WO2011045625A1 - Using cell debris generated from pha recovery for enhanced cell growth and biopolyester formation - Google Patents
Using cell debris generated from pha recovery for enhanced cell growth and biopolyester formation Download PDFInfo
- Publication number
- WO2011045625A1 WO2011045625A1 PCT/IB2009/007142 IB2009007142W WO2011045625A1 WO 2011045625 A1 WO2011045625 A1 WO 2011045625A1 IB 2009007142 W IB2009007142 W IB 2009007142W WO 2011045625 A1 WO2011045625 A1 WO 2011045625A1
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- WO
- WIPO (PCT)
- Prior art keywords
- pha
- cell debris
- solution
- cell
- process according
- Prior art date
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Classifications
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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
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
Definitions
- the present invention relates to a process for producing biodegradable polymeric materials from an organic carbon source, e.g. sugar beet pulp and molasses. Particularly, the present invention relates to a process for producing biodegradable polymeric materials including polyhydroxyalkanoates with cell debris, a type of organic waste left from recovery and purification of polyhydroxyalkanoates from cells.
- an organic carbon source e.g. sugar beet pulp and molasses.
- biodegradable polymeric materials including polyhydroxyalkanoates with cell debris, a type of organic waste left from recovery and purification of polyhydroxyalkanoates from cells.
- Polyhydroxyalkanoates are homopolymers or copolymers of hydroxyalkanoates, such as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4- hydroxyvalerate (4HV) and 3-hydroxyhexanoate (3HH). These thermoplastic or elastic biopolymers are synthesized and accumulated by many microorganisms, bacteria in particular, as carbon and energy storage materials. PHAs are conveniently synthesized by cultivating the microbial cells in an aqueous medium on a carbon source, including sugars, organic acids and alcohols. Depending on strains and growth conditions, the biopolyesters in a form of small granules (0.3-0.5 ⁇ ) may account for 50-80 wt% of dry cell mass.
- the PHA granules should be separated from the rest non-PHA cell mass so that the bioplastics have the desired purity and material property.
- the residual cell debris from PHA recovery consisting of proteins, nucleic acids, lipids and wall fragments, has no market value and is either discarded as a solid waste or discharged in process wastewater.
- U.S. Patent No. 7,514,525 relates to a method to recover, purify and isolate PHA biopolymers from PHA-containing cell mass, which includes: (a) solubilizing the non- pHA cell mass in an acidic solution, leaving a suspension of partially crystallized PHA granules; (b) adjusting the pH of the suspension to 7-1 1 and separating the PHA solids from the dissolved non-PHA cellular mass; (c) resuspending the PHA solids in a bleaching solution for decolorization; and (d) drying the resulting PHA solids. About 95% or greater of original PHA in cell mass is recovered, and the purity of PHA solids is about 97% or above.
- the Applicant has faced a problem of improving the yield of a process for producing PHAs from an organic carbon source by microbial fermentation.
- the Applicant has also faced the problem of disposing the cell debris left from PHA recovery and purification.
- the Applicant has found that the above problems can be solved by using the cell debris obtained upon solubilizing the non-PHA cell, mass to further feed the PHA producing microorganisms. Reuse of the above cell debris as nutrients remarkably increases the yields of cell growth and PHA synthesis on carbonaceous substrates such as glucose, fructose and sucrose, since it can be readily assimilated by the microbial cells.
- the present invention relates to a process for producing biodegradable polymeric materials including polyhydroxyalkanoates (PHAs) from an organic carbon source, which comprises:
- organic carbon source any organic compound or mixtures thereof which can be metabolized by PHA-producing microbial cells, such as glucose, fructose, sucrose or similar carbohydrates or any organic mixtures of carbohydrates such as sugar beet pulp, sugar beet molasses, sugar cane molasses.
- the medium solution besides the organic carbon source, may contain additional organic growth factors, N, P and/or other minerals as nutrients for the cell growth.
- the solubilizing step (b) is carried out by: (bl) solubilizing the non-PHA cell mass in an acidic solution to obtain a first suspension of a PHA solid in an acidic cell debris solution;
- the cells containing PHA granules are first treated in an acidic solution releasing a substantial portion of proteins (acidic cell debris).
- the PHA granules are partially crystallized from the original labile structure, which makes the polyesters tough and resistant to chemical digestion.
- the acidic treatment may be carried out by adding an aqueous solution of a strong acid, such as sulfuric acid.
- the aqueous solution of a strong acid is added to the non-PHA cell mass in an amount so as to achieve a concentration of hydrogen ions (H + ) from 0.01 to 0.5 mole/L.
- the solubilizing step (bl) is preferably carried out at a temperature from 80° to 130°C, for a time from 0.5 to 5 hours.
- the cells with PHA granules are further treated in a base solution to dissolve the rest non-PHA cell mass.
- the base solution is an aqueous solution of at least one strong base, such as sodium hydroxide or potassium hydroxide.
- the alkaline treatment may be assisted by adding at least one surfactant, preferably at least one ionic surfactant, preferably a C 6 -Ci8 alkyl sulfate, such as sodium dodecyl sulfate (SDS).
- the at least one surfactant promotes cell disruption and membrane decomposition.
- the at least one surfactant is preferably added in an amount of from 2 to 10 g/L, preferably from 4 to 7 g/L.
- the above two-step treatment digests and dissolves most of non-PHA cell mass while little PHA is lost. Equally important, the treatments can convert the cell debris into appropriate forms that can be directly assimilated by microbial cells as nutrients for growth and PHA formation.
- ACDS Acidic cell debris solution
- BCDS Basic cell debris solution
- surfactant that contains am io acids, fatty acids and other digested cell components derived from the acid-treated cell residues under alkaline conditions.
- Acid-base cell debris solution optionally including at least one surfactant, that contains amino acids, fatty acids, peptides and other digested cell - components in both acid and base conditions. This aqueous waste solution is generated when the sequential acid and base treatments are performed without cell separation.
- these three types of cell debris may be reused as nutrients in microbial production of PHA biopolyester.
- a separating step (cl ) is carried out on the first suspension and the resulting acidic cell debris solution is fed according to step (d).
- a separating step (c2) is carried out on the second suspension and the resulting basic cell debris solution is fed according to step (d).
- both the separating step (cl ) and the separating step (c2) are carried out on the first suspension and on the second suspension respectively, and the resulting cell debris solutions are fed according to step (d).
- a separating step (c3) is carried out only on the second suspension and the resulting acid-base cell debris solution is fed according to step (d).
- the total amount of cell debris that is co-fed with the organic carbon source to the cultivation step (a) is from 5 to 50% by weight, preferably from 10 to 40% by weight, with respect to the weight of the glucose-equivalent carbon substrate.
- weight of the glucose-equivalent carbon substrate it is meant the amount of the carbon organic source expressed by the corresponding amount of glucose as substrate for the microbial fermentation.
- the amount of cell debris is preferably from 5 to 20% by weight with respect to the weight of the glucose-equivalent carbon substrate.
- an inhibition effect by the ACDS has been observed on cell growth and PHA formation when the above amount is greater than 20% by weight. Such an effect has not been observed when feeding the BCDS or the ABCDS.
- the PHA solid obtained from the separation step (c) may be subjected to further treatments, such as decolorization, washing and/or drying for achieving the desired product purity and quality according to known techniques, such as those reported in the above-cited U.S. Patent 7,514,525, whose disclosure is herein incorporated by reference.
- the cells containing PHA granules were harvested from medium with centrifugation at 5,000 g for 10 min.
- the wet cell pellets had a density of 450-500 g dry mass/L depending on medium volume, centrifugation force and time.
- PHA were recovered and purified from the cells by removing the non-PHA cell mass (about 30% w/w) in three steps: (1) acid pretreatment, (2) base treatment, (3) hypochlorite decolorization, and (4) washing and drying. They are detailed as follows.
- Acid pretreatment The wet cell pellets from fermentation broth were re- suspended in an equivalent volume of 0.2M H 2 S0 4 aqueous solution. The slurry of 200- 250 g dry matter/L was heated to boil and then maintained for one hour under ambient conditions. After cooling to room temperature, the acid pretreated pellets were separated from the acidic solution with centrifugation at 5,000 g for 10 min. The clean supernatant had a brownish color and a solid content between 35-65 g/L depending on the density of slurry and treatment conditions. It is referred as acidic cell debris solution (ACDS) thereafter.
- ACDS acidic cell debris solution
- hypochlorite decolorization The wet pellets from base treatment were re-suspended in a commercially available bleaching solution containing 6% w/w of hypochlorite. The volume of bleaching solution was estimated based 1 part of hypochlorite for 1 part of PHA-containing dry solid. The amount of PHA solid was estimated from the wet mass and its dry solid content of 60% w/w. The slurry was stirred for 2 hours under ambient conditions. A white PHA pellets were recovered after centrifugation at 5,000 g for 20-30 min. The supernatant solution contained residual hypochtonte was reused for bleaching after fresh hypochlorite was added.
- the following scheme shows an example of PHA recovery and purification and discharge of acidic and basic cell debris solutions. It started with 0.3 L cell slurry containing 83.4 g dry cell mass (DM) with a PHA content of 72% w/w. After treatment with removal cell debris, the final PHA powder contained 96.4% of PHA.
- DM dry cell mass
- the non-PHA cell mass including proteins, nucleic acids, membrane lipids and cell wall fragments were decomposed to cell debris that was dissolved as soluble solids in aqueous solutions.
- the overall cell growth yield (Y x/s) and PHA formation yield (Yp/s) are calculated from the amounts of cell mass and PHA formed and the amount of sugar initially added, regardless the amount of sugar unused.
- the benefits of reusing cell debris in PHA fermentation are clear and substantial when the yields are compared with those of controls as shown in Table 2.
- the differences of the relative yields are statistically meaningful.
- the cell growth has a gain of 40-50% and PHA formation has a gain of 45-65% w/w in-comparison with the controls.
- the yields are relative to the controls without cell debris addition.
- Surfactants such as SDS are optionally used in PHA recovery to disrupt cells and remove lipids and pigments from polyesters. They are soluble and left in the cell debris solutions, which may cause adverse effect on cell growth when the cell debris solution is reused in PHA fermentation.
- An acid-base cell debris solution containing 48.5 g/L of soluble solids and 5 g/L of SDS was used in this case.
- 8 mL of ABCDS was added to give 1.94 g/L of cell debris and 0.2 g/L of SDS. Additional SDS was also added to increase SDS concentration to 0.4, 0.6 and 0.8 g/L. Table 4 gives the results of cell growth and PHA formation at different surfactant levels.
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- 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)
- Polyesters Or Polycarbonates (AREA)
- Biological Depolymerization Polymers (AREA)
Abstract
Description
Claims
Priority Applications (13)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR112012008850-5A BR112012008850B1 (en) | 2009-10-16 | 2009-10-16 | process for producing biodegradable polymeric materials |
PT97488480T PT2488653E (en) | 2009-10-16 | 2009-10-16 | Using cell debris generated from pha recovery for enhanced cell growth |
PL09748848T PL2488653T3 (en) | 2009-10-16 | 2009-10-16 | Using cell debris generated from PHA recovery for enhanced cell growth |
PCT/IB2009/007142 WO2011045625A1 (en) | 2009-10-16 | 2009-10-16 | Using cell debris generated from pha recovery for enhanced cell growth and biopolyester formation |
AU2009354139A AU2009354139B2 (en) | 2009-10-16 | 2009-10-16 | Using cell debris generated from PHA recovery for enhanced cell growth and biopolyester formation |
ES09748848.0T ES2437340T3 (en) | 2009-10-16 | 2009-10-16 | Use of cellular particles generated from PHA recovery for enhanced cell growth |
DK09748848.0T DK2488653T3 (en) | 2009-10-16 | 2009-10-16 | USING CELL BRIDGE FROM PHA RECOVERY TO IMPROVED CELL GROWTH |
EP09748848.0A EP2488653B1 (en) | 2009-10-16 | 2009-10-16 | Using cell debris generated from PHA recovery for enhanced cell growth |
US13/502,323 US20120252081A1 (en) | 2009-10-16 | 2009-10-16 | Using cell debris generated from pha recovery for enhanced cell growth and biopolyester formation |
JP2012533702A JP5735970B2 (en) | 2009-10-16 | 2009-10-16 | Use of cell debris generated from PHA recovery to promote cell growth and biopolyester formation |
ZA2012/02607A ZA201202607B (en) | 2009-10-16 | 2012-04-11 | Using cell debris generated form pha recovery for enhanced cell growth and biopolyester formation |
HRP20131138AT HRP20131138T1 (en) | 2009-10-16 | 2013-11-28 | Using cell debris generated from pha recovery for enhanced cell growth |
US15/172,071 US20170002385A1 (en) | 2009-10-16 | 2016-06-02 | Using cell debris generated from pha recovery for enhanced cell growth and biopolyester formation |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/IB2009/007142 WO2011045625A1 (en) | 2009-10-16 | 2009-10-16 | Using cell debris generated from pha recovery for enhanced cell growth and biopolyester formation |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/502,323 A-371-Of-International US20120252081A1 (en) | 2009-10-16 | 2009-10-16 | Using cell debris generated from pha recovery for enhanced cell growth and biopolyester formation |
US15/172,071 Continuation US20170002385A1 (en) | 2009-10-16 | 2016-06-02 | Using cell debris generated from pha recovery for enhanced cell growth and biopolyester formation |
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WO2011045625A1 true WO2011045625A1 (en) | 2011-04-21 |
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PCT/IB2009/007142 WO2011045625A1 (en) | 2009-10-16 | 2009-10-16 | Using cell debris generated from pha recovery for enhanced cell growth and biopolyester formation |
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US (2) | US20120252081A1 (en) |
EP (1) | EP2488653B1 (en) |
JP (1) | JP5735970B2 (en) |
AU (1) | AU2009354139B2 (en) |
BR (1) | BR112012008850B1 (en) |
DK (1) | DK2488653T3 (en) |
ES (1) | ES2437340T3 (en) |
HR (1) | HRP20131138T1 (en) |
PL (1) | PL2488653T3 (en) |
PT (1) | PT2488653E (en) |
WO (1) | WO2011045625A1 (en) |
ZA (1) | ZA201202607B (en) |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2013072723A1 (en) | 2011-11-17 | 2013-05-23 | Bio-On S.R.L. | Process for producing microbial copolyesters from sucrose-containing feedstocks |
WO2013156930A1 (en) | 2012-04-17 | 2013-10-24 | Bio On S.R.L. | Composition comprising at least one biodegradable polymer and at least one plasticiser |
ITMI20131276A1 (en) * | 2013-07-30 | 2015-01-31 | Bio On S R L | PROCESS TO RECOVER AND PURIFY POLYIDROSSIALCANOATES FROM A CELL CULTURE |
WO2019175725A1 (en) | 2018-03-12 | 2019-09-19 | Bio-On S.P.A. | Polyhydroxyalkanoates for use in the prevention of colorectal cancer |
IT201900002149A1 (en) | 2019-02-14 | 2020-08-14 | Bio On Spa | SLOW RELEASE FERTILIZER COMPOSITION, AND RELATED PRODUCTION PROCESS. |
IT201900003981A1 (en) | 2019-03-19 | 2020-09-19 | Bio On Spa | PROCESS TO PRODUCE POROUS PARTICLES OF A POLYHYDROXIALKANOATE (PHA), POROUS PARTICLES OBTAINED FROM THIS PROCESS AND COSMETIC COMPOSITIONS THAT INCLUDE THEM. |
WO2020240439A1 (en) | 2019-05-31 | 2020-12-03 | Bio-On S.P.A. | Filter element suitable for use in a smoking article and process for producing the same |
WO2021009624A1 (en) | 2019-07-15 | 2021-01-21 | Bio-On S.P.A. | Aerosol-generating article suitable for use in an aerosol-generating device |
IT201900013866A1 (en) | 2019-08-02 | 2021-02-02 | Bio On Spa | SMOKING DEVICE INCLUDING AT LEAST ONE FRANGIBLE AROMATIZING CAPSULE. |
US11021382B2 (en) | 2016-09-26 | 2021-06-01 | Bio-On S.P.A. | Methods for bioremediation of waters contaminated with hydrocarbons |
US11173218B2 (en) | 2016-06-29 | 2021-11-16 | Bio-On S.P.A. | Biocompatible polymeric nanoparticles containing functional metal nanostructures, preparation processes, and related uses in diagnostic and/or therapeutic fields |
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EP3728615A4 (en) | 2017-12-21 | 2021-10-06 | Genecis Bioindustries Inc. | Method for producing polyhydroxyalkanoates (pha) from organic waste |
CN111363126B (en) * | 2020-04-29 | 2021-02-02 | 南京钛净流体技术有限公司 | Ceramic membrane reactor and method for extracting polyhydroxyalkanoate by using ceramic membrane reactor |
KR20230097635A (en) * | 2021-12-24 | 2023-07-03 | 씨제이제일제당 (주) | A monitoring method of cell disruption and a manufacturing method for polyhydroxyalkanoate using the same |
Family Cites Families (2)
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US7141400B2 (en) * | 2002-01-16 | 2006-11-28 | University Of Hawaii | Production of biodegradable thermoplastic materials from organic wastes |
US7514525B2 (en) * | 2007-03-09 | 2009-04-07 | University Of Hawaii | Recovery and purification of polyhydroxyalkanoates |
-
2009
- 2009-10-16 BR BR112012008850-5A patent/BR112012008850B1/en active IP Right Grant
- 2009-10-16 DK DK09748848.0T patent/DK2488653T3/en active
- 2009-10-16 ES ES09748848.0T patent/ES2437340T3/en active Active
- 2009-10-16 EP EP09748848.0A patent/EP2488653B1/en active Active
- 2009-10-16 US US13/502,323 patent/US20120252081A1/en not_active Abandoned
- 2009-10-16 AU AU2009354139A patent/AU2009354139B2/en not_active Ceased
- 2009-10-16 JP JP2012533702A patent/JP5735970B2/en active Active
- 2009-10-16 WO PCT/IB2009/007142 patent/WO2011045625A1/en active Application Filing
- 2009-10-16 PL PL09748848T patent/PL2488653T3/en unknown
- 2009-10-16 PT PT97488480T patent/PT2488653E/en unknown
-
2012
- 2012-04-11 ZA ZA2012/02607A patent/ZA201202607B/en unknown
-
2013
- 2013-11-28 HR HRP20131138AT patent/HRP20131138T1/en unknown
-
2016
- 2016-06-02 US US15/172,071 patent/US20170002385A1/en not_active Abandoned
Non-Patent Citations (2)
Title |
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RAHAYU ET AL: "Production of copolymer poly(3-hydroxybutyrate-co-4-hydroxybutyrate) through a one-step cultivation process", WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, vol. 24, 6 May 2008 (2008-05-06), pages 2403 - 2409, XP019650334 * |
ZHANG ET AL: "Microbial production of 4-hydroxybutyrate, poly-4-hydroxybutyrate, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by recombinant microorganisms", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 84, 12 May 2009 (2009-05-12), pages 909 - 916, XP019737741 * |
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US9796988B2 (en) | 2011-11-17 | 2017-10-24 | Bio-On S.P.A. | Processes for producing microbial copolyesters from sucrose-containing feedstocks |
WO2013072723A1 (en) | 2011-11-17 | 2013-05-23 | Bio-On S.R.L. | Process for producing microbial copolyesters from sucrose-containing feedstocks |
WO2013156930A1 (en) | 2012-04-17 | 2013-10-24 | Bio On S.R.L. | Composition comprising at least one biodegradable polymer and at least one plasticiser |
ITMI20131276A1 (en) * | 2013-07-30 | 2015-01-31 | Bio On S R L | PROCESS TO RECOVER AND PURIFY POLYIDROSSIALCANOATES FROM A CELL CULTURE |
WO2015015395A1 (en) | 2013-07-30 | 2015-02-05 | Bio On S.R.L. | Process for recovering and purifying polyhydroxyalkanoates from a cell culture |
US9683076B2 (en) | 2013-07-30 | 2017-06-20 | Bio-On S.P.A. | Processes for recovering and purifying polyhydroxyalkanoates from cell cultures |
AU2014298034B2 (en) * | 2013-07-30 | 2017-12-14 | Bio-On S.P.A. | Process for recovering and purifying polyhydroxyalkanoates from a cell culture |
US11173218B2 (en) | 2016-06-29 | 2021-11-16 | Bio-On S.P.A. | Biocompatible polymeric nanoparticles containing functional metal nanostructures, preparation processes, and related uses in diagnostic and/or therapeutic fields |
US11021382B2 (en) | 2016-09-26 | 2021-06-01 | Bio-On S.P.A. | Methods for bioremediation of waters contaminated with hydrocarbons |
WO2019175725A1 (en) | 2018-03-12 | 2019-09-19 | Bio-On S.P.A. | Polyhydroxyalkanoates for use in the prevention of colorectal cancer |
IT201900002149A1 (en) | 2019-02-14 | 2020-08-14 | Bio On Spa | SLOW RELEASE FERTILIZER COMPOSITION, AND RELATED PRODUCTION PROCESS. |
IT201900003981A1 (en) | 2019-03-19 | 2020-09-19 | Bio On Spa | PROCESS TO PRODUCE POROUS PARTICLES OF A POLYHYDROXIALKANOATE (PHA), POROUS PARTICLES OBTAINED FROM THIS PROCESS AND COSMETIC COMPOSITIONS THAT INCLUDE THEM. |
WO2020240439A1 (en) | 2019-05-31 | 2020-12-03 | Bio-On S.P.A. | Filter element suitable for use in a smoking article and process for producing the same |
US11832644B2 (en) | 2019-05-31 | 2023-12-05 | Bio-On S.P.A. | Processes for producing filter elements suitable for use in smoking articles |
WO2021009624A1 (en) | 2019-07-15 | 2021-01-21 | Bio-On S.P.A. | Aerosol-generating article suitable for use in an aerosol-generating device |
IT201900013866A1 (en) | 2019-08-02 | 2021-02-02 | Bio On Spa | SMOKING DEVICE INCLUDING AT LEAST ONE FRANGIBLE AROMATIZING CAPSULE. |
Also Published As
Publication number | Publication date |
---|---|
HRP20131138T1 (en) | 2014-01-17 |
DK2488653T3 (en) | 2013-12-09 |
AU2009354139A1 (en) | 2012-05-10 |
ES2437340T3 (en) | 2014-01-10 |
PT2488653E (en) | 2013-12-02 |
JP5735970B2 (en) | 2015-06-17 |
EP2488653B1 (en) | 2013-08-28 |
BR112012008850B1 (en) | 2018-11-21 |
BR112012008850A2 (en) | 2017-07-25 |
US20170002385A1 (en) | 2017-01-05 |
AU2009354139B2 (en) | 2014-11-27 |
US20120252081A1 (en) | 2012-10-04 |
PL2488653T3 (en) | 2014-03-31 |
JP2013507908A (en) | 2013-03-07 |
EP2488653A1 (en) | 2012-08-22 |
ZA201202607B (en) | 2013-06-26 |
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