JP6135144B2 - Oil production method - Google Patents
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- 238000000605 extraction Methods 0.000 claims description 84
- 244000005700 microbiome Species 0.000 claims description 57
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 27
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- 239000003495 polar organic solvent Substances 0.000 claims description 17
- 241001306132 Aurantiochytrium Species 0.000 claims description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 9
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- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical compound COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 claims 2
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- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
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Description
本発明は、従属栄養性藻類から油脂を回収する油脂製造方法に関する。 The present invention relates to a method for producing fats and oils that recovers fats and oils from heterotrophic algae.
公共分野あるいは産業排水分野において有機性排水を処理する場合、活性汚泥法が広く採用されている。活性汚泥法を用いることで、下水や産業排水は、有機物、リン、窒素などの含有量が低減された処理水と、活性汚泥処理に利用された微生物を含む余剰汚泥とに分離される。このうち下水からの余剰汚泥は、全産業廃棄物中の約２０％を占め、年間で８，０００万ｔにも達するが、これら下水汚泥の約７割が焼却処分されているのが現状である。 In the case of treating organic wastewater in the public field or industrial wastewater field, the activated sludge method is widely adopted. By using the activated sludge method, sewage and industrial wastewater are separated into treated water in which the contents of organic matter, phosphorus, nitrogen, etc. are reduced, and surplus sludge containing microorganisms used for activated sludge treatment. Of this, surplus sludge from sewage accounts for about 20% of all industrial waste, reaching 80 million tons per year. Currently, about 70% of sewage sludge is incinerated. is there.
一般に、活性汚泥処理法で生じる余剰汚泥の含水率は８０〜９０％であり、その処分には脱水・乾燥、焼却、運搬等に多大なエネルギーが要される。特にその焼却の際には、重油や天然ガスなどの燃料が多量に消費される。 In general, the water content of surplus sludge generated by the activated sludge treatment method is 80 to 90%, and the disposal requires a lot of energy for dehydration / drying, incineration, transportation, and the like. In particular, a large amount of fuel such as heavy oil or natural gas is consumed during the incineration.
そこで、環境負荷への配慮や汚泥減量化の観点から、余剰汚泥の再資源化への取組が行われている。 Therefore, efforts are being made to recycle excess sludge from the viewpoint of environmental impact and sludge reduction.
例えば、特許文献１には、有機汚泥を栄養源として、従属栄養性藻類を培養し、培養した従属栄養性藻類を、ダイノミルや超音波等で破砕した後、有機溶媒を用いて油脂を抽出することが開示されている。 For example, in Patent Document 1, heterotrophic algae are cultured using organic sludge as a nutrient source, and the cultured heterotrophic algae are crushed with dynomill or ultrasonic waves, and then oils and fats are extracted using an organic solvent. It is disclosed.
また、特許文献２には、ナビクラ属に属する微細藻類を培養し、ノルマルヘキサンからなる溶媒、ノルマルヘキサン及びメタノールからなる溶媒、又は、ノルマルヘキサン及びエタノールからなる溶媒を用いて油脂を抽出することが開示されている。また、段落番号００４１には、油分の抽出効率を高めるために、藻体を懸濁した有機溶媒を超音波ホモジナイザー等にかけて藻体を物理的に破壊することが好ましいと記載されている。 In Patent Document 2, fine algae belonging to the genus Navikura are cultured, and fats and oils are extracted using a solvent composed of normal hexane, a solvent composed of normal hexane and methanol, or a solvent composed of normal hexane and ethanol. It is disclosed. Paragraph No. 0041 describes that it is preferable to physically destroy the algal bodies by applying an organic solvent in which the algal bodies are suspended to an ultrasonic homogenizer or the like in order to increase the oil extraction efficiency.
また、特許文献３には、イカダモ属に属する微細藻類の藻細胞壁を、機械的な破壊処理、酵素処理、アルカリ化合物による処理、酸性化合物による処理、酸素又は窒素を有する有機化合物による処理から選ばれた少なくとも一種の方法で破壊処理した後、処理物から油脂類を有機溶媒抽出することが開示されている。 Patent Document 3 selects the algal cell walls of microalgae belonging to the genus Ichadamo from mechanical destruction treatment, enzyme treatment, treatment with alkali compounds, treatment with acidic compounds, and treatment with organic compounds containing oxygen or nitrogen. In addition, it has been disclosed to extract fats and oils from a treated product after the destruction treatment by at least one method.
従来は、従属栄養性藻類から、抽出溶媒を用いて油脂を抽出処理するにあたり、油脂の回収率を高めるため、菌体の細胞壁を破壊又は溶解処理した後、抽出処理を行っていた。 Conventionally, when extracting fats and oils from heterotrophic algae using an extraction solvent, in order to increase the recovery rate of the fats and oils, the cell walls of the bacterial cells have been destroyed or dissolved and then extracted.
しかしながら、菌体の細胞壁を破壊又は溶解処理するための設備が別途必要となるので、装置がより大型化、複雑化する傾向にあった。また、これらの処理を行うには、エネルギーを導入する必要があるので、油脂の製造コストが嵩む問題があった。 However, since a separate facility for destroying or dissolving the cell walls of the bacterial cells is required, the apparatus tends to be larger and more complicated. Moreover, in order to perform these processes, since it is necessary to introduce energy, there existed a problem that the manufacturing cost of fats and oils increased.
本発明は、従属栄養性藻類から油脂を、低コストで、効率よく回収できる油脂製造方法を提供することを目的とする。 An object of the present invention is to provide a method for producing fats and oils that can efficiently collect fats and oils from heterotrophic algae at low cost.
本発明者は、従属栄養性藻類による油脂生産について鋭意研究を進めた結果、オーランチオキトリウム属に属する微生物、スロストキトリウム属に属する微生物、シゾキトリウム属に属する微生物、及びパリエチキトリウム属に属する微生物は、その細胞壁を破壊又は溶解処理を行わなくても、無極性有機溶媒を含有する抽出溶媒と混合することで、菌体内の油脂を効率よく抽出できることを見出し、本発明を完成させるに至った。 As a result of diligent research on the production of fats and oils by heterotrophic algae, the inventor of the present invention belongs to the microorganisms belonging to the genus Aurantiochytrium, the microorganisms belonging to the genus Sulostyttrium, the microorganisms belonging to the genus Schizochytrium, and the genus Pariechichytrium. It has been found that microorganisms can efficiently extract oils and fats in cells by mixing with an extraction solvent containing a nonpolar organic solvent without disrupting or lysing the cell wall, and the present invention has been completed. It was.
すなわち、本発明は、菌体内に油脂を生成及び蓄積する能力を有する従属栄養性藻類から、抽出溶媒を用いて油脂を抽出処理し、油脂を回収する油脂製造方法であって、
前記従属栄養性藻類が、オーランチオキトリウム属に属する微生物、スロストキトリウム属に属する微生物、シゾキトリウム属に属する微生物、及びパリエチキトリウム属に属する微生物からなる群から選ばれる１種又は２種以上であり、
前記従属栄養性藻類を、その細胞壁を破壊又は溶解処理していない状態で、無極性有機溶媒を含有する抽出溶媒と混合することにより、前記抽出処理を行うことを特徴とする。
That is, the present invention is a method for producing fats and oils by extracting fats and oils using an extraction solvent from heterotrophic algae having the ability to produce and accumulate fats and oils in the fungus body,
The heterotrophic algae is one or more selected from the group consisting of a microorganism belonging to the genus Aurantiochytrium, a microorganism belonging to the genus Throstochytrium, a microorganism belonging to the genus Schizochytrium, and a microorganism belonging to the genus Palychychytrium. And
The extraction treatment is performed by mixing the heterotrophic algae with an extraction solvent containing a nonpolar organic solvent without destroying or dissolving the cell walls.
オーランチオキトリウム属に属する微生物、スロストキトリウム属に属する微生物、シゾキトリウム属に属する微生物及びパリエチキトリウム属に属する微生物からなる群の従属栄養性藻類は、細胞壁の合成速度が、菌体の増殖速度に比べ遅いため、細胞壁が薄いあるいは脆弱という特徴がある。例えば、オーランチオキトリウム属に属する微生物は、菌体サイズが３０〜１００μｍで、細胞壁の厚さが２〜３μｍである。また、シゾキトリウム属に属する微生物は、菌体サイズが２０〜３０μｍで、細胞壁の厚さが４〜６μｍである。また、パリエチキトリウム属に属する微生物は、菌体サイズが２０〜３０μｍで、細胞壁の厚さが２〜３μｍである。また、スロストキトリウム属に属する微生物は、菌体サイズが３０〜４０μｍで、細胞壁の厚さが２０〜３０μｍである。このため、上記従属栄養性藻類の細胞壁を、破壊又は溶解処理していない状態で、ノルマルヘキサンやトルエンなどの無極性有機溶媒を含有する溶媒と接触させると、無極性有機溶媒によって細胞壁の一部が損傷したり、無極性有機溶媒が細胞壁を通過して、細胞膜へ達するといった現象が生じる。細胞膜は、リン脂質やたんぱく質で主に構成されているが、リン脂質は無極性有機溶媒によって分解され易いため、無極性有機溶媒は細胞内へ容易に浸透することができる。また、菌体内で生成される油脂は疎水性であるので、菌体内の水分に対して強い反発力が生じると共に、菌体内に浸透した無極性有機溶媒に対して親和力を生じる。その結果、菌体内で生成した油脂は、無極性有機溶媒に引き寄せられる形で細胞外へと移動し、最終的には、無極性有機溶媒に溶解した形で菌体から脱離する。このため、本発明によれば、従属栄養性藻類の細胞壁の破壊又は溶解処理を行わなくても油脂を効率よく回収でき、ランニングコストを低減できる。 The heterotrophic algae of the group consisting of the microorganisms belonging to the genus Aurantiochytrium, the microorganisms belonging to the genus Throstochytrium, the microorganisms belonging to the genus Schizochytrium, and the microorganisms belonging to the genus Palychychytrium have a cell wall synthesis rate that increases the cell growth. Since it is slower than the speed, the cell wall is thin or fragile. For example, a microorganism belonging to the genus Aurantiochytrium has a cell size of 30 to 100 μm and a cell wall thickness of 2 to 3 μm. The microorganism belonging to the genus Schizochytrium has a cell size of 20 to 30 μm and a cell wall thickness of 4 to 6 μm. Moreover, the microorganisms which belong to the genus Pariechichytrium have a cell size of 20 to 30 μm and a cell wall thickness of 2 to 3 μm. Moreover, the microorganisms which belong to the genus Throstochytrium have a microbial cell size of 30 to 40 μm and a cell wall thickness of 20 to 30 μm. For this reason, when the cell wall of the heterotrophic algae is brought into contact with a solvent containing a nonpolar organic solvent such as normal hexane or toluene in a state where it has not been destroyed or dissolved, a part of the cell wall is caused by the nonpolar organic solvent. Damage occurs or nonpolar organic solvents pass through the cell wall and reach the cell membrane. The cell membrane is mainly composed of phospholipids and proteins. Since phospholipids are easily decomposed by nonpolar organic solvents, the nonpolar organic solvents can easily penetrate into cells. Moreover, since the fats and oils produced in the microbial cells are hydrophobic, a strong repulsive force is generated against the moisture in the microbial cells, and an affinity is generated for the nonpolar organic solvent that has penetrated into the microbial cells. As a result, the fats and oils produced in the microbial cells move out of the cell in a form attracted to the nonpolar organic solvent, and finally desorb from the microbial cells in a form dissolved in the nonpolar organic solvent. For this reason, according to this invention, fats and oils can be efficiently collect | recovered even if it does not destroy or dissolve the cell wall of heterotrophic algae, and a running cost can be reduced.
本発明の油脂製造方法は、前記従属栄養性藻類が、オーランチオキトリウム属に属する微生物、シゾキトリウム属に属する微生物、及びパリエチキトリウム属に属する微生物からなる群から選ばれる１種又は２種以上であることが好ましい。これらの微生物は、細胞壁が特に薄いので、油脂を効率よく回収できる。 In the method for producing fats and oils of the present invention, the heterotrophic algae is one or more selected from the group consisting of a microorganism belonging to the genus Aurantiochytrium, a microorganism belonging to the genus Schizochytrium, and a microorganism belonging to the genus Palychychytrium. It is preferable that Since these microorganisms have a particularly thin cell wall, oils and fats can be efficiently recovered.
本発明の油脂製造方法は、前記抽出溶媒として、無極性有機溶媒と極性有機溶媒とを含有するものを用いることが好ましい。そして、前記抽出処理前の前記従属栄養性藻類を含む培養物と前記抽出溶媒との混合液中に、前記極性有機溶媒が１０〜５０質量％含有するように、前記抽出溶媒と前記従属栄養性藻類とを混合して前記抽出処理を行うことが好ましい。 In the method for producing fats and oils of the present invention, it is preferable to use a solvent containing a nonpolar organic solvent and a polar organic solvent as the extraction solvent. And the extraction solvent and the heterotrophic so that the polar organic solvent is contained in the mixed solution of the culture containing the heterotrophic algae before the extraction treatment and the extraction solvent. It is preferable to perform the extraction process by mixing with algae.
本発明の油脂製造方法は、前記無極性有機溶媒が、ノルマルヘキサン、ベンゼン、トルエン、ジエチルエーテルからなる群から選ばれる１種又は２種以上であることが好ましい。 In the method for producing fats and oils of the present invention, the nonpolar organic solvent is preferably one or more selected from the group consisting of normal hexane, benzene, toluene, and diethyl ether.
本発明の油脂製造方法は、前記極性有機溶媒が、アセトン、アセトニトリル、Ｎ，Ｎ−ジメチルホルミアミド、ジメチルスルホキシド、メタノール、エタノール及びプロパノールからなる群から選ばれる１種又は２種以上であることが好ましい。 In the method for producing fats and oils of the present invention, the polar organic solvent is one or more selected from the group consisting of acetone, acetonitrile, N, N-dimethylformamide, dimethyl sulfoxide, methanol, ethanol and propanol. Is preferred.
本発明の油脂製造方法は、前記従属栄養性藻類に、前記従属栄養性藻類の乾燥質量１００質量部に対して前記無極性有機溶媒を１０〜２０００質量部添加して前記抽出処理を行うことが好ましい。 In the method for producing fats and oils of the present invention, the extraction treatment may be performed by adding 10 to 2000 parts by mass of the nonpolar organic solvent to the heterotrophic algae with respect to 100 parts by mass of the dry mass of the heterotrophic algae. preferable.
本発明の油脂製造方法は、前記抽出溶媒を複数回に分けて前記従属栄養性藻類に添加し、前記従属栄養性藻類の乾燥質量１００質量部に対して前記無極性有機溶媒を積算して１０〜２０００質量部添加して前記抽出処理を行うことが好ましい。 In the method for producing fats and oils according to the present invention, the extraction solvent is added to the heterotrophic algae in a plurality of times, and the nonpolar organic solvent is added to 100 parts by mass of the dry mass of the heterotrophic algae. It is preferable to perform the extraction treatment by adding ~ 2000 parts by mass.
上記各態様によれば、従属栄養性藻類から油脂を、より効率よく回収できる。 According to each said aspect, fats and oils can be more efficiently collect | recovered from heterotrophic algae.
本発明によれば、従属栄養性藻類から油脂を、低コストで、効率よく回収できる。 According to the present invention, fats and oils can be efficiently recovered from heterotrophic algae at a low cost.
本発明の油脂製造方法の一実施形態について、図１に示す油脂製造装置を用いて説明する。 One Embodiment of the fats and oils manufacturing method of this invention is described using the fats and oils manufacturing apparatus shown in FIG.
図１に示す油脂製造装置は、従属栄養性藻類を前培養するための藻類培養タンク１と、前培養した従属栄養性藻類を培養するための藻類培養槽２と、藻類培養槽２で培養した培養物を固液分離するための分離槽３と、抽出溶媒を貯留するための抽出溶媒貯留槽４と、分離槽３で固液分離して採取した固相から油脂を抽出するための油脂抽出槽５と、油脂抽出槽５で抽出した油脂を精製するための精製装置６とを備えている。これらは配管により連通し、図示しないポンプやバルブによって、所定の処理が成されるまでそれぞれ内容物を留め置いたり、他に移動させたりすることができるようになっている。 1 is cultured in an algal culture tank 1 for preculturing heterotrophic algae, an algae culture tank 2 for culturing precultured heterotrophic algae, and an algae culture tank 2. Separation tank 3 for solid-liquid separation of the culture, extraction solvent storage tank 4 for storing the extraction solvent, and fat extraction for extracting fat from the solid phase collected by solid-liquid separation in the separation tank 3 The tank 5 and the refiner | purifier 6 for refine | purifying the fats and oils extracted with the fat and oil extraction tank 5 are provided. These are communicated by piping, and the contents can be retained or moved by a pump or a valve (not shown) until a predetermined processing is performed.
藻類培養タンク１では、オーランチオキトリウム属に属する微生物、スロストキトリウム属に属する微生物、シゾキトリウム属に属する微生物及びパリエチキトリウム属に属する微生物からなる群から選ばれる１種又は２種以上の従属栄養性藻類が前培養されている。すなわち、藻類培養タンク１では、前記従属栄養性藻類の前培養に必要な有機化合物、栄養塩などが供給されて、従属栄養性藻類の活性や菌体量の最適化が行われる。 In the algae culture tank 1, one or more subordinates selected from the group consisting of microorganisms belonging to the genus Aurantiochytrium, microorganisms belonging to the genus Throstochytrium, microorganisms belonging to the genus Schizochytrium, and microorganisms belonging to the genus Palychychytrium Nutrient algae are pre-cultured. That is, the algal culture tank 1 is supplied with organic compounds, nutrient salts, and the like necessary for the preculture of the heterotrophic algae to optimize the activity and the amount of cells of the heterotrophic algae.
本発明において、「オーランチオキトリウム属に属する微生物」とは、従属栄養性藻類（生育において炭酸ガス同化を行わず、糖、脂肪酸、アミノ酸などの有機化合物を必要とする藻類）であって、オーランチオキトリウム（Ａｕｒａｎｔｉｏｃｈｙｔｒｉｕｍ）属に属し、その菌体に脂肪を生成蓄積するものである。例えば、オーランチオキトリウム ＮＢＲＣ１０２６１４、ＮＢＲＣ１０３２６８、ＮＢＲＣ１０３２６９等が挙げられる。特にオーランチオキトリウム ＮＢＲＣ１０２６１４が好ましい。（ＮＢＲＣの番号が付された菌株は、独立行政法人 製品評価技術基盤機構から分譲を受けることが可能な菌株である。以下同様。）オーランチオキトリウム属に属する微生物は、菌体サイズが３０〜１００μｍで、細胞壁の厚さが２〜３μｍである。 In the present invention, the “microorganism belonging to the genus Aurantiochytrium” is a heterotrophic algae (algae that does not undergo assimilation of carbon dioxide in growth and requires organic compounds such as sugars, fatty acids, amino acids), It belongs to the genus Aurantiochytrium and produces and accumulates fat in its cells. For example, auranthiochytrium NBRC102614, NBRC103268, NBRC103269, etc. are mentioned. In particular, auranthiochytrium NBRC102614 is preferable. (The NBRC numbered strain is a strain that can be sold from the National Institute of Technology and Evaluation, the same applies hereinafter.) Microorganisms belonging to the genus Aurantiochytrium have a cell size of 30. ˜100 μm and cell wall thickness is 2-3 μm.
本発明において、「シゾキトリウム属に属する微生物」とは、従属栄養性藻類であって、シゾキトリウム（Ｓｃｈｉｚｏｃｈｙｒｉｕｍ）属に属し、アミラーゼ、セルラーゼ、プロテアーゼ等、澱粉やタンパク質を分解する酵素を分泌する特性を有しつつその菌体に脂肪を生成蓄積するものである。例えば、シゾキトリウム ＡＴＣＣ２０８８８、２０８８９、２８２０９、シゾキトリウム ＭＹＡ―１３９１（ＡＴＣＣより入手可能）等が挙げられる。シゾキトリウム属に属する微生物は、菌体サイズが２０〜３０μｍで、細胞壁の厚さが４〜６μｍである。 In the present invention, the term “microorganism belonging to the genus Schizochytrium” is a heterotrophic alga that belongs to the genus Schizochytrium and has the characteristic of secreting enzymes that degrade starch and proteins, such as amylase, cellulase, and protease. However, it produces and accumulates fat in the cells. For example, Schizochytrium ATCC 20888, 20889, 28209, Schizochytrium MYA-1391 (available from ATCC) and the like. A microorganism belonging to the genus Schizochytrium has a cell size of 20 to 30 μm and a cell wall thickness of 4 to 6 μm.
本発明において、「パリエチキトリウム属に属する微生物」とは、従属栄養性藻類であって、パリエチキトリウム（Ｐａｒｉｅｔｉｃｈｙｔｒｉｕｍ）属に属し、アミラーゼ、セルラーゼ、プロテアーゼ等、澱粉やタンパク質を分解する酵素を分泌する特性を有しつつその菌体に脂肪を生成蓄積するものである。例えば、Ｐａｒｉｅｔｉｃｈｙｔｒｉｕｍ ｓａｒｋａｒｉａｎｕｍ ＮＢＲＣ１０４１０８、１０２９８４等が挙げられる。パリエチキトリウム属に属する微生物は、菌体サイズが２０〜３０μｍで、細胞壁の厚さが２〜３μｍである。 In the present invention, the term “microorganism belonging to the genus Pariethichytrium” is a heterotrophic algae that belongs to the genus Parieticitrium and secretes enzymes that degrade starch and proteins, such as amylase, cellulase, and protease. It produces and accumulates fat in the cells while having the characteristics of For example, Parietichytrium sarkarianum NBRC104108,102984 etc. are mentioned. Microorganisms belonging to the genus Parietichitrium have a cell size of 20 to 30 μm and a cell wall thickness of 2 to 3 μm.
本発明において、「スロストキトリウム属に属する微生物」とは、従属栄養性藻類であって、スロストキトリウム（Ｔｈｒａｕｓｔｏｃｈｙｔｒｉｕｍ）属に属し、アミラーゼ、セルラーゼ、プロテアーゼ等、澱粉やタンパク質を分解する酵素を分泌する特性を有しつつその菌体に脂肪を生成蓄積するものである。例えば、スロストキトリウム ＡＴＣＣ１８９０７、ＡＴＣＣ２０８９０、ＡＴＣＣ２０８９１、ＡＴＣＣ２０９８２、ＡＴＣＣ２４４７３、ＡＴＣＣ２６１８５、ＡＴＣＣ２８２１０、ＡＴＣＣ３４３０４等が挙げられる。（ＡＴＣＣの番号が付された菌株は、Ａｍｅｒｉｃａｎ Ｔｙｐｅ Ｃｕｌｔｕｒｅ Ｃｏｌｌｅｃｔｉｏｎ（ＡＴＣＣ）から分譲を受けることが可能な菌株である。以下同様。）スロストキトリウム属に属する微生物は、菌体サイズが２０〜３０μｍで、細胞壁の厚さが３０〜４０μｍである。 In the present invention, the term “microorganism belonging to the genus Throstochytrium” is a heterotrophic algae that belongs to the genus Thraustochytrium and secretes enzymes that degrade starch and proteins, such as amylase, cellulase, and protease. It produces and accumulates fat in the cells while having the characteristics of Examples thereof include Throstochytrium ATCC 18907, ATCC 20890, ATCC 20891, ATCC 20982, ATCC 24473, ATCC 26185, ATCC 28210, ATCC 34304, and the like. (The strain with the ATCC number is a strain that can be sold from the American Type Culture Collection (ATCC). The same applies hereinafter.) Microorganisms belonging to the genus Throstochytrium have a cell size of 20 to 30 μm. And the thickness of a cell wall is 30-40 micrometers.
藻類培養槽２には、従属栄養性藻類の栄養源となる基質と、藻類培養タンク１にて前培養した従属栄養性藻類とが供給される。従属栄養性藻類の栄養源となる基質としては、有機性排水、汚泥や食品廃棄物等の有機性廃棄物が挙げられる。藻類培養槽２に供給された従属栄養性藻類は、前記基質を栄養源として生育及び増殖し、菌体内に油脂を蓄積する。 The algae culture tank 2 is supplied with a substrate serving as a nutrient source for heterotrophic algae and heterotrophic algae pre-cultured in the algae culture tank 1. Examples of the substrate serving as a nutrient source for heterotrophic algae include organic wastewater, organic waste such as sludge and food waste. The heterotrophic algae supplied to the algae culture tank 2 grow and proliferate using the substrate as a nutrient source, and accumulate fats and oils in the cells.
藻類培養槽２における従属栄養性藻類の培養条件は特に限定は無い。従属栄養性藻類の種類に応じて適宜調整することが好ましい。例えば、オーランチオキトリウムＮＢＲＣ１０２６１４とスロストキトリウムＡＴＣＣ３４３０４とを混合培養する場合、２０〜４０℃、汚泥濃度０．１〜５．０質量％、溶存酸素濃度（ＤＯ）１．０〜５．０ｍｇ／Ｌの条件が好ましい。 The culture conditions for heterotrophic algae in the algae culture tank 2 are not particularly limited. It is preferable to adjust appropriately according to the kind of heterotrophic algae. For example, in the case where aurantiochytrium NBRC102614 and Throstochytrium ATCC34304 are mixed and cultured, 20 to 40 ° C., sludge concentration 0.1 to 5.0 mass%, dissolved oxygen concentration (DO) 1.0 to 5.0 mg / The condition of L is preferable.
藻類培養槽２にて所定時間培養した培養物は、分離槽３に導入され、固相と液相とに固液分離される。藻類培養槽２では、基質は従属栄養性藻類によって資化及び分解されるが、消化されずに残留する未消化物も存在する。従属栄養性藻類は、汚泥等の基質表面に付着して基質を資化及び分解し、生育及び増殖すると同時に菌体に油脂を生成蓄積するので、その未消化物を回収すれば、油脂を蓄積した従属栄養性藻類も回収できる。分離手段としては特に限定は無い。フィルターブレス式、スクリュープレス式などの脱水装置、膜ろ過、沈降槽などが挙げられる。例えば沈殿槽を利用すれば、自然沈降により、ほとんどエネルギーを消費することなく、未消化物とそこに付着した従属栄養性藻類を回収できる。 The culture cultured for a predetermined time in the algae culture tank 2 is introduced into the separation tank 3 and is solid-liquid separated into a solid phase and a liquid phase. In the algae culture tank 2, the substrate is assimilated and decomposed by heterotrophic algae, but there are also undigested substances that remain without being digested. Heterotrophic algae adhere to the substrate surface such as sludge, assimilate and decompose the substrate, and grow and proliferate, and at the same time produce and accumulate fats and oils in the microbial cells. Heterotrophic algae can be recovered. There is no limitation in particular as a separation means. Examples include a filter breath type and a screw press type dehydrator, membrane filtration, a sedimentation tank, and the like. For example, if a sedimentation tank is used, undigested matter and heterotrophic algae attached thereto can be recovered by natural sedimentation with little energy consumption.
培養物の固液分離後の液相には、難分解性物質などが含まれないので、水処理施設などへ導入して処理した後、放流することができる。 Since the liquid phase after the solid-liquid separation of the culture does not contain a hardly decomposable substance, it can be discharged after being introduced into a water treatment facility or the like.
培養物の固液分離後の固相には、培養後の油脂を蓄積した従属栄養性藻類が多く含まれている。油脂抽出槽５に、分離槽３での固液分離で得られた固相を供給すると共に、抽出溶媒貯留槽４から抽出溶媒を供給して、油脂の抽出処理を行う。本発明では、この抽出処理を、従属栄養性藻類の細胞壁を破壊又は溶解処理していない状態で、無極性有機溶媒を含有する抽出溶媒と混合することにより行うことを特徴としている。なお、本発明において、「従属栄養性藻類の細胞壁を破壊又は溶解処理していない状態で、無極性有機溶媒を含有する抽出溶媒と混合する」とは、培養後の従属栄養性藻類に対し、物理処理（加圧減圧、加熱、凍結、圧搾など）、化学処理（酸処理、アルカリ処理など）、生物処理（酵素など）等の処理を行うことなく、無極性有機溶媒を含有する抽出溶媒と混合させることを意味する。 The solid phase after the solid-liquid separation of the culture is rich in heterotrophic algae that have accumulated fat after the cultivation. While the solid phase obtained by solid-liquid separation in the separation tank 3 is supplied to the oil / fat extraction tank 5, the extraction solvent is supplied from the extraction solvent storage tank 4 to perform the oil / fat extraction process. The present invention is characterized in that this extraction treatment is performed by mixing with an extraction solvent containing a nonpolar organic solvent in a state where the cell walls of heterotrophic algae are not destroyed or dissolved. In the present invention, `` mixed with an extraction solvent containing a nonpolar organic solvent in a state where the cell walls of heterotrophic algae have not been destroyed or dissolved, '' for heterotrophic algae after culture, An extraction solvent containing a nonpolar organic solvent without performing physical treatment (pressure reduction, heating, freezing, pressing, etc.), chemical treatment (acid treatment, alkali treatment, etc.), biological treatment (enzyme, etc.) It means mixing.
前述したように、従属栄養性藻類から、抽出溶媒を用いて油脂を抽出処理するに際し、従来では、菌体の細胞壁を破壊又は溶解処理した後、抽出処理を行っていた。 As described above, when extracting fats and oils from heterotrophic algae using an extraction solvent, conventionally, extraction processing is performed after cell walls of cells have been destroyed or dissolved.
本発明者は、従属栄養性藻類による油脂生産について鋭意研究を進めた結果、オーランチオキトリウム属に属する微生物、スロストキトリウム属に属する微生物、シゾキトリウム属に属する微生物、及びパリエチキトリウム属に属する微生物は、その細胞壁を破壊又は溶解処理を行わなくても、ノルマルヘキサンやトルエン等の無極性有機溶媒を含有する抽出溶媒と混合することで、菌体内の油脂を効率よく抽出できることを見出した。 As a result of diligent research on the production of fats and oils by heterotrophic algae, the inventor of the present invention belongs to the microorganisms belonging to the genus Aurantiochytrium, the microorganisms belonging to the genus Sulostyttrium, the microorganisms belonging to the genus Schizochytrium, and the genus Pariechichytrium. It was found that the microorganisms can efficiently extract the fats and oils in the cells by mixing with an extraction solvent containing a nonpolar organic solvent such as normal hexane or toluene without destroying or dissolving the cell wall.
この理由について説明すると、オーランチオキトリウム属に属する微生物、スロストキトリウム属に属する微生物、シゾキトリウム属に属する微生物及びパリエチキトリウム属に属する微生物からなる群の従属栄養性藻類は、細胞壁の合成速度が、菌体の増殖速度に比べ遅いため、細胞壁が薄いあるいは脆弱という特徴がある。上記従属栄養性藻類の細胞壁を、破壊又は溶解処理していない状態で、無極性有機溶媒を含有する抽出溶媒と接触させると、無極性有機溶媒によって細胞壁の一部が損傷したり、無極性有機溶媒が細胞壁を通過して、細胞膜へ達するといった現象が生じる。細胞膜は、リン脂質やたんぱく質で主に構成されているが、リン脂質は無極性有機溶媒によって分解され易いため、無極性有機溶媒は細胞内へ容易に浸透することができる。また、菌体内で生成される油脂は疎水性であるので、菌体内の水分に対して強い反発力が生じると共に、菌体内に浸透した無極性有機溶媒に対して親和力を生じる。このため、菌体内で生成した油脂は、無極性有機溶媒に引き寄せられる形で細胞外へと移動し、最終的には、無極性有機溶媒に溶解した形で菌体から脱離するので、菌体の細胞壁を破壊又は溶解処理しなくても、油脂を効率よく回収できる。なかでも、オーランチオキトリウム属に属する微生物、シゾキトリウム属に属する微生物及びパリエチキトリウム属に属する微生物からなる群の従属栄養性藻類は、特に細胞壁が薄いあるいは脆弱であるため、油脂を効率よく回収できる。 The reason for this is that the heterotrophic algae of the group consisting of the microorganisms belonging to the genus Aurantiochytrium, the microorganism belonging to the genus Throstochyttrium, the microorganism belonging to the genus Schizochytrium and the microorganism belonging to the genus Palychychytrium are subject to the rate of cell wall synthesis. However, since it is slower than the growth rate of the bacterial cells, the cell wall is thin or fragile. When the cell wall of the heterotrophic algae is brought into contact with an extraction solvent containing a nonpolar organic solvent in a state where the heterotrophic algae has not been destroyed or dissolved, a part of the cell wall is damaged by the nonpolar organic solvent. A phenomenon occurs in which the solvent passes through the cell wall and reaches the cell membrane. The cell membrane is mainly composed of phospholipids and proteins. Since phospholipids are easily decomposed by nonpolar organic solvents, the nonpolar organic solvents can easily penetrate into cells. Moreover, since the fats and oils produced in the microbial cells are hydrophobic, a strong repulsive force is generated against the moisture in the microbial cells, and an affinity is generated for the nonpolar organic solvent that has penetrated into the microbial cells. For this reason, the fats and oils produced in the cells move to the outside of the cells in a form attracted to the nonpolar organic solvent, and finally are detached from the cells in a form dissolved in the nonpolar organic solvent. Oils and fats can be efficiently recovered without destroying or dissolving the cell walls of the body. Among them, the heterotrophic algae of the group consisting of microorganisms belonging to the genus Aurantiochytrium, microorganisms belonging to the genus Schizochytrium and microorganisms belonging to the genus Palychychytrium are particularly effective in recovering oils and fats because their cell walls are thin or fragile. it can.
本発明において、無極性有機溶媒としては、ノルマルヘキサン、ベンゼン、トルエン、ジエチルエーテル等が挙げられる。なかでも溶媒の回収が容易であるという理由からノルマルヘキサン、トルエンが好ましい。 In the present invention, examples of the nonpolar organic solvent include normal hexane, benzene, toluene, diethyl ether and the like. Of these, normal hexane and toluene are preferable because the solvent can be easily recovered.
本発明において、油脂の抽出処理は、従属栄養性藻類の乾燥質量１００質量部に対して、好ましくは１０〜２０００質量部、より好ましくは１００〜１０００質量部、特に好ましくは３００〜６００質量部の無極性有機溶媒を含有する抽出溶媒を、従属栄養性藻類に混合して行う。無極性有機溶媒の添加量が、前記割合で１０質量部未満であると、油脂の回収率が低下する傾向にあり、２０００質量部を超えて添加しても、油脂の回収率は向上しない。上記範囲内であると油脂を効率よく回収できる。 In the present invention, the fat and oil extraction treatment is preferably 10 to 2000 parts by mass, more preferably 100 to 1000 parts by mass, and particularly preferably 300 to 600 parts by mass with respect to 100 parts by mass of the dry mass of the heterotrophic algae. An extraction solvent containing a non-polar organic solvent is mixed with heterotrophic algae. If the addition amount of the nonpolar organic solvent is less than 10 parts by mass in the above-mentioned ratio, the oil recovery rate tends to decrease, and even if the addition amount exceeds 2000 parts by mass, the oil recovery rate does not improve. Oils and fats can be efficiently recovered within the above range.
本発明において、抽出溶媒には、無極性有機溶媒を含有するものを用いるが、無極性有機溶媒と極性有機溶媒とを含有するものを用いることが好ましい。無極性有機溶媒と極性有機溶媒とを併用することで、油脂の回収率を高めることができる。この場合、無極性有機溶媒、極性有機溶媒をそれぞれの種類ごとに貯留した抽出溶媒貯留槽４を用意しておき、各抽出溶媒貯留槽から無極性有機溶媒、極性有機溶媒をそれぞれ個別に油脂抽出槽５に供給して、油脂抽出槽５内で各有機溶媒を混合して抽出処理を行ってもよい。また、無極性有機溶媒と極性有機溶媒とを予め混合した混合溶媒を抽出溶媒貯留槽４に貯留しておき、該混合溶媒を油脂抽出槽５に供給して抽出処理を行ってもよい。 In the present invention, a solvent containing a nonpolar organic solvent is used as the extraction solvent, but a solvent containing a nonpolar organic solvent and a polar organic solvent is preferably used. By using a nonpolar organic solvent and a polar organic solvent in combination, the oil and fat recovery rate can be increased. In this case, the extraction solvent storage tank 4 which stored the nonpolar organic solvent and the polar organic solvent for each kind is prepared, and the nonpolar organic solvent and the polar organic solvent are separately extracted from each extraction solvent storage tank. You may supply to the tank 5 and mix each organic solvent in the fat extraction tank 5, and may perform an extraction process. Alternatively, a mixed solvent in which a nonpolar organic solvent and a polar organic solvent are mixed in advance may be stored in the extraction solvent storage tank 4, and the mixed solvent may be supplied to the oil and fat extraction tank 5 to perform the extraction process.
極性有機溶媒としては、アセトン、アセトニトリル、Ｎ，Ｎ−ジメチルホルミアミド、ジメチルスルホキシド等の極性非プロトン性有機溶媒、メタノール、エタノール、プロパノール等の極性プロトン性有機溶媒が挙げられる。これらを１種又は２種以上を用いることができる。なかでも、生物分解性が高いという理由からメタノールもしくはエタノールが好ましい。 Examples of the polar organic solvent include polar aprotic organic solvents such as acetone, acetonitrile, N, N-dimethylformamide, and dimethyl sulfoxide, and polar protic organic solvents such as methanol, ethanol, and propanol. One or more of these can be used. Of these, methanol or ethanol is preferred because of its high biodegradability.
抽出溶媒として、無極性有機溶媒と極性有機溶媒とを含有するものを用いる場合、培養物と抽出溶媒との混合液中に、極性有機溶媒が好ましくは０．１〜５０質量％、より好ましくは５〜３０質量％、特に好ましくは１０〜２０質量％含有するように、抽出溶媒を培養物に添加して抽出処理を行う。極性有機溶媒の含有量が０．１質量％未満であると油脂の回収率は向上せず、５０質量％を超えて添加しても、油脂の回収率は向上しない。上記範囲内であれば効率的に油脂の回収率を高めることができる。 In the case where a solvent containing a nonpolar organic solvent and a polar organic solvent is used as the extraction solvent, the polar organic solvent is preferably 0.1 to 50% by mass, more preferably in the mixed solution of the culture and the extraction solvent. The extraction solvent is added to the culture so as to contain 5 to 30% by mass, particularly preferably 10 to 20% by mass, and the extraction treatment is performed. If the content of the polar organic solvent is less than 0.1% by mass, the oil / fat recovery rate does not improve, and even if it exceeds 50% by mass, the oil / fat recovery rate does not improve. If it is in the said range, the collection | recovery rate of fats and oils can be raised efficiently.
油脂抽出槽５で抽出処理した後、有機溶剤相を精製装置６に導入し、精製処理を行って油脂を製造する。精製処理方法は特に限定は無い。例えば低温蒸留によって高純度化を行う方法が挙げられる。また、エステル交換反応によるバイオディーゼル燃料化、水素化分解による軽油化といった処理を行なってもよい。また、油脂抽出槽５での抽出処理後の水相及び沈殿物は、引き抜いて水処理施設等に導入して処理を行う。 After the extraction process in the oil / fat extraction tank 5, the organic solvent phase is introduced into the refining device 6, and the refining process is performed to produce the oil / fat. There is no particular limitation on the purification treatment method. For example, there is a method of performing high purity by low temperature distillation. Moreover, you may perform processes, such as biodiesel fuel conversion by transesterification, and light oil conversion by hydrocracking. Further, the aqueous phase and the precipitate after the extraction treatment in the oil and fat extraction tank 5 are extracted and introduced into a water treatment facility or the like for treatment.
このようにして、本発明では従属栄養性藻類から油脂を回収できる。 Thus, in this invention, fats and oils can be collect | recovered from heterotrophic algae.
本発明の油脂製造方法の他の実施形態について、図２に示す油脂製造装置を用いて説明する。 Another embodiment of the oil and fat manufacturing method of the present invention will be described using the oil and fat manufacturing apparatus shown in FIG.
この油脂製造装置は、複数の油脂抽出槽５ａ，５ｂ，５ｃが直列に接続して配設されている。図２では油脂抽出槽５ａは３個配設されているが、その数は特に限定されない。 In this oil and fat manufacturing apparatus, a plurality of oil and fat extraction tanks 5a, 5b and 5c are connected in series. In FIG. 2, three oil / fat extraction tanks 5 a are arranged, but the number is not particularly limited.
この実施形態では、抽出溶媒を複数回に分けて従属栄養性藻類に添加して抽出処理を行うことができる。抽出溶媒を複数回に分けて従属栄養性藻類に添加して抽出処理を行うことで、後述する実施例に示すように、油脂の回収率を高めることができる。 In this embodiment, the extraction solvent can be divided into a plurality of times and added to the heterotrophic algae for extraction. By extracting the extraction solvent into a plurality of times and adding it to the heterotrophic algae and performing the extraction treatment, the recovery rate of fats and oils can be increased as shown in the examples described later.
この実施形態において、油脂の抽出処理は、抽出溶媒を複数回に分けて従属栄養性藻類に添加し、従属栄養性藻類の乾燥質量１００質量部に対して無極性有機溶媒を積算して１０〜２０００質量部添加することが好ましく、１００〜１０００質量部がより好ましく、３００〜５００質量部が特に好ましい。無極性有機溶媒の積算量が上記範囲内であると油脂の回収率を高めることができる。 In this embodiment, the oil and fat extraction process is carried out by adding the extraction solvent to the heterotrophic algae in several batches, adding the nonpolar organic solvent to 100 parts by mass of the heterotrophic algae and adding 10 to 10 parts by weight. It is preferable to add 2000 mass parts, 100-1000 mass parts is more preferable, 300-500 mass parts is especially preferable. When the integrated amount of the nonpolar organic solvent is within the above range, the oil and fat recovery rate can be increased.
抽出処理の回数は、２〜５回が好ましく、２〜３回がより好ましい。抽出処理の回数を多くすると油脂抽出槽の数が嵩むので、装置が大型化や複雑化する恐れがある。 The number of extraction processes is preferably 2 to 5 times, more preferably 2 to 3 times. If the number of extraction processes is increased, the number of oil extraction tanks increases, which may increase the size and complexity of the apparatus.
（試験例１）
従属栄養性藻類として、オーランチオキトリウム ＮＢＲＣ１０２６１４を用いた。
人工海水（塩化ナトリウム３．０質量％、塩化カリウム０．０７質量％、塩化マグネシウム１．０８質量％、硫酸マグネシウム０．５４質量％、塩化カルシウム０．１質量％の組成）を、終濃度が５０質量％になるように添加して調製した人工海水５０％含有−ＬＢ培地を滅菌した後、その１０００ｍＬに対しビタミン剤（和光純薬社製、ビタミンＢ１、Ｂ２、Ｂ１２含有）を１ｇ添加した液体培地を用いて、３０℃、曝気量１Ｌ／分で曝気を行いながら２５０ｒｐｍでスターラー攪拌することにより従属栄養性藻類の培養を行った。この培養で菌体重量は１０ｇ／Ｌ程度となった。遠心分離により含水率８０％程度まで濃縮し、ここから乾燥菌体重量が１ｇとなるように濃縮菌液を採取した。濃縮菌液に対し、菌体の細胞壁を破壊又は溶解処理することなく、菌体の乾燥質量の０．１〜２０倍量のノルマルヘキサンを添加し、抽出処理を行った。抽出処理は、１０秒間ボルテックスミキサーによる攪拌を行ったのちに３０分間静置により油脂の抽出を行い、次いで、４８００ｒｐｍで５分間遠心分離を行ったのちにピペッティングにより有機溶剤相を回収した。これを７０℃で一晩乾燥させ、回収できた油脂の重量を測定した。
(Test Example 1)
Aurantiochytrium NBRC102614 was used as a heterotrophic algae.
Artificial seawater (composition of sodium chloride 3.0 mass%, potassium chloride 0.07 mass%, magnesium chloride 1.08 mass%, magnesium sulfate 0.54 mass%, calcium chloride 0.1 mass%) After sterilizing 50% artificial seawater containing LB medium prepared by adding 50% by mass, 1 g of vitamin preparation (containing Wako Pure Chemical Industries, vitamins B1, B2, and B12) was added to 1000 mL of the LB medium. Heterotrophic algae were cultured by using a liquid medium and stirring with a stirrer at 250 rpm while aeration was performed at 30 ° C. and an aeration rate of 1 L / min. In this culture, the cell weight became about 10 g / L. The water content was concentrated to about 80% by centrifugation, and a concentrated bacterial solution was collected from this so that the dry cell weight would be 1 g. To the concentrated bacterial solution, normal hexane was added in an amount of 0.1 to 20 times the dry mass of the cells without destroying or dissolving the cell walls of the cells. In the extraction process, after stirring with a vortex mixer for 10 seconds, the fats and oils were extracted by standing for 30 minutes, then centrifuged at 4800 rpm for 5 minutes, and then the organic solvent phase was recovered by pipetting. This was dried at 70 ° C. overnight, and the weight of the recovered fat was measured.
従属栄養性藻類の乾燥質量あたりのノルマルヘキサンの使用量（ｍｌ（ＨＥＸ）／ｇ（ｄｒｙ ｃｅｌｌ））と、ノルマルヘキサンの使用量あたりの油脂回収量（ｍｌ（ｏｉｌ）／ｍｌ（ＨＥＸ））との関係を、図２に記す。 The amount of normal hexane used per dry mass of heterotrophic algae (ml (HEX) / g (dry cell)), and the amount of oil recovered per amount of normal hexane used (ml (oil) / ml (HEX)) The relationship is shown in FIG.
図２に示すように、従属栄養性藻類の乾燥質量に対し、０．１〜２０倍量のノルマルヘキサンを使用することで、油脂を効率よく回収できた。 As shown in FIG. 2, fats and oils could be efficiently recovered by using 0.1 to 20 times the amount of normal hexane with respect to the dry mass of heterotrophic algae.
なお、オーランチオキトリウム ＮＢＲＣ１０２６１４の代わりに、細胞壁の厚さが、オーランチオキトリウムに属する微生物と同等である、シゾキトリウム属に属する微生物、パリエチキトリウム属に属する微生物を使用した場合でも、同様の結果が得られ、従属栄養性藻類の乾燥質量に対し、０．１〜２０倍量のノルマルヘキサンを使用することで、油脂を効率よく回収できた。 In addition, even when a microorganism belonging to the genus Schizochytrium or a microorganism belonging to the genus Parietichitrium is used in place of the auranthiochytrium NBRC102614, the thickness of the cell wall is equivalent to that of the microorganism belonging to the auranthiochytrium. The result was obtained, and the fats and oils could be efficiently recovered by using 0.1 to 20 times the amount of normal hexane with respect to the dry mass of the heterotrophic algae.
（試験例２）
試験例１と同様の条件で従属栄養性藻類（オーランチオキトリウム ＮＢＲＣ１０２６１４）を培養した。培養後の従属栄養性藻類に対し、その細胞壁を破壊又は溶解処理することなく、従属栄養性藻類の乾燥質量の４倍量のノルマルヘキサンを添加し、２５℃で１０分間混合して、抽出処理を行った。そして、試験例１と同様の条件で蒸留処理を行い、油脂を回収した。従属栄養性藻類の乾燥質量あたりの油脂の回収量は、０．１８ｍｌ（ｏｉｌ）／ｇ（ｄｒｙ ｃｅｌｌ）であった。
(Test Example 2)
Heterotrophic algae (Auranthiochytrium NBRC102614) were cultured under the same conditions as in Test Example 1. Extraction treatment is performed on heterotrophic algae after cultivation without disrupting or dissolving the cell walls, adding 4 times the amount of normal hexane algae dry mass, and mixing for 10 minutes at 25 ° C. Went. And the distillation process was performed on the conditions similar to Test Example 1, and fats and oils were collect | recovered. The amount of recovered oil per dry mass of the heterotrophic algae was 0.18 ml (oil) / g (dry cell).
（試験例３）
試験例２において、培養後の従属栄養性藻類に対し、その細胞壁を破壊又は溶解処理することなく、従属栄養性藻類の乾燥質量の２倍量のノルマルヘキサンを添加し、２５℃で１０分間混合した後に有機溶媒層のみ回収した。次いで、従属栄養性藻類の乾燥質量の２倍量のノルマルヘキサンを添加し、２５℃で１０分時間混合した。このようにして抽出処理を行った。そして、試験例１と同様の条件で蒸留処理を行い、油脂を回収した。従属栄養性藻類の乾燥質量あたりの油脂の回収量は、０．２２ｍｌ（ｏｉｌ）／ｇ（ｄｒｙ ｃｅｌｌ）であった。
(Test Example 3)
In Test Example 2, normal hexane twice the dry mass of heterotrophic algae was added to the cultured heterotrophic algae without disrupting or dissolving the cell walls, and mixed at 25 ° C. for 10 minutes. After that, only the organic solvent layer was recovered. Next, normal hexane twice the dry mass of heterotrophic algae was added and mixed for 10 minutes at 25 ° C. The extraction process was performed in this way. And the distillation process was performed on the conditions similar to Test Example 1, and fats and oils were collect | recovered. The amount of recovered oil per dry mass of the heterotrophic algae was 0.22 ml (oil) / g (dry cell).
（試験例４）
試験例２において、培養後の従属栄養性藻類に対し、その細胞壁を破壊又は溶解処理することなく、従属栄養性藻類の乾燥質量の１．３３倍量のノルマルヘキサンを添加し、２５℃で１０分時間混合した後に有機溶媒層のみ回収した。次いで、従属栄養性藻類の乾燥質量の１．３３倍量のノルマルヘキサンを添加し、２５℃で１０分時間混合した後に有機溶媒層のみ回収した。次いで、従属栄養性藻類の乾燥質量の１．３３倍量のノルマルヘキサンを添加し、２５℃で１０分時間混合した。このようにして抽出処理を行った。そして、試験例１と同様の条件で蒸留処理を行い、油脂を回収した。従属栄養性藻類の乾燥質量あたりの油脂の回収量は、０．２７ｍｌ（ｏｉｌ）／ｇ（ｄｒｙ ｃｅｌｌ）であった。
(Test Example 4)
In Test Example 2, normal hexane of 1.33 times the dry mass of the heterotrophic algae was added to the cultured heterotrophic algae without destroying or dissolving the cell walls. Only the organic solvent layer was recovered after mixing for minutes. Subsequently, normal hexane of 1.33 times the dry mass of heterotrophic algae was added, and after mixing at 25 ° C. for 10 minutes, only the organic solvent layer was recovered. Then, normal hexane of 1.33 times the dry mass of heterotrophic algae was added and mixed for 10 minutes at 25 ° C. The extraction process was performed in this way. And the distillation process was performed on the conditions similar to Test Example 1, and fats and oils were collect | recovered. The amount of recovered oil per dry mass of the heterotrophic algae was 0.27 ml (oil) / g (dry cell).
試験例２〜４の結果を図４に示す。 The results of Test Examples 2 to 4 are shown in FIG.
図４に示されるように、複数回に分けて抽出処理を行うことで油脂の回収量を高めることができた。 As shown in FIG. 4, it was possible to increase the amount of recovered oil by performing the extraction process in a plurality of times.
（試験例５）
試験例１と同様の条件で従属栄養性藻類（オーランチオキトリウム ＮＢＲＣ１０２６１４）を培養し、濃縮菌液を調製した。
濃縮菌液（乾燥菌体重量１ｇ）に対し、ノルマルヘキサン（和光純薬社製）１０ｍｌ、ノルマルヘキサン１０ｍｌとアセトン（和光純薬社製）との混合溶媒、ノルマルヘキサン１０ｍｌとメタノール（和光純薬社製）との混合溶媒をそれぞれ抽出溶媒として用いて抽出処理を行った。抽出処理は、菌体の細胞壁を破壊又は溶解処理することなく抽出溶媒に接触させて行った。抽出処理は、１０秒間ボルテックスミキサーによる攪拌を行ったのちに３０分間静置により油脂の抽出を行い、次いで、４８００ｒｐｍで５分間遠心分離を行ったのちにピペッティングにより有機溶剤相を回収した。これを７０℃で一晩乾燥させ、回収できた油脂の重量を測定した。
(Test Example 5)
Heterotrophic algae (Auranthiochytrium NBRC102614) were cultured under the same conditions as in Test Example 1 to prepare a concentrated bacterial solution.
For concentrated bacterial solution (dry cell weight 1 g), normal hexane (manufactured by Wako Pure Chemical Industries) 10 ml, mixed solvent of normal hexane 10 ml and acetone (manufactured by Wako Pure Chemical Industries), normal hexane 10 ml and methanol (Wako Pure Chemical Industries) The extraction process was performed using each of the mixed solvents as the extraction solvent. The extraction treatment was performed by contacting the cell wall of the cells with the extraction solvent without destroying or dissolving the cell walls. In the extraction process, after stirring with a vortex mixer for 10 seconds, the fats and oils were extracted by standing for 30 minutes, then centrifuged at 4800 rpm for 5 minutes, and then the organic solvent phase was recovered by pipetting. This was dried at 70 ° C. overnight, and the weight of the recovered fat was measured.
図５に、横軸にアセトンもしくはメタノールの終濃度（％）をとり、縦軸にノルマルヘキサン１０ｍｌのみで抽出処理を行った油脂回収量を１００％とした場合における油脂回収量の比率を比回収量（％）として示した図を示す。なお、終濃度は、アセトン又はエタノールの質量を、濃縮菌液と抽出溶媒との混合液の質量で割った値を百分率で表記した値である。 In FIG. 5, the ratio of the recovered amount of oil and fat when the final concentration (%) of acetone or methanol is taken on the horizontal axis and the recovered amount of oil and fat extracted with only 10 ml of normal hexane is taken as the vertical axis is shown as the ratio recovery. The figure shown as amount (%) is shown. In addition, final concentration is the value which expressed the value which divided the mass of acetone or ethanol by the mass of the liquid mixture of a concentrated microbe liquid and an extraction solvent in percentage.
図５に示されるように、アセトンおよびメタノールどちらの場合においても、終濃度１０％から回収量は急激に増加し、１０〜３０％の範囲で高い回収量が得られた。 As shown in FIG. 5, in both cases of acetone and methanol, the recovery amount increased rapidly from the final concentration of 10%, and a high recovery amount was obtained in the range of 10 to 30%.
なお、オーランチオキトリウム ＮＢＲＣ１０２６１４の代わりに、シゾキトリウム属に属する微生物、パリエチキトリウム属に属する微生物、スロストキトリウム属に属する微生物を使用した場合でも、同様の結果が得られ、抽出溶媒として無極性有機溶媒と極性有機溶媒とを併用することで、油脂を効率よく回収できた。 Similar results were obtained even when a microorganism belonging to the genus Schizochytrium, a microorganism belonging to the genus Pariechichytrium, or a microorganism belonging to the genus Throstochytrium was used instead of auranthiochytrium NBRC102614. By using the organic solvent and the polar organic solvent in combination, the fats and oils could be recovered efficiently.
（試験例６）
試験例５において、濃縮菌液の含水量を調整して、アセトンの終濃度は１０％又は２０％で一定とするが、終濃度１０％条件では乾燥菌体重量１ｇ当り０．５〜２．５ｍｌ、終濃度２０％条件では乾燥菌体重量１ｇ当り１．０〜２．５ｍｌと変化させて抽出処理を行い、各条件における油脂回収量を測定した。なお、終濃度は、アセトンの質量を、濃縮菌液と抽出溶媒との混合液の質量で割った値を百分率で表記した値である。
(Test Example 6)
In Test Example 5, the water content of the concentrated bacterial solution is adjusted so that the final concentration of acetone is constant at 10% or 20%. However, under the condition of 10% final concentration, 0.5-2. Under the conditions of 5 ml and a final concentration of 20%, the extraction treatment was carried out with 1.0 to 2.5 ml per 1 g of dry cell weight, and the amount of oil recovered under each condition was measured. In addition, final concentration is the value which expressed the value which divided the mass of acetone by the mass of the liquid mixture of a concentrated microbe liquid and an extraction solvent in percentage.
図６に、横軸にアセトンの添加量（ｍｌ）をとり、縦軸にノルマルヘキサン１０ｍｌのみで抽出処理を行った油脂回収量を１００％とした場合における各混合溶媒における油脂回収量の比率を比回収量（％）として示した図を示す。 In FIG. 6, the horizontal axis represents the amount of acetone added (ml), and the vertical axis represents the ratio of the amount of collected oil and fat in each mixed solvent when the amount of collected oil and fat extracted with only 10 ml of normal hexane is 100%. The figure shown as specific recovery (%) is shown.
図６に示されるように、アセトンの添加量を増加させてもアセトンの終濃度が同じであれば、油脂の回収率はほぼ一定であった。 As shown in FIG. 6, even when the amount of acetone added was increased, if the final concentration of acetone was the same, the oil and fat recovery rate was almost constant.
１：藻類培養タンク
２：藻類培養槽
３：分離槽
４：抽出溶媒貯留槽
５，５ａ，５ｂ，５ｃ：油脂抽出槽
６：精製装置
1: Algal culture tank 2: Algal culture tank 3: Separation tank 4: Extraction solvent storage tank 5, 5a, 5b, 5c: Oil extraction tank 6: Purification device
Claims (6)
前記従属栄養性藻類が、オーランチオキトリウム属に属する微生物、スロストキトリウム属に属する微生物、シゾキトリウム属に属する微生物、及びパリエチキトリウム属に属する微生物からなる群から選ばれる１種又は２種以上であり、
前記抽出溶媒として、無極性有機溶媒と極性有機溶媒とを含有するものを用い、
前記抽出処理前の前記従属栄養性藻類を含む培養物と前記抽出溶媒との混合液中に、前記極性有機溶媒が１０〜５０質量％含有するように、前記抽出溶媒と前記従属栄養性藻類とを混合して、
前記従属栄養性藻類を、その細胞壁を破壊又は溶解処理していない状態で、前記抽出処理を行うことを特徴とする油脂製造方法。 A method for producing fats and oils by extracting fats and oils using an extraction solvent from heterotrophic algae having the ability to generate and accumulate fats and oils in the fungus body,
The heterotrophic algae is one or more selected from the group consisting of a microorganism belonging to the genus Aurantiochytrium, a microorganism belonging to the genus Throstochytrium, a microorganism belonging to the genus Schizochytrium, and a microorganism belonging to the genus Palychychytrium. And
As the extraction solvent, a solvent containing a nonpolar organic solvent and a polar organic solvent is used,
The extraction solvent and the heterotrophic algae are contained in a mixed solution of the culture containing the heterotrophic algae before the extraction treatment and the extraction solvent so that the polar organic solvent is contained in an amount of 10 to 50% by mass. Mix
The method for producing fats and oils , wherein the extraction treatment is performed on the heterotrophic algae in a state where the cell wall is not destroyed or dissolved.
The extraction solvent is added to the heterotrophic algae in a plurality of times, and the nonpolar organic solvent is added to the dry mass of 100 parts by mass of the heterotrophic algae and added to 10 to 2000 parts by mass. The fats and oils manufacturing method of any one of Claims 1-5 which performs an extraction process.
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