41g/L of glucose, 1.4-1.6g/L of KH 2PO4 1.4-1.6g/L,MgSO4, 11-13g/L of molasses, 24-26g/L of corn steep liquor, 11-13g/L of ammonium sulfate and 0.0015-0.0025g/L of MnSO 4 0.0015-0.0025g/L,FeSO4.
In the present invention, when the production of fermented lysine is performed, the fermentation culture conditions further include: the fermentation temperature is 36-38 ℃, the fermentation pressure is controlled to be 0.07-0.09MPa, and the ventilation rate is 0.8-1.2vvm; and (3) controlling the residual sugar in the fermentation process to be 0.8-1.2g/L from the beginning to the end of the fermentation, wherein the mass ratio of threonine addition to glucose addition is (0.0014-0.0016) 1, the concentration of ammonia nitrogen in the fermentation is 0.8-1.2g/L, and the pH value of the fermentation is 6.7-7.1 by regulating and controlling the pH value by ammonia water.
From the beginning of feeding to the end of fermentation, the fermentation conditions are: the ventilation is 0.5-0.8vvm, the rotating speed is 300-700rpm, the pressure is controlled to be 0.06-0.08MPa, the pH value is 7.0-7.2, the temperature is 36-38 ℃, and the dissolved oxygen is 18-22%.
And when the residual sugar content in the fermentation liquid is 0.1-0.2g/L, starting to feed glucose solution, so that the concentration of glucose is controlled to be 0.8-1.2g/L, and taking nitrogen source ammonia water as a pH regulator.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
The fermentation strain used in the following examples is Escherichia coli with a preservation number of CGMCC No. 22648.
The experimental procedure used in the following examples is as follows:
(1) Gradient reducing fermentation 0h medium components, and performing an experiment on the influence of lysine fermentation and acetic acid;
(2) And (3) feeding fermentation medium components with different concentrations, and performing experiments on the influence of lysine fermentation and acetic acid.
The specific flow is as follows:
① The first-level seed liquid is inoculated into a 10L fermentation tank for secondary seed culture;
② And (3) inoculating the secondary seed liquid into a fermentation medium, and fermenting and culturing in a 50L fermentation tank.
And (3) condition control:
① Ammonia concentration control: the ammonia concentration as a nitrogen source required for amino acid production cannot be in a low state in the medium, which would otherwise lead to a decrease in the productivity of basic amino acids. During the fermentation of the strain MHZ-0914 of L-lysine, ammonia water is fed in, and simultaneously, ammonium sulfate is fed in to maintain the ammonia concentration in the fermentation broth at 0.8-1.2g/L, preferably 1g/L.
② Sugar concentration and pH control: the proportional relationship between the two is obtained by examining the consumption conditions of acid production, sugar and ammonia of thalli in the fermentation process. According to the method, pH feedback signals are used as control conditions, zero sugar is used for control in fermentation liquor, so that the pH feedback system can realize the supplement of sugar while ammonia is fed into a fermentation tank, a Kjeldahl nitrogen analyzer is used for monitoring the content of free ammonia in the fermentation process, and recording is carried out every 6 hours.
Reducing the production of acetic acid in the fermentation process, relieving the influence of acetic acid on the growth and metabolism of thalli, determining the optimal addition concentration and realizing the improvement of fermentation acid production.
Example 1 method for producing lysine by fermentation
The present example provides a method for producing lysine by fermentation, using a strain producing L-lysine: MHZ-0914.
The method comprises the following steps:
1. Preparing culture medium
Seed culture medium: 40g/L of glucose, 2PO4 1.5g/L,MgSO4 1.5.5 g/L of KH, 12g/L of molasses, 25g/L of corn steep liquor, 12g/L of ammonium sulfate and 0.002g/L of MnSO 4 0.002g/L,FeSO4.
Control group: 100% fermentation medium: glucose 20g/L, molasses 10g/L, corn steep liquor 60g/L, soybean meal hydrolysate 30g/L,H3PO4 1.5g/L,KCl 0.5g/L,MgSO4 0.7g/L,MnSO4 0.002g/L,FeSO4 0.002g/L, biotin 200 mug/L and VB 1 mug/L.
Experiment group 1:85% fermentation medium: glucose 20g/L, molasses 10g/L, corn steep liquor 60g/L, soybean meal hydrolysate 30g/L,H3PO4 1.275g/L,KCl 0.425g/L,MgSO4 0.595g/L,MnSO4 0.0017g/L,FeSO4 0.0017g/L, biotin 170 mug/L and VB 1 mug/L.
Experiment group 2:70% fermentation medium: glucose 20g/L, molasses 10g/L, corn steep liquor 60g/L, soybean meal hydrolysate 30g/L,H3PO4 1.05g/L,KCl 0.35g/L,MgSO4 0.49g/L,MnSO4 0.0014g/L,FeSO40.0014g/L, biotin 140 mug/L and VB 1 mug/L.
Experiment group 3:55% fermentation medium: glucose 20g/L, molasses 10g/L, corn steep liquor 60g/L, soybean meal hydrolysate 30g/L,H3PO4 0.825g/L,KC1 0.275g/L,MgSO4 0.385g/L,MnSO4 0.0011g/L,FeSO4 0.0011g/L, biotin 110 mug/L and VB 1 mug/L.
And (3) material supplementing liquid: glucose 600g/L, ammonium sulfate 500g/L, ammonia water 26%, threonine 4g/L.
2. Experimental method
The above fermentation substrates were sterilized at 121℃for 20min.
The L-lysine production strains are respectively connected into a seed tank, when the OD 600 value reaches 0.8, the seed tank is connected into a fermentation tank (15L/50L of liquid loading) for fermentation, the fermentation inoculation ratio is 20%, the initial fermentation bottom sugar (glucose) concentration is 20g/L, the fermentation temperature is 36-38 ℃, the fermentation pressure is controlled to be 0.08MPa, the ventilation quantity is 1vvm, the fermentable sugar is continuously fed into the fermentation tank when the residual sugar content in the liquid to be fermented is 0.2g/L, the fermentable sugar is 600g/L of high-concentration glucose liquid, the fed ammonium sulfate concentration is 500g/L, the fed ammonia water concentration is 26%, the fed threonine concentration is 4g/L, the threonine addition amount accounts for 22% of the volume of the sugar liquid, the residual sugar in the fermentation process is 1g/L, the fermentation ammonia nitrogen (inorganic free ammonia NH 4 +, the residual sugar concentration is provided by ammonium sulfate and ammonia water) is 0.8-1.2g/L, the pH value is regulated and controlled by ammonia water, the fermentation pH value is 6.7-7.1, and the volume of the liquid to be discharged to be 70% of the volume of the fermentation tank when the volume of the culture medium in the fermentation tank is 70% of the volume of the fermentation tank is discharged for 36h. During fermentation, the acid and free ammonia are measured during fermentation.
3. Fermentation results
Experimental results, the fermentation results corresponding to the control group, the experimental groups 1, 2 and 3 are respectively: 186g/L,188g/L,190g/L and 181g/L of acid are produced. Acetic acid was 1.0g/L,0.3g/L,0.2g/L,0.4g/L, respectively. 70% of the fermentation medium has the best effect, the concentration of the fermentation medium for 0h is reduced, the growth of thalli is not affected, and the concentration of the substrate in the culture medium is maintained at a lower level, so that the fermentation medium is favorable for mass accumulation of metabolites of escherichia coli, and the fermentation acid production in the middle stage of fermentation can be improved.
FIGS. 1 and 2 show the fermentation acid production and process acetic acid conditions of the control and experimental groups, respectively, in example 1 of the present invention. Wherein, the control group, the experimental group 1 and the experimental group 2 and the experimental group 3 respectively represent the nutrition proportion of the bottom materials of 100%,85%, 70% and 55% respectively.
Example 2 method for producing lysine by fermentation
The present example provides a method for producing lysine by fermentation, using a strain producing L-lysine: MHZ-0914. The specific method is the same as in example 1, except that: the experimental group 2 is adopted as the base material culture medium (initial fermentation culture medium), and the experimental groups 4, 5 and 6 are adopted as the fed-batch to prepare 600mL.
Experiment group 4: nutrient solution I (15% of culture medium )：H3PO4 0.225g/L,KCl 0.075g/L,MgSO4 0.105g/L,MnSO4 0.0003g/L,FeSO4 0.0003g/L, biotin 30. Mu.g/L, VB 1. Mu.g/L. Added in 600 mL).
Experimental group 5: nutrient solution II (30% of culture medium )：H3PO4 0.45g/L,KCl 0.15g/L,MgSO40.21g/L,MnSO4 0.0006g/L,FeSO4 0.0006g/L, biotin 60. Mu.g/L, VB 1. Mu.g/L. Added in 600 mL).
Experiment group 6: nutrient solution III (45% of culture medium )：H3PO4 0.675g/L,KCl 0.225g/L,MgSO4 0.315g/L,MnSO4 0.0009g/L,FeSO4 0.0009g/L, biotin 90. Mu.g/L, VB 1. Mu.g/L. Added in 600 mL).
The fermentation results corresponding to the control group and the experimental groups 4, 5 and 6 are as follows: the fermentation index corresponding to 15% of the initial culture medium is 192g/L of acidogenesis and 0.4g/L of acetic acid. 30% of the corresponding fermentation indexes are 200g/L of acidogenesis and 0.2g/L of acetic acid, and 45% of the corresponding fermentation indexes are 193g/L of acidogenesis and 0.2g/L of acetic acid. Experimental results show that the thallus grows rapidly into the logarithmic phase in the earlier stage of fermentation, the growth is not affected, nutrition starts from 18-24h, 70% +30% of the bottom material is fed-batch fermented to produce acid, the conversion rate is higher than that of a control, and the acetic acid is reduced to the lowest value, so that the effect is optimal.
FIGS. 3 and 4 show the fermentation acid production and process acetic acid in the control and experimental groups, respectively, of example 2 according to the present invention. Wherein, experiment group 2 in example 1 is used as a control group, experiment group 4, experiment group 5 and experiment group 6 respectively show that the fed-batch nutrition ratio is 15%, 30% and 45%.
EXAMPLE 3 method for producing lysine by fermentation
The present example provides a method for producing lysine by fermentation, using a strain producing L-lysine: MHZ-0914. The specific method was identical to the nutrient concentration of experimental group 5 in example 2, except that: the feeding time is different, and the feeding is started by respectively selecting 0-6h,6-12h,12-18h,18-24h and 24-30h, and 600mL is prepared by feeding.
The fermentation results corresponding to the feeding time of 0-6h,6-12h,12-18h,18-24h and 24-30h are respectively as follows: acid production is 192g/L, 196g/L, 199g/L, 202g/L and 198g/L. Acetic acid 0.8g/L, 0.5g/L, 0.3g/L, 0.1g/L. Experimental results show that the thallus grows rapidly into the logarithmic phase in the earlier stage of fermentation, the growth is not affected, the nutrient solution starts to be fed in 18-24h and 24-30h, the acetic acid is reduced to the minimum value, and the lysine concentration is highest in 18-24 h. Therefore, fermentation for 18 to 24 hours is preferable as the fed-batch period.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (6)
1. A method for increasing lysine fermentation production and reducing acetic acid as a byproduct, the method comprising: the method comprises the steps of utilizing escherichia coli to ferment and produce lysine, wherein in an initial fermentation culture medium, other nutrient substances in the fermentation culture medium except a carbon source and a nitrogen source use C 0 concentration, feeding is started in a middle fermentation period, and meanwhile, other nutrient substances in the fermentation culture medium with the concentration of (100% -C 0) are fed into a fermentation system;
Wherein, C 0 is 55-85%.
2. The method according to claim 1, wherein C 0 is 55%, 70% or 85%, preferably 70%.
3. The method according to claim 1, comprising the steps of:
(1) Preparing primary seed liquid;
(2) Preparing secondary seed liquid;
(3) Fermentation culture: when the OD 600 value reaches 0.8, inoculating the secondary seed liquid into a fermentation tank for fermentation, wherein an initial fermentation medium is filled in the fermentation tank, the liquid loading amount of the fermentation tank is 15L/50L, and inoculating according to the volume ratio of 18-22%;
After the secondary seed liquid is inoculated into an initial fermentation culture medium, the fermentation is carried out for 18-24 hours under the conditions of 36-38 ℃ of temperature, 0.07-0.09MPa of pressure of a fermentation tank, 0.8-1.2vvm of ventilation rate and 6.7-7.1 of pH, then the feed liquid is pumped into the fermentation system, meanwhile, the feeding culture medium is pumped in, the flow rate is 90-110mL/h, the fermentation is continued until the fermentation is finished under the conditions of 36-38 ℃ of temperature, 0.06-0.08MPa of pressure of the fermentation tank, 300-700rpm of rotation speed, 0.5-0.8vvm of ventilation rate, 18-22% of dissolved oxygen and 7.0-7.2 of pH, the ammonia nitrogen concentration is controlled to be 0.8-1.2g/L, the pH is controlled to be 6.7-7.1, and the feeding culture medium is 600mL; the total fermentation period is 35-37 hours; placing the fermentation tank when the volume of the fermentation liquid is 65-75% of the volume of the fermentation tank;
Wherein, the initial fermentation culture medium comprises the following components: 19-21g/L of glucose, 9-11g/L of molasses, 58-62g/L of corn steep liquor, 29-31g/L,H3PO4(1.2-1.7)×C0 g/L,KCl(0.4-0.6)×C0g/L,MgSO4(0.6-0.8)×C0 g/L,MnSO4(0.0018-0.0022)×C0 g/L,FeSO4(0.0018-0.0022)×C0 g/L,VB1(40-80)×C0μg/L of bean pulp hydrolysate and 190-210 of biotin (C 0 mu g/L);
The ingredients of the feed supplement liquid are as follows: 550-650g/L of glucose solution, 450-550g/L of ammonium sulfate solution, 3-5g/L of threonine solution and 25% -28% of ammonia water; wherein the mass ratio of threonine fed-batch amount to glucose fed-batch amount is (0.0014-0.0016): 1;
The components of the fed-batch culture medium are ：H3PO4(1.2-1.7)×(100％-C0)g/L,KCl(0.4-0.6)×(100％-C0)g/L,MgSO4(0.6-0.8)×(100％-C0)g/L,MnSO4(0.0018-0.0022)×(100％-C0)g/L,FeSO4(0.0018-0.0022)×(100％-C0)g/L,VB1(40-80)×(100％-C0)μg/L and biotin (190-210) x (100% -C 0) mug/L.
4. A method according to claim 3, characterized in that the seed medium used for the preparation of the primary seed liquid, the secondary seed liquid is: 39-41g/L of glucose, 1.4-1.6g/L of KH 2PO4 1.4-1.6g/L,MgSO4, 11-13g/L of molasses, 24-26g/L of corn steep liquor, 11-13g/L of ammonium sulfate, 0.0015-0.0025g/L of MnSO 4 and 0.0015-0.0025g/L of FeSO 4.
5. A method according to claim 3, wherein the pH is adjusted with aqueous ammonia.
6. The method according to any one of claims 1 to 5, wherein the escherichia coli is escherichia coli with a preservation number of CGMCC No. 22648.
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