JP2015173624A - Method for manufacturing bioethanol - Google Patents
Method for manufacturing bioethanol Download PDFInfo
- Publication number
- JP2015173624A JP2015173624A JP2014051930A JP2014051930A JP2015173624A JP 2015173624 A JP2015173624 A JP 2015173624A JP 2014051930 A JP2014051930 A JP 2014051930A JP 2014051930 A JP2014051930 A JP 2014051930A JP 2015173624 A JP2015173624 A JP 2015173624A
- Authority
- JP
- Japan
- Prior art keywords
- solution
- growth
- inhibitory substance
- saccharified
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
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
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
本発明は、バイオエタノールの製造方法に関する。 The present invention relates to a method for producing bioethanol.
近年、地球温暖化の一因と考えられている二酸化炭素の排出量削減が求められており、ガソリン等の液体炭化水素とエタノールとの混合燃料を自動車燃料に用いることが検討されている。前記エタノールとしては、サトウキビ、トウモロコシ等の農作物等の植物性物質の発酵により得られたエタノールを用いることができる。 In recent years, reduction of carbon dioxide emission, which is considered to be a cause of global warming, has been demanded, and use of a mixed fuel of liquid hydrocarbons such as gasoline and ethanol as an automobile fuel has been studied. As said ethanol, the ethanol obtained by fermentation of plant substances, such as agricultural products, such as sugarcane and corn, can be used.
かかる植物性物質から得られたエタノールにおいては、原料となる植物自体が既に光合成により二酸化炭素を吸収しているので、該エタノールを燃焼したときに排出される二酸化炭素の量は、該植物自体が吸収した二酸化炭素の量に等しい。即ち、総計としての二酸化炭素の排出量は理論的にはゼロになるという、所謂カーボンニュートラル効果を得ることができる。 In ethanol obtained from such plant substances, since the plant itself as a raw material has already absorbed carbon dioxide by photosynthesis, the amount of carbon dioxide emitted when the ethanol is burned is determined by the plant itself. Equal to the amount of carbon dioxide absorbed. That is, it is possible to obtain a so-called carbon neutral effect that the total amount of carbon dioxide emission is theoretically zero.
ところが、前記サトウキビ、トウモロコシ等の農作物は、本来食糧とされるものであるので、エタノールの原料として大量に消費されると、食糧として供給される量が減少するという問題がある。 However, since the agricultural products such as sugar cane and corn are originally used as food, there is a problem that the amount supplied as food decreases when consumed in large quantities as a raw material for ethanol.
そこで、原料として、サトウキビ、トウモロコシ等の農作物に代えて、食用ではないリグノセルロース系バイオマス（以下、バイオマスと略記することがある）を用いてエタノールを製造する技術が検討されている。リグノセルロース系バイオマスは、セルロースとヘミセルロース（以下、セルロース類と略記することがある）を含んでおり、セルロース類を糖化酵素によってグルコースやキシロース等の糖に分解し、得られた糖を発酵させることによりエタノールを得ることができる。 Therefore, a technique for producing ethanol using, as a raw material, lignocellulosic biomass that is not edible (hereinafter sometimes abbreviated as biomass) instead of sugarcane, corn and other agricultural crops has been studied. Lignocellulosic biomass contains cellulose and hemicellulose (hereinafter sometimes abbreviated as celluloses), decomposes celluloses into sugars such as glucose and xylose by saccharifying enzymes, and ferments the resulting sugars. To obtain ethanol.
リグノセルロース系バイオマスとしては、例えば、木材、稲藁、麦藁、バガス、竹、トウモロコシの茎や葉や芯、パルプ、及びこれらから生じる廃棄物、例えば古紙等を挙げることができる。 Examples of lignocellulosic biomass include wood, rice straw, wheat straw, bagasse, bamboo, corn stalks, leaves and cores, pulp, and waste products such as waste paper.
ところが、リグノセルロース系バイオマスは、セルロースの他にヘミセルロース及びリグニンを主な構成成分としており、通常セルロース及びヘミセルロースは、リグニンに強固に結合しているため、そのままではセルロースに対する糖化反応を行うことが難しい。 However, lignocellulosic biomass is mainly composed of hemicellulose and lignin in addition to cellulose, and usually cellulose and hemicellulose are firmly bound to lignin, so that it is difficult to perform a saccharification reaction on cellulose as it is. .
そこで、従来、リグノセルロース系バイオマス由来のエタノールは、次のようにして製造される。まず、リグノセルロース系バイオマスを前処理することにより、リグノセルロース系バイオマスからリグニンを解離し、又はリグノセルロース系バイオマスを膨潤させた前処理物を得る。本明細書において、解離とは、リグノセルロース系バイオマスのセルロース又はヘミセルロースに結合しているリグニンの結合部位のうち、少なくとも一部の結合を切断することをいう。また、膨潤とは、液体の浸入により結晶性セルロースを構成するセルロース又はヘミセルロースに空隙が生じ、又は、セルロース繊維の内部に空隙が生じて膨張することをいう。 Therefore, conventionally, ethanol derived from lignocellulosic biomass is produced as follows. First, pretreatment of lignocellulosic biomass is obtained by pretreating lignocellulosic biomass to dissociate lignin from lignocellulosic biomass or swell lignocellulosic biomass. In the present specification, dissociation means that at least a part of the binding sites of lignin binding to cellulose or hemicellulose of lignocellulosic biomass is broken. Swelling means that voids are generated in cellulose or hemicellulose constituting crystalline cellulose by the intrusion of liquid, or voids are generated inside cellulose fibers to expand.
次に、前記前処理物を前記糖化酵素を用いて糖化処理することにより、糖化溶液を得る。次に、前記糖化溶液を発酵槽で微生物によって発酵させてエタノールを生成させた後、蒸留することにより、バイオエタノールを製造する。このとき、前記発酵における発酵効率が低いことがあるという問題がある。 Next, a saccharification solution is obtained by saccharifying the pretreated product using the saccharifying enzyme. Next, the saccharification solution is fermented by microorganisms in a fermentor to produce ethanol, and then distilled to produce bioethanol. At this time, there exists a problem that the fermentation efficiency in the said fermentation may be low.
前記問題を解決するために、従来、前記糖化溶液にサトウキビの廃糖蜜を混合して糖濃度を高くし、得られた混合液を発酵させる技術が提案されている（例えば特許文献１参照）。前記従来技術によれば、前記発酵における発酵効率を向上できるとされている。 In order to solve the above problems, conventionally, a technique has been proposed in which sugarcane waste molasses is mixed with the saccharified solution to increase the sugar concentration, and the resulting mixed liquid is fermented (see, for example, Patent Document 1). According to the said prior art, it is supposed that the fermentation efficiency in the said fermentation can be improved.
しかし、前記従来技術においても、前記発酵効率を向上させることができない場合があるという問題がある。 However, even in the prior art, there is a problem that the fermentation efficiency may not be improved.
本発明者らは、前記発酵効率を向上させることができない原因について検討した。その結果、前記糖化溶液が、前記前処理及び前記糖化処理の際に副生成物として生成した酢酸、蟻酸、ｐ−クマル酸等の化合物を含み、該化合物によって前記発酵の際に前記微生物の増殖が阻害されていることを見出した。本願では、前記微生物の増殖を阻害する化合物を「増殖阻害物質」と言う。 The present inventors examined the cause that the fermentation efficiency cannot be improved. As a result, the saccharification solution contains a compound such as acetic acid, formic acid or p-coumaric acid generated as a by-product during the pretreatment and the saccharification treatment, and the growth of the microorganism by the compound during the fermentation. Was found to be inhibited. In the present application, a compound that inhibits the growth of the microorganism is referred to as a “growth inhibitor”.
そこで、前記増殖阻害物質の影響を低減するために、前記糖化溶液と前記廃糖蜜とを混合した混合液を、ナノ透過膜又は逆浸透膜を用いて濾過することにより、該混合液から前記増殖阻害物質を除去する技術が提案されている（例えば特許文献２参照）。 Accordingly, in order to reduce the influence of the growth inhibitory substance, the mixture solution obtained by mixing the saccharification solution and the waste molasses is filtered using a nano-permeable membrane or a reverse osmosis membrane, whereby the growth solution is removed from the mixture solution. A technique for removing an inhibitory substance has been proposed (see, for example, Patent Document 2).
しかしながら、前記糖化溶液に対して前記ナノ透過膜又は逆浸透膜を用いて濾過する場合には、濾過工程が増えるので、製造工程が煩雑になる上にバイオエタノール製造に要する時間が長くなり、さらには濾過を行うための設備が必要となるという不都合がある。 However, when the saccharified solution is filtered using the nano-permeable membrane or reverse osmosis membrane, the number of filtration steps increases, so the manufacturing process becomes complicated and the time required for bioethanol production increases. Has the disadvantage of requiring equipment for filtration.
本発明は、かかる不都合を解消して、増殖阻害物質を除去することなく発酵の際に微生物の増殖阻害を回避することができるバイオエタノールの製造方法を提供することを目的とする。前記除去とは、濾過、中和、吸着等により糖化溶液に含まれる増殖阻害物質を取り除いたり、その効力を失わせたりすることをいう。 An object of the present invention is to provide a method for producing bioethanol capable of eliminating such inconvenience and avoiding microbial growth inhibition during fermentation without removing the growth inhibitory substance. The term “removal” means that the growth inhibitory substance contained in the saccharification solution is removed or its efficacy is lost by filtration, neutralization, adsorption or the like.
本発明は、リグノセルロース系バイオマスを前処理した前処理物を糖化酵素によって糖化処理し、得られた糖化溶液を微生物によって発酵させてエタノールを生成させるバイオエタノールの製造方法において、該微生物の増殖を阻害する増殖阻害物質を含む該糖化溶液を、該糖化溶液よりも増殖阻害物質濃度が小さい糖液によって該増殖阻害物質濃度が低下するように希釈することにより、混合糖化溶液を得る工程と、該混合糖化溶液を該微生物によって発酵させてエタノールを生成させる工程とを備えることを特徴とする。 The present invention relates to a method for producing bioethanol in which a pretreated product obtained by pretreating lignocellulosic biomass is saccharified with a saccharification enzyme, and the resulting saccharification solution is fermented with microorganisms to produce ethanol. Diluting the saccharification solution containing a growth inhibitory substance to be inhibited with a sugar solution having a growth inhibitory substance concentration lower than that of the saccharification solution so that the growth inhibitory substance concentration is lowered, and obtaining a mixed saccharification solution; And a step of fermenting the mixed saccharified solution with the microorganism to produce ethanol.
本発明の製造方法では、まず、糖化溶液を、該糖化溶液よりも増殖阻害物質濃度が小さい糖液によって希釈することにより、増殖阻害物質濃度が低下された混合糖化溶液を得る。次に、前記混合糖化溶液を微生物によって発酵させてエタノールを生成させる。前記発酵の際、前記混合糖化溶液は増殖阻害物質濃度が低下されているので、前記増殖阻害物質によって前記微生物の増殖が阻害されることを防ぐことができる。 In the production method of the present invention, first, a saccharified solution is diluted with a saccharide solution having a growth inhibitory substance concentration lower than that of the saccharified solution to obtain a mixed saccharified solution having a reduced growth inhibitory substance concentration. Next, the mixed saccharified solution is fermented with microorganisms to produce ethanol. During the fermentation, since the concentration of the growth inhibitory substance in the mixed saccharified solution is reduced, it is possible to prevent the growth of the microorganism from being inhibited by the growth inhibitory substance.
したがって、本発明の製造方法によれば、増殖阻害物質を除去することなく、発酵の際に微生物の増殖阻害を回避することができる。これにより、発酵効率を向上することができる。 Therefore, according to the production method of the present invention, growth inhibition of microorganisms can be avoided during fermentation without removing the growth inhibitory substance. Thereby, fermentation efficiency can be improved.
ところで、前記増殖阻害物質としては、例えば酢酸、フルフラール、ｐ−クマル酸を挙げることができる。本発明の製造方法において、前記糖化溶液を、前記酢酸の非解離分の濃度が７００ｍｇ／Ｌ以下となり、前記フルフラールの濃度が７００ｍｇ／Ｌ以下となり、且つ前記ｐ−クマル酸の濃度が５００ｍｇ／Ｌ以下となるように希釈することが好ましい。各増殖阻害物質濃度が前記範囲である前記混合糖化溶液とすることにより、前記発酵の際に前記微生物の増殖阻害を回避し、発酵効率を向上することができる。 By the way, examples of the growth inhibitor include acetic acid, furfural, and p-coumaric acid. In the production method of the present invention, the concentration of the non-dissociated portion of acetic acid is 700 mg / L or less, the concentration of furfural is 700 mg / L or less, and the concentration of p-coumaric acid is 500 mg / L. It is preferable to dilute so that: By using the mixed saccharified solution in which the concentration of each growth inhibitory substance is within the above range, it is possible to avoid inhibition of the growth of the microorganism during the fermentation and improve the fermentation efficiency.
各増殖阻害物質濃度が前記範囲を上回る場合には、前記発酵の際に前記微生物を十分に増殖させることができないことがある。 When the concentration of each growth inhibitor exceeds the above range, the microorganism may not be able to grow sufficiently during the fermentation.
また、本発明の製造方法において、前記糖液としては、例えば、サトウキビの廃糖蜜、
サトウキビの絞り汁、該サトウキビの絞り汁の濃縮液、米を糖化した液、麦を糖化した液、トウモロコシを糖化した液からなる群から選択される１種以上の液体を用いることができる。前記廃糖蜜とは、サトウキビの絞り汁から砂糖を結晶化させた後に残る残液であり、モラセス又は糖蜜と呼ばれることがある。以下、サトウキビの廃糖蜜を「廃糖蜜」と略記することがある。
In the production method of the present invention, as the sugar liquid, for example, sugarcane molasses,
One or more liquids selected from the group consisting of sugarcane juice, concentrated sugarcane juice, rice saccharified liquid, wheat saccharified liquid, and corn saccharified liquid can be used. The waste molasses is a residual liquid remaining after crystallization of sugar from sugarcane juice, and is sometimes referred to as molasses or molasses. Hereinafter, sugarcane molasses may be abbreviated as “molasses”.
前記糖液として前記液体を用いることにより、低コストで前記増殖阻害物質濃度を低下させることができる。 By using the liquid as the sugar solution, the concentration of the growth inhibitory substance can be reduced at a low cost.
また、本発明の製造方法では、次のようにして、前記微生物の増殖阻害を回避することができる糖化溶液率を備える混合糖化溶液を得ることができる。本願では、「糖化溶液率」とは、糖化溶液を糖液で希釈して混合糖化溶液を得るときの該混合糖化溶液の全質量に対する該糖化溶液の質量の割合を意味する。この方法では、前記糖化溶液に含まれる増殖阻害物質の濃度に着目して前記糖化溶液率を決定するが、はじめに、１種類の増殖阻害物質に着目する方法について説明する。 Moreover, in the manufacturing method of this invention, the mixed saccharification solution provided with the saccharification solution rate which can avoid the growth inhibition of the said microorganisms can be obtained as follows. In the present application, the “saccharified solution ratio” means the ratio of the mass of the saccharified solution to the total mass of the mixed saccharified solution when the mixed saccharified solution is obtained by diluting the saccharified solution with the saccharified solution. In this method, the ratio of the saccharified solution is determined by paying attention to the concentration of the growth inhibitory substance contained in the saccharified solution. First, a method focusing on one kind of growth inhibitory substance will be described.
まず、第１の工程として、試験用糖液に、前記増殖阻害物質をそれぞれ異なる増殖阻害物質濃度で添加してなる複数の培養液を調製する。そして、前記複数の培養液を用いて前記微生物を培養し、各培養液に含まれる該増殖阻害物質濃度毎の微生物増殖数を測定する。 First, as a first step, a plurality of culture solutions are prepared by adding the growth inhibitory substance at different growth inhibitory substance concentrations to the test sugar solution. Then, the microorganism is cultured using the plurality of culture solutions, and the number of microorganisms grown for each concentration of the growth inhibitory substance contained in each culture solution is measured.
次に、第２の工程として、前記増殖阻害物質を無添加の前記試験用糖液を培養液に用いて前記微生物を培養し、該無添加の試験用糖液における微生物増殖数を測定して標準微生物増殖数とする。 Next, as a second step, the microorganism is cultured using the test sugar solution to which the growth inhibitor is not added as a culture solution, and the number of microbial growth in the additive test sugar solution is measured. The standard microbial growth number.
次に、第３の工程として、前記第１の工程で得られた前記増殖阻害物質濃度毎の微生物増殖数を、前記第２の工程で得られた前記標準微生物増殖数で除することにより、各培養液の増殖阻害物質濃度における微生物増殖率を算出する。 Next, as a third step, by dividing the microbial growth number for each growth inhibitor concentration obtained in the first step by the standard microbial growth number obtained in the second step, The microbial growth rate at the growth inhibitor concentration of each culture solution is calculated.
次に、第４の工程として、前記第３の工程で得られた各培養液の前記増殖阻害物質濃度と前記微生物増殖率との関係から、第１の増殖阻害曲線を作成する。 Next, as a fourth step, a first growth inhibition curve is created from the relationship between the growth inhibitory substance concentration of each culture solution obtained in the third step and the microorganism growth rate.
次に、第５の工程として、本発明のバイオエタノール製造に用いる前記糖化溶液に含まれる増殖阻害物質濃度を測定する。 Next, as a fifth step, the concentration of the growth inhibitory substance contained in the saccharification solution used for the bioethanol production of the present invention is measured.
次に、第６の工程として、前記第４の工程で得られた前記第１の増殖阻害曲線から、前記第５の工程で得られた前記糖化溶液に含まれる増殖阻害物質濃度に対応する微生物増殖率を求める。 Next, as a sixth step, from the first growth inhibition curve obtained in the fourth step, a microorganism corresponding to the concentration of the growth inhibitory substance contained in the saccharification solution obtained in the fifth step Determine the growth rate.
次に、第７の工程として、まず、未希釈の前記糖化溶液の糖化溶液率を１とする。次に、前記未希釈の糖化溶液を本発明のバイオエタノール製造で用いる前記糖液によってそれぞれ異なる量で希釈することにより得られる各混合糖化溶液について、前記第４の工程で得られた前記第１の増殖阻害曲線から、各混合糖化溶液の糖化溶液率に対応する微生物増殖率を求める。このとき、前記糖液の増殖阻害物質濃度は、前記糖化溶液の増殖阻害物質濃度よりも非常に小さく実質的に無視できるとして、ゼロとみなすこととする。 Next, as a seventh step, first, the saccharified solution ratio of the undiluted saccharified solution is set to 1. Next, for each mixed saccharified solution obtained by diluting the undiluted saccharified solution with a different amount depending on the saccharified solution used in the bioethanol production of the present invention, the first saccharified solution obtained in the fourth step is used. From the growth inhibition curve, the microbial growth rate corresponding to the saccharification solution rate of each mixed saccharification solution is obtained. At this time, the concentration of the growth inhibitory substance in the sugar solution is considered to be zero because it is much smaller than the concentration of the growth inhibitory substance in the saccharified solution and can be substantially ignored.
次に、第８の工程として、前記第７の工程で得られた各混合糖化溶液における前記糖化溶液率と前記微生物増殖率との関係から、第２の増殖阻害曲線を作成する。 Next, as an eighth step, a second growth inhibition curve is created from the relationship between the saccharified solution rate and the microbial growth rate in each mixed saccharified solution obtained in the seventh step.
次に、第９の工程として、前記第８の工程で得られた前記第２の増殖阻害曲線から、所望の微生物増殖率に対応する糖化溶液率を求める。 Next, as a ninth step, a saccharified solution rate corresponding to a desired microbial growth rate is obtained from the second growth inhibition curve obtained in the eighth step.
次に、第１０の工程として、第９の工程で得られた前記糖化溶液率となるように、前記糖化溶液を本発明のバイオエタノール製造で用いる前記糖液によって希釈することにより、混合糖化溶液を得る。 Next, as a tenth step, a mixed saccharified solution is obtained by diluting the saccharified solution with the saccharide solution used in the bioethanol production of the present invention so that the saccharified solution ratio obtained in the ninth step is obtained. Get.
以上により、前記増殖阻害物質を除去することなく発酵の際に微生物の増殖阻害を回避することができる前記混合糖化溶液を得ることができる。 As described above, the mixed saccharified solution that can avoid the growth inhibition of microorganisms during fermentation can be obtained without removing the growth inhibitory substance.
前記糖化溶液に含まれる増殖阻害物質の濃度に着目して前記糖化溶液率を決定する方法によれば、前記微生物の増殖阻害に最も大きい影響を与える増殖阻害物質に着目して前記希釈を行うことができる。 According to the method of determining the saccharification solution ratio by paying attention to the concentration of the growth inhibitory substance contained in the saccharification solution, the dilution is performed by paying attention to the growth inhibitory substance that has the greatest influence on the growth inhibition of the microorganism. Can do.
また、次のようにすることにより、前記糖化溶液に含まれる複数の増殖阻害物質に着目して前記糖化溶液率を決定することが可能である。 In addition, by performing the following, it is possible to determine the saccharified solution ratio by paying attention to a plurality of growth inhibitory substances contained in the saccharified solution.
まず、第１の工程として、本発明のバイオエタノール製造に用いる前記糖化溶液に含まれる複数の増殖阻害物質の各増殖阻害物質毎に、試験用糖液に該増殖阻害物質をそれぞれ異なる増殖阻害物質濃度で添加してなる複数の培養液を調製する。そして、各増殖阻害物質毎に前記複数の培養液を用いて前記微生物を培養し、各増殖阻害物質毎の各培養液に含まれる該増殖阻害物質濃度毎の微生物増殖数を測定する。 First, as a first step, for each growth inhibitory substance of a plurality of growth inhibitory substances contained in the saccharification solution used for the bioethanol production of the present invention, the growth inhibitory substance is different in the test sugar solution. A plurality of culture broths added at a concentration are prepared. Then, the microorganisms are cultured for each growth inhibitory substance using the plurality of culture solutions, and the number of microorganism growth for each growth inhibitory substance concentration contained in each culture solution for each growth inhibitory substance is measured.
次に、第２の工程として、各増殖阻害物質を無添加の前記試験用糖液を培養液に用いて前記微生物を培養し、該無添加の試験用糖液における微生物増殖数を測定して標準微生物増殖数とする。 Next, as a second step, the microorganism is cultured using the test sugar solution to which each growth inhibitor is not added as a culture solution, and the number of microorganism growth in the additive-free test sugar solution is measured. The standard microbial growth number.
次に、第３の工程として、前記第１の工程で得られた各増殖阻害物質毎の各増殖阻害物質濃度毎の微生物増殖数を、前記第２の工程で得られた前記標準微生物増殖数で除することにより、各増殖阻害物質毎に、各培養液の増殖阻害物質濃度における微生物増殖率を算出する。 Next, as a third step, the microbial growth number for each growth inhibitory substance concentration for each growth inhibitory substance obtained in the first step is used as the standard microbial growth number obtained in the second step. By dividing by the above, the microbial growth rate at the growth inhibitor concentration of each culture solution is calculated for each growth inhibitor.
次に、第４の工程として、前記第３の工程で得られた各増殖阻害物質毎の各培養液の前記増殖阻害物質濃度と前記微生物増殖率との関係から、各増殖阻害物質毎に第１の増殖阻害曲線を作成する。 Next, as a fourth step, from the relationship between the growth inhibitory substance concentration of each culture solution for each growth inhibitory substance obtained in the third step and the microbial growth rate, the growth inhibitory substance is One growth inhibition curve is generated.
次に、第５の工程として、前記糖化溶液に含まれる各増殖阻害物質濃度を測定する。 Next, as a fifth step, the concentration of each growth inhibitor contained in the saccharification solution is measured.
次に、第６の工程として、前記第４の工程で得られた各増殖阻害物質毎の第１の増殖阻害曲線から、前記第５の工程で得られた各増殖阻害物質毎の前記糖化溶液に含まれる増殖阻害物質濃度に対応する微生物増殖率を求める。 Next, as a sixth step, the saccharification solution for each growth inhibitory substance obtained in the fifth step is obtained from the first growth inhibition curve for each growth inhibitory substance obtained in the fourth step. The microbial growth rate corresponding to the concentration of the growth inhibitory substance contained in is determined.
次に、第７の工程として、まず、未希釈の前記糖化溶液の糖化溶液率を１とする。次に、前記未希釈の糖化溶液を本発明のバイオエタノール製造で用いる前記糖液によってそれぞれ異なる量で希釈することにより得られる各混合糖化溶液について、前記第４の工程で得られた各増殖阻害物質毎の前記第１の増殖阻害曲線から、各混合糖化溶液の糖化溶液率に対応する微生物増殖率を求める。次に、前記糖化溶液率が同一である混合糖化溶液における各増殖阻害物質の微生物増殖率をそれぞれ乗じることにより、各混合糖化溶液の糖化溶液率に対応する微生物増殖率を算出する。 Next, as a seventh step, first, the saccharified solution ratio of the undiluted saccharified solution is set to 1. Next, for each mixed saccharified solution obtained by diluting the undiluted saccharified solution with different amounts of the saccharified solution used in the bioethanol production of the present invention, each growth inhibition obtained in the fourth step is performed. From the first growth inhibition curve for each substance, the microorganism growth rate corresponding to the saccharification solution rate of each mixed saccharification solution is obtained. Next, the microbial growth rate corresponding to the saccharification solution rate of each mixed saccharification solution is calculated by multiplying the microbial growth rate of each growth inhibitory substance in the mixed saccharification solution having the same saccharification solution rate.
次に、第８の工程として、前記第７の工程で得られた各混合糖化溶液における前記糖化溶液率と前記微生物増殖率との関係から、第２の増殖阻害曲線を作成する。 Next, as an eighth step, a second growth inhibition curve is created from the relationship between the saccharified solution rate and the microbial growth rate in each mixed saccharified solution obtained in the seventh step.
次に、第９の工程として、前記第８の工程で得られた前記第２の増殖阻害曲線から、所望の微生物増殖率に対応する糖化溶液率を求める。 Next, as a ninth step, a saccharified solution rate corresponding to a desired microbial growth rate is obtained from the second growth inhibition curve obtained in the eighth step.
次に、第１０の工程として、前記第９の工程で得られた前記糖化溶液率となるように前記糖化溶液を本発明のバイオエタノール製造で用いる前記糖液によって希釈することにより、混合糖化溶液を得る。 Next, as a tenth step, the saccharified solution is diluted with the saccharified solution used in the bioethanol production of the present invention so as to achieve the saccharified solution rate obtained in the ninth step, thereby obtaining a mixed saccharified solution. Get.
以上により、前記増殖阻害物質を除去することなく発酵の際に微生物の増殖阻害を回避することができる前記混合糖化溶液を得ることができる。 As described above, the mixed saccharified solution that can avoid the growth inhibition of microorganisms during fermentation can be obtained without removing the growth inhibitory substance.
前記複数種類の増殖阻害物質に着目して前記糖化溶液率を決定する方法によれば、１種類の増殖阻害物質のみに着目する方法と比較して、該糖化溶液率をより正確に決定することができる。 According to the method of determining the saccharification solution rate by paying attention to the plurality of types of growth inhibitory substances, the saccharification solution rate can be determined more accurately than the method of focusing on only one type of growth inhibitory substance. Can do.
ところで、上記２つの方法では、前記糖液の増殖阻害物質濃度は、前記糖化溶液の増殖阻害物質濃度よりも非常に小さく実質的に無視できるのでゼロとみなして、該糖化溶液の増殖阻害物質濃度のみを考慮して前記糖化溶液率を決定する。そこで、次のようにして、前記糖化溶液の増殖阻害物質濃度だけでなく、前記糖液の増殖阻害物質濃度も考慮するようにしてよい。 By the way, in the above two methods, the growth inhibitor concentration of the saccharified solution is much smaller than the growth inhibitor concentration of the saccharified solution and can be substantially ignored. The saccharification solution rate is determined considering only the above. Therefore, not only the growth inhibitor concentration of the saccharified solution but also the growth inhibitor concentration of the sugar solution may be considered as follows.
まず、前記糖液に含まれる増殖阻害物質濃度を測定する。次に、前記第１の工程において、前記試験用糖液として前記糖液を用いて、該糖液に前記増殖阻害物質を添加した複数の培養液を用いて前記微生物を培養する。そして、前記糖液について測定された前記増殖阻害物質濃度と、前記試験用糖液としての前記糖液に添加された増殖阻害物質濃度との和を、前記培養液に含まれる増殖阻害物質濃度として、該増殖阻害物質濃度毎の微生物増殖数を測定する。 First, the growth inhibitor concentration contained in the sugar solution is measured. Next, in the first step, the microorganism is cultured using a plurality of culture solutions obtained by adding the growth inhibitory substance to the sugar solution using the sugar solution as the test sugar solution. Then, the sum of the growth inhibitory substance concentration measured for the sugar solution and the growth inhibitory substance concentration added to the sugar solution as the test sugar solution is used as the growth inhibitory substance concentration contained in the culture solution. Then, the number of microbial growth for each concentration of the growth inhibitory substance is measured.
その後は、前記糖化溶液の増殖阻害物質濃度のみを考慮する上記の方法と同様にして、前記第２の工程〜前記第９の工程を行って前記糖化溶液率を決定する。次に、前記第１０の工程において、前記第９の工程で得られた前記糖化溶液率となるように前記糖化溶液を前記糖液によって希釈することにより前記混合糖化溶液を得る。 Thereafter, in the same manner as in the above method considering only the growth inhibitor concentration of the saccharified solution, the saccharified solution rate is determined by performing the second to ninth steps. Next, in the tenth step, the mixed saccharified solution is obtained by diluting the saccharified solution with the saccharified solution so as to achieve the saccharified solution rate obtained in the ninth step.
前記糖化溶液の増殖阻害物質濃度と前記糖液の増殖阻害物質濃度との両方を考慮する方法によれば、該糖化溶液の増殖阻害物質濃度のみを考慮する方法と比較して、前記糖化溶液率をさらに正確に決定することができる。 According to the method that considers both the growth inhibitory substance concentration of the saccharified solution and the growth inhibitory substance concentration of the sugar solution, the saccharified solution rate is higher than the method that considers only the growth inhibitory substance concentration of the saccharified solution. Can be determined more accurately.
また、前記糖液に含まれる前記増殖阻害物質としては、例えば、有機酸、アルデヒド基を有する化合物、フェノール基を有する化合物、ケトン基を有する化合物、メラノイジンからなる群から選択される１又は複数の化合物を挙げることができる。 In addition, as the growth inhibitory substance contained in the sugar solution, for example, one or more selected from the group consisting of organic acids, compounds having an aldehyde group, compounds having a phenol group, compounds having a ketone group, and melanoidins A compound can be mentioned.
次に、本発明の実施の形態についてさらに詳しく説明する。本実施形態のバイオエタノールの製造方法は、リグノセルロース系バイオマスを前処理した前処理物を糖化酵素によって糖化処理し、得られた糖化溶液を所定のｐＨで微生物によって発酵させてエタノールを生成させることによりバイオエタノールを製造する方法である。 Next, embodiments of the present invention will be described in more detail. In the bioethanol production method of the present embodiment, a pretreated product obtained by pretreating lignocellulosic biomass is saccharified with a saccharifying enzyme, and the resulting saccharified solution is fermented with a microorganism at a predetermined pH to produce ethanol. To produce bioethanol.
本実施形態のバイオエタノールの製造方法では、まず、リグノセルロース系バイオマスを前処理することにより、リグノセルロース系バイオマスからリグニンを解離し、又はリグノセルロース系バイオマスを膨潤させた前処理物を得る。前記リグノセルロース系バイオマスとしては、木材、稲藁、麦藁、バガス、竹、トウモロコシの茎や葉や芯、パルプ、及びこれらから生じる廃棄物、例えば古紙等を挙げることができる。前記前処理としては、湿式粉砕、乾式粉砕、爆砕、水蒸気処理、酸又はアルカリによる処理等を挙げることができる。 In the bioethanol production method of the present embodiment, first, lignocellulosic biomass is pretreated to obtain a pretreated product that dissociates lignin from lignocellulosic biomass or swells lignocellulosic biomass. Examples of the lignocellulosic biomass include wood, rice straw, wheat straw, bagasse, bamboo, corn stalks, leaves and cores, pulp, and wastes such as waste paper. Examples of the pretreatment include wet pulverization, dry pulverization, explosion, steam treatment, treatment with acid or alkali, and the like.
次に、得られた前処理物を酵素を用いて糖化処理することにより、糖化溶液を得る。前記糖化処理は、例えば、前記前処理物に糖化酵素を添加して撹拌することにより行うことができる。得られた糖化溶液は、２０〜３００ｇ／Ｌ、好ましくは５０〜２００ｇ／Ｌの糖を含むとともに、副生成物として前記微生物の増殖を阻害する増殖阻害物質を含んでいる。 Next, the saccharification solution is obtained by saccharifying the obtained pre-processed product using an enzyme. The saccharification treatment can be performed, for example, by adding a saccharification enzyme to the pretreated product and stirring. The obtained saccharified solution contains 20 to 300 g / L, preferably 50 to 200 g / L of sugar, and also contains a growth inhibitory substance that inhibits the growth of the microorganism as a by-product.
前記糖としては、グルコース、キシロース、アラビノースを挙げることができる。 Examples of the sugar include glucose, xylose, and arabinose.
前記増殖阻害物質としては、例えば、酢酸、蟻酸、ｐ−クマル酸、フェルラ酸、安息香酸等の有機酸、フルフラール、５−ヒドロキシメチルフルフラール（ＨＭＦ）、バニリン、シリンガアルデヒド、４−ヒドロキシアセトフェノン等のアルデヒド基を有する化合物、フェノール、グアイアコール等のフェノール基を有する化合物、ケトン基を有する化合物、メラノイジンからなる群から選択される１又は複数の化合物を挙げることができる。 Examples of the growth inhibitory substance include organic acids such as acetic acid, formic acid, p-coumaric acid, ferulic acid, benzoic acid, furfural, 5-hydroxymethylfurfural (HMF), vanillin, syringaldehyde, 4-hydroxyacetophenone, etc. Or a compound having a phenol group such as phenol or guaiacol, a compound having a ketone group, or one or more compounds selected from the group consisting of melanoidins.
次に、前記糖化溶液を、該糖化溶液よりも増殖阻害物質濃度が小さい糖液によって該増殖阻害物質濃度が低下するように希釈することにより、混合糖化溶液を得る。前記糖液としては、例えば、サトウキビの絞り汁を濃縮させて結晶を分離した後に副産物として生じる廃糖蜜（モラセス）、サトウキビの絞り汁、該サトウキビの絞り汁の濃縮液、米を糖化した液、麦を糖化した液、トウモロコシを糖化した液等を用いることができる。 Next, the saccharified solution is diluted with a sugar solution having a growth inhibitory substance concentration lower than that of the saccharified solution so that the growth inhibitory substance concentration is lowered, thereby obtaining a mixed saccharified solution. As the sugar liquid, for example, molasses produced as a by-product after concentrating sugarcane juice and separating crystals, sugarcane juice, concentrated sugarcane juice, saccharified rice, A liquid obtained by saccharification of wheat, a liquid obtained by saccharification of corn, or the like can be used.
前記希釈は、例えば、前記混合糖化溶液において、酢酸の非解離分の濃度が７００ｍｇ／Ｌ以下となり、フルフラールの濃度が７００ｍｇ／Ｌ以下となり、且つ該ｐ−クマル酸の濃度が５００ｍｇ／Ｌ以下となるように行う。 For example, in the mixed saccharified solution, the dilution is such that the concentration of non-dissociated acetic acid is 700 mg / L or less, the concentration of furfural is 700 mg / L or less, and the concentration of p-coumaric acid is 500 mg / L or less. Do so.
得られた前記混合糖化溶液は、前記糖液によって希釈されたことにより、前記増殖阻害物質濃度が前記糖化溶液よりも低下されている。 The obtained mixed saccharified solution is diluted with the saccharified solution, so that the growth inhibitor concentration is lower than that of the saccharified solution.
次に、前記混合糖化溶液を所定のｐＨで微生物によって発酵させることによりエタノールを生成させてエタノール水溶液を得た後、該エタノール水溶液を蒸留して濃縮することにより、バイオエタノールを製造する。前記微生物としては、例えば、ピキア属酵母又は他の酵母、ザイモモナス、ザイモバクター、コリネバクテリウム、大腸菌、これらの微生物の遺伝子改変体を挙げることができる。 Next, ethanol is produced by fermenting the mixed saccharified solution with microorganisms at a predetermined pH to obtain an ethanol aqueous solution, and then the ethanol aqueous solution is distilled and concentrated to produce bioethanol. Examples of the microorganism include Pichia yeast or other yeasts, Zymomonas, Zymobacter, Corynebacterium, Escherichia coli, and genetic variants of these microorganisms.
前記混合糖化溶液を発酵させる際、該混合糖化溶液は増殖阻害物質濃度が低下されているので、前記増殖阻害物質によって前記微生物の増殖が阻害されることを低減することができる。 When the mixed saccharified solution is fermented, the concentration of the growth inhibitory substance in the mixed saccharified solution is lowered, so that the growth inhibition of the microorganism by the growth inhibitory substance can be reduced.
したがって、本実施形態の製造方法によれば、増殖阻害物質を濾過、中和、吸着等の除去を行うことなく、前記発酵の際に前記微生物の増殖阻害を回避することができ、発酵効率を向上することができる。 Therefore, according to the production method of the present embodiment, growth inhibition of the microorganism can be avoided during the fermentation without performing filtration, neutralization, adsorption or the like removal of the growth inhibitory substance, and fermentation efficiency can be reduced. Can be improved.
また、前記糖液として、前記廃糖蜜を用いる場合には、低コストで前記増殖阻害物質濃度を低下させることができる。 In addition, when the molasses is used as the sugar solution, the concentration of the growth inhibitory substance can be reduced at a low cost.
次に、図１を参照しながら、前記微生物の増殖阻害を回避することができる糖化溶液率を備える混合糖化溶液の製造方法について説明する。前記糖化溶液率は、前記糖化溶液に含まれる増殖阻害物質α，β，γ，δ，ε，…のうちの１種類又は複数種類の増殖阻害物質の濃度に着目して決定することができる。 Next, a method for producing a mixed saccharification solution having a saccharification solution rate that can avoid the growth inhibition of the microorganism will be described with reference to FIG. The saccharification solution ratio can be determined by paying attention to the concentration of one or a plurality of types of growth inhibitory substances among the growth inhibitory substances α, β, γ, δ, ε,.
はじめに、前記糖化溶液に含まれる増殖阻害物質α，β，γ，δ，ε，…のうちの１種の増殖阻害物質αに着目する方法について説明する。前記糖化溶液として稲藁を糖化した溶液を用い、前記糖液としてサトウキビの廃糖蜜を用いる。前記廃糖蜜は、増殖阻害物質を含むものの、その濃度は、前記稲藁を糖化した溶液と比較して非常に小さく実質的に無視できるので、ゼロとみなすことができる。 First, a method of paying attention to one type of growth inhibitory substance α among the growth inhibitory substances α, β, γ, δ, ε,... Contained in the saccharified solution will be described. A solution obtained by saccharifying rice straw is used as the saccharification solution, and sugarcane waste molasses is used as the sugar solution. Although the waste molasses contains a growth inhibitory substance, the concentration thereof is very small compared to the solution obtained by saccharifying rice straw and can be substantially ignored, and thus can be regarded as zero.
まず、糖としてのグルコース及びキシロースと、微生物の栄養源としてのペプトンとを水に溶解することにより、試験用糖液を調製する。前記試験用糖液は、糖濃度が１００〜１５０ｇ／Ｌである一方、増殖阻害物質を全く含んでおらず、前記糖液を実験的に調製したものである。 First, a test sugar solution is prepared by dissolving glucose and xylose as sugars and peptone as a nutrient source of microorganisms in water. The sugar solution for test has a sugar concentration of 100 to 150 g / L, but does not contain any growth inhibitory substance, and is prepared experimentally.
次に、前記試験用糖液に、増殖阻害物質αとしての例えばフルフラールを、それぞれ異なる増殖阻害物質濃度Ｃ１，Ｃ２，Ｃ３，…で添加することにより、複数の培養液を調製する。次に、得られた各培養液を用いて前記微生物を培養する。前記培養は、例えば、前記培養液に、微生物として酵母を添加し、ｐＨ４〜７で温度３０℃で２０時間撹拌、振盪することにより行うことができる。前記酵母は、例えば、濁度０．０５〜５の範囲で前記培養液に添加することができる。 Next, a plurality of culture solutions are prepared by adding, for example, furfural as a growth inhibitory substance α to the test sugar solution at different growth inhibitory substance concentrations C 1 , C 2 , C 3 ,. Next, the microorganisms are cultured using the obtained culture solutions. The culture can be performed, for example, by adding yeast as a microorganism to the culture solution and stirring and shaking at pH 4 to 7 at a temperature of 30 ° C. for 20 hours. The said yeast can be added to the said culture solution in the range of turbidity 0.05-5, for example.
そして、各培養液に含まれる増殖阻害物質αの濃度Ｃ１，Ｃ２，Ｃ３，…毎に、前記培養によって増殖された前記微生物の数を測定して、微生物増殖数Ｎα１，Ｎα２，Ｎα３，…とする（ステップ（以下、ＳＴと略記する）１）。前記微生物の数の測定は、前記培養液の濁度を測定することにより行うことができ、該濁度は前記微生物の数に比例する。 Then, for each concentration C 1 , C 2 , C 3 ,... Of the growth inhibitory substance α contained in each culture solution, the number of the microorganisms grown by the culture is measured, and the microorganism growth number N α1 , N α2 , N α3 ,... (Step (hereinafter abbreviated as ST) 1). The number of microorganisms can be measured by measuring the turbidity of the culture solution, and the turbidity is proportional to the number of microorganisms.
次に、増殖阻害物質を全く添加しない以外は、ＳＴ１と全く同一にして前記微生物を培養し、該無添加の試験用糖液における微生物増殖数を測定して標準微生物増殖数Ｎ０とする（ＳＴ２）。 Next, the microorganism is cultured in exactly the same manner as ST1 except that no growth inhibitor is added, and the number of microorganism growth in the additive-free test sugar solution is measured to obtain the standard microorganism growth number N 0 ( ST2).
次に、ＳＴ１で得られた微生物増殖数Ｎα１，Ｎα２，Ｎα３，…をＳＴ２で得られた標準微生物増殖数Ｎ０で除すことにより、各増殖阻害物質濃度Ｃ１，Ｃ２，Ｃ３，…における微生物増殖率Ｇα１（Ｇα１＝Ｎα１／Ｎ０），Ｇα２，Ｇα３，…を算出する（ＳＴ３）。 Next, by dividing the microorganism growth number N α1 , N α2 , N α3 ,... Obtained in ST1 by the standard microorganism growth number N 0 obtained in ST2, each growth inhibitor concentration C 1 , C 2 , C 3, microbial growth rate G [alpha] 1 in ... (G α1 = N α1 / N 0), G α2, G α3, calculates a ... (ST3).
次に、各増殖阻害物質濃度Ｃ１，Ｃ２，Ｃ３，…と、ＳＴ３で得られた微生物増殖率Ｇα１，Ｇα２，Ｇα３，…との関係から、図２（ａ）に示す第１の増殖阻害曲線としての増殖阻害曲線Ｌα１を作成する（ＳＴ４）。図２（ａ）に示すグラフは、横軸が増殖阻害物質濃度を示し、縦軸が微生物増殖率を示す。微生物増殖率は、糖化溶液における微生物増殖係数を表し、１に近づくほど微生物が増殖し易いことを示している。 Next, from the relationship between each growth inhibitory substance concentration C 1 , C 2 , C 3 ,... And the microorganism growth rate G α1 , G α2 , G α3,. A growth inhibition curve L α1 as a first growth inhibition curve is created (ST4). In the graph shown in FIG. 2A, the horizontal axis indicates the growth inhibitor concentration, and the vertical axis indicates the microorganism growth rate. The microbial growth rate represents the microbial growth coefficient in the saccharified solution, and indicates that the closer to 1, the easier the microorganisms grow.
次に、本実施形態のバイオエタノール製造方法に用いる前記糖化溶液に含まれる阻害物質濃度Ｃαａを測定する（ＳＴ５）。前記測定には、高速液体クロマトグラフィーを用いることができる。 Next, the inhibitor concentration C αa contained in the saccharification solution used in the bioethanol production method of the present embodiment is measured (ST5). For the measurement, high performance liquid chromatography can be used.
次に、図２（ｂ）に示すように、ＳＴ４で得られた増殖阻害曲線Ｌα１から、前記糖化溶液に含まれる増殖阻害物質濃度Ｃαａに対応する微生物増殖率Ｇαａを求める（ＳＴ６）。 Next, as shown in FIG. 2 (b), the microbial growth rate G αa corresponding to the growth inhibitor concentration C αa contained in the saccharification solution is obtained from the growth inhibition curve L α1 obtained in ST4 (ST6). .
次に、全く希釈していない糖化溶液の糖化溶液率をＳａ＝１とする。そして、全く希釈していない糖化溶液を糖液によってそれぞれ異なる添加量で希釈することにより得られる各混合糖化溶液について、増殖阻害曲線Ｌα１から、各混合糖化溶液の糖化溶液率Ｓｂ，Ｓｃ，Ｓｄ，…に対応する各増殖阻害物質濃度Ｃαｂ，Ｃαｃ，Ｃαｄ…を求める。 Next, the saccharified solution ratio of a saccharified solution that has not been diluted at all is set to S a = 1. And about each mixed saccharified solution obtained by diluting the saccharified solution which is not diluted at all with each addition amount with a saccharified solution, from the growth inhibition curve Lα1 , the saccharified solution ratios S b and S c of each mixed saccharified solution are obtained. , S d ,... Corresponding to each growth inhibitory substance concentration C αb , C αc , C αd .
上述したように前記糖液における増殖阻害物質濃度はゼロとみなされるので、例えば、糖化溶液率をＳｂ＝０．７５とするとき、この糖化溶液率は増殖阻害物質濃度Ｃαａの前記糖化溶液０．７５質量部を前記糖液０．２５質量部で希釈することを意味する。糖化溶液率Ｓｂ＝０．７５のときに得られる混合糖化溶液の増殖阻害物質濃度Ｃαｂは０．７５Ｃαａ（＝０．７５×Ｃαａ）である。 As described above, since the growth inhibitor concentration in the sugar solution is regarded as zero, for example, when the saccharification solution rate is S b = 0.75, the saccharification solution rate is the saccharification solution having the growth inhibitor concentration C αa. It means that 0.75 part by mass is diluted with 0.25 part by mass of the sugar solution. Growth inhibitor concentration C .alpha.b mixed saccharified solution obtained when saccharification solution ratio S b = 0.75 is 0.75C αa (= 0.75 × C αa ).
同様に、糖化溶液率をＳｂ＝０．５として前記糖化溶液０．５質量部を前記糖液０．５質量部で希釈するとき、得られる混合糖化溶液の増殖阻害物質濃度Ｃαｃは０．５Ｃαａ（＝０．５×Ｃαａ）である。同様に、糖化溶液率をＳｄ＝０．２５として前記糖化溶液０．２５質量部を前記糖液０．７５質量部で希釈するとき、得られる混合糖化溶液の増殖阻害物質濃度Ｃαｄは０．２５Ｃαａ（＝０．２５×Ｃαａ）である。 Similarly, when the saccharified solution ratio is S b = 0.5 and 0.5 part by mass of the saccharified solution is diluted with 0.5 part by mass of the saccharified liquid, the growth inhibitor concentration C αc of the obtained mixed saccharified solution is 0. .5C is αa (= 0.5 × C αa) . Similarly, when the saccharified solution ratio is S d = 0.25 and 0.25 part by mass of the saccharified solution is diluted with 0.75 part by mass of the saccharified liquid, the growth inhibitor concentration C αd of the obtained mixed saccharified solution is 0. .25C an αa (= 0.25 × C αa) .
すなわち、所定の糖化溶液率となるように前記糖化溶液を前記糖液によって希釈することにより得られる混合糖化溶液の増殖阻害物質濃度は、該糖化溶液の増殖阻害物質濃度Ｃαａに該糖化溶液率を乗じた値と等しくなる。 That is, the growth inhibitory substance concentration of the mixed saccharified solution obtained by diluting the saccharified solution with the saccharified solution so as to obtain a predetermined saccharified solution ratio is the growth inhibitory substance concentration C αa of the saccharified solution. It is equal to the value multiplied by.
そして、図２（ｂ）において、ＳＴ４で得られた増殖阻害曲線Ｌα１から、糖化溶液率がＳａ，Ｓｂ，Ｓｃ，Ｓｄ，…であって増殖阻害物質濃度がＣαａ，Ｃαｂ，Ｃαｃ，Ｃαｄ，…である各混合糖化溶液における微生物増殖率Ｇαａ，Ｇαｂ，Ｇαｃ，Ｇαｄ，…を求める（ＳＴ７）。 2B, from the growth inhibition curve L α1 obtained in ST4, the saccharification solution rate is S a , S b , S c , S d ,... And the growth inhibitor concentration is C αa , C αb, C αc, C αd, microbial growth rate G .alpha.a in each mixture saccharified solution is ..., G αb, G αc, G αd, seek ... (ST7).
次に、図２（ｂ）の増殖阻害曲線Ｌα１の横軸について、増殖阻害物質濃度Ｃαａ，Ｃαｂ，Ｃαｃ，Ｃαｄ，…を糖化溶液率Ｓａ，Ｓｂ，Ｓｃ，Ｓｄ，…に読み替えることにより、該糖化溶液率Ｓａ，Ｓｂ，Ｓｃ，Ｓｄ，…と、微生物増殖率Ｇαａ，Ｇαｂ，Ｇαｃ，Ｇαｄ，…との関係から、図３（ａ）に示す第２の増殖阻害曲線としての総合増殖阻害曲線Ｌα２を作成する（ＳＴ８）。 Next, the horizontal axis of the growth inhibition curve L [alpha] 1 in FIG. 2 (b), the growth inhibitor concentration C αa, C αb, C αc , C αd, ... saccharified solution ratio S a, S b, S c , S d, the replaced things ..., sugar solution ratio S a, S b, S c , S d, ... and, microbial growth rate G αa, G αb, G αc , G αd, ... from the relationship between FIG. 3 A total growth inhibition curve L α2 is created as a second growth inhibition curve shown in (a) (ST8).
次に、図３（ｂ）に示すように、ＳＴ８で得られた総合増殖阻害曲線Ｌα２から、所望の微生物増殖率Ｇｘに対応する糖化溶液率Ｓｘを求める（ＳＴ９）。ここで、前記所望の微生物増殖率Ｇｘは、前記糖液の価格、必要な微生物量、微生物増殖に要する時間、エネルギー及びコスト等を考慮して決定される。 Next, as shown in FIG. 3B, the saccharification solution rate S x corresponding to the desired microbial growth rate G x is obtained from the total growth inhibition curve L α2 obtained in ST8 (ST9). Here, the desired microorganism growth rate Gx is determined in consideration of the price of the sugar solution, the amount of microorganisms required, the time required for microorganism growth, energy, cost, and the like.
次に、ＳＴ９で得られた糖化溶液率Ｓｘとなるように、前記糖化溶液を前記糖液によって希釈することにより、混合糖化溶液を得る（ＳＴ１０）。 Next, the saccharified solution is diluted with the saccharified solution so that the saccharified solution rate Sx obtained in ST9 is obtained, thereby obtaining a mixed saccharified solution (ST10).
以上により、増殖阻害物質を除去することなく発酵の際に微生物の増殖阻害を回避することができる前記混合糖化溶液を得ることができる。前記混合糖化溶液を発酵するとき、前記微生物の増殖率はＧｘとなる。 As described above, the mixed saccharified solution that can avoid the growth inhibition of microorganisms during fermentation can be obtained without removing the growth inhibitory substance. When fermenting the mixed saccharified solution, growth rate of the microorganism becomes G x.
ところで、前記増殖阻害物質αが酢酸、蟻酸等の弱酸の有機酸である場合には、該増殖阻害物質の酸解離定数を考慮して、前記糖化溶液に含まれる阻害物質濃度Ｃαａを補正することにより、増殖阻害物質濃度Ｃαａをより正確に求めることができる。 When the growth inhibitor α is a weak acid organic acid such as acetic acid or formic acid, the inhibitor concentration C αa contained in the saccharification solution is corrected in consideration of the acid dissociation constant of the growth inhibitor. Thus, the growth inhibitory substance concentration C αa can be obtained more accurately.
前記補正は、前記ＳＴ５で測定された前記糖化溶液に含まれる阻害物質濃度Ｃαａに、該糖化溶液のｐＨにおける非解離度を乗ずることにより行うことができる。前記非解離度は、次の式に従うことが知られている。 The correction can be performed by multiplying the inhibitor concentration C αa contained in the saccharified solution measured in ST5 by the degree of non-dissociation at the pH of the saccharified solution. It is known that the degree of non-dissociation follows the following formula.
前記式において、ｐＫａは、前記糖化溶液における前記増殖阻害物質の酸解離定数である。例えば、酢酸の酸解離定数ｐＫａは４．５６であり、蟻酸の酸解離定数ｐＫａは３．５５である。 In the above formula, pKa is the acid dissociation constant of the growth inhibitor in the saccharification solution. For example, the acid dissociation constant pKa of acetic acid is 4.56, and the acid dissociation constant pKa of formic acid is 3.55.
また、次のようにすることにより、前記糖化溶液に含まれる１種類の増殖阻害物質αのみではなく、複数の増殖阻害物質α，βに着目して前記糖化溶液率を決定することも可能である。以下、増殖阻害物質αについての説明は省略し、主に、増殖阻害物質βについて説明する。 In addition, the saccharification solution ratio can be determined by focusing on not only one kind of growth inhibitory substance α contained in the saccharification solution but also a plurality of growth inhibitory substances α and β by doing as follows. is there. Hereinafter, description of the growth inhibitory substance α will be omitted, and the growth inhibitory substance β will be mainly described.
まず、ＳＴ１において、増殖阻害物質αのときと同様にして、増殖阻害物質βとしての例えばフェルラ酸を、それぞれ異なる増殖阻害物質濃度Ｃ１，Ｃ２，Ｃ３，…で添加した複数の培養液を調製し、前記微生物を培養する。そして、各培養液に含まれる増殖阻害物質濃度Ｃ１，Ｃ２，Ｃ３，…毎の微生物増殖数Ｎβ１，Ｎβ２，Ｎβ３，…を測定する。 First, in ST1, as in the case of the growth inhibitory substance α, a plurality of culture solutions to which, for example, ferulic acid as a growth inhibitory substance β is added at different growth inhibitory substance concentrations C 1 , C 2 , C 3 ,. And culturing the microorganism. Then, the number of growth of microorganisms N β1 , N β2 , N β3 ,... For each growth inhibitor concentration C 1 , C 2 , C 3 ,.
次に、ＳＴ２において、標準微生物増殖数Ｎ０を測定する。 Then, in ST2, it measures the standard microbial growth number N 0.
次に、ＳＴ３において、増殖阻害物質αのときと同様にして、各培養液の増殖阻害物質濃度Ｃ１，Ｃ２，Ｃ３，…毎の微生物増殖率Ｇβ１（Ｇβ１＝Ｎβ１／Ｎ０），Ｇβ２，Ｇβ３，…を算出する。 Next, in ST3, as in the case of the growth inhibitory substance α, the microorganism growth rate G β1 (G β1 = N β1 / N for each growth inhibitor concentration C 1 , C 2 , C 3 ,. 0 ), Gβ2 , Gβ3 ,...
次に、ＳＴ４において、増殖阻害物質αのときと同様にして、各増殖阻害物質濃度Ｃ１，Ｃ２，Ｃ３，…と各微生物増殖率Ｇβ１，Ｇβ２，Ｇβ３，…との関係から、図４（ａ）に示す増殖阻害物質βの増殖阻害曲線Ｌβ１を作成する。 Next, in ST4, similarly to the case of the growth inhibitory substance α, the relationship between the growth inhibitory substance concentrations C 1 , C 2 , C 3 ,... And the respective microorganism growth rates G β1 , G β2 , G β3,. From this, a growth inhibition curve L β1 of the growth inhibitory substance β shown in FIG.
次に、ＳＴ５において、増殖阻害物質αのときと同様にして、前記糖化溶液に含まれる増殖阻害物質βの濃度Ｃβａを測定する。 Next, in ST5, similarly to the case of the growth inhibitory substance α, the concentration Cβa of the growth inhibitory substance β contained in the saccharification solution is measured.
次に、ＳＴ６において、増殖阻害物質αのときと同様にして、図４（ｂ）に示すように、前記糖化溶液に含まれる増殖阻害物質濃度Ｃβａに対応する微生物増殖率Ｇβａを求める。 Next, in ST6, as in the case of the growth inhibitory substance α, as shown in FIG. 4B, the microbial growth rate Gβa corresponding to the growth inhibitory substance concentration Cβa contained in the saccharification solution is obtained.
次に、ＳＴ７において、まず、増殖阻害物質αのときと同様にして、増殖阻害曲線Ｌβ１から、糖化溶液率がＳａ，Ｓｂ，Ｓｃ，Ｓｄ，…であって増殖阻害物質濃度がＣβａ，Ｃβｂ，Ｃβｃ，Ｃβｄ，…である各混合糖化溶液における微生物増殖率Ｇβａ，Ｇβｂ，Ｇβｃ，Ｇβｄ，…を求める。 Next, in ST7, first, as in the case of the growth inhibitory substance α, from the growth inhibition curve Lβ1 , the saccharification solution ratio is S a , S b , S c , S d ,. Are determined as microbial growth rates G βa , G βb , G βc , G βd ,... In each of the mixed saccharified solutions where C βa , C βb , C βc , C βd,.
次に、前記糖化溶液率が同一である混合糖化溶液における増殖阻害物質α，βの各微生物増殖率をそれぞれ乗じることにより、各混合糖化溶液の糖化溶液率に対応する微生物増殖率を算出する。例えば、糖化溶液率Ｓａにおける微生物増殖率Ｇαβａは、Ｇαａ×Ｇβａにより算出され、糖化溶液率Ｓｂにおける微生物増殖率Ｇαβｂは、Ｇαｂ×Ｇβｂにより算出される。 Next, the microbial growth rate corresponding to the saccharified solution rate of each mixed saccharified solution is calculated by multiplying each microbial saccharified growth rate of the growth inhibitory substances α and β in the mixed saccharified solution having the same saccharified solution rate. For example, microbial growth rate G Arufabetaei in saccharification solution ratio S a is calculated by G αa × G βa, microbial growth rate G Arufabetabi in saccharification solution ratio S b is calculated by G αb × G βb.
次に、ＳＴ８において、得られた各混合糖化溶液における糖化溶液率Ｓａ，Ｓｂ，Ｓｃ，Ｓｄ，…と微生物増殖率Ｇαβａ，Ｇαβｂ，Ｇαβｃ，Ｇαβｄ，…との関係から、図５（ａ）に示す総合増殖阻害曲線Ｌαβ２を作成する。 Next, in ST8, the relationship between the saccharified solution ratios S a , S b , S c , S d ,... And the microbial growth rates G αβa , G αβb , G αβc , G αβd,. From this, an overall growth inhibition curve L αβ2 shown in FIG.
次に、ＳＴ９において、図５（ｂ）に示すように、総合増殖阻害曲線Ｌαβ２から、所望の微生物増殖率Ｇｘに対応する糖化溶液率Ｓｘを求める。 Next, in ST9, as shown in FIG. 5B, a saccharification solution rate S x corresponding to a desired microbial growth rate G x is obtained from the total growth inhibition curve L αβ2 .
次に、ＳＴ１０において、ＳＴ９で得られた糖化溶液率Ｓｘとなるように、前記糖化溶液を前記糖液によって希釈することにより、混合糖化溶液を得る。 Next, in ST10, the saccharified solution is diluted with the saccharified solution so that the saccharified solution rate Sx obtained in ST9 is obtained, thereby obtaining a mixed saccharified solution.
上記複数種類の増殖阻害物質α，βに着目して糖化溶液率Ｓｘを決定する方法によれば、１種類の増殖阻害物質αのみに着目する方法と比較して、該糖化溶液率Ｓｘをより正確に決定することができる。 The plurality of types of growth inhibitors alpha, according to the method of determining the glycosylated solution ratio S x in view of the beta, compared with a method of focusing only on one type of growth inhibitor alpha, sugar solution ratio S x Can be determined more accurately.
また、上記２つの方法では、前記糖液の増殖阻害物質濃度は、前記糖化溶液の増殖阻害物質濃度よりも非常に小さく実質的に無視できるのでゼロとみなして、該糖化溶液の増殖阻害物質濃度のみを考慮して前記糖化溶液率Ｓｘを決定している。これに対して、次のようにして、前記糖化溶液の増殖阻害物質濃度だけでなく、前記糖液の増殖阻害物質濃度も考慮するようにしてもよい。ここでは、１種類の増殖阻害物質αのみに着目する方法を用いて説明する。 In the above two methods, the growth inhibitor concentration of the saccharified solution is much smaller than the growth inhibitor concentration of the saccharified solution and can be substantially ignored. The saccharified solution rate S x is determined only by considering On the other hand, not only the growth inhibitor concentration of the saccharified solution but also the growth inhibitor concentration of the sugar solution may be considered as follows. Here, a description will be made using a method that focuses on only one type of growth inhibitor α.
まず、ＳＴ１において、前記試験用糖液として前記糖液を用いて、該糖液に前記増殖阻害物質αをそれぞれ異なる増殖阻害物質濃度Ｃ１，Ｃ２，Ｃ３，…で添加した複数の培養液を用いて前記微生物を培養する。次に、前記糖液に含まれる増殖阻害物質αの濃度Ｃα０を測定する。 First, in ST1, using the sugar solution as the test sugar solution, a plurality of cultures in which the growth inhibitory substance α is added to the sugar solution at different growth inhibitory substance concentrations C 1 , C 2 , C 3 ,. The microorganism is cultured using the liquid. Next, the concentration C α0 of the growth inhibitor α contained in the sugar solution is measured.
次に、測定された前記増殖阻害物質濃度Ｃα０と、前記試験用糖液としての前記糖液に添加された増殖阻害物質αの濃度Ｃ１，Ｃ２，Ｃ３，…との和を、前記培養液に含まれる増殖阻害物質濃度Ｃ´１（Ｃ´１＝Ｃα０＋Ｃ１），Ｃ´２，Ｃ´３，…とする。そして、前記培養液に含まれる増殖阻害物質濃度Ｃ´１，Ｃ´２，Ｃ´３，…毎の微生物増殖数Ｎα１，Ｎα２，Ｎα３，…を測定する。 Next, the sum of the measured growth inhibitory substance concentration C α0 and the concentration C 1 , C 2 , C 3 ,... Of the growth inhibitory substance α added to the sugar liquid as the test sugar liquid, It is assumed that the growth inhibitor concentration C ′ 1 (C ′ 1 = C α0 + C 1 ), C ′ 2 , C ′ 3 ,. Then, the growth inhibitory substance concentrations C ′ 1 , C ′ 2 , C ′ 3 ,... Contained in the culture solution are measured for the number of microorganism growth N α1 , N α2 , N α3,.
その後は、前記糖化溶液の増殖阻害物質濃度のみを考慮する方法におけるＳＴ２〜ＳＴ９と全く同一にして、前記糖化溶液率Ｓｘを決定する。次に、ＳＴ１０において、ＳＴ９で得られた糖化溶液率Ｓｘとなるように前記糖化溶液を前記糖液によって希釈することにより前記混合糖化溶液を得る。 Thereafter, the saccharification solution ratio Sx is determined in the same manner as ST2 to ST9 in the method that considers only the growth inhibitor concentration of the saccharification solution. Next, in ST10, the mixed saccharified solution is obtained by diluting the saccharified solution with the sugar solution so that the saccharified solution rate Sx obtained in ST9 is obtained.
前記糖化溶液の増殖阻害物質濃度Ｃαａと前記糖液の増殖阻害物質濃度Ｃα０との両方を考慮する方法によれば、該糖化溶液の増殖阻害物質濃度Ｃαａのみを考慮する方法と比較して、前記糖化溶液率Ｓｘをさらに正確に決定することができる。 According to the method considering both the growth inhibitory substance concentration C αa of the saccharified solution and the growth inhibitory substance concentration C α0 of the sugar solution, it is compared with the method considering only the growth inhibitory substance concentration C αa of the saccharified solution. Thus, the saccharification solution rate S x can be determined more accurately.
また、本実施形態では、前記微生物を培養したときの各微生物増殖数Ｎα１，Ｎα２，Ｎα３，…，Ｎβ１，Ｎβ２，Ｎβ３，…から作成した増殖阻害曲線Ｌα１,Ｌβ１に、前記糖化溶液の増殖阻害物質濃度Ｃαａを当てはめて総合増殖阻害曲線Ｌαβ２を作成する。総合増殖阻害曲線Ｌαβ２は、各糖化溶液率を備える混合糖化溶液を前記微生物を用いて発酵させるときの微生物増殖数を示している。 Further, in this embodiment, the growth inhibition curves L α1 , L β1 created from the numbers of microorganism growth N α1 , N α2 , N α3 ,..., N β1 , N β2 , N β3 ,. to, to create a total growth inhibition curves L Arufabeta2 by applying a growth inhibitor concentration C .alpha.a of the glycated solution. The total growth inhibition curve L αβ2 indicates the number of microbial growth when a mixed saccharified solution having each saccharified solution rate is fermented using the microorganism.
次に、本実施形態のバイオエタノール製造方法について、実施例を示す。 Next, an Example is shown about the bioethanol manufacturing method of this embodiment.
〔実施例１〕
まず、リグノセルロース系バイオマスを前処理することにより、リグノセルロース系バイオマスからリグニンを解離し、又はリグノセルロース系バイオマスを膨潤させた前処理物を得た。本実施例では、リグノセルロース系バイオマスとして、粉砕された乾燥コーンストーバを用い、該乾燥コーンストーバに希硫酸を含浸させて１５０℃以上の温度で５分間保持した後で大気開放することにより、前処理を行った。
[Example 1]
First, by pretreating lignocellulosic biomass, a pretreated product was obtained in which lignin was dissociated from lignocellulosic biomass or lignocellulosic biomass was swollen. In this example, as a lignocellulosic biomass, a pulverized dry corn stover was used, and the dry corn stover was impregnated with dilute sulfuric acid, held at a temperature of 150 ° C. or higher for 5 minutes, and then released into the atmosphere to perform pretreatment. went.
次に、得られた前処理物に、水を添加した後に至適ｐＨに調整し、セルロース及びヘミセルロース分解酵素を所定量添加した後に、温度５０℃に保持して７２時間撹拌し、その後、遠心分離により固形分を除去することにより、糖化溶液を得た。 Next, after adding water to the obtained pretreated product, the pH is adjusted to an optimum pH, and after adding a predetermined amount of cellulose and hemicellulose-degrading enzyme, the temperature is maintained at 50 ° C. and stirred for 72 hours, and then centrifuged. The saccharified solution was obtained by removing solid content by separation.
次に、得られた糖化溶液について、増殖阻害物質αとしての酢酸、増殖阻害物質βとしての蟻酸、増殖阻害物質γとしてのフルフラール、増殖阻害物質δとしてのＨＭＦ、増殖阻害物質εとしてのバニリン、増殖阻害物質ζとしてのシリンガアルデヒド、増殖阻害物質ηとしての４−ヒドロキシアセトフェノン、増殖阻害物質θとしてのフェノール、増殖阻害物質ιとしてのグアイアコール、増殖阻害物質κとしてのフェルラ酸、増殖阻害物質λとしてのｐ−クマル酸の各増殖阻害物質濃度を測定した。結果を表１に示す。尚、酢酸の増殖阻害物質濃度Ｃα０及び蟻酸の増殖阻害物質濃度Ｃβ０は、非解離度による補正はしていない値である。 Next, with respect to the obtained saccharified solution, acetic acid as growth inhibitory substance α, formic acid as growth inhibitory substance β, furfural as growth inhibitory substance γ, HMF as growth inhibitory substance δ, vanillin as growth inhibitory substance ε, Syringaldehyde as growth inhibitor ζ, 4-hydroxyacetophenone as growth inhibitor η, phenol as growth inhibitor θ, guaiacol as growth inhibitor ι, ferulic acid as growth inhibitor κ, growth inhibitor λ Each growth inhibitor concentration of p-coumaric acid was measured. The results are shown in Table 1. The acetic acid growth inhibitory substance concentration C α0 and the formic acid growth inhibitory substance concentration C β0 are values that are not corrected by the degree of non-dissociation.
一方、混合糖化溶液の糖化溶液率を決定するために、試験用糖液に、上記１１種類の増殖阻害物質を種々の濃度で添加することにより、培養液を調製した。前記試験用糖液は、糖としてのグルコース８０ｇ／Ｌ及びキシロース４０ｇ／Ｌと、微生物の栄養源としてのイーストエクストラクト１０ｇ／Ｌとペプトン２０ｇ／Ｌとを水に溶解してなり、糖液としてのサトウキビの廃糖蜜を実験的に調製したものである。 On the other hand, in order to determine the saccharified solution ratio of the mixed saccharified solution, a culture solution was prepared by adding the above 11 kinds of growth inhibitory substances at various concentrations to the test sugar solution. The sugar solution for testing is obtained by dissolving 80 g / L of glucose as sugar and 40 g / L of xylose, 10 g / L of yeast extract as a nutrient source of microorganisms and 20 g / L of peptone in water. The sugarcane waste molasses was experimentally prepared.
次に、得られた培養液をｐＨ５に調製した後、微生物として酵母（ピキア属酵母）を添加し、温度３０℃で２０時間撹拌、振盪することにより、該微生物を培養した。前記微生物の添加量は、濁度０．５であった。 Next, after adjusting the obtained culture solution to pH 5, yeast (Pichia genus yeast) was added as a microorganism, and the microorganism was cultured by stirring and shaking at a temperature of 30 ° C. for 20 hours. The amount of the microorganism added was turbidity 0.5.
次に、前記培養によって増殖した微生物増殖数を、前記１１種類の各増殖阻害物質の各増殖阻害物質濃度毎に測定して増殖阻害曲線Ｌα１,Ｌβ１,…Ｌλ１を作成した。 Next, the growth inhibition curves L α1 , L β1 ,... L λ1 were prepared by measuring the number of microorganisms grown by the culture for each growth inhibitor concentration of each of the 11 types of growth inhibitory substances.
次に、得られた各増殖阻害曲線Ｌα１,Ｌβ１,…Ｌλ１から、総合増殖阻害曲線Ｌαβ…λ２_ｐＨ５を作成した。得られた総合増殖阻害曲線Ｌαβ…λ２_ｐＨ５を図６に示す。 Then, each growth inhibition was obtained curve L α1, L β1, from ... L λ1, was to create a comprehensive growth inhibition curve L αβ ... λ2_pH5. The obtained overall growth inhibition curve L αβ... Λ2_pH5 is shown in FIG.
次に、前記糖化溶液を糖液としての試験用糖液によって希釈し、糖化溶液率が１，０．７５，０．５，０．２５である混合糖化溶液を得た。前記試験用糖液は、糖液としてのサトウキビの廃糖蜜と作用が同一とみなすことができるので、前記糖化溶液を該試験用糖液で希釈した混合糖化溶液は、該糖化溶液をサトウキビの廃糖蜜で希釈した混合糖化溶液と同一とみなすことができる。 Next, the saccharification solution was diluted with a test saccharide solution as a saccharide solution to obtain a mixed saccharification solution having a saccharification solution ratio of 1,0.75, 0.5, 0.25. Since the test sugar solution can be regarded as having the same action as the sugarcane molasses as the sugar solution, the mixed saccharification solution obtained by diluting the saccharification solution with the test sugar solution is used to remove the saccharification solution from the sugarcane waste. It can be regarded as the same as the mixed saccharification solution diluted with molasses.
次に、得られた混合糖化溶液をｐＨ５に調整した後に、前記酵母を濁度０．５で添加し、温度３０℃に保持して２０時間撹拌して培養することにより、エタノール水溶液を得た。そして、培養前後において、前記混合糖化溶液の濁度を測定し、微生物増殖率を算出した。図６に、各混合糖化溶液の糖化溶液率に対応する微生物増殖率を×印で示す。 Next, after adjusting the obtained mixed saccharified solution to pH 5, the yeast was added at a turbidity of 0.5, and maintained at a temperature of 30 ° C. and stirred for 20 hours to obtain an aqueous ethanol solution. . And before and after culture | cultivation, the turbidity of the said mixed saccharification solution was measured and the microorganism growth rate was computed. In FIG. 6, the microbial growth rate corresponding to the saccharified solution rate of each mixed saccharified solution is indicated by x.
次に、得られたエタノール水溶液のエタノール濃度をガスクロマトグラフィーにより測定した。 Next, the ethanol concentration of the obtained aqueous ethanol solution was measured by gas chromatography.
表２に、糖化溶液率が１，０．７５，０．５，０．２５である混合糖化溶液の増殖阻害物質濃度、微生物増殖率、及びエタノール濃度を示す。 Table 2 shows the growth inhibitory substance concentration, microbial growth rate, and ethanol concentration of the mixed saccharified solution with saccharified solution rates of 1,0.75, 0.5, and 0.25.
図６から、総合増殖阻害曲線Ｌαβ…λ２_ｐＨ５は、×印にほぼ対応しており、前記糖化溶液率の決定に用いることが可能であることが明らかである。また、総合増殖阻害曲線Ｌαβ…λ２_ｐＨ５は、混合糖化溶液の糖化溶液率が小さいほど微生物増殖率が大きいことから、前記糖化溶液を前記糖液で希釈した混合糖化溶液は、微生物の増殖阻害を回避できることが明らかである。 From FIG. 6, it is clear that the total growth inhibition curve L αβ... Λ2_pH5 substantially corresponds to the x mark and can be used to determine the saccharification solution ratio. Further, since the overall growth inhibition curve L αβ... Λ2_pH5 has a higher microorganism growth rate as the saccharification solution rate of the mixed saccharification solution is smaller, the mixed saccharification solution obtained by diluting the saccharification solution with the sugar solution inhibits the growth of microorganisms. Obviously it can be avoided.
また、表２から、前記混合糖化溶液の糖化溶液率を小さくするほど、微生物増殖率が大きくなり、該混合糖化溶液の発酵によって得られるエタノール濃度が大きくなることが明らかである。 Further, it is clear from Table 2 that the smaller the saccharified solution ratio of the mixed saccharified solution, the higher the microorganism growth rate and the higher the ethanol concentration obtained by fermentation of the mixed saccharified solution.
〔実施例２〕
本実施例では、まず、前記培養液を用いた培養をｐＨ６で行うとともに、混合糖化溶液を用いた培養をｐＨ６で行う以外は、実施例１と全く同一にして総合増殖阻害曲線Ｌαβ…λ２を作成した。得られた総合増殖阻害曲線Ｌαβ…λ２_ｐＨ６を図６に示す。
[Example 2]
In this example, first, the overall growth inhibition curve L αβ... Λ2 was exactly the same as in Example 1 except that the culture using the culture solution was performed at pH 6 and the culture using the mixed saccharification solution was performed at pH 6 . It was created. The obtained overall growth inhibition curve L αβ... Λ2_pH6 is shown in FIG.
次に、糖化溶液率が１，０．７５，０．５，０．２５である混合糖化溶液を、ｐＨ６で培養した以外は、実施例１と全く同一にしてエタノール水溶液を得た。図６に、各混合糖化溶液の糖化溶液率に対応する微生物増殖率を●印で示す。 Next, an ethanol aqueous solution was obtained in exactly the same manner as in Example 1 except that the mixed saccharified solution having a saccharified solution ratio of 1,0.75, 0.5, 0.25 was cultured at pH 6. In FIG. 6, the microbial growth rate corresponding to the saccharified solution rate of each mixed saccharified solution is indicated by ●.
〔実施例３〕
本実施例では、まず、培養液を用いた培養をｐＨ４で行うとともに、混合糖化溶液を用いた培養をｐＨ４で行う以外は、実施例１と全く同一にして総合増殖阻害曲線Ｌαβ…λ２を作成した。得られた総合増殖阻害曲線Ｌαβ…λ２_ｐＨ４を図６に示す。
Example 3
In this example, first, the total growth inhibition curve L αβ... Λ2 is set to be exactly the same as in Example 1 except that the culture using the culture solution is performed at pH 4 and the culture using the mixed saccharification solution is performed at pH 4. Created. The obtained overall growth inhibition curve L αβ... Λ2_pH4 is shown in FIG.
次に、糖化溶液率が１，０．７５，０．５，０．２５である混合糖化溶液を、ｐＨ４で培養した以外は、実施例１と全く同一にしてエタノール水溶液を得た。図６に、各混合糖化溶液の糖化溶液率に対応する微生物増殖率を○印で示す。 Next, an ethanol aqueous solution was obtained in exactly the same manner as in Example 1 except that the mixed saccharified solution having a saccharified solution ratio of 1,0.75, 0.5, 0.25 was cultured at pH 4. In FIG. 6, the microbial growth rate corresponding to the saccharified solution rate of each mixed saccharified solution is indicated by ◯.
図６から、総合増殖阻害曲線Ｌαβ…λ２_ｐＨ６及びＬαβ…λ２_ｐＨ４についても、●印及び○印にほぼ対応しており、目的とする混合糖化溶液の糖化溶液率の決定に用いることが可能であることが明らかである。 From FIG. 6, the total growth inhibition curves L αβ... Λ2_pH6 and L αβ... Λ2_pH4 also substantially correspond to the marks ● and ○, and can be used to determine the saccharified solution ratio of the target mixed saccharified solution. It is clear that there is.
Ｃα０…糖液の増殖阻害物質濃度、 Ｃ１，Ｃ２，Ｃ３…試験用糖液の増殖阻害物質濃度、 Ｃαａ，Ｃβａ…糖化溶液の増殖阻害物質濃度、 Ｃαｂ，Ｃαｃ，Ｃαｄ，Ｃβｂ，Ｃβｃ，Ｃβｄ，…混合糖化溶液の増殖阻害物質濃度、 Ｓａ，Ｓｂ，Ｓｃ，Ｓｄ，Ｓｘ…糖化溶液率、Ｇｘ…所望の微生物増殖率、 Ｇα１，Ｇα２，Ｇα３，Ｇβ１，Ｇβ２，Ｇβ３…試験用糖液における微生物増殖率、 Ｇαａ，Ｇαｂ，Ｇαｃ，Ｇαｄ，Ｇβａ，Ｇβｂ，Ｇβｃ，Ｇβｄ，Ｇαβａ，Ｇαβｂ，Ｇαβｃ，Ｇαβｄ…混合糖化溶液における微生物増殖率、Ｌα１，Ｌβ１…第１の増殖阻害曲線、Ｌα２，Ｌαβ２，Ｌαβ…λ２_ｐＨ４，Ｌαβ…λ２_ｐＨ５，Ｌαβ…λ２_ｐＨ６…第２の増殖阻害曲線、 Ｎ０…標準微生物増殖数、 Ｎα１，Ｎα２，Ｎα３，Ｎβ１，Ｎβ２，Ｎβ３…微生物増殖数、 α，β，γ,δ，ε…増殖阻害物質。 Growth inhibitor concentration C .alpha.0 ... molasses, C 1, C 2, C 3 ... growth inhibitor concentration of the test sugar solution, C .alpha.a, growth inhibitor concentration C .beta.a ... saccharified solution, C αb, C αc, C αd, C βb, C βc , C βd, ... growth inhibitor concentration in the mixed saccharified solution, S a, S b, S c, S d, S x ... saccharification solution ratio, G x ... desired microbial growth rate, G α1, G α2, G α3 , G β1, G β2, microbial growth rate in G .beta.3 ... test molasses, G αa, G αb, G αc, G αd, G βa, G βb, G βc, G βd , G αβa , G αβb , G αβc, G αβd ... microbial growth rate in the mixed saccharification solution, L α1 , L β1 ... first growth inhibition curve, L α2 , L αβ2, L αβ ... λ2_pH4 , L αβ ... λ2_pH5 , L αβ ... λ2_pH6 ... the second of the growth inhibition curve, N 0 Standard microbial growth number, N α1, N α2, N α3, N β1, N β2, N β3 ... microbial growth number, α, β, γ, δ , ε ... growth inhibitor.
Claims (7)
該微生物の増殖を阻害する増殖阻害物質を含む該糖化溶液を、該糖化溶液よりも増殖阻害物質濃度が小さい糖液によって該増殖阻害物質濃度が低下するように希釈することにより、混合糖化溶液を得る工程と、
該混合糖化溶液を該微生物によって発酵させてエタノールを生成させる工程とを備えることを特徴とするバイオエタノールの製造方法。 In a bioethanol production method in which a pretreated product obtained by pretreating lignocellulosic biomass is saccharified with a saccharification enzyme, and the resulting saccharification solution is fermented by a microorganism to produce ethanol.
By diluting the saccharification solution containing a growth inhibitory substance that inhibits the growth of the microorganism so that the growth inhibitory substance concentration is lowered by a sugar solution having a growth inhibitory substance concentration lower than that of the saccharification solution, a mixed saccharification solution is obtained. Obtaining a step;
And a step of producing ethanol by fermenting the mixed saccharified solution with the microorganism.
前記増殖阻害物質は、酢酸、フルフラール、ｐ−クマル酸であり、
前記糖化溶液を、該酢酸の非解離分の濃度が７００ｍｇ／Ｌ以下となり、該フルフラールの濃度が７００ｍｇ／Ｌ以下となり、且つ該ｐ−クマル酸の濃度が５００ｍｇ／Ｌ以下となるように希釈することを特徴とするバイオエタノールの製造方法。 In the manufacturing method of the bioethanol of Claim 1,
The growth inhibitor is acetic acid, furfural, p-coumaric acid,
The saccharified solution is diluted so that the concentration of non-dissociated portion of acetic acid is 700 mg / L or less, the concentration of furfural is 700 mg / L or less, and the concentration of p-coumaric acid is 500 mg / L or less. A method for producing bioethanol characterized by the above.
前記糖液は、サトウキビの廃糖蜜、サトウキビの絞り汁、該サトウキビの絞り汁の濃縮液、米を糖化した液、麦を糖化した液、トウモロコシを糖化した液からなる群から選択される１種以上の液体であることを特徴とするバイオエタノールの製造方法。 In the manufacturing method of the bioethanol of Claim 1 or Claim 2,
The sugar solution is selected from the group consisting of sugarcane molasses, sugarcane juice, concentrated sugarcane juice, rice saccharified liquid, wheat saccharified liquid, and corn saccharified liquid A method for producing bioethanol, which is a liquid as described above.
試験用糖液に、前記増殖阻害物質をそれぞれ異なる増殖阻害物質濃度で添加してなる複数の培養液を用いて前記微生物を培養し、各培養液に含まれる該増殖阻害物質濃度毎の微生物増殖数を測定する第１の工程と、
該増殖阻害物質を無添加の該試験用糖液を培養液に用いて該微生物を培養し、該無添加の試験用糖液における微生物増殖数を測定して標準微生物増殖数とする第２の工程と、
該増殖阻害物質濃度毎の微生物増殖数を該標準微生物増殖数で除することにより、各培養液の増殖阻害物質濃度における微生物増殖率を算出する第３の工程と、
各培養液の該増殖阻害物質濃度と該微生物増殖率との関係から第１の増殖阻害曲線を作成する第４の工程と、
前記糖化溶液に含まれる増殖阻害物質濃度を測定する第５の工程と、
該第１の増殖阻害曲線から、該糖化溶液に含まれる増殖阻害物質濃度に対応する微生物増殖率を求める第６の工程と、
未希釈の前記糖化溶液の糖化溶液率を１とし、該未希釈の糖化溶液をそれぞれ異なる量の前記糖液によって希釈した各混合糖化溶液について、該第１の増殖阻害曲線から各混合糖化溶液の糖化溶液率に対応する微生物増殖率を求める第７の工程と、
各混合糖化溶液における該糖化溶液率と該微生物増殖率との関係から第２の増殖阻害曲線を作成する第８の工程と、
該第２の増殖阻害曲線から所望の微生物増殖率に対応する糖化溶液率を求める第９の工程と、
該第９の工程で得られた糖化溶液率となるように前記糖化溶液を前記糖液によって希釈することにより前記混合糖化溶液を得る第１０の工程と
を備えることを特徴とするバイオエタノールの製造方法。 In the manufacturing method of the bioethanol of any one of Claims 1-3,
The microorganism is cultured using a plurality of culture solutions obtained by adding the growth inhibitory substance to the test sugar solution at different growth inhibitory substance concentrations, and the microorganisms are grown for each concentration of the growth inhibitory substance contained in each culture solution. A first step of measuring a number;
The microorganism is cultured using the test sugar solution to which the growth inhibitor is not added as a culture solution, and the number of microorganism growth in the additive-free test sugar solution is measured to obtain a standard microorganism growth number. Process,
A third step of calculating the microbial growth rate at the growth inhibitory substance concentration of each culture solution by dividing the microbial growth number for each growth inhibitory substance concentration by the standard microorganism growth number;
A fourth step of creating a first growth inhibition curve from the relationship between the concentration of the growth inhibitory substance in each culture solution and the microbial growth rate;
A fifth step of measuring the concentration of a growth inhibitory substance contained in the saccharification solution;
A sixth step of obtaining a microbial growth rate corresponding to the growth inhibitor concentration contained in the saccharification solution from the first growth inhibition curve;
For each mixed saccharified solution in which the saccharified solution ratio of the undiluted saccharified solution is 1, and each undiluted saccharified solution is diluted with a different amount of the saccharified solution, each mixed saccharified solution is determined from the first growth inhibition curve. A seventh step for obtaining a microbial growth rate corresponding to the saccharified solution rate;
An eighth step of creating a second growth inhibition curve from the relationship between the saccharified solution rate and the microbial growth rate in each mixed saccharified solution;
A ninth step of obtaining a saccharified solution rate corresponding to a desired microbial growth rate from the second growth inhibition curve;
And a tenth step of obtaining the mixed saccharified solution by diluting the saccharified solution with the saccharified solution so as to achieve the saccharified solution rate obtained in the ninth step. Method.
試験用糖液に、前記糖化溶液に含まれる複数の増殖阻害物質の各増殖阻害物質毎に、該増殖阻害物質をそれぞれ異なる増殖阻害物質濃度で添加してなる複数の培養液を用いて前記微生物を培養し、各増殖阻害物質毎に、各培養液に含まれる該増殖阻害物質濃度毎の微生物増殖数を測定する第１の工程と、
各増殖阻害物質を無添加の該試験用糖液を培養液に用いて該微生物を培養し、該無添加の試験用糖液における微生物増殖数を測定して標準微生物増殖数とする第２の工程と、
各増殖阻害物質毎に、該増殖阻害物質濃度毎の微生物増殖数を該標準微生物増殖数で除することにより、各増殖阻害物質毎に、各増殖阻害物質濃度における微生物増殖率を算出する第３の工程と、
各増殖阻害物質毎に、各培養液の該増殖阻害物質濃度と該微生物増殖率との関係から第１の増殖阻害曲線を作成する第４の工程と、
前記糖化溶液に含まれる各増殖阻害物質濃度を測定する第５の工程と、
各増殖阻害物質毎の該第１の増殖阻害曲線から、各増殖阻害物質毎に該糖化溶液に含まれる該増殖阻害物質濃度に対応する微生物増殖率を求める第６の工程と、
未希釈の前記糖化溶液の糖化溶液率を１とし、該未希釈の糖化溶液をそれぞれ異なる量の前記糖液によって希釈した各混合糖化溶液について、各増殖阻害物質毎の該第１の増殖阻害曲線から各混合糖化溶液の糖化溶液率に対応する微生物増殖率を各増殖阻害物質毎に求めた後に、
該糖化溶液率が同一である混合糖化溶液における各増殖阻害物質の微生物増殖率をそれぞれ乗じることにより、各混合糖化溶液の糖化溶液率に対応する微生物増殖率を算出する第７の工程と、
各混合糖化溶液における該糖化溶液率と該微生物増殖率との関係から第２の増殖阻害曲線を作成する第８の工程と、
該第２の増殖阻害曲線から所望の微生物増殖率に対応する糖化溶液率を求める第９の工程と、
該第９の工程で得られた糖化溶液率となるように前記糖化溶液を前記糖液によって希釈することにより前記混合糖化溶液を得る第１０の工程と
を備えることを特徴とするバイオエタノールの製造方法。 In the manufacturing method of the bioethanol of any one of Claims 1-3,
The microorganism using a plurality of culture solutions obtained by adding the growth inhibitory substances at different growth inhibitory substance concentrations for each growth inhibitory substance of the plurality of growth inhibitory substances contained in the saccharified solution to the test sugar solution A first step of measuring, for each growth inhibitory substance, the number of microbial growth for each growth inhibitory substance concentration contained in each culture solution;
The microorganism is cultured using the test sugar solution to which each growth inhibitor is not added as a culture solution, and the number of microbial growth in the additive-free test sugar solution is measured to obtain a standard microorganism growth number. Process,
For each growth inhibitory substance, the microbial growth rate at each growth inhibitory substance concentration is calculated for each growth inhibitory substance by dividing the number of microbial growths at each growth inhibitory substance concentration by the standard microorganism growth number. And the process of
For each growth inhibitory substance, a fourth step of creating a first growth inhibition curve from the relationship between the growth inhibitory substance concentration of each culture solution and the microorganism growth rate;
A fifth step of measuring the concentration of each growth inhibitor contained in the saccharification solution;
A sixth step of determining, from the first growth inhibition curve for each growth inhibitory substance, the microbial growth rate corresponding to the concentration of the growth inhibitory substance contained in the saccharification solution for each growth inhibitory substance;
The first growth inhibition curve for each growth inhibitory substance for each of the mixed saccharification solutions obtained by setting the saccharification solution ratio of the undiluted saccharification solution to 1 and diluting the undiluted saccharification solution with different amounts of the saccharide solution. After obtaining the microbial growth rate corresponding to the saccharified solution rate of each mixed saccharified solution from each growth inhibitory substance,
A seventh step of calculating a microbial growth rate corresponding to the saccharification solution rate of each mixed saccharification solution by multiplying the microbial growth rate of each growth inhibitory substance in the mixed saccharification solution having the same saccharification solution rate;
An eighth step of creating a second growth inhibition curve from the relationship between the saccharified solution rate and the microbial growth rate in each mixed saccharified solution;
A ninth step of obtaining a saccharified solution rate corresponding to a desired microbial growth rate from the second growth inhibition curve;
And a tenth step of obtaining the mixed saccharified solution by diluting the saccharified solution with the saccharified solution so as to achieve the saccharified solution rate obtained in the ninth step. Method.
前記第１の工程は、前記試験用糖液として前記糖液を用いるとともに、該糖液に含まれる増殖阻害物質濃度を測定し、測定された該増殖阻害物質濃度と、該試験用糖液としての該糖液に添加された増殖阻害物質濃度との和を、前記培養液に含まれる増殖阻害物質濃度とすることを特徴とするバイオエタノールの製造方法。 In the method for producing bioethanol according to claim 4 or 5,
In the first step, the sugar solution is used as the test sugar solution, the growth inhibitor concentration contained in the sugar solution is measured, and the measured growth inhibitor concentration and the test sugar solution are measured. A method for producing bioethanol, characterized in that a concentration of the growth inhibitory substance added to the sugar solution is a growth inhibitory substance concentration contained in the culture solution.
前記増殖阻害物質は、有機酸、アルデヒド基を有する化合物、ケトン基を有する化合物、フェノール基を有する化合物、メラノイジンからなる群から選択される１又は複数の化合物であることを特徴とするバイオエタノールの製造方法。 In the manufacturing method of the bioethanol of any one of Claims 1-6,
The growth inhibitory substance is one or more compounds selected from the group consisting of organic acids, compounds having an aldehyde group, compounds having a ketone group, compounds having a phenol group, and melanoidins. Production method.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014051930A JP6324768B2 (en) | 2014-03-14 | 2014-03-14 | Bioethanol production method |
| US14/643,149 US9732363B2 (en) | 2014-03-14 | 2015-03-10 | Growth method for microbe and bioethanol production method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2014051930A JP6324768B2 (en) | 2014-03-14 | 2014-03-14 | Bioethanol production method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2015173624A true JP2015173624A (en) | 2015-10-05 |
| JP6324768B2 JP6324768B2 (en) | 2018-05-16 |
Family
ID=54253426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2014051930A Expired - Fee Related JP6324768B2 (en) | 2014-03-14 | 2014-03-14 | Bioethanol production method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6324768B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349565A (en) * | 2018-12-21 | 2020-06-30 | 中国科学院青岛生物能源与过程研究所 | Low-cost, high-biomass and high-protein-content chlorella pyrenoidosa culture method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011167096A (en) * | 2010-02-17 | 2011-09-01 | Kobe Univ | Method for producing ethanol from biomass |
| JP2012170444A (en) * | 2011-02-24 | 2012-09-10 | Tsukishima Kikai Co Ltd | Method for producing ethanol |
| JP2012170443A (en) * | 2011-02-24 | 2012-09-10 | Tsukishima Kikai Co Ltd | Method for producing ethanol |
| JP2013226113A (en) * | 2012-03-30 | 2013-11-07 | Honda Motor Co Ltd | Highly efficient process for producing bioethanol |
| JP2014003914A (en) * | 2012-06-21 | 2014-01-16 | Tsukishima Kikai Co Ltd | Treatment system and treatment method for biomass |
-
2014
- 2014-03-14 JP JP2014051930A patent/JP6324768B2/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2011167096A (en) * | 2010-02-17 | 2011-09-01 | Kobe Univ | Method for producing ethanol from biomass |
| JP2012170444A (en) * | 2011-02-24 | 2012-09-10 | Tsukishima Kikai Co Ltd | Method for producing ethanol |
| JP2012170443A (en) * | 2011-02-24 | 2012-09-10 | Tsukishima Kikai Co Ltd | Method for producing ethanol |
| JP2013226113A (en) * | 2012-03-30 | 2013-11-07 | Honda Motor Co Ltd | Highly efficient process for producing bioethanol |
| JP2014003914A (en) * | 2012-06-21 | 2014-01-16 | Tsukishima Kikai Co Ltd | Treatment system and treatment method for biomass |
Non-Patent Citations (3)
| Title |
|---|
| CRISTINA TOQUERO, SILVIA BOLADO: "Effect of four pretereatments on enzymatic hydrolysis and ethanol fermentation of wheat straw. Influ", BIORESOURCE TECHNOLOGY, vol. Vol.157, JPN6017036296, 29 January 2014 (2014-01-29), pages 68 - 76 * |
| MARIA CANTARELLA, ET AL.: "Effect of Inhibitors Released during Steam-Explosion Treatment of Poplar Wood on Subsequent Enzymati", BIOTECHNOL. PROG, vol. Vol.20, JPN6017036295, 2004, pages 200 - 206 * |
| PATRIK R. LENNARTSSON, ET AL.: "Integration of the first and second generation bioethanol processes and the importance of by-product", BIORESOURCE TECHNOLOGY, vol. Vol.165, JPN6017036294, 8 February 2014 (2014-02-08), pages 3 - 8 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349565A (en) * | 2018-12-21 | 2020-06-30 | 中国科学院青岛生物能源与过程研究所 | Low-cost, high-biomass and high-protein-content chlorella pyrenoidosa culture method |
| CN111349565B (en) * | 2018-12-21 | 2022-04-15 | 中国科学院青岛生物能源与过程研究所 | Method for culturing chlorella pyrenoidosa with high biomass and high protein content |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6324768B2 (en) | 2018-05-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2708962C (en) | Non-sterile fermentation of bioethanol | |
| US20190032094A1 (en) | Method for preparing sugar, bioethanol or microbial metabolite from lignocellulosic biomas | |
| Kim | Evaluation of Alkali‐Pretreated Soybean Straw for Lignocellulosic Bioethanol Production | |
| US10407700B2 (en) | Surfactant-improved simultaneous saccharification and co-fermentation method for lignocellulose | |
| US9732363B2 (en) | Growth method for microbe and bioethanol production method | |
| Xu et al. | Enzymatic hydrolysis and fermentability of corn stover pretreated by lactic acid and/or acetic acid | |
| Guan et al. | Effects of Tween 80 on fermentative butanol production from alkali-pretreated switchgrass | |
| US9441249B2 (en) | Method for producing ethanol from biomass | |
| Moshi et al. | High bioethanol titre from Manihot glaziovii through fed-batch simultaneous saccharification and fermentation in Automatic Gas Potential Test System | |
| CA3027374A1 (en) | Methods and systems for starch based propagation of a microorganism | |
| JP2011167096A (en) | Method for producing ethanol from biomass | |
| JP6532248B2 (en) | Method of growing microorganisms | |
| JP6324768B2 (en) | Bioethanol production method | |
| US20220315886A1 (en) | Methods for propagating microorganisms for fermentation & related methods & systems | |
| JP6014426B2 (en) | Highly efficient bioethanol production method | |
| JP5935020B2 (en) | New production method of ethanol | |
| AU2017254816B2 (en) | Method of producing ethanol using continuous culture and continuous culture apparatus | |
| Zhang et al. | Isolation of xylose fermentation strains for ethanol production and xylose fermentation research | |
| US10280437B2 (en) | Method for producing ethanol | |
| KR101447534B1 (en) | Method for producing fermentable sugar solution containing less toxic acetate from lignocellulosic biomass | |
| Pecar et al. | The influence of chicken manure pretreatment with wood decay fungi on biogas production | |
| KR101642056B1 (en) | Method for producing microbial metabolites from starch-containing lignocellulosic biomass | |
| Panagiotopoulos et al. | Biomass crops for fermentative biofuel production | |
| TWI496890B (en) | The method for improving efficiency of monosaccharide fermentation | |
| WO2019170788A1 (en) | Improved anaerobic fermentation process |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20161129 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20170926 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20171107 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20180403 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20180411 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6324768 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| LAPS | Cancellation because of no payment of annual fees |