JP2011152079A - Saccharifying fermentation system of cellulose-based biomass - Google Patents

Saccharifying fermentation system of cellulose-based biomass Download PDF

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JP2011152079A
JP2011152079A JP2010015868A JP2010015868A JP2011152079A JP 2011152079 A JP2011152079 A JP 2011152079A JP 2010015868 A JP2010015868 A JP 2010015868A JP 2010015868 A JP2010015868 A JP 2010015868A JP 2011152079 A JP2011152079 A JP 2011152079A
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saccharification
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cellulose
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Jun Sugiura
純 杉浦
Gahin Cho
雅蘋 趙
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Abstract

<P>PROBLEM TO BE SOLVED: To obtain a saccharifying fermentation system widely improving ethanol production efficiency to raise economical property in producing the ethanol from a cellulose-based biomass. <P>SOLUTION: This saccharifying fermentation system using the cellulose-based biomass as a raw material, and comprises a saccharifying fermentation reaction process for performing the simultaneous saccharifying fermentation reaction by adding a cellulose-decomposing enzyme and an alcohol fermentative yeast to biomass raw material-containing liquid, an alcohol-separation process for separating the reaction liquid from the saccharifying fermentation reaction process to a produced alcohol-containing liquid fraction and residues containing the cellulose-decomposing enzyme, alcohol fermentative yeast and various components originated from the biomass raw material, and a process for circulating/supplying a part or the whole of the residues to the saccharifying fermentation reaction process, wherein Issatchenkia orientalis is used as the alcohol fermentative yeast. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

本発明は、セルロース系バイオマスを酵素で糖化し、アルコール発酵させる糖化発酵システムにおいて、糖化と発酵を同時に行う併行糖化発酵システムに関する。   The present invention relates to a parallel saccharification and fermentation system that simultaneously performs saccharification and fermentation in a saccharification and fermentation system in which cellulosic biomass is saccharified with an enzyme and subjected to alcohol fermentation.

再生可能資源であるセルロース系バイオマスから糖を製造する技術は、この糖を微生物の発酵基質として用いることによりアルコールのようなガソリンの代替となる燃料、コハク酸や乳酸などのプラスチック原料を製造することができ、循環型社会の形成に役立つ技術である。
バイオマス資源中の多糖類から発酵基質となる単糖や少糖類を作る方法として、酵素やその酵素を生産する微生物を用いて、セルロースやヘミセルロースを加水分解する酵素糖化法が、環境負荷の小さい方法として検討されている。 The technology for producing sugar from cellulosic biomass, which is a renewable resource, uses this sugar as a fermentation substrate for microorganisms to produce fuels that replace gasoline such as alcohol, and plastic raw materials such as succinic acid and lactic acid. It is a technology that can help create a recycling-oriented society.
Enzymatic saccharification that hydrolyzes cellulose and hemicellulose using enzymes and microorganisms that produce them as a fermentation substrate from polysaccharides in biomass resources is a method that has a low environmental impact. As being considered.

酵素糖化法では、近年酵素の価格が下がってはいるものの、まだ酵素自体の価格が高く、実用化には一層の酵素コストの低減が必要である。そのため、一度使用した酵素を回収し再利用する技術が検討されている。   In the enzymatic saccharification method, although the price of the enzyme has been decreasing in recent years, the price of the enzyme itself is still high, and it is necessary to further reduce the cost of the enzyme for practical use. Therefore, a technique for collecting and reusing an enzyme once used has been studied.

Scott, C. D.らは、古紙の糖化装置として、連続的な磨砕と膜を用いた分離と酵素の再利用、固定化菌体による酵素の生産、高濃度のスラリー状態で処理することにより、低コスト化が可能であると予測している。摩砕しながら高い酵素濃度で処理することにより、糖化率は25時間で100％であった。この方法では酵素の回収率は24時間で95％以上であるが、酵素が残渣に吸着するため、これをpHや温度を変えて酵素を基質からはがして回収するとしている（非特許文献1参照）。   Scott, CD, et al., As a waste paper saccharification device, continuously grinded and separated using membranes and reused enzymes, produced enzymes by immobilized cells, and processed in a high concentration slurry state. It is predicted that costs can be reduced. By treating with high enzyme concentration while grinding, the saccharification rate was 100% in 25 hours. In this method, the recovery rate of the enzyme is 95% or more in 24 hours, but since the enzyme is adsorbed to the residue, the enzyme is removed from the substrate by changing the pH and temperature (see Non-Patent Document 1). ).

Woodらは、オフィス混合古紙をKlebsiella oxytocaとカビ由来の酵素Spezyme CP(Genencor社)、Novozyme 188で併行糖化発酵を行う際に、240分に15分の割合で超音波を照射すると、酵素の使用量をパルプ1gに対して、濾紙分解活性で5単位に半減することが出来たと報告している。糖化が促進される理由として、単に超音波によって繊維がほぐれるためではなく、酵素がセルロース繊維に単に吸着して作用できない状態のものを引き剥がして、再度新しい作用点で作用できるようにする効果があるためであると考察している（非特許文献２、非特許文献３参照）。   Wood et al. Used a mixed saccharification and fermentation of Klebsiella oxytoca and mold-derived enzymes Spezyme CP (Genencor) and Novozyme 188 when irradiated with ultrasonic waves at a rate of 15 minutes in 240 minutes. It is reported that the amount can be halved to 5 units by filter paper decomposition activity for 1 g of pulp. The reason that saccharification is promoted is not simply that the fiber is loosened by ultrasonic waves, but the effect that the enzyme is simply adsorbed on the cellulose fiber and peeled off so that it can act again at a new point of action. It is considered that this is because there are (see Non-Patent Document 2 and Non-Patent Document 3).

蒸煮・爆砕処理したシラカンバ材を5％の濃度で糖化槽に加え、2万単位のセルラーゼを添加して、限外濾過により糖液と酵素液とを分離し、酵素を回収再利用しながら、8日間で2kgのシラカンバ材から単糖類を630g得ている。（非特許文献４参照）。しかしこの方法ではコストは20％程度しか削減できていない。   Steamed and crushed birch wood is added to the saccharification tank at a concentration of 5%, 20,000 units of cellulase are added, the sugar solution and the enzyme solution are separated by ultrafiltration, and the enzyme is recovered and reused. In 8 days, 630 g of monosaccharides are obtained from 2 kg of birch wood. (Refer nonpatent literature 4). However, this method can only reduce the cost by about 20%.

これに対し、セルロース系バイオマスを酵素による糖化の原料とし、糖化酵素を反応後の糖液から分離回収して再利用する連続糖化反応において、リグニンの除去操作を施したセルロース系バイオマスを基質とし、糖化反応槽中における糖化酵素の濃度を、投入した糖質資源の96％以上を滞留時間内に糖化するのに必要な高い濃度に維持することによって、残渣の蓄積を防止しつつ連続的に糖化を行うことで、酵素の回収率を高める方法が報告されている（特許文献１参照）。   In contrast, in a continuous saccharification reaction in which cellulosic biomass is used as a raw material for enzymatic saccharification, and the saccharification enzyme is separated and recovered from the sugar solution after the reaction, the cellulosic biomass subjected to lignin removal operation is used as a substrate, By maintaining the concentration of saccharifying enzyme in the saccharification reaction tank at a high level necessary for saccharifying more than 96% of the input sugar resources within the residence time, continuous saccharification is possible while preventing accumulation of residues. There has been reported a method for increasing the enzyme recovery rate by performing (see Patent Document 1).

一方、糖化発酵に要する時間を短縮し、糖化発酵効率を高める方法として、併行糖化発酵が注目されている（非特許文献２、非特許文献３参照）。併行糖化発酵は麹菌の糖化酵素と日本酒酵母による日本酒の製造に古くから利用されている技術であり、現在は糖化発酵の時間短縮など効率化に向けて検討されている。   On the other hand, concurrent saccharification and fermentation has attracted attention as a method for shortening the time required for saccharification and fermentation and increasing the efficiency of saccharification and fermentation (see Non-Patent Documents 2 and 3). Parallel saccharification and fermentation is a technology that has been used for a long time in the production of sake using saccharifying enzymes of koji mold and sake yeast, and is currently being studied for efficiency such as shortening the time for saccharification and fermentation.

併行糖化発酵では、糖化酵素の至適温度、至適pHと酵母の増殖の最適温度、pHが一致することが望ましいが、市販のカビ類の糖化酵素の至適温度は一般に50〜60℃であるのに対し、酵母の増殖は30℃前後に過ぎない。そのため糖化酵素の働きを十分に発揮させることができない。
セルロース系バイオマスの糖化、発酵では糖化酵素のコストが高いことが課題であり、糖化酵素の糖化効率を高めることが重要であるにもかかわらず、至適温度で反応できないという課題があった。そこで酒酵母より高い温度で増殖し、発酵することができる微生物の開発は重要である。この様な微生物としてイサチェンキア・オリエンタリスが単離されている(特許文献２参照)。
また、特許文献３では、高温で同時糖化発酵できる酵母について記載されている。
その他、酵素の有効利用については、特許文献４、５などにも記載されている。 In parallel saccharification and fermentation, it is desirable that the optimum temperature and pH of the saccharifying enzyme coincide with the optimum temperature and pH of yeast growth, but the optimum temperature of commercially available fungal saccharifying enzymes is generally 50-60 ° C. In contrast, yeast growth is only around 30 ° C. Therefore, the function of saccharifying enzyme cannot be fully exhibited.
In the saccharification and fermentation of cellulosic biomass, the problem is that the cost of the saccharifying enzyme is high, and it is important to increase the saccharifying efficiency of the saccharifying enzyme. Therefore, it is important to develop microorganisms that can grow and ferment at higher temperatures than sake yeast. Isachenchia orientalis has been isolated as such a microorganism (see Patent Document 2).
Patent Document 3 describes a yeast capable of simultaneous saccharification and fermentation at high temperatures.
In addition, the effective use of enzymes is also described in Patent Documents 4 and 5.

ところで、これまで検討されてきた併行糖化発酵は、古くから日本酒の製造に使用されているサッカロマイセス・セレビシエ（Saccharomyces cerevisiae）を発酵微生物として使用する技術であるが、酒酵母は菌体外に分泌するプロテアーゼの活性が実質的にほとんど無いことから、糖化酵素の活性が失われずに再利用できるものである。同様に大腸菌も菌体外にプロテアーゼを分泌しないことが知られているが、多くの微生物は菌体外にプロテアーゼを分泌生産する。
一般に自然界より分離される微生物の多くは、プロテアーゼを菌体外に分泌生産すること、または増殖後の死滅期に、溶菌により菌体内のプロテアーゼが溶出することが知られており、これらの微生物を発酵菌として用いる場合には、糖化酵素が分解されるため、併行糖化発酵には好ましくない。特に酵素を回収再利用しながら行う連続的に併行糖化発酵の場合、少量のプロテアーゼであっても、回収再利用する間に著しく糖化酵素活性が損なわれ、実用的ではない。 By the way, parallel saccharification and fermentation, which has been studied so far, is a technique that uses Saccharomyces cerevisiae, which has been used in the production of sake for a long time, as a fermentation microorganism, but liquor yeast is secreted outside the cell. Since there is substantially no activity of protease, the activity of saccharifying enzyme can be reused without loss. Similarly, it is known that E. coli does not secrete protease outside the cell, but many microorganisms secrete and produce protease outside the cell.
In general, many microorganisms isolated from nature are known to secrete and produce proteases outside the cells, or to elute proteases in the cells by lysis during the death phase after growth. When used as a fermenting bacterium, the saccharification enzyme is decomposed, which is not preferable for concurrent saccharification and fermentation. In particular, in the case of continuous parallel saccharification and fermentation carried out while collecting and reusing the enzyme, even a small amount of protease is not practical because the saccharifying enzyme activity is significantly impaired during the collection and reuse.

更には、連続的に併行糖化発酵を進めるためには、酵素活性が前記プロテアーゼにより阻害されないことだけではなく、プロテアーゼ以外の作用などにより酵素活性が低下しないことが必要と考えられるが、高温で連続的に併行糖化発酵を行ったという実例や報告は一つもない。
また、酵素糖化にあたっては、バイオマスの前処理を必要とすることが多く、酸やアルカリで前処理した後には、糖化に適したpH領域にまで中和して糖化させるが、酵素を循環再利用することにより、反応系内にイオンが蓄積し、発酵反応を阻害するという問題がある。 Furthermore, in order to continuously proceed with concurrent saccharification and fermentation, it is considered necessary not only that the enzyme activity is not inhibited by the protease, but also that the enzyme activity does not decrease due to an action other than the protease. There are no actual examples or reports of simultaneous saccharification and fermentation.
Enzymatic saccharification often requires pretreatment of biomass, and after pretreatment with acid or alkali, it is neutralized to a pH range suitable for saccharification and saccharified, but the enzyme is recycled and reused. By doing so, there is a problem that ions accumulate in the reaction system and inhibit the fermentation reaction.

特開2006-087319号公報JP 2006-087319 A 特開2004-344084号公報JP 2004-344084 A 特開昭63-042690号公報JP 63-042690 A 特開昭60-244294号公報JP 60-244294 A 特開昭62-087096号公報JP 62-087096 A

Scott, C.D., Rothrock, D.S., Appl. Biochem. Biotechnol., 45/46, pp.641-653(1994)Scott, C.D., Rothrock, D.S., Appl.Biochem.Biotechnol., 45/46, pp.641-653 (1994) Wood, B.E.,Aldrich, H.C., Ingram, L.O., Biotechnol. Prog., 13, 232-237(1997)Wood, B.E., Aldrich, H.C., Ingram, L.O., Biotechnol. Prog., 13, 232-237 (1997) Tomme, P., Warren, A.J., Miller, T.C.J., Kilburn, D.G., Gilkes, M. R., In “Enzymatic Degradation of Insoluble Carbohydrates” (J.N. Saddler ed.) ACS Symposium Ser. Vol 618, pp. 145-163, ACS, San Diego, CA(1995)Tomme, P., Warren, AJ, Miller, TCJ, Kilburn, DG, Gilkes, MR, In “Enzymatic Degradation of Insoluble Carbohydrates” (JN Saddler ed.) ACS Symposium Ser. Vol 618, pp. 145-163, ACS, San Diego, CA (1995) Ishihara, M., et al., Biotechnol. Bioeng., 37, 948-954(1991)Ishihara, M., et al., Biotechnol. Bioeng., 37, 948-954 (1991)

セルロース系バイオマスから発酵生産物を製造する場合に、酵素を回収しながら併行糖化発酵を行うプロセスに使用する微生物として、高温で高い発酵能力を示し、かつ長期間の酵素の繰り返し回収再利用時にも、プロテアーゼ等によりセルロース分解活性を損なうことのない酵母が必要である。更に、系内のイオン濃度が上昇しても高い発酵能力を示す酵母が必要である。   When producing fermented products from cellulosic biomass, as a microorganism used in the process of performing simultaneous saccharification and fermentation while recovering enzymes, it exhibits high fermentative ability at high temperatures, and can be used for repeated recovery and reuse of enzymes over a long period of time. In addition, a yeast that does not impair cellulolytic activity by protease or the like is required. Furthermore, a yeast that exhibits high fermentation ability even when the ion concentration in the system increases is required.

本発明者らは、併行糖化発酵後に酵素を回収し再利用しながら行う糖化発酵システムにおいて、糖化酵素の活性を損なうことなく、より高温で発酵を行うことができる微生物の開発を鋭意行った結果、本発明に至った。   In the saccharification and fermentation system in which the enzyme is recovered and reused after the concurrent saccharification and fermentation, the present inventors have intensively developed a microorganism capable of performing fermentation at a higher temperature without impairing the activity of the saccharification enzyme. The present invention has been reached.

すなわち本発明は、「セルロース系バイオマスを原料とする糖化発酵システムであって、該バイオマス原料含有液にセルロース分解酵素及びアルコール発酵性酵母を加えて併行糖化発酵反応を行う糖化醗酵反応工程、該糖化発酵反応工程からの反応液を生成アルコール含有液体留分と、セルロース分解酵素、アルコール発酵性酵母及びバイオマス原料由来の各種物質を含有する残留分とに分離するアルコール分離工程、該残留分の一部または全部を前記糖化醗酵反応工程に循環供給する工程を有するシステムにおいて、アルコール発酵性酵母としてイサチェンキア・オリエンタリス（Issatchenkia orientalis）を用いることを特徴とする、セルロース系バイオマスを原料とする糖化醗酵システム」である。   That is, the present invention is a saccharification and fermentation system using cellulosic biomass as a raw material, a saccharification and fermentation reaction step in which a cellulose-degrading enzyme and an alcohol-fermenting yeast are added to the biomass raw material-containing liquid, Alcohol separation step for separating the reaction liquid from the fermentation reaction step into a produced alcohol-containing liquid fraction and a residue containing cellulose-degrading enzymes, alcohol-fermenting yeast and various materials derived from biomass raw materials, a part of the residue Alternatively, in a system having a step of circulating and supplying the whole to the saccharification and fermentation reaction step, Isacatchia orientalis is used as the alcohol-fermenting yeast, and the saccharification and fermentation system using cellulosic biomass as a raw material ” is there.

本発明により、酵素のコストを大幅に削減することができ、セルロース系バイオマスから糖類を製造する経済性を高めることが可能となる。従って、糖類を発酵基質としてアルコールを供給する経済性を高めることが可能となる。   By this invention, the cost of an enzyme can be reduced significantly and it becomes possible to improve the economical efficiency which manufactures saccharides from cellulosic biomass. Therefore, it is possible to increase the economy of supplying alcohol using saccharides as a fermentation substrate.

図１は本発明の糖化発酵システムの代表的なフローを示す。FIG. 1 shows a typical flow of the saccharification and fermentation system of the present invention. 図２は本発明の糖化発酵システムの別の例のフローを示す。FIG. 2 shows the flow of another example of the saccharification and fermentation system of the present invention.

本発明で使用する酵母はイサチェンキア・オリエンタリスであって、37℃から45℃の範囲であっても増殖することが可能で、実質的にプロテアーゼを生産しない株であればいずれも用いることができるが、特に好ましくはＩｓｓａｔｃｈｅｎｋｉａ属ｏｒｉｅｎｔａｌｉｓ種のアルコール発酵性酵母ＭＦ−１２１が例示される。本菌株は平成１５年５月２２日に独立行政法人産業技術総合研究所特許生物寄託センターに寄託され、受託番号ＦＥＲＭ Ｐ−１９３６８が付与された。   The yeast used in the present invention is Isachenchia orientalis, which can grow even in the range of 37 ° C. to 45 ° C., and any strain that does not substantially produce protease can be used. Particularly preferably, alcohol fermentable yeast MF-121 of the genus Issatchenkia orientalis is exemplified. This strain was deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on May 22, 2003, and was assigned the deposit number FERM P-19368.

本発明の酵母を増殖するには単糖を有機基質として含むものであればいずれも用いることができる。単糖としてはグルコースが特に好ましい。窒素源としては特に制限されるものではないが、硫安等のアンモニウム塩、コーンスティープリカー等を用いることができる。また酵母エキス、マルツエキスなどを含む培地でも増殖させることができる。培地のpHは弱酸性が好ましく、pH3ないし2でも増殖する。   In order to grow the yeast of the present invention, any can be used as long as it contains a monosaccharide as an organic substrate. Glucose is particularly preferred as the monosaccharide. The nitrogen source is not particularly limited, but ammonium salts such as ammonium sulfate, corn steep liquor and the like can be used. It can also be grown in a medium containing yeast extract, malt extract and the like. The pH of the medium is preferably weakly acidic and grows even at pH 3 to 2.

併行糖化発酵に用いるセルロース分解酵素の種類については、セルロースを分解できるものであれば特に限定されるものではないが、セロビオヒドロラーゼ活性、エンドグルカナーゼ活性、ベータグルコシダーゼ活性を有する、所謂セルラーゼと総称される酵素であれば良い。セルラーゼは、トリコデルマ（Trichoderma）属、アスペルギルス（Aspergillus）属、フミコーラ（Humicola）属、イルペックス（Irpex）属などに属する菌が生産する糖化酵素や、商業的に生産される酵素を、単独でもしくは組み合わせて用いることができる。好ましくはプロテアーゼを含まないものを使用する。特にバイオマスの糖化用に開発された酵素が好適である。
このようなセルラーゼ製剤の市販品としては、全て商品名で、例えば、セルロイシンＴ２（エイチピィアイ社製）、メイセラーゼ（明治製菓社製）、ノボザイム１８８（ノボザイム社製）、マルティフェクトＣＸ１０Ｌ（ジェネンコア社製）等が挙げられる。 The type of cellulolytic enzyme used for concurrent saccharification and fermentation is not particularly limited as long as it is capable of degrading cellulose, but is generically referred to as so-called cellulase having cellobiohydrolase activity, endoglucanase activity, and betaglucosidase activity. Any enzyme can be used. Cellulase is a saccharification enzyme produced by bacteria belonging to the genus Trichoderma, Aspergillus, Humicola, and Irpex, and commercially produced enzymes, either alone or in combination. Can be used. Preferably, a protease-free product is used. In particular, enzymes developed for biomass saccharification are preferred.
Commercially available products of such cellulase preparations are all trade names, for example, cellulosin T2 (manufactured by HIPI), mecerase (manufactured by Meiji Seika Co., Ltd.), Novozyme 188 (manufactured by Novozyme), multifect CX10L (manufactured by Genencor) ) And the like.

本発明で原料とするセルロース系バイオマスとしては、針葉樹、広葉樹、林地残材、建築廃材、剪定廃棄物、ソーダスト、ケナフ、稲藁、麦わらなどの農産破棄物等のセルロース系バイオマスからアルカリ抽出、アルカリ蒸解等の化学パルプ製造法、オルガノソルブなどの方法により酵素が作用しやすいように処理を施されたセルロース、ヘミセルロースを主成分とする繊維、もしくはセルロース系バイオマスから機械的磨砕と熱的、化学的前処理を施して得た処理物が好ましく、例えば古紙、パルプ工場のスラッジ、林地残材の処理物、ユーカリ樹皮のメカノケミカル処理物などを挙げることができる。特に化学パルプを含む古紙が好適である。   Cellulose biomass used as a raw material in the present invention includes alkali extraction from cellulose biomass such as conifers, hardwoods, forest residue, building waste, pruning waste, agricultural waste such as sawdust, kenaf, rice straw, straw, etc. Mechanical milling and thermal, chemical treatment from cellulose, fibers mainly composed of hemicellulose, or cellulosic biomass that have been treated so that enzymes can easily act by chemical pulp production methods such as cooking and organosolv A treated product obtained by subjecting to a pre-treatment is preferable, and examples include waste paper, pulp factory sludge, treated forest residue, and mechanochemical treated eucalyptus bark. In particular, waste paper containing chemical pulp is preferred.

以下、本発明のセルロース系バイオマスの糖化発酵システムを、図面を参照して具体的に説明する。   Hereinafter, the saccharification and fermentation system for cellulosic biomass of the present invention will be specifically described with reference to the drawings.

図１は、本発明のセルロース系バイオマスの糖化発酵システムの基本実施形態を示す工程図である。
図１に示すセルロース系バイオマスを原料とする糖化発酵システムにおいて、セルロース系バイオマス原料調製工程からのセルロース系バイオマス原料は、経路（イ）を経由して糖化醗酵反応工程に供給され、同時にセルロース分解酵素と、アルコール発酵性酵母及び培地が糖化醗酵反応工程に供給されてセルロースの糖化とアルコール発酵とが同時に行われる。 FIG. 1 is a process diagram showing a basic embodiment of a saccharification and fermentation system for cellulosic biomass of the present invention.
In the saccharification and fermentation system using cellulosic biomass as a raw material shown in FIG. 1, the cellulosic biomass raw material from the cellulosic biomass raw material preparation step is supplied to the saccharification and fermentation reaction step via route (i), and at the same time cellulolytic enzyme And alcohol fermentable yeast and a culture medium are supplied to a saccharification fermentation reaction process, and saccharification of cellulose and alcohol fermentation are performed simultaneously.

糖化醗酵反応工程から得られる糖化醗酵反応液は、経路（ロ）を経由して生成アルコール含有液体留分（以下アルコール留分と略称）と、セルロース分解酵素、アルコール発酵性酵母及びバイオマス原料由来の各種物質を含有する残留分とに分離するアルコール分離工程に送られて、アルコール留分と残留分とに分離され、アルコール留分は経路（ニ）を経由してアルコール貯槽に送られる。アルコール分離工程は、蒸留による分離、または限外濾過膜による濾過分離、あるいはパーベーパーレーションにより分離される。
一方、アルコール分離工程からの残留分は経路（ハ）を経由して、直接に糖化醗酵反応工程に循環供給される。あるいは、必要に応じて、五炭糖の処理、リグニンの処理、アルカリ分の処理などの工程を経ても良い。 The saccharification and fermentation reaction liquid obtained from the saccharification and fermentation reaction step is derived from the alcohol-containing liquid fraction (hereinafter abbreviated as “alcohol fraction”), cellulolytic enzyme, alcohol-fermentable yeast, and biomass raw material via the route (b). It is sent to an alcohol separation step that separates it into a residue containing various substances, and is separated into an alcohol fraction and a residue, and the alcohol fraction is sent to an alcohol storage tank via a route (d). In the alcohol separation step, separation by distillation, filtration separation by an ultrafiltration membrane, or pervaporation is performed.
On the other hand, the residue from the alcohol separation step is directly circulated and supplied to the saccharification and fermentation reaction step via the route (c). Or you may pass processes, such as a process of pentose, a process of lignin, a process of an alkali, as needed.

本発明のセルロース系バイオマス糖化醗酵システムは、上記したように、基本的には、バイオマス原料調製工程→糖化醗酵反応工程→アルコール分離工程→糖化醗酵反応工程で一巡する循環系によって構成されており、高価な酵素等の循環利用を可能とした商業的に価値の高いアルコール等の生産システムである。本発明においては、耐塩性の酵母を使用することにより、酵素を循環する際に系内のイオン濃度が高くなっても発酵効率が落ちない。また、耐熱性の酵母を用いることにより、糖化発酵反応の温度を高くすることができる。更には、プロテアーゼを全く生成しない酵母を用いることにより、酵素の循環再利用が何度でもできる、などの利点がある。   As described above, the cellulose-based biomass saccharification and fermentation system of the present invention is basically composed of a circulation system that makes a round in the biomass raw material preparation step → saccharification and fermentation reaction step → alcohol separation step → saccharification and fermentation reaction step, It is a production system for alcohol and the like with high commercial value that enables the recycling of expensive enzymes and the like. In the present invention, by using a salt-tolerant yeast, the fermentation efficiency does not decrease even if the ion concentration in the system increases when circulating the enzyme. Moreover, the temperature of saccharification and fermentation reaction can be raised by using heat-resistant yeast. Furthermore, the use of yeast that does not produce any protease has the advantage that the enzyme can be recycled and reused any number of times.

本発明のセルロース系バイオマス糖化醗酵システムは基本的には上記した循環系によって構成されているが、上記糖化醗酵システムをより一層商業的に価値の高いシステムとするために、さらに、図２のような工程を付加したシステムとすることができる。   The cellulosic biomass saccharification and fermentation system of the present invention is basically constituted by the circulation system described above, but in order to make the saccharification and fermentation system more commercially valuable, as shown in FIG. It can be set as the system which added the process.

図２のシステムについて説明する。糖化醗酵反応工程とアルコール分離工程の間に、固液分離工程を配置して糖化醗酵反応工程からの反応液を、生成アルコールと使用酵素等を含有する液体留分と、酵母や未反応物質等を含有する固形分含有留分とに分離し、液体留分は経路（ホ）を経由して次のアルコール分離工程に送り、固形分含有留分は、経路（ヘ）を経由して糖化醗酵反応工程に循環し、再利用する。
固液分離工程からの液体留分は、アルコール分離工程により生成アルコール分が経路（ニ）を経て回収されアルコール貯槽に貯蔵され、残留分は経路（ハ）から取り出されて糖化醗酵反応工程に循環し、再利用する。 The system of FIG. 2 will be described. Between the saccharification and fermentation reaction step and the alcohol separation step, a solid-liquid separation step is arranged, the reaction solution from the saccharification and fermentation reaction step, the liquid fraction containing the produced alcohol and the enzyme used, yeast, unreacted substances, etc. The liquid fraction is sent to the next alcohol separation step via the route (e), and the solid content fraction is saccharified and fermented via the route (f). It is recycled to the reaction process and reused.
The liquid fraction from the solid-liquid separation process is recovered by the alcohol separation process via the route (d) and stored in the alcohol storage tank, and the residue is taken out from the route (c) and recycled to the saccharification and fermentation reaction step. And reuse it.

以上のように、酵素を含む留分を糖化発酵工程に再使用するが、一連の操作により失われた酵素活性を補填するために、新たに酵素を加え、併行糖化発酵槽中の糖化力を維持することが好ましい。新たに加える酵素は、当初用いた酵素でもよいし、特定の酵素成分でもよい。特に酵素活性が失われやすいβグルコシダーゼの追加が好ましい。   As described above, the fraction containing the enzyme is reused in the saccharification and fermentation process. To compensate for the enzyme activity lost by a series of operations, a new enzyme is added to increase the saccharification power in the parallel saccharification and fermentation tank. It is preferable to maintain. The enzyme to be newly added may be the enzyme used initially or a specific enzyme component. In particular, addition of β-glucosidase which easily loses enzyme activity is preferable.

併行糖化発酵液は用いる酵素に適した条件でかつイサチェンキア・オリエンタリスが生理的に発酵可能な条件とする。トリコデルマなど一般の糸状菌の生産する酵素の場合、pH3ないし7で、温度は35ないし45℃が好ましい。
所定の滞留時間で併行糖化発酵を行うために、イサチェンキア・オリエンタリスの濃度は１０６〜１０８個/mlが好ましい。酵素の活性は所望の滞留時間で所定の糖化率となるような濃度が好ましく、滞留時間が48時間として、基質1gに対して濾紙崩壊活性で10単位が例示されるが、基質の種類、前処理の方法によって適宜酵素の添加量を調節する。特に滞留時間を15ないし35時間となるように大量の酵素を添加し、反応効率を高めて、酵素あたりの基質分解量をなるべく高く維持し、その酵素を回収再利用して有効に利用することが好ましい。 The parallel saccharification and fermentation broth should be under conditions suitable for the enzyme used and conditions under which Isachenchia orientalis can be fermented physiologically. In the case of enzymes produced by common filamentous fungi such as Trichoderma, the pH is preferably 3 to 7 and the temperature is preferably 35 to 45 ° C.
In order to perform parallel saccharification and fermentation with a predetermined residence time, the concentration of Isachenchia orientalis is preferably 10 6 to 10 8 pieces / ml. The enzyme activity is preferably at a concentration such that a predetermined saccharification rate is obtained at a desired residence time. The residence time is 48 hours, and 10 units of filter paper disintegration activity is exemplified for 1 g of the substrate. The amount of the enzyme added is appropriately adjusted according to the treatment method. In particular, add a large amount of enzyme so that the residence time is 15 to 35 hours, increase the reaction efficiency, maintain the substrate degradation amount per enzyme as high as possible, and recover and reuse the enzyme effectively. Is preferred.

本発明において、糖化率は基質の有機分当りの生成糖量と定義する。基質の有機分は基質中の水分、灰分を除いた質量とする。   In the present invention, the saccharification rate is defined as the amount of sugar produced per organic component of the substrate. The organic content of the substrate is the mass excluding moisture and ash in the substrate.

また酵素活性を代表してCBH Iの活性を以下のように測定する。
（１）CBH Ｉ活性
1.25 mM 4-Methyl-umberiferyl-cellobiosideを含む125 mM 酢酸緩衝液(pH4.0) 16μlに、酵素液4 μlを加え、50℃、10min反応を行ったのち、500 mM glycine-NaOH緩衝液(pH10.0)100 μlを添加し、反応を停止させた。これを350 nmの励起光での460 nmの蛍光を測定し、１分間に１μmolのウンベリフェロンを生成する酵素の量を１単位とした。 In addition, CBH I activity is measured as follows, representing enzyme activity.
(1) CBH I activity
4 μl of enzyme solution is added to 16 μl of 125 mM acetate buffer (pH 4.0) containing 1.25 mM 4-Methyl-umberiferyl-cellobioside, and the reaction is carried out at 50 ° C. for 10 min, followed by 500 mM glycine-NaOH buffer (pH 10 .0) 100 μl was added to stop the reaction. This was measured for fluorescence at 460 nm with 350 nm excitation light, and the amount of enzyme producing 1 μmol of umbelliferone per minute was defined as 1 unit.

（３）エタノールおよびグルコース濃度
溶液中のエタノールおよびグルコースの濃度はグルコースセンサー（王子計測機器製BF-400型）で定量した。 (3) Ethanol and glucose concentrations Ethanol and glucose concentrations in the solution were quantified with a glucose sensor (BF-400 manufactured by Oji Scientific Instruments).

以下、本発明について、実施例を挙げて説明する。
＜実施例１＞
図２に示す糖化醗酵システムにより木質バイオマスを原料とするエタノール生産を行った。
原料として、上質紙のアルミニウム蒸着ラベル古紙を苛性ソーダで処理して、蒸着金属を溶解させて水分60％に濃縮した古紙パルプを使用した。
酵素としては、ジェネンコア社製のセルラーゼ「マルティフェクトＣＸ１０Ｌ」（ろ紙崩壊活性が１２０ＦＰＵ／ｍｌ）を使用した。
エタノール発酵を行う微生物として酵母「イサチェンキア・オリエンタリス」（Issatchenkia orientalis）のＭＦ−１２１株を使用した。この酵母を、液体培地〔2質量％グルコース、1質量％ＣＳＬ（コーンスティープリカー）、0.5質量％硫酸アンモニウム、pH4.5〕で、130rpmで振とうしながら、24時間37℃で培養し、得られた菌体を発酵に供した。 Hereinafter, the present invention will be described with reference to examples.
<Example 1>
Ethanol production using woody biomass as a raw material was performed by the saccharification and fermentation system shown in FIG.
As the raw material, used paper pulp obtained by treating high-quality aluminum vapor-deposited label waste paper with caustic soda to dissolve the deposited metal and concentrating it to 60% moisture was used.
As the enzyme, cellulase “Multifect CX10L” (filter paper disintegration activity of 120 FPU / ml) manufactured by Genencor was used.
As a microorganism for ethanol fermentation, MF-121 strain of yeast “Issatchenkia orientalis” (Issatchenkia orientalis) was used. The yeast is obtained by culturing at 37 ° C. for 24 hours in a liquid medium [2% by mass glucose, 1% by mass CSL (corn steep liquor), 0.5% by mass ammonium sulfate, pH 4.5] while shaking at 130 rpm. The microbial cells were subjected to fermentation.

（１）糖化発酵反応工程
糖化発酵反応工程は、5L容量の攪拌翼付培養槽を用いて行った。始めに、前記古紙パルプを水で希釈し、固形分8質量％の分散液とし、次いで塩酸を添加し、pHを7.0に調整して原料パルプスラリーとした。
前記原料パルプスラリー3kg(パルプ固形分250 g)に酵素を200g、酵母を１×１０８ｃｅｌｌｓ／ｍｌとなるように加えた。また培地成分としてＣＳＬ（コーンスティープリカー）25g、硫酸アンモニウム12.5gを加えた。150〜250rpmで攪拌しながら、37℃で20時間反応を行った。反応終了後、反応液の一部をとり、反応液中の固形分量を測定し、糖化率を算出した。 (1) Saccharification and fermentation reaction process The saccharification and fermentation reaction process was performed using a 5 L capacity culture tank with a stirring blade. First, the waste paper pulp was diluted with water to obtain a dispersion having a solid content of 8% by mass, and then hydrochloric acid was added to adjust the pH to 7.0 to obtain a raw material pulp slurry.
200 g of enzyme and 1 × 10 8 cells / ml of enzyme were added to 3 kg of the raw pulp slurry (pulp solid content 250 g). Further, 25 g of CSL (corn steep liquor) and 12.5 g of ammonium sulfate were added as medium components. The reaction was carried out at 37 ° C. for 20 hours while stirring at 150 to 250 rpm. After completion of the reaction, a part of the reaction solution was taken, the solid content in the reaction solution was measured, and the saccharification rate was calculated.

糖化率は以下のように算出した。
糖化率＝（供給した固形分−反応後の反応液中の固形分量）÷供給した固形分×100 The saccharification rate was calculated as follows.
Saccharification rate = (supplied solid content−solid content in reaction solution after reaction) ÷ supplied solid content × 100

（２）固液分離工程
得られた糖化発酵反応液について、遠心分離機を用い、固形分40質量％に濃縮して固液分離を行った。固形分含有留分(パルプ分28g、水分42g)は糖化発酵反応工程に戻し、これに新たに前記原料パルプを加え固形分8質量％のパルプスラリーとし、塩酸を添加してpHを7.0に調整して、第２回目の糖化発酵反応に供する。 (2) Solid-liquid separation process About the obtained saccharification fermentation reaction liquid, it concentrated to 40 mass% of solid content using the centrifuge, and solid-liquid separation was performed. The solid content fraction (pulp content 28g, moisture content 42g) is returned to the saccharification and fermentation reaction step, and the raw material pulp is newly added to this to obtain a pulp slurry with a solid content of 8% by mass, and hydrochloric acid is added to adjust the pH to 7.0. Then, it is used for the second saccharification and fermentation reaction.

（３）アルコール分離工程
遠心分離機により得られた液体分中のエタノールは、フラッシュエバポレーター（東京理化器械）を用いた蒸留工程で分離した。エタノールとともに、水分も一定量蒸発させた。エタノール分離後の濃縮液のエタノール濃度は0.1質量％以下であった。蒸留液のエタノール濃度をバイオセンサー（王子計測機器）で測定し、エタノール生産量を算出した。 (3) Alcohol separation process Ethanol in the liquid obtained by the centrifuge was separated in a distillation process using a flash evaporator (Tokyo Rika Kikai). A certain amount of water was evaporated along with ethanol. The ethanol concentration of the concentrated solution after ethanol separation was 0.1% by mass or less. The ethanol concentration of the distillate was measured with a biosensor (Oji Scientific Instruments), and the ethanol production was calculated.

（４）アルコール分離残留分をリサイクルする工程
蒸留後、残留した液(若干の固形物も含む)をそのまま第２回目の糖化発酵工程に投入する。 (4) Step of recycling alcohol separation residue After distillation, the remaining liquid (including some solid matter) is directly put into the second saccharification and fermentation step.

以上の（１）〜（４）の各工程を１サイクルとして、５サイクルの反応を行った結果の糖化率、エタノール生産量、残留酵素活性を表１に示す。但し、糖化率は第１回の処理の89％から第5回の81％まで暫減しているため、そのたびごとに、残留固形分から、新規追加パルプの量を調整し、１ロットのパルプ量を250gとなるように調整した。
残留酵素活性は、第１回目の反応開始前の系内の液と、第5回目の反応終了時の系内の液について、CBH I活性を測定し、その比率で示した。 Table 1 shows the saccharification rate, the amount of ethanol production, and the residual enzyme activity as a result of carrying out the reaction of 5 cycles with each step of (1) to (4) as one cycle. However, since the saccharification rate has decreased for a while from 89% in the first treatment to 81% in the fifth treatment, each time, the amount of newly added pulp is adjusted from the residual solid content, and one lot of pulp The amount was adjusted to 250 g.
The residual enzyme activity was expressed as a ratio of CBHI activity measured for the solution in the system before the start of the first reaction and the solution in the system at the end of the fifth reaction.

＜比較例１＞
酵母をＭＦ−１２１株に変えて、サッカロミセス・セレビシエ２４４株とし、酵母の増殖ならびに糖化発酵温度を３０℃とした他は実施例と同様にした。 <Comparative Example 1>
The yeast was changed to the MF-121 strain to give Saccharomyces cerevisiae 244 strain, and the same procedure as in Example was carried out except that the yeast growth and saccharification and fermentation temperature were 30 ° C.

Figure 2011152079
Figure 2011152079

＜結果の考察＞
両酵母とも、酵素活性については同様の傾向を示すが、糖化率はＭＦ−１２１株の方が高くなった。これは、酵素活性を十分に生かせる温度で糖化発酵を行えたためであると考えられる。また、糖化率の差より、エタノール生産量の差が大きくなっているのは、ＭＦ−１２１株は２４４株に比較して、系内のイオン濃度が上昇しても発酵の効率が下がらないためと考えられる。なお、両酵母とも、繰り返し使用による酵素活性の著しい低下をもたらすことはなく、プロテアーゼなど、糖化酵素に悪影響を及ぼす酵素を発生していないことが推定される。 <Consideration of results>
Both yeasts showed the same tendency for enzyme activity, but the saccharification rate was higher in the MF-121 strain. This is considered to be because saccharification and fermentation were performed at a temperature at which enzyme activity could be fully utilized. In addition, the difference in ethanol production is greater than the difference in saccharification rate because the MF-121 strain does not lower the efficiency of fermentation even if the ion concentration in the system increases compared to the 244 strain. it is conceivable that. Both yeasts do not cause a significant decrease in enzyme activity due to repeated use, and it is presumed that no enzymes such as proteases that adversely affect saccharifying enzymes have been generated.

本発明により、エタノール生産効率を大幅に高めることができ、セルロース系バイオマスからエタノールを製造する経済性を高めることが可能となる。   By this invention, ethanol production efficiency can be raised significantly and it becomes possible to improve the economical efficiency which manufactures ethanol from cellulosic biomass.

Claims (1)

セルロース系バイオマスを原料とする糖化発酵システムであって、該バイオマス原料含有液にセルロース分解酵素及びアルコール発酵性酵母を加えて併行糖化発酵反応を行う糖化醗酵反応工程、該糖化発酵反応工程からの反応液を生成アルコール含有液体留分と、セルロース分解酵素、アルコール発酵性酵母及びバイオマス原料由来の各種物質を含有する残留分とに分離するアルコール分離工程、該残留分の一部または全部を前記糖化醗酵反応工程に循環供給する工程を有するシステムにおいて、アルコール発酵性酵母としてイサチェンキア・オリエンタリス（Issatchenkia orientalis）を用いることを特徴とする、セルロース系バイオマスを原料とする糖化醗酵システム。   A saccharification and fermentation system using cellulosic biomass as a raw material, a saccharification and fermentation reaction step in which a cellulose-degrading enzyme and an alcohol-fermentable yeast are added to the biomass raw material-containing liquid to perform a parallel saccharification and fermentation reaction, a reaction from the saccharification and fermentation reaction step An alcohol separation step of separating the liquid into a produced alcohol-containing liquid fraction and a residue containing cellulose-degrading enzyme, alcohol-fermenting yeast, and various materials derived from biomass raw materials, and a part or all of the residue is subjected to saccharification and fermentation A saccharification and fermentation system using cellulosic biomass as a raw material, characterized in that Issatchenkia orientalis is used as an alcohol-fermenting yeast in a system having a step of circulating and supplying the reaction step.
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JP2011139686A (en) * 2010-01-08 2011-07-21 Oji Paper Co Ltd Continuous method for producing ethanol by parallel saccharifying fermentation reaction
JP2013188202A (en) * 2012-02-13 2013-09-26 Oji Holdings Corp Method for producing ethanol from lignocellulose-containing biomass
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KR101511370B1 (en) 2013-08-13 2015-04-10 주식회사 배상면주가 Method for Preparing Ethanol Using Vegetable Waste
JP2015042145A (en) * 2013-08-26 2015-03-05 王子ホールディングス株式会社 Method for producing ethanol
JP2015159755A (en) * 2014-02-27 2015-09-07 王子ホールディングス株式会社 Method for producing ethanol from lignocellulose-containing biomass
WO2020096013A1 (en) 2018-11-09 2020-05-14 ユニ・チャーム株式会社 Method for producing recycled pulp fibers, recycled pulp fibers, and use for ozone
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