CN116926134B - Method for producing D-lactic acid by fermentation - Google Patents
Method for producing D-lactic acid by fermentation Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 201
- 230000004151 fermentation Effects 0.000 title claims abstract description 196
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 62
- 229930182843 D-Lactic acid Natural products 0.000 title claims abstract description 60
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 title claims abstract description 60
- 229940022769 d- lactic acid Drugs 0.000 title claims abstract description 60
- 238000011218 seed culture Methods 0.000 claims abstract description 45
- 238000000034 method Methods 0.000 claims abstract description 29
- 238000012807 shake-flask culturing Methods 0.000 claims abstract description 23
- 241000588724 Escherichia coli Species 0.000 claims abstract description 18
- 239000001963 growth medium Substances 0.000 claims description 28
- 239000002609 medium Substances 0.000 claims description 26
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 25
- 239000008103 glucose Substances 0.000 claims description 25
- 230000008569 process Effects 0.000 claims description 19
- 238000009423 ventilation Methods 0.000 claims description 18
- 239000002131 composite material Substances 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- 238000005273 aeration Methods 0.000 claims description 12
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 claims description 12
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 11
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 11
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 9
- 238000011081 inoculation Methods 0.000 claims description 7
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 6
- 239000004475 Arginine Substances 0.000 claims description 6
- 241001061264 Astragalus Species 0.000 claims description 6
- 239000005696 Diammonium phosphate Substances 0.000 claims description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 6
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims description 6
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims description 6
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 6
- 239000004473 Threonine Substances 0.000 claims description 6
- 240000008042 Zea mays Species 0.000 claims description 6
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 6
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 6
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 6
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 6
- 235000006533 astragalus Nutrition 0.000 claims description 6
- 229960003403 betaine hydrochloride Drugs 0.000 claims description 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 6
- 239000004327 boric acid Substances 0.000 claims description 6
- 229940041514 candida albicans extract Drugs 0.000 claims description 6
- HOPSCVCBEOCPJZ-UHFFFAOYSA-N carboxymethyl(trimethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CC(O)=O HOPSCVCBEOCPJZ-UHFFFAOYSA-N 0.000 claims description 6
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 claims description 6
- 235000020415 coconut juice Nutrition 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 6
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 6
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 6
- 229940044631 ferric chloride hexahydrate Drugs 0.000 claims description 6
- 150000004676 glycans Chemical class 0.000 claims description 6
- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 239000011565 manganese chloride Substances 0.000 claims description 6
- 235000002867 manganese chloride Nutrition 0.000 claims description 6
- 229940099607 manganese chloride Drugs 0.000 claims description 6
- 229930182817 methionine Natural products 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 6
- 239000006012 monoammonium phosphate Substances 0.000 claims description 6
- 229920001282 polysaccharide Polymers 0.000 claims description 6
- 239000005017 polysaccharide Substances 0.000 claims description 6
- 235000010413 sodium alginate Nutrition 0.000 claims description 6
- 229940005550 sodium alginate Drugs 0.000 claims description 6
- 239000000661 sodium alginate Substances 0.000 claims description 6
- 239000011684 sodium molybdate Substances 0.000 claims description 6
- 235000015393 sodium molybdate Nutrition 0.000 claims description 6
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 210000004233 talus Anatomy 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000012138 yeast extract Substances 0.000 claims description 6
- 239000011592 zinc chloride Substances 0.000 claims description 6
- 235000005074 zinc chloride Nutrition 0.000 claims description 6
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 4
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 229960003280 cupric chloride Drugs 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 238000001694 spray drying Methods 0.000 claims description 3
- 239000012137 tryptone Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 235000009697 arginine Nutrition 0.000 claims 2
- 235000006109 methionine Nutrition 0.000 claims 2
- 235000008521 threonine Nutrition 0.000 claims 2
- 239000003607 modifier Substances 0.000 claims 1
- 238000004321 preservation Methods 0.000 claims 1
- 239000002253 acid Substances 0.000 abstract description 27
- 241001052560 Thallis Species 0.000 abstract description 13
- 230000003287 optical effect Effects 0.000 abstract description 12
- 125000005842 heteroatom Chemical group 0.000 abstract description 9
- 238000006243 chemical reaction Methods 0.000 abstract description 8
- 239000004251 Ammonium lactate Substances 0.000 abstract description 7
- 229940059265 ammonium lactate Drugs 0.000 abstract description 7
- 235000019286 ammonium lactate Nutrition 0.000 abstract description 7
- RZOBLYBZQXQGFY-HSHFZTNMSA-N azanium;(2r)-2-hydroxypropanoate Chemical compound [NH4+].C[C@@H](O)C([O-])=O RZOBLYBZQXQGFY-HSHFZTNMSA-N 0.000 abstract description 7
- 238000009825 accumulation Methods 0.000 abstract description 3
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 18
- 230000000052 comparative effect Effects 0.000 description 11
- 239000004310 lactic acid Substances 0.000 description 9
- 235000014655 lactic acid Nutrition 0.000 description 9
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 238000012258 culturing Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229920000747 poly(lactic acid) Polymers 0.000 description 4
- 239000004626 polylactic acid Substances 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 3
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 3
- 239000000920 calcium hydroxide Substances 0.000 description 3
- 229910001861 calcium hydroxide Inorganic materials 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 150000007524 organic acids Chemical class 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- PKAUICCNAWQPAU-UHFFFAOYSA-N 2-(4-chloro-2-methylphenoxy)acetic acid;n-methylmethanamine Chemical compound CNC.CC1=CC(Cl)=CC=C1OCC(O)=O PKAUICCNAWQPAU-UHFFFAOYSA-N 0.000 description 1
- 241000534000 Berula erecta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- -1 ammonium ions Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 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
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical group [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 1
- 239000001527 calcium lactate Substances 0.000 description 1
- 235000011086 calcium lactate Nutrition 0.000 description 1
- 229960002401 calcium lactate Drugs 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000019504 cigarettes Nutrition 0.000 description 1
- 229920006237 degradable polymer Polymers 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000010985 leather Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
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- 238000003756 stirring Methods 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
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
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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
A method for producing D-lactic acid by fermentation belongs to the field of D-lactic acid production. The method for producing D-lactic acid by fermentation comprises the following steps: shake flask culture, seed culture, and fermentation culture. The method for producing D-lactic acid by fermentation can effectively solve the problem that the continuous accumulation of ammonium lactate inhibits the growth of thalli and the capacity of metabolizing and producing acid in the fermentation process of escherichia coli, and avoid the problems of slow growth, low production strength and low yield of the escherichia coli; and the problem of high content of hetero acid in the anaerobic and micro-aerobic combined fermentation process; the method can effectively improve the conversion rate of sugar in fermentation liquor, shorten the production period and improve the yield of D-lactic acid while ensuring the optical purity and the yield of the D-lactic acid.
Description
Technical Field
The invention relates to the field of D-lactic acid production, in particular to a method for producing D-lactic acid by fermentation.
Background
Lactic acid is one of the three recognized organic acids in the world and is extremely widely used. Plays an important role in the industries of food, brewing, spice, medicine, leather, cigarette, chemical industry, printing and dyeing and the like. In particular, polylactic acid, which is a polymer of lactic acid in recent years, is used as a nontoxic and degradable polymer material with biocompatibility, and has been widely applied to the fields of manufacturing biodegradable plastics, green packaging materials, medical repair materials and the like, and has attracted wide attention in the world, and has very broad application prospects. However, the production cost of polylactic acid products is high, and the problem is always an important factor for limiting the comprehensive replacement of traditional plastic products. Therefore, reducing the production cost of the monomer lactic acid of the polylactic acid and improving the production efficiency are the problems to be solved at present.
In the prior art, microbial fermentation is the main method for producing lactic acid. Compared with chemical synthesis methods, enzyme conversion methods and the like, the microbial fermentation method for producing the D-lactic acid has the advantages of wide sources of fermentation raw materials, recycling and the like, and therefore, the microbial fermentation method has become a main method for industrially producing the D-lactic acid. The existing industrial lactobacillus fermentation to produce lactic acid is performed under anaerobic condition, but researches find that a proper amount of oxygen environment is also helpful for the growth of thalli. Yang Wenge et al report a process for producing D-lactic acid by combined fermentation (Chinese patent CN 1952164A), wherein lactobacillus is used for producing D-lactic acid by fermentation, the fermentation time of the strain is 25-38 hours, and the acid yield can reach 75-131g/L of lactic acid. Qian Zhiliang and the like report that lactobacillus chrysanthemi and a method for preparing D-lactic acid by adopting the strain to ferment (Chinese patent CN 101974447B) have the fermentation state between anaerobic and micro-oxygen, the strain fermentation time is 40-50 hours, the fermentation acid production is 12-14%, and the sugar acid conversion rate is 92-94%. However, the aforementioned D-lactic acid production process has disadvantages: the method has the advantages that a rich culture medium is needed in the fermentation process, the operation and control of a micro-oxygen control stage are complex, the ventilation quantity is large, the content of hetero acid in a fermentation product is high, and calcium hydroxide or calcium carbonate is needed to be used as a neutralizer for controlling the fermentation pH, so that the final fermentation product is calcium lactate; sulfuric acid is required to be added in the subsequent separation and extraction process, so that a large amount of calcium sulfate waste is generated, effective utilization is difficult, and finally the production cost and the separation and extraction cost are high; and the optical purity of the prepared lactic acid is low, so that the requirement of producing polylactic acid can not be met.
Furthermore, the escherichia coli has the advantages of clear genetic background, easiness in genetic engineering transformation, short fermentation period, simple nutrition requirement and the like, and plays an important role in lactic acid production. However, at present, an industrial production process for preparing D-lactic acid by using escherichia coli generally adopts calcium hydroxide or potassium hydroxide as a neutralizing agent. Calcium hydroxide is used as a neutralizer, and a large amount of calcium sulfate waste which is difficult to utilize is generated in the subsequent extraction process; and potassium hydroxide is used as a neutralizer, so that the cost is high, the economical efficiency is poor, and the large-scale industrial production is not facilitated. The inventor adopts ammonia water as a neutralizer for large-scale production of D-lactic acid, but the inventor discovers that although the use of ammonia water as the neutralizer can reduce the fermentation cost, as the concentration of ammonium ions in fermentation liquor increases, ammonium lactate can be generated, and continuous accumulation of ammonium lactate can inhibit the growth of thalli and the acid-producing capacity of metabolism, so that the growth of escherichia coli is slow, the production intensity is reduced, and finally the acid-producing concentration is low and the fermentation period is long.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides a method for producing D-lactic acid by fermentation, which can effectively solve the problem that the continuous accumulation of ammonium lactate inhibits the growth and the metabolic acid production capacity of thalli in the fermentation process of escherichia coli, and avoid the problems of slow growth, low production intensity and low yield of the escherichia coli; and the problem of high content of hetero acid in the anaerobic and micro-aerobic combined fermentation process; the method can effectively improve the conversion rate of sugar in fermentation liquor, shorten the production period and improve the yield of D-lactic acid while ensuring the optical purity and the yield of the D-lactic acid.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for producing D-lactic acid by fermentation, which comprises the following steps: shake flask culture, seed culture, and fermentation culture.
And (3) culturing in a shaking bottle, inoculating escherichia coli thalli on a flat plate into a shaking bottle culture medium, controlling the shaking bottle culture temperature to be 37 ℃, controlling the shaking table frequency to be 200rpm, and culturing for 16 hours to obtain a shaking bottle culture solution.
In the shake flask culture, a shake flask culture medium consists of 10g/L of tryptone, 5g/L of yeast powder and 10g/L of NaCl, and the pH value is 7.0.
The seed culture is carried out, shake flask culture solution obtained by shake flask culture is inoculated into a seed culture medium according to the inoculum size of 1-2%, and anaerobic fermentation is carried out; controlling the seed culture temperature to 37 ℃, the rotating speed to 200rpm, the seed culture pressure to 0.05Mpa, and keeping anaerobic in the seed culture process without introducing any air; in the seed culture process, ammonia water is adopted to control the PH value to 7.0, and the seed culture time is 16 hours, so as to prepare the seed culture solution.
In the seed culture, the seed culture medium consists of 90g/L of glucose, 3.5g/L of compound regulator, 0.82g/L of monoammonium phosphate, 2.35g/L of diammonium phosphate, 0.25g/L of magnesium sulfate, 0.2g/L of betaine hydrochloride, 2.1 mu g/L of ferric chloride hexahydrate, 0.3 mu g/L of cobalt chloride hexahydrate, 0.2 mu g/L of copper chloride, 0.15 mu g/L of sodium molybdate, 0.25 mu g/L of zinc chloride, 0.3 mu g/L of manganese chloride and 0.08 mu g/L of boric acid, and the solvent is water.
The composite regulator is prepared by putting corn steep liquor dry powder, yeast extract, coconut water and sodium alginate into a mixer, mixing at 120rpm for 20min, continuously putting threonine, methionine, arginine and astragalus polysaccharide into the mixer, mixing at 150rpm for 20min, and spray drying to obtain the composite regulator. In the composite regulator, the weight ratio of corn steep liquor dry powder to yeast extract to coconut water to sodium alginate to threonine to methionine to arginine to astragalus polysaccharide is 12:4:1:60:0.3:0.08:0.05:0.5.
The fermentation culture is carried out, seed culture solution obtained by seed culture is inoculated into a fermentation culture medium according to the inoculation amount of 3-4%, and fermentation culture is carried out; controlling the concentration of glucose in the fermentation medium to 150g/L, the fermentation temperature to 37 ℃, the rotating speed to 100rpm and the culture pressure to 0.05Mpa; in the fermentation culture process, a staged microporous aeration mode is adopted, and sterile air is continuously introduced into a fermentation tank; in the fermentation culture process, ammonia water is adopted to control the pH value of the fermentation liquor to 7.0;
in the fermentation culture, the stage type microporous aeration comprises a first stage, a second stage and a third stage;
the first stage is that fermentation culture is carried out for 0-9h, and the volume ratio of ventilation quantity per minute to fermentation culture medium is 0.02:1;
the second stage is 9-18h of fermentation culture, and the volume ratio of ventilation quantity per minute to fermentation culture medium is 0.05:1;
the third stage is that after 18 hours of fermentation culture, the volume ratio of ventilation quantity per minute to fermentation culture medium is 0.03:1;
and in the third stage, monitoring the glucose content in the fermentation culture solution, and ending the fermentation culture when the glucose content is 0g/L to obtain the fermentation culture solution.
In the fermentation culture, a fermentation culture medium consists of 150g/L of glucose, 4.2g/L of a compound regulator, 0.82g/L of monoammonium phosphate, 2.35g/L of diammonium phosphate, 0.4g/L of magnesium sulfate, 0.5g/L of betaine hydrochloride, 2.1 mu g/L of ferric chloride hexahydrate, 0.3 mu g/L of cobalt chloride hexahydrate, 0.2 mu g/L of copper chloride, 0.15 mu g/L of sodium molybdate, 0.25 mu g/L of zinc chloride, 0.3 mu g/L of manganese chloride and 0.08 mu g/L of boric acid, and the solvent is water. The composite regulator is the same as the composite regulator of the seed culture medium.
In the fermentation culture, a disc-type air distributor is adopted for microporous aeration, and the air distributor is arranged at the bottom of a fermentation tank; sterile air is conveyed to a disc-type air distributor through an air inlet pipe, and the air is discharged into micro bubbles through ventilation micropores arranged on the disc-type air distributor and dispersed into a fermentation medium; the hole area of all ventilation micropores on the disc-type air distributor is 60% of the circular area of the cross section of the air inlet pipe.
Compared with the prior art, the invention has the beneficial effects that:
(1) The method for producing the D-lactic acid by fermentation can be suitable for industrialized mass production of the D-lactic acid, can effectively solve the problems of slow growth, low production strength and low yield of the escherichia coli due to the inhibition of the ammonium lactate on the growth and metabolism of thalli in the fermentation process of the escherichia coli; and the problem of high content of hetero acid in the anaerobic and micro-aerobic combined fermentation process; the method can effectively improve the conversion rate of sugar in fermentation liquor, shorten the production period and improve the yield of D-lactic acid while ensuring the optical purity and the yield of the D-lactic acid.
(2) The method for producing the D-lactic acid by fermentation has the fermentation period of 29 hours, the concentration of the D-lactic acid produced by fermentation can reach 114.7g/L, the acid production rate can reach 3.96 g/L.h, and the content of the mixed acid is reduced to 0.2g/L; meanwhile, the optical purity of the D-lactic acid in the fermentation broth is 99.95%, the yield is 97.8%, the optical purity and the yield of the D-lactic acid can be ensured, the conversion rate of sugar in the fermentation broth can be effectively improved, the production period is shortened, and the yield of the D-lactic acid is improved; the method for producing the D-lactic acid by fermentation is far lower than the fermentation period (48 hours) of the D-lactic acid in the prior art, obviously improves the fermentation efficiency and improves the quality of the D-lactic acid product.
(3) According to the method for producing the D-lactic acid by fermentation, stage type micropore aeration is adopted in the fermentation culture process, and the growth speed of thalli is dynamically adjusted, so that the inhibition effect of ammonium lactate on the growth of thalli and the capacity of metabolizing to produce acid is relieved.
Detailed Description
Specific embodiments of the present invention will now be described in order to provide a clearer understanding of the technical features, objects and effects of the present invention.
Example 1
A method for producing D-lactic acid by fermentation specifically comprises the following steps:
(1) Shake flask culture
And inoculating a proper amount of escherichia coli thalli on a flat plate into 100ml of shake flask culture medium, and culturing for 16 hours in an incubator with the culture temperature of 37 ℃ and the shaking table frequency of 200rpm to prepare a shake flask culture solution.
Shake flask medium: tryptone 10g/L, yeast powder 5g/L, naCl10g/L, pH=7.0.
In this example, the E.coli cells used were obtained by the institute of Biotechnology, tianjin, national academy of sciences, and were designated as strain Dlac-206, classified: escherichia coli (Escherichia coli) with a collection number of CGMCC7679.
(2) Seed culture
The volume of the seed culture medium in the 4L fermentation tank is 2.5L, the shake flask culture solution is inoculated into the seed culture medium according to the inoculation amount of 1 percent for anaerobic fermentation, the culture temperature is 37 ℃, the rotating speed is 200rpm, the tank pressure is maintained at 0.05Mpa, and the anaerobic is kept in the culture process without introducing any air. In the seed culture process, ammonia water with the concentration of 10% is adopted to control the PH value to be 7.0, and the culture time is 16 hours, so as to prepare the seed culture solution.
Seed culture medium: glucose 90g/L, a compound regulator 3.5g/L, monoammonium phosphate 0.82g/L, diammonium phosphate 2.35g/L, magnesium sulfate 0.25g/L, betaine hydrochloride 0.2g/L, ferric chloride hexahydrate 2.1 μg/L, cobalt chloride hexahydrate 0.3 μg/L, cupric chloride 0.2 μg/L, sodium molybdate 0.15 μg/L, zinc chloride 0.25 μg/L, manganese chloride 0.3 μg/L, boric acid 0.08 μg/L, and water as a solvent.
The composite regulator is prepared by putting corn steep liquor dry powder, yeast extract, coconut water and sodium alginate into a mixer, mixing at 120rpm for 20min, continuously adding threonine, methionine, arginine and astragalus polysaccharide, mixing at 150rpm for 20min, and spray drying to obtain the composite regulator.
Wherein, the weight ratio of the corn steep liquor dry powder to the yeast extract to the coconut water to the sodium alginate to the threonine to the methionine to the arginine to the astragalus polysaccharide is 12:4:1:60:0.3:0.08:0.05:0.05:0.5.
(3) Fermentation culture
The volume of the fermentation medium in the 4L fermentation tank is 2.5L, the seed culture solution is inoculated into the fermentation medium according to the inoculation amount of 4 percent for fermentation culture, the concentration of glucose in the fermentation medium is 150g/L, the fermentation temperature is 37 ℃, the rotating speed is 100rpm, and the tank pressure is maintained at 0.05Mpa. In the fermentation culture process, a staged microporous aeration mode is adopted, and sterile air is continuously introduced into a fermentation tank; and in the fermentation culture process, ammonia water with the concentration of 10% is adopted to control the pH value of the fermentation liquor to be 7.0. After fermentation culture for 18 hours, monitoring the glucose content in the fermentation culture solution in real time, and ending the fermentation culture when the glucose content is 0g/L to obtain the fermentation culture solution.
The stage type microporous aeration comprises the following three stages:
(1) fermenting and culturing for 0-9h, wherein the volume ratio of ventilation quantity per minute to fermentation culture medium is 0.02:1;
(2) fermenting and culturing for 9-18h, wherein the volume ratio of ventilation quantity per minute to fermentation culture medium is 0.05:1;
(3) after 18h of fermentation culture, the volume ratio of ventilation per minute to the fermentation medium was 0.03:1.
Wherein, the specific mode of micropore aeration is, set up disk air distributor in the fermentation cylinder bottom, and aseptic air is carried to disk air distributor through the intake pipe, and the micropore of ventilating of passing through disk air distributor upper portion and lateral part is given vent to anger and is the tiny bubble, disperses in the zymotic fluid. The hole area of all ventilation micropores on the disc-type air distributor is 60% of the circular area of the cross section of the air inlet pipe.
Fermentation medium: 150g/L of glucose, 4.2g/L of compound regulator, 0.82g/L of monoammonium phosphate, 2.35g/L of diammonium phosphate, 0.4g/L of magnesium sulfate, 0.5g/L of betaine hydrochloride, 2.1 mu g/L of ferric chloride hexahydrate, 0.3 mu g/L of cobalt chloride hexahydrate, 0.2 mu g/L of copper chloride, 0.15 mu g/L of sodium molybdate, 0.25 mu g/L of zinc chloride, 0.3 mu g/L of manganese chloride, 0.08 mu g/L of boric acid and water as a solvent.
In the fermentation medium, the adopted composite regulator is the same as that of the seed medium.
Comparative example 1
Shake flask culture was the same as seed culture and example 1.
The fermentation culture modification is as follows: the volume of a fermentation medium in a 4L fermentation tank is 2.5L, a seed culture solution is inoculated into the fermentation medium according to the inoculation amount of 4 percent for anaerobic fermentation, the concentration of glucose in the fermentation medium is 150g/L, the fermentation temperature is 37 ℃, the rotating speed is 100rpm, and the tank pressure is maintained at 0.05Mpa. During the fermentation culture, no air is introduced. In the fermentation culture process, ammonia water with the concentration of 10% is adopted to control the PH value to be 7.0. Detecting the glucose content in the fermentation culture solution, and ending the fermentation culture when the glucose content is 0g/L to obtain the fermentation culture solution.
Comparative example 2
Shake flask culture was the same as seed culture and example 1.
The fermentation culture modification is as follows: the volume of the fermentation medium in the 4L fermentation tank is 2.5L, the seed culture solution is inoculated into the fermentation medium according to the inoculation amount of 4 percent for fermentation culture, the concentration of glucose in the fermentation medium is 150g/L, the fermentation temperature is 37 ℃, the rotating speed is 200rpm, and the tank pressure is maintained at 0.05Mpa. In the fermentation culture process, the volume ratio of ventilation quantity per minute to the fermentation culture medium is controlled to be 0.1:1, and sterile air is continuously introduced. In the fermentation culture process, ammonia water with the concentration of 10% is adopted to control the PH value to be 7.0. Detecting the glucose content in the fermentation culture solution, and ending the fermentation culture when the glucose content is 0g/L to obtain the fermentation culture solution.
Comparative example 3
Shake flask culture was the same as seed culture and example 1.
The fermentation culture modification is as follows: the volume of the fermentation medium in the 4L fermentation tank is 2.5L, the seed culture solution is inoculated into the fermentation medium according to the inoculation amount of 4 percent for fermentation culture, the concentration of glucose in the fermentation medium is 150g/L, the fermentation temperature is 37 ℃, the rotating speed is 150rpm, and the tank pressure is maintained at 0.05Mpa. In the fermentation culture process, the ventilation quantity per minute and the volume ratio of a fermentation culture medium are controlled to be 0.1:1 in the first 12 hours, and sterile air is introduced; after 12h, the introduction of sterile air was stopped for anaerobic fermentation. In the fermentation culture process, ammonia water with the concentration of 10% is adopted to control the PH value to be 7.0. Detecting the glucose content in the fermentation culture solution, and ending the fermentation culture when the glucose content is 0g/L to obtain the fermentation culture solution.
Comparative example 4
Shake flask culture was the same as in example 1.
In the seed culture, the seed medium was not prepared using a complex regulator, and the other operations were the same as in example 1.
In the fermentation culture, the complex regulator was not used for the fermentation medium, and the other operations were the same as in example 1.
After completion of the fermentation culture of example 1 and comparative examples 1 to 4, the fermentation culture solutions thus obtained were measured. Wherein, each component in the fermentation culture solution is measured by using an Agilent-1260 high performance liquid chromatograph. Glucose and organic acid concentrations in the fermentation broth were measured using an Aminex HPX-87H organic acid analytical column from Berle (Biorad). The optical purity of lactic acid was measured by SUMICHIRALOA-6000 chiral column analysis of Japanese Sumika Chemical Analysis Service Co. The specific results are shown in Table 1:
TABLE 1 summary of D lactic acid production results
According to the method for producing the D-lactic acid by fermentation, the staged microporous aeration is adopted, the seed culture is carried out by adopting the seed culture medium, and the fermentation culture is carried out by adopting the fermentation culture medium, so that the problems of slow growth, low production strength and low yield of the escherichia coli can be effectively solved by inhibiting the growth and the metabolism of ammonium lactate on the acid production capacity of the thalli in the fermentation process of the escherichia coli; and the problem of high content of hetero acid in the anaerobic and micro-aerobic combined fermentation process; the method can effectively improve the conversion rate of sugar in fermentation liquor, shorten the production period and improve the yield of D-lactic acid while ensuring the optical purity and the yield of the D-lactic acid. Compared with comparative example 1, the method for producing D-lactic acid by fermentation effectively shortens the fermentation period, improves the fermentation effect, effectively prepares the fermentation liquor with D-lactic acid content and yield, and reduces the content of hetero acid; meanwhile, the acid production rate is improved, and the high optical purity of the D-lactic acid is maintained. As can be seen from the result of comparative example 2, although the fermentation period of comparative example 2 is shorter, the fermentation speed is fast, the acid production rate is higher, the content of D-lactic acid is lower, the content of hetero acid is high, the subsequent separation and purification are not facilitated, and the yield and the optical purity of D-lactic acid are reduced to a certain extent; as can be seen from the results of comparative example 3, the fermentation period, D-lactic acid content, hetero acid content, acid production rate, D-lactic acid yield and optical purity were further improved. Compared with comparative examples 2-3, the method for producing D-lactic acid by fermentation of the invention adopts staged microporous aeration fermentation to prepare D-lactic acid, which not only can improve the conversion rate of sugar in fermentation liquor, improve the acid production rate, ensure the yield and optical purity index of D-lactic acid, and effectively reduce the content of hetero acid; meanwhile, the utilization efficiency of oxygen can be improved, the ventilation and stirring frequency can be reduced, the fermentation process is easy to control, the production cost is effectively reduced, and the production benefit is improved. As can be seen from comparative example 4, the method for producing D-lactic acid by fermentation of the invention can effectively promote the early growth of thalli and promote the later acid production of thalli by adopting the composite regulator in seed culture and fermentation culture, simultaneously avoid the adverse effect of too fast early growth of thalli on the later acid production, further improve the fermentation and acid production rate and shorten the fermentation period.
The percentages used in the present invention are mass percentages unless otherwise indicated.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A method for producing D-lactic acid by fermentation, comprising the steps of: shake flask culture, seed culture and fermentation culture;
the method for producing the D-lactic acid by fermentation adopts an escherichia coli strain Dlac-206, and the preservation number is CGMCC NO.7679;
the fermentation culture is carried out, seed culture solution obtained by seed culture is inoculated into a fermentation culture medium according to the inoculation amount of 3-4%, and fermentation culture is carried out; controlling the concentration of glucose in the fermentation medium to 150g/L, the fermentation temperature to 37 ℃, the rotating speed to 100rpm and the culture pressure to 0.05Mpa; in the fermentation culture process, a staged microporous aeration mode is adopted, and sterile air is continuously introduced into a fermentation tank; in the fermentation culture process, ammonia water is adopted to control the pH value of the fermentation liquor to 7.0;
in the fermentation culture, the stage type microporous aeration comprises a first stage, a second stage and a third stage;
the first stage is that fermentation culture is carried out for 0-9h, and the volume ratio of ventilation quantity per minute to fermentation culture medium is 0.02:1;
the second stage is 9-18h of fermentation culture, and the volume ratio of ventilation quantity per minute to fermentation culture medium is 0.05:1;
the third stage is that after 18 hours of fermentation culture, the volume ratio of ventilation quantity per minute to fermentation culture medium is 0.03:1;
and in the third stage, monitoring the glucose content in the fermentation culture solution, and ending the fermentation culture when the glucose content is 0g/L to obtain the fermentation culture solution.
2. The method for producing D-lactic acid by fermentation according to claim 1, wherein in the fermentation culture, the fermentation medium is composed of glucose 150g/L, a complex regulator 4.2g/L, monoammonium phosphate 0.82g/L, diammonium phosphate 2.35g/L, magnesium sulfate 0.4g/L, betaine hydrochloride 0.5g/L, ferric chloride hexahydrate 2.1. Mu.g/L, cobalt chloride hexahydrate 0.3. Mu.g/L, cupric chloride 0.2. Mu.g/L, sodium molybdate 0.15. Mu.g/L, zinc chloride 0.25. Mu.g/L, manganese chloride 0.3. Mu.g/L, boric acid 0.08. Mu.g/L, and water as a solvent;
the composite regulator is prepared by putting corn steep liquor dry powder, yeast extract, coconut water and sodium alginate into a mixer, mixing at 120rpm for 20min, continuously putting threonine, methionine, arginine and astragalus polysaccharide into the mixer, mixing at 150rpm for 20min, and spray drying to obtain the composite regulator.
3. The method for producing D-lactic acid by fermentation according to claim 2, wherein the weight ratio of corn steep liquor dry powder, yeast extract, coconut water, sodium alginate, threonine, methionine, arginine and astragalus polysaccharide in the composite modifier is 12:4:1:60:0.3:0.08:0.05:0.05:0.5.
4. The method for producing D-lactic acid by fermentation according to claim 1, wherein in the fermentation culture, a tray-type air distributor is used for the aeration of the micropores, and the air distributor is arranged at the bottom of the fermentation tank; sterile air is conveyed to a disc-type air distributor through an air inlet pipe, and the air is discharged into micro bubbles through ventilation micropores arranged on the disc-type air distributor and dispersed into a fermentation medium;
the hole area of all ventilation micropores on the disc-type air distributor is 60% of the circular area of the cross section of the air inlet pipe.
5. The method for producing D-lactic acid by fermentation according to claim 2, wherein the seed culture is performed by inoculating a shake flask culture solution obtained by shake flask culture in a seed culture medium in an inoculum size of 1-2% for anaerobic fermentation; controlling the seed culture temperature to 37 ℃, the rotating speed to 200rpm, the seed culture pressure to 0.05Mpa, and keeping anaerobic in the seed culture process without introducing any air; in the seed culture process, ammonia water is adopted to control the PH value to 7.0, and the seed culture time is 16 hours, so as to prepare the seed culture solution.
6. The method for producing D-lactic acid by fermentation according to claim 5, wherein in the seed culture, the seed culture medium is composed of glucose 90g/L, a complex regulator 3.5g/L, monoammonium phosphate 0.82g/L, diammonium phosphate 2.35g/L, magnesium sulfate 0.25g/L, betaine hydrochloride 0.2g/L, ferric chloride hexahydrate 2.1. Mu.g/L, cobalt chloride hexahydrate 0.3. Mu.g/L, cupric chloride 0.2. Mu.g/L, sodium molybdate 0.15. Mu.g/L, zinc chloride 0.25. Mu.g/L, manganese chloride 0.3. Mu.g/L, boric acid 0.08. Mu.g/L, and a solvent of water;
the composite regulator is the same as that of the fermentation medium.
7. The method for producing D-lactic acid by fermentation according to claim 1, wherein the shake flask culture is carried out by inoculating Escherichia coli cells on a plate into a shake flask culture medium, controlling the shake flask culture temperature to 37 ℃, and controlling the shake flask culture frequency to 200rpm for 16 hours to obtain a shake flask culture solution.
8. The method for producing D-lactic acid by fermentation according to claim 7, wherein in the shake flask culture, the shake flask medium is composed of 10g/L of tryptone, 5g/L of yeast powder, 10g/L of NaCl and the pH value is 7.0.
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