WO2015133887A1 - Process for the production of biopolymer from waste fish oil or waste palm oil - Google Patents
Process for the production of biopolymer from waste fish oil or waste palm oil Download PDFInfo
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- WO2015133887A1 WO2015133887A1 PCT/MY2015/000012 MY2015000012W WO2015133887A1 WO 2015133887 A1 WO2015133887 A1 WO 2015133887A1 MY 2015000012 W MY2015000012 W MY 2015000012W WO 2015133887 A1 WO2015133887 A1 WO 2015133887A1
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- WIPO (PCT)
- Prior art keywords
- biopolymer
- production
- waste
- hydroxybutyrate
- fish oil
- Prior art date
Links
- 239000002699 waste material Substances 0.000 title claims abstract description 61
- 235000021323 fish oil Nutrition 0.000 title claims abstract description 42
- 238000000034 method Methods 0.000 title claims abstract description 42
- 229920001222 biopolymer Polymers 0.000 title claims abstract description 38
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 34
- 235000019482 Palm oil Nutrition 0.000 title claims abstract description 18
- 239000002540 palm oil Substances 0.000 title claims abstract description 18
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 19
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 16
- -1 poly(3-hydroxybutyrate) Polymers 0.000 claims abstract description 8
- 229920000070 poly-3-hydroxybutyrate Polymers 0.000 claims abstract description 5
- 229920000520 poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Polymers 0.000 claims abstract description 4
- 229920001013 poly(3-hydroxybutyrate-co-4-hydroxybutyrate) Polymers 0.000 claims abstract description 4
- 229920000903 polyhydroxyalkanoate Polymers 0.000 claims description 44
- 239000002609 medium Substances 0.000 claims description 26
- 239000000203 mixture Substances 0.000 claims description 21
- 239000002054 inoculum Substances 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 11
- LHYPLJGBYPAQAK-UHFFFAOYSA-M sodium;pentanoate Chemical compound [Na+].CCCCC([O-])=O LHYPLJGBYPAQAK-UHFFFAOYSA-M 0.000 claims description 10
- 229920001577 copolymer Polymers 0.000 claims description 9
- 244000215068 Acacia senegal Species 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 8
- 229920000084 Gum arabic Polymers 0.000 claims description 8
- 239000000205 acacia gum Substances 0.000 claims description 8
- 235000010489 acacia gum Nutrition 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 8
- 239000002243 precursor Substances 0.000 claims description 8
- 239000011347 resin Substances 0.000 claims description 8
- 229920005989 resin Polymers 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- 235000015097 nutrients Nutrition 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 6
- 239000003599 detergent Substances 0.000 claims description 6
- 239000007003 mineral medium Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- XYGBKMMCQDZQOZ-UHFFFAOYSA-M sodium;4-hydroxybutanoate Chemical compound [Na+].OCCCC([O-])=O XYGBKMMCQDZQOZ-UHFFFAOYSA-M 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 5
- QJRVOJKLQNSNDB-UHFFFAOYSA-N 4-dodecan-3-ylbenzenesulfonic acid Chemical compound CCCCCCCCCC(CC)C1=CC=C(S(O)(=O)=O)C=C1 QJRVOJKLQNSNDB-UHFFFAOYSA-N 0.000 claims description 4
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 claims description 4
- 238000002156 mixing Methods 0.000 claims description 4
- 235000013619 trace mineral Nutrition 0.000 claims description 4
- 239000011573 trace mineral Substances 0.000 claims description 4
- 241001528480 Cupriavidus Species 0.000 claims description 3
- 230000003287 optical effect Effects 0.000 claims description 3
- 241000588986 Alcaligenes Species 0.000 claims description 2
- 239000001888 Peptone Substances 0.000 claims description 2
- 108010080698 Peptones Proteins 0.000 claims description 2
- 241000589516 Pseudomonas Species 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 235000013372 meat Nutrition 0.000 claims description 2
- 235000019319 peptone Nutrition 0.000 claims description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 239000012138 yeast extract Substances 0.000 claims description 2
- REKYPYSUBKSCAT-UHFFFAOYSA-N 3-hydroxypentanoic acid Chemical compound CCC(O)CC(O)=O REKYPYSUBKSCAT-UHFFFAOYSA-N 0.000 claims 2
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims 1
- 238000005453 pelletization Methods 0.000 claims 1
- 229910052698 phosphorus Inorganic materials 0.000 claims 1
- 239000011574 phosphorus Substances 0.000 claims 1
- 238000010298 pulverizing process Methods 0.000 claims 1
- 239000002904 solvent Substances 0.000 claims 1
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 description 42
- 210000004027 cell Anatomy 0.000 description 30
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 12
- 244000005700 microbiome Species 0.000 description 11
- 229920000642 polymer Polymers 0.000 description 11
- 239000000243 solution Substances 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 239000008188 pellet Substances 0.000 description 9
- 239000003921 oil Substances 0.000 description 8
- 235000019198 oils Nutrition 0.000 description 8
- 238000004108 freeze drying Methods 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000004817 gas chromatography Methods 0.000 description 6
- JGHZJRVDZXSNKQ-UHFFFAOYSA-N methyl octanoate Chemical compound CCCCCCCC(=O)OC JGHZJRVDZXSNKQ-UHFFFAOYSA-N 0.000 description 6
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 6
- 241001528539 Cupriavidus necator Species 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 5
- 229920002988 biodegradable polymer Polymers 0.000 description 5
- 239000004621 biodegradable polymer Substances 0.000 description 5
- 239000000178 monomer Substances 0.000 description 5
- YEJRWHAVMIAJKC-UHFFFAOYSA-N 4-Butyrolactone Chemical compound O=C1CCCO1 YEJRWHAVMIAJKC-UHFFFAOYSA-N 0.000 description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005227 gel permeation chromatography Methods 0.000 description 4
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 150000004665 fatty acids Chemical class 0.000 description 3
- 235000021588 free fatty acids Nutrition 0.000 description 3
- 229920001519 homopolymer Polymers 0.000 description 3
- 229920003023 plastic Polymers 0.000 description 3
- 239000004033 plastic Substances 0.000 description 3
- 229940005605 valeric acid Drugs 0.000 description 3
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 2
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 2
- SJZRECIVHVDYJC-UHFFFAOYSA-M 4-hydroxybutyrate Chemical compound OCCCC([O-])=O SJZRECIVHVDYJC-UHFFFAOYSA-M 0.000 description 2
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000005642 Oleic acid Substances 0.000 description 2
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 2
- 235000021314 Palmitic acid Nutrition 0.000 description 2
- 241000481518 Ralstonia eutropha H16 Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 239000003995 emulsifying agent Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000006140 methanolysis reaction Methods 0.000 description 2
- 150000004702 methyl esters Chemical class 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- WQEPLUUGTLDZJY-UHFFFAOYSA-N n-Pentadecanoic acid Natural products CCCCCCCCCCCCCCC(O)=O WQEPLUUGTLDZJY-UHFFFAOYSA-N 0.000 description 2
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 2
- 238000000643 oven drying Methods 0.000 description 2
- 239000003346 palm kernel oil Substances 0.000 description 2
- 235000019865 palm kernel oil Nutrition 0.000 description 2
- 239000004800 polyvinyl chloride Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 159000000000 sodium salts Chemical class 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- WHBMMWSBFZVSSR-GSVOUGTGSA-N (R)-3-hydroxybutyric acid Chemical compound C[C@@H](O)CC(O)=O WHBMMWSBFZVSSR-GSVOUGTGSA-N 0.000 description 1
- HGUFODBRKLSHSI-UHFFFAOYSA-N 2,3,7,8-tetrachloro-dibenzo-p-dioxin Chemical compound O1C2=CC(Cl)=C(Cl)C=C2OC2=C1C=C(Cl)C(Cl)=C2 HGUFODBRKLSHSI-UHFFFAOYSA-N 0.000 description 1
- 241000589152 Azotobacter chroococcum Species 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 description 1
- 241000320117 Pseudomonas putida KT2440 Species 0.000 description 1
- WHBMMWSBFZVSSR-UHFFFAOYSA-N R3HBA Natural products CC(O)CC(O)=O WHBMMWSBFZVSSR-UHFFFAOYSA-N 0.000 description 1
- 241000589196 Sinorhizobium meliloti Species 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 241000700566 Swinepox virus (STRAIN KASZA) Species 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 235000020778 linoleic acid Nutrition 0.000 description 1
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000010773 plant oil Substances 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000011148 porous material Substances 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
- 239000002244 precipitate Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 239000007320 rich medium Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000021309 simple sugar Nutrition 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002910 solid waste Substances 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 229920001169 thermoplastic Polymers 0.000 description 1
- 239000004416 thermosoftening plastic Substances 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000004056 waste incineration Methods 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
- C12P7/62—Carboxylic acid esters
- C12P7/625—Polyesters of hydroxy carboxylic acids
Definitions
- the present invention relates to a process for the production of biopolymer such as poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and derivatives thereof by using waste fish oil or waste palm oil as carbon feedstock for the biosynthesis process.
- PHA polyhydroxyalkanoates
- PHA polymer Different structural analogs of PHA polymer can be produced, depending on the microorganism and substrate used as feedstock.
- Poly (3-hydroxybuty rate), i.e. P(3HB) is the most abundant polymer produced in nature, a linear unbranched polymer built up of (R)-3 hydroxybutyric acid monomers.
- PHA was produced from microorganisms using simple sugars, free fatty acids and triacylglycerols.
- the cost of the carbon substrate reportedly contributes to more than 50% of the production cost of most bioproducts. Therefore, there is a need to source for inexpensive renewable materials such as agriculture and industrial coproducts as feedstocks for PHA production.
- the present invention uses microorganisms of genus Cupriavidus necator for biosynthesis of PHAs or any other biodegradable polymers production.
- Fish oil has high amount of palmitic acid and oleic acid, while Cupriavidus necator is known to well utilize these fatty acids for PHA conversion.
- waste fish oil or waste palm oil is used as the carbon feedstock in the production of biodegradable polymer.
- Yet another object of the present invention is to provide a process for the production of biopolymer from waste fish oil or waste palm oil to reduce cost for handling disposal of conventional petrochemical-based plastics such as polyvinyl chloride (PVC).
- PVC polyvinyl chloride
- Yet another object of the present invention is to provide a process for the production of biopolymer from waste fish oil or waste palm oil as another alternative carbon feedstock to reduce the cost of PHAs production.
- a process for the production of biopolymer comprising the steps of, i. culruring biopolymer producing bacteria in nutrient medium; ii. forming and accumulating the biopolymer by adding carbon feedstock to said culture; iii. recovering the biopolymer from said culture; characterised in that said added carbon feedstock is waste fish oil or waste palm oil.
- FIG. 1 shows the fatty acids compositions of waste fish oil used in the present invention.
- FIG. 2- A shows the composition of nutrient rich medium.
- FIG. 2-B shows the composition mineral medium broth.
- FIG. 2-C shows the composition of trace element solution.
- FIG. 3 shows a flow chart of a method of producing PHA resin.
- FIG. 4 shows the results obtained from two different concentration of waste fish oil on the biosynthesis of P(3HB).
- FIG. 5-A shows the appearance of waste fish oil that is in liquid form at 37 ° C.
- FIG. 5-B shows the appearance of waste fish oil that crystallizes at 30 ° C.
- FIG. 6 shows the effects of two different method of carbon source preparation on the biosynthesis of P(3HB).
- FIG. 7 shows the results obtained when various concentration of waste fish oil are used.
- FIG. 8-A and FIG. 8-B show the results obtained when various concentration of sodium valerate are used. 5. DETAILED DESCRIPTION OF THE DRAWINGS
- the present invention describes a process for the production of biopolymer by using waste fish oil or waste palm oil as carbon feedstock for bacterial cell growth and polyhydroxylalkanoate (PH A) assimilation.
- the microorganisms of the present invention is not subject to limitation, as long as it is a microorganism of the genus Cupriavidus, Burklialderia, Alcaligenes or Pseudomonas, which is capable of synthesizing the polymer as described above.
- Examples thereof include Cupriavidus necator H16 (formerly known as Ralstonia eutropha, Alcaligenes eutrophus and Wautersia eutroplia) for the biosynthesis of poly(3hydroxybutyrate), i.e. P(3HB) homopolymer and poly(3- hydroxybutyrate-co-3-hydroxyvalerate), i.e.
- Numerous other strains such as Bacillus cereus SPV, Sinorhizobium meliloti, Azotobacter chroococcum G-3, Pseudomonas putida KT2440 and Metylobacterium sp V49 also gaining attention for the PHA production.
- Cupriavidus necator H16 is an example of microorganism used in the present invention.
- the maintenance of bacterial strains can be carried out by using of mineral medium (MM) agar plate and 20 % glycerol.
- the strain is consistently streaked on a MM agar plate with 10 g/L of fructose as the carbon source and 0.54 g/L of ammonium chloride (NH4CI) as the nitrogen source and incubated at 30 C for 72 hours before being kept in 4 ° C chiller.
- the strain can be maintained in a 20 % v/v glycerol stock solution and kept in -20 0 C freezer for a longer period of storage.
- FIG. 1 there is shown the fatty acids compositions of waste fish oil.
- the waste fish oil generally contains 36.8 % oleic acid, 31.3 % palmitic acid, 8.1 % stearic acid and 7.6 % linoleic acid.
- waste palm oil with free fatty acid (FFA) in the range of 50% - 80% also can be used as carbon feedstock for bacterial cell growth.
- the oil is sterilized by autoclave procedure before it is used in the PHA biosynthesis process.
- the production of biopolymer of the present invention is generally being carried out by culturing microorganisms in a medium using one-stage cultivation, forming and accumulating the biopolymer of the present invention in the microorganism or in the culture and thereafter recovering the biopolymer from cultured microorganism or from the culture.
- the PHA biosynthesis for the production of biopolymer is carried out by inoculating an amount of bacterial cells into nutrient rich (NR) medium and cultured for approximately 5 to 12 hours at 25 ° C to 35 0 C on a rotary shaker at a speed of 150 - 250 rotation per minute (rpm) for inoculum preparation.
- NR nutrient rich
- NR medium Doi et al., 1995b
- Said NR medium may be composed of 10 g/L peptone (enzymatic digest from gelatin), 10 g/L meat extract and 2 g/L yeast extract.
- Said NR medium is used to activate and to enrich the bacterial cells so that sufficient cell biomass or inoculums is prepared for the subsequent culturing step in PHA production.
- the optical density (OD) of bacterial cultures is then determined with UV/ Visible spectrophotometer at the wavelength of 600 nm. This can be carried out by using non-inoculated NR broth as blank, aliquot approximately 500 ⁇ of the bacterial inoculums into a clean cuvette for OD reading (ODeoo).
- FIG. 2-B shows the composition MM broth (Doi et al., 1995b).
- Said MM is prepared by dissolving approximate 2.8 g/L of monopotassium phosphate (KH 2 PO 4 ), 3.32 g/L of disodium phosphate (Na 2 HPO 4 ) and 0.54 g/L of ammonium chloride (NH 4 C1) as nitrogen source in distilled water and the pH of the medium is adjusted to 7.0.
- KH 2 PO 4 monopotassium phosphate
- Na 2 HPO 4 disodium phosphate
- NH 4 C1 ammonium chloride
- a desired concentration of waste fish oil or waste palm oil and approximately 3% v/v of the prepared inoculum are added aseptically into the MM broth in an inoculated flask. Said inoculated flask is then incubated at 25 - 35 ° C, 150 - 250 rpm for 36 - 72 hours.
- sodium valerate and sodium-4-hydroxybutyrate (Na4HB) which are the precursors for the 3HV and 4HB biosynthesis respectively are added into the culture medium at specific interval within 32 hours to 72 hours at various concentrations. 20 % w/v of stock solutions of said both precursors are prepared and autoclaved separately. Said stock solution of sodium valerate can be converted from valeric acid.
- the Na4HB is kept in an airtight container.
- said precursors are added into MM broth at 48 hours and / or 60 hours to produce copolymer of P(3HB-co-3HV) and P(3HB-co-4HB) respectively and derivatives thereof.
- the PHA compositions produced in the present invention can be recovered from the PHA-producing microorganism by harvesting the cultured cells through centrifugation such as tubular centrifugation. After centrifugation, the supernatant is decanted. Cell pellet is washed and vortexed with hexane to remove the residual oil. The washed cell pellets are resuspended in distilled water, transferred into Bijoux bottles and frozen at -20 0 C for 24 hours prior to lyophilisation.
- the harvested cell pellets are subjected to freeze drying for approximately 2 days by using of freeze dryer. Said harvested cell pellets also can be dried by means of oven drying to obtain dried cells. The lyophilized cells are then prepared for gas chromatography (GC) analysis or other characterization studies.
- GC gas chromatography
- the dried cells are pulverized to a predetermined size by using grinder to obtain cell powder.
- Detergents such as linear alkylbenzene sulfonic acid (LAS-99), Triton x-100, and sodium dodecyl sulfate (SDS) can be used in the mixing process.
- LAS-99 linear alkylbenzene sulfonic acid
- Triton x-100 Triton x-100
- SDS sodium dodecyl sulfate
- FIG. 3 shows a flow chart of a method of producing PHA resin.
- the PHA biosynthesis is carried out by inoculating substantially two loopfull of bacterial cells into 50 mL nutrient rich (NR) medium as described above and cultured for approximately 5 hours at 30 C on a rotary shaker at a speed of 200 rotation per minute (rpm) for inoculum preparation.
- the optical density (OD) of bacterial cultures is determined with UV/ Visible spectrophotometer at the wavelength of 600 nm. This can be carried out by using non-inoculated NR broth as blank, aliquot approximately 500 ⁇ _ of the bacterial inoculums into a clean cuvette for OD reading (ODeoo).
- the prepared inoculum Upon reaching an ODeoo of 4.0 - 5.0, the prepared inoculum is then cultured in mineral medium (MM) broth as described above with nitrogen limitation to initiate PHA production.
- MM mineral medium
- waste fish oil with concentration range of 2.5 - 12.5 g/L or waste palm oil with concentration range of 5 - 10 wt% and approximately 3% v/v of the prepared inoculum are added aseptically into the MM broth in an inoculated flask. Said inoculated flask is then incubated at 30 0 C, 200 rpm for 48 hours.
- sodium valerate at concentration range of 1 - 13 g/L and sodium-4-hydroxybut rate (Na4HB) at concentration range of 1 - 13 g/L which are the precursors for the 3HV and 4HB biosynthesis respectively are added into the culture medium at specific intervals within 32 hours to 72 hours. Said precursors are added into MM broth at 48 hours and / or 60 hours to produce copolymer of P(3HB-co-3HV) and P(3HB-co-4HB) respectively.
- the cultured cells are harvested by centrifugation (8000 rpm, 7 minutes, 4 0 C). After centrifugation, the supernatant is decanted. Cell pellet is washed and vortexed with approximately 20 mL of hexane to remove the residual oil. The cell pellet is then re-centrifuged and the hexane is discarded. The cell pellet is resuspended with about 50 mL of distilled water, centrifuged and decanted to remove the remaining hexane. The washed cell pellets are resuspended in 1 mL of distilled water, transferred into Bijoux bottles and frozen at -20 ° C for 24 hours prior to lyophilisation.
- the harvested cells are subjected to freeze drying for approximately 2 days by using of freeze dryer.
- the lyophilized cells can be prepared for gas chromatography (GC) analysis.
- polymers can be extracted and purified by having the lyophilized cells to be stirred for 5 days in chloroform with ratio of cells to chloroform, lg : lOOmL. The cells are then filtered with filter paper to remove cell debris and the filtrate is concentrated tc f about 20 mL. The polymer is precipitated and purified by dripping the concentrated solution into 100 mL of vigorously stirred cool methanol. The purified polymer is obtained after removal of the excessive methanol and dried in oven. The molecular weights of the purified polymers are determined by using gel permeation chromatography (GPC).
- GPC gel permeation chromatography
- the dry Bijoux bottles are pre-weighed before being used to store the harvested cell pellets. After lyophilisation, said Bijoux bottles are weighed to acquire the CDW.
- the PHA content is determined by subjecting approximately 18-21 mg of the obtained lyophilised cells to methanolysis by means of heating at 100 °C for 140 minutes in 2 mL of methanolysis solution [compose of mixture of methanol and concentrated sulphuric acid at a ratio of 85 : 15 (v/v)] and 2 mL of chloroform.
- methanolysis solution Compose of mixture of methanol and concentrated sulphuric acid at a ratio of 85 : 15 (v/v)] and 2 mL of chloroform.
- the reaction mixture is cooled to room temperature and then followed by adding 1 mL of distilled water.
- the solution is vortexed for 1 minute to separate the mixture into two heterogeneous layers: hydrophilic layer (water dissolvable material) with cell debris on the top and hydrophobic layer (chloroform with resulting methyl esters) at the bottom.
- the hydrophobic layer is transferred with Pasteur pipette to a clean universal bottle containing sodium sulphate anhydrous to remove excess water. Then 0.5 mL of the chloroform with resulting methyl esters is added with 0.5 mL of internal standard, i.e. caprylic acid methyl ester (CME) solution (CME : chloroform at a ratio of 1 : 500) for GC analysis.
- CME caprylic acid methyl ester
- the molecular weights of the extracted and purified polymers are determined by using gel permeation chromatography (GPC) system connected to a refractive index detector. Polymers are dissolved in chloroform at concentration of 1 mg/mL and filtered through 0.45 ⁇ PTFE membrane. Chloroform is used as the eluent with a flow rate of 0.8 mL/min at 40 °C.
- GPC gel permeation chromatography
- Example 3 Effects of waste fish oil homogenized with gum Arabic on P(3HB) biosynthesis
- FIG. 4 there is shown the results obtained from two different concentration of waste fish oil on the biosynthesis of P(3HB). It was found that the results obtained for CDW is considered low when compared with plant oil such as crude palm kernel oil with 67 wt% of PHA content and 6 g/L CDW with just 5 g/L of crude palm kernel oil (CPKO) (Lee at al., 2008). The low CDW could be contributed by the solid state of the oil as the waste fish oil crystallizes at 30 °C. The bacterium is unable to completely utilize the clumped oil for bioconversion into P(3HB).
- FIG. 6 shows the effects of two different method of carbon source preparation on the biosynthesis of P(3HB). In first method, the fish oil is homogenized with 2.5 g/L of gum Arabic using homogenizer. In second method, the gum Arabic is added separately into the MM medium before addition of oil. Results showed that no significant different between the two methods employed.
- gum Arabic as emulsifier aid the bacterium in utilization of the waste fish oil or waste palm oil for bioconversion into polyhydroxyalkanoate.
- gum Arabic at concentration of 2.5 g/L - 20 g/L can be used.
- FIG. 7 there is shown the results obtained when various concentration of waste fish oil are used.
- the cultivation is carried out by having incubation at 30 ° C, 200 rpm for a period of 48 hours. It was found that the P(3HB) accumulation and CDW increased as waste fish oil concentration increased from 2.5 g/L to 12.5 g/L. This indicated that the oil was consumed by the cells for cell growth and P(3HB) accumulation.
- the CDW (3.05 g/L) was the highest when 15 g/L of waste fish oil was used.
- the P(3HB) content (74 wt%) and total PHA (2.1 g/L) was the highest when 12.5 g/L of waste fish oil was used.
- the PHA content and total P(3HB) accumulation was constant after the 12.5 g/L concentration.
- FIG. 8-A and FIG. 8-B there is shown the results obtained when various concentration of sodium valerate are used.
- the cultivation is carried out by having incubation at 30 ° C, 200 rpm for a period of 48 hours. It was found that the PHA content (wt%) accumulation trend was high at the lowest concentration of sodium valerate at lg/L and decreased as the concentration of the precursor was increased.
- the 3HV monomer composition increased gradually with the increase in the concentration of sodium valerate with 13 g/L of sodium valerate producing 63 mol% of 3HV.
- the waste fish oil was fed at a lower concentration of 5 g/L.
- the cultivation is carried out at various concentration of sodium 4- hydroxybutyrate (Na4HB) by having incubation at 30 0 C, 200 rpm for a period of 48 hours.
- the PHA content (wt%) accumulation tends to be decreased considerably as concentration of sodium 4-hydroxybutyrate is increased. While the 4HB monomer composition will increase gradually with increasing concentration of sodium 4-hydroxybutyrate.
Abstract
The present invention relates to a process for the production of biopolymer such as poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and derivatives thereof by using waste fish oil or waste palm oil as carbon feedstock for the biosynthesis process.
Description
PROCESS FOR THE PRODUCTION OF BIOPOLYMER FROM WASTE FISH
OIL OR WASTE PALM OIL
1. TECHNICAL FIELD OF THE INVENTION
The present invention relates to a process for the production of biopolymer such as poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and derivatives thereof by using waste fish oil or waste palm oil as carbon feedstock for the biosynthesis process.
2. BACKGROUND OF THE INVENTION
As environmental awareness is increasing, more concerns have been raised on problems due to using of non-biodegradable polymers which tend to affect ecological systems by their long term presence. The cost for handling disposal of conventional petrochemical-based plastics such as polyvinyl chloride (PVC) is increasing and release of hazards from waste incineration such as dioxin emission makes synthetic waste management an arduous task. Thus there is a growing interest to source for a method of producing inexpensive alternatives such as producing of biodegradable polymers or biocompatible polymers. Among those biodegradable polymers, polyhydroxyalkanoates (PHAs) are of great interest and have resulted in an
interesting new source of commodity polymers. PHAs are produced by microorganisms and stored in the cell cytoplasm as water-insoluble inclusions. Different structural analogs of PHA polymer can be produced, depending on the microorganism and substrate used as feedstock. Poly (3-hydroxybuty rate), i.e. P(3HB) is the most abundant polymer produced in nature, a linear unbranched polymer built up of (R)-3 hydroxybutyric acid monomers.
In the past, PHA was produced from microorganisms using simple sugars, free fatty acids and triacylglycerols. The cost of the carbon substrate reportedly contributes to more than 50% of the production cost of most bioproducts. Therefore, there is a need to source for inexpensive renewable materials such as agriculture and industrial coproducts as feedstocks for PHA production.
It would hence be advantageous if the above shortcomings can be alleviated by having waste fish oil or waste palm oil as a promising alternative carbon feedstock for the production of PHA. The present invention uses microorganisms of genus Cupriavidus necator for biosynthesis of PHAs or any other biodegradable polymers production. Fish oil has high amount of palmitic acid and oleic acid, while Cupriavidus necator is known to well utilize these fatty acids for PHA conversion.
3. SUMMARY OF THE INVENTION
Accordingly, it is the primary aim of the present invention to provide a process for the production of biopolymer from waste fish oil or waste palm oil wherein waste fish oil or waste palm oil is used as the carbon feedstock in the production of biodegradable polymer.
It is yet another object of the present invention to provide a process for the production of biopolymer from waste fish oil or waste palm oil wherein the biopolymer produced resembles the characteristic of a thermoplastic.
It is yet a further object of the present invention to provide a process for the production of biopolymer from waste fish oil or waste palm oil to replace conventional petrochemical-based plastics so as to reduce non-biodegradable solid wastes.
Yet another object of the present invention is to provide a process for the production of biopolymer from waste fish oil or waste palm oil to reduce cost for handling disposal of conventional petrochemical-based plastics such as polyvinyl chloride (PVC).
Yet another object of the present invention is to provide a process for the production of biopolymer from waste fish oil or waste palm oil as another alternative carbon feedstock to reduce the cost of PHAs production.
Other and further objects of the invention will become apparent with an understanding of the following detailed description of the invention or upon employment of the invention in practice.
According to a preferred embodiment of the present invention there is provided,
A process for the production of biopolymer comprising the steps of, i. culruring biopolymer producing bacteria in nutrient medium; ii. forming and accumulating the biopolymer by adding carbon feedstock to said culture; iii. recovering the biopolymer from said culture; characterised in that said added carbon feedstock is waste fish oil or waste palm oil.
4. BRIEF DESCRIPTION OF THE DRAWINGS
Other aspect of the present invention and their advantages will be discerned after studying the Detailed Description in conjunction with the accompanying drawings in which:
FIG. 1 shows the fatty acids compositions of waste fish oil used in the present invention.
FIG. 2- A shows the composition of nutrient rich medium.
FIG. 2-B shows the composition mineral medium broth.
FIG. 2-C shows the composition of trace element solution.
FIG. 3 shows a flow chart of a method of producing PHA resin. FIG. 4 shows the results obtained from two different concentration of waste fish oil on the biosynthesis of P(3HB).
FIG. 5-A shows the appearance of waste fish oil that is in liquid form at 37 ° C. FIG. 5-B shows the appearance of waste fish oil that crystallizes at 30 ° C.
FIG. 6 shows the effects of two different method of carbon source preparation on the biosynthesis of P(3HB).
FIG. 7 shows the results obtained when various concentration of waste fish oil are used.
FIG. 8-A and FIG. 8-B show the results obtained when various concentration of sodium valerate are used. 5. DETAILED DESCRIPTION OF THE DRAWINGS
In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be
understood by those of ordinary skill in the art that the invention may be practised without these specific details. In other instances, well known methods, procedures and/ or components have not been described in detail so as not to obscure the invention. The invention will be more clearly understood from the following description of the embodiments thereof, given by way of example only with reference to the accompanying drawings, which are not drawn to scale.
The present invention describes a process for the production of biopolymer by using waste fish oil or waste palm oil as carbon feedstock for bacterial cell growth and polyhydroxylalkanoate (PH A) assimilation.
The microorganisms of the present invention is not subject to limitation, as long as it is a microorganism of the genus Cupriavidus, Burklialderia, Alcaligenes or Pseudomonas, which is capable of synthesizing the polymer as described above. Examples thereof include Cupriavidus necator H16 (formerly known as Ralstonia eutropha, Alcaligenes eutrophus and Wautersia eutroplia) for the biosynthesis of poly(3hydroxybutyrate), i.e. P(3HB) homopolymer and poly(3- hydroxybutyrate-co-3-hydroxyvalerate), i.e. P(3HB-co-3HV) copolymer and poly(3-hydroxybutyrate-co-4- hydroxybutyrate), i.e. P(3HB-co-4HB) copolymer and derivatives thereof. Numerous other strains such as Bacillus cereus SPV, Sinorhizobium meliloti, Azotobacter chroococcum G-3, Pseudomonas putida KT2440 and Metylobacterium sp V49 also gaining attention for the PHA production.
Hereinafter, Cupriavidus necator H16 is an example of microorganism used in the present invention.
The maintenance of bacterial strains can be carried out by using of mineral medium (MM) agar plate and 20 % glycerol. The strain is consistently streaked on a MM agar plate with 10 g/L of fructose as the carbon source and 0.54 g/L of ammonium chloride (NH4CI) as the nitrogen source and incubated at 30 C for 72 hours before being kept in 4 ° C chiller. The strain can be maintained in a 20 % v/v glycerol stock solution and kept in -20 0 C freezer for a longer period of storage. Referring now to FIG. 1, there is shown the fatty acids compositions of waste fish oil. The waste fish oil generally contains 36.8 % oleic acid, 31.3 % palmitic acid, 8.1 % stearic acid and 7.6 % linoleic acid. Alternatively, waste palm oil with free fatty acid (FFA) in the range of 50% - 80% also can be used as carbon feedstock for bacterial cell growth. The oil is sterilized by autoclave procedure before it is used in the PHA biosynthesis process.
The production of biopolymer of the present invention is generally being carried out by culturing microorganisms in a medium using one-stage cultivation, forming and accumulating the biopolymer of the present invention in the microorganism or in the culture and thereafter recovering the biopolymer from cultured microorganism or from the culture.
The PHA biosynthesis for the production of biopolymer is carried out by inoculating an amount of bacterial cells into nutrient rich (NR) medium and cultured for approximately 5 to 12 hours at 25 ° C to 35 0 C on a rotary shaker at a speed of 150 - 250 rotation per minute (rpm) for inoculum preparation. FIG. 2- A shows the composition of NR medium (Doi et al., 1995b) whereby the pH of NR medium is adjusted to 7.0 prior to autoclave. Said NR medium may be composed of 10 g/L peptone (enzymatic digest from gelatin), 10 g/L meat extract and 2 g/L yeast extract. Said NR medium is used to activate and to enrich the bacterial cells so that sufficient cell biomass or inoculums is prepared for the subsequent culturing step in PHA production. Before the prepared inoculums is proceeded to subsequent culturing step, the optical density (OD) of bacterial cultures is then determined with UV/ Visible spectrophotometer at the wavelength of 600 nm. This can be carried out by using non-inoculated NR broth as blank, aliquot approximately 500 μί of the bacterial inoculums into a clean cuvette for OD reading (ODeoo).
Upon reaching an ODeoo of 4.0 - 5.0, the prepared inoculum is then cultured in mineral medium (MM) broth with nitrogen limitation to initiate PHA production. FIG. 2-B shows the composition MM broth (Doi et al., 1995b). Said MM is prepared by dissolving approximate 2.8 g/L of monopotassium phosphate (KH2PO4), 3.32 g/L of disodium phosphate (Na2HPO4) and 0.54 g/L of ammonium chloride (NH4C1) as nitrogen source in distilled water and the pH of the medium is adjusted to 7.0. Meanwhile, stock solution of magnesium sulphate heptahydrate (MgSO4 · 7H2O) at 50 % (w/v) is prepared and
autoclaved separately. Trace elements solution with the composition as shown in FIG. 2-C (Kahar et al., 2004) is prepared by dissolving in 0.1 N hydrochloric acid (HC1) and filter-sterilized with sterilized cellulose acetate membrane filter (pore size of 0.2 μτη). Approximately 0.25 g/L of MgSO · 7H20, 1 mL/L of trace elements and 10 g/L of fructose are added aseptically into the MM solution.
During the cultivation, a desired concentration of waste fish oil or waste palm oil and approximately 3% v/v of the prepared inoculum are added aseptically into the MM broth in an inoculated flask. Said inoculated flask is then incubated at 25 - 35 ° C, 150 - 250 rpm for 36 - 72 hours. Additionally, sodium valerate and sodium-4-hydroxybutyrate (Na4HB) which are the precursors for the 3HV and 4HB biosynthesis respectively are added into the culture medium at specific interval within 32 hours to 72 hours at various concentrations. 20 % w/v of stock solutions of said both precursors are prepared and autoclaved separately. Said stock solution of sodium valerate can be converted from valeric acid. 40 g of sodium hydroxide (NaOH) was dissolved in 1 L of (95%) absolute ethanol and stirred. 102 ml of valeric acid is added slowly into the NaOH solution. The salt precipitate of valeric acid is then recovered and dried completely in the oven at 45 C until a constant weight is achieved. The sodium salt of 4-hydroxybutyrate is prepared by the reaction of γ- butyrolactone with NaOH solution. A total of 60 g NaOH is stirred in 1 L of (95%) absolute ethanol until complete dissolution. Approximately 120 mL γ- butyrolactone is poured into the NaOH solution. The precipitated sodium salt of
4-hydroxybutyrate is placed in 45 0 C oven until constant weight. After the drying process, the Na4HB is kept in an airtight container. In the present invention, said precursors are added into MM broth at 48 hours and / or 60 hours to produce copolymer of P(3HB-co-3HV) and P(3HB-co-4HB) respectively and derivatives thereof.
Upon completion of the cultivation process, the PHA compositions produced in the present invention can be recovered from the PHA-producing microorganism by harvesting the cultured cells through centrifugation such as tubular centrifugation. After centrifugation, the supernatant is decanted. Cell pellet is washed and vortexed with hexane to remove the residual oil. The washed cell pellets are resuspended in distilled water, transferred into Bijoux bottles and frozen at -20 0 C for 24 hours prior to lyophilisation.
During the lyophilisation process, the harvested cell pellets are subjected to freeze drying for approximately 2 days by using of freeze dryer. Said harvested cell pellets also can be dried by means of oven drying to obtain dried cells. The lyophilized cells are then prepared for gas chromatography (GC) analysis or other characterization studies.
Meanwhile, the dried cells are pulverized to a predetermined size by using grinder to obtain cell powder. The obtained cell powder is then mixed with 3 - 5 % w/v detergent (cells : detergents = 1 : 2.5 w/w) for approximately 3 - 5 hours at 50 - 60 C water bath. Detergents such as linear alkylbenzene sulfonic
acid (LAS-99), Triton x-100, and sodium dodecyl sulfate (SDS) can be used in the mixing process. Upon completion of mixing, the mixture is subjected to centrifugation and washing with distilled water for 10 - 15 minutes by means of tubular centrifuge to remove unwanted impurities. Said mixture is then transferred for oven drying to obtain PHA powder and followed by pelletize the PHA in resin form by extruder. FIG. 3 shows a flow chart of a method of producing PHA resin. By having the above mentioned method of producing PHA resin, the purity of said produced PHA resin can be determined and therefore also able to determine the biodegradability of said resin to be used in particular application such as the application of said resin in slow release fertilizer.
Hereinafter, the present invention is described in more details with reference to the following examples which should not be construed to limit the scope of the present invention. Example 1 - PHA biosynthesis by Cupriavidus necator
The PHA biosynthesis is carried out by inoculating substantially two loopfull of bacterial cells into 50 mL nutrient rich (NR) medium as described above and cultured for approximately 5 hours at 30 C on a rotary shaker at a speed of 200 rotation per minute (rpm) for inoculum preparation. Before the prepared inoculum is proceeded to subsequent culturing step, the optical density (OD) of bacterial cultures is determined with UV/ Visible spectrophotometer at the wavelength of 600 nm. This can be carried out by using non-inoculated NR
broth as blank, aliquot approximately 500 μΙ_ of the bacterial inoculums into a clean cuvette for OD reading (ODeoo).
Upon reaching an ODeoo of 4.0 - 5.0, the prepared inoculum is then cultured in mineral medium (MM) broth as described above with nitrogen limitation to initiate PHA production. During the cultivation, waste fish oil with concentration range of 2.5 - 12.5 g/L or waste palm oil with concentration range of 5 - 10 wt% and approximately 3% v/v of the prepared inoculum are added aseptically into the MM broth in an inoculated flask. Said inoculated flask is then incubated at 30 0 C, 200 rpm for 48 hours. Meanwhile, sodium valerate at concentration range of 1 - 13 g/L and sodium-4-hydroxybut rate (Na4HB) at concentration range of 1 - 13 g/L which are the precursors for the 3HV and 4HB biosynthesis respectively are added into the culture medium at specific intervals within 32 hours to 72 hours. Said precursors are added into MM broth at 48 hours and / or 60 hours to produce copolymer of P(3HB-co-3HV) and P(3HB-co-4HB) respectively.
Upon completion of the cultivation process, the cultured cells are harvested by centrifugation (8000 rpm, 7 minutes, 4 0 C). After centrifugation, the supernatant is decanted. Cell pellet is washed and vortexed with approximately 20 mL of hexane to remove the residual oil. The cell pellet is then re-centrifuged and the hexane is discarded. The cell pellet is resuspended with about 50 mL of distilled water, centrifuged and decanted to remove the remaining hexane. The washed cell pellets are resuspended in 1 mL of distilled
water, transferred into Bijoux bottles and frozen at -20 ° C for 24 hours prior to lyophilisation.
During the lyophilisation process, the harvested cells are subjected to freeze drying for approximately 2 days by using of freeze dryer. The lyophilized cells can be prepared for gas chromatography (GC) analysis.
Meanwhile, polymers can be extracted and purified by having the lyophilized cells to be stirred for 5 days in chloroform with ratio of cells to chloroform, lg : lOOmL. The cells are then filtered with filter paper to remove cell debris and the filtrate is concentrated tcf about 20 mL. The polymer is precipitated and purified by dripping the concentrated solution into 100 mL of vigorously stirred cool methanol. The purified polymer is obtained after removal of the excessive methanol and dried in oven. The molecular weights of the purified polymers are determined by using gel permeation chromatography (GPC). Example 2 - Characterization of PHAs
Measurement of cell dry weight (CDW)
The dry Bijoux bottles are pre-weighed before being used to store the harvested cell pellets. After lyophilisation, said Bijoux bottles are weighed to acquire the CDW. The CDW is calculated as follow:
CDW (g/L) = (weight of bottle with lyophilized cells - pre-weighed empty bottle) x 1000
Volume of culture (mL)
Determination of PHA content and composition by Gas Chromatography
The PHA content is determined by subjecting approximately 18-21 mg of the obtained lyophilised cells to methanolysis by means of heating at 100 °C for 140 minutes in 2 mL of methanolysis solution [compose of mixture of methanol and concentrated sulphuric acid at a ratio of 85 : 15 (v/v)] and 2 mL of chloroform. Upon completion of the heating process, the reaction mixture is cooled to room temperature and then followed by adding 1 mL of distilled water. The solution is vortexed for 1 minute to separate the mixture into two heterogeneous layers: hydrophilic layer (water dissolvable material) with cell debris on the top and hydrophobic layer (chloroform with resulting methyl esters) at the bottom. The hydrophobic layer is transferred with Pasteur pipette to a clean universal bottle containing sodium sulphate anhydrous to remove excess water. Then 0.5 mL of the chloroform with resulting methyl esters is added with 0.5 mL of internal standard, i.e. caprylic acid methyl ester (CME) solution (CME : chloroform at a ratio of 1 : 500) for GC analysis. The calculation of PHA content and monomer composition are based on the comparison of their peak areas of certain retention time to those of the internal standard. The CDW is needed for the calculation of total PHA and residual biomass. The calculations are as follows:
P(3HB) homopolymer
Total P(3HB) (g/L) = P(3HB) content (wt%) x CDW (g/L) P(3HB-co-3HV) copolymer
Total PHA (g/L) = PHA content (wt%) x CDW (g/L) P(3HB-co-4HB) copolymer
Total PHA (g/L) = PHA content (wt%) x CDW (g/L) Determination of molecular weight
The molecular weights of the extracted and purified polymers are determined by using gel permeation chromatography (GPC) system connected to a refractive index detector. Polymers are dissolved in chloroform at concentration of 1 mg/mL and filtered through 0.45 μιη PTFE membrane. Chloroform is used as the eluent with a flow rate of 0.8 mL/min at 40 °C.
Example 3 - Effects of waste fish oil homogenized with gum Arabic on P(3HB) biosynthesis Referring now to FIG. 4, there is shown the results obtained from two different concentration of waste fish oil on the biosynthesis of P(3HB). It was found that the results obtained for CDW is considered low when compared with plant oil such as crude palm kernel oil with 67 wt% of PHA content and 6 g/L
CDW with just 5 g/L of crude palm kernel oil (CPKO) (Lee at al., 2008). The low CDW could be contributed by the solid state of the oil as the waste fish oil crystallizes at 30 °C. The bacterium is unable to completely utilize the clumped oil for bioconversion into P(3HB). FIG. 5-A and FIG. 5-B show that the appearance of waste fish oil that is in liquid form at 37 ° C but crystallizes at 30 C. However, the bacterium grows optimally at 30 ° C and therefore, gum Arabic is added as emulsifier to homogenize with the waste fish oil. Cultivation is conducted by having incubation at 30 ° C, 200 rpm for a period of 48 hours. FIG. 6 shows the effects of two different method of carbon source preparation on the biosynthesis of P(3HB). In first method, the fish oil is homogenized with 2.5 g/L of gum Arabic using homogenizer. In second method, the gum Arabic is added separately into the MM medium before addition of oil. Results showed that no significant different between the two methods employed. However, PHA content increased from 48 wt% to 70 wt% when comparing with results without using of gum Arabic. Therefore, the use of gum Arabic as emulsifier aid the bacterium in utilization of the waste fish oil or waste palm oil for bioconversion into polyhydroxyalkanoate. In the present invention, gum Arabic at concentration of 2.5 g/L - 20 g/L can be used.
Example 4 - Effects of adding different concentration of waste fish oil on P(3HB) biosynthesis
Referring now to FIG. 7, there is shown the results obtained when various concentration of waste fish oil are used. The cultivation is carried out by having
incubation at 30 ° C, 200 rpm for a period of 48 hours. It was found that the P(3HB) accumulation and CDW increased as waste fish oil concentration increased from 2.5 g/L to 12.5 g/L. This indicated that the oil was consumed by the cells for cell growth and P(3HB) accumulation. The CDW (3.05 g/L) was the highest when 15 g/L of waste fish oil was used. The P(3HB) content (74 wt%) and total PHA (2.1 g/L) was the highest when 12.5 g/L of waste fish oil was used. The PHA content and total P(3HB) accumulation was constant after the 12.5 g/L concentration.
Example 5 - Biosynthesis of P(3HB-co-3HV) copolymer from mixtures of waste fish oil with different concentration of sodium valerate
Referring now to FIG. 8-A and FIG. 8-B, there is shown the results obtained when various concentration of sodium valerate are used. The cultivation is carried out by having incubation at 30 ° C, 200 rpm for a period of 48 hours. It was found that the PHA content (wt%) accumulation trend was high at the lowest concentration of sodium valerate at lg/L and decreased as the concentration of the precursor was increased. The 3HV monomer composition increased gradually with the increase in the concentration of sodium valerate with 13 g/L of sodium valerate producing 63 mol% of 3HV. The waste fish oil was fed at a lower concentration of 5 g/L. This was done to force the bacterium to uptake the precursor for second monomer assimilation rather than using the waste fish oil to produce 3HB homopolymer.
Example 6 - Biosynthesis of P(3HB-co-4HB) copolymer from mixtures of waste fish oil with different concentration of sodium 4-hydroxybutyrate
The cultivation is carried out at various concentration of sodium 4- hydroxybutyrate (Na4HB) by having incubation at 30 0 C, 200 rpm for a period of 48 hours. The PHA content (wt%) accumulation tends to be decreased considerably as concentration of sodium 4-hydroxybutyrate is increased. While the 4HB monomer composition will increase gradually with increasing concentration of sodium 4-hydroxybutyrate.
While the preferred embodiment of the present invention and its advantages has been disclosed in the above Detailed Description, the invention is not limited thereto but only by the scope of the appended claim.
Claims
1. culturing biopolymer producing bacteria in nutrient medium;
11. forming and accumulating the biopolymer by adding carbon feedstock to said culture; ui. recovering the biopolymer from said culture; characterised in that said added carbon feedstock is waste fish oil or waste palm oil.
2. A process for the production of biopolymer as claimed in Claim 1 wherein said step of culturing biopolymer producing bacteria in nutrient medium is carried out at a speed of 150 - 250 rpm at 25 - 35 0 C for 5 - 12 hours for inoculums preparation, said nutrient medium contains peptone, meat extract and yeast extract; wherein the optical density (ODeoo) of bacterial cultures is determined to have 4.0 - 5.0 before proceeding to step ii.
3. A process for the production of biopolymer as claimed in Claim 2 wherein said prepared inoculums is cultured in mineral medium broth with nitrogen limitation by adding waste fish oil at concentration range of 2.5 - 12.5 g/L or adding waste palm oil at concentration range of 5-10 wt% to said culture, said mineral medium contains nitrogen, phosphorus and trace elements;
wherein said culture is incubated at 25 - 35 0 C, 150 - 250 rpm for 36 - 72 hours.
A process for the production of biopolymer as claimed in Claim 1 or Claim 3 wherein sodium valerate and/ or sodium-4-hydroxybutyrate (Na4HB) are added to said culture as precursors for 3-hydroxyvalerate and 4- hydroxybutyrate copolymer biosynthesis.
A process for the production of biopolymer as claimed in Claim 4 wherein said produced biopolymer is poly(3-hydroxybutyrate), poly(3- hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxybutyrate-co-4- hydroxybutyrate) and derivatives thereof.
A process for the production of biopolymer as claimed in Claim 3 or Claim 4 wherein gum Arabic at concentration of 2.5 g/L - 20 g/L are used by either homogenized with said waste fish oil or adding into said irtineral medium before addition of said waste fish oil.
A process for the production of biopolymer as claimed in Claim 1 wherein said biopolymer producing bacteria is of the genus Cupriavidus, Burldialderia, Alcaligenes or Pseudomonas.
8. A process for the production of biopolymer as claimed in Claim 1 wherein said carbon feedstock is sterilized before being added to said culture.
9. A process for the production of biopolymer as claimed in Claim 1 wherein said step of recovering the biopolymer from said culture is carried out by centrifugation, then washed with solvent and distilled water and freeze- dried.
10. A process for the production of biopolymer as claimed in Claim 1 or Claim 9 further comprising the following steps after step iii: iv. pulverizing the recovered biopolymer to a predetermined size by using grinder to obtain cell powder; v. mixing the obtained cell powder with 3 - 5 % w/v detergent (cells : detergents = 1 : 2.5 w/w) for approximately 3 - 5 hours at 50 - 60 0 C water bath; vi. centrifuging and washing the mixture with distilled water for 10 - 15 minutes by means of tubular centrifuge; vii. drying said mixture by using oven to obtain PHA powder; viii. pelletizing the PHA in resin form by extruder.
11. A process for the production of biopolymer as claimed in Claim 10 wherein said detergent such as linear alkylbenzene sulfonic acid (LAS-99), Triton x- 100 and sodium dodecyl sulfate (SDS) can be used in mixing with the obtained cell powder.
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