DE2603889C3 - Verfahren zur enzymatischen Hydrolyse von Cellulose zu wasserlöslichen Zuckern - Google Patents
Verfahren zur enzymatischen Hydrolyse von Cellulose zu wasserlöslichen ZuckernInfo
- Publication number
- DE2603889C3 DE2603889C3 DE2603889A DE2603889A DE2603889C3 DE 2603889 C3 DE2603889 C3 DE 2603889C3 DE 2603889 A DE2603889 A DE 2603889A DE 2603889 A DE2603889 A DE 2603889A DE 2603889 C3 DE2603889 C3 DE 2603889C3
- Authority
- DE
- Germany
- Prior art keywords
- cellulose
- enzyme
- hydrolysis
- aqueous
- water
- 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.)
- Expired
Links
- 239000001913 cellulose Substances 0.000 title claims description 39
- 229920002678 cellulose Polymers 0.000 title claims description 39
- 235000000346 sugar Nutrition 0.000 title claims description 17
- 230000007071 enzymatic hydrolysis Effects 0.000 title claims description 12
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 title claims description 12
- 150000008163 sugars Chemical class 0.000 title claims description 11
- 238000000034 method Methods 0.000 title claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 49
- 102000004190 Enzymes Human genes 0.000 claims description 49
- 238000006460 hydrolysis reaction Methods 0.000 claims description 17
- 230000007062 hydrolysis Effects 0.000 claims description 16
- 230000001461 cytolytic effect Effects 0.000 claims description 12
- 241000223261 Trichoderma viride Species 0.000 claims description 9
- 244000005700 microbiome Species 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 235000015097 nutrients Nutrition 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 5
- 239000002699 waste material Substances 0.000 claims description 5
- 229940088598 enzyme Drugs 0.000 description 43
- 235000010980 cellulose Nutrition 0.000 description 34
- 239000000835 fiber Substances 0.000 description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 239000007787 solid Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 108010059892 Cellulase Proteins 0.000 description 4
- 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 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 241000218657 Picea Species 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- WDMUXYQIMRDWRC-UHFFFAOYSA-N 2-hydroxy-3,4-dinitrobenzoic acid Chemical compound OC(=O)C1=CC=C([N+]([O-])=O)C([N+]([O-])=O)=C1O WDMUXYQIMRDWRC-UHFFFAOYSA-N 0.000 description 2
- 244000144725 Amygdalus communis Species 0.000 description 2
- 241000427940 Fusarium solani Species 0.000 description 2
- 241000577872 Fusarium striatum Species 0.000 description 2
- 229920001131 Pulp (paper) Polymers 0.000 description 2
- 241000378866 Trichoderma koningii Species 0.000 description 2
- 239000008351 acetate buffer Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000011491 glass wool Substances 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000006286 aqueous extract Substances 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 235000021186 dishes Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009837 dry grinding Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000012065 filter cake Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000001965 potato dextrose agar Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000004537 pulping Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 235000021309 simple sugar Nutrition 0.000 description 1
- 235000020374 simple syrup Nutrition 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000001238 wet grinding Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
-
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
- C13K1/00—Glucose; Glucose-containing syrups
- C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
-
- 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
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/911—Microorganisms using fungi
- Y10S435/945—Trichoderma
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Emergency Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Biological Depolymerization Polymers (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US57242875A | 1975-04-28 | 1975-04-28 | |
| US05/614,490 US3990945A (en) | 1975-04-28 | 1975-09-18 | Enzymatic hydrolysis of cellulose |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| DE2603889A1 DE2603889A1 (de) | 1976-11-04 |
| DE2603889B2 DE2603889B2 (de) | 1977-10-06 |
| DE2603889C3 true DE2603889C3 (de) | 1978-08-17 |
Family
ID=27075834
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| DE2603889A Expired DE2603889C3 (de) | 1975-04-28 | 1976-02-02 | Verfahren zur enzymatischen Hydrolyse von Cellulose zu wasserlöslichen Zuckern |
Country Status (8)
| Country | Link |
|---|---|
| US (1) | US3990945A (enExample) |
| JP (1) | JPS51128491A (enExample) |
| CA (1) | CA1051803A (enExample) |
| DE (1) | DE2603889C3 (enExample) |
| FR (1) | FR2309637A1 (enExample) |
| GB (1) | GB1489145A (enExample) |
| IT (1) | IT1055857B (enExample) |
| NL (1) | NL7601080A (enExample) |
Families Citing this family (42)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4110475A (en) * | 1977-05-11 | 1978-08-29 | Chevron Research Company | Cellulose fermentation process |
| US4237226A (en) * | 1979-02-23 | 1980-12-02 | Trustees Of Dartmouth College | Process for pretreating cellulosic substrates and for producing sugar therefrom |
| US4220721A (en) * | 1979-04-27 | 1980-09-02 | University Of Arkansas Foundation | Method for enzyme reutilization |
| DE2953302A1 (de) * | 1979-09-28 | 1982-02-04 | Vsesoyuzny Ni Biotek Inst | Method of production of ethyl alcohol from starch raw material |
| JPS56127601A (en) * | 1980-03-10 | 1981-10-06 | Baiorisaac Center:Kk | Treating method of substance containing cellulose |
| US5302611A (en) * | 1980-10-07 | 1994-04-12 | Klaus Keplinger | Oxindole alkaloids having properties stimulating the immunologic system & preparation containing the same |
| US4409329A (en) * | 1981-03-23 | 1983-10-11 | Gulf Research & Development Company | Saccharification method |
| GB2116977B (en) * | 1981-12-22 | 1985-07-03 | Novo Industri As | An agent for decomposition of vegetable remanence especially soy remanence a method for production of a purified vegetable protein product and a purified vegetable protein product |
| US4609624A (en) * | 1984-02-03 | 1986-09-02 | Les Services De Consultation D.B. Plus Limitee | Process for producing isopropyl alcohol from cellulosic substrates |
| US5348871A (en) * | 1992-05-15 | 1994-09-20 | Martin Marietta Energy Systems, Inc. | Process for converting cellulosic materials into fuels and chemicals |
| US5407817A (en) * | 1993-12-23 | 1995-04-18 | Controlled Environmental Systems Corporation | Municipal solid waste processing facility and commercial ethanol production process |
| US5571703A (en) * | 1993-12-23 | 1996-11-05 | Controlled Environmental Systems Corporation | Municipal solid waste processing facility and commercial ethanol production process |
| AU5099599A (en) * | 1998-07-14 | 2000-02-07 | Colorado State University Research Foundation | Bio-reaction process and product |
| US20030044951A1 (en) * | 1998-07-14 | 2003-03-06 | Sporleder Robert A. | Bio-reaction process and product |
| US8093456B2 (en) | 2000-10-20 | 2012-01-10 | Board Of Trustees Of Michigan State University | Transgenic cover plants containing hemicellulase and cellulase which degrade lignin and cellulose to fermentable sugars |
| AU2002211798A1 (en) | 2000-10-20 | 2002-05-06 | Michigan State University | Transgenic plants containing ligninase and cellulase which degrade lignin and cellulose to fermentable sugars |
| US20050198704A1 (en) * | 2000-10-20 | 2005-09-08 | Board Of Trustees Of Michigan State University | Chloroplast transgenesis of monocots: bioconfined genetically engineered monocot crops that will eliminate transgene flow |
| US20070192900A1 (en) * | 2006-02-14 | 2007-08-16 | Board Of Trustees Of Michigan State University | Production of beta-glucosidase, hemicellulase and ligninase in E1 and FLC-cellulase-transgenic plants |
| ES2810026T3 (es) | 2004-03-25 | 2021-03-08 | Novozymes Inc | Métodos para la degradación o la conversión de polisacáridos de la pared celular vegetal |
| US7259231B2 (en) | 2004-10-12 | 2007-08-21 | Yulex Corporation | Extraction and fractionation of biopolymers and resins from plant materials |
| US7923039B2 (en) | 2005-01-05 | 2011-04-12 | Yulex Corporation | Biopolymer extraction from plant materials |
| FR2881753B1 (fr) * | 2005-02-09 | 2009-10-02 | Inst Francais Du Petrole | Procede de production d'enzymes cellulolytiques et hemicellulolytiques utilisant les residus de distillation de fermentation ethanolique d'hydrolysats enzymatiques de materiaux (ligno-)cellulosique |
| UA100662C2 (uk) * | 2005-07-19 | 2013-01-25 | Інбікон А/С | Спосіб перетворення целюлозного матеріалу в етанол |
| US7815741B2 (en) | 2006-11-03 | 2010-10-19 | Olson David A | Reactor pump for catalyzed hydrolytic splitting of cellulose |
| US7815876B2 (en) | 2006-11-03 | 2010-10-19 | Olson David A | Reactor pump for catalyzed hydrolytic splitting of cellulose |
| WO2008133742A2 (en) * | 2006-12-06 | 2008-11-06 | Musc Foundation For Research Development | Apparatus and methods for the production of ethanol, hydrogen and electricity |
| WO2009076760A1 (en) * | 2007-12-19 | 2009-06-25 | Fpinnovations | Conversion of knot rejects from chemical pulping |
| US8495149B2 (en) * | 2008-05-15 | 2013-07-23 | International Business Machines Corporation | Off-line smartphone file system snapshots |
| CA2757040C (en) | 2009-03-31 | 2016-02-09 | Codexis, Inc. | Improved endoglucanases |
| CH700770A2 (de) | 2009-04-15 | 2010-10-15 | Philippe Saint Ger Ag | Verfahren zum Unterstützen und/oder Intensivieren einer physikalischen und/oder chemischen Reaktion und eine Reaktionseinrichtung zum Ausführen des Verfahrens. |
| WO2010124944A1 (en) * | 2009-04-28 | 2010-11-04 | Heli Inovatio Handelsbolag | Process for the hydrolysis of cellulose |
| US9493802B2 (en) | 2010-08-20 | 2016-11-15 | Codexis, Inc. | Use of glycohydrolase 61 protein variants with improved thermostability for processing cellulose |
| EP2471940A1 (en) | 2010-12-31 | 2012-07-04 | Süd-Chemie AG | Efficient lignocellulose hydrolysis with integrated enzyme production |
| US8759050B2 (en) | 2011-02-14 | 2014-06-24 | Quad County Corn Processors | Process and system for producing ethanol from a byproduct of an ethanol production facility |
| US8633003B2 (en) | 2011-02-14 | 2014-01-21 | Quad County Corn Processors | Process and system for producing ethanol from a byproduct of an ethanol production facility |
| EP2748317B1 (en) | 2011-08-22 | 2017-04-19 | Codexis, Inc. | Gh61 glycoside hydrolase protein variants and cofactors that enhance gh61 activity |
| FR2984353B1 (fr) | 2011-12-14 | 2014-11-21 | IFP Energies Nouvelles | Procede de production d'un cocktail enzymatique utilisant les residus solides d'un procede de conversion biochimique de materiaux ligno-cellulosiques |
| EP2794871A4 (en) | 2011-12-20 | 2015-12-30 | Codexis Inc | VARIANTS OF ENDOGLUCANASE-1B (EG1B) |
| DK2859099T3 (da) | 2012-06-11 | 2019-07-29 | Codexis Inc | Svampe-beta-xylosidase-varianter |
| FR2991998B1 (fr) | 2012-06-18 | 2014-07-11 | IFP Energies Nouvelles | Utilisation du co2 pour la desactivation d'un microorganisme cellulolytique utilise dans l a conversion biochimique de materiaux ligno-cellulosiques |
| FR3014903B1 (fr) | 2013-12-17 | 2017-12-01 | Ifp Energies Now | Procede d'hydrolyse enzymatique avec production in situ de glycosides hydrolases par des microorganismes genetiquement modifies (mgm) et non mgm |
| US9752165B2 (en) | 2014-02-10 | 2017-09-05 | Cellulosic Ethanol Technologies, Llc | Processes and systems for recovering oil from fermentation products |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3462275A (en) * | 1968-01-31 | 1969-08-19 | Gen Electric | Waste conversion process and product |
| DE1908225C3 (de) * | 1969-02-14 | 1975-08-14 | 1000 Berlin | Biotechnisches Verfahren zur Herstellung von Enzymen, die stärkeartige Polysaccharide und Dextrane sowie Oligosaccharide der entsprechenden Bindungstypen hydrolytisch spalten |
| US3734831A (en) * | 1970-11-27 | 1973-05-22 | Canadian Patents Dev | Production of cellulase |
| US3764475A (en) * | 1971-12-22 | 1973-10-09 | Us Army | Enzymatic hydrolysis of cellulose to soluble sugars |
| US3812013A (en) * | 1972-03-01 | 1974-05-21 | Gen Electric | Soluble cellulase enzyme production |
| JPS531834B2 (enExample) * | 1973-03-28 | 1978-01-23 | ||
| JPS5548799B2 (enExample) * | 1973-03-31 | 1980-12-08 |
-
1975
- 1975-09-18 US US05/614,490 patent/US3990945A/en not_active Expired - Lifetime
-
1976
- 1976-01-23 CA CA244,162A patent/CA1051803A/en not_active Expired
- 1976-01-28 GB GB3224/76A patent/GB1489145A/en not_active Expired
- 1976-02-02 IT IT19808/76A patent/IT1055857B/it active
- 1976-02-02 DE DE2603889A patent/DE2603889C3/de not_active Expired
- 1976-02-03 NL NL7601080A patent/NL7601080A/xx not_active Application Discontinuation
- 1976-03-12 JP JP51027608A patent/JPS51128491A/ja active Granted
- 1976-04-05 FR FR7609774A patent/FR2309637A1/fr active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| DE2603889B2 (de) | 1977-10-06 |
| JPS573357B2 (enExample) | 1982-01-21 |
| JPS51128491A (en) | 1976-11-09 |
| CA1051803A (en) | 1979-04-03 |
| GB1489145A (en) | 1977-10-19 |
| DE2603889A1 (de) | 1976-11-04 |
| US3990945A (en) | 1976-11-09 |
| FR2309637B1 (enExample) | 1981-08-07 |
| FR2309637A1 (fr) | 1976-11-26 |
| IT1055857B (it) | 1982-01-11 |
| NL7601080A (nl) | 1976-11-01 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C3 | Grant after two publication steps (3rd publication) | ||
| 8339 | Ceased/non-payment of the annual fee |