JP6730582B2 - A method for separating Botryococcus, a high oil producer, using radiation. - Google Patents
A method for separating Botryococcus, a high oil producer, using radiation. Download PDFInfo
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Description
本発明は、放射線を用いたボトリオコッカスのオイル高生産体の分離方法に関する。 TECHNICAL FIELD The present invention relates to a method for separating a Botryococcus high oil producer using radiation.
ボトリオコッカス（Botryococcus）をはじめとするオイルを高生産する微細藻類がバイオマス資源として注目されている。
このような藻体の回収技術としては、ナノバブルを用いて藻体を物理的に浮遊させる方法が知られている（特許文献１）。しかし、この方法によると、オイル含量の低い藻体も同時に浮遊してしまうという欠点があった。また、藻体の状態によってはナノバブルでは回収できない場合もあった。
Microalgae that produce high amounts of oil such as Botryococcus are attracting attention as biomass resources.
As a technique for recovering such algal bodies, a method of physically suspending algal bodies using nanobubbles is known (Patent Document 1). However, this method has a drawback in that algal cells having a low oil content also float at the same time. In addition, depending on the state of the algal cells, it may not be possible to collect them with nanobubbles.
一方、ボトリオコッカスと同じ緑藻であるクラミドモナスにおいて、窒素欠乏培地に交換することでオイルの生産量を高め浮遊させるという現象が報告されている（非特許文献１）。しかし、ボトリオコッカスの場合は、藻体回収が困難であり培地交換による方法を適用するのは困難であった。また、遠心分離法で藻体を回収しようとすると、オイル生産量が低く比重の重い藻体が優先的に回収されてしまう。そのため、培地交換を経ずに藻体を浮遊させる技術が求められていた。 On the other hand, it has been reported that in Chlamydomonas, which is the same green alga as Botryococcus, the production of oil is increased and suspended by exchanging it with a nitrogen-deficient medium (Non-Patent Document 1). However, in the case of Botryococcus, it was difficult to recover the algal cells and it was difficult to apply the method by changing the medium. In addition, when attempting to collect algal cells by the centrifugation method, algal cells with low oil production and high specific gravity are preferentially recovered. Therefore, there has been a demand for a technique for suspending algal cells without changing the medium.
かように、オイル生産量が高いボトリオコッカスのみを分離回収できる技術はこれまでになかった。したがって、本発明は、ボトリオコッカスの回収工程において、オイル生産量の高い藻体のみを選択的に分離する方法を提供することを目的とする。 Thus, there has not been a technology that can separate and collect only Botryococcus that produces a large amount of oil. Therefore, it is an object of the present invention to provide a method for selectively separating only algal cells with high oil production in a Botryococcus recovery step.
本願発明者らは、上記課題を解決するために鋭意研究を行った結果、下記の方法により上述した課題を解決できることを見出し、本発明に想到するに至った。 As a result of earnest research for solving the above-mentioned problems, the inventors of the present application have found that the above-mentioned problems can be solved by the following method, and have arrived at the present invention.
即ち、本発明は、次の発明を提供するものである。 That is, the present invention provides the following inventions.
＜１＞
液中のボトリオコッカス藻体に、重イオンビーム又はＸ線を照射する工程および浮遊したオイル高生産ボトリオコッカス藻体を採取する工程を含む、オイル高生産ボトリオコッカス藻体の分離方法。
<1>
A method for separating an oil-producing high Botryococcus alga body, which comprises a step of irradiating a Botryococcus alga body in a liquid with a heavy ion beam or X-rays and a step of collecting floating oil-producing high Botryococcus alga body.
＜２＞
前記重イオンビーム又はＸ線の線量が25〜150Gyである＜１＞に記載の方法。
<2>
The method according to <1>, wherein the dose of the heavy ion beam or X-ray is 25 to 150 Gy.
＜３＞
前記重イオンビームが、炭素イオンビーム又はアルゴンイオンビームである＜１＞又は＜２＞に記載の方法。
<3>
The method according to <1> or <2>, wherein the heavy ion beam is a carbon ion beam or an argon ion beam.
＜４＞
液中のボトリオコッカス藻体に、重イオンビーム又はＸ線を照射する工程および浮遊したオイル高生産ボトリオコッカス藻体を採取し、採取したオイル高生産ボトリオコッカス藻体からオイルを分取する工程を含む、オイルの製造方法。
<4>
The step of irradiating the Botryococcus alga in the liquid with a heavy ion beam or X-ray and the suspended oil-rich Botryococcus alga is collected, and the oil is fractionated from the collected oil-rich Botryococcus alga. A method for producing oil, including the step of:
本発明の方法によれば、オイルを多量に生産した藻体のみを採取することができる。
また、増殖状態にある浮遊しなかった藻体は、さらに培養することで、オイル高生産ボトリオコッカス藻体とし、連続的にオイル高生産ボトリオコッカス藻体を回収でき、オイルを有利に製造することができる。
According to the method of the present invention, it is possible to collect only algal cells that produce a large amount of oil.
In addition, the non-floating algal cells in a growing state can be further cultured to obtain oil-rich Botryococcus algal cells that can be continuously collected to produce oil-rich Botryococcus algal cells. can do.
以下、本発明を詳細に説明する。
本発明では、ボトリオコッカスに放射線、具体的には重イオンビーム又はＸ線を照射する。
Hereinafter, the present invention will be described in detail.
In the present invention, Botryococcus is irradiated with radiation, specifically, a heavy ion beam or X-ray.
本発明で照射する重イオンビームとしては、例えば、アルゴンイオンビーム、炭素イオンビームなどが挙げられる。なかでも、アルゴンイオンビームが特に好ましい。 Examples of the heavy ion beam irradiated in the present invention include an argon ion beam and a carbon ion beam. Of these, an argon ion beam is particularly preferable.
重イオンビーム又はＸ線照射の照射線量は、本発明の効果が得られ、かつ照射対象であるボトリオコッカスの生長に大きな影響を与えない範囲内である。この範囲は、25〜150Gyであるが、25〜100Gyが好ましく、50〜75Gyが特に好ましい。 The irradiation dose of heavy ion beam or X-ray irradiation is within a range in which the effects of the present invention are obtained and the growth of Botryococcus as an irradiation target is not significantly affected. This range is 25 to 150 Gy, preferably 25 to 100 Gy, particularly preferably 50 to 75 Gy.
本発明で照射対象として用いるボトリオコッカスは、ボトリオコッカス（Botryococcus）属の微細藻類であれば特に限定されず、湖沼や池などの野生で入手可能なものを使用することができる。通常、ボトリオコッカスといえばボトリオコッカス・ブラウニー（学名：Botryococcus braunii）を指す。
また、ボトリオコッカスは常法に従って培養したものを使用すればよい。例えば、培地としては、Chu13改変培地やChu10培地、BG11、BBM、BBMa培地、AF-6培地などが使用され、その中でもAF-6培地は多くのBotryococcus株で安定した増殖を示すためよく使われている。また、培養条件としては以下のような条件が挙げられる。
・温度:15〜40℃（至適温度は30℃前後）
・培地pH：5-9
・光強度：10-160μmol/m2/s
・明暗周期：連続光で光を与えることも可能だが、一日のうち数時間（2〜12時間）の暗期を設けることが望ましい。
・培養期間：窒素源となっている硝酸態窒素が培地中より枯渇すると増殖が停止するため、培養期間はこの濃度に依存することが多い。培地中の硝酸態窒素濃度が150mg/L程度の場合、3-4週間程度の培養期間を要する。
・エアレーション：空気あるいは10％程度までの二酸化炭素濃度になるように圧縮空気と混合して培地に与える。
The Botryococcus used as an irradiation target in the present invention is not particularly limited as long as it is a microalga of the genus Botryococcus, and those available in the wild such as lakes and ponds can be used. Normally, Botryococcus refers to Botryococcus braunii.
In addition, Botryococcus may be used after being cultured according to a conventional method. For example, as the medium, Chu13 modified medium or Chu10 medium, BG11, BBM, BBMa medium, AF-6 medium, etc. are used, and among them, AF-6 medium is often used because it shows stable growth in many Botryococcus strains. ing. Moreover, the following conditions can be mentioned as culture conditions.
・Temperature: 15-40℃ (The optimum temperature is around 30℃)
・Media pH: 5-9
・Light intensity: 10-160μmol/m 2 /s
-Brightness cycle: It is possible to give light by continuous light, but it is desirable to provide a dark period of several hours (2 to 12 hours) in one day.
-Culture period: Since the growth stops when nitrate nitrogen, which is a nitrogen source, is depleted from the medium, the culture period often depends on this concentration. When the nitrate nitrogen concentration in the medium is about 150 mg/L, a culture period of about 3-4 weeks is required.
・Aeration: Air or mixed with compressed air to a carbon dioxide concentration of up to about 10% and fed to the medium.
上述の方法で培養したボトリオコッカスに重イオンビーム又はＸ線を照射する。照射は、定常期にある藻体に対して行ってもよく、増殖期にある藻体に対して行ってもよく、特に限定されない。 Botryococcus cultivated by the above method is irradiated with a heavy ion beam or X-ray. Irradiation may be performed on the algal cells in the stationary phase or may be performed on the algal cells in the growth phase and is not particularly limited.
照射は液中、特に培養液中のボトリオコッカスに対して行うことが好ましく、重イオンビーム又はＸ線を照射後、暫く静置すればオイル高生産ボトリオコッカス藻体が浮遊する。ここで、オイル高生産ボトリオコッカス藻体とは、乾燥した藻体中、オイルを40重量％以上含有する藻体をさす。すなわち、照射から一定時間経過後、通常3時間程度でオイル高生産ボトリオコッカス藻体の浮遊が確認される。このときの条件は、特に限定されないが、培養時の条件、例えば、光量約100μmol/m2/secのもと、室温で静置することが好ましい。 Irradiation is preferably performed on Botryococcus in a liquid, particularly in a culture solution. After irradiation with heavy ion beams or X-rays, the oil-producing high Botryococcus algal cells float if left standing for a while. Here, the high oil-producing Botryococcus algal cells refer to algal cells containing 40% by weight or more of oil in the dried algal cells. That is, after a certain period of time has elapsed from the irradiation, the high oil-producing Botryococcus algal cells are confirmed to float in about 3 hours. The conditions at this time are not particularly limited, but it is preferable to stand at room temperature under the conditions for culturing, for example, under a light amount of about 100 μmol/m 2 /sec.
浮遊したボトリオコッカスは、常法により採取すればよい。例えば、ピペットを用いて浮遊したボトリオコッカスを採取したり、デカンテーションにより行うことができる。 The floating Botryococcus may be collected by a conventional method. For example, floating Botryococcus can be collected using a pipette or can be decanted.
採取したボトリオコッカスから常法に従ってオイルを抽出することができる。ここで本発明において、ボトリオコッカスから得られる「オイル」は、多くの藻類が生産するトリグリセリド（いわゆる植物油）ではなく、炭化水素（いわゆる石油系オイル）を主成分とするものである。オイルの抽出方法としては、例えば、採取した藻体を凍結乾燥し、オイルを抽出することができる有機溶媒に浸漬し、遠心分離し、溶媒を濃縮する方法などが挙げられる。このとき、必要に応じてシリカゲルクロマトグラフィーなどでオイルの精製を行ってもよい。 Oil can be extracted from the collected Botryococcus according to a conventional method. Here, in the present invention, the "oil" obtained from Botryococcus is not a triglyceride (so-called vegetable oil) produced by many algae, but a hydrocarbon (so-called petroleum oil) as a main component. Examples of the method for extracting the oil include a method in which the collected algal cells are freeze-dried, immersed in an organic solvent capable of extracting the oil, centrifuged, and the solvent is concentrated. At this time, the oil may be purified by silica gel chromatography or the like, if necessary.
以下、実施例および比較例を挙げて、本願発明をさらに詳細に説明するが、本願発明は下記の例に制限されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to the following examples.
野外の池から採取したボトリオコッカス（Hojo株）を培養し、実験に供した。
培養は、下記の通り行った。
・光条件；蛍光灯３本（約100μmol/m2/sec）
・明暗周期；明：暗＝17H：7H
・エアレーション；5% CO2（暗条件下ではCO2を含まない空気を通気）
・培養温度；25℃
・培地；組成を表１に示す。
Botryococcus (Hojo strain) collected from an outdoor pond was cultured and used for the experiment.
Culture was performed as follows.
・Light conditions: 3 fluorescent lamps (about 100 μmol/m 2 /sec)
・Light-dark cycle; light:dark = 17H:7H
・Aeration: 5% CO 2 (air that does not contain CO 2 is aerated under dark conditions)
・Culturing temperature: 25℃
-Media: The composition is shown in Table 1.
重イオンビームとしては、理化学研究所仁科加速器研究センター施設内のリングサイクロトロンにおいて、アルゴンイオンビームを用いた。
Ｘ線の照射は、Ｘ線照射装置（RIGAKU）を用いた。
As a heavy ion beam, an argon ion beam was used in a ring cyclotron in the Nishina Accelerator Research Center facility of RIKEN.
An X-ray irradiation device (RIGAKU) was used for X-ray irradiation.
実施例１〜４
上記条件で培養した培養開始から12日後のボトリオコッカス培養液を200μLの８連チューブに入れ、表２に示す線量のアルゴンイオンビームをそれぞれ照射した。
アルゴンイオンビーム照射後、光量約100μmol/m2/sec、25℃で3時間静置した後チューブを観察したところ、アルゴンイオンビームの線量に依存して、藻体の浮遊がみられた。
Examples 1 to 4
After 12 days from the start of the culture under the above conditions, the Botryococcus culture solution was placed in a 200 μL 8-tube and irradiated with argon ion beams at the doses shown in Table 2.
After irradiation with an argon ion beam, the tube was observed after standing still at 25°C for 3 hours at a light intensity of 100 μmol/m 2 /sec, and the alga was found to float depending on the dose of the argon ion beam.
実施例５
上記条件で培養した培養開始から12日後のボトリオコッカス培養液を200μLの８連チューブに入れ、4Gy/分の線量率で12.5分、50GyのＸ線を照射し、実施例１〜４と同様に光量約100μmol/m2/sec、25℃で3時間静置した後観察したところ、藻体の浮遊がみられた。
Example 5
After 12 days from the start of the culture under the above conditions, the Botryococcus culture solution was placed in a 200 μL 8-strip tube and irradiated with 50 Gy of X-ray for 12.5 minutes at a dose rate of 4 Gy/min, and the same as in Examples 1 to 4. When observed for 3 hours at 25°C with a light intensity of about 100 μmol/m 2 /sec, the algal cells were found to be floating.
比較例１
アルゴンイオンビームおよびＸ線いずれも照射しなかった以外は同様にして培養液の観察を行った。
Comparative Example 1
The culture solution was observed in the same manner except that neither the argon ion beam nor the X-ray was irradiated.
静置後の各チューブを撮影した写真を図１に示す。また、図１の写真から以下の方法で算出した浮遊指数を表２に示す。 A photograph of each tube after standing is shown in FIG. In addition, Table 2 shows the floating index calculated from the photograph of FIG. 1 by the following method.
［浮遊指数算出方法］
（１）画像解析ソフト ImageJに画像を取り込む。
（２）8-bit グレースケールに変換する。
（３）同一面積内の沈殿藻体の輝度を、各例につき3チューブずつ測定し平均値を算出する。
（４）（３）で算出した平均値に対し、比較例１の値を0、実施例３の値を100として、各例について浮遊指数を算出する。
[Floating index calculation method]
(1) Import images into image analysis software ImageJ.
(2) Convert to 8-bit grayscale.
(3) The brightness of the precipitated algal cells in the same area is measured in 3 tubes for each example, and the average value is calculated.
(4) Taking the value of Comparative Example 1 as 0 and the value of Example 3 as 100 with respect to the average value calculated in (3), the floating index is calculated for each example.
アルゴンイオンビームの線量を一定以上にすると、オイル含量の高い藻体の浮遊がみられた。100Gyおよび150Gyを照射した場合には、藻体が浮遊した。Ｘ線50Gyを照射した場合でも、藻体が浮遊した。線質を問わず放射線照射により、藻体の浮遊現象が起こることを見出した。以後の解析をＸ線50Gyで行うこととした。 When the dose of the argon ion beam was above a certain level, floating of algal cells with high oil content was observed. When irradiated with 100 Gy and 150 Gy, algal cells floated. Even when irradiated with 50 Gy of X-rays, algal cells floated. It was found that the floating phenomenon of algal cells occurs by irradiation regardless of the radiation quality. It was decided to carry out the subsequent analysis with 50 Gy of X-ray.
次に、浮遊藻体と沈殿藻体に脂溶性蛍光色素を作用させて顕微鏡観察を行った。
実施例５と同様の条件でボトリオコッカス培養液にＸ線を照射した。照射後、光量約100μmol/m2/sec、25℃で3時間静置し、浮遊藻体と沈殿藻体と分取した。浮遊藻体と沈殿藻体それぞれに脂溶性蛍光色素BODIPYを終濃度1μg/mLになるよう加えて10分間染色し、蛍光顕微鏡で観察した。図２に明視野顕微鏡写真（左側）及び蛍光顕微鏡写真を示す。
浮遊藻体にはBODIPYで染色される（図２の蛍光顕微鏡写真の白く明るい部分）藻体が多くみられ、沈殿藻体ではBODIPYの蛍光強度が弱かった。本実験において、浮遊藻体ではオイルが高生産していることが分かった。
Next, a lipid-soluble fluorescent dye was allowed to act on the floating algal cells and the precipitated algal cells, and microscopic observation was performed.
The Botryococcus culture medium was irradiated with X-rays under the same conditions as in Example 5. After the irradiation, the cells were allowed to stand at 25°C for 3 hours with a light amount of about 100 µmol/m 2 /sec for 3 hours to separate suspended algal cells and precipitated algal cells. The lipid-soluble fluorescent dye BODIPY was added to each of the floating algal cells and the precipitated algal cells at a final concentration of 1 μg/mL, and the cells were stained for 10 minutes and observed with a fluorescence microscope. FIG. 2 shows a bright field microscope photograph (left side) and a fluorescence microscope photograph.
Many floating algal cells were stained with BODIPY (white and bright areas in the fluorescence micrograph of FIG. 2), and precipitated algal cells had weak BODIPY fluorescence intensity. In this experiment, it was found that oil was highly produced in floating algal cells.
最後に、定常期と増殖期にある藻体に対してＸ線照射を行い、オイル含量を分析した。 Finally, X-ray irradiation was performed on the algal cells in the stationary phase and the growth phase, and the oil content was analyzed.
［定常期］
4日間培養した前培養液を1Lデュラン瓶に植菌（最終培地量800mL）し本培養を行い、培養開始から18日目にサンプリングした。培養5日目と18日目（藻体回収日サンプル）で増殖率速度μ(day-1)を求めると、0.038であった。
［増殖期］
7日間培養した前培養液を1L三角フラスコに植菌（最終培地量700mL）し本培養とした。本培養では、培養開始から13日目に栄養塩を添加し、培養最終日まで増殖状態を保たせた。培養13日目と26日目（藻体回収日サンプル）での増殖率速度μ(day-1)は、0.104であり、定常期に比べ約3倍の増殖能をもっていた。
[Stationary phase]
The pre-cultured solution that had been cultivated for 4 days was inoculated into a 1 L Duran bottle (final medium amount 800 mL), main culture was performed, and sampling was performed on the 18th day from the start of culture. The proliferation rate μ (day −1 ) was calculated on the 5th day and the 18th day of culture (the sample on the day when the algal cells were collected), and it was 0.038.
[Growth phase]
The pre-cultured liquid that had been cultivated for 7 days was inoculated into a 1 L Erlenmeyer flask (final medium amount: 700 mL) to make a main culture. In the main culture, nutrient salts were added on the 13th day from the start of the culture to keep the growth state until the last day of the culture. The growth rate μ (day −1 ) on day 13 and day 26 of culture (the sample on the day when the algal cells were collected) was 0.104, which was about 3 times that of the stationary phase.
次に、定常期にある培養18日目の藻体および増殖期にある培養26日目の藻体に対して、それぞれ培養液の濃縮を行った。各培養液は孔径20μmのナイロンメッシュフィルターを取り付けたロート（オートクレーブ滅菌済み）を使用して濃縮した。滅菌済み培地で3回洗浄後、50mL平型培養フラスコに移し、Ｘ線の照射試験に供した。 Next, the culture solution was concentrated for each of the algal cells on the 18th day of culture in the stationary phase and the algal cells on the 26th day of culture in the growth phase. Each culture solution was concentrated using a funnel (autoclave sterilized) equipped with a nylon mesh filter having a pore size of 20 μm. After washing three times with a sterilized medium, the medium was transferred to a 50 mL flat culture flask and subjected to an X-ray irradiation test.
上述した培養液に対して、4Gy/分の線量率で12.5分、50GyのＸ線を照射した。照射後は、光量約100μmol/m2/sec、25℃で3時間静置した。その後、浮遊藻体と沈殿藻体とを分取した。 The above culture solution was irradiated with 50 Gy of X-ray at a dose rate of 4 Gy/min for 12.5 minutes. After the irradiation, it was allowed to stand at 25° C. for 3 hours with a light amount of about 100 μmol/m 2 /sec. Then, the floating algal cells and the precipitated algal cells were separated.
浮遊藻体と沈殿藻体それぞれに対して、以下の操作を行い、オイル含量を算出した。まず、分取した藻体を凍結乾燥し、一定量（1g前後）を正確に秤量し、10mLのヘキサン／エチルアルコール（3:1, v/v）に2時間浸漬した。次に、その藻体懸濁液を2,800rpm、20分間遠心分離後、溶媒部分を分取した。残渣に10mLのヘキサン／エチルアルコール（3:1, v/v）を加え、同様の操作で溶媒を分取した。抽出溶媒を合わせ、ロータリーエバポレーターで濃縮乾固した。残留物をn-ヘキサンに懸濁し、2mLのシリカゲルカラムに添加した。10mLのn-ヘキサン／クロロホルム（1:1, v/v）で溶出する画分を炭化水素画分とし、溶媒を窒素ガス気流下で完全に除いた後、残留したオイル画分の重量を秤量した。
得られたオイルの組成は、主にC33H56の炭化水素であった。
The following operations were performed on the floating algal cells and the precipitated algal cells to calculate the oil content. First, the separated algal cells were freeze-dried, a certain amount (around 1 g) was accurately weighed, and immersed in 10 mL of hexane/ethyl alcohol (3:1, v/v) for 2 hours. Next, the algal suspension was centrifuged at 2,800 rpm for 20 minutes, and then the solvent portion was separated. 10 mL of hexane/ethyl alcohol (3:1, v/v) was added to the residue, and the solvent was fractionated by the same operation. The extraction solvents were combined and concentrated to dryness on a rotary evaporator. The residue was suspended in n-hexane and added to a 2 mL silica gel column. The fraction eluted with 10 mL of n-hexane/chloroform (1:1, v/v) was used as the hydrocarbon fraction, the solvent was completely removed under a nitrogen gas stream, and the residual oil fraction was weighed. did.
The composition of the obtained oil was mainly C 33 H 56 hydrocarbons.
結果を表３に示す。定常期および増殖期いずれの場合も、浮遊藻体は沈殿藻体や非照射藻体よりもオイル含量が高かった。比重が小さくなって藻体が浮上すると考えられる。 The results are shown in Table 3. In both the stationary phase and the growth phase, the suspended algal bodies had higher oil contents than the precipitated algal bodies and the non-irradiated algal bodies. It is considered that the specific gravity decreases and the algal body floats.
本発明の方法を用いることにより、オイル含量の高い藻体のみを選択的に分離できる。また、増殖状態にある浮遊しなかったオイル含量の低い藻体をさらに培養することで、連続的にオイル高含量藻体を得ることができるため、多くのオイルを効率よく得られる。 By using the method of the present invention, only algal cells having a high oil content can be selectively separated. Further, by further culturing the algal cells with a low oil content, which are in a proliferating state and did not float, it is possible to continuously obtain algal cells with a high oil content, so that many oils can be efficiently obtained.
Claims (3)
The step of irradiating the Botryococcus algal cells in the liquid with X-rays at a dose of 50 to 75 Gy and the floating oil-rich Botryococcal algal cells were collected, and the collected oil-rich Botryococcus algal cells were used as oil A method for producing oil, comprising the step of collecting oil.
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