JP2011055715A - Method for producing ethanol by simultaneous saccharifying fermentation - Google Patents
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Abstract
Description
本発明は、同時糖化発酵時の原料処理方法およびこの処理方法を利用したエタノールの生産方法に関する。 The present invention relates to a raw material processing method at the time of simultaneous saccharification and fermentation and a method for producing ethanol using this processing method.
同時糖化発酵は、酵素による原料の分解と、生成された分解物の発酵用微生物の資化による目的物質の生産を同一槽内で行う方法である。同時糖化発酵は１９７６年にGaussらによって初めて考案された（非特許文献１）。現在この方法は、原料にセルロース系バイオマスを用い、酵素にセルラーゼを用いて生成されるグルコースをはじめとする分解産物を酵母の資化によりエタノールを製造する方法として注目されており、他に乳酸を製造する方法としても検討されている。 The simultaneous saccharification and fermentation is a method in which the decomposition of the raw material by the enzyme and the production of the target substance by utilization of the microorganism for fermentation of the generated decomposition product are performed in the same tank. Simultaneous saccharification and fermentation was first devised by Gauss et al. In 1976 (Non-patent Document 1). At present, this method is attracting attention as a method for producing ethanol by utilizing yeast as a decomposition product including glucose produced using cellulosic biomass as a raw material and cellulase as an enzyme. It is also considered as a manufacturing method.
一般的に酵素は基質に対する特異的な触媒活性を持ち反応物を生成するが、その反応生成物により酵素の触媒活性が阻害されるという競争阻害が起こる。同時糖化発酵では、酵素により生成した反応生成物を発酵微生物により資化することで競争阻害を抑制することができ、全体の反応性が向上すると考えられる。さらに同一の反応槽で反応を行うことで、反応プロセスを簡略化することができ、設備コストを低減することができる。 In general, an enzyme has a specific catalytic activity with respect to a substrate and produces a reaction product, but competitive inhibition occurs in which the catalytic activity of the enzyme is inhibited by the reaction product. In simultaneous saccharification and fermentation, competition inhibition can be suppressed by assimilating a reaction product produced by an enzyme with a fermentation microorganism, and the overall reactivity is considered to be improved. Furthermore, by performing the reaction in the same reaction vessel, the reaction process can be simplified and the equipment cost can be reduced.
近年、地球規模での環境意識の高まりや、いずれ直面する石油資源の枯渇問題に対応するために、セルロース系バイオマスからの有価物生産が世界的に検討されており、前述の同時糖化発酵方法は、効率的にセルロース系バイオマスから有価物を生産する方法として注目されている。さらに発酵用微生物にセルラーゼなどのバイオマスを分解する酵素の機能を付加させた遺伝子改変体を用いて糖化発酵同時進行（consolidated bio-process）といった検討もされている。 In recent years, the production of valuable materials from cellulosic biomass has been studied globally in order to respond to the growing environmental awareness on a global scale and the depletion problem of petroleum resources that will eventually face. It is attracting attention as a method for efficiently producing valuable materials from cellulosic biomass. Furthermore, studies have also been conducted on a consolidated bio-process using a genetically modified product obtained by adding a function of an enzyme that degrades biomass such as cellulase to a fermentation microorganism.
このようなバイオマスを原料として微生物による変換を経る各種有用物質の生産は、前処理、糖化、前培養、発酵、蒸留といった要素技術があり、各要素技術およびその連携、組み合わせなどの開発によって低コスト化、最適化が図られている。特にバイオマスの原料として利用される植物系バイオマスはセルロースとヘミセルロースがリグニンと共に複雑に絡み合い、結晶化することで強固な構造を保っており、下流の発酵に不適なリグニン成分の除去も含めた全体的なプロセスを決定することは困難である。 There are elemental technologies such as pretreatment, saccharification, preculture, fermentation, and distillation for the production of various useful substances that are converted from microorganisms using such biomass as a raw material. And optimization. In particular, plant biomass used as a raw material for biomass has a strong structure because cellulose and hemicellulose are intertwined with lignin in complex and crystallized, and the entire process including removal of lignin components that are inappropriate for downstream fermentation is included. It is difficult to determine the correct process.
ところで、現在検討されているバイオマス資源中の有望な原料の１つとして紙が挙げられる。紙は、製紙工程において脱リグニン化され、結晶構造が破壊もしくは緩慢になっていることから、同時糖化発酵を行うに適した原料である。バイオマスの賦存量としては、リサイクルが可能として収集された古紙以外にも、製紙工場で発生するペーパースラッジや、一般廃棄物、事業系廃棄物として焼却処分等されてしまっている紙がある。 By the way, paper is mentioned as one of the promising raw materials in the biomass resources currently being studied. Paper is a raw material suitable for simultaneous saccharification and fermentation because it is delignified in the papermaking process and its crystal structure is destroyed or slowed down. In addition to waste paper collected as being recyclable, the biomass content includes paper sludge generated at paper mills, paper that has been incinerated as general waste, and business waste.
これらの紙を原料として利用することは産業的にも有用である。 It is industrially useful to use these papers as raw materials.
現在同時糖化発酵について多くの研究が多く行われているが、実際の産業として応用されているものはない。考えられるその主要な理由として、プロセス調整の困難さが挙げられる。酵素による加水分解と発酵用微生物による発酵を同時に行うため、複雑な反応になり、単純な発酵に比べると反応時間が長いため調節が難しい。さらに、反応時間が長いことから雑菌汚染の危険性が高く、その影響も大きくなる。このように産業化するには困難な方法ではあるが、各プロセスの最適化の集積によって実現可能となることは言うまでもない。 Currently, many studies on simultaneous saccharification and fermentation have been conducted, but none have been applied as actual industries. The main reason for this is the difficulty of process adjustment. Since hydrolysis by an enzyme and fermentation by a microorganism for fermentation are performed at the same time, the reaction becomes complicated, and the reaction time is longer than that of simple fermentation, making adjustment difficult. Furthermore, since the reaction time is long, there is a high risk of contamination and the effect is also great. Although it is a difficult method for industrialization in this way, it goes without saying that it can be realized by integrating optimization of each process.
研究レベルで検討を行う場合、ｐＨ調整には、その調節が容易であることから緩衝溶液が用いられる。しかし、この方法では実際の産業下では明らかにコストが高くなるため、ｐＨ調整にかかる部分についての検討は喫緊の課題の１つである。 When studying at the research level, a buffer solution is used for pH adjustment because it is easy to adjust. However, in this method, the cost is obviously high in an actual industry, and therefore, examination of a portion related to pH adjustment is one of urgent issues.
以上の背景に鑑みて、同時糖化発酵反応用に調整する研究を鋭意重ねた結果、紙を主な原料とする同時糖化発酵を行う場合に、紙に含まれている填料や、同時に処理する物質中に含まれる成分により原料依存的なｐＨの値を、同時糖化発酵反応の原料を調整する段階で同時糖化発酵反応時に目的とするｐＨの値より高い値に予め設定することで同時糖化発酵中ｐＨ調整用薬剤の低減化を実現する同時糖化発酵によるエタノール生産方法を発見するに至った。 In view of the above background, as a result of earnest research to adjust for simultaneous saccharification and fermentation reaction, when performing simultaneous saccharification and fermentation using paper as the main raw material, fillers contained in paper and substances to be processed simultaneously During the simultaneous saccharification and fermentation by presetting the pH value dependent on the raw material depending on the components contained therein to a value higher than the target pH value during the simultaneous saccharification and fermentation reaction at the stage of adjusting the raw material of the simultaneous saccharification and fermentation reaction It came to discover the ethanol production method by simultaneous saccharification fermentation which implement | achieves reduction of the chemical | medical agent for pH adjustment.
すなわち、本発明は、
（１）セルロース系原料を用いた同時糖化発酵によりエタノールを生産する方法において、同時糖化発酵反応の原料を調整する段階でｐＨの値が同時糖化発酵反応の目的とするｐＨの値よりも高い値に予め設定することを特徴とするエタノール生産方法；
（２）同時糖化発酵反応の目的とするｐＨの値が３．５〜５である、前記（１）のエタノール生産方法；
（３）予め設定されるｐＨの値が５〜６である、前記（１）のエタノール生産方法；
（４）前記セルロース系原料は紙を含む、前記（１）〜（３）のいずれか１つのエタノール生産方法；
（５）前記セルロース系原料は、乾燥重量で５０％以上の紙を含む、前記（１）のエタノールの生産方法；
（６）前記セルロース系原料は、セルロースおよびヘミセルロースの合計であるホロセルロースを、乾燥重量で５０％以上含む、前記（１）の生産方法
である。
That is, the present invention
(1) In a method for producing ethanol by simultaneous saccharification and fermentation using cellulosic raw materials, the pH value is higher than the target pH value in the simultaneous saccharification and fermentation reaction at the stage of adjusting the raw material for simultaneous saccharification and fermentation reaction An ethanol production method characterized in that the ethanol production method is set in advance;
(2) The ethanol production method according to (1), wherein the target pH value of the simultaneous saccharification and fermentation reaction is 3.5 to 5;
(3) The ethanol production method of (1) above, wherein the preset pH value is 5-6;
(4) The ethanol production method according to any one of (1) to (3), wherein the cellulosic material includes paper;
(5) The method for producing ethanol according to (1) above, wherein the cellulosic material includes paper of 50% or more by dry weight;
(6) The said cellulosic raw material is a production method of said (1) which contains the holocellulose which is the sum total of a cellulose and
本発明は、同時糖化発酵反応の原料を調整する段階で同時糖化発酵反応の目的とするｐＨより高い値に予めｐＨを設定するものであるが、このことにより、以下の効果を得ることができる。 In the present invention, the pH is set in advance to a value higher than the target pH of the simultaneous saccharification and fermentation reaction at the stage of adjusting the raw material for the simultaneous saccharification and fermentation reaction, and this can provide the following effects. .
（１）ｐＨ調整用の薬剤の低減もしくは削減が図れる。 (1) It is possible to reduce or reduce the pH adjusting chemical.
（２）プロセスを省力化することができる。 (2) The process can be labor-saving.
（３）酵素の失活もしくは活性低下の危険性を低下させることができる。 (3) The risk of enzyme deactivation or reduced activity can be reduced.
（４）発酵用微生物の活性低下の危険性を低下させることができる。 (4) The risk of a decrease in the activity of the microorganism for fermentation can be reduced.
次に本発明についてさらに説明する。 Next, the present invention will be further described.
通常、エタノール発酵を行う場合、グルコースを基質とするならば、１モルのグルコースから２モルのエタノールと２モルの炭酸ガスが生成する。さらに発酵用微生物の増殖もしくは維持のために行う生命活動下で、グルコースは他の物質に変換され、有機酸をはじめとする有機化合物の菌体外への排出入、および無機塩の排出入が起こる。これらの影響で、培養の進行に伴ってｐＨ値が低下する現象が観測される。このとき発酵用微生物の増殖もしくは維持に適したｐＨ値に培養液のｐＨを調節することは安定的な培養のために重要である。 Usually, when performing ethanol fermentation, if glucose is used as a substrate, 2 mol of ethanol and 2 mol of carbon dioxide gas are generated from 1 mol of glucose. Furthermore, glucose is converted into other substances under the vital activities for the growth or maintenance of fermentation microorganisms, and organic compounds such as organic acids are discharged outside the cells and inorganic salts are discharged. Occur. Due to these effects, a phenomenon is observed in which the pH value decreases as the culture progresses. At this time, adjusting the pH of the culture solution to a pH value suitable for the growth or maintenance of the microorganism for fermentation is important for stable culture.
これは同時糖化発酵を行った場合も同様に、同時糖化発酵の進行に伴ってｐＨ値の低下が観測され、培養および酵素活性に影響を及ぼすｐＨ値に反応液のｐＨが達すると、これにより同時糖化発酵の反応性に影響が与えられる。同時糖化発酵に用いられる酵素としてはデンプン系原料の場合は主にアミラーゼが用いられ、さらにその他の原料が混在しているような生ゴミを原料とする場合にはプロテアーゼやリパーゼといった酵素も併用することが想定される。また、セルロース系原料の場合は主にセルラーゼが用いられるが、セルラーゼを構成する酵素群であるエンドグルカナーゼ、セロビオハイドラーゼ、ベータグルコシダーゼの量的比率や、追加で用いられるキシラーゼの使用、および上記デンプン系原料が混入している場合には前述の酵素群を使用することが考えられる。しかし、各酵素の至適ｐＨは異なることが容易に想像でき、発酵用微生物の活性と共に、ｐＨの値によって同時糖化発酵の効率は変化する。 Similarly, when simultaneous saccharification and fermentation are performed, a decrease in pH value is observed with the progress of simultaneous saccharification and fermentation, and when the pH of the reaction solution reaches a pH value that affects culture and enzyme activity, The reactivity of simultaneous saccharification and fermentation is affected. As the enzyme used for simultaneous saccharification and fermentation, amylase is mainly used in the case of starch-based materials, and enzymes such as protease and lipase are also used in combination when raw garbage containing other materials is used as a raw material. It is assumed that In the case of cellulosic materials, cellulase is mainly used. However, the quantitative ratio of endoglucanase, cellobiohydrase and betaglucosidase, which are enzyme groups constituting cellulase, use of additional xylase, and the above When starch-based raw materials are mixed, it is conceivable to use the aforementioned enzyme group. However, it can be easily imagined that the optimum pH of each enzyme is different, and the efficiency of simultaneous saccharification and fermentation varies depending on the value of pH along with the activity of the microorganisms for fermentation.
一般的に同時糖化発酵中はその流動性が原因でｐＨを調整することが困難であり、緩衝溶液を用いてｐＨの大きな変化が起こらないようにしている。 In general, during simultaneous saccharification and fermentation, it is difficult to adjust the pH due to its fluidity, and a large change in pH is prevented by using a buffer solution.
そこで、本発明では予め低下するｐＨ値の分を想定して、同時糖化発酵反応に影響が少ない範囲での高いｐＨに設定し、その同時糖化発酵反応の原料を調整する段階でｐＨの値を使用酵素の反応至適ｐＨより高い値に設定することを特徴とする。高く設定する値はｐＨ４．５〜７が望ましく、より望ましくはｐＨ５〜６である。 Therefore, in the present invention, assuming a pH value that decreases in advance, the pH value is set at a stage where a high pH is set in a range where there is little influence on the simultaneous saccharification and fermentation reaction, and the raw material of the simultaneous saccharification and fermentation reaction is adjusted. It is characterized by being set to a value higher than the optimum reaction pH of the enzyme used. The value to be set high is preferably pH 4.5-7, more preferably pH 5-6.
本発明は、同時糖化発酵におけるプロセスの省力化、使用薬剤の低減に関するものであり、原料は紙に含まれるセルロース系バイオマスであるが、同時糖化発酵を行ってエタノールを生成し、そのｐＨが５以上であれば他の条件については特に何らの限定を課すこともなく適応することができる。他のセルロース系、デンプン系、糖類やこれらの混合物、さらに他の有機物質が含まれていてもよく、同時糖化発酵に顕著な影響を与えない無機成分や有機合成品をはじめとする未反応成分を含んでいてもよい。 The present invention relates to labor saving of processes in simultaneous saccharification and fermentation and reduction of chemicals used. The raw material is cellulosic biomass contained in paper, but ethanol is produced by simultaneous saccharification and fermentation, and the pH is 5 As described above, other conditions can be applied without any particular limitation. Other cellulose-based, starch-based, saccharides and mixtures thereof, and other organic substances may be included, and unreacted components such as inorganic components and organic synthetic products that do not significantly affect simultaneous saccharification and fermentation May be included.
使用酵素は同時糖化発酵原料の分解を行いその構成成分を得るために必要な酵素であれば如何なるものであってもよいが、原料にセルロース系原料が含まれていればエンドグルカナーゼ、セロビオハイドラーゼ、ベータグルコシダーゼの少なくとも１つを含むセルラーゼであることが望ましく、これにキシラーゼが含まれていればなお望ましいが、特にここに挙げられたものに限定されるものではない。また、原料にデンプン系のものが含まれていれば、アミラーゼ、厨芥であればセルラーゼ、アミラーゼ、プロテアーゼ、リパーゼを含んでいることが望ましいが、これらのものに限定されない。 The enzyme used may be any enzyme as long as it is necessary for decomposing the raw material for simultaneous saccharification and fermentation to obtain its constituent components. However, if the raw material contains a cellulosic material, endoglucanase and cellobiohydride are used. A cellulase containing at least one of a lyase and a beta glucosidase is desirable, and it is more desirable if it contains a xylase, but it is not particularly limited to those listed here. In addition, if the raw material contains starch-based materials, it is desirable to contain amylase, and if it is koji, it contains cellulase, amylase, protease, and lipase, but is not limited thereto.
使用発酵用微生物は、発酵によりエタノールを産生する微生物であれば、特定のものに限定する必要はなく、遺伝子改変によりエタノール生成能の付加もしくは増強された菌体を用いてもよく、また一般的に資化しやすいグルコースやスクロース以外のヘキソースおよびそのオリゴマーの資化能の付加もしくは増強を行った菌体でもよく、さらにキシロースやアラビノースといったペントース資化能の付加もしくは増強を行った菌体を用いてもよく、前述の酵素を付加もしくは増強を行った菌体を用いてもよい。 As long as the microorganism used for fermentation is a microorganism that produces ethanol by fermentation, it is not necessary to be limited to a specific microorganism, and a bacterial cell with an ethanol production ability added or enhanced by genetic modification may be used. Bacteria that have added or enhanced hexoses other than glucose or sucrose and their oligomers, or those that have added or enhanced pentose utilization such as xylose and arabinose. Alternatively, bacterial cells to which the above-described enzyme has been added or enhanced may be used.
本発明は反応液のｐＨを目的とする値より予め高い値に設定することを特徴とし、温度、溶存酸素量、攪拌速度、攪拌方法、栄養塩添加量およびその種類といった他の条件について特段の制限が課されることはない。 The present invention is characterized in that the pH of the reaction solution is set to a value that is higher than the target value in advance, and other conditions such as temperature, dissolved oxygen amount, stirring speed, stirring method, nutrient salt addition amount and type thereof are special. There are no restrictions.
次に実施例に基づいて本発明を具体的に説明するが、本発明は、以下の実施例に何ら限定されるものではない。 EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited to a following example at all.
（実施例）
本実施例では、同時糖化発酵用の酵素はジェネンコア社製セルラーゼ製剤であるアクセララーゼを用いた。使用量はＦＰＵで表わされる。１ＦＰＵは６０分間にろ紙からグルコースを１０．８ｍｇ生成するセルラーゼ活性の単位である。
(Example)
In this example, an enzyme for simultaneous saccharification and fermentation was Accelerase, a cellulase preparation manufactured by Genencor. The amount used is expressed in FPU. 1FPU is a unit of cellulase activity that produces 10.8 mg of glucose from filter paper in 60 minutes.
発酵用微生物は酵母（サッカロマイセスセルビジエ）を用い、前培養はＹＰ培地にグルコースを１５％加えたものを用いた。また、菌体は遠心分離により回収したものを用いた。原料は調整した模擬紙ゴミを用いた。模擬紙ゴミは、ゴミ中に存在する紙成分の重量比率を調べ、別途材料を調達し、同比率で混合し、同様に処理したものを用いた。表１に模擬紙ゴミの重量比率を示す。 As a microorganism for fermentation, yeast (Saccharomyces cerevisiae) was used, and for preculture, a YP medium added with 15% glucose was used. Moreover, what was collect | recovered by centrifugation was used for the microbial cell. The prepared simulated paper waste was used. For the simulated paper waste, the weight ratio of the paper components present in the waste was examined, materials were separately procured, mixed at the same ratio, and treated in the same manner. Table 1 shows the weight ratio of simulated paper waste.
上記模擬紙ゴミに水を加えてミキサーで離解後、原料濃度を調整することにより同時糖化発酵用原料とした。紙はその填量に含まれる物質により中性から塩基性を示すことが多く、使用した模擬紙ゴミを水でミキシングすることで約ｐＨ７．４の値を示した。 Water was added to the simulated paper waste and disintegrated with a mixer, and the raw material concentration was adjusted to obtain a raw material for simultaneous saccharification and fermentation. The paper is often neutral to basic depending on the substance contained in the filling amount, and the used paper waste was mixed with water to show a value of about pH 7.4.
ミキシングした模擬紙ゴミを含水率８５重量％に調整し、乾燥紙１ｇ当たりセルラーゼ８ＦＰＵおよび酵母を０．０３ｇとなるように加え、３５℃で同時糖化発酵を行った。 The mixed simulated paper waste was adjusted to a moisture content of 85% by weight, cellulase 8FPU and yeast were added to 0.03 g per 1 g of dry paper, and simultaneous saccharification and fermentation were performed at 35 ° C.
エタノール濃度はグルコースセンサー（王子計測機製ＢＦ−５）にて計測した。 The ethanol concentration was measured with a glucose sensor (BF-5 manufactured by Oji Scientific Instruments).
対照区として、通常、同時糖化発酵方法でよく用いられるｐＨ３、３．５、４、４．５、５．５、６、７程度に調整した酢酸緩衝溶液を用いて、模擬紙ゴミを原料に同時糖化発酵を行った。その結果を図１に示す。
As a control group, using an acetic acid buffer solution adjusted to about
実施区として、緩衝能のない酸である硫酸を用いて模擬紙ゴミをｐＨ３、３．５、４、４．５、５、６、７程度に調整し、同時糖化発酵を行った。結果を図２に示す。
As an execution group, the simulated paper waste was adjusted to about
対照区および実施区の同時糖化発酵反応前後のｐＨの推移を下記の表２にまとめた。 The changes in pH before and after the simultaneous saccharification and fermentation reaction in the control group and the execution group are summarized in Table 2 below.
対照区ではｐＨ４〜４．５に調整した場合で同時糖化発酵反応によるエタノール生成が優れていることが観測された。一方、実施区である、緩衝能を持たない酸である硫酸のみでｐＨ調整を行った場合、ｐＨ４．５〜７、特にｐＨ５〜６の場合にエタノール生成が優れていた。
In the control group, it was observed that the ethanol production by the simultaneous saccharification and fermentation reaction was excellent when the pH was adjusted to 4 to 4.5. On the other hand, when the pH was adjusted only with sulfuric acid, which was an acid having no buffering capacity, which was an implementation zone, ethanol production was excellent at pH 4.5 to 7, particularly
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| CN102242158A (en) * | 2011-05-26 | 2011-11-16 | 湘潭大学 | Method for producing ethanol with lotus peel powder as raw material by synchronization of saccharification and fermentation |
| WO2015053364A1 (en) * | 2013-10-09 | 2015-04-16 | 新日鉄住金エンジニアリング株式会社 | Ethanol production facility, ethanol production method, and facility for producing ethanol and biopellets |
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| WO2015053364A1 (en) * | 2013-10-09 | 2015-04-16 | 新日鉄住金エンジニアリング株式会社 | Ethanol production facility, ethanol production method, and facility for producing ethanol and biopellets |
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