JP2005261239A - Method for producing lower alcohol - Google Patents
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- JP2005261239A JP2005261239A JP2004075590A JP2004075590A JP2005261239A JP 2005261239 A JP2005261239 A JP 2005261239A JP 2004075590 A JP2004075590 A JP 2004075590A JP 2004075590 A JP2004075590 A JP 2004075590A JP 2005261239 A JP2005261239 A JP 2005261239A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
Description
本発明はセルロース系バイオマス資源から石油代替エネルギーとして有用な低級アルコールを効率よく製造する方法、及びその生産菌に関する。 The present invention relates to a method for efficiently producing a lower alcohol useful as an alternative energy for petroleum from cellulosic biomass resources, and a microorganism producing the same.
石油に代表される化石燃料の消費を抑制し、地球温暖化ガスの排出を抑制するために新エネルギーの導入が進められている。その中で、バイオマス由来の原料から生産されるエタノールが注目されている。 The introduction of new energy is being promoted to reduce the consumption of fossil fuels typified by oil and to suppress the emission of greenhouse gases. Among them, ethanol produced from biomass-derived raw materials has attracted attention.
再生可能なバイオマスを原料とするエタノールの製造方法としては、古くから行われている、糖質から酵母を用いてアルコール発酵を行う方法；セルロース等を原料とし、まず酸を用いて加水分解して糖を得、次いで酵母を利用してアルコール発酵を行う方法が知られている。しかしながら、酸を用いる方法では、装置の腐食やランニングコストがかかるという問題があった。
そこで、クロストリジウム属に属する微生物を用いて、セルロース等のバイオマスから直接エタノールを製造する方法が提案されている（特許文献１〜５）。
Then, the method of manufacturing ethanol directly from biomass, such as a cellulose, using the microorganisms which belong to Clostridium genus is proposed (patent documents 1-5).
しかしながら、これら従来のセルロースから直接エタノールを生産する能力を有する微生物は、いずれもクロストリジウム属に属する偏性嫌気性菌であるため、嫌気的条件下での培養、すなわち、培養装置内を窒素ガス等の不活性ガスで置換する必要があり、取り扱いが煩雑であって大量生産には適さなかった。
従って、本発明の目的は、容易な操作でセルロースから直接エタノール等の低級アルコールを効率良く生産する方法を提供することにある。
However, since these microorganisms having the ability to directly produce ethanol directly from cellulose are obligate anaerobic bacteria belonging to the genus Clostridium, they are cultured under anaerobic conditions, that is, the inside of the culture apparatus is nitrogen gas or the like. It was necessary to substitute with an inert gas, and the handling was complicated and not suitable for mass production.
Accordingly, an object of the present invention is to provide a method for efficiently producing a lower alcohol such as ethanol directly from cellulose by an easy operation.
そこで本発明者は、セルロースから直接エタノールを生産する能力を有する新たな微生物を探索したところ、ジオバシラス属に属する微生物の中にセルロースから直接エタノール、１−プロパノール、１−ブタノール及び２−ブタノールの生産能力を有する微生物を見出した。さらに、この微生物は、好熱性の通性嫌気性菌であるため、酸素があっても失活したり、死滅することがなく、好気的条件下で培養した後に酸素の供給を停止すれば酸素を消費して嫌気的条件になるため、取り扱いが容易であり、低級アルコールの大量生産に適していることを見出し、本発明を完成するに至った。 Then, when this inventor searched for the new microorganism which has the capability to produce ethanol directly from a cellulose, production of ethanol, 1-propanol, 1-butanol, and 2-butanol directly from a cellulose in the microorganisms which belong to Geobacillus genus. We have found a microorganism with capacity. Furthermore, since this microorganism is a thermophilic facultative anaerobe, it will not be inactivated or killed even if oxygen is present, and if the supply of oxygen is stopped after culturing under aerobic conditions. Since oxygen is consumed and anaerobic conditions are obtained, it is easy to handle and suitable for mass production of lower alcohols, and the present invention has been completed.
すなわち、本発明は、セルロース系物質及び／又はセルロース系物質由来の糖質を含む培地で、ジオバシラス属に属し、セルロース系物質及び／又はセルロース系物質由来の糖質から低級アルコールを生産する能力を有する微生物を培養し、培養物から低級アルコールを採取することを特徴とする低級アルコールの製造法を提供するものである。
また、本発明は、ジオバシラス エスピー．ＢＳ−００１（ＦＥＲＭ Ｐ−１９７０６）を提供するものである。
That is, the present invention is a medium containing a cellulosic material and / or a saccharide derived from a cellulosic material, belonging to the genus Geobacillus, and having the ability to produce a lower alcohol from a saccharide derived from a cellulosic material and / or a cellulosic material. The present invention provides a method for producing a lower alcohol, which comprises culturing a microorganism having the microorganism and collecting the lower alcohol from the culture.
The present invention also relates to Geobacillus sp. BS-001 (FERM P-19706) is provided.
本発明によれば、培養装置内を窒素置換する等の煩雑な操作をすることなく、セルロース系物質又はセルロース系物質由来の糖質から直接エタノール等の低級アルコールを効率良く生産することができる。 According to the present invention, a lower alcohol such as ethanol can be efficiently produced directly from a cellulosic material or a saccharide derived from a cellulosic material without complicated operations such as substituting the inside of the culture apparatus with nitrogen.
本発明方法に用いられる微生物としては、ジオバシラス属（geobacillus）に属し、セルロース系物質及び／又はセルロース系物質由来の糖質から低級アルコールを生産する能力を有する微生物であれば特に制限されないが、甲虫の幼虫を飼育した培養土から採取されたジオバシラス エスピー．ＢＳ−００１株又はその類縁株が好ましい。 The microorganism used in the method of the present invention is not particularly limited as long as it is a microorganism belonging to the genus Geobacillus and having the ability to produce a lower alcohol from a cellulosic material and / or a sugar derived from the cellulosic material. Geobacillus sp. Collected from culture soil rearing larvae. The BS-001 strain or its related strain is preferred.
本発明に用いられるＢＳ−００１株の分離方法及び菌学的性質について説明する。 The isolation method and mycological properties of the BS-001 strain used in the present invention will be described.
１．菌体の分離源
甲虫の幼虫を飼育した培養土を分離源とした。分離培地に下記の培地成分に分離源を添加し、６０℃で２週間保温した。その後、セルロースパウダーを炭素源とした培地（pH７．０）の入った３００mL三角フラスコに分離源を１mL投入し、ブチルゴム栓をして、６０℃で３〜４日静置培養した。この操作を数回行い、セルロースを分解し、エタノール等のアルコールを生産する微生物を得た。
1. Isolation source of bacterial cells The culture soil in which beetle larvae were reared was used as the isolation source. A separation source was added to the following medium components in the separation medium, and kept at 60 ° C. for 2 weeks. Thereafter, 1 mL of a separation source was put into a 300 mL Erlenmeyer flask containing a medium (pH 7.0) using cellulose powder as a carbon source, and a butyl rubber stopper was attached, followed by stationary culture at 60 ° C. for 3 to 4 days. This operation was performed several times to decompose the cellulose and obtain a microorganism producing alcohol such as ethanol.
２．分離培地の組成を以下に示す。
ＫＨ2ＰＯ4・Ｈ2Ｏ及びＫ2ＨＰＯ4：５ｇ
尿素：２ｇ
（ＮＨ4）2ＳＯ4：２ｇ
酵母抽出物：５ｇ
炭素源（セルロース）：５ｇ
蒸留水：１Ｌ
2. The composition of the separation medium is shown below.
KH 2 PO 4 .H 2 O and K 2 HPO 4 : 5 g
Urea: 2g
(NH 4 ) 2 SO 4 : 2 g
Yeast extract: 5g
Carbon source (cellulose): 5g
Distilled water: 1L
３．菌学的性質
３−１．細菌第一段階試験
細菌第一段階試験として、光学顕微鏡Ｕ−ＬＨ１０００（オリンパス，日本）による細胞形態、グラム染色性、胞子の有無、鞭毛による運動性の有無を観察した。Trypticase Soy Agar(Becton Dickinson, NJ, U.S.A)平板培地上でのコロニー形態を観察した。カタラーゼ反応、オキシダーゼ反応、ブドウ糖からの酸／ガス産生、ブドウ糖の酸化／発酵（Ｏ／Ｆ）について試験を行った。結果を表１に示す。
3. 3. Mycological properties 3-1. Bacteria first-stage test As a bacteria first-stage test, cell morphology, gram staining, presence or absence of spores, and flagellar motility were observed with an optical microscope U-LH1000 (Olympus, Japan). The colony morphology on Trypticase Soy Agar (Becton Dickinson, NJ, USA) plate medium was observed. Tests were conducted for catalase reaction, oxidase reaction, acid / gas production from glucose, oxidation / fermentation of glucose (O / F). The results are shown in Table 1.
３−２．細菌第二段階試験
細菌第二段階試験として、ＡＰＩシステム（bioMeneux社）を使い、その測定方法に従い生化学的性状試験を実施した。
至適生育温度試験として、３７℃、４２℃、４５℃、５０℃、６０℃の各条件でＢＳ−００１株をTrypticase Soy Agar液体培地で培養した。また、至適生育pH試験としてpH６、７、８、９、１０の各条件でＢＳ−００１株をTrypticase Soy Agar液体培地で培養した。結果を表２及び表３に示す。
3-2. Bacteria second stage test As a bacteria second stage test, an API system (bioMeneux) was used, and a biochemical property test was performed according to the measurement method.
As an optimal growth temperature test, the BS-001 strain was cultured in Trypticase Soy Agar liquid medium under conditions of 37 ° C., 42 ° C., 45 ° C., 50 ° C. and 60 ° C. As an optimum growth pH test, the BS-001 strain was cultured in a Trypticase Soy Agar liquid medium under each condition of
至適生育温度試験では４２℃から６０℃で生育性を示し、至適生育pH試験ではpH７からpH９（pH９では弱い生育性）で生育性を示した。至適生育温度試験と至適生育pH試験は好気及び嫌気の条件で実施し、同様の結果を得た。このことからＢＳ−００１の通性嫌気性が確認された。これらの性状はGeobacillusの記載と一致する。 The optimum growth temperature test showed growth at 42 ° C. to 60 ° C., and the optimum growth pH test showed growth at pH 7 to pH 9 (weak growth at pH 9). The optimum growth temperature test and the optimum growth pH test were carried out under aerobic and anaerobic conditions, and similar results were obtained. From this, the facultative anaerobic property of BS-001 was confirmed. These properties are consistent with the description of Geobacillus.
４．１６ＳｒＤＮＡ（１６ＳｒＲＮＡ遺伝子）の解析
ＢＳ−００１株を前記分離培地に植菌し、６０℃で１日間培養し、この菌体をＤＮＡ抽出の供試菌体とした。ゲノムＤＮＡの抽出にはPrepMan Method(Applied Biosystems.社)を使用した。抽出したゲノムＤＮＡを鋳型として、ＰＣＲにより１６Ｓ Ribosomal ＲＮＡ遺伝子（１６ＳｒＤＮＡ）のうち５′末端側約５００bpの領域を増幅した。その後、増幅された塩基配列をシーケンシングし、検体の１６ＳｒＤＮＡ部分塩基配列を得た（配列番号１）。
4. Analysis of 16SrDNA (16SrRNA gene) The BS-001 strain was inoculated into the separation medium and cultured at 60 ° C for 1 day, and this cell was used as a test cell for DNA extraction. PrepMan Method (Applied Biosystems.) Was used for genomic DNA extraction. Using the extracted genomic DNA as a template, a region of about 500 bp on the 5 ′ end side of the 16S Ribosomal RNA gene (16SrDNA) was amplified by PCR. Thereafter, the amplified base sequence was sequenced to obtain a 16S rDNA partial base sequence of the sample (SEQ ID NO: 1).
以上の結果から、ＢＳ−００１株は、ジオバシラス属に属する新規菌株を判断した。そこで本菌株をジオバシラス エスピー．ＢＳ−００１と命名し、特許微生物寄託センターにＦＥＲＭ Ｐ−１９７０６として寄託した。 From the above results, the BS-001 strain was judged as a novel strain belonging to the genus Geobacillus. Therefore, this strain was designated as Geobacillus sp. It was named BS-001 and deposited at the Patent Microorganism Deposit Center as FERM P-19706.
本菌株を用いて低級アルコールを製造するには、セルロース系物質及び／又はセルロース由来の糖質を含む培地で、本菌株又はその類縁株を培養し、培養物から低級アルコールを採取すればよい。 In order to produce a lower alcohol using this strain, the strain or a related strain thereof is cultured in a medium containing a cellulosic material and / or a sugar derived from cellulose, and the lower alcohol is collected from the culture.
セルロース系物質及び／又はセルロース由来の糖質としては、古紙・製紙スラッジ、セルロースを含有する草木等が挙げられるが、効率よく反応させるためには、破砕やその他の前処理を施してから炭素源として供給することが望ましい。その他に培地成分として、窒素源、無機塩、その他発酵に必要な成分の種類、添加量はエタノール発酵における既知の条件の中から、適宜選定すればよい。 Examples of cellulosic substances and / or cellulose-derived saccharides include waste paper, paper sludge, and plants containing cellulose. In order to react efficiently, the carbon source must be subjected to crushing and other pretreatments. It is desirable to supply as In addition, as a medium component, the nitrogen source, inorganic salt, other kinds of components necessary for fermentation, and the addition amount may be appropriately selected from known conditions in ethanol fermentation.
培養に際して、温度、pH等の条件は使用する微生物が低級アルコールを生産しうる範囲であればよく、通常は５０℃〜６５℃、pH５．８〜９より好ましくは、６．２〜７．５のpHの範囲である。また本発明の菌株は、通性嫌気性菌であるため、好気的条件で培養した後に酸素の供給を停止すればよく、培養条件の管理が容易である。 In culturing, conditions such as temperature and pH may be in a range in which the microorganism to be used can produce a lower alcohol, and is usually 50 ° C. to 65 ° C., preferably pH 5.8 to 9, more preferably 6.2 to 7.5. PH range. Moreover, since the strain of the present invention is a facultative anaerobic bacterium, it is only necessary to stop the supply of oxygen after culturing under aerobic conditions, and management of the culture conditions is easy.
本発明方法によって得られる低級アルコールとしては、エタノールの他、１−プロパノール、１−ブタノール、２−ブタノールが挙げられる。このうち、エタノールの生産性が最も高く、本発明方法は、特にエタノールの生産に好適である。培養物からの低級アルコールの採取は、常法に従えばよい。 Examples of the lower alcohol obtained by the method of the present invention include ethanol, 1-propanol, 1-butanol, and 2-butanol. Among these, ethanol productivity is the highest, and the method of the present invention is particularly suitable for ethanol production. The collection of the lower alcohol from the culture may be performed according to a conventional method.
次に実施例を挙げて本発明を説明するが、本発明はこれにより何ら限定されるものではない。 EXAMPLES Next, the present invention will be described with reference to examples, but the present invention is not limited thereto.
実施例１
下記の成分培地に、ＢＳ−００１株を接種し、温度６０℃、pH７．０、回転数１５０rpmで撹拌して培養した。
Example 1
The following component medium was inoculated with the BS-001 strain and cultured with stirring at a temperature of 60 ° C., pH 7.0, and a rotational speed of 150 rpm.
（表４）
ＫＨ2ＰＯ4 ４．４ｇ
尿素 １．５ｇ
（ＮＨ4）2ＳＯ4 ０．５ｇ
酵母エキス ３．０ｇ
ＣａＣｌ2・Ｈ2Ｏ ０．０５ｇ
システイン塩酸塩 １．０ｇ
ＦｅＳＯ4 １．０mg
セルロース ５０ｇ
蒸留水 １Ｌ
(Table 4)
4.4 g of KH 2 PO 4
1.5g of urea
(NH 4 ) 2 SO 4 0.5 g
Yeast extract 3.0g
CaCl 2 · H 2 O 0.05g
Cysteine hydrochloride 1.0g
FeSO 4 1.0mg
Cellulose 50g
1L of distilled water
培養時間と培養物中の低級アルコール濃度との関係を図１に示す。図１から明らかなように、本発明方法によればセルロースから直接エタノール、１−プロパノール、１−ブタノール及び２−ブタノールを生産できる。 The relationship between the culture time and the lower alcohol concentration in the culture is shown in FIG. As apparent from FIG. 1, according to the method of the present invention, ethanol, 1-propanol, 1-butanol and 2-butanol can be produced directly from cellulose.
Claims (4)
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008038019A3 (en) * | 2006-09-28 | 2008-09-25 | Tmo Renewables Ltd | Thermophilic microorganisms for ethanol production |
| US20110097775A1 (en) * | 2008-04-03 | 2011-04-28 | Green Biologics Limited | Production of butanol |
| JP2011522543A (en) * | 2008-06-04 | 2011-08-04 | ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー | Method for producing butanol using two-phase extractive fermentation |
| US8021865B2 (en) | 2007-08-13 | 2011-09-20 | Tmo Renewables Limited | Thermophilic micro-organisms for ethanol production |
| US8486687B2 (en) | 2008-11-05 | 2013-07-16 | Tmo Renewables Limited | Sporulation-deficient thermophilic microorganisms for the production of ethanol |
| US8541222B2 (en) | 2005-06-07 | 2013-09-24 | Tmo Renewables Limited | Modified microorganisms with inactivated lactate dehydrogenase gene |
| US9249431B2 (en) | 2008-02-28 | 2016-02-02 | Green Biologics Limited | Production process |
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2004
- 2004-03-17 JP JP2004075590A patent/JP2005261239A/en active Pending
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8541222B2 (en) | 2005-06-07 | 2013-09-24 | Tmo Renewables Limited | Modified microorganisms with inactivated lactate dehydrogenase gene |
| EA017548B1 (en) * | 2006-09-28 | 2013-01-30 | Тмо Реньюаблз Лимитед | Thermophilic microorganism modified for increased ethanol production, and method for production of ethanol using same |
| JP2010504747A (en) * | 2006-09-28 | 2010-02-18 | ティーエムオー リニューアブルズ リミティド | Thermophilic microorganisms for ethanol production |
| US8932841B2 (en) | 2006-09-28 | 2015-01-13 | Tmo Renewables Limited | Thermophilic microorganisms for ethanol production |
| WO2008038019A3 (en) * | 2006-09-28 | 2008-09-25 | Tmo Renewables Ltd | Thermophilic microorganisms for ethanol production |
| US8852906B2 (en) | 2007-08-13 | 2014-10-07 | Tmo Renewables Limited | Thermophilic micro-organisms for ethanol production |
| US8143038B2 (en) | 2007-08-13 | 2012-03-27 | Tmo Renewables Limited | Thermophilic micro-organisms for ethanol production |
| US8021865B2 (en) | 2007-08-13 | 2011-09-20 | Tmo Renewables Limited | Thermophilic micro-organisms for ethanol production |
| US9249431B2 (en) | 2008-02-28 | 2016-02-02 | Green Biologics Limited | Production process |
| US20110097775A1 (en) * | 2008-04-03 | 2011-04-28 | Green Biologics Limited | Production of butanol |
| JP2011522543A (en) * | 2008-06-04 | 2011-08-04 | ビュータマックス・アドバンスド・バイオフューエルズ・エルエルシー | Method for producing butanol using two-phase extractive fermentation |
| US8486687B2 (en) | 2008-11-05 | 2013-07-16 | Tmo Renewables Limited | Sporulation-deficient thermophilic microorganisms for the production of ethanol |
| US9469858B2 (en) | 2008-11-05 | 2016-10-18 | Tmo Renewables Limited | Sporulation-deficient thermophilic microorganisms for the production of ethanol |
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