JP6327822B2 - Method for producing ethanol from woody biomass using filamentous fungi - Google Patents
Method for producing ethanol from woody biomass using filamentous fungi Download PDFInfo
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- JP6327822B2 JP6327822B2 JP2013202539A JP2013202539A JP6327822B2 JP 6327822 B2 JP6327822 B2 JP 6327822B2 JP 2013202539 A JP2013202539 A JP 2013202539A JP 2013202539 A JP2013202539 A JP 2013202539A JP 6327822 B2 JP6327822 B2 JP 6327822B2
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims description 56
- 241000233866 Fungi Species 0.000 title claims description 20
- 238000004519 manufacturing process Methods 0.000 title claims description 15
- 239000002028 Biomass Substances 0.000 title description 12
- 108010059892 Cellulase Proteins 0.000 claims description 19
- 229940106157 cellulase Drugs 0.000 claims description 19
- 241000235389 Absidia Species 0.000 claims description 5
- 230000003248 secreting effect Effects 0.000 claims description 3
- 241000293029 Absidia caerulea Species 0.000 claims description 2
- 241001570673 Absidia cylindrospora Species 0.000 claims description 2
- 238000000855 fermentation Methods 0.000 description 13
- 230000004151 fermentation Effects 0.000 description 13
- 244000005700 microbiome Species 0.000 description 9
- 239000010802 sludge Substances 0.000 description 9
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 4
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 150000002972 pentoses Chemical class 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 239000002023 wood Substances 0.000 description 4
- 229920002488 Hemicellulose Polymers 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 3
- 235000007164 Oryza sativa Nutrition 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 235000009566 rice Nutrition 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 235000000346 sugar Nutrition 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000235395 Mucor Species 0.000 description 2
- 241000235648 Pichia Species 0.000 description 2
- 241000235402 Rhizomucor Species 0.000 description 2
- 240000000111 Saccharum officinarum Species 0.000 description 2
- 235000007201 Saccharum officinarum Nutrition 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000002154 agricultural waste Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- 150000002402 hexoses Chemical class 0.000 description 2
- 239000002440 industrial waste Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000010893 paper waste Substances 0.000 description 2
- 239000010902 straw Substances 0.000 description 2
- 241000233788 Arecaceae Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241001450909 Gongronella Species 0.000 description 1
- 241001450899 Gongronella butleri Species 0.000 description 1
- 241001305961 Lichtheimia hyalospora Species 0.000 description 1
- 241000218657 Picea Species 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 241001215623 Talaromyces cellulolyticus Species 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 241000758405 Zoopagomycotina Species 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
- 239000002551 biofuel Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001461 cytolytic effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- 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
Description
本発明は、特定の糸状菌を用いる木質系バイオマスまたはその加水分解物から、エタノールを製造する方法に関するものである。 The present invention relates to a method for producing ethanol from a woody biomass using a specific filamentous fungus or a hydrolyzate thereof.
現在、ブラジルやアメリカなどで行われているエタノールの製造は、トウモロコシやサトウキビから得られた６炭糖を主成分とする糖液を、酵母（Ｓ．ｃｅｒｅｖｉｓｉａｅ）を用いる発酵法により行われる。トウモロコシやサトウキビは生産が容易で加工がしやすく豊富な糖が得られ、酵母は高濃度の糖存在下で優れた成長能力を持ち、エタノール生産収率も高い。しかし食料を燃料に替えるという倫理観の問題に始まり、食料としての供給の減少などの重大な問題を抱えている。このような背景から、未利用なバイオマス資源である農産廃棄物（稲わら、もみ殻など）、林産廃棄物（間伐材、廃木材）や産業廃棄物（ペーパースラッジなど）からエタノールを高収率で得る研究が進められている。
未利用なバイオマス資源である農産廃棄物からのエタノール生産は、成分であるセルロースやヘミセルロースなどを分解・発酵させてエタノールを生産する微生物が利用される。
Currently, ethanol production performed in Brazil, the United States, and the like is performed by fermentation using yeast (S. cerevisiae) with a sugar solution mainly composed of hexose obtained from corn and sugarcane. Corn and sugarcane are easy to produce, easy to process, and abundant sugars are obtained. Yeast has excellent growth ability in the presence of high concentrations of sugar and has a high ethanol production yield. However, it begins with the ethical problem of replacing food with fuel, and has serious problems such as a decline in the supply of food. Against this background, high yields of ethanol from unused biomass resources such as agricultural waste (rice straw, rice husk, etc.), forest waste (thinned wood, waste wood) and industrial waste (paper sludge, etc.) The research gained in is underway.
Ethanol production from agricultural waste, which is an unused biomass resource, uses microorganisms that produce ethanol by decomposing and fermenting components such as cellulose and hemicellulose.
しかし、これらＳ．ｃｅｒｅｖｉｓｉａｅを代表するエタノール発酵微生物を用いて５炭糖の代表であるキシロースからエタノール発酵は不可能であり、キシロース発酵酵母であるＣａｎｄｉｄａ ｓｈｅａｔａｅやＰｉｃｈｉａ ｓｔｉｐｉｔｉｓにおいても培養が難しい、副生成物が生成する、エタノール耐性が低い、発酵阻害物質により強く発酵が阻害されるなど多くの問題を抱えている。さらに、組換え微生物（例えば、特許文献１、２）を用いた場合には、高いエタノール生産性を達成できるものの、組換え菌を使用する際の安全性の問題や倫理的問題を解決していく必要がある。 However, these S.P. Ethanol fermentation is impossible from xylose, which is representative of pentose, using ethanol-fermenting microorganisms that represent cerevisiae, and by-products are produced that are difficult to culture even in Candida sheatae and Pichia stipis, which are xylose-fermenting yeasts. It has many problems such as low ethanol resistance and strong inhibition of fermentation by fermentation inhibitors. Furthermore, when recombinant microorganisms (for example, Patent Documents 1 and 2) are used, although high ethanol productivity can be achieved, the problems of safety and ethical problems when using recombinant bacteria are solved. We have to go.
これらのことから、本発明者らエタノール微生物として考えられていなかった糸状菌、特にケカビの検索を行い、Ｍｕｃｏｒ属とＲｈｉｚｏｍｕｃｏｒ属の糸状菌を用いて我が国の主要な未利用バイオマスとして稲わらからのバイオエタノール製造システムの開発を行ってきた(特許文献３)。 From these facts, the present inventors searched for filamentous fungi that were not considered as ethanol microorganisms, especially fungi, and from rice straw as the main unused biomass in Japan using the fungi of the genus Mucor and Rhizomucor. A bioethanol production system has been developed (Patent Document 3).
一方、Ｍｕｃｏｒ属やＲｈｉｚｏｍｕｃｏｒ属の特定の菌株を用いて、主にトウヒに由来する森林残渣の加水分解物からエタノールを製造できることが知られている（非特許文献１、２）。 On the other hand, it is known that ethanol can be produced from a hydrolyzate of a forest residue mainly derived from spruce using a specific strain of the genus Mucor or Rhizomucor (Non-patent Documents 1 and 2).
製紙製造事業所から排出されるペーパースラッジやスラッジテールなど製紙廃棄物は、木質系バイオマスであり、バイオマス成分である糖質(セルロースとヘミセルロース)を含み、さらに、 リグニンおよび無機分を多量に含んでいる。この成分中の糖質を有効利用し生物変換によりエタノールを製造するためには、セルロースおよびヘミセルロース成分を加水分解する酵素群の分泌と、加水分解液中の５炭糖および６炭糖ともに効率よくエタノールへ変換することが望まれる。しかし、一般の発酵瀬微生物
Ｓ．ｃｅｒｅｖｉｓｉａｅは、セルロース分解酵素（セルラーゼ）を分泌せず、さらに、５炭糖を資化できるものの発酵性を有しない。また、キシロース発酵酵母であるＣａｎｄｉｄａ ｓｈｅａｔａｅやＰｉｃｈｉａ ｓｔｉｐｉｔｉｓは、５炭糖は発酵するもののセルラーゼ等は分泌しない。さらに、セルラーゼを多量に分泌する糸状菌Ｔｒｉｃｈｏｄｅｒｍａ ｒｅｓｓｅｉやＡｃｒｅｍｏｎｉｕｍ ｃｅｌｌｕｌｏｌｙｔｉｃｕｓはエタノール発酵能を有していない。
Paper waste such as paper sludge and sludge tail discharged from the paper manufacturing plant is a woody biomass that contains carbohydrates (cellulose and hemicellulose) as biomass components, and also contains a large amount of lignin and inorganic components. Yes. In order to produce ethanol by bioconversion using the carbohydrates in this component effectively, both the secretion of enzymes that hydrolyze the cellulose and hemicellulose components and the pentose and hexose in the hydrolyzed solution are both efficient. It is desirable to convert to ethanol. However, general fermented microorganism S. cerevisiae does not secrete cellulolytic enzyme (cellulase), and can assimilate pentose, but has no fermentability. In addition, Candida sheatae and Pichia stititis, which are xylose-fermenting yeasts, ferment pentose but do not secrete cellulase or the like. Furthermore, the filamentous fungi Trichoderma ressei and Acremonium cellulolyticus that secrete a large amount of cellulase do not have ethanol fermentation ability.
そこで、近年、セルラーゼおよびキシロース代謝酵素遺伝子を組換えたＳ．ｃｅｒｅｖｉｓｉａｅやＺ．ｐａｌｍａｅ、地球環境産業技術研究機構（ＲＩＴＥ）による組換えコリネ菌などが開発されてきているが、遺伝子組換え菌であることからカルタヘナル法の適用を受けて規制が厳しく、さらに、実際の製造において拡散防止などの設備を必要とする製造施設に多大なコストが負担となり商業化されていない。 Therefore, in recent years, S. cerevisiae having cellulase and xylose metabolizing enzyme genes recombined has been developed. cerevisiae and Z. Recombinant corynebacteria have been developed by Palmae, Institute for Global Environmental Industrial Technology (RITE), but since it is a genetically engineered bacterium, regulations are severe under the application of the Cartagenal Act, and actual production In manufacturing facilities that require equipment such as diffusion prevention, large costs are incurred and are not commercialized.
木質系バイオマスからの少ない工程数でエタノール生産を実用化させるためには、セルラーゼを分泌生産し、さらに、キシロースも発酵できる野性の菌株を見出すことが必要である。そこで、発明者らは、当研究室に保存している接合菌ライブラリーからセルラーゼを分泌生産すると共に、キシロースも発酵できる菌株の検索を行い、有用な糸状菌株を見出し、本発明を完成させるに至った。
以下、本発明を詳細に説明する。
In order to put ethanol production into practical use with a small number of steps from woody biomass, it is necessary to find a wild strain capable of secreting and producing cellulase and fermenting xylose. Therefore, the inventors searched for a strain that can secrete and produce cellulase from a zygote library stored in our laboratory and can also ferment xylose, find a useful filamentous strain, and complete the present invention. It came.
Hereinafter, the present invention will be described in detail.
本発明において使用される糸状菌は、接合菌門・接合菌綱・ケカビ目に属する糸状菌であり、アブシディア（Ａｂｓｉｄｉａ）、バクセラ（Ｂａｃｋｕｓｅｌｌａ）およびゴングロネリア（Ｇｏｎｇｒｏｎｅｌｌａ）に属する糸状菌である。具体的には、例えば以下のような種を示すことができる。 The filamentous fungus used in the present invention is a filamentous fungus belonging to the zygomycota, zygomycetes, and fungi, and is a filamentous fungus belonging to Absidia, Baccusella, and Gongronella. Specifically, for example, the following species can be shown.
アブシディア・コエルレア（Ａｂｓｉｄｉａ ｃｏｅｒｕｌｅａ）
アブシディア・シリンドロスポラ（Ａｂｓｉｄｉａ ｃｙｌｉｎｄｒｏｓｐｏｒａ）
アブシディア・ヒアロスポラ（Ａｂｓｉｄｉａ ｈｙａｌｏｓｐｏｒａ）
バクセラ・シリシナ（Ｂａｃｋｕｓｅｌｌａ ｃｉｒｃｉｎａ）
ゴングロネリア・ブトレリ（Ｇｏｎｇｒｏｎｅｌｌａ ｂｕｔｌｅｒｉ）
Absidia coerulea
Absidia cylindrospora
Absidia hyalospora
Baxella circina
Gongronella butleri
上記の糸状菌は、湿気の多い有機物上に出現する、ごく普通のカビである。これら微生物の、土壌、河川、あるいは湖沼などの材料からの単離・同定法は公知である。たとえば単離および同定方法については以下のような文献を参照することができる。
カビ：”The Genera of Hyphomycetes from soil”， G.L.Barron, Baltimore, Maryland, Williams and Wilkins(1968)．
”Compendium of soil Fungi”， K.H.
Domsh, W. Gams, T. Anderson, New York, Academic Press(1980)．
The above-mentioned filamentous fungus is an ordinary mold that appears on moist organic matter. Methods for isolating and identifying these microorganisms from materials such as soil, rivers, and lakes are well known. For example, the following documents can be referred to for isolation and identification methods.
Mold: “The Genera of Hyphomycetes from soil”, GL Barron, Baltimore, Maryland, Williams and Wilkins (1968).
“Compendium of soil Fungi”, KH
Domsh, W. Gams, T. Anderson, New York, Academic Press (1980).
より具体的には、以下の微生物菌株を示すことができる。
アブシディア・コエルレア（ＮＢＲＣ４０１３，４４２３）
アブシディア・シリンドロスポラ（ＮＢＲＣ４００１）
アブシディア・ヒアロスポラ（ＮＢＲＣ８０９２）
バクセラ・シリシナ（ＮＢＲＣ９２３１）
ゴングロネリア・ブトレリ（ＮＢＲＣ１０１１１１）
これらの糸状菌は、野生株または変異株のいずれの株も用いることができる。また、これらの糸状菌は、単独または混合して使用することができる。
More specifically, the following microbial strains can be shown.
Absidia Coellea (NBRC 4013, 4423)
Absidia Cylindrosspora (NBRC4001)
Absidia Hiarospora (NBRC8092)
Baccera Siricina (NBRC9231)
Gongnerelia butleri (NBRC101111)
As these filamentous fungi, either wild strains or mutant strains can be used. These filamentous fungi can be used alone or in combination.
上記の菌株は、独立行政法人製品評価技術基盤機構 バイオテクノロジー本部 生物遺伝資源部門（ＮＢＲＣ）
発行の微生物カタログ第１版（２００５年）に記載されており、それぞれのアクセション番号をもとにＮＢＲＣなどのセルバンクから入手することができる。
The above strains are from the National Institute of Product Evaluation Technology Biotechnology Headquarters Biogenetic Resources Division (NBRC)
It is described in the published microorganism catalog 1st edition (2005) and can be obtained from cell banks such as NBRC based on the respective accession numbers.
上記した糸状菌は、エンド−β−グルカナーゼを分泌し、エタノールの発酵生産のみならず、セルラーゼも分泌生産することができる。 The above-mentioned filamentous fungus secretes endo-β-glucanase and can secrete and produce cellulase as well as fermentative production of ethanol.
上記した糸状菌を作用させる木質系バイオマスとしては、廃材、間伐材、樹皮、パルプ、古紙およびそれらの加水分解物が挙げられるが、例えば、製紙工程から排出されるペーパースラッジ、スラッジテールも木質系バイオマスに含まれる。 Examples of the woody biomass that causes the above fungi to act include waste wood, thinned wood, bark, pulp, waste paper, and hydrolysates thereof. For example, paper sludge and sludge tail discharged from the papermaking process are also woody. Included in biomass.
上記した糸状菌のセルラーゼの分泌生産能を利用してエタノールの製造を行う場合、発酵を糖化発酵同時進行で行うシステムを構築することができる。 When ethanol is produced using the above-described ability to produce cellulase of filamentous fungi, a system can be constructed in which fermentation is performed simultaneously with saccharification and fermentation.
上記した糸状菌とセルラーゼ剤を用いて、同時糖化発酵によりエタノールを製造することができる。ここで用いられるセルラーゼ剤は特に限定されず、市販のセルラーゼ剤を用いればよい。セルラーゼ剤は複数組み合わせでカクテルとすることができ、例えば、アクセラーゼ・メイセラーゼ・ペクチナーゼのセルラーゼカクテルなどが挙げられる。 Ethanol can be produced by simultaneous saccharification and fermentation using the above-mentioned filamentous fungus and cellulase agent. The cellulase agent used here is not particularly limited, and a commercially available cellulase agent may be used. The cellulase agent can be used as a cocktail by combining a plurality of cellulase agents, and examples thereof include cellulase cocktails of accelerators, mecerases, and pectinases.
本発明のセルラーゼの分泌能を有するエタノール発酵微生物を使用することから、同時糖化発酵においては、使用酵素の添加量の軽減に繋がり、製造コストの削減が可能である。
また、糖化発酵同時進行の場合、本発明のセルラーゼの分泌能を有するエタノール発酵微生物のみを発酵槽へ投入し、ペーパースラッジなどへの糖化と発酵を行うことから、酵素製造に係わるコストは必要としない。すなわち、本発明により、ペーパースラッジのような産業廃棄物を含む未利用木質系バイオマスからバイオ燃料として利用されているエタノールを安価に製造することが可能となる。
Since the ethanol-fermenting microorganism having the secretory ability of the cellulase of the present invention is used, in the simultaneous saccharification and fermentation, the amount of the enzyme used is reduced, and the production cost can be reduced.
In the case of simultaneous progress of saccharification and fermentation, only the ethanol-fermenting microorganism having the ability to secrete the cellulase of the present invention is introduced into the fermentor, and saccharification and fermentation to paper sludge and the like are necessary. do not do. That is, according to the present invention, ethanol used as biofuel can be produced at low cost from unused woody biomass containing industrial waste such as paper sludge.
本発明について、以下の実施例に基づきさらに詳細に説明するが、本発明は以下の実施例に制限されるものではない。 The present invention will be described in more detail based on the following examples, but the present invention is not limited to the following examples.
［実施例１］
硫酸アンモニウム（０．７５％）、リン酸水素二カリウム（０．３５％）、塩化カルシウム（０．１％）、硫酸マグネシウム・７水和物（０．０７５％）、酵母エキス（０．５％）、ｐＨ７．５の液体培地に、グルコース（２％）を含む寒天プレートで、糸状菌株を２８℃で３〜５日培養した後、寒天プレートを粉砕し生理食塩液に懸濁させた。
生ペーパースラッジ（５％）を含有する上記の２５ｍＬに、上記懸濁液１ｍＬを加え、２８℃、１２０時間嫌気下で振とう培養した。
[Example 1]
Ammonium sulfate (0.75%), dipotassium hydrogen phosphate (0.35%), calcium chloride (0.1%), magnesium sulfate heptahydrate (0.075%), yeast extract (0.5% ) After culturing filamentous strains at 28 ° C. for 3 to 5 days on an agar plate containing glucose (2%) in a liquid medium at pH 7.5, the agar plate was pulverized and suspended in physiological saline.
1 mL of the above suspension was added to the above 25 mL containing raw paper sludge (5%), and cultured with shaking at 28 ° C. for 120 hours under anaerobic conditions.
培養終了後、定性濾紙(Ａｄｖａｎｔｅｃ製、Ｎｏ．１３１)を用いて濾過を行うことにより菌体を除去し、各培養液の培養上清液を調製した。濾紙上に得られた菌体は、蒸留水で十分洗浄した後、９０℃で２４時間乾燥させた後、重量を測定し、乾燥菌体重量を求めた。一方、ＧＣにより、培養により得られた培養液中のエタノールを定量した。
本発明の糸状菌株のエタノールの発酵能を表１に示す。また、エタノール生産とセルラーゼ（エンド−β−グルカナーゼ）生産を表２に示す。
After completion of the culture, the cells were removed by filtration using qualitative filter paper (manufactured by Advantec, No. 131), and a culture supernatant of each culture was prepared. The bacterial cells obtained on the filter paper were sufficiently washed with distilled water and then dried at 90 ° C. for 24 hours, and then the weight was measured to obtain the dry bacterial cell weight. On the other hand, the ethanol in the culture solution obtained by culture was quantified by GC.
Table 1 shows the ethanol fermentability of the filamentous strain of the present invention. Table 2 shows ethanol production and cellulase (endo-β-glucanase) production.
本発明により、例えば、製紙業界から排出される製紙廃棄物の問題点である化石燃料の多量消費、それに伴う炭酸ガスの発生を軽減でき、さらに、備蓄性の燃料であるエタノールを安価かつ効率よく製造することが可能となる。この分野において、エタノール製造の実用化は、エタノールの発酵効率の向上とコスト削減に係っている。本発明に使用される糸状菌は、セルラーゼの分必能を有していることから、例えば、バイオマスであるペーパースラッジの発酵処理に必要な酵素添加量の軽減あるいは酵素無添加状態でエタノールを生産でき、大幅なコスト削減が可能である。 According to the present invention, for example, a large amount of fossil fuel, which is a problem of papermaking waste discharged from the paper industry, can be reduced, and the generation of carbon dioxide gas associated therewith can be reduced. Further, ethanol as a stockpile fuel can be inexpensively and efficiently used. It can be manufactured. In this field, the practical use of ethanol production is related to the improvement of ethanol fermentation efficiency and cost reduction. Since the filamentous fungus used in the present invention has the necessary ability of cellulase, for example, it reduces the amount of enzyme required for fermentation treatment of biomass paper sludge or produces ethanol in the absence of enzyme. And significant cost reduction is possible.
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