JP2004254668A - Method for producing pha - Google Patents
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- JP2004254668A JP2004254668A JP2003052226A JP2003052226A JP2004254668A JP 2004254668 A JP2004254668 A JP 2004254668A JP 2003052226 A JP2003052226 A JP 2003052226A JP 2003052226 A JP2003052226 A JP 2003052226A JP 2004254668 A JP2004254668 A JP 2004254668A
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- 238000004519 manufacturing process Methods 0.000 title claims abstract description 15
- 241000894006 Bacteria Species 0.000 claims abstract description 20
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims abstract description 19
- 229910052739 hydrogen Inorganic materials 0.000 claims abstract description 19
- 239000001257 hydrogen Substances 0.000 claims abstract description 19
- 239000002699 waste material Substances 0.000 claims abstract description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 10
- 239000007864 aqueous solution Substances 0.000 claims abstract description 7
- 239000008157 edible vegetable oil Substances 0.000 claims abstract description 7
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 230000002934 lysing effect Effects 0.000 claims description 4
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 abstract description 40
- 229920000903 polyhydroxyalkanoate Polymers 0.000 abstract description 40
- 239000000243 solution Substances 0.000 abstract description 5
- 238000010276 construction Methods 0.000 abstract description 3
- 230000001413 cellular effect Effects 0.000 abstract 1
- 230000009089 cytolysis Effects 0.000 abstract 1
- 229920000704 biodegradable plastic Polymers 0.000 description 7
- 239000008162 cooking oil Substances 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 150000007524 organic acids Chemical class 0.000 description 4
- 235000015112 vegetable and seed oil Nutrition 0.000 description 4
- 239000008158 vegetable oil Substances 0.000 description 4
- 238000010571 fourier transform-infrared absorption spectrum Methods 0.000 description 3
- 230000005484 gravity Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 235000019198 oils Nutrition 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 229920000331 Polyhydroxybutyrate Polymers 0.000 description 2
- 235000019484 Rapeseed oil Nutrition 0.000 description 2
- 125000000217 alkyl group Chemical group 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000005015 poly(hydroxybutyrate) Substances 0.000 description 2
- 238000011085 pressure filtration Methods 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 239000008159 sesame oil Substances 0.000 description 2
- 235000011803 sesame oil Nutrition 0.000 description 2
- 235000002639 sodium chloride Nutrition 0.000 description 2
- 238000010792 warming Methods 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 241000252867 Cupriavidus metallidurans Species 0.000 description 1
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 1
- 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 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 235000019482 Palm oil Nutrition 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 235000019486 Sunflower oil Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000003916 acid precipitation Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000011260 aqueous acid Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 239000010495 camellia oil Substances 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 239000010849 combustible waste Substances 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 235000012343 cottonseed oil Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000001938 differential scanning calorimetry curve Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000002540 palm oil Substances 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000010802 sludge Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 239000002600 sunflower oil Substances 0.000 description 1
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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Abstract
Description
【０００１】
【発明の属する技術分野】
本発明は、ＰＨＡの製造方法に関する。
【０００２】
【従来の技術】
現在、一般家庭で年間約４２万トンの食用油が消費されているが、食品産業や外食産業と異なり、そのほとんどが回収されることなく可燃ゴミとして廃棄されている。そして、ゴミ焼却の際に大量の熱量と二酸化炭素（ＣＯ２ ）を発生するため、地球温暖化や酸性雨の原因として、また、廃食用油の一部は下水に流れて河川や湖沼・海洋汚染の原因になるなど、地球規模での環境問題となっている。
【０００３】
植物油廃液からの生分解性プラスチックの製造方法として、植物油廃液を嫌気処理し、酸発酵により植物油廃液中の有機物を有機酸に変換し、次いで固液分離手段により嫌気処理液からスラッジを分離除去し、固形物を除去した有機酸液を濃縮し、この濃縮有機酸液から水素細菌によりＰＨＡを生成する方法が公知である（例えば、特許文献１参照）。
【０００４】
【特許文献１】
特開２０００−１８９１８３号公報
【０００５】
【発明が解決しようとする課題】
しかし、上記発明では、その工程が多段階にわたっており、手間がかかっていた。また、プラント建設コスト及びランニングコストが高いという欠点があった。また、上記特許文献１には、ＰＨＡの単離および精製について明確に記載されていない。
【０００６】
そこで、本発明は、簡便にＰＨＡを製造することができるＰＨＡの製造方法を提供することを目的とする。また、プラント建設コスト及びランニングコストを低減することができるＰＨＡの製造方法を提供することを他の目的とする。さらに、菌体を凍結乾燥することなく、エーテルやヘキサン等の有機溶媒を使用せず、簡易にかつ低コストにてＰＨＡを単離・精製することができるＰＨＡの製造方法を提供することを別の目的とする。
【０００７】
【課題を解決するための手段】
上述の目的を達成するために、本発明に係るＰＨＡの製造方法は、廃食用油を炭素源として、水素細菌と混ぜ、該水素細菌の菌体内にＰＨＡを産生させる方法である。
【０００８】
また、廃食用油を炭素源として、水素細菌と混ぜ、該水素細菌の菌体内にＰＨＡを産生させ、次に、上記菌体を回収し、回収した該菌体と次亜塩素酸水溶液とを混合して溶菌し、その後、ＰＨＡを単離する方法である。
【０００９】
【発明の実施の形態】
以下、実施の形態を示す図面に基づき、本発明を詳説する。
【００１０】
図１は、本発明の実施の一形態を示したフローチャート図であって、このＰＨＡの製造方法は、廃食用油───大豆油、ごま油、オリーブ油、ヤシ油、パーム油、こめ油、綿実油、ひまわり油、コーン油、べにばな油、なたね油、椿油等から成る植物油の廃油───を原料として、生分解性プラスチックの一種であるＰＨＡ（すなわち、ポリヒドロキシアルカノエート）を製造する方法である。ＰＨＡの化学構造式を、化学式（１） に示す。
【００１１】
【化１】
【００１２】
このＰＨＡの製造方法は、廃食用油を唯一の炭素源として、水素細菌と混ぜ、水素細菌の菌体内にＰＨＡを産生させ、次に、菌体を回収し、回収した菌体と次亜塩素酸水溶液とを混合して溶菌し、その後、ＰＨＡを単離する方法である。
【００１３】
具体的には、まず、水素細菌を定法により培養し、炭素源及び窒素源を制限した培地に交換する。次に、廃食用油を添加し、３０℃で所定時間好気性条件化で培養する。水素細菌は、汎用培地（例えば、ＬＢ培地）で容易に培養することができる。また、単糖類（例えば、果糖）やアルキル鎖長の短い有機酸を炭素源として、窒素源を欠乏させた飢餓状態で培養したとき、生分解性プラスチックのＰＨＡを産生することは公知である。培養中、酸性度（ＰＨ）の上昇が観測されるが、特に制御装置（例えば、ＰＨセンサー）を使用することなく通常の回分式でＰＨＡは効率よく産生することができる。
【００１４】
培養終了後、自然沈降、濾過、あるいは遠心分離により菌体を分離する。濾過方法については重力濾過、減圧濾過、加圧濾過等の濾過方式が挙げられる。次に、分離した菌体に次亜塩素酸水溶液を滴下し、菌体を溶菌させる。この際、次亜塩素酸水溶液に不溶のＰＨＡのみが沈殿し、他の菌体由来成分は水溶液中で可溶状態にある。
【００１５】
ＰＨＡは、自然沈降、濾過、あるいは遠心分離により単離する。このようにして得られたＰＨＡは、水（蒸留水）やエタノールで洗浄することにより精製することができる。
【００１６】
なお、本発明は、設計変更可能であって、例えば、廃食用油に、水素細菌を添加するも良い。
【００１７】
【実施例】
ＬＢ培地で前培養した水素細菌（Ｒａｌｓｔｏｎｉａ ｅｕｔｒｏｐｈａ）を遠心分離により集菌し、 ５００ｍｌのさかぐちフラスコに ３００ｍｇの菌体と炭素源及び窒素源を欠いた培地を加え、微量塩類を添加する。ここへ、唯一の炭素源として、ごま油の廃油を１ｍｌ加えて、３０℃で３日間振とう培養を行なう。培養終了後、遠心分離によって菌体を分離し、１０ｍｌの３０％次亜塩素酸水溶液を滴下した。３０℃で９０分溶菌させた後、重力濾過によりＰＨＡを分離した。このようにして得られたＰＨＡを蒸留水とエタノールで洗浄し、減圧乾燥により精製した。
【００１８】
分析は、以下の通りに行なった。
１）核磁気共鳴（ １Ｈ−ＮＭＲ）；Ｖａｒｉａｎ Ｇｅｍｉｎｉ−２００（２００ＭＨｚ） を用い、溶媒は重水素化クロロホルムであり、内部基準にテトラメチルシランを用いて化学シフトを求めた。
２）フーリエ変換赤外吸収スペクトル（ＦＴ−ＩＲ）；島津製作所社製 ＦＴＩＲ−８２００を用い、岩塩板を用いたキャスト法で行なった。
【００１９】
３）熱示差走査測定（ＤＳＣ）；島津製作所社製ＤＳＣ−５０を用い、窒素雰囲気下、昇温速度１０℃／ｍｉｎで行なった。
４）サイズ排除クロマトグラフィー（ＧＰＣ）；本体は島津製作所社製ＬＣ−１０ＡＤ 型ポンプ、同 ＣＲ−７Ａ型データ処理器、同ＲＩＤ−６Ａ型示差屈折率検出器を用い、カラムは東ソー社製 ＴＳＫｇｅｌＧ４０００Ｈ８と Ｇ２５００Ｈ８（いずれも内径 ７．５ｍｍ、長さ ３００ｍｍ）とを直列連結して用いた。溶離液はエタノールを ０．５％含有クロロホルム、カラム温度３５℃、流速０．７ ｍｌ／ｍｉｎの条件で測定した。検量線は、定法通りに標準ポリスチレンを用いて作成した。
【００２０】
測定結果を、図２〜図８に示す。
これらの測定結果より、次の各値を得た。
▲１▼ １Ｈ−ＮＭＲ（重水素化クロロホルム）；デルタ値／ｐｐｍ；１．３０（ｄ、３Ｈ、−ＣＨ３ ）、２．５５（ｍ、２Ｈ、−ＣＨ２ −）、５．２７（ｍ、１Ｈ、−ＣＨ−）
▲２▼ １Ｃ−ＮＭＲ（重水素化クロロホルム）；デルタ値／ｐｐｍ；１９．８（−ＣＨ３ ）、４０．８（−ＣＨ２ −）、６７．６（−ＣＨ−）、１６９．２ （＞Ｃ＝Ｏ）
▲３▼ＦＴ−ＩＲ；カルボニル基の伸縮振動１７２７ｃｍ−１
▲４▼ＤＳＣ；融点 １７０．０℃
▲５▼ＧＰＣ；重量平均分子量 ２２８ｋＤ
すなわち、本発明のＰＨＡの製造方法によって、上述のようなＰＨＡが得られたことが分かる。具体的には、ポリヒドロキシブチレート（ＰＨＢ）が得られた。
【００２１】
【発明の効果】
本発明は、上述の如く構成されるので、次に記載する効果を奏する。
【００２２】
（請求項１，２によれば）廃食用油からＰＨＡが簡便にかつ非常に安価で製造することが可能となる。例えば、製造コストを従来の製造方法の約６０分の１に低減することができる。また、地球温暖化の原因となる二酸化炭素の削減はもとより、ゴミ減量、河川・湖沼・海洋の汚染防止などの環境問題を克服できる有力な解決策の一つとなる。また、これまでＰＨＡの問題であったコスト面でも解決可能であり、石油代替材料としての用途や利用分野が拡大される。さらに、資源循環型社会への転換に大きく寄与することができる。また、廃食用油の種類によって、種々のＰＨＡを製造することができる。
【００２３】
（請求項２によれば）菌体を凍結乾燥することなく、かつ、エーテルやヘキサン等の有機溶媒を使用せず、簡易にかつ低コストにてＰＨＡを単離・精製することができる。そして、環境への負荷を低くすることができる。
【図面の簡単な説明】
【図１】本発明の実施の一形態を示すフローチャート図である。
【図２】水素細菌と生分解性プラスチックの重量変化の関係を示すグラフである。
【図３】図２における生分解性プラスチックの菌体中の割合を示すグラフである。
【図４】廃食用油濃度と水素細菌及び生分解性プラスチックの重量変化の関係を示すグラフである。
【図５】図４における生分解性プラスチックの菌体中の割合を示すグラフである。
【図６】実施例で得られたＰＨＡの１Ｈ−ＮＭＲスペクトルを示すグラフである。
【図７】実施例で得られたＰＨＡのＦＴ−ＩＲスペクトルを示すグラフである。
【図８】実施例で得られたＰＨＡのＤＳＣ曲線を示すグラフである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for producing a PHA.
[0002]
[Prior art]
At present, about 420,000 tons of edible oil is consumed annually in ordinary households, but unlike the food and restaurant industries, most of them are discarded as combustible waste without being collected. In addition, since a large amount of heat and carbon dioxide (CO 2 ) are generated during garbage incineration, it causes global warming and acid rain, and a part of waste cooking oil flows into sewage and flows into rivers, lakes and oceans. It is an environmental problem on a global scale, such as causing pollution.
[0003]
As a method for producing a biodegradable plastic from a vegetable oil waste liquid, the vegetable oil waste liquid is subjected to anaerobic treatment, the organic matter in the vegetable oil waste liquid is converted into an organic acid by acid fermentation, and then sludge is separated and removed from the anaerobic treatment liquid by solid-liquid separation means. A method of concentrating an organic acid solution from which solid matter has been removed and producing PHA from the concentrated organic acid solution by hydrogen bacteria is known (for example, see Patent Document 1).
[0004]
[Patent Document 1]
JP 2000-189183 A
[Problems to be solved by the invention]
However, in the above-mentioned invention, the process is multi-step, and it is troublesome. Further, there is a disadvantage that the plant construction cost and the running cost are high. Further,
[0006]
Therefore, an object of the present invention is to provide a method of manufacturing a PHA that can easily manufacture a PHA. Another object of the present invention is to provide a method of manufacturing a PHA that can reduce plant construction costs and running costs. Furthermore, it is another object of the present invention to provide a method for producing PHA that can easily and inexpensively isolate and purify PHA without freeze-drying the cells and without using an organic solvent such as ether or hexane. The purpose of.
[0007]
[Means for Solving the Problems]
In order to achieve the above object, a method for producing PHA according to the present invention is a method in which waste edible oil is used as a carbon source, mixed with hydrogen bacteria, and PHA is produced in the cells of the hydrogen bacteria.
[0008]
In addition, waste edible oil is used as a carbon source, mixed with a hydrogen bacterium to produce PHA in the cells of the hydrogen bacterium, then the cells are collected, and the collected cells and an aqueous solution of hypochlorous acid are added. This is a method of mixing and lysing, and then isolating the PHA.
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail with reference to the drawings showing the embodiments.
[0010]
FIG. 1 is a flow chart showing an embodiment of the present invention. The method for producing PHA uses waste cooking oil───soybean oil, sesame oil, olive oil, coconut oil, palm oil, rice oil, cottonseed oil. For producing PHA (ie, polyhydroxyalkanoate), which is a kind of biodegradable plastic, from waste oil of vegetable oil consisting of sunflower oil, corn oil, rapeseed oil, rapeseed oil, camellia oil, etc. It is. The chemical structural formula of PHA is shown in chemical formula (1).
[0011]
Embedded image
[0012]
In this method of producing PHA, waste edible oil is used as a sole carbon source, mixed with hydrogen bacteria, PHA is produced in the cells of the hydrogen bacteria, and then the cells are collected. This is a method of lysing bacteria by mixing with an aqueous acid solution, and then isolating the PHA.
[0013]
Specifically, first, a hydrogen bacterium is cultured by a standard method, and is replaced with a medium in which the carbon source and the nitrogen source are restricted. Next, waste cooking oil is added, and the cells are cultured at 30 ° C. for a predetermined time under aerobic conditions. Hydrogen bacteria can be easily cultured in a general-purpose medium (for example, LB medium). It is known that when a monosaccharide (for example, fructose) or an organic acid having a short alkyl chain length is used as a carbon source and cultured in a starvation state deficient in a nitrogen source, PHA as a biodegradable plastic is produced. During the cultivation, an increase in the acidity (PH) is observed, but PHA can be efficiently produced by a usual batch system without using a control device (for example, a PH sensor).
[0014]
After completion of the culture, the cells are separated by natural sedimentation, filtration, or centrifugation. Examples of the filtration method include a filtration method such as gravity filtration, reduced pressure filtration, and pressure filtration. Next, an aqueous solution of hypochlorous acid is added dropwise to the separated cells to lyse the cells. At this time, only the PHA insoluble in the hypochlorous acid aqueous solution precipitates, and the other bacterial components are in a soluble state in the aqueous solution.
[0015]
PHA is isolated by gravity sedimentation, filtration, or centrifugation. The PHA thus obtained can be purified by washing with water (distilled water) or ethanol.
[0016]
In the present invention, the design can be changed. For example, hydrogen bacteria may be added to waste cooking oil.
[0017]
【Example】
Hydrogen bacteria (Ralstonia eutropha) pre-cultured in LB medium are collected by centrifugation, and 300 mg of cells and a medium lacking a carbon source and a nitrogen source are added to a 500 ml flank flask, and trace salts are added. Here, 1 ml of sesame oil waste oil is added as a sole carbon source, and shaking culture is performed at 30 ° C. for 3 days. After completion of the culture, the cells were separated by centrifugation, and 10 ml of a 30% aqueous solution of hypochlorous acid was added dropwise. After lysing at 30 ° C. for 90 minutes, PHA was separated by gravity filtration. The PHA thus obtained was washed with distilled water and ethanol and purified by drying under reduced pressure.
[0018]
The analysis was performed as follows.
1) Nuclear magnetic resonance ( 1 H-NMR); Varian Gemini-200 (200 MHz) was used, the solvent was deuterated chloroform, and the chemical shift was determined using tetramethylsilane as an internal standard.
2) Fourier transform infrared absorption spectrum (FT-IR): FTIR-8200 manufactured by Shimadzu Corporation was used and cast by using a rock salt plate.
[0019]
3) Thermal Differential Scanning Measurement (DSC): Using DSC-50 manufactured by Shimadzu Corporation under nitrogen atmosphere at a heating rate of 10 ° C./min.
4) Size Exclusion Chromatography (GPC): The main unit used was an LC-10AD pump manufactured by Shimadzu Corporation, a CR-7A data processor, and a RID-6A differential refractive index detector manufactured by Shimadzu Corporation, and the column was TSKgelG4000H8 manufactured by Tosoh Corporation. And G2500H8 (both inner diameter 7.5 mm, length 300 mm) were used in series. The eluent was measured under the conditions of chloroform containing 0.5% ethanol, a column temperature of 35 ° C., and a flow rate of 0.7 ml / min. The calibration curve was prepared using standard polystyrene as usual.
[0020]
The measurement results are shown in FIGS.
The following values were obtained from these measurement results.
▲ 1 ▼ 1 H-NMR (deuterated chloroform); delta value /ppm;1.30(d,3H,-CH 3), 2.55 (m, 2H, -CH 2 -), 5.27 ( m, 1H, -CH-)
{Circle around ( 2 )} 1 C-NMR (deuterated chloroform); delta value / ppm; 19.8 (—CH 3 ), 40.8 (—CH 2 —), 67.6 (—CH—), 169.2 (> C = O)
(3) FT-IR; stretching vibration of carbonyl group at 1727 cm -1
(4) DSC; melting point 170.0 ° C
(5) GPC; weight average molecular weight 228 kD
That is, it can be seen that the PHA as described above was obtained by the method for producing a PHA of the present invention. Specifically, polyhydroxybutyrate (PHB) was obtained.
[0021]
【The invention's effect】
The present invention is configured as described above, and has the following effects.
[0022]
(According to
[0023]
According to claim 2, PHA can be easily isolated and purified at low cost without freeze-drying the cells and without using an organic solvent such as ether or hexane. And the load on the environment can be reduced.
[Brief description of the drawings]
FIG. 1 is a flowchart illustrating an embodiment of the present invention.
FIG. 2 is a graph showing the relationship between the weight change of hydrogen bacteria and biodegradable plastic.
FIG. 3 is a graph showing the ratio of biodegradable plastic in cells in FIG.
FIG. 4 is a graph showing the relationship between waste cooking oil concentration and changes in weight of hydrogen bacteria and biodegradable plastics.
FIG. 5 is a graph showing the ratio of the biodegradable plastic in the cells in FIG. 4;
FIG. 6 is a graph showing a 1H-NMR spectrum of PHA obtained in an example.
FIG. 7 is a graph showing an FT-IR spectrum of PHA obtained in an example.
FIG. 8 is a graph showing a DSC curve of PHA obtained in the example.
Claims (2)
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014032633A1 (en) | 2012-08-27 | 2014-03-06 | Vysoke Uceni Technicke V Brne | Method of producing polyhydroxyalkanoates (pha) from oil substrate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014032633A1 (en) | 2012-08-27 | 2014-03-06 | Vysoke Uceni Technicke V Brne | Method of producing polyhydroxyalkanoates (pha) from oil substrate |
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