CN108410912B - Method for directly preparing butanol by using starch-containing waste - Google Patents
Method for directly preparing butanol by using starch-containing waste Download PDFInfo
- Publication number
- CN108410912B CN108410912B CN201810187132.8A CN201810187132A CN108410912B CN 108410912 B CN108410912 B CN 108410912B CN 201810187132 A CN201810187132 A CN 201810187132A CN 108410912 B CN108410912 B CN 108410912B
- Authority
- CN
- China
- Prior art keywords
- starch
- fermentation
- butanol
- containing waste
- culture medium
- 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.)
- Active
Links
Images
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
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/16—Butanols
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention relates to the technical field of biological fermentation, provides a method for preparing butanol by using starch-containing waste, and solves the problem that the cost for producing butanol by adding amylase or saccharifying enzyme into the starch-containing waste is too high. The invention takes starch-containing waste as a fermentation raw material to be sorted and ground, prepares a fermentation culture medium after high-temperature sterilization, and inoculates Clostridium saccharoperbutylacetonicum N1-4 bacterial strain to directly ferment and prepare the butanol. The invention directly utilizes the waste containing starch to ferment and produce the butanol, has simple process and no need of adding other auxiliary substances, and reduces the production cost by 20 to 30 percent compared with the conventional method for preparing the butanol by adding amylase or saccharifying enzyme for saccharification and fermentation.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a method for preparing butanol by directly fermenting starch-containing waste by adding Clostridium Clostridium for producing butanol through Saccharomyces cerevisiae N1-4.
Background
In recent years, the annual decline in petrochemical resource reserves, global warming and climate change, the constant conflict of petroleum supply areas leading to fluctuations in petroleum supply, and the restrictions of current laws on the use of non-renewable energy sources have made the use of biotechnology to produce renewable biomass energy sources increasingly important. Compared with the traditional biomass energy fuel ethanol, the butanol has the advantages of high energy, good combustion performance, small corrosivity, easy transportation, safe storage and transportation and the like. In addition, butanol can be mixed with gasoline in any proportion and even used as fuel by itself without any modification to the existing engine, so that in recent years, many researches consider that the biomass butanol is a novel biofuel with great potential.
At present, the traditional industrial production of biomass butanol mainly uses grain crops or other starchy agricultural byproducts as raw materials, and the mixture of acetone, butanol and ethanol is obtained under the anaerobic ABE fermentation action of clostridium butyricum after hydrolysis, and then the corresponding product is obtained by rectification. However, the current biomass butanol production process has high cost and lacks commercial competitiveness, so that the development of the process is limited mainly due to the following reasons: (1) the price of the raw materials is high: with the shortage of global food resources and the rising of grain prices, butanol fermentation using traditional crops such as corn and wheat as raw materials is strongly impacted; (2) the process route for fermenting by taking lignocellulose as a raw material is complex, the pretreatment condition is harsh, and the use of various hydrolases and how to solve the equal utilization of pentose and hexose are difficult points to be solved. In order to solve the above limitation problems and improve the market competitiveness of biomass butanol, cheap production raw materials are selected, and efficient and simple production processes are developed and established, which become hot spots of butanol fermentation research at present.
The starch-containing waste generally comprises kitchen waste in food waste, starch processing industry residue in industrial production, starch-containing food production residue such as bread and the like, agricultural starch-containing crops and the like. The traditional starch-containing waste is subjected to liquefaction and saccharification steps after being pretreated, and then is inoculated with related strains for fermentation to produce butanol. The food waste is fermented to produce butanol under the condition of adding saccharifying enzyme in the patent CN102250967B, and the kitchen waste treated by a plurality of enzyme preparations in the patent CN102586382B is inoculated with mixed bacterial liquid to ferment and produce butanol. The invention effectively solves the problems by directly fermenting the starch-containing waste to produce the butanol under the condition of not adding enzyme, simplifies the production process, saves the production cost, reduces the energy consumption and has obvious economic, social and environmental benefits.
Disclosure of Invention
Technical problem to be solved
Aiming at the problems in the prior art, the invention aims to develop a method for directly producing butanol by fermentation under the condition of widely using cheap production raw materials and not adding exogenous enzyme preparations.
(II) technical scheme
In order to solve the technical problem, the invention provides a method for preparing butanol by directly fermenting starch-containing waste by adding Clostridium butyricum Clostridium saccharoperbutylacetonicum N1-4(Al-Shorgani et Al, 2012), namely, the waste containing starchy materials are used as fermentation raw materials to be pretreated, and fermentation seed liquid is directly inoculated for anaerobic fermentation to prepare the butanol.
Further, starchy waste materials including bread crumbs, kitchen waste, industrial flour processing residues, starch residues of starch processing industries such as raw flour slurry and potatoes, and grain products such as corn and cassava are not added with any nutrient salt solution and amylase or saccharifying enzyme.
Further, the starch-containing waste is sorted, purified, crushed and sterilized at 115-121 ℃ for 15-20 min to prepare the fermentation medium.
Further, the fermentation strain is Clostridium saccharoperbutylacetonicum N1-4, the inoculation amount is 10% (v/v), and the anaerobic fermentation temperature is 30-35 ℃.
Further, the fermentation strain is obtained by heat shock and activation of sandy soil containing spores of Clostridium saccharoperbutylacetonicum N1-4 strain preserved under the condition of normal temperature drying, and the steps are as follows: (1) culture medium: preparing fresh mashed potato 150g/L, (NH)4)2SO40.5g/L glucose 10g/L, CaCO3Sterilizing 3g/L Potato Glucose (PG) culture medium at 121 deg.C for 60 min; (2) preparing a spore suspension: weighing 1.0-2.0 g of preserved sandy soil containing bacterial strain spores under an aseptic condition, placing the preserved sandy soil into a PG culture medium, carrying out hot shock on the spores in a boiling water bath for 1min, rapidly cooling to room temperature, and carrying out anaerobic culture for 24h to obtain the strain spores; (3) and (3) activation: inoculating 1mL of strain spore suspension into a fresh 9mL of PG culture medium, carrying out hot shock in a boiling water bath for 1min, and carrying out anaerobic culture for 24h to obtain an activated seed solution for subsequent production.
Further, the activated seed solution is inoculated into a tryptone-yeast powder-acetate (TYA) culture medium rich in nitrogen source according to the inoculation amount of 10% (v/v) for enrichment culture, and the initial pH value is adjusted to be 6.0-6.5, and the culture is carried out for 15-17 h under anaerobic condition, so as to prepare the enriched and efficient logarithmic phase fermentation seed solution for the main ABE fermentation of the biomass substrate.
Further, inoculating the fermentation seed liquid into a fermentation culture medium with the inoculation amount of 10% (v/v), and culturing for 60-72 h under an anaerobic condition to produce butanol through fermentation.
(III) advantageous effects
The technical scheme of the invention has the following beneficial effects: the method utilizes clostridium saccharoacetobutylicum to directly utilize starch-containing waste to carry out anaerobic ABE fermentation to produce butanol; the method does not need additional saccharifying enzyme and other enzymes or auxiliary substances, has wide raw material sources, low cost and simple production process, effectively solves the problems of low raw material cost, high cost of enzyme preparation addition and high energy consumption in the traditional butanol production, and provides a new method for butanol production.
Drawings
FIG. 1 shows the substrates glucose and starch, respectively, at an initial concentration of (a) glucose 40 g/L; (b) 50g/L of glucose; (c) glucose 60 g/L; (d) glucose 80g/L (e) starch 40 g/L; (f) 50g/L of starch; (g) 60g/L of starch; (h) starch 80g/L, comparison of butanol yield when inoculated with Clostridium saccharoperbutylacetonicum N1-4 fermentation. Wherein the total solvent refers to the total yield of acetone-butanol-ethanol
FIG. 2 shows the butanol yield of the strain Clostridium saccharoperbutylacetonicum N1-4 in the saccharification and direct fermentation processes using the starch-containing waste kitchen garbage with a solid-to-liquid ratio of 1:2 (w/v). (a) Saccharifying and fermenting the kitchen waste; (b) kitchen waste direct fermentation
Detailed Description
The following examples further describe embodiments of the present invention in detail. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
In this example, fermentation media with glucose concentration of 40, 50, 60, 80g/L were prepared, i.e. 12, 15, 18, 24g of glucose was dissolved in 70mL of deionized water, 200mL of TYA medium (containing no glucose carbon source) was prepared, the glucose solution and TYA medium were sterilized at 115 ℃ for 15min, after they were naturally cooled, the glucose solution and TYA medium were mixed under aseptic conditions, then 30mL of fermentation seed solution of strain Clostridium saccharophagaceae family N1-4 was inoculated therein, the inoculum size was 10% (v/v), sterile nitrogen was introduced for 10min, and anaerobic fermentation was carried out at 32 ℃ for 72h to obtain butanol, the fermentation results of which are shown in table 1 and fig. 1.
Example 2
In the embodiment, 100g of kitchen waste is taken, and large bones, napkin paper, disposable tableware and the like are removed and then crushed. The ratio of the treated kitchen waste to water in a solid-liquid ratio is 1:2(w/v), the pH value is adjusted to be 4.2, 200U/g of saccharifying enzyme is added at 60 ℃, the pH value is adjusted to be 6.2 after 6 hours of saccharification, the following steps of sterilization, inoculation, fermentation and the like are the same as those in example 1, and the fermentation results are shown in table 1 and figure 2.
Example 3
This example prepares 20g/L pure 100% (w/w) amylose, 27% (w/w) amylose-containing corn starch, and 15% (w/w) amylose-containing potato starch fermentation media by adding 0.2g of each different starch to 2mL of deionized water to prepare 7mL of a TYA medium (containing no glucose carbon source), sterilizing the starch solution and TYA medium at 115 ℃ for 15min, mixing the starch solution and TYA medium under aseptic conditions after natural cooling, inoculating 1mL of fermentation seed solution of Clostridium saccharophagyllotylacetonicum N1-4 therein, inoculating 10% (v/v), and anaerobically fermenting at 30 ℃ for 72h to obtain butanol, the fermentation results of which are shown in Table 1.
Comparative example 1
In this comparative example, fermentation media containing starch at concentrations of 40, 50, 60, and 80g/L were prepared by mixing 12, 15, 18, and 24g of starch with 70mL of deionized water, respectively, to prepare 200mL of TYA medium (containing no glucose carbon source), sterilizing the starch solution and TYA medium at 115 deg.C for 15min, cooling naturally, sterilizing, inoculating, and fermenting the same as in example 1, and the fermentation results are shown in Table 1 and FIG. 1.
The results of example 1 and comparative example 1 show that the strain Clostridium saccharoperbutylacetonicum N1-4 can directly and efficiently utilize starch as a substrate to ferment and produce butanol, and the direct utilization of starch as a substrate can obtain higher carbon conversion rate and butanol production rate.
Comparative example 2
100g of kitchen waste is taken in the comparative example, and large bones, napkin paper, disposable tableware and the like are removed and crushed. The ratio of the treated kitchen waste to water in solid-liquid ratio is 1:2(w/v), the initial pH value is adjusted to be 6.2, the following steps of sterilization, inoculation, fermentation and the like are the same as those in example 1, and the fermentation results are shown in table 1 and fig. 2.
Results of the embodiment 2 and the comparative example 2 show that the strain Clostridium saccharoperbutylacetonicum N1-4 can directly and efficiently utilize the starchy waste kitchen waste to ferment and produce butanol, the starch-containing waste kitchen waste can be directly fermented to produce butanol with higher carbon conversion rate without saccharification, and the butanol production rate can reach 2 times of saccharification and fermentation.
The butanol yields in the examples and comparative examples are shown in table 1:
TABLE 1 fermentation kinetics parameters in butanol production Process
In the above examples and comparative examples, the selection of specific disclosed data, the selection of any raw material, and the like are not intended to limit the scope of protection in the claims, and any specific numerical value, raw material, and the like in each numerical range of the claims can be selected to completely implement the technical scheme of the present invention.
References
1.Al-Shorgani,N.K.N.,Ali,E.,Kalil,M.S.,Yusoff,W.M.W.,2012.Bioconversion of Butyric Acid to Butanol by Clostridium saccharoperbutylacetonicum N1-4(ATCC 13564)in a Limited Nutrient Medium.BioEnergy Research,5,287-293.
Claims (6)
1. A method for preparing butanol by using starch-containing waste is characterized in that starch-containing waste raw materials are subjected to sorting, impurity removal, crushing and high-temperature sterilization, then inoculated with fermentation seed liquid of a fermentation strain Clostridium saccharoperbutylacetonicum N1-4, and subjected to ABE fermentation directly under a certain temperature condition, wherein the fermentation seed liquid is obtained by strain activation and enrichment, and no nutrient salt solution and amylase or glucoamylase are added into the starch-containing waste; the method specifically comprises the following steps:
sorting the starch-containing waste, removing non-fermentable impurities, crushing, and sterilizing at 115-121 ℃ for 15-20 min to prepare a substrate culture medium as a fermentation raw material; the concentration of starch in a substrate is 40-80%, and the initial pH value is 6.0-6.5;
inoculating a proper amount of sandy soil containing Clostridium saccharoperbutylacetonicum N1-4 strain spores preserved under the normal-temperature drying condition into a potato glucose culture medium, and performing activation culture after hot shock in a boiling water bath to obtain activated seed liquid;
inoculating the activated seed liquid into a tryptone-yeast powder-acetate culture medium rich in nitrogen source for enrichment culture, adjusting the pH value to be 6.0-6.5 initially, and culturing for 15-17 h under anaerobic condition to prepare enriched and efficient logarithmic phase fermentation seed liquid for main ABE fermentation of a biomass substrate;
inoculating the fermentation seed liquid into a fermentation culture medium with the inoculation amount of 10%, and culturing for 60-72 h under an anaerobic condition to produce butanol through fermentation.
2. The method for preparing butanol using starch-containing waste according to claim 1, wherein the starch-containing waste is used as a raw material, and comprises bread crumbs, kitchen waste, industrial flour processing residues, raw flour slurry, starch residues of potato starch processing industry, and corn, tapioca grain products or their processing residues.
3. The method for preparing butanol using starch-containing waste according to claim 1, wherein said strain Clostridium saccharoperbutylacetonicum N1-4 performs butanol-producing fermentation using amylose and amylopectin.
4. The method for preparing butanol using starch-containing waste according to claim 1, wherein the fermentation temperature is 30 to 35 ℃.
5. The method for preparing butanol using starch-containing waste according to claim 1, wherein the fermentation strain activation method comprises: the potato glucose culture medium contains proper amount of fresh mashed potato and (NH)4)2SO4Glucose and CaCO3Sterilizing at 121 deg.C for 60 min.
6. The method for preparing butanol using starch-containing waste according to claim 1, wherein the fermentation strain activation method comprises: inoculating the strain spore suspension into a fresh potato glucose culture medium, performing hot shock in a boiling water bath for 1min, and performing anaerobic culture for 24h to obtain an activated seed solution for subsequent production.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810187132.8A CN108410912B (en) | 2018-03-07 | 2018-03-07 | Method for directly preparing butanol by using starch-containing waste |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810187132.8A CN108410912B (en) | 2018-03-07 | 2018-03-07 | Method for directly preparing butanol by using starch-containing waste |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108410912A CN108410912A (en) | 2018-08-17 |
| CN108410912B true CN108410912B (en) | 2021-08-03 |
Family
ID=63130484
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810187132.8A Active CN108410912B (en) | 2018-03-07 | 2018-03-07 | Method for directly preparing butanol by using starch-containing waste |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108410912B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113215204A (en) * | 2021-03-01 | 2021-08-06 | 北京科技大学 | Method for efficiently preparing butanol from perishable garbage by combining lactic acid pre-fermentation with ABE fermentation |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101358187A (en) * | 2008-09-26 | 2009-02-04 | 清华大学 | Method for obtaining butanol high yield strain using <60+>Co gamma-ray irradiation |
| CN101845413A (en) * | 2010-05-05 | 2010-09-29 | 华北制药集团有限责任公司 | Clostridium acetobutylicum and construction method and purposes thereof |
| WO2014122449A1 (en) * | 2013-02-05 | 2014-08-14 | Green Biologics Ltd | Cyclodextrin glucanotransferase |
-
2018
- 2018-03-07 CN CN201810187132.8A patent/CN108410912B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101358187A (en) * | 2008-09-26 | 2009-02-04 | 清华大学 | Method for obtaining butanol high yield strain using <60+>Co gamma-ray irradiation |
| CN101845413A (en) * | 2010-05-05 | 2010-09-29 | 华北制药集团有限责任公司 | Clostridium acetobutylicum and construction method and purposes thereof |
| WO2014122449A1 (en) * | 2013-02-05 | 2014-08-14 | Green Biologics Ltd | Cyclodextrin glucanotransferase |
Non-Patent Citations (7)
| Title |
|---|
| A Novel Process for Direct Production of Acetone–Butanol–Ethanol from Native Starches Using Granular Starch Hydrolyzing Enzyme by Clostridium saccharoperbutylacetonicum N1-4;Vu Hong Thang等;《Appl Biochem Biotechnol》;20131126(第172期);第1818页摘要 * |
| Bioconversion of Butyric Acid to Butanol by Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564) in a Limited Nutrient Medium;Najeeb Kaid Nasser Al-Shorgani等;《Bioenerg. Res.》;20110503;第2012卷(第5期);第287-293页 * |
| Fermentation of sago starch to biobutanol in a batch culture using Clostridium saccharoperbutylacetonicum N1-4 (ATCC 13564);Najeeb Kaid Nasser Al-Shorgani等;《Ann Microbiol》;20110924;第2012卷(第62期);第1060-1061页 * |
| Metabolic analysis of butanol production from acetate 1 in Clostridium;Ming Gao等;《RSC Advances》;20141222;第2015卷(第5期);第1-29页 * |
| Production of Acetone–Butanol–Ethanol (ABE) in Direct Fermentation of Cassava by Clostridium saccharoperbutylacetonicum N1-4;Vu Hong Thang等;《Appl Biochem Biotechnol》;20090922;第2010卷(第161期);第158页第1段、第158页第3段-159页 * |
| 餐厨垃圾发酵制备生物燃料丁醇的研究;石姗姗等;《环境工程》;20170222;第35卷(第2期);第117-122页 * |
| 餐厨垃圾直接发酵生产生物质丁醇的研究;王永林等;《环境工程》;20190415;第37卷(第4期);第1-6页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108410912A (en) | 2018-08-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Bioethanol production from taro waste using thermo-tolerant yeast Kluyveromyces marxianus K21 | |
| Virunanon et al. | Cassava pulp enzymatic hydrolysis process as a preliminary step in bio-alcohols production from waste starchy resources | |
| Thatoi et al. | Bioethanol production from tuber crops using fermentation technology: a review | |
| Lin et al. | Fermentative hydrogen production from wastewaters: a review and prognosis | |
| US7449313B2 (en) | Systems and processes for cellulosic ethanol production | |
| Cheng et al. | Fungal pretreatment enhances hydrogen production via thermophilic fermentation of cornstalk | |
| CN100478437C (en) | Method of producing fuel alcohol by kitchen garbage fermentation | |
| CN102453614B (en) | New method for comprehensively utilizing food wastes | |
| Shen et al. | Succinic acid production from duckweed (Landoltia punctata) hydrolysate by batch fermentation of Actinobacillus succinogenes GXAS137 | |
| Waqas et al. | Conversion of food waste to fermentation products | |
| Ghaleb | Biorefinery of industrial potato wastes to ethanol by solid state fermentation | |
| CN101638673B (en) | Method for manufacturing alcohol by utilizing fermentation of plant straws | |
| WO2010072093A1 (en) | Method for producing cellulosic ethanol | |
| Wang et al. | High-efficient production of citric acid by Aspergillus niger from high concentration of substrate based on the staged-addition glucoamylase strategy | |
| Harde et al. | Continuous lignocellulosic ethanol production using Coleus forskohlii root hydrolysate | |
| CN102041235A (en) | High-temperature resistant saccharomyces cerevisiae and application thereof | |
| Ungureanu et al. | Capitalization of wastewater-grown algae in bioethanol production | |
| Moshi et al. | Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of inedible wild cassava flour to bioethanol | |
| CN102250967B (en) | Method for preparing biofuel butanol from foodstuff wastes | |
| Ebrahimian et al. | Coproduction of hydrogen, butanol, butanediol, ethanol, and biogas from the organic fraction of municipal solid waste using bacterial cocultivation followed by anaerobic digestion | |
| Bhatia et al. | Banana peel waste as substrate for ethanol production | |
| CN101878308B (en) | A process for the preparation of ethanol from starch | |
| CN108410912B (en) | Method for directly preparing butanol by using starch-containing waste | |
| CN111394397A (en) | Method for producing caproic acid by fermenting kitchen waste | |
| CN106893682B (en) | Method for expanding culture of saccharomycetes by using liquefied mash and application of saccharomycetes and method for fermenting 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |