JP2015167480A - Method for enzymatic saccharification of biomass containing lignocellulose - Google Patents
Method for enzymatic saccharification of biomass containing lignocellulose Download PDFInfo
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Abstract
Description
本発明は、リグノセルロース系原料を酵素で糖化処理する方法において、糖化処理で分解されなかった未分解残渣に吸着した酵素を回収して長期間にわたって循環利用することを可能とするリグノセルロース系バイオマスの酵素糖化処理方法に関する。 The present invention relates to a method for saccharifying lignocellulosic raw material with an enzyme, and lignocellulosic biomass that enables the enzyme adsorbed to an undegraded residue that has not been decomposed by saccharification to be recycled for a long period of time. The present invention relates to an enzymatic saccharification treatment method.
糖化に適した処理を施したリグノセルロース原料から糖を製造する技術は、この糖を微生物の発酵基質として用いることによりガソリンの代替燃料となるアルコールや、プラスチック原料となるコハク酸や乳酸などの化成品原料を製造することができることから、循環型社会の形成に有益な技術である。
植物系バイオマス中の多糖類から発酵基質となる単糖や小糖類を製造する方法は2つに大別できる。一つは鉱酸を用いて加水分解する酸糖化法であり、もう一つは酵素やその酵素を生産する微生物を用いて加水分解する酵素糖化法である。
The technology to produce sugar from lignocellulose raw material that has been treated suitable for saccharification is the use of this sugar as a fermentation substrate for microorganisms to convert alcohol as an alternative fuel for gasoline, succinic acid and lactic acid as plastic raw materials. Since it can produce raw materials for products, it is a useful technology for the formation of a recycling society.
Methods for producing monosaccharides and small saccharides as fermentation substrates from polysaccharides in plant biomass can be broadly classified into two. One is an acid saccharification method in which hydrolysis is performed using a mineral acid, and the other is an enzyme saccharification method in which hydrolysis is performed using an enzyme and a microorganism that produces the enzyme.
酸糖化法は酵素糖化法に比べて技術的に完成されているが、リグノセルロース系バイオマスを原料とする方法の場合は、澱粉や廃糖蜜などを原料とする方法に比べて糖収率が低いことに加えて、処理工程から排出される廃酸の処理設備や、酸による腐食に耐え得る大型の設備が必要となること等が製品コストの増大原因となっていて実用化の大きな障壁となっている。 The acid saccharification method has been technically completed compared to the enzymatic saccharification method, but in the case of the method using lignocellulosic biomass as a raw material, the sugar yield is lower than the method using starch or waste molasses as a raw material. In addition, the need for a treatment facility for waste acid discharged from the treatment process and a large facility that can withstand the corrosion caused by the acid is the cause of the increase in product cost and becomes a major barrier to practical use. ing.
一方、酵素糖化法は、近年酵素の価格が下がってきていることと技術の進歩から、後処理まで含めた全体のコストで酸糖化法のコストに近づいてきてはいるが、酵素糖化法の全体コストに占める割合が高い酵素の価格は依然として高いことから、酵素糖化法の実用化のためには酵素にかかる費用の一層の低減が重要である。 On the other hand, the enzymatic saccharification method has been approaching the cost of acid saccharification method at the total cost including post-treatment due to the recent decrease in enzyme prices and technological progress. Since the price of an enzyme that accounts for a high percentage of the cost is still high, it is important to further reduce the cost of the enzyme for practical use of the enzymatic saccharification method.
酵素糖化法のコストを下げる技術としては、セルロース繊維への酵素のアクセスを容易にする前処理の方法の開発や、結晶性セルロースを効率よく糖化する方法の開発、更には酵素の効率的な回収、再利用方法の開発などが考えられる。 Technologies to reduce the cost of enzymatic saccharification include the development of pretreatment methods that facilitate enzyme access to cellulose fibers, the development of efficient saccharification of crystalline cellulose, and the efficient recovery of enzymes. Development of a reuse method can be considered.
リグニンを除去していないリグノセルロース材料は、リグニンを除去したリグノセルロース材料と比べて酵素によって分解されにくく、糖化されずに樹脂、金属などの不純物と一緒に糖化液中に残渣として残る。一般に、この残渣はスクリーン、遠心分離等により分離し廃棄される。この残渣には酵素糖化法におけるコストの中で大きな比重を占めている酵素が多量に吸着されているため、反応液から分離した残渣をそのまま廃棄してしまうと高価な酵素も廃棄されてしまうという問題があった。 The lignocellulose material from which lignin has not been removed is less susceptible to degradation by enzymes than the lignocellulose material from which lignin has been removed, and remains as a residue in the saccharified solution together with impurities such as resin and metal without being saccharified. In general, the residue is separated and discarded by screen, centrifugation, or the like. This residue is adsorbed with a large amount of enzyme that occupies a large specific gravity in the cost of enzymatic saccharification, so if the residue separated from the reaction solution is discarded as it is, the expensive enzyme is also discarded. There was a problem.
上記のような残渣中の酵素の回収手段として、残渣の洗浄が考えられる。しかし、酵素は、その分子内に有しているセルロースに特異的に吸着するセルロースバインディングドメイン(CBD)等によりセルロースと強固に結合しているため、単なる水洗浄ではセルロースに吸着した酵素を十分に回収することは困難であった。
そこで、酵素の回収率の改善を目的として界面活性剤を添加して処理する方法(特許文献1参照)などが提案されている。しかし、界面活性剤処理法でも、酵素の回収率が十分であるとはいえず、また、薬品添加による酵素の失活や、処理工程付加に伴うコストアップ及び後の発酵段階における微生物への悪影響などが懸念されることなどから実用的ではない。
As a means for recovering the enzyme in the residue as described above, washing of the residue can be considered. However, since the enzyme is strongly bound to cellulose by the cellulose binding domain (CBD) that specifically adsorbs to the cellulose contained in the molecule, the enzyme adsorbed to the cellulose can be sufficiently obtained by simple water washing. It was difficult to recover.
Therefore, a method of adding a surfactant to treat the enzyme for the purpose of improving the enzyme recovery rate (see Patent Document 1) has been proposed. However, even with the surfactant treatment method, it cannot be said that the enzyme recovery rate is sufficient. In addition, the enzyme is deactivated by the addition of chemicals, the cost increases due to the addition of the treatment process, and the adverse effects on microorganisms in the subsequent fermentation stage. It is not practical because of concerns.
糖液からの酵素の回収法としては、限外濾過を用いた方法(特許文献2参照)、糖液に再度セルロースを添加して酵素を吸着回収する方法(特許文献3参照)などが提案されている。しかし、限外濾過法は微少な不純物がろ過膜につまり十分な処理速度及び酵素回収率が得られない問題があるし、セルロース添加による回収法では十分な酵素回収が困難であった。 As a method for recovering an enzyme from a sugar solution, a method using ultrafiltration (see Patent Document 2), a method of adding cellulose to the sugar solution again and adsorbing and recovering the enzyme (see Patent Document 3), and the like have been proposed. ing. However, the ultrafiltration method has a problem that minute impurities are present in the filtration membrane, that is, a sufficient processing speed and enzyme recovery rate cannot be obtained, and sufficient recovery of the enzyme is difficult by the recovery method by adding cellulose.
吸着した酵素を剥離させる工程を経ずに、酵素が吸着しているリグノセルロース残渣を次回分の酵素糖化に再利用する方法が提案されている(特許文献4)。この方法では、残渣の蓄積は避けられないので反応効率が低下することが懸念される。また、CBH(セロビオハイドラーゼ)等、CBDを有する酵素に関してはリグノセルロース残渣を次回分で再処理することで酵素の循環利用が可能であるが、ベータ−グルコシダーゼ等は上清中に遊離している場合もあるので、添加したセルラーゼの全てを循環利用することは困難である。 There has been proposed a method of reusing the lignocellulose residue adsorbed by the enzyme for the next enzymatic saccharification without going through the step of peeling off the adsorbed enzyme (Patent Document 4). In this method, accumulation of residue is unavoidable, so there is a concern that the reaction efficiency may be lowered. In addition, for enzymes having CBD, such as CBH (cellobiohydrase), the lignocellulose residue can be recycled by reprocessing the next time, but beta-glucosidase and the like are released into the supernatant. In some cases, it is difficult to recycle all of the added cellulase.
酵素のコストを低減する方法として、酵素を循環利用する方法が報告されている。Scott,C.D.らの方法(非特許文献1)によると、酵素を大量(濾紙分解活性で基質1gに対して80−160単位)に添加して古紙原料を酵素加水分解する主反応槽に、酵素加水分解液中の未反応古紙面から高剪断力で生成グルコースやセロビオース成分を除いて常に新しいセルロース繊維表面を露出させる高速遠心ポンプによる磨砕装置と、磨砕装置からの処理液から未反応原料と加水分解液を分離して未反応原料のみを主反応槽に循環する膜分離装置と、膜分離装置からの加水分解液から酵素と生成グルコース及びセロビオースを分離して酵素のみを主反応槽に循環する限外濾過装置とを有する循環ラインを設けた連続システムを想定してコストを予測している。このシステムにより、糖化率は25時間で100%であり、酵素の残存率は24時間で95%以上であるとされている。また、酵素が残渣に吸着されて失われること、残渣の酵素の吸着機能はpHを5〜7に高めることで低下可能な場合があること、温度を5℃に下げることで低減できるという報告もあることが記載されている。 As a method for reducing the cost of the enzyme, a method of recycling the enzyme has been reported. Scott, C.I. D. According to these methods (Non-Patent Document 1), an enzyme hydrolyzate is added to a main reaction tank in which enzyme is hydrolyzed by adding enzyme in a large amount (80-160 units per 1 g of substrate with filter paper decomposition activity). A high-speed centrifugal pump that always exposes the surface of new cellulose fibers except for the glucose and cellobiose components generated from the unreacted old paper surface with high shear force, and unreacted raw materials and hydrolysis from the processing liquid from the grinding device A membrane separator that separates the liquid and circulates only unreacted raw materials to the main reaction tank, and a limit that circulates only the enzyme to the main reaction tank by separating the enzyme, produced glucose and cellobiose from the hydrolyzate from the membrane separator. Costs are estimated assuming a continuous system with a circulation line having an external filtration device. According to this system, the saccharification rate is 100% in 25 hours, and the residual rate of the enzyme is 95% or more in 24 hours. There are also reports that the enzyme is adsorbed on the residue and lost, the adsorption function of the enzyme in the residue may be lowered by raising the pH to 5-7, and can be reduced by lowering the temperature to 5 ° C. It is described that there is.
酵素を回収再利用する方法として、蒸煮・爆砕処理したシラカンバ材を5%の濃度で糖化槽に加え、2万単位のセルラーゼを添加して、限外濾過により糖液と酵素液とを分離し、酵素を回収再利用しながら、8日間で2kgのシラカンバ材から単糖類を630g得ている方法も報告されており、この方法で酵素の使用量を20%節約できたとされている(非特許文献2)。 As a method of recovering and reusing enzymes, steamed and crushed birch wood is added to a saccharification tank at a concentration of 5%, 20,000 units of cellulase are added, and the sugar solution and the enzyme solution are separated by ultrafiltration. In addition, a method of obtaining 630 g of monosaccharides from 2 kg of birch wood in 8 days while collecting and reusing the enzyme has been reported, and it is said that this method saved 20% of the amount of enzyme used (non-patented) Reference 2).
リグノセルロース系原料への酵素の吸着を抑制する方法として、リグノセルロース系原料に水溶性塩類を添加する方法(特許文献5)、リグノセルロース系原料に炭酸カルシウムを添加する方法(特許文献6)が報告されている。しかし、リグノセルロース系原料を酵素で糖化処理した後のリグニンを多く含む未分解残渣に吸着している酵素を効率的に回収する方法の開発が望まれている。 As a method for suppressing the adsorption of the enzyme to the lignocellulosic material, a method of adding a water-soluble salt to the lignocellulosic material (Patent Document 5) and a method of adding calcium carbonate to the lignocellulosic material (Patent Document 6). It has been reported. However, development of a method for efficiently recovering an enzyme adsorbed on an undegraded residue containing a large amount of lignin after saccharification treatment of a lignocellulosic raw material with an enzyme is desired.
本発明の課題は、リグノセルロース系原料を酵素(セルラーゼ)で糖化処理する方法において、糖化処理で分解されなかった残渣(リグニン含有)に吸着した酵素を効率的に回収する方法を提供する。 An object of the present invention is to provide a method for efficiently recovering an enzyme adsorbed on a residue (containing lignin) that has not been decomposed by a saccharification treatment in a method for saccharifying a lignocellulosic raw material with an enzyme (cellulase).
本発明者らは、上記の課題を解決するために鋭意検討した結果、リグノセルロース系原料を酵素で糖化処理した後の固液分離装置で分離された残渣をオゾン処理することにより、残渣に吸着している酵素を効率的に回収できることを見出し、下記発明を完成した。 As a result of intensive studies to solve the above-mentioned problems, the present inventors adsorbed the residue separated by the solid-liquid separation apparatus after the saccharification treatment of the lignocellulosic raw material with an enzyme to the residue. The inventors have found that the enzyme can be efficiently recovered, and have completed the following invention.
(1)リグノセルロース系原料を酵素で糖化処理し、糖化処理した処理懸濁液を固液分離装置で固形分(残渣)と液体分に分離し、前記固液分離装置で分離された残渣をオゾン処理することを特徴とするリグノセルロース系原料の糖化処理方法。 (1) The lignocellulose raw material is saccharified with an enzyme, the saccharified suspension is separated into a solid (residue) and a liquid by a solid-liquid separator, and the residue separated by the solid-liquid separator is separated. A method for saccharification of a lignocellulosic material, characterized by performing ozone treatment.
(2)前記糖化処理に発酵用微生物を添加して併行糖化発酵処理し、前記併行糖化発酵処理した処理懸濁液を固液分離装置で固形分(残渣)と液体分に分離し、前記固液分離装置で分離された残渣をオゾン処理することを特徴とする(1)に記載のリグノセルロース系原料の糖化処理方法。 (2) Fermentation microorganisms are added to the saccharification treatment and subjected to concurrent saccharification and fermentation treatment, and the suspension treated by the concurrent saccharification and fermentation treatment is separated into a solid (residue) and a liquid by a solid-liquid separator, The residue separated by the liquid separation device is subjected to ozone treatment, The method for saccharification of a lignocellulose-based raw material according to (1).
(3)前記オゾン処理が、前記固液分離装置で分離した残渣に吸着している酵素を遊離させる方法であることを特徴とする請求項1または請求項2に記載の糖化処理方法。 (3) The saccharification treatment method according to claim 1 or 2, wherein the ozone treatment is a method of liberating an enzyme adsorbed on the residue separated by the solid-liquid separator.
本発明により、糖化処理で分解されなかった未分解残渣に吸着した酵素を効率的に回収することができる。酵素糖化処理工程内における糖化酵素の循環率が長期にわたって高い水準に維持されるため、リグノセルロース原料の酵素糖化処理による糖類やエタノール等を工業的に生産することが可能となる。 According to the present invention, the enzyme adsorbed on the undecomposed residue that has not been decomposed by the saccharification treatment can be efficiently recovered. Since the circulation rate of the saccharifying enzyme in the enzymatic saccharification treatment process is maintained at a high level over a long period of time, it becomes possible to industrially produce saccharides, ethanol and the like by the enzymatic saccharification treatment of the lignocellulose raw material.
以下、本発明をさらに詳しく説明する。 Hereinafter, the present invention will be described in more detail.
<リグノセルロース系原料>
本発明の方法で原料として使用するリグノセルロース系原料としては、木質系として、製紙用樹木、林地残材、間伐材等のチップ又は樹皮、木本性植物の切株から発生した萌芽、製材工場等から発生する鋸屑又はおがくず、街路樹の剪定枝葉、建築廃材等が挙げられ、草本系としてケナフ、稲藁、麦わら、コーンコブ、バガス等の農産廃棄物、油用作物やゴム等の工芸作物の残渣及び廃棄物(例えば、EFB: Empty Fruit Bunch)、草本系エネルギー作物のエリアンサス、ミスカンサスやネピアグラス等が挙げられる。
また、バイオマスとしては、木材由来の紙、古紙、パルプ、パルプスラッジ、スラッジ、下水汚泥等、食品廃棄物、等を原料として利用することができる。これらのバイオマスは、単独、あるいは複数を組み合わせて使用することができる。また、バイオマスは、乾燥固形物であっても、水分を含んだ固形物であっても、スラリーであっても用いることができる。
<Lignocellulose raw material>
As a lignocellulosic raw material used as a raw material in the method of the present invention, as a woody system, chips or bark of papermaking trees, forest land residual materials, thinned wood, etc., sprouts generated from stumps of woody plants, sawmills, etc. Sawdust or sawdust that is generated, pruned branches and leaves of street trees, construction waste, etc. Examples include wastes (for example, EFB: Empty Fruit Bunch), herbaceous energy crops Eliansus, Miscanthus, and Napiergrass.
Further, as biomass, food waste such as paper derived from wood, waste paper, pulp, pulp sludge, sludge, sewage sludge, and the like can be used as raw materials. These biomasses can be used alone or in combination. The biomass can be used as a dry solid, a solid containing water, or a slurry.
前記木質系のリグノセルロース系原料としては、ユーカリ(Eucalyptus)属植物、ヤナギ(Salix)属植物、ポプラ属植物、アカシア(Acacia)属植物、スギ(Cryptomeria)属植物等が利用できるが、ユーカリ属植物、アカシア属、ヤナギ属植物が原料として大量に採取し易いため好ましい。木本性植物由来のリグノセルロース系原料の中では、林地残材(樹皮、枝葉を含む)、樹皮が好ましい。例えば、製紙原料用として一般に用いられるユーカリ(Eucalyptus)属又はアカシア(Acacia)属等の樹種の樹皮は、製紙原料用の製材工場やチップ工場等から安定して大量に入手可能であるため、特に好適に用いられる。 Examples of the woody lignocellulosic material include Eucalyptus, Salix, Poplar, Acacia, and Cryptomeria plants. Plants, genus Acacia and willow genus are preferable because they can be easily collected in large quantities as raw materials. Among the lignocellulosic raw materials derived from woody plants, forest land remnants (including bark and leaves) and bark are preferable. For example, bark of tree species such as Eucalyptus genus or Acacia genus commonly used for papermaking raw materials can be obtained in large quantities stably from lumber mills and chip factories for papermaking raw materials. Preferably used.
<機械的処理>
本発明では、前記リグノセルロース原料に機械的処理を施すことができる。機械的処理としては、切断、裁断、破砕、磨砕等の任意の機械的手段が挙げられ、リグノセルロースを次工程の化学的処理工程で糖化され易い状態にすることである。使用する機械装置については特に限定されないが、例えば、切出し装置、一軸破砕機、二軸破砕機、ハンマークラッシャー、レファイナー、ニーダー、ボールミル等を用いることができる。
<Mechanical processing>
In the present invention, the lignocellulose raw material can be subjected to mechanical treatment. Examples of the mechanical treatment include any mechanical means such as cutting, cutting, crushing, and grinding, and making lignocellulose easy to be saccharified in the next chemical treatment step. Although it does not specifically limit about the mechanical apparatus to be used, For example, a cutting device, a uniaxial crusher, a biaxial crusher, a hammer crusher, a refiner, a kneader, a ball mill etc. can be used.
前記機械的処理の前工程又は後工程として、異物(石、ゴミ、金属、プラステック等のリグノセルロース以外の異物)を除去するための洗浄などによる異物除去工程を導入することもできる。
原料を洗浄する方法としては、例えば、原料に水を噴射して原料に混合されている異物を除く方法、あるいは、原料を水中に浸漬し異物を沈降させて取り除く方法等が挙げられる。また、メタルトラップ等の装置を用いて、異物を原料から分離する方法が挙げられる。
原料に異物が含まれていると、リファイナーのディスク(プレート)等の機械的処理で用いる装置の部品を破損させる可能性があるし、配管が詰まる等の製造工程内でトラブルを起こす等の問題が発生するため、異物除去工程を導入することが望ましい。
As a pre-process or post-process of the mechanical treatment, a foreign matter removing step by washing or the like for removing foreign matter (foreign matter other than lignocellulose such as stone, dust, metal, plastic) can be introduced.
Examples of the method for washing the raw material include a method of removing water from the foreign material mixed with the raw material by spraying water on the raw material, or a method of removing the foreign material by immersing the raw material in water and sedimenting the foreign material. Moreover, the method of isolate | separating a foreign material from a raw material using apparatuses, such as a metal trap, is mentioned.
If foreign materials are included in the raw materials, there is a possibility of causing damage to equipment parts used in mechanical processing such as refiner discs (plates), and causing problems in the manufacturing process such as clogging of piping. Therefore, it is desirable to introduce a foreign substance removing step.
<化学的処理>
前記、機械的処理を施したリグノセルロース原料を次に化学的処理する。化学的処理で用いる化学薬品としては、水酸化ナトリウム、水酸化カリウム、水酸化カルシウム、炭酸ナトリウム及び炭酸水素ナトリウムから選ばれる1種以上のアルカリ薬品、又は、亜硫酸ナトリウムと前記アルカリ薬品の中から選ばれる1種以上のアルカリ薬品を含有する溶液に浸漬する化学的処理を含む前処理である。また、オゾン、二酸化塩素などの酸化剤による化学的処理も可能である。
化学的処理は、前記機械的処理と組み合わせてそれらの前処理の後処理として行うことが好適である。
<Chemical treatment>
Next, the mechanically treated lignocellulose raw material is then chemically treated. The chemical used in the chemical treatment is selected from one or more alkaline chemicals selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate and sodium hydrogen carbonate, or sodium sulfite and the above alkaline chemicals A pretreatment including a chemical treatment of immersing in a solution containing one or more alkaline chemicals. Further, chemical treatment with an oxidizing agent such as ozone or chlorine dioxide is also possible.
The chemical treatment is preferably performed as a post-treatment of the pretreatment in combination with the mechanical treatment.
化学的処理で使用する化学薬品の添加量は、状況に応じて任意に調整可能であるが、薬品コスト低下の面から、またセルロースの溶出・過分解による収率低下防止の面から、リグノセルロース系原料の絶乾100質量部に対して50質量部以下であることが望ましい。化学的処理における薬品の水溶液への浸漬時間及び処理温度は、使用する原料や薬品によって任意に設定可能であるが、処理時間20〜120分、処理温度80〜230℃が好ましい。処理条件を厳しくすることで、原料中のセルロースの液側への溶出又は過分解が起こる場合もあるため、処理時間は90分以下、処理温度は200℃以下であることが好ましい。 The amount of chemicals used in the chemical treatment can be arbitrarily adjusted according to the situation, but lignocellulose can be used to reduce the cost of chemicals and to prevent the yield from decreasing due to cellulose dissolution and over-decomposition. It is desirable that it is 50 parts by mass or less with respect to 100 parts by mass of the absolutely dry material. Although the immersion time and the processing temperature of the chemical | medical solution in chemical processing can be arbitrarily set with the raw material and chemical | medical agent to be used, processing time 20-120 minutes and processing temperature 80-230 degreeC are preferable. By tightening the processing conditions, elution or excessive decomposition of cellulose in the raw material may occur, so that the processing time is preferably 90 minutes or less and the processing temperature is preferably 200 ° C. or less.
(磨砕処理)
本発明では、前記化学的処理により得られたリグノセルロース原料を磨砕処理することが望ましい。磨砕処理で用いる磨砕処理装置としては、レファイナー、ボールミル等が挙げられる。レファイナーを用いる場合、レファイナーのディスク(プレート)のクリアランスを0.1〜2.0mmの範囲で磨砕することが好ましく、0.1〜1.0mmの範囲で磨砕することがさらに好ましい。使用するレファイナーとしては、シングルディスクレファイナー、ダブルディスクレファイナー等を使用することができ相対するディスクのクリアランスを0.1〜2.0mmの範囲に設定できるレファイナーであれば特に制限なく使用することができる。ディスクのクリアランスが2.0mmを超えると糖化または併行糖化発酵で得られる糖収率が添加するため好ましくない。一方、ディスクのクリアランスが0.1mmより低いとレファイナーで磨砕処理した後の加水分解物(固形分)の収率が低下するため好ましくない。また、ディスクのクリアランスが0.1mmより低いとレファイナーの運転に要する電気消費量が増大するため好ましくない。
(Grinding treatment)
In the present invention, it is desirable to grind the lignocellulose raw material obtained by the chemical treatment. Examples of the grinding device used in the grinding treatment include a refiner and a ball mill. When a refiner is used, the refiner disk (plate) clearance is preferably ground within a range of 0.1 to 2.0 mm, and more preferably within a range of 0.1 to 1.0 mm. As a refiner to be used, a single disk refiner, a double disk refiner, or the like can be used, and any refiner that can set the clearance of the opposing disk within a range of 0.1 to 2.0 mm can be used without particular limitation. it can. If the disc clearance exceeds 2.0 mm, the sugar yield obtained by saccharification or concurrent saccharification and fermentation is added, which is not preferable. On the other hand, if the disc clearance is lower than 0.1 mm, the yield of the hydrolyzate (solid content) after grinding with a refiner is not preferable. Also, if the disc clearance is lower than 0.1 mm, the electricity consumption required for the operation of the refiner increases, which is not preferable.
レファイナーのディスク(プレート)の材質、ディスクの型、ディスク面の刃の型、ディスク面に対する刃の方向等のディスクの形状については効果が得られる材質、形状であれば、特に制限なく使用することができる。 The material of the refiner disk (plate), the disk mold, the disk surface blade mold, the direction of the blade with respect to the disk surface, etc. should be used without any limitation as long as the material and shape are effective. Can do.
前記の磨砕処理が施されたリグノセルロース系原料を水溶液と固形分に固液分離し、固形分を糖化または併行糖化発酵の原料として用いることができる。固液分離する方法としては、例えば、スクリュープレス等を用いて水溶液と固形分に分離することができ、水溶液と固形分に分離することができる装置であれば制限なく使用することができる。 The lignocellulosic raw material subjected to the above-mentioned grinding treatment can be solid-liquid separated into an aqueous solution and a solid content, and the solid content can be used as a raw material for saccharification or concurrent saccharification and fermentation. As a method for solid-liquid separation, for example, an apparatus that can be separated into an aqueous solution and a solid content using a screw press or the like and can be used without limitation as long as it can be separated into an aqueous solution and a solid content.
前記の固形分離後の原料を用いて糖化または併行糖化発酵を行う前に殺菌処理を行うことが好ましい。リグノセルロース系バイオマス原料中に雑菌が混入していると、酵素による糖化を行う際に雑菌が糖を消費して生成物の収量が低下してしまうという問題が発生する。
殺菌処理は、酸やアルカリなど、菌の生育困難なpHに原料を晒す方法でも良いが、高温下で処理する方法でも良く、両方を組み合わせても良い。酸、アルカリ処理後の原料については、中性付近、もしくは、糖化及び/又は糖化発酵工程に適したpHに調整した後に原料として使用することが好ましい。また、高温殺菌した場合も、室温もしくは糖化発酵工程に適した温度まで降温させてから原料として使用することが好ましい。このように、温度やpHを調整してから原料を送り出すことで、好適pH、好適温度外に酵素が晒されて、失活することを防ぐことができる。
It is preferable to perform sterilization treatment before saccharification or parallel saccharification fermentation using the raw material after the solid separation. When miscellaneous bacteria are mixed in the lignocellulosic biomass raw material, there is a problem that the miscellaneous bacteria consume sugar when the enzyme is saccharified and the yield of the product decreases.
The sterilization treatment may be a method in which the raw material is exposed to a pH at which bacteria are difficult to grow, such as acid or alkali, but may be a method in which the raw material is treated at a high temperature, or a combination of both. About the raw material after an acid and an alkali treatment, it is preferable to use as a raw material, after adjusting to neutrality vicinity or pH suitable for a saccharification and / or saccharification fermentation process. In addition, even when pasteurized at a high temperature, it is preferably used as a raw material after being cooled to room temperature or a temperature suitable for the saccharification and fermentation process. Thus, by feeding out the raw material after adjusting the temperature and pH, it is possible to prevent the enzyme from being exposed to the outside of the preferred pH and the preferred temperature and being deactivated.
前記前処理が施されているリグノセルロース原料が、糖化工程又は一次併行糖化発酵工程へ供給される。 The lignocellulose raw material subjected to the pretreatment is supplied to the saccharification step or the primary concurrent saccharification and fermentation step.
<糖化工程>
酵素糖化反応に適した前処理が施されたリグノセルロース系原料は、適量の水と酵素と混合されて原料懸濁液とされ、糖化工程へ供給される。リグノセルロース系原料は酵素(セルラーゼ、ヘミセルラーゼ)により糖化(セルロース→グルコース、ヘミセルロース→グルコース、キシロース)される。
<Saccharification process>
The lignocellulosic raw material that has been subjected to pretreatment suitable for the enzymatic saccharification reaction is mixed with an appropriate amount of water and an enzyme to form a raw material suspension, which is supplied to the saccharification step. Lignocellulose-based raw materials are saccharified (cellulose → glucose, hemicellulose → glucose, xylose) by enzymes (cellulase, hemicellulase).
<併行糖化発酵工程>
酵素糖化反応に適した前処理が施されたリグノセルロース系原料は、適量の水と酵素と混合されて原料懸濁液とされ、さらに酵母等の微生物と混合されて併行糖化発酵工程へ供給される。リグノセルロース系原料は酵素により糖化され、生成された糖類が発酵微生物(酵母など)によりエタノールに発酵される。
<Concurrent saccharification and fermentation process>
The lignocellulosic raw material that has been pretreated suitable for the enzymatic saccharification reaction is mixed with an appropriate amount of water and enzyme to form a raw material suspension, and further mixed with microorganisms such as yeast and supplied to the parallel saccharification and fermentation process. The The lignocellulosic raw material is saccharified by an enzyme, and the produced saccharide is fermented to ethanol by a fermentation microorganism (such as yeast).
糖化工程又は併行糖化発酵工程で用いるリグノセルロース系原料の懸濁濃度は、1〜30質量%であることが好ましい。1質量%未満であると、最終的に生産物の濃度が低すぎて生産物の濃縮のコストが高くなるという問題が発生する。また、30質量%を超えて高濃度となるにしたがって原料の攪拌が困難になり、生産性が低下するという問題が発生する。 It is preferable that the suspension concentration of the lignocellulosic material used in the saccharification process or the concurrent saccharification and fermentation process is 1 to 30% by mass. If it is less than 1% by mass, there is a problem in that the concentration of the product is ultimately too low and the cost for concentrating the product becomes high. Moreover, as the concentration exceeds 30% by mass, it becomes difficult to stir the raw materials, resulting in a problem that productivity is lowered.
糖化工程又は併行糖化発酵で使用するセルロース分解酵素は、セロビオヒドロラーゼ活性、エンドグルカナーゼ活性、ベータグルコシダーゼ活性を有する、所謂セルラーゼと総称される酵素である。
各セルロース分解酵素は、夫々の活性を有する酵素を適宜の量で添加しても良いが、市販されているセルラーゼ製剤は、上記の各種のセルラーゼ活性を有すると同時に、ヘミセルラーゼ活性も有しているものが多いので市販のセルラーゼ製剤を用いれば良い。
Cellulolytic enzymes used in the saccharification step or the concurrent saccharification and fermentation are enzymes collectively called cellulases having cellobiohydrolase activity, endoglucanase activity, and betaglucosidase activity.
Each cellulolytic enzyme may be added with an appropriate amount of an enzyme having the respective activity. However, commercially available cellulase preparations have the above-mentioned various cellulase activities and also have hemicellulase activity. Since many products are available, a commercially available cellulase preparation may be used.
市販のセルラーゼ製剤としては、トリコデルマ(Trichoderma)属、アクレモニウム(Acremonium)属、アスペルギルス(Aspergillus)属、ファネロケエテ(Phanerochaete)属、トラメテス(Trametes)属、フーミコラ(Humicola)属、バチルス(Bacillus)属などに由来するセルラーゼ製剤がある。このようなセルラーゼ製剤の市販品としては、全て商品名で、例えば、セルロイシンT2(エイチピィアイ社製)、メイセラーゼ(明治製菓社製)、ノボザイム188(ノボザイム社製)、マルティフェクトCX10L(ジェネンコア社製)、GC220(ジェネンコア社製)等が挙げられる。
原料固形分100質量部に対するセルラーゼ製剤の使用量は、0.5〜100質量部が好ましく、1〜50質量部が特に好ましい。
Commercial cellulase preparations include the genus Trichoderma, the genus Acremonium, the genus Aspergillus, the genus Phanerochaete, the genus Trametes, the genus Humicola, and the like. There are cellulase formulations derived from Commercially available products of such cellulase preparations are all trade names, for example, cellulosin T2 (manufactured by HIPI), mecerase (manufactured by Meiji Seika Co., Ltd.), Novozyme 188 (manufactured by Novozyme), multifect CX10L (manufactured by Genencor) ), GC220 (manufactured by Genencor).
0.5-100 mass parts is preferable and, as for the usage-amount of the cellulase formulation with respect to 100 mass parts of raw material solid content, 1-50 mass parts is especially preferable.
糖化工程又は併行糖化発酵工程での反応液のpHは3.5〜10.0の範囲に維持することが好ましく、4.0〜7.5の範囲に維持することがより好ましい。 The pH of the reaction solution in the saccharification step or the concurrent saccharification and fermentation step is preferably maintained in the range of 3.5 to 10.0, more preferably in the range of 4.0 to 7.5.
糖化工程または併行糖化発酵工程での反応液の温度は、酵素の至適温度の範囲内であれば特に制限はなく、25〜50℃が好ましく、30〜40℃がさらに好ましい。反応は、連続式が好ましいが、セミバッチ式、バッチ式でも良い。反応時間は、酵素濃度によっても異なるが、バッチ式の場合は10〜240時間、さらに好ましくは15〜160時間である。連続式の場合も、平均滞留時間が、10〜150時間、さらに好ましくは15〜100時間である。 The temperature of the reaction solution in the saccharification step or the concurrent saccharification and fermentation step is not particularly limited as long as it is within the optimum temperature range of the enzyme, preferably 25 to 50 ° C, and more preferably 30 to 40 ° C. The reaction is preferably continuous, but may be semi-batch or batch. The reaction time varies depending on the enzyme concentration, but in the case of a batch system, it is 10 to 240 hours, more preferably 15 to 160 hours. Also in the case of a continuous type, the average residence time is 10 to 150 hours, more preferably 15 to 100 hours.
<発酵工程>
糖化工程と発酵工程を別の反応槽で行う場合は、前記糖化工程後の処理液は、発酵工程へ移送し発酵微生物を用いて発酵を行う。
<Fermentation process>
When performing a saccharification process and a fermentation process with another reaction tank, the process liquid after the said saccharification process is transferred to a fermentation process, and fermentation is performed using a fermentation microorganism.
発酵工程、又は併行糖化発酵工程では、糖類(六炭糖、五炭糖)が発酵できる発酵微生物を用いる。発酵微生物としては、サッカロマイセス・セラビシエ(Saccharomyces cerevisiae)、ピキア・スティピティス(Pichia stipitis)、キャンディダ・シハタエ(Candida shihatae)、パチソレン・タノフィルス(Pachysolen tannophilus)、イサチェンキア・オリエンタリス(Issatchenkia orientalis)等の酵母やザイモモナス・モビリス(Zymomonas mobilis)等の細菌が挙げられる。また、遺伝子組換技術を用いて作製した遺伝子組換微生物(酵母、細菌等)を用いることもできる。遺伝子組換微生物としては、六炭糖と五炭糖を同時に発酵できる微生物を特に制限なく用いることができる。 In the fermentation process or the concurrent saccharification and fermentation process, fermented microorganisms that can ferment sugars (hexose sugar, pentose sugar) are used. Examples of fermenting microorganisms include Saccharomyces cerevisiae, Pichia stipais, and Candida shihatae sapiens. -Bacteria such as mobilis (Zymomonas mobilis). In addition, genetically modified microorganisms (yeast, bacteria, etc.) produced using genetic recombination techniques can also be used. As the genetically modified microorganism, a microorganism capable of simultaneously fermenting hexose and pentose can be used without particular limitation.
微生物は固定化しておいても良い。微生物を固定化しておくと、次工程で微生物を分離して再回収する工程を省くことができるため、少なくとも回収工程に要する負担を軽減することができ、微生物のロスが軽減できるというメリットがある。また、凝集性のある微生物を選択することにより微生物の回収を容易にすることができる。 Microorganisms may be immobilized. Immobilizing microorganisms can eliminate the step of separating and re-recovering microorganisms in the next step, so that at least the burden required for the recovery step can be reduced, and the loss of microorganisms can be reduced. . Moreover, the collection of microorganisms can be facilitated by selecting microorganisms having aggregating properties.
<第一固液分離工程>
前記併行糖化発酵工程又は発酵工程から排出された培養液は、固液分離装置へ移送し液体分(濾液)と固形分(残渣)に分離する。固液分離を行う装置としては、スクリュープレス、スクリーン、フィルタープレス、ベルトプレス、ロータリープレス等を用いることができる。スクリーンとしては、振動装置が付加された振動スクリーンなどを用いることができる。固液分離で用いるメッシュサイズは、1.25〜600メッシュが好ましく、60〜600メッシュがさらに好ましい。
<First solid-liquid separation process>
The culture solution discharged from the parallel saccharification and fermentation process or the fermentation process is transferred to a solid-liquid separator and separated into a liquid component (filtrate) and a solid component (residue). As an apparatus for performing solid-liquid separation, a screw press, a screen, a filter press, a belt press, a rotary press, or the like can be used. As the screen, a vibrating screen to which a vibrating device is added can be used. The mesh size used in the solid-liquid separation is preferably 1.25 to 600 mesh, more preferably 60 to 600 mesh.
<オゾン処理>
本発明では、前記固液分離装置で分離した残渣をオゾン処理する。オゾン処理の方法としては一般に用いられるオゾン発生装置等によりオゾンを製造し、水溶液に懸濁した状態の残渣懸濁液にオゾンを吹き込む。残渣懸濁液の固形分濃度は、1〜30質量%が好ましく、5〜20質量%がさらに好ましい。残渣懸濁液中のオゾン濃度は、1〜300mg/Lが好ましく、20〜200mg/Lがさらに好ましい。オゾン処理中の残渣懸濁液を攪拌することが残渣とオゾンの接触が良くなるため望ましい。固形分濃度が30質量%を超えると残渣の攪拌が困難になるため好ましくない。上記の方法で糖化(又は併行糖化発酵)処理後の未分解残渣(リグニン含有)にオゾン処理を行うことにより、未分解残渣に吸着している酵素が遊離されて酵素を効率的に回収することができる。オゾン処理により、未分解残渣に含まれるリグニンが酸化し帯電することにより残渣に吸着している酵素と反発し合い酵素が遊離される。
オゾン処理した後の残渣懸濁液は、酵素を含有する水溶液の状態で糖化工程又は併行糖化発酵工程へ循環させて糖化又は糖化発酵の原料として用いることもできる。残渣懸濁液を第二固液分離工程で固液分離装置により固形分(残渣)と液体分(酵素含有)に分離し、固形分と液体分を別々に糖化工程又は併行糖化発酵工程へ循環させることもできるし、液体分のみを糖化工程又は併行糖化発酵工程へ循環させても良い。
<Ozone treatment>
In the present invention, the residue separated by the solid-liquid separator is subjected to ozone treatment. As an ozone treatment method, ozone is produced by a generally used ozone generator or the like, and ozone is blown into a residue suspension suspended in an aqueous solution. The solid content concentration of the residue suspension is preferably 1 to 30% by mass, and more preferably 5 to 20% by mass. The ozone concentration in the residue suspension is preferably 1 to 300 mg / L, more preferably 20 to 200 mg / L. It is desirable to stir the residue suspension during the ozone treatment because the contact between the residue and ozone is improved. If the solid content concentration exceeds 30% by mass, it is not preferable because stirring of the residue becomes difficult. By performing ozone treatment on the undegraded residue (containing lignin) after saccharification (or concurrent saccharification and fermentation) treatment by the above method, the enzyme adsorbed on the undegraded residue is released and the enzyme is efficiently recovered. Can do. By the ozone treatment, the lignin contained in the undegraded residue is oxidized and charged, and repels the enzyme adsorbed on the residue and releases the enzyme.
The residual suspension after the ozone treatment can also be used as a raw material for saccharification or saccharification and fermentation by circulating it to the saccharification step or concurrent saccharification and fermentation step in the state of an aqueous solution containing an enzyme. The residue suspension is separated into solid (residue) and liquid (enzyme-containing) by the solid-liquid separator in the second solid-liquid separation process, and the solid and liquid are separately circulated to the saccharification process or parallel saccharification and fermentation process. Alternatively, only the liquid component may be circulated to the saccharification process or the concurrent saccharification and fermentation process.
第一固液分離工程で分離された液体分(濾液)は蒸留工程へ移送される。 The liquid component (filtrate) separated in the first solid-liquid separation step is transferred to the distillation step.
<エタノール蒸留工程>
併行糖化発酵工程、又は発酵工程から排出された培養液は、蒸留工程で減圧蒸留装置等のエタノール分離装置により発酵生成物(エタノール等)を蒸留分離することができる。減圧下では低い温度で発酵生成物を分離できるため、酵素の失活を防ぐことができる。減圧蒸留装置としては、ロータリーエバポレーター、フラッシュエバポレーターなどを用いることができる。
蒸留温度は25〜60℃が好ましい。25℃未満であると、生成物の蒸留に時間がかかって生産性が低下する。また、60℃より高いと、酵素が熱変性して失活してしまい、新たに追加する酵素量が増加するため経済性が悪くなる。
蒸留後の蒸留残渣留分中に残る発酵生成物濃度は0.1質量%以下であることが好ましい。このような濃度にすることによって、後段の固液分離工程において固形物とともに排出される発酵生成物量を低減することができ、収率を向上させることができる。
<Ethanol distillation process>
The culture solution discharged from the concurrent saccharification and fermentation process or the fermentation process can be subjected to distillation separation of a fermentation product (ethanol or the like) by an ethanol separation apparatus such as a vacuum distillation apparatus in the distillation process. Since the fermentation product can be separated at a low temperature under reduced pressure, inactivation of the enzyme can be prevented. As the vacuum distillation apparatus, a rotary evaporator, a flash evaporator, or the like can be used.
The distillation temperature is preferably 25 to 60 ° C. If it is lower than 25 ° C., it takes time to distill the product, and the productivity is lowered. On the other hand, when the temperature is higher than 60 ° C., the enzyme is heat-denatured and deactivated, and the amount of newly added enzyme increases, resulting in poor economic efficiency.
The concentration of the fermentation product remaining in the distillation residue fraction after distillation is preferably 0.1% by mass or less. By setting it as such a density | concentration, the amount of fermentation products discharged | emitted with a solid substance in a subsequent solid-liquid separation process can be reduced, and a yield can be improved.
蒸留後の発酵生成物(エタノール等)を分離した後の蒸留残液は、固液分離装置により残渣と液体分に分離することができる。分離された残渣には、酵素、リグニン、発酵微生物が含まれている。残渣に吸着している酵素を遊離させて回収し、再利用することもできる。リグニンは、燃焼原料として回収しエネルギーとして利用することもできるし、リグニンを回収し有効利用することもできる。発酵微生物(酵母など)を残渣から分離して、糖化又は併行糖化発酵工程で再利用することもできる。 The distillation residue after separation of the fermentation product (such as ethanol) after distillation can be separated into a residue and a liquid by a solid-liquid separator. The separated residue contains enzymes, lignin, and fermenting microorganisms. The enzyme adsorbed on the residue can be released and recovered and reused. Lignin can be recovered as a combustion raw material and used as energy, or lignin can be recovered and used effectively. Fermentation microorganisms (such as yeast) can be separated from the residue and reused in the saccharification or concurrent saccharification and fermentation process.
次に実施例を示して本発明を更に詳細に説明するが、本発明はこれらの実施例等によって限定されるものではない。 EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited by these Examples.
[実施例1]
図1に示す製造フローで実施した。
[前処理]
チップ状のユーカリ・グロブラスの林地残材(樹皮70%、枝葉30%)を20mmの丸孔スクリーンを取り付けた一軸破砕機(西邦機工社製、SC−15)で破砕し原料として用いた。
[化学的処理]
上記原料1kg(絶乾重量)に対して97%亜硫酸ナトリウム200g及び水酸化ナトリウム10gを添加後、水を添加し水溶液の容量を8Lに調製した。前記原料懸濁液を混合後、120℃で1時間加熱した。加熱処理後の原料懸濁液をレファイナー(熊谷理器工業製、KRK高濃度ディスクレファイナー)でディスク(プレート)のクリアランスを1.0mmに設定し磨砕した。次に60メッシュ(250μm)のスクリーンを用いて磨砕処理後の原料懸濁液を固液分離(脱水)することにより溶液の電気伝導度が30μS/cmになるまで水で洗浄した。固液分離後の固形分を原料として併行糖化発酵を行った。
[Example 1]
The manufacturing flow shown in FIG.
[Preprocessing]
Chip-like eucalyptus and globula woodland residues (70% bark, 30% branches and leaves) were crushed with a uniaxial crusher (SC-15, manufactured by Saiho Kiko Co., Ltd.) equipped with a 20 mm round hole screen and used as a raw material.
[Chemical treatment]
After adding 200 g of 97% sodium sulfite and 10 g of sodium hydroxide to 1 kg (absolute dry weight) of the raw material, water was added to adjust the volume of the aqueous solution to 8 L. The raw material suspension was mixed and then heated at 120 ° C. for 1 hour. The raw material suspension after the heat treatment was crushed with a refiner (manufactured by Kumagai Riki Kogyo Co., Ltd., KRK high concentration disc refiner) with the disc (plate) clearance set to 1.0 mm. Next, the raw material suspension after the grinding treatment was subjected to solid-liquid separation (dehydration) using a 60 mesh (250 μm) screen, and washed with water until the electric conductivity of the solution reached 30 μS / cm. Simultaneous saccharification and fermentation was performed using the solid content after solid-liquid separation as a raw material.
[併行糖化発酵]
予め、液体培地(グルコース30g/L、ポリペプトン5g/L、酵母エキス3g/L、麦芽エキス3g/L、pH5.6)50Lで酵母としてSaccharomyces cerevisiae (市販酵母、商品名:商品名:Maurivin: Mauri Yeast Australia Pty Limited)を30℃で24時間培養した。
図1に示す糖化発酵槽BR1にポリペプトン5g/L、酵母エキス3g/L、麦芽エキス3g/Lとなるように各々を添加後,水を添加し最終容量を0.8m3に調整した。酵母菌体を含む培養液を糖化発酵槽BR1に添加し24時間培養した。酵母の密度が、1x108/mlに増殖した時点で、市販セルラーゼ溶液(マルティフェクトCX10L、ジェネンコア社製)50L、及び原料100kg(乾燥重量)を糖化発酵槽BR1に添加した。次に、糖化発酵槽BR1に水を添加し培養液の最終容量を1m3に調製した。培養液のpHを5.0に調整し30℃で併行糖化発酵を開始した。糖化発酵槽BR1内での培養液の滞留時間(原料懸濁液が糖化発酵槽BR1を通過する時間:糖化発酵槽BR1の容量/流速)を20時間に設定し糖化発酵行った。すなわち、糖化発酵を開始した時点から、固形分濃度(乾燥重量当たり)が10質量%の原料懸濁液(原料を水に懸濁)を流速50L/hで糖化発酵槽BR1の原料供給口1から連続的に添加した。一方、原料供給開始と同時に糖化発酵槽BR1の培養液排出口2より原料懸濁液を50L/hで排出し、固液分離工程へ移送した。また、前記セルラーゼ溶液を2.5L/hで糖化発酵槽BR1に連続的に添加した。尚、連続運転中に培養液が減少した場合、自動的に培地を添加することにより培養液の最終容量を1m3に維持した。培養中の培養液のpHを5.0に維持した。
[Concurrent saccharification and fermentation]
Saccharomyces cerevisiae (commercially available yeast, trade name: trade name: Maurivin: Mauri) as yeast in a liquid medium (glucose 30 g / L, polypeptone 5 g / L, yeast extract 3 g / L, malt extract 3 g / L, pH 5.6) in advance. Yeast Australia Pty Limited) was cultured at 30 ° C. for 24 hours.
1 was added to the saccharification and fermentation tank BR1 shown in FIG. 1 so that the polypeptone was 5 g / L, the yeast extract was 3 g / L, and the malt extract was 3 g / L, and water was added to adjust the final volume to 0.8 m 3 . A culture solution containing yeast cells was added to the saccharification and fermentation tank BR1 and cultured for 24 hours. When the density of yeast grew to 1 × 10 8 / ml, 50 L of a commercially available cellulase solution (Multifect CX10L, Genencor) and 100 kg (dry weight) of raw material were added to the saccharification and fermentation tank BR1. Next, water was added to the saccharification and fermentation tank BR1 to adjust the final volume of the culture solution to 1 m 3 . The pH of the culture solution was adjusted to 5.0, and concurrent saccharification and fermentation was started at 30 ° C. Saccharification and fermentation were carried out by setting the retention time of the culture solution in the saccharification and fermentation tank BR1 (time for the raw material suspension to pass through the saccharification and fermentation tank BR1: capacity / flow rate of the saccharification and fermentation tank BR1) to 20 hours. That is, from the start of saccharification and fermentation, a raw material suspension (raw material suspended in water) having a solid content concentration (per dry weight) of 10% by mass at a flow rate of 50 L / h is a raw material supply port 1 of the saccharification and fermentation tank BR1. From continuously. On the other hand, the raw material suspension was discharged at 50 L / h from the culture liquid outlet 2 of the saccharification and fermentation tank BR1 simultaneously with the start of the raw material supply, and transferred to the solid-liquid separation process. The cellulase solution was continuously added to the saccharification and fermentation tank BR1 at 2.5 L / h. When the culture solution decreased during continuous operation, the final volume of the culture solution was maintained at 1 m 3 by automatically adding a medium. The pH of the culture medium during the culture was maintained at 5.0.
[固液分離]
前記一次併行糖化発酵工程から排出された原料懸濁液を、固液分離装置S:スクリュープレス(富国工業株式会社製SHX−200 x 1500L、スクリーンサイズ1.2mm(14メッシュ))で固液分離して固形分と液体分(濾液)を分離した。固形分については前記併行糖化発酵工程へ供給し糖化の原料として再利用した。一方、液体分については、蒸留工程へ移送した。
[Solid-liquid separation]
The raw material suspension discharged from the primary saccharification and fermentation step is subjected to solid-liquid separation using a solid-liquid separator S: screw press (SHX-200 x 1500 L, screen size 1.2 mm (14 mesh) manufactured by Togoku Industry Co., Ltd.). Thus, the solid content and the liquid content (filtrate) were separated. The solid content was supplied to the parallel saccharification and fermentation process and reused as a raw material for saccharification. On the other hand, the liquid component was transferred to the distillation process.
[エタノール蒸留]
前記固液分離で分離した液体分を減圧蒸留装置EV(エバポールCEP−1、大川原製作所)で蒸留温度:40℃、加熱温度:80℃、供給液量:95L/hの条件でエタノールを含む水溶液と濃縮液に分離した。
[Ethanol distillation]
The liquid component separated by the solid-liquid separation is an aqueous solution containing ethanol at a distillation temperature of 40 ° C., a heating temperature of 80 ° C., and a supply liquid amount of 95 L / h using a vacuum distillation apparatus EV (Evapor PEP-1, Okawara Seisakusho). And concentrated.
[残渣分離]
減圧蒸留装置EVから分離された濃縮液をデカンタ式遠心機(IHI製、HS−204L形)で回転数4500rpm、差速5.0rpmで運転し、固形分(残渣A)と液体分(濾液)に分離した。液体分はライン7を経由して糖化発酵槽BR1へ移送した。
[Residue separation]
The concentrated liquid separated from the vacuum distillation apparatus EV is operated at a rotational speed of 4500 rpm and a differential speed of 5.0 rpm in a decanter centrifuge (IHI, HS-204L type), and the solid content (residue A) and liquid content (filtrate) Separated. The liquid was transferred to the saccharification and fermentation tank BR1 via line 7.
[残渣のオゾン処理]
前記で分離した残渣Aを水に懸濁し10質量%濃度の残渣懸濁液を調製した。オゾン発生装置により製造したオゾンを原料懸濁液に吹き込み、原料懸濁液に含まれるオゾン濃度が100mg/Lになるようにし30分間処理した。次に400メッシュ(32μm)のスクリーンを用いて残渣懸濁液を固液分離により固形分(残渣B)と液体分に分離することにより溶液の電気伝導度が30μS/cmになるまで水で洗浄した。固液分離後の残渣Bを原料として下記の方法で酵素(セルラーゼ)吸着抑制試験を実施した。結果を表1に示す。
[Ozone treatment of residue]
Residue A separated above was suspended in water to prepare a 10% strength by weight residue suspension. Ozone produced by the ozone generator was blown into the raw material suspension, and the treatment was performed for 30 minutes such that the ozone concentration contained in the raw material suspension was 100 mg / L. Next, the residue suspension is separated into solid (residue B) and liquid by solid-liquid separation using a 400 mesh (32 μm) screen, and washed with water until the electric conductivity of the solution reaches 30 μS / cm. did. Using the residue B after solid-liquid separation as a raw material, an enzyme (cellulase) adsorption inhibition test was carried out by the following method. The results are shown in Table 1.
[酵素(セルラーゼ)吸着抑制試験]
原料(残渣B)の最終濃度5質量%、CSL(コーンスティープリカー)を最終濃度1質量%、硫酸アンモニウムを最終濃度0.5質量%、となるように調製した水溶液にセルラーゼ10ml(商品名、GC220:ジェネンコア社製)を添加した。30℃、120rpmの攪拌下で糖化反応を行い、24時間後の反応液1mlを回収し、10,000rpmで5分間遠心分離した上清の酵素活性を測定した。
[Enzyme (cellulase) adsorption inhibition test]
10 ml of cellulase (trade name, GC220) was added to an aqueous solution prepared such that the final concentration of the raw material (residue B) was 5% by mass, the final concentration of CSL (corn steep liquor) was 1% by mass, and the final concentration of ammonium sulfate was 0.5% by mass. : Genencor Corporation) was added. The saccharification reaction was performed under stirring at 30 ° C. and 120 rpm, and 1 ml of the reaction solution after 24 hours was collected, and the enzyme activity of the supernatant centrifuged at 10,000 rpm for 5 minutes was measured.
酵素回収で最も重要とされるベータ−グルコシダーゼの活性を指標にして回収率を算出した。活性測定は以下に示す方法で行った。結果を表1に示す。
(ベータ−グルコシダーゼ活性)
ベータ−グルコシダーゼ活性の測定は、1.25mM 4−Methyl−umberiferyl−glucosideを含む125mM酢酸緩衝液(pH5.0)16μlに、酵素液4ul加え、37℃、10分間反応を行った後、500mM glycine−NaOH緩衝液(pH10.0)100μlを添加して反応を停止させ、350nmの励起光での460nmの蛍光強度を測定することで行った。酵素回収率は以下の計算式から求めた。
酵素回収率(%)=(上清の酵素活性/添加した酵素活性)x 100
The recovery rate was calculated using the activity of beta-glucosidase, which is most important in enzyme recovery, as an index. Activity measurement was performed by the method shown below. The results are shown in Table 1.
(Beta-glucosidase activity)
The beta-glucosidase activity was measured by adding 4 μl of enzyme solution to 16 μl of 125 mM acetate buffer (pH 5.0) containing 1.25 mM 4-methyl-mberiferyl-glucoside, followed by reaction at 37 ° C. for 10 minutes, and then 500 mM glycine. -The reaction was stopped by adding 100 µl of NaOH buffer (pH 10.0), and the fluorescence intensity at 460 nm with 350 nm excitation light was measured. The enzyme recovery rate was determined from the following formula.
Enzyme recovery rate (%) = (enzyme activity of supernatant / added enzyme activity) × 100
[実施例2]
実施例1([化学的処理])において、原料1kg(絶乾重量)に対して水酸化カルシウム200gを添加し化学的処理を行った以外は全て実施例1と同様の方法で試験した。結果を表1に示す。
[Example 2]
In Example 1 ([chemical treatment]), all tests were performed in the same manner as in Example 1 except that 200 g of calcium hydroxide was added to 1 kg (absolute dry weight) of the raw material and subjected to chemical treatment. The results are shown in Table 1.
[実施例3]
実施例1([化学的処理])において、原料1kg(絶乾重量)に対して水酸化ナトリウム200gを添加し化学的処理を行った以外は全て実施例1と同様の方法で試験した。結果を表1に示す。
[Example 3]
In Example 1 ([Chemical treatment]), all tests were performed in the same manner as in Example 1 except that 200 g of sodium hydroxide was added to 1 kg (absolute dry weight) of the raw material to perform chemical treatment. The results are shown in Table 1.
[実施例4]
実施例1([化学的処理])において、原料1kg(絶乾重量)に対して水酸化ナトリウム100gを添加し化学的処理を行った以外は全て実施例1と同様の方法で試験した。結果を表1に示す。
[Example 4]
In Example 1 ([chemical treatment]), all tests were performed in the same manner as in Example 1 except that 100 g of sodium hydroxide was added to 1 kg (absolute dry weight) of the raw material for chemical treatment. The results are shown in Table 1.
[比較例1]
実施例1において、残渣Aのオゾン処理を行わない試験を比較例1とした。尚、実施例1で調製した残渣Aを用いて酵素(セルラーゼ)吸着抑制試験を実施した。それ以外の操作は全て実施例1と同様の方法で試験した。結果を表1に示す。
[Comparative Example 1]
In Example 1, the test in which the residue A was not subjected to ozone treatment was referred to as Comparative Example 1. In addition, the enzyme (cellulase) adsorption | suction suppression test was implemented using the residue A prepared in Example 1. FIG. All other operations were tested in the same manner as in Example 1. The results are shown in Table 1.
[比較例2]
実施例2において、残渣Aのオゾン処理を行わない試験を比較例2とした。尚、実施例2で調製した残渣Aを用いて酵素(セルラーゼ)吸着抑制試験を実施した。それ以外の操作は全て実施例2と同様の方法で試験した。結果を表1に示す。
[Comparative Example 2]
In Example 2, the test in which the residue A was not subjected to ozone treatment was referred to as Comparative Example 2. In addition, the enzyme (cellulase) adsorption | suction suppression test was implemented using the residue A prepared in Example 2. FIG. All other operations were tested in the same manner as in Example 2. The results are shown in Table 1.
[比較例3]
実施例3において、残渣Aのオゾン処理を行わない試験を比較例3とした。尚、実施例3で調製した残渣Aを用いて酵素(セルラーゼ)吸着抑制試験を実施した。それ以外の操作は全て実施例3と同様の方法で試験した。結果を表1に示す。
[Comparative Example 3]
In Example 3, a test in which the residue A was not subjected to ozone treatment was defined as Comparative Example 3. In addition, the enzyme (cellulase) adsorption | suction suppression test was implemented using the residue A prepared in Example 3. FIG. All other operations were tested in the same manner as in Example 3. The results are shown in Table 1.
[比較例4]
実施例4において、残渣Aのオゾン処理を行わない試験を比較例4とした。尚、実施例4で調製した残渣Aを用いて酵素(セルラーゼ)吸着抑制試験を実施した。それ以外の操作は全て実施例4と同様の方法で試験した。結果を表1に示す。
[Comparative Example 4]
In Example 4, the test in which the residue A was not subjected to ozone treatment was referred to as Comparative Example 4. In addition, the enzyme (cellulase) adsorption | suction suppression test was implemented using the residue A prepared in Example 4. FIG. All other operations were tested in the same manner as in Example 4. The results are shown in Table 1.
表1に示すように、糖化処理で分解されなかった未分解残渣をオゾン処理した試験では未分解残渣に吸着している酵素が遊離された。 As shown in Table 1, in the test in which the undecomposed residue that was not decomposed by the saccharification treatment was treated with ozone, the enzyme adsorbed on the undegraded residue was released.
本発明により、糖化処理で分解されなかった未分解残渣に吸着した酵素を効率的に回収することができる。酵素糖化処理工程内における糖化酵素の循環率が長期にわたって高い水準に維持されるため、リグノセルロース原料の酵素糖化処理による糖類やエタノール等を工業的に生産することが可能となる。 According to the present invention, the enzyme adsorbed on the undecomposed residue that has not been decomposed by the saccharification treatment can be efficiently recovered. Since the circulation rate of the saccharifying enzyme in the enzymatic saccharification treatment process is maintained at a high level over a long period of time, it becomes possible to industrially produce saccharides, ethanol and the like by the enzymatic saccharification treatment of the lignocellulose raw material.
1:原料供給口
2:併行糖化発酵槽排出口
3:固形分移送ライン
4:液体分移送ライン
5:蒸留後濃縮液移送ライン
6:残渣排出ライン
7:液体分循環ライン
BR1:併行糖化発酵槽
S:固液分離装置
EV:減圧蒸留装置
C:残渣分離装置
1: Raw material supply port 2: Parallel saccharification and fermentation tank discharge port 3: Solid content transfer line 4: Liquid content transfer line 5: Concentrated liquid transfer line after distillation 6: Residue discharge line 7: Liquid content circulation line BR1: Parallel saccharification and fermentation tank S: Solid-liquid separator EV: Vacuum distillation apparatus C: Residue separator
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KR20190027282A (en) | 2017-09-06 | 2019-03-14 | 한국화학연구원 | Preparing method of saccharification residue for biopolymer |
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