CN112608951A - Fermentation method for improving butyric acid yield - Google Patents
Fermentation method for improving butyric acid yield Download PDFInfo
- Publication number
- CN112608951A CN112608951A CN202110057687.2A CN202110057687A CN112608951A CN 112608951 A CN112608951 A CN 112608951A CN 202110057687 A CN202110057687 A CN 202110057687A CN 112608951 A CN112608951 A CN 112608951A
- Authority
- CN
- China
- Prior art keywords
- fermentation
- butyric acid
- stage
- added
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 title claims abstract description 108
- 230000004151 fermentation Effects 0.000 title claims abstract description 74
- 238000000855 fermentation Methods 0.000 title claims abstract description 74
- 238000000034 method Methods 0.000 title claims abstract description 13
- 239000001963 growth medium Substances 0.000 claims abstract description 17
- 101710088194 Dehydrogenase Proteins 0.000 claims abstract description 10
- 239000002243 precursor Substances 0.000 claims abstract description 9
- 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 abstract description 7
- 239000008103 glucose Substances 0.000 claims abstract description 7
- 230000035040 seed growth Effects 0.000 claims abstract description 5
- CRFNGMNYKDXRTN-CITAKDKDSA-N butyryl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CCC)O[C@H]1N1C2=NC=NC(N)=C2N=C1 CRFNGMNYKDXRTN-CITAKDKDSA-N 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 6
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 6
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 6
- 229910052564 epsomite Inorganic materials 0.000 claims description 6
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 6
- 229910052603 melanterite Inorganic materials 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 239000011686 zinc sulphate Substances 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 229910052799 carbon Inorganic materials 0.000 claims description 3
- 125000001477 organic nitrogen group Chemical group 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 230000012010 growth Effects 0.000 claims description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 abstract description 16
- 235000014655 lactic acid Nutrition 0.000 abstract description 8
- 239000004310 lactic acid Substances 0.000 abstract description 8
- 238000006243 chemical reaction Methods 0.000 abstract description 7
- 239000000126 substance Substances 0.000 abstract description 6
- 239000002609 medium Substances 0.000 abstract description 5
- 239000006227 byproduct Substances 0.000 abstract description 4
- 230000034659 glycolysis Effects 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 description 7
- 238000012258 culturing Methods 0.000 description 5
- 229910021645 metal ion Inorganic materials 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000010899 nucleation Methods 0.000 description 2
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 1
- PTFCDOFLOPIGGS-UHFFFAOYSA-N Zinc dication Chemical compound [Zn+2] PTFCDOFLOPIGGS-UHFFFAOYSA-N 0.000 description 1
- PASOAYSIZAJOCT-UHFFFAOYSA-N butanoic acid Chemical compound CCCC(O)=O.CCCC(O)=O PASOAYSIZAJOCT-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 229910001437 manganese ion Inorganic materials 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
Classifications
-
- 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
- C12P7/52—Propionic acid; Butyric acids
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of butyric acid fermentation, and particularly discloses a fermentation method for improving butyric acid yield, which comprises a seed growth culture stage and a fermentation culture stage, in the fermentation culture stage, cofactor butyric acid is added into the culture medium at the early stage of fermentation to synthesize precursor substance, the fermentation promoter is added into the fermentation medium, and as the cofactor butyric acid is added into the medium at the early stage of the fermentation in the butyric acid fermentation process to synthesize the precursor substance, the fermentation promoter is added into the fermentation medium, so that the fermentation period of the butyric acid is shortened from 50 hours to 40 hours, the conversion rate of the butyric acid is improved, the thallus produces butyric acid by glucose through glycolysis, but lactic acid is accumulated in the fermentation process, so that the content of butyric acid is low, the number of byproducts is large, and the conversion of lactic acid into butyric acid can be promoted by adding lactic dehydrogenase and the like, so that the yield of the final butyric acid is increased by 9.9%.
Description
Technical Field
The invention belongs to the technical field of butyric acid fermentation, and particularly relates to a fermentation method for improving the yield of butyric acid.
Background
Butyric acid (butyric acid) is a platform compound, is widely applied to industries such as food, chemical industry, pharmacy and the like, and has a global market demand of about 80000t every year. There are two main ways of butyric acid production at present: the chemical synthesis method and the microbial fermentation method have high yield and good product specificity, but the production operation is carried out under the conditions of high temperature and high pressure, and non-renewable fossil raw materials are used, so that the cost is very high, and the microbial fermentation method can also produce the butyric acid.
Disclosure of Invention
The invention aims to provide a fermentation method for improving the yield of butyric acid, which increases the yield of butyric acid by 9.9 percent on the basis of the original conventional butyric acid microbial fermentation.
In order to achieve the purpose, the invention adopts the technical scheme that:
a fermentation method for improving the yield of butyric acid comprises a seed growth culture stage and a fermentation culture stage, wherein in the fermentation culture stage, a cofactor butyric acid synthesis precursor butyryl COA is added into a culture medium at the early stage of fermentation, a fermentation promoter lactic dehydrogenase is added into the fermentation culture medium, the addition amount of the butyryl COA is 0.001-0.005g/L and the addition amount of the lactic dehydrogenase is 0.01-0.1 g/L, glucose is utilized by the thallus to produce butyric acid through glycolysis, but lactic acid is accumulated in the fermentation process, so that the content of butyric acid is low and the byproducts are more, and the conversion of the lactic acid into the butyric acid can be promoted by adding the lactic dehydrogenase and the like.
Further, the culture medium used in the seed growth culture stage comprises 20g/L of glucose, 2g/L of yeast extract, 4g/L of peptone and (NH)4)2SO4 2g/L、K2HPO4 1g/L、KH2PO4 1g/L、MgSO4·7H2O 0 .1g/L、FeSO4·7H2O 0 .015g/L、CaCl2·2H2O 0 .015g/L、MnSO4·H2O 0 .01g/L、CoCl2·6H2O 0 .02g/L、ZnSO4·7H2O0.002 g/L, and the balance being water.
Further, each liter of fermentation medium in the fermentation culture stage comprises 50-60 g/L of carbon source, 2-8 g/L of organic nitrogen source, (NH)4)2SO4 2g/L、K2HPO4 1g/L、KH2PO4 1g/L、MgSO4·7H2O 0 .1g/L、FeSO4·7H2O 0 .015g/L、CaCl2·2H2O 0 .015g/L、MnSO4·H2O 0 .01g/L、CoCl2·6H2O 0 .02g/L、ZnSO4·7H2O0.002 g/L, and the balance being water.
The invention has the advantages that: in the process of butyric acid fermentation, a cofactor butyric acid synthesis precursor material butyryl COA is added into a culture medium at the early stage of fermentation, and because bacteria exist in the culture medium at the early stage of fermentation and an intermediate metabolite for synthesizing butyric acid, the precursor material is added in advance to serve as an inducer, so that a butyric acid production path is formed by inducing bacteria to metabolize, the improvement of the yield of the butyric acid at the later stage is facilitated, glucose is used for producing butyric acid through a glycolysis path, but lactic acid is accumulated in the fermentation process, so that the content of the butyric acid is low, a large amount of byproducts are produced, the conversion of the lactic acid into the butyric acid can be promoted by adding lactic dehydrogenase, in addition, a fermentation promoter is added into the fermentation culture medium, the fermentation period of the butyric acid is shortened, the conversion rate of the butyric acid is improved, and the yield of the final butyric.
Detailed Description
Examples
A fermentation method for improving the yield of butyric acid comprises a seed growth culture stage and a fermentation culture stage, wherein in the fermentation culture stage, a cofactor butyric acid synthesis precursor substance butyryl COA is added into a culture medium at the early stage of fermentation, a fermentation promoter lactic dehydrogenase is added into the fermentation culture medium, the addition amount of the butyryl COA is 0.001-0.005g/L and the addition amount of the lactic dehydrogenase is 0.01-0.1 g/L, glucose is utilized by the thallus to produce butyric acid through glycolysis, but lactic acid is accumulated in the fermentation process, so that the content of butyric acid is low and the number of byproducts is large, and the conversion of lactic acid into butyric acid can be promoted by adding the lactic dehydrogenase; used in the growth and culture stage of the seedsComprises 20g/L of glucose, 2g/L of yeast extract, 4g/L of peptone and (NH)4)2SO42g/L、K2HPO4 1g/L、KH2PO4 1g/L、MgSO4·7H2O 0 .1g/L、FeSO4·7H2O 0 .015g/L、CaCl2·2H2O 0 .015g/L、MnSO4·H2O 0 .01g/L、CoCl2·6H2O 0 .02g/L、ZnSO4·7H20.002 g/L of O and the balance of water; in the fermentation culture stage, each liter of fermentation culture medium comprises 50-60 g/L of carbon source, 2-8 g/L of organic nitrogen source and (NH)4)2SO4 2g/L、K2HPO4 1g/L、KH2PO4 1g/L、MgSO4·7H2O 0 .1g/L、FeSO4·7H2O 0 .015g/L、CaCl2·2H2O 0 .015g/L、MnSO4·H2O 0 .01g/L、CoCl2·6H2O 0 .02g/L、ZnSO4·7H20.002 g/L of O and the balance of water; in the process of butyric acid fermentation, the cofactor butyric acid is added into the culture medium in the early stage of the fermentation to synthesize the precursor substance, and the fermentation promoter is added into the fermentation culture medium, so that the fermentation period of the butyric acid is shortened from 50 hours to 40 hours, and the conversion rate of the butyric acid is improved, so that the yield of the final butyric acid is increased by 9.9 percent.
Effect test
And (3) determining effectiveness of the addition amount of the cofactor and the metal ions through a shake flask experiment, and verifying addition time in a small experiment.
And (3) contrast: culturing a seed tank and a fermentation tank by using the original normal culture medium, and respectively measuring the content of butyric acid after the fermentation is finished;
experiment one: normally culturing in a seeding tank, and directly adding a certain fermentation cofactor (such as butyric acid synthesis precursor substance) or a certain metal ion (such as manganese ion, magnesium ion and zinc ion) into a fermentation medium, and respectively measuring the content of butyric acid after the fermentation is finished;
experiment two: normally culturing in a seed tank, adding the fermentation cofactor or the metal ions in the early fermentation stage of a fermentation tank, and respectively measuring the content of butyric acid after the fermentation is finished;
experiment three: normally culturing in a seed tank, adding the fermentation cofactor or the metal ion promoter in the middle fermentation stage of a fermentation tank, and respectively measuring the content of butyric acid after the fermentation is finished;
experiment four: and (4) normally culturing in a seeding tank, and determining the period for adding the cofactor and the metal ions according to the previous three experiments and in consideration of the operation convenience and feasibility.
The results are shown in Table 1
Claims (3)
1. A fermentation method for improving the yield of butyric acid comprises a seed growth culture stage and a fermentation culture stage, and is characterized in that: in the fermentation culture stage, a cofactor butyric acid synthesis precursor butyryl COA is added to a culture medium at the early stage of fermentation, a fermentation promoter lactic dehydrogenase is added to the fermentation culture medium, the addition amount of the butyryl COA is 0.001-0.005g/L, and the addition amount of the lactic dehydrogenase is 0.01g/L-0.1 g/L.
2. The fermentation process for increasing butyric acid production according to claim 1, wherein: the culture medium used in the growth and culture stage of the seeds comprises 20g/L of glucose, 2g/L of yeast extract, 4g/L of peptone and (NH)4)2SO4 2g/L、K2HPO4 1g/L、KH2PO4 1g/L、MgSO4·7H2O 0 .1g/L、FeSO4·7H2O 0 .015g/L、CaCl2·2H2O 0 .015g/L、MnSO4·H2O 0 .01g/L、CoCl2·6H2O 0 .02g/L、ZnSO4·7H2O0.002 g/L, and the balance being water.
3. The fermentation process for increasing butyric acid production according to claim 2, wherein: in the fermentation culture stage, each liter of fermentation culture medium comprises 50-60 g/L of carbon source, 2-8 g/L of organic nitrogen source and (NH)4)2SO4 2g/L、K2HPO4 1g/L、KH2PO4 1g/L、MgSO4·7H2O 0 .1g/L、FeSO4·7H2O 0 .015g/L、CaCl2·2H2O 0 .015g/L、MnSO4·H2O 0 .01g/L、CoCl2·6H2O 0 .02g/L、ZnSO4·7H2O0.002 g/L, and the balance being water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110057687.2A CN112608951A (en) | 2021-01-15 | 2021-01-15 | Fermentation method for improving butyric acid yield |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110057687.2A CN112608951A (en) | 2021-01-15 | 2021-01-15 | Fermentation method for improving butyric acid yield |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112608951A true CN112608951A (en) | 2021-04-06 |
Family
ID=75253897
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110057687.2A Pending CN112608951A (en) | 2021-01-15 | 2021-01-15 | Fermentation method for improving butyric acid yield |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112608951A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540435A (en) * | 2022-04-27 | 2022-05-27 | 驻马店华中正大有限公司 | Method for improving yield of butyric acid by using metabolic enzyme |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160068919A1 (en) * | 2014-09-05 | 2016-03-10 | Green Cellulosity Corporation | Microorganism co-culture system and uses of the same |
| WO2017223025A1 (en) * | 2016-06-21 | 2017-12-28 | University Of Florida Research Foundation, Incorporated | Engineering escherichia coli for production of butyric acid |
| CN111363707A (en) * | 2020-04-26 | 2020-07-03 | 驻马店华中正大有限公司 | Method for improving acid production rate and conversion rate of butyric acid fermentation |
-
2021
- 2021-01-15 CN CN202110057687.2A patent/CN112608951A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20160068919A1 (en) * | 2014-09-05 | 2016-03-10 | Green Cellulosity Corporation | Microorganism co-culture system and uses of the same |
| WO2017223025A1 (en) * | 2016-06-21 | 2017-12-28 | University Of Florida Research Foundation, Incorporated | Engineering escherichia coli for production of butyric acid |
| CN111363707A (en) * | 2020-04-26 | 2020-07-03 | 驻马店华中正大有限公司 | Method for improving acid production rate and conversion rate of butyric acid fermentation |
Non-Patent Citations (1)
| Title |
|---|
| 邓名荣等: ""微生物催化生产丁酸研究进展"", 《中国生物工程杂志》, vol. 29, no. 3, pages 118 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540435A (en) * | 2022-04-27 | 2022-05-27 | 驻马店华中正大有限公司 | Method for improving yield of butyric acid by using metabolic enzyme |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xu et al. | Two-stage utilization of corn straw by Rhizopus oryzae for fumaric acid production | |
| Zhang et al. | Recent advances in microbial production of mannitol: utilization of low-cost substrates, strain development and regulation strategies | |
| RU2009107208A (en) | A NEW DESIGNED MICROORGANISM PRODUCING HOMO AMBERIC ACID AND A METHOD FOR PRODUCING AMBER ACID WITH ITS APPLICATION | |
| KR20170091773A (en) | Continuous culture for 1,3-propanediol production using high glycerine concentration | |
| Yang et al. | Production of erythritol from glucose by an osmophilic mutant of Candida magnoliae | |
| CN111363707A (en) | Method for improving acid production rate and conversion rate of butyric acid fermentation | |
| CN112608951A (en) | Fermentation method for improving butyric acid yield | |
| US8187847B2 (en) | Process for producing erythritol using Moniliella tomentosa strains in the presence of neutral inorganic nitrates, such as potassium nitrate, ammonium nitrate or sodium nitrate, as nitrogen source | |
| WO2009048202A1 (en) | Method for preparing succinic acid using glycerol as carbon source | |
| CN110004202B (en) | Method for synthesizing hexanoic acid by catalyzing carbohydrate through microbial co-culture | |
| CN110029134B (en) | Process for producing and extracting glutamic acid | |
| Liang et al. | Bioconversion of volatile fatty acids from organic wastes to produce high-value products by photosynthetic bacteria: A review | |
| CN112626136A (en) | Method for producing butyric acid by fermenting distiller's grain water | |
| JP6857022B2 (en) | Manufacturing method of organic acid | |
| JP2017507673A (en) | Method for producing lactones from strains of AUREOBASIDIUM PULLULANS | |
| KR20050072066A (en) | Method for producing optically active 3-chloro-2-methyl-1,2-propanediol taking advantage of microorganism | |
| JP2009148212A (en) | Method for fermentatively producing mannitol and microorganism used for performance thereof | |
| CN105073999A (en) | Method for producing 5-aminolevulinic acid or salt thereof | |
| CN112626135A (en) | Method for producing butyric acid by fermenting corn starch | |
| JP6121226B2 (en) | Method for producing lactic acid | |
| US20230055635A1 (en) | Organic Acid Production Process Using Aspergillus Strains Consuming Methanol | |
| CN111378693A (en) | Method for preparing 1, 3-propylene glycol by microbial fermentation method | |
| CN111500486B (en) | Strain capable of directly synthesizing butanol by using inulin as unique carbon source and application thereof | |
| CN116970658A (en) | Method for producing succinic acid based on charcoal fermentation | |
| JP2005052020A (en) | Method for producing ethanol |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |