METHOD FOR THE TREATMENT OF ENZYMATIC SACARIFICATION OF A RAW MATERIAL BASED ON LIGNOCELLULOSIS

专利摘要:
method for the treatment of enzymatic saccharification of a raw material based on lignocellulose a method for the treatment of enzymatic saccharification of a raw material based on lignocellulose is provided, in which a raw material based on lignocellulose which has been subjected to a pretreatment to produce the appropriate raw material for an enzymatic saccharification reaction is added, along with an electrolyte containing a water-soluble salt, to the water containing cellulose saccharification enzyme; the raw material based on lignocellulose as a suspension of raw material whose electrical conductivity has been adjusted from 5 ms / cm to 25 ms / cm, is subjected to an enzymatic saccharification treatment through an enzymatic saccharification reaction; the reaction product and an enzyme-containing solution are separated and recovered from the treated suspension after the enzymatic saccharification treatment; and the recovered enzyme-containing solution is recycled as an enzyme for the enzymatic saccharification treatment process.
公开号:BR112013004261B1
申请号:R112013004261-3
申请日:2011-08-31
公开日:2021-04-06
发明作者:Kotaro Ishikawa；Atsushi Furujyo；Yaping Chao；Hisako Tokuno；Jun Sugiura；Motohiro Matsumura
申请人:Oji Holdings Corporation；
IPC主号:

专利说明:

[0001] The present invention relates to a method for the treatment of enzymatic saccharification of a raw material based on lignocellulose or a method for the production of ethanol from a raw material based on lignocellulose, both methods with a step of pre-treatment of subjecting a lignocellulose-based raw material to a treatment to convert the lignocellulose-based raw material into an appropriate raw material for an enzymatic saccharification reaction; and an enzymatic saccharification step of saccharifying a raw material based on lignocellulose pretreated with an enzyme. More particularly, the invention relates to a method for the treatment of enzymatic saccharification of a lignocellulose-based biomass, the method including a reaction of saccharifying a biomass containing lignocellulose which has undergone an appropriate treatment for saccharification, using a group of enzymes which consists of cellulolytic enzymes or hemicellulolytic enzymes, and the method making it possible to recover the group of enzymes used with a high recovery rate and to recycle the group of enzymes for a long period of time; or a method for the production of ethanol from a biomass containing lignocellulose, the method making it possible to increase ethanol production by recovering fine fibers that remain in the treated suspension after a parallel fermentation and saccharification treatment, and submitting the recovered fine fibers a saccharification treatment or a parallel fermentation and saccharification treatment.
[0002] Priority is claimed in Japanese patent application No. 2010-193310, filed on August 31, 2010; Japanese patent application No. 2010-254441, filed on November 15, 2010; Japanese patent application No. 2010-274235, filed on December 9, 2010; Japanese patent application No. 2011-075772, filed on March 30, 2011; Japanese patent application No. 2011-107820, filed on May 13, 2011; and Japanese patent application No. 2011-123976, filed on June 2, 2011, the contents of which are incorporated herein by reference. BACKGROUND OF THE INVENTION
[0003] A technology for producing saccharides from a lignocellulose raw material that has undergone an appropriate treatment for saccharification is a beneficial technology for the formation of a recycling-oriented society, since alcohols that can be used as a substitute fuel gasoline, or chemical raw materials such as succinic acid and lactic acid that can be used with raw materials for plastics, can be produced using saccharides as the fermentation substrates for microorganisms.
[0004] Methods to produce monosaccharides or oligosaccharides that can be used as substrates for fermentation of polysaccharides in a plant-based biomass can be roughly classified into two types. One type is an acid saccharification method of hydrolyzing polysaccharides using a mineral acid, and the other type is an enzymatic saccharification method of hydrolyzing polysaccharides using an enzyme or microorganism that produces the enzyme.
[0005] The acid saccharification method is technically complete, compared to the enzymatic saccharification method. However, in the case of the method using a biomass based on lignocellulose as a raw material, the saccharide yield is low compared to the method of using starch or molasses as a raw material, and also the fact that a facility is required to treat residual acids discharged from the treatment processes, or a large facility capable of withstanding acid corrosion causes an increase in the cost of the product, so this presents a serious problem for practical application.
[0006] On the other hand, with regard to the enzymatic saccharification method, due to the decrease in the price of enzymes in recent years and the progress of technology, the total cost including post-treatments is becoming closer to the cost of the acid saccharification method. However, because the price of enzymes which is responsible for a high proportion of the total cost of the enzymatic saccharification method is still high, in order to obtain practical application of the enzymatic saccharification method, an additional decrease in the cost of enzymes is important.
[0007] As a technology to lower the cost of the enzymatic saccharification method, the development of a method for a pretreatment that facilitates the access of an enzyme in cellulose fibers, or the development of a method of efficiently saccharifying crystalline cellulose, and the development of a method for efficient enzyme recovery and reuse can be considered.
[0008] A lignocellulose material from which lignin has not been removed is not easily degraded by enzymes, compared to a lignocellulose material from which lignin has been removed, and is not saccharified so that the lignocellulose material from which lignin has not been removed remains as a residue in the saccharification liquid together with impurities such as resins and metals. In general, this residue is separated by sieving, centrifuging or the like, and is discarded. Since this residue contains a large amount of adsorbed enzymes, which occupy a large proportion of the cost in the enzymatic saccharification method, there is a problem that, if the residue separated from the reaction liquid is directly discarded, the highly expensive enzymes will also be discarded. In other words, it is desired to recover and effectively use the residue in order to reduce the cost of the enzymatic saccharification method. Regarding the technology of reusing the waste recovered in the enzymatic saccharification method, there are reports of a method of combustion of the waste and obtaining thermal energy (PTL 5), a method of subjecting the residue to hydrothermal gasification, and synthesizing ethanol from synthesis gas produced using an ethanol synthesis catalyst (PTL 6), a method of using the residue as a fuel or fertilizer (PTL 7), and a method of using the residue as thermal energy (PTL 8). However, since these methods cause a large increase in cost due to the addition of treatment processes, in the case of designing a practically used facility, it cannot be said that these methods are satisfactory as methods to address the problem of cost reduction.
[0009] As a means of recovering enzymes in the residue as described above, washing of the residue can be taken into account. However, since the enzymes are firmly bound in cellulose through the cellulose binding domain (CBD) that specifically adsorbes on cellulose, which is carried by the enzymes in the molecules, it has been difficult to sufficiently recover the enzymes that have been adsorbed on cellulose, for example. washing of natural water.
[0010] Thus, in order to improve the recovery rate of the enzymes, a method of treating the enzymes has been suggested by adding a surfactant (see PTL 1), and the like. However, even in the surfactant treatment method, the enzyme recovery rate cannot be said to be satisfactory, and the method is not practical from the point of view that there is an interest in deactivating the enzymes due to the addition of substances chemicals, an increase in cost due to the addition of treatment processes, and the adverse effects on microorganisms in the fermentation step that follows.
[0011] As the method of recovering enzymes from a saccharide solution, a method of using ultrafiltration (see PTL 2), a method of adsorbing and recovering enzymes by adding cellulose back to the saccharide solution (see PTL 3), and the like, has been suggested. However, the ultrafiltration method has a problem that fine impurities clog the filtration membrane and thus sufficient treatment speed and sufficient enzyme recovery rate cannot be achieved, and it is difficult to achieve sufficient enzyme recovery with recovery method by adding cellulose.
[0012] A method of reusing a lignocellulose residue in which the enzymes are adsorbed in the subsequent batch of enzymatic saccharification has been suggested, without going through a step of separating the adsorbed enzymes (PTL 4).
[0013] In this method, since the accumulation of the residue cannot be avoided, there is a concern for a decrease in the efficiency of the reaction. In addition, along with enzymes such as CBD, such as CBH (cellobiohydrase), recycling of the enzymes can be achieved by pretreating the lignocellulose residue in the subsequent batch; however, since there are times when β-glucosidases and the like are released into the supernatant, it is difficult to recycle all the cellulases that have been added.
[0014] Also, in this method, because the non-degraded residue itself is in a state that is not easily degradable even if mixed again with an enzyme solution, it is desired to bring the non-degraded residue to a state that is easily saccharifiable. The inventors of the present invention have observed that when an undegraded waste recovered by solid-liquid separation is mechanically treated and subjected to saccharification and fermentation again, ethanol production is increased (PTL 9). However, this method has a problem that the waste that was used as a raw material is a waste recovered using a 420 mesh sieve (38-μm) and includes fibers of a wide range of sizes. Large-sized fibers with a large amount of adsorbed lignin are not easily degraded by enzymes, and therefore are not sufficiently saccharified if not subjected to a pretreatment (mechanical treatment or the like). If only those fibers of small sizes with a small amount of adsorbed lignin can be selectively recovered and enzymatically saccharified again as a raw material without undergoing a pretreatment, an improvement in ethanol production can be expected with high efficiency.
[0015] As a method of lowering the cost of enzymes, methods of recycling enzymes have been reported. According to the method of Scott, CD and collaborators (Not PTL 1), a continuous system that is contemplated, with a recycling line with a grinding device based on a high speed centrifugal pump that, in a reaction tank main enzyme that enzymatically hydrolyzes an old paper raw material, adding a large amount of an enzyme (80 to 160 units relative to 1 g of a substrate in terms of filter paper degradation activity), removes the glucose and cellobiose components produced with high shear strength on the surface of unreacted old paper in the enzymatic hydrolyzate and, thereby, always exposes new surfaces of cellulose fibers; a membrane separation apparatus that separates the unreacted raw material and the hydrolyzate from the treatment liquid from the grinding apparatus, and circulates only the unreacted raw material in the main reaction tank; and a filtering apparatus that separates the enzymes and glucose and cellobiose produced from the hydrolyzate from the membrane separation apparatus, and circulates only the enzymes to the main reaction tank, and the cost is predictable. According to this system, the saccharification ratio is 100% in 25 hours, and the residual enzyme ratio is 95% or greater in 24 hours. Also, it is described that enzymes adsorb in the residue and are eliminated, that the adsorbable function of the enzymes in the residue can be decreased by increasing the pH from 5 to 7, and it is reported that the adsorbable function of the enzymes can be decreased by lowering the temperature to 5 °. Ç.
[0016] As a method of recovering and reusing enzymes, a method has been reported in which birch wood that has been treated with steam blasting is added to a saccharification tank at a concentration of 5%, 20,000 units of a cellulase are added to it, a saccharide solution and enzyme solution are separated by ultrafiltration and, while the enzyme is recovered and reused, 630 g of monosaccharides are obtained from 2 kg of birch wood for 8 days. It is considered that the amount of the enzyme used can be saved by 20% by this method (Not PTL 2). CITATION LIST PATENT LITERATURE
[0017] [PTL 1] JP-A-63-87994 [PTL 2] JP-A-61-234790 [PTL 3] JP-A-55-144885 [PTL 4] JP-A-2010-98951 [PTL 5] Japanese patent No. 4447148 [PTL 6] JP-A-2005-168335 [PTL 7] JP-A-2008-54676 [PTL 8] JP-A-2009-106932 [PTL 9] Japanese patent application No. 2009-190862 Non-patent literature [NPL 1] Scott, CD, Rothrock, DS, Appl. Biochem. Biotechnol., 45/46, pp. 641-653 (1994) [NPL 2] Ishihara, M., et al., Biotechnol. Bioeng., 37, 948-954 (1991) SUMMARY OF THE INVENTION TECHNICAL PROBLEM
[0018] The technology of producing saccharides from a biomass of lignocellulose and the like is a technology capable of supplying new raw materials for fuels or plastics that were hitherto dependent on fossil resources, and is a technology that is particularly useful for the establishment of a recycling oriented society. As previously described, several technologies have been developed until then, but there is a problem that the technology is not economical, basically due to the high cost of the enzymes needed in saccharification.
[0019] As previously described, several attempts have been made to reduce the amount of enzymes used, recovering and repeatedly using the enzymes used in saccharification. However, due to the fact that the enzymes strongly adsorb in the residue generated at the time of saccharification, the recovery rate decreased, and the problem could not be solved. As such, the strong adsorption of enzymes in the residue generated at the time of enzymatic saccharification is the biggest problem at the time of enzyme recovery and, if this can be resolved, the recyclability of the enzymes can be improved, the cost can be reduced, and the Economic efficiency of the enzymatic saccharification treatment method can be much better. Therefore, it is an objective of the present invention to provide a method that can effectively use, without waste, the enzymes introduced for the treatment of enzymatic saccharification of a lignocellulose material, and to provide a method for producing ethanol with high ethanol yield in the production process. of ethanol using lignocellulose as a raw material. SOLUTION TO THE PROBLEM
[0020] In order to address the aforementioned problems, the inventors of the present invention conducted an investigation into the method of decreasing the cost, with respect to a process of continuously carrying out an enzymatic saccharification reaction, using repeatedly, with a higher recovery rate, an enzyme that is expensive is responsible for a very large proportion of the total cost, and as a result, the inventors obtained the following invention. The present invention is based on the idea that employing means to suppress the adsorption of enzymes in the lignocellulose raw material or the reaction residue in an enzymatic saccharification reaction liquid, is the means to facilitate the separation of enzymes from the liquid of the enzyme reaction after the enzymatic saccharification reaction is facilitated, and also to prevent the discharge of enzymes out of the system along with the waste that is discarded. Also, the inventors observed that when only the fine fibers that are easily saccharified from the residue included in the culture fluid of a parallel fermentation and saccharification process are recovered, and parallel fermentation and saccharification or saccharification is performed using the recovered fine fibers as a raw material, ethanol production is improved, and the amount of waste discharged in the process is reduced. Thus, the inventors have completed the present invention as described below.
[0021] (1) Um método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose, o método incluindo adicionar uma matéria-prima a base de lignocelulose que foi submetida a um pré-tratamento para produzir a matéria-prima apropriada para uma reação de sacarificação enzimática, junto com um eletrólito contendo um sal solúvel em água, em água contendo enzima de sacarificação de celulose; submeter à matéria-prima a base de lignocelulose como uma suspensão de matéria-prima, cuja condutividade elétrica foi ajustada em 5 mS/cm a 25 mS/cm, a um tratamento de sacarificação enzimática através de uma reação de sacarificação enzimática; separar e recuperar o produto da reação e uma solução contendo enzima da suspensão tratada depois do tratamento de sacarificação enzimática; e reciclar a solução contendo enzima recuperada como uma enzima para o processo de tratamento de sacarificação enzimática. (2) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em (1), em que o pré-tratamento para produzir a matéria-prima apropriada para uma reação de sacarificação enzimática é um pré-tratamento incluindo um tratamento químico de imergir a matéria-prima a base de lignocelulose em uma solução contendo uma substância química alcalina selecionada de hidróxido de sódio, hidróxido de potássio, hidróxido de cálcio, carbonato de sódio e hidrogenocarbonato de sódio, ou uma mistura destes, ou uma mistura de sulfito de sódio e um álcali como esse. (3) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em (1) ou (2), em que a matéria-prima a base de lignocelulose é resíduo de floresta. (4) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em (1) ou (2), em que a matéria-prima a base de lignocelulose é casca de árvore. (5) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em qualquer um de (1) a (4), em que o sal solúvel em água é pelo menos um sal solúvel em água selecionado de sais de metal alcalino e sais de metal alcalino terrosos. (6) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em qualquer um de (1) a (5), em que o sal solúvel em água é um sal selecionado do grupo que consiste em haletos, sulfatos, sulfitos, tiossulfatos, carbonatos, hidrogenocarbonatos, fosfatos, di-idrogenofosfatos, hidrogenodifosfatos, acetatos, e citratos de metais alcalinos e metais alcalinos terrosos. (7) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em qualquer um de (1) a (6), em que o método de tratamento de sacarificação enzimática é um método de submeter uma matéria-prima a base de lignocelulose a um tratamento de sacarificação enzimática, de acordo com uma série de processos incluindo uma etapa de pré-tratamento de submeter uma matéria-prima a base de lignocelulose a um tratamento para produzir a matéria-prima a base de lignocelulose apropriada para uma reação de sacarificação enzimática; uma etapa de tratamento de sacarificação enzimática de adicionar a matéria-prima a base de lignocelulose pré-tratada, junto com um eletrólito contendo um sal solúvel em água, em água contendo enzima de sacarificação de celulose, e tratar a matéria-prima a base de lignocelulose como uma suspensão de matéria-prima, cuja condutividade elétrica foi ajustada em 5 mS/cm a 25 mS/cm, através de uma reação de sacarificação enzimática; uma etapa de separação sólido-líquido de remover um resíduo sólido da suspensão tratada proveniente da etapa de tratamento de sacarificação enzimática; uma etapa de centrifugação de centrifugar a fração líquida proveniente da etapa de separação sólido-líquido, e, por meio disso, obtendo uma fração líquida que contém enzimas e sacarídeos e tem todo o resíduo restante removido; uma etapa de separação de membrana de separar a fração líquida proveniente da etapa de centrifugação em uma solução contendo enzima e uma solução contendo sacarídeo produzida; e uma etapa de reciclagem de enzima de reciclar e suprir a solução contendo enzima obtenível da etapa de separação de membrana à etapa de tratamento de sacarificação de enzima como uma fonte de enzima. (8) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em qualquer um de (1) a (7), em que a etapa de tratamento de sacarificação enzimática é uma etapa de tratamento de fermentação e sacarificação paralelas de realizar um tratamento com base em uma reação de sacarificação enzimática de uma matéria-prima a base de lignocelulose e um tratamento de fermentação de sacarídeos produzidos por um micro-organismo para fermentação em combinação usando uma preparação de celulase e um micro-organismo para fermentação que usa sacarídeos como um substrato de fermentação (= matéria-prima) em combinação, e, por meio disso, produzir um produto de fermentação junto com sacarídeos. (9) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em (8), em que o método de sacarificação enzimática é um método de submeter uma matéria-prima a base de lignocelulose a um tratamento de fermentação e sacarificação paralelas de acordo com uma série de processos incluindo uma etapa de pré-tratamento de submeter uma matéria-prima a base de lignocelulose a um tratamento para produzir a matéria-prima a base de lignocelulose apropriada para uma reação de sacarificação enzimática; uma etapa de tratamento de fermentação e sacarificação paralelas de adicionar a matéria-prima a base de lignocelulose pré-tratada, junto com um micro-organismo para fermentação que usa sacarídeos como um substrato de fermentação, e com um eletrólito contendo um sal solúvel em água, na água contendo enzima de sacarificação de celulose, e submeter a matéria-prima a base de lignocelulose como uma suspensão de matéria-prima, cuja condutividade elétrica foi ajustada em 5 mS/cm a 25 mS/cm, tanto a um tratamento de sacarificação enzimática quanto a um tratamento de fermentação de usar os sacarídeos produzidos como um substrato; uma etapa de separação sólido-líquido de remover um resíduo sólido da suspensão tratada proveniente da etapa de tratamento de sacarificação enzimática; uma etapa de destilação de separar e recuperar o produto de fermentação da fração líquida proveniente da etapa de separação sólido-líquido através de destilação; uma etapa de centrifugação de centrifugar o destilado residual proveniente da etapa de destilação para remover qualquer resíduo restante, e, por meio disso, obtendo uma fração líquida contendo enzimas e sacarídeos; uma etapa de separação de membrana de separar a fração líquida proveniente da etapa de centrifugação em uma solução contendo enzima e uma solução contendo sacarídeo; e uma etapa de reciclagem de enzima de reciclar e suprir a solução contendo enzima obtenível da etapa de separação de membrana à etapa de tratamento de sacarificação de enzima como uma fonte de enzima. (10) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em (9), em que a solução contendo sacarídeo separado e recuperado da etapa de separação de membrana é um líquido contendo sacarídeos que inclui oligossacarídeos como componentes principais. (11) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em (9), em que a fração líquida proveniente da etapa de centrifugação é reciclada e suprida à etapa de tratamento de sacarificação enzimática como uma solução contendo enzima contendo sacarídeos, sem passar pela etapa de separação de membrana. (12) O método para o tratamento de sacarificação enzimática de uma matéria-prima a base de lignocelulose conforme descrito em (8), em que o método de tratamento de sacarificação enzimática é um método de submeter uma matéria-prima a base de lignocelulose a um tratamento de fermentação e sacarificação paralelas de acordo com uma série de processos incluindo uma etapa de pré-tratamento de submeter uma matéria-prima a base de lignocelulose a um tratamento para produzir a matéria-prima a base de lignocelulose apropriada para uma reação de sacarificação enzimática; uma etapa de tratamento de fermentação e sacarificação paralelas de adicionar a matéria-prima a base de lignocelulose pré-tratada, junto com um microorganismo para fermentação que usa sacarídeos como um substrato de fermentação, e com um eletrólito contendo um sal solúvel em água, na água contendo enzima de sacarificação de celulose, e submeter a matéria-prima a base de lignocelulose como uma suspensão de matéria-prima cuja condutividade elétrica foi ajustada em 5 mS/cm a 25 mS/cm, tanto a um tratamento de sacarificação enzimática de tratar a matéria-prima a base de lignocelulose através de uma reação de sacarificação enzimática quanto a um tratamento de fermentação de usar os sacarídeos produzidos como um substrato; uma etapa de separação sólido-líquido de separar a suspensão tratada proveniente da etapa de tratamento de fermentação e sacarificação paralelas em um resíduo e uma fração líquida usando uma prensa de parafuso com um tamanho de peneira de 1,0 mm a 2,0 mm; uma etapa de tratamento de peneira de separar a fração líquida proveniente da etapa de separação sólido-líquido nas fibras finas e uma fração líquida através de um tratamento de peneira usando uma peneira de malha 80 a 600; uma etapa de destilação de separar e recuperar um produto de fermentação da fração líquida obtida depois do tratamento de peneira excluindo fibras finas, através de destilação; uma etapa de centrifugação de centrifugar o destilado residual proveniente da etapa de destilação para remover qualquer resíduo restante, e, por meio disso, obtendo uma fração líquida contendo enzimas e sacarídeos; e uma etapa de reciclar e suprir a fração líquida proveniente da etapa de centrifugação à etapa de tratamento de sacarificação de enzima como uma solução contendo enzima contendo sacarídeos, sem passar pela etapa de separação de membrana. The present invention is based on the observations described above, and includes the following modalities. (1) A method for the treatment of enzymatic saccharification of a lignocellulose-based raw material, the method including adding a lignocellulose-based raw material that has been subjected to a pre-treatment to produce the appropriate raw material for a enzymatic saccharification reaction, together with an electrolyte containing a water-soluble salt, in water containing cellulose saccharification enzyme; submit the lignocellulose base to the raw material as a suspension of raw material, whose electrical conductivity was adjusted from 5 mS / cm to 25 mS / cm, to an enzymatic saccharification treatment through an enzymatic saccharification reaction; separating and recovering the reaction product and an enzyme-containing solution from the treated suspension after enzymatic saccharification treatment; and recycling the solution containing recovered enzyme as an enzyme for the enzymatic saccharification treatment process. (2) The method for the treatment of enzymatic saccharification of a lignocellulose-based raw material as described in (1), in which the pre-treatment to produce the appropriate raw material for an enzymatic saccharification reaction is a pre- treatment including a chemical treatment of immersing the raw material based on lignocellulose in a solution containing an alkaline chemical substance selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate and sodium hydrogen carbonate, or a mixture of these, or a mixture of sodium sulfite and an alkali like that. (3) The method for the treatment of enzymatic saccharification of a raw material based on lignocellulose as described in (1) or (2), in which the raw material based on lignocellulose is forest waste. (4) The method for the treatment of enzymatic saccharification of a raw material based on lignocellulose as described in (1) or (2), in which the raw material based on lignocellulose is tree bark. (5) The method for treating enzymatic saccharification of a lignocellulose-based raw material as described in any one of (1) to (4), in which the water-soluble salt is at least one water-soluble salt selected of alkali metal salts and alkaline earth metal salts. (6) The method for the treatment of enzymatic saccharification of a raw material based on lignocellulose as described in any one of (1) to (5), in which the water-soluble salt is a salt selected from the group consisting of halides, sulphates, sulphites, thiosulphates, carbonates, hydrogen carbonates, phosphates, dihydrogen phosphates, hydrogen phosphates, acetates, and citrates of alkali metals and alkaline earth metals. (7) The method for the treatment of enzymatic saccharification of a raw material based on lignocellulose as described in any one of (1) to (6), wherein the method of treating enzymatic saccharification is a method of submitting a material -lignocellulose-based raw material to an enzymatic saccharification treatment, according to a series of processes including a pre-treatment step of subjecting a lignocellulose-based raw material to a treatment to produce the lignocellulose-based raw material suitable for an enzymatic saccharification reaction; an enzymatic saccharification treatment step of adding the raw material based on pre-treated lignocellulose, together with an electrolyte containing a water-soluble salt in water containing cellulose saccharification enzyme, and treating the raw material based on lignocellulose as a suspension of raw material, whose electrical conductivity was adjusted from 5 mS / cm to 25 mS / cm, through an enzymatic saccharification reaction; a solid-liquid separation step of removing a solid residue from the treated suspension from the enzymatic saccharification treatment step; a centrifugation step of centrifuging the liquid fraction from the solid-liquid separation step, and thereby obtaining a liquid fraction that contains enzymes and saccharides and has all the remaining residue removed; a membrane separation step of separating the liquid fraction from the centrifugation step into a solution containing enzyme and a solution containing saccharide produced; and an enzyme recycling step of recycling and supplying the enzyme-containing solution obtainable from the membrane separation step to the enzyme saccharification treatment step as an enzyme source. (8) The method for the treatment of enzymatic saccharification of a raw material based on lignocellulose as described in any one of (1) to (7), in which the enzymatic saccharification treatment step is a fermentation treatment step parallel saccharification to carry out a treatment based on an enzymatic saccharification reaction of a raw material based on lignocellulose and a treatment of fermentation of saccharides produced by a microorganism for fermentation in combination using a cellulase preparation and a micro- fermentation organism that uses saccharides as a fermentation substrate (= raw material) in combination, and thereby produce a fermentation product together with saccharides. (9) The method for the treatment of enzymatic saccharification of a raw material based on lignocellulose as described in (8), in which the method of enzymatic saccharification is a method of subjecting a raw material based on lignocellulose to a treatment of parallel fermentation and saccharification according to a series of processes including a pre-treatment step of subjecting a lignocellulose-based raw material to a treatment to produce the lignocellulose-based raw material suitable for an enzymatic saccharification reaction; a parallel fermentation and saccharification treatment step of adding the raw material based on pre-treated lignocellulose, together with a micro-organism for fermentation that uses saccharides as a fermentation substrate, and with an electrolyte containing a water-soluble salt , in water containing cellulose saccharification enzyme, and subject the lignocellulose-based raw material as a suspension of raw material, whose electrical conductivity was adjusted from 5 mS / cm to 25 mS / cm, both to a saccharification treatment enzymatic as to a fermentation treatment to use the saccharides produced as a substrate; a solid-liquid separation step of removing a solid residue from the treated suspension from the enzymatic saccharification treatment step; a distillation step of separating and recovering the fermentation product from the liquid fraction from the solid-liquid separation step through distillation; a centrifugation step of centrifuging the residual distillate from the distillation step to remove any remaining residue, and thereby obtaining a liquid fraction containing enzymes and saccharides; a membrane separation step of separating the liquid fraction from the centrifugation step into a solution containing enzyme and a solution containing saccharide; and an enzyme recycling step of recycling and supplying the enzyme-containing solution obtainable from the membrane separation step to the enzyme saccharification treatment step as an enzyme source. (10) The method for the treatment of enzymatic saccharification of a raw material based on lignocellulose as described in (9), in which the solution containing saccharide separated and recovered from the membrane separation step is a liquid containing saccharides that includes oligosaccharides as major components. (11) The method for treating enzymatic saccharification of a lignocellulose-based raw material as described in (9), in which the liquid fraction from the centrifugation step is recycled and supplied to the enzymatic saccharification treatment step as a enzyme-containing solution containing saccharides, without going through the membrane separation step. (12) The method for the treatment of enzymatic saccharification of a raw material based on lignocellulose as described in (8), in which the method of treatment of enzymatic saccharification is a method of subjecting a raw material based on lignocellulose to a parallel fermentation and saccharification treatment according to a series of processes including a pre-treatment step of subjecting a lignocellulose-based raw material to a treatment to produce the lignocellulose-based raw material suitable for a saccharification reaction enzymatic; a parallel fermentation and saccharification treatment step of adding the raw material based on pre-treated lignocellulose, together with a microorganism for fermentation that uses saccharides as a fermentation substrate, and with an electrolyte containing a water-soluble salt, in the water containing cellulose saccharification enzyme, and submit the raw material based on lignocellulose as a suspension of raw material whose electrical conductivity has been adjusted from 5 mS / cm to 25 mS / cm, both to a treatment of enzymatic saccharification to treat the raw material based on lignocellulose through an enzymatic saccharification reaction regarding a fermentation treatment using the saccharides produced as a substrate; a solid-liquid separation step of separating the treated suspension from the parallel fermentation and saccharification treatment step into a residue and a liquid fraction using a screw press with a sieve size of 1.0 mm to 2.0 mm; a sieve treatment step of separating the liquid fraction from the solid-liquid separation step in the fine fibers and a liquid fraction through a sieve treatment using an 80 to 600 mesh sieve; a distillation step of separating and recovering a fermentation product from the liquid fraction obtained after the sieve treatment excluding fine fibers, through distillation; a centrifugation step of centrifuging the residual distillate from the distillation step to remove any remaining residue, and thereby obtaining a liquid fraction containing enzymes and saccharides; and a step of recycling and supplying the liquid fraction from the centrifugation step to the step of treating enzyme saccharification as a solution containing enzyme containing saccharides, without going through the membrane separation step.
[0022] According to the enzymatic saccharification treatment method of the present invention, a method is provided for the treatment of continuous enzymatic saccharification of a lignocellulose-based biomass, which has a very small enzyme loss and high economic efficiency, as a result of adsorption suppressed of saccharification enzymes in an unreacted portion or a residue from the reaction of the raw material based on lignocellulose, and facilitated separation and recovery of saccharification enzymes from the enzyme-treated suspension.
[0023] In addition, according to the present invention, ethanol production can be increased by selectively recovering fine fibers included in the culture fluid obtained after parallel fermentation and saccharification using lignocellulose as a raw material, and submitting the recovered fine fibers as a material -press fermentation and saccharification or parallel saccharification again. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 is a flow chart of the process showing a modality of the method for the treatment of enzymatic saccharification of a lignocellulose-based raw material of the present invention. Figure 2 is a flowchart of the process showing the enzymatic saccharification treatment process of a lignocellulose-based raw material of the present invention, carried out as a parallel fermentation and saccharification treatment process of carrying out an enzymatic saccharification treatment in combination with a fermentation treatment using saccharides produced as a raw material. Figure 3 is a diagram showing the production process flow for example Bl. Figure 4 is a diagram showing the production process flow of example B2. Figure 5 is a diagram showing the production process flow of comparative example B1. Figure 6 is a diagram showing the production process flow of example B5. Figure 7 is a diagram showing the production process flow of example B6. DESCRIPTION OF MODALITIES
[0025] In the following, the present invention will be described in more detail. <Raw material based on lignocellulose>
[0026] Examples of the raw material based on lignocellulose used as the raw material for the method of the present invention include, as woody materials, chips or bark of construction wood for the production of paper, forest waste, thinning construction wood, and the like; sawdust generated from sawmills and the like; pruned branches and leaves, and construction waste materials. Examples of herbaceous materials include agricultural residues such as kenaf, rice straw, wheat straw and bagasse; industrial waste and refuse such as oil and rubber remnants (eg EFB: Empty Fruit Bunch); and lignocellulose-based biomasses of remnants of herbal energy such as Erianthus, Miscanthus, and Napier grass. In addition, as the raw material based on lignocellulose according to the present invention, paper that originates from wood, old paper, pulp, pulp sludge, and the like can also be used.
[0027] Among the raw materials based on the aforementioned woody lignocellulose, tree bark is substantially of actual use today, large quantities of bark with uniform quality is available at sawmills or chip mills, and soft and soluble components are present in large proportions in the part of the wooden trunk. Thus, husk is particularly preferable as a raw material for a saccharification treatment or a parallel fermentation and saccharification treatment.
[0028] For example, the bark of trees belonging to the genus Eucalyptus or to the genus Acacia, which are generally used for papermaking raw materials, are available in large quantities in a stable manner from sawmills or chip mill for raw materials. raw materials for papermaking, and therefore bark is particularly suitably used. <Pretreatment to produce raw material suitable for enzymatic saccharification treatment>
[0029] The pre-treatment to produce a raw material suitable for the enzymatic saccharification treatment of the present invention is a treatment whereby the following pre-treatment is carried out on the lignocellulose-based raw material, as previously described, and thereby lignocellulose is taken to a state where it is enzymatically saccharifiable: a chemical treatment, a hydrothermal treatment, a pressurized hot water treatment, a hydrothermal treatment with added carbon dioxide, a steam treatment, mechanical treatments such as a wet crush treatment, a mechanical grinding treatment, and a formation treatment shredded fibers, a diluted sulfuric acid treatment, a steam blast treatment, an ammonia blast treatment, a carbon dioxide blast treatment, an ultrasonic irradiation treatment, a microwave irradiation treatment, a treatment with electronic beam irradiation, treatment with γ-ray irradiation, supercritical treatment, subcritical treatment, treatment with organic solvent, treatment with phase separation, treatment with wood decay fungus, treatment with activation of ecological solvent, several catalytic treatments, a treatment with radical reaction, and a treatment with ozone oxidation.
[0030] These treatments can be carried out alone or in combinations of multiple treatments.
[0031] Among them, it is preferable to subject biomass based on lignocellulose to one or more pre-treatments selected from a chemical treatment, a treatment with pressurized hot water, a treatment with the formation of shredded fibers, and a treatment with mechanical grinding.
[0032] Chemical treatment is a treatment of immersing a raw material based on lignocellulose in an aqueous solution of a chemical substance such as an acid or an alkali, and thereby bringing the raw material based on lignocellulose to an appropriate state for the treatment of enzymatic saccharification of the subsequent step.
[0033] The chemical substance and the like used in chemical treatment are not particularly limited, but, for example, one or more are selected from hydroxides, sulfides such as sulfuric acid and dilute sulfuric acid, carbonates, sulfates and sulfites, alkali metals and alkaline earth metals . An alkaline treatment obtained by immersing the raw material based on lignocellulose in an aqueous solution of one or more chemical substances selected from sodium hydroxide, calcium hydroxide, sodium sulfide, sodium carbonate, calcium carbonate and sodium sulfite, is suitable as a chemical treatment. In addition, a chemical treatment based on an oxidizing agent such as ozone or chlorine dioxide can also be performed.
[0034] It is appropriate to carry out the chemical treatment in combination with a mechanical treatment such as a shredding fiber treatment or a mechanical grinding treatment previously described, such as a post-treatment of those pre-treatments.
[0035] The amount of the chemical substance added to be used in the chemical treatment can be arbitrarily adjusted according to the circumstances, but, from the point of view of reducing the cost of the chemical, and from the point of view of preventing a decrease in yield due to elution and cellulose overdegradation, the amount is preferably 50 parts by weight or less based on 100 parts by weight of raw material based on dry lignocellulose. The immersion time in the aqueous solution of a chemical substance and the treatment temperature in the chemical treatment can be arbitrarily adjusted according to the raw material or chemical used, but, in general, the chemical treatment can be carried out for a treatment time. 20 to 90 minutes and at a treatment temperature of 80 ° C to 200 ° C. Since elution on the liquid side or cellulose overdegradation can occur when severe treatment conditions are applied, it is preferable to adjust the treatment time to 70 minutes or less and the treatment temperature to 180 ° C or lower. Most preferably, the treatment time is 30 minutes to 1 hour, and the treatment temperature is 80 ° C to 130 ° C.
[0036] Like the mechanical treatment, any mechanical means, such as shredding, cutting and grinding can be used, and lignocellulose, thereby becoming prone to be saccharified by the saccharification and fermentation treatment process of the subsequent step. There is no particular limitation with respect to the machine apparatus used, but, for example, a single screw shredder, a twin screw shredder, a hammer shredder, a refiner, and a kneader can be used.
[0037] For a lignocellulose-based raw material that has been subjected to a pretreatment to produce the appropriate raw material for treatment based on an enzymatic saccharification reaction, it is preferable to perform a disinfection treatment first on the base raw material of lignocellulose in the preparation of a suspension of raw material based on lignocellulose. If unwanted bacteria are incorporated into the biomass raw material based on lignocellulose, there is a problem that the unwanted bacteria consume the saccharides when saccharification by enzymes is carried out, and the yield of the product is decreased.
[0038] Disinfection treatment can be carried out by a method of exposing the raw material to a pH at which the growth of bacteria is difficult, such as an acidic pH or an alkaline pH, but it can also be carried out by a method of treating the material press at a high temperature, or both methods can be combined. For the raw material after an acid or alkaline treatment, it is preferable to adjust the treated raw material to a neutral pH, or to an appropriate pH for a saccharification treatment or a saccharification and fermentation treatment, and then use it as a raw material. Also, even in the case of high-temperature disinfection, it is preferable to cool the treated raw material to room temperature or to an appropriate temperature for the treatment in the fermentation and saccharification process, and then use it as a raw material. As such, when the raw material is taken out after the temperature or pH is adjusted, the enzymes cannot be exposed to a pH or temperature other than a suitable pH or a suitable temperature, and be deactivated.
[0039] The raw material based on lignocellulose which has been subjected to a pretreatment to produce the appropriate raw material for treatment based on an enzymatic saccharification reaction, is additionally mixed with an appropriate amount of water, an enzyme, and a water-soluble salt, and optionally with a micro-organism necessary for fermentation, such as yeast, and thus a suspension of raw material is prepared. The raw material suspension is supplied in the enzymatic saccharification treatment step after the electrical conductivity is adjusted to a predetermined value. A representative process for carrying out the enzymatic saccharification treatment method is shown in figure 1. <Enzymatic saccharification treatment>
[0040] In the case of the enzymatic saccharification treatment method according to the process in figure 1, in the enzymatic saccharification treatment step indicated as "saccharification" in figure 1, a suspension of raw material that was prepared by adding the raw material to the base of lignocellulose supplied from the pretreatment step indicated as "pretreatment", a saccharification enzyme, and a water-soluble salt as an electrolyte for an appropriate amount of water, is subjected to enzymatic saccharification under agitation. The concentration of the lignocellulose feedstock in the feedstock suspension is preferably 1 wt% to 30 wt%. If the concentration is less than 1% by mass, the final concentration of the product is very low, and there is a problem that the cost for the concentration of the product increases. Also, when the concentration is greater than 30% by mass and the raw material is highly concentrated, agitation of the raw material becomes difficult, and there is a problem that productivity is reduced.
[0041] The electrical conductivity of the raw material suspension in the enzymatic saccharification treatment step is preferably maintained in the range of 5 mS / cm to 25 mS / cm.
[0042] The pH is selected in the range of 3.5 to 10.0, in which the enzyme used is unlikely to be deactivated, but it is more preferable to maintain the pH in the range of 3.5 to 7.5.
[0043] The pH in the saccharification step or in the parallel fermentation and saccharification step is preferably maintained in the range of 3.5 to 10.0, and more preferably in the range of 4.0 to 7.5.
[0044] The temperature for the treatment of enzymatic saccharification is not particularly limited as long as the temperature is in the ideal enzyme temperature range, and the temperature is generally 25 ° C to 50 ° C, and preferably 30 ° C to 40 ° C.
[0045] In addition, the enzyme saccharification reaction mode is preferably a continuous mode, but a semi-lot or batch mode can also be used.
[0046] The reaction time may vary with the enzyme concentration, but in the case of a batch mode, the reaction time is generally 10 to 240 hours, and preferably 15 to 160 hours. Also, in the case of a continuous mode, the general average retention time is 10 to 150 hours, and preferably 15 to 100 hours.
[0047] The cellulolytic enzyme used in the treatment of enzymatic saccharification or parallel fermentation and saccharification is appropriately selected from a group of enzymes that are collectively referred to as so-called cellulases, which have cellobiohydrolase activity, endoglucanase activity, and beta-glucosidase activity.
[0048] With respect to the various cellulolytic enzymes, enzymes with the respective activities can be added in an appropriate amount; however, since many of these commercially available cellulase preparations have the various cellulase activities described above as well as hemicellulase activity, commercially available cellulase preparations can also be used.
[0049] Examples of commercially available cellulase preparations include cellulase preparations that originate from the genus Trichoderma, the genus Acrremonium, the genus Aspergillus, the genus Phanerochaete, the genus Trametes, the genus Humicola, and the genus Bacillus. Examples of commercial products from such cellulase preparations include Cellucine T2 (manufactured by HPI Co., Ltd.), Meicelase (manufactured by Meiji Seika Kaisha, Ltd.), Novozyme 188 (manufactured by Novozymes A / S), Multifect CX10L (manufactured by Genencor International, Inc.), and GC220 (manufactured by Genencor International, Inc.) (all registered trademarks).
[0050] The amount of the cellulase preparation used based on 100 parts by weight of the solids content of the raw material is preferably 0.5 to 100 parts by weight, and particularly preferably 1 to 50 parts by weight.
[0051] As the water-soluble salt that is added as an electrolyte, those selected from acid salts, basic salts, neutral salts, and buffer solutions containing salt such as an acetate buffer solution and a citrate buffer solution can be used alone or in combination . The concentration of the water-soluble salt can be freely established in a range that has no adverse effects on the enzymatic saccharification reaction.
[0052] Among them, a water-soluble salt selected from alkali metal salts and alkaline earth metal salts is preferable. Examples of the alkali metal salts and alkaline earth metal salts include water-soluble salts selected from the group consisting of halides, sulphates, sulphites, thiosulphates, carbonates, hydrogen carbonates, phosphates, dihydrogen phosphates, hydrogen phosphates, acetates, and alkali metal citrates and alkaline earth metals.
[0053] In the present invention, a water-soluble salt can be added as an electrolyte in the enzymatic saccharification treatment process.
[0054] In the enzymatic saccharification treatment process, it is preferable to add an electrolyte to the raw material suspension and maintain the electrical conductivity of the raw material suspension in the range of 5 mS / cm to 25 mS / cm. When the electrical conductivity is maintained in the range of 5 mS / cm to 25 mS / cm, the adsorption of enzymes in the unreacted components or in the reaction residue of the lignocellulose raw material and, therefore, the recycling rate of the enzymes in the process of enzymatic saccharification treatment can be maintained at a high level for a long period of time. The electrolyte can be added without any limitation at any stage, as long as the stage is in the enzymatic saccharification treatment process, and the stage allows the operation to add the electrolyte. It is preferable to add the electrolyte during the saccharification and primary fermentation step because the operation is easy. <Solid-liquid separation>
[0055] The treatment suspension discharged from the "saccharification step" is taken to the solid-liquid separation process with a filtration apparatus indicated as "solid-liquid separation" in figure 1, and a solid residue is eliminated. The solid residue separated by the filtration apparatus in the solid-liquid separation process includes lignin, hemicellulose and cellulose. However, cellulose exists in a state in which it is protected by lignin and the like, and in a state in which additional saccharification cannot be promoted and, therefore, cellulose is normally discharged outside the process.
[0056] In addition, the culture fluid discharged from the primary parallel fermentation and saccharification stage is transferred to the solid-liquid separation stage and is separated into a liquid fraction (filtrate) and a residue (primary residue). As the apparatus for carrying out solid-liquid separation, a screw press with a sieve size of 1.0 mm to 2.0 mm is used. The screw press is a device that does not easily pass through clogging caused by fibers due to the structure and is capable of efficiently carrying out solid-liquid separation with a relatively smaller amount of energy. In order to improve the efficiency of solid-liquid separation, back pressure can be applied.
[0057] The residue separated in the solid-liquid separation step includes lignin, hemicellulose and cellulose, and cellulose has lignin and the like adsorbed on it and is thus in a state in which enzyme saccharification is difficult. The residue obtained after the solid-liquid separation step includes a large amount of fibrous components that were not degraded in the primary parallel fermentation and saccharification step, so that when the residue is subjected to mechanical treatment or chemical treatment, saccharification is facilitated (PTL 6).
[0058] The filtrate (liquid fraction) separated in the solid-liquid separation step is transferred to the subsequent sieve treatment step. <Centrifugation step>
[0059] The liquid fraction from which a solid residue was excluded in the solid-liquid separation step is subsequently taken to the centrifugation step indicated as "centrifugation", and the remaining residue present in the liquid fraction from the solid separation step is removed. The liquid fraction is taken to the process to recover a saccharide solution and an enzyme solution, which is indicated as "membrane separation" in figure 1.
[0060] In addition, the residual distillate is transferred to the centrifugation step, where the residue remaining in it (secondary residue) is removed by centrifugation, and then the liquid fraction is recycled to the primary parallel fermentation and saccharification process (see figure 3). This liquid fraction contains enzymes, e.g. enzymes are reused in the primary parallel fermentation and saccharification stage. However, the waste contains lignin, so the waste can be recovered as a fuel for combustion and can be used as energy, or lignin can be recovered and used effectively. <Membrane separation step>
[0061] The liquid fraction from which the remaining residue was excluded in the centrifugation step is a liquid fraction containing enzymes and saccharides produced, and the liquid fraction is separated into a solution containing enzyme and a solution containing saccharide in the membrane separation step indicated as "separation membrane "in figure 1 and is taken to an enzyme solution storage tank indicated as" recovered enzyme ", where the enzyme-containing solution is recycled as an enzyme source. The saccharide-containing solution is extracted directly as a product.
[0062] Since the saccharide-containing solution contains monosaccharides such as hexoses and pentoses, as well as oligosaccharides, if the intention is to produce monosaccharides, oligosaccharides that can be separated and supplied in the "saccharification step", so that the oligosaccharides can be further enzymatically treated and degraded to monosaccharides. <Primary parallel fermentation and saccharification treatment step>
[0063] The enzymatic saccharification treatment step indicated as the "saccharification step" in figure 1 can be performed as a so-called parallel fermentation and saccharification treatment process that simultaneously performs a fermentation treatment using a microorganism that uses the saccharides produced by the enzymatic saccharification reaction as a raw material (fermentation substrate). In this case, a microorganism for fermentation that uses the saccharides produced as a fermentation substrate (fermentation of the raw material) is added to the suspension of the raw material, along with the saccharification enzyme. A typical process for carrying out the parallel fermentation and saccharification treatment method is shown in figure 2.
[0064] In figure 2, the raw material based on lignocellulose that has been treated in an appropriate state for the treatment of enzymatic saccharification in the pre-treatment step is added to an appropriate amount of water together with a water-soluble salt as an electrolyte, a cellulolytic enzyme, and a microorganism for fermentation such as an alcohol yeast, and is subjected, in the form of a suspension of raw material with the electrical conductivity adjusted to a predetermined value, both to a treatment of cellulose saccharification by an enzymatic saccharification reaction for a fermentation treatment such as fermentation alcohol using the saccharides produced as a fermentation substrate in the parallel fermentation and saccharification step.
[0065] The raw material based on lignocellulose which has been subjected to an appropriate pretreatment for fermentation and saccharification is mixed with an appropriate amount of water, an enzyme, and a micro-organism required for fermentation, such as yeast, and the mixture is supplied in the primary parallel fermentation and saccharification stage. A typical process of the parallel fermentation and saccharification treatment method is shown in figure 1.
[0066] In figure 3, the raw material based on lignocellulose which was treated in an appropriate state for the treatment of saccharification and fermentation in the pre-treatment step, is saccharified (cellulose ⟶ glucose) by enzymes and is subsequently fermented (glucose ⟶ ethanol ) by a yeast.
[0067] As the micro-organism used for fermentation, a yeast and the like are used. The microorganism can be added together with the medium and the like used in their culture. Like yeast, well-known yeasts that are described in PTL 3, for example, yeasts such as Sacharomiyces cerevisae, Pichia stipitis, Issatchenkia orientalis, Candida brassicae, and Rhizopus javanicus.
[0068] The microorganism can be immobilized. If the microorganism is immobilized, the step of recovering the microorganism in the subsequent step can be omitted, or at least the burden presented by the recovery step can be reduced, and the risk of losing the microorganism can be reduced. Also, although it is not as advantageous as the immobilization of the microorganism, the recovery of the microorganism can be facilitated by selecting a microorganism with a cohesive property.
[0069] In the processes of figure 2, the treated suspension discharged from the parallel fermentation and saccharification step is taken to the solid-liquid separation step where the solids are removed from the suspension, and then the liquid fraction containing a fermentation product and saccharides is taken to the "distillation step" indicated as "distillation" in figure 2 in order to separate and recover the fermentation product. In the distillation stage, the fermentation product is distilled and separated by a low pressure distillation apparatus. When low pressure distillation is used, the fermentation product can be separated at a low temperature and therefore deactivation of the enzymes can be prevented. As the low pressure distillation apparatus, a rotary evaporator, a flash evaporator and the like can be used.
[0070] The distillation temperature is preferably 25 ° C to 60 ° C. If the distillation temperature is lower than 25 ° C, distillation of the product takes time, and productivity decreases. Also, if the distillation temperature is higher than 60 ° C, the enzymes are thermally denatured and are deactivated, and the amount of enzymes to be added again increases. Thus, economic efficiency is deteriorated.
[0071] The concentration of the fermentation product remaining in the residual distillate after distillation is preferably 0.1% by weight or less. When the fermentation product is adjusted to such a concentration, the amount of fermentation product discharged together with the residue remaining in the downstream centrifugation step can be reduced, and the yield can be increased.
[0072] The residual distillate from the distillation step is subsequently taken to the centrifugation step indicated as "centrifugation" in figure 2, and the remaining residue present in the residual distillate is excluded. Thus, a liquid fraction containing enzymes and saccharides is obtained.
[0073] This liquid fraction containing enzymes and saccharides is taken to the membrane separation step indicated as "membrane separation" in figure 2, and is separated into a solution containing enzyme and a solution containing saccharide. The enzyme-containing solution is recycled and supplied in the "parallel fermentation and saccharification step" through the enzyme solution storage tank indicated as "recovered enzyme" in figure 2. Also, the saccharide-containing solution is collected in the storage tank of saccharide solution indicated as "saccharides" in figure 2 and is processed into saccharide products.
[0074] In the parallel fermentation and saccharification stage, hexoses are produced which are the enzymatic degradation products of cellulose, namely glucose, mannose, galactose and the like, and pentoses originating from hemicellulose, namely xylose and the like, as well as oligosaccharides . Hexoses such as glucose are mainly used as the fermentation substrate, and alcohols such as ethanol are produced. Also, because pentoses and oligosaccharides are not used as fermentation substrates, pentoses and oligosaccharides are taken directly to the enzyme recovery step along with the enzymes. In this case, the pentoses can be added to the raw material suspension together with a yeast that certainly uses these pentoses as fermentation substrates, or they can be subjected to a fermentation treatment in a separate process. Also, if necessary, pentoses can be recovered as products.
[0075] In addition, oligosaccharides can be recovered as products according to need, or they can be used as a raw material to be degraded into monosaccharides by enzymes in the parallel fermentation and saccharification step as discussed in the enzymatic saccharification treatment method according to with the process in figure 1.
[0076] The concentration of the lignocellulose based raw material suspension is preferably 1 wt% to 30 wt%. If the concentration of the suspension is less than 1% by mass, the final concentration of the product is very low, and there is a problem that the cost for the concentration of the product increases. In addition, if the concentration is greater than 30% by mass and the raw material is highly concentrated, the agitation of the raw material becomes difficult, and there is a problem that productivity is reduced. <Sieve treatment step>
[0077] The filtrate obtained after the solid-liquid separation is subjected to a sieving treatment, and is separated into the fine fibers and a filtrate (liquid fraction). Like the sieve treatment method, any sieve treatment apparatus capable of separating fine fibers can be used without particular limitations. As the sieve treatment apparatus, a sieve, a filter press, and the like can be used. The sieve mesh size is preferably 80 mesh to 600 mesh (28 μm at 182 pm), and more preferably 150 mesh to 400 mesh (39 μm at 97 pm). In order to increase the efficiency of the treatment, a sieve vibrator can be attached, and vibration can be applied. The fine fibers separated by the treatment described above have a low lignin content compared to the primary or secondary waste, and can be easily saccharified by enzymes. In addition, when fine fibers are excluded by the sieve treatment, there is an advantage that long-term operation of the apparatus is possible, which can reduce the amount of solids adhering to the low pressure distillation apparatus used in the step downstream distillation. The recovered fine fibers can be transferred to the saccharification and primary fermentation stage and can be used as the raw material for fermentation and saccharification (see figure 3). In addition, the recovered fine fibers can be transferred to a secondary saccharification and fermentation step (a fermentation and saccharification process different from the primary fermentation and saccharification process) which will be described below and can be used as a raw material for the fermentation and saccharification (see figure 4). In addition, the recovered fine fibers can also be subjected to saccharification in a separate process. When the fine fibers are treated by saccharification or fermentation and saccharification as such, the enzymes adsorbed on the fine fibers can be effectively used.
[0078] On the other hand, the filtrate separated by the sieve treatment is transferred to the distillation step. <Secondary parallel fermentation and saccharification treatment step>
[0079] The secondary parallel fermentation and saccharification treatment step is a saccharification and fermentation step independent of the primary parallel fermentation and saccharification treatment step, and fermentation and saccharification can be performed using fresh lignocellulose as a raw material, or fermentation and saccharification can be carried out using the waste discharged during the process as a raw material. In addition, saccharides that were not fermented in ethanol in the primary parallel fermentation and saccharification stage, can also be fermented in the secondary parallel fermentation and saccharification treatment. In the primary parallel fermentation and saccharification stage, hexoses that originate from cellulose, namely, glucose, mannose, galactose and the like are fermented in ethanol, but xylose, which is a pentose that originates from hemicellulose, can remain unreacted. In this case, a yeast that most certainly ferment pentoses can be added in the secondary parallel fermentation and saccharification treatment step to ferment the pentoses. In the present invention, the fine fibers recovered by the sieve treatment can be transferred to the secondary parallel fermentation and saccharification treatment step and can be treated by fermentation and saccharification. <Distillation step>
[0080] The filtrate obtained after the sieve treatment, or the treated liquid (culture fluid) obtained after the secondary parallel fermentation and saccharification treatment step is transferred to the distillation step (see figure 3 and figure 4).
[0081] In the distillation stage, the fermentation product is distilled and separated by a low pressure distillation apparatus. Since the fermentation product can be separated at a low temperature under low pressure, deactivation of the enzymes can be prevented. As the low pressure distillation apparatus, a rotary evaporator, a flash evaporator and the like can be used. EXAMPLES
[0082] In the following, the present invention will be described on the basis of the examples, but the present invention is not intended to be limited by those examples. In the respective examples and comparative examples, the unit "%" indicates "% by mass", and the unit "parts" indicates "parts by mass". (Example A1)
[0083] 100 g of crushed forest residues were introduced into 1,000 mL of water containing 20 g of 48% caustic soda, and the forest residues were treated at 90 ° C for 30 minutes. Subsequently, the mixture was ground with a refiner (gap 0.5 mm). This product was dehydrated and washed with a screw press, and the resulting product was used as the raw material for the substrate.
[0084] The substrate raw material was added to a final concentration of 5%, CSL (maceration liquor) was added to a final concentration of 1%, ammonium sulfate was added to a final concentration of 0.5%, and sodium chloride was added to a final concentration of 100 mM. Thus, 400 mL of a suspension of lignocellulose with an electrical conductivity of 11.8 mS / cm was prepared.
[0085] The lignocellulose suspension prepared as previously described was steam sterilized at 120 ° C for 20 minutes and was cooled to 40 ° C.
[0086] Subsequently, 10 ml of an enzyme (trademark: GC220; manufactured by Genencor International, Inc.) was added to it.
[0087] A saccharification reaction was performed at 30 ° C and stirred at 120 rpm, and 1 mL of the reaction liquid was collected after every 24 hours and 48 hours and was centrifuged for 5 minutes at 10,000 rpm. The enzyme activity of the supernatant obtained thereby was measured.
[0088] The recovery ratio was calculated using β-glucosidase activity, which is the most important in enzyme recovery, as an index, the activity measurement was performed by the method described below. (β-glucosidase activity)
[0089] Measurement of β-glucosidase activity was performed by adding 4 μL of an enzyme solution to 16 μL of a 125 mM acetate buffer solution (pH 5.0) containing 1.25 mM 4-methyl-umberiferyl-glycoside, carrying out a reaction for 10 minutes at 37 ° C, subsequently adding 100 μL of a 500 mM glycine-NaOH buffer solution (pH 10.0) to end the reaction, and measuring the fluorescence intensity at 460 nm using an excitation light at 350 nm . The enzyme recovery ratio was determined using the following calculation formula.
[0090] Enzyme recovery ratio (%) = (Supernatant enzyme activity / added enzyme activity) x 100 (Example A2)
[0091] The experiment was carried out in the same way as in example A1, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A1, sodium hydrogen carbonate was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 8.6 mS / cm. (Example A3)
[0092] 100 g of crushed forest residues were introduced into 1,000 mL of water containing 20 g of 48% caustic soda, and the forest residues were treated at 90 ° C for 30 minutes. Subsequently, the mixture was ground with a refiner (gap 0.5 mm). This product was dehydrated and washed with a screw press, and the resulting product was used as a raw material for the substrate.
[0093] The substrate raw material was added to a final concentration of 5%, CSL (maceration liquor) was added to a final concentration of 1%, ammonium sulfate was added to a final concentration of 0.5%, and sodium chloride was added to a final concentration of 100 mM. Thus, 400 mL of a suspension of lignocellulose with an electrical conductivity of 12.0 mS / cm was prepared.
[0094] The lignocellulose suspension prepared as previously described was steam sterilized at 120 ° C for 20 minutes and was cooled to 40 ° C. Subsequently, 10 ml of an enzyme (trademark: GC220; manufactured by Genencor International, Inc.) was added to it.
[0095] In addition, a commercially available yeast (trademark: Maurivin: Mauri Yeast Australia Pty, Limited) was added to the raw material suspension prepared as previously described, and the yeast was subjected to saccharification, fermentation and culture at 30 ° C under agitation at 120 rpm, and 1 mL of the reaction liquid was collected after every 24 hours and 48 hours and was centrifuged for 5 minutes at 10,000 rpm. The enzyme activity of the supernatant obtained thereby was measured. (Example A4)
[0096] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, potassium chloride was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 13.3 mS / cm. (Example A5)
[0097] The experiment was carried out in the same way as in example A3, except that instead of the sodium chloride used in the method of example A3 added to a final concentration of 100 mM, potassium iodide was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 14.5 mS / cm. (Example A6)
[0098] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, sodium sulfate was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 14.7 mS / cm. (Example A7)
[0099] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, sodium sulfite was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 13.6 mS / cm. (Example A8)
[0100] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, sodium thiosulfate was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 16.9 mS / cm. (Example A9)
[0101] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, sodium carbonate was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 12.6 mS / cm. (Example A10)
[0102] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, dipotassium hydrogen phosphate was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 15.0 mS / cm. (Example A11)
[0103] The experiment was carried out in the same way as in example A3, except that, instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, disodium hydrogen phosphate was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 11.9 mS / cm. (Example A12)
[0104] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, sodium hydrogen carbonate was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 8.0 mS / cm. (Example A13)
[0105] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, trisodium citrate was added to a final concentration of 100 mM. The electrical conductivity in the reaction system at this time was 15.4 mS / cm. (Example A14)
[0106] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, an acetate buffer (pH 5.0) was added to a concentration end of 100 mM. The electrical conductivity in the reaction system at this time was 7.1 mS / cm. (Example A15)
[0107] The experiment was carried out in the same way as in example A3, except that, instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, a citrate buffer (pH 5.0) was added to a concentration end of 100 mM. The electrical conductivity in the reaction system at this time was 10.9 mS / cm. (Example A16)
[0108] 100 g of crushed bark of Eucalyptus globulus were introduced into 1,000 mL of water containing 20 g of 48% caustic soda, and the forest residues were treated at 90 ° C for 30 minutes. Subsequently, the mixture was ground with a refiner (gap 0.5 mm). This product was dehydrated and washed with a screw press, and the resulting product was used as a raw material for the substrate.
[0109] The substrate raw material was added to a final concentration of 5%, CSL (maceration liquor) was added to a final concentration of 1%, ammonium sulfate was added to a final concentration of 0.5%, and sodium chloride was added to a final concentration of 100 mM. Thus, 400 mL of a suspension of lignocellulose with an electrical conductivity of 11.8 mS / cm was prepared.
[0110] The lignocellulose suspension prepared as previously described was steam sterilized at 120 ° C for 20 minutes and was cooled to 40 ° C. Subsequently, 10 ml of an enzyme (trademark: GC220; manufactured by Genencor International, Inc.) was added to it.
[0111] A saccharification reaction was carried out at 30 ° C with agitation at 120 rpm, and 1 mL of the reaction liquid was collected after every 24 hours and 48 hours and was centrifuged for 5 minutes at 10,000 rpm. The enzyme activity of the supernatant obtained thereby was measured. (Example A17)
[0112] 100 g of crushed bark of Eucalyptus globulus were introduced in 1,000 mL of water containing 20 g of caustic soda 48%, and the crushed bark was treated at 90 ° C for 30 minutes. Subsequently, the mixture was ground with a refiner (gap 0.5 mm). This product was dehydrated and washed with a screw press, and the resulting product was used as a raw material for the substrate.
[0113] The substrate raw material was added to a final concentration of 5%, CSL (maceration liquor) was added to a final concentration of 1%, ammonium sulfate was added to a final concentration of 0.5%, and sodium chloride was added to a final concentration of 100 mM. Thus, 400 mL of a suspension of lignocellulose with an electrical conductivity of 11.8 mS / cm was prepared.
[0114] The lignocellulose suspension prepared as previously described was steam sterilized at 120 ° C for 20 minutes and was cooled to 40 ° C. Subsequently, 10 ml of an enzyme (trademark: GC220; manufactured by Genencor International, Inc.) was added to it. In addition, a commercially available yeast (trademark: Maurivin: Mauri Yeast Australia Pty, Limited) was added to the raw material suspension prepared as previously described, and the yeast was subjected to saccharification, fermentation and culture at 30 ° C under agitation at 120 rpm, and 1 mL of the reaction liquid was collected after every 24 hours and 48 hours and was centrifuged for 5 minutes at 10,000 rpm. The enzyme activity of the supernatant obtained thereby was measured. (Example A18)
[0115] 100 g of crushed forest residues were introduced into 700 mL of water containing 20 g of 97.0% sodium sulfite and 1 g of caustic soda, and the forest residues were treated at 170 ° C for 60 minutes. Subsequently, the mixture was ground with a refiner (gap 0.5 mm). This product was dehydrated and washed with a screw press, and the resulting product was used as a raw material for the substrate.
[0116] The substrate raw material was added to a final concentration of 5%, CSL (maceration liquor) was added to a final concentration of 1%, ammonium sulfate was added to a final concentration of 0.5%, and sodium chloride was added to a final concentration of 100 mM. Thus, 400 mL of a lignocellulose suspension with an electrical conductivity of 8.0 mS / cm was prepared.
[0117] The lignocellulose suspension prepared as previously described was steam sterilized at 120 ° C for 20 minutes and was cooled to 40 ° C. Subsequently, 10 ml of an enzyme (trademark: GC220; manufactured by Genencor International, Inc.) was added to it.
[0118] A saccharification reaction was carried out at 30 ° C with agitation at 120 rpm, and 1 mL of the reaction liquid was collected after every 24 hours and 48 hours and was centrifuged for 5 minutes at 10,000 rpm. The enzyme activity of the supernatant obtained thereby was measured. (Example A19)
[0119] 100 g of crushed forest residues were introduced into 700 mL of water containing 20 g of 97.0% sodium sulfite and 1 g of caustic soda, and the forest residues were treated at 170 ° C for 60 minutes. Subsequently, the mixture was ground with a refiner (gap 0.5 mm). This product was dehydrated and washed with a screw press, and the resulting product was used as a raw material for the substrate.
[0120] The substrate raw material was added to a final concentration of 5%, CSL (maceration liquor) was added to a final concentration of 1%, ammonium sulfate was added to a final concentration of 0.5%, and sodium chloride was added to a final concentration of 100 mM. Thus, 400 mL of a lignocellulose suspension with an electrical conductivity of 9.4 mS / cm was prepared.
[0121] The lignocellulose suspension prepared as previously described was steam sterilized at 120 ° C for 20 minutes and was cooled to 40 ° C. Subsequently, 10 ml of an enzyme (trademark: GC220; manufactured by Genencor International, Inc.) was added to it.
[0122] In addition, a commercially available yeast (trademark: Maurivin: Mauri Yeast Australia Pty, Limited) was added to the raw material suspension prepared as previously described, and the yeast was subjected to saccharification, fermentation and culture at 30 ° C under agitation at 120 rpm, and 1 mL of the reaction liquid was collected after every 24 hours and 48 hours and was centrifuged for 5 minutes at 10,000 rpm. The enzyme activity of the supernatant obtained thereby was measured. (Example A20)
[0123] The experiment was carried out in the same way as in example A16, except that 700 ml of water containing 20 g of 97.0% sodium sulfite and 1 g of used caustic soda were used, instead of 1,000 ml of water containing 20 g of caustic soda 48% used in the method of example A16. The electrical conductivity in the reaction system at this time was 11.2 mS / cm (Example A21)
[0124] The experiment was carried out in the same way as in example A17, except that 700 ml of water containing 20 g of 97.0% sodium sulfite and 1 g of caustic soda were used, instead of 1,000 ml of water containing 20 g of soda caustic 48% used in the method of example A17. The electrical conductivity in the reaction system at this time was 11.2 mS / cm. (Comparative example A1)
[0125] The experiment was carried out in the same way as in example A1, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A1, sulfuric acid was added to adjust the electrical conductivity of the reaction system to 6 , 5 mS / cm. (Comparative example A2)
[0126] The experiment was carried out in the same way as in example A1, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A1, sodium hydroxide was added to adjust the electrical conductivity of the reaction system to 8.0 mS / cm. (Comparative example A3)
[0127] The experiment was carried out in the same way as in example 3, except that sodium chloride was not added in the method of example A3. The electrical conductivity in the reaction system at this time was 4.2 mS / cm. (Comparative example A4)
[0128] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, sulfuric acid was added to adjust the electrical conductivity of the reaction system to 6 , 3 mS / cm. (Comparative example A5)
[0129] The experiment was carried out in the same way as in example A3, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, hydrochloric acid was added to adjust the electrical conductivity of the reaction system to 6 , 6 mS / cm. (Comparative example A6)
[0130] The experiment was carried out in the same way as in example A3 except that, instead of sodium chloride added to a final concentration of 100 mM in the method of example A3, sodium hydroxide was added to adjust the electrical conductivity of the reaction system to 8 , 2 mS / cm. (Comparative example A7)
[0131] The experiment was carried out in the same way as in example A1, except that instead of sodium chloride added to a final concentration of 100 mM in the method of example A1, sodium chloride was added to a final concentration of 5 mM to adjust the electrical conductivity of the reaction system at 4.6 mS / cm.
[0132] The results of examples A1 to A21 and comparative examples A1 to A7 are shown in table A1.
[0133] According to the results in Table A1, the method for treating enzymatic saccharification of a lignocellulose-based raw material in the examples shows that when a suspension of lignocellulose-based raw material in which a water-soluble salt was added to an enzymatic saccharification reaction system and the electrical conductivity has been adjusted in a predetermined range of values is subjected to enzymatic saccharification, not only the enzyme recovery rate of the liquid treated with saccharification is high in the initial stage, but also the ratio enzyme recovery is stable at a high level, even after a lapse of time.
[0134] Conversely, when electrical conductivity is adjusted using sulfuric acid (Comparative example A1, Comparative example A4), hydrochloric acid (Comparative example A5), or sodium hydroxide (Comparative example A2, Comparative example A6) without adding a water-soluble salt. to the enzymatic saccharification reaction system, the enzyme recovery ratio of the liquid treated with saccharification is low in the initial stage, and the decrease in the recovery ratio after a period of time is also significant. In addition, even in the case where the electrical conductivity of the enzyme reaction system is low (Comparative example A3, Comparative example A7) without or with the addition of a salt, the enzyme recovery ratio of the liquid treated with saccharification is low in the stage initial, and is further decreased after a period of time. (Example B1)
[0135] Ethanol production was carried out by the process flow shown in figure 3 [Pretreatment]
[0136] Chipped bark of Eucalyptus globulus was ground with a single screw shredder equipped with a 20 mm round hole sieve (manufactured by Seiho Kiko Co., Ltd., SC-15) and was used as a raw material.
[0137] A calcium hydroxide suspended in the calcium hydroxide solution in water was added to the raw material at a concentration of 12.5% by mass relative to 100 kg (absolute dry weight) of the raw material (ratio of liquid to raw material). press 8), and then the mixture was heated to 120 ° C for 1 hour (alkaline treatment). The raw material after the alkaline treatment was ground with a refiner (manufactured by Kumagai Riki Kogyo Co., Ltd., KRK High Concentration Disk Refiner: clearance 0.5 mm). An equal amount of pure water was added to the raw material after the milling treatment, and then the mixture was adjusted to pH 5 using sulfuric acid under stirring. Subsequently, the mixture was subjected to solid-liquid separation (dehydration) using a 20 mesh sieve (847 μm), and then the solid was washed with water until the electrical conductivity of the solution reached 30 pS / cm. The solid obtained after the solid-liquid separation (pre-treated product) was supplied as a raw material in the saccharification and fermentation stage. [Primary parallel fermentation and saccharification]
[0138] 100 kg (absolute dry weight) of the raw material, 5 g / L of polypeptone, 3 g / L of a yeast extract, and 3 g / L of a wheat germ extract were respectively added to the fermentation and saccharification tank primary parallels so that the concentration of the raw material was 10% by mass, and then water was added to it to adjust the final volume a1m3. A culture fluid containing yeast cells that have been pre-cultured in 50 L of a liquid medium (glucose 30 g / L, polypeptone 5 g / L, yeast extract 3 g / L, wheat germ extract 3 g / L , pH 5.6) at 30 ° C for 24 hours, and 50 L of a commercially available cellulase (Accellerase DUET, manufactured by Genencor International, Inc.) were added to the fermentation tank, and primary parallel fermentation and saccharification were performed at 30 ° C for 24 hours. The pH of the culture fluid during fermentation and saccharification was adjusted to 5.0. [Solid-liquid separation]
[0139] The culture fluid obtained by primary parallel fermentation and saccharification was subjected to solid-liquid separation with a paraffin press (SHX-200 x 1500 L, manufactured by Fukoku Kogyo Co., Ltd., sieve size 1.2 mm), and a residue (primary residue) and a filtrate were separated. The primary residue thus recovered was 19.4 kg (absolute dry weight). [Sieve treatment]
[0140] The filtrate obtained after the solid-liquid separation was passed through a 400 mesh screen (39 pm), and, thereby, fine fibers in the culture fluid were recovered. The amount of fine fibers recovered thus obtained was 15.6 kg (absolute dry weight) in total. The total amount (15.6 kg) of the recovered fine fibers was transferred to the primary parallel fermentation and saccharification tank. [Ethanol production]
[0141] The filtrate obtained by the sieve treatment was separated into an aqueous solution containing ethanol and a concentrated culture fluid using a low pressure distillation apparatus (Evapor CEP-1, Okawara Corp.) under the conditions of distillation temperature: 40 ° C, heating temperature: 80 ° C, and the amount of liquid fed: 150 L / h. The volume and concentration of ethanol in the aqueous solution containing ethanol thus obtained were measured, and the amount of ethanol recovered was calculated. The concentration of ethanol in the solution was measured with a glucose sensor (Model BF-400, manufactured by Oji Scientific Instruments Co., Ltd.). [Centrifugation]
[0142] The concentrated culture fluid separated from the low pressure distillation apparatus was separated into a residue (secondary residue) and a filtrate operating a centrifugal type decanter (manufactured by ΙΕΠ Corp., Model HS-204L) at a rotation speed of 4,500 rpm and a differential speed of 5.0 rpm. The filtrate was transferred to the primary parallel fermentation and saccharification tank. 18.6 kg (absolute dry weight) of the secondary residue were recovered. (Example B2)
[0143] Ethanol production was carried out using the production flow shown in figure 4. Pre-treatment
[0144] Pre-treatment was carried out by the same method used in example B1. [Primary parallel fermentation and saccharification]
[0145] The primary parallel fermentation and saccharification were carried out by the same method used in example Bl. [Solid-liquid separation]
[0146] The solid-liquid separation was carried out by the same method used in the example Bl. 19.2 kg (absolute dry weight) of the primary waste were recovered. [Sieve treatment]
[0147] The sieve treatment was carried out by the same method used in example Bl. The amount of fine fibers recovered obtained by subjecting 100 kg of raw material to primary parallel fermentation and saccharification was 15.5 kg (absolute dry weight) in total. [Secondary parallel fermentation and saccharification]
[0148] 15.5 kg (absolute dry weight) of the fine fibers obtained by the sieve treatment were introduced into the secondary parallel fermentation and saccharification tank as a raw material. 5 g / L of polypeptone, 3 g / L of a yeast extract, and 3 g / L of a wheat germ extract were respectively introduced into the secondary parallel fermentation and saccharification tank, and the final volume was adjusted to 150 L with water. A commercially available yeast (trademark: Maurivin: Mauri Yeast Australia Pty Limited) was grown in 50 L of a liquid medium (glucose 30 g / L, polypeptone 5 g / L, yeast extract 3 g / L, germ extract of wheat 3 g / L, pH 5.6) at 30 ° C for 24 hours. 50 L of the culture fluid containing yeast obtained after cultivation, and 10 L of a commercially available cellulase (Accellerase DUET, manufactured by Genencor International, Inc.) were introduced into the fermentation tank, and secondary parallel fermentation and saccharification was performed at 30 ° C. ° C for 24 hours. The pH of the culture fluid during fermentation and saccharification was adjusted to 5.0. [Ethanol production]
[0149] Ethanol production was carried out using the same method used in example B1 [Centrifugation]
[0150] Centrifugation was performed by the same method used in example B1. 18.6 kg (absolute dry weight) of the secondary residue were recovered. <Comparative example B1>
[0151] Ethanol production was carried out by the process flow shown in figure 5. A test excluding the [sieve treatment] of example Bl was carried out as a comparative example Bl (described below). [Pretreatment]
[0152] Pre-treatment was carried out by the same method used in example Bl. [Primary parallel fermentation and saccharification]
[0153] The primary parallel fermentation and saccharification were carried out by the same method used in example B1. [Solid-liquid separation]
[0154] The solid-liquid separation was carried out by the same method used in example B1. 19.3 kg (absolute dry weight) of the primary waste were recovered. [Ethanol production]
[0155] The filtrate obtained by separating the solid was separated into an aqueous solution containing ethanol and a concentrated culture fluid by the same method as described in example B1. The volume and concentration of ethanol in the aqueous solution containing ethanol thus obtained were measured, and the amount of ethanol recovered was calculated. [Centrifugation]
[0156] Centrifugation was performed by the same method used in example Bl. 34.2 kg (absolute dry weight) of the secondary residue were recovered.
[0157] The results of ethanol production are shown in table B1. In example B1 (the case in which fine fibers were recovered and transferred to the primary parallel fermentation and saccharification tank) and example B2 (the case in which fine fibers were recovered and transferred to the secondary parallel fermentation and saccharification tank), the Ethanol production was increased, compared with the comparative example Bl (the case in which fine fibers were not recovered). (Example B3) [Fermentation and saccharification test]
[0158] A fermentation and saccharification test was carried out in a test tube using the fine fibers obtained in example Bl as a raw material, and the ethanol production was measured by the method described below.
[0159] A commercially available yeast (trademark: Maurivin: Mauri Yeast Australia Pty Limited) was grown in a prepared medium by mixing 100 mL of liquid medium A (polypeptone 5 g / L, yeast extract 3 g / L, wheat germ extract 3 g / L, glucose 30 g / L, dissolved in distilled water, pH 5.6) and 20 mL of liquid medium B (polypeptone 15 g / L, yeast extract 10 g / L, wheat germ extract 10 g / L: dissolved in distilled water) at 30 ° C for 24 hours. 100 ml of the culture fluid obtained after the culture was centrifuged (5,000 rpm, for 20 minutes), and the supernatant was removed. The volume of the residual culture fluid was adjusted to 10 ml (yeast cells were collected) (concentrated yeast cells).
[0160] The raw material (fine fibers) was introduced into a conical flask with a capacity of 300 mL, at a final concentration of 5% by mass. Subsequently, 10 ml of concentrated yeast cells and 2.5 ml of a commercially available cellulase (Accellerase DUET, manufactured by Genencor International, Inc.) were added to it, and the final volume was made up to 100 ml with distilled water. This liquid mixture was grown at 30 ° C for 24 hours (fermentation and saccharification). The culture fluid obtained after the culture was centrifuged (5000 rpm, for 20 minutes), and the concentration of ethanol in the supernatant was measured. In addition, the Kappa number (lignin content index) of fine fibers was measured by a measurement method equivalent to JIS P8211. <Comparative example B2>
[0161] A fermentation and saccharification test was carried out by the same method used in example B3, using the primary residue obtained in example BI as a raw material. Ethanol production and the Kappa number of the primary waste were measured. <Comparative example B3>
[0162] A fermentation and saccharification test was carried out by the same method used in example B3, using the secondary residue obtained in example BI as a raw material. Ethanol production and the Kappa number of the secondary waste were measured.
[0163] Ethanol concentrations and Kappa numbers are shown in table B2. When the fine fibers (Example B3) were used as the raw material, the concentration of ethanol in the culture fluid was higher, compared to the cases of using the primary waste (comparative example B2) and the secondary waste (comparative example B3) as the raw material. In addition, the Kappa number of fine fibers (Example B3) had a lower value compared to the Kappa numbers of the primary residue (comparative example B2) and the secondary residue (comparative example B3). From the results described above, it was observed that the fine fibers had a low lignin content compared to the primary and secondary waste and, as a result, when the fine fibers were used as the raw material for fermentation and saccharification, ethanol production increased, compared to primary and secondary waste. The fine fibers allowed fermentation and saccharification to proceed easily, even if the fine fibers are not pre-treated (mechanical treatment or the like), and thus, it was observed that the fine fibers are suitable as the raw material for fermentation and saccharification . (Example B4)
[0164] A test in which Eucalyptus globulus forest waste (bark 70%, branches and leaves 30%) was used as the raw material, instead of the Eucalyptus globulus bark used as the raw material in example Bl, was carried out as example B4.
[0165] The test was carried out completely in the same way as in example Bl, except that forest waste was used (the process flow was the same as shown in figure 1). <Comparative example B4>
[0166] A test in which the [screen treatment] of example B4 was omitted, was performed as a comparative example B4. The test was carried out completely in the same way as in example B4, except that the sieve treatment was omitted (the process flow was the same as shown in figure 5).
[0167] The results of ethanol production are shown in table B3. In example B4 (the case in which the fine fibers were collected and transferred to the primary parallel fermentation and saccharification tank), ethanol production increased compared to the comparative example B4 (the case in which the fine fibers were not recovered). (Example B5)
[0168] Ethanol production was carried out using the process flow shown in figure 6. [Pretreatment]
[0169] Pre-treatment was carried out in the same way as in example Bl. [Primary parallel fermentation and saccharification]
[0170] The primary parallel fermentation and saccharification were carried out by the same method used in example Bl, except that sodium chloride was added as an electrolyte in the culture fluid. Sodium chloride (electrolyte) was added to the culture fluid which was adjusted by the same method used in example Bl, to a final concentration of 100 mM (electrical conductivity of the raw material suspension: 12.2 mS / cm). Subsequently, yeast cells and a commercially available cellulase were introduced into the fermentation tank by the same method used in example Bl, and thus, the primary parallel fermentation and saccharification were carried out. [Solid-liquid separation]
[0171] The solid-liquid separation was carried out by the same method used in example Bl. 15.3 kg (absolute dry weight) of the primary residue were recovered. [Sieve treatment]
[0172] The sieve treatment was carried out by the same method used in example Bl. 13.4 kg (absolute dry weight) in the total of the fine fibers were recovered. The total amount (13.4 kg) of the recovered fine fibers was transferred to the primary parallel fermentation and saccharification tank. [Ethanol production]
[0173] Ethanol production was carried out by the same method used in example Bl. [Centrifugation]
[0174] Centrifugation was performed by the same method used in example Bl. 14.7 kg (absolute dry weight) of the secondary residue were recovered.
[0175] The results of ethanol production are shown in table B4. In the case of adding sodium chloride to the culture fluid (Example B5), ethanol production increased, compared to the case in which sodium chloride was not added (Example Bl). (Example B6)
[0176] Ethanol production was carried out using the same process flow shown in figure 7. [Pretreatment]
[0177] Pre-treatment was carried out in the same way as in example B1. [Primary parallel fermentation and saccharification]
[0178] The primary parallel fermentation and saccharification were carried out by the same method used in example Bl, except that sodium chloride was added as an electrolyte in the culture fluid. Sodium chloride (electrolyte) was added to the culture fluid, which was adjusted by the same method used in example Bl, to a final concentration of 100 mM (electrical conductivity of the raw material suspension: 12.2 mS / cm). Subsequently, yeast cells and a commercially available cellulase were introduced into the fermentation tank by the same method used in example Bl, and thus, the primary parallel fermentation and saccharification were carried out. [Solid-liquid separation]
[0179] The solid-liquid separation was carried out by the same method used in example Bl. 14.8 kg (absolute dry weight) of the primary residue were recovered. [Sieve treatment]
[0180] The sieve treatment was carried out by the same method used in example B1. 13.0 kg (absolute dry weight) of the total fine fibers were recovered. The total amount (13.0 kg) of the recovered fine fibers was transferred to the primary parallel fermentation and saccharification tank. [Secondary parallel fermentation and saccharification]
[0181] Secondary parallel fermentation and saccharification were carried out by the same method used in example B2. [Ethanol production]
[0182] Ethanol production was carried out by the same method used in example B1. [Centrifugation]
[0183] Centrifugation was performed by the same method used in example B1. 14.2 kg (absolute dry weight) of the secondary residue were recovered.
[0184] The results of ethanol production are shown in table 5. In the case of adding sodium chloride to the culture fluid (Example B6), ethanol production increased compared to the case in which sodium chloride was not added (Example B2). INDUSTRIAL APPLICABILITY
[0185] According to the enzymatic saccharification treatment method of the present invention, the adsorption of saccharification enzymes on the unreacted components or reaction residue of a lignocellulose-based raw material is suppressed, the separation of the enzymes from the treated enzyme saccharification liquid it is facilitated, and the recycling ratio of saccharification enzymes is maintained at a high level for a long period of time during the enzymatic saccharification treatment process. Therefore, it is possible to industrially produce saccharides, ethanol and the like through the treatment of enzymatic saccharification of a raw material based on lignocellulose.
[0186] In addition, according to the present invention, ethanol production can be increased by reusing the fine fibers included in the culture fluid obtained after fermentation and saccharification as a raw material for fermentation and saccharification. In addition, ethanol production can be increased by adding an electrolyte to the culture fluid.

权利要求:
Claims (12)
[0001]
Method for the treatment of enzymatic saccharification of a raw material based on lignocellulose, characterized by the fact that the method comprises: add a raw material based on lignocellulose that has been subjected to a pre-treatment to bring the lignocellulose to an state of being enzymatically saccharifiable, together with an electrolyte containing a water-soluble salt, water containing cellulose saccharification enzyme; submit the lignocellulose base to the raw material as a suspension of raw material whose electrical conductivity was adjusted from 5 mS / cm to 25 mS / cm, to an enzymatic saccharification treatment through an enzymatic saccharification reaction; separating and recovering the reaction product and an enzyme-containing solution from the treated suspension after enzymatic saccharification treatment; and recycle the solution containing recovered enzyme as an enzyme for the enzymatic saccharification treatment process.
[0002]
Method for the treatment of enzymatic saccharification of a raw material based on lignocellulose according to claim 1, characterized by the fact that the pre-treatment comprises a chemical treatment of immersing the raw material based on lignocellulose in a solution containing an alkali selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate and sodium hydrogen carbonate, or a mixture of these, or a mixture of sodium sulfite and an alkali like that.
[0003]
Method for the treatment of enzymatic saccharification of a lignocellulose-based raw material according to claim 1 or 2, characterized by the fact that the lignocellulose-based raw material is forest waste.
[0004]
Method for the treatment of enzymatic saccharification of a lignocellulose-based raw material according to claim 1 or 2, characterized by the fact that the lignocellulose-based raw material is tree bark.
[0005]
Method for the treatment of enzymatic saccharification of a lignocellulose-based raw material according to any one of claims 1 to 4, characterized in that the water-soluble salt is at least one water-soluble salt selected from metal salts alkali and alkaline earth metal salts.
[0006]
Method for the treatment of enzymatic saccharification of a raw material based on lignocellulose according to any one of claims 1 to 5, characterized by the fact that the water-soluble salt is a salt selected from the group consisting of halides, sulfates, sulfites, thiosulfates, carbonates, hydrogen carbonates, phosphates, di-diogenes phosphates, hydrogen diphosphates, acetates, and citrates of alkali metals and alkaline earth metals.
[0007]
Method for the treatment of enzymatic saccharification of a raw material based on lignocellulose according to any one of claims 1 to 6, characterized in that the method of treatment of enzymatic saccharification is a method of subjecting a raw material to base of lignocellulose to an enzymatic saccharification treatment according to a series of processes including: a pre-treatment step of subjecting a lignocellulose-based raw material to a treatment to bring the lignocellulose to an state of being enzymatically saccharifiable; an enzymatic saccharification treatment step of adding the raw material based on pre-treated lignocellulose, together with an electrolyte containing a water-soluble salt in the water containing cellulose saccharification enzyme, and treating the raw material based on lignocellulose as a suspension of raw material whose electrical conductivity was adjusted from 5 mS / cm to 25 mS / cm, through an enzymatic saccharification reaction; a solid-liquid separation step of removing a solid residue from the treated suspension from the enzymatic saccharification treatment step; a centrifugation step of centrifuging the liquid fraction from the solid-liquid separation step and thereby obtaining a liquid fraction that contains enzymes and saccharides and has all the remaining residue removed; a membrane separation step of separating the liquid fraction from the centrifugation step into a solution containing enzyme and a solution containing saccharide produced; and an enzyme recycling step of recycling and supplying the enzyme-containing solution obtainable from the membrane separation step in the enzyme saccharification treatment step as an enzyme source.
[0008]
Method for the treatment of enzymatic saccharification of a lignocellulose-based raw material according to any one of claims 1 to 7, characterized by the fact that the enzymatic saccharification treatment step is a parallel fermentation and saccharification treatment step of perform a treatment based on an enzymatic saccharification reaction of a raw material based on lignocellulose and a treatment of fermentation of saccharides produced by a micro-organism for fermentation in combination using a cellulase preparation and a micro-organism for fermentation uses saccharides as a fermentation substrate (raw material) in combination and thereby produces a fermentation product together with saccharides.
[0009]
Method for the treatment of enzymatic saccharification of a raw material based on lignocellulose according to claim 8, characterized by the fact that the method of enzymatic saccharification is a method of subjecting a raw material based on lignocellulose to a treatment of parallel fermentation and saccharification according to a series of processes including: a pretreatment step of subjecting a lignocellulose-based raw material to a treatment to produce the lignocellulose-based raw material suitable for an enzymatic saccharification reaction; a parallel fermentation and saccharification treatment step of adding the raw material based on pre-treated lignocellulose, together with a micro-organism for fermentation that uses saccharides as a fermentation substrate, and with an electrolyte containing a water-soluble salt , in water containing cellulose saccharification enzyme, and subjecting the raw material to a lignocellulose base as a suspension of raw material whose electrical conductivity was adjusted from 5 mS / cm to 25 mS / cm, both to an enzymatic saccharification treatment as for a fermentation treatment to use the saccharides produced as a substrate; a solid-liquid separation step of removing a solid residue from the treated suspension from the enzymatic saccharification treatment step; a distillation step of separating and recovering the fermentation product from the liquid fraction from the solid-liquid separation step through distillation; a centrifugation step of centrifuging the residual distillate from the distillation step to remove any remaining residue and thereby obtain a liquid fraction containing enzymes and saccharides; a membrane separation step of separating the liquid fraction from the centrifugation step into a solution containing enzyme and a solution containing saccharide; and an enzyme recycling step of recycling and supplying the enzyme-containing solution obtainable from the membrane separation step in the enzyme saccharification treatment step as an enzyme source.
[0010]
Method for the treatment of enzymatic saccharification of a lignocellulose-based raw material according to claim 9, characterized in that the solution containing saccharide separated and recovered from the membrane separation step is a liquid containing saccharides that includes oligosaccharides such as main components.
[0011]
Method for the treatment of enzymatic saccharification of a lignocellulose-based raw material according to claim 9, characterized by the fact that the liquid fraction from the centrifugation step is recycled and supplied to the enzymatic saccharification treatment step as a solution containing enzyme containing saccharides, without going through the membrane separation step.
[0012]
Method for the treatment of enzymatic saccharification of a raw material based on lignocellulose according to claim 8, characterized by the fact that the method of treatment of enzymatic saccharification is a method of subjecting a raw material based on lignocellulose to a parallel fermentation and saccharification treatment according to a series of processes including: a pre-treatment step of subjecting a lignocellulose-based raw material to a treatment to bring the lignocellulose to an state of being enzymatically saccharifiable; a parallel fermentation and saccharification treatment step of adding the raw material based on pre-treated lignocellulose, together with a micro-organism for fermentation that uses saccharides as a fermentation substrate, and with an electrolyte containing a water-soluble salt , in water containing cellulose saccharification enzyme, and subjecting the raw material to a lignocellulose base as a suspension of raw material whose electrical conductivity was adjusted from 5 mS / cm to 25 mS / cm, both to an enzymatic saccharification treatment to treat the raw material based on lignocellulose through an enzymatic saccharification reaction for a fermentation treatment using the saccharides produced as a substrate; a solid-liquid separation step of separating the treated suspension from the parallel fermentation and saccharification treatment step into a residue and a liquid fraction using a screw press with a sieve size of 1.0 mm to 2.0 mm; a sieve treatment step to separate the liquid fraction from the solid-liquid separation step in the fine fibers and a liquid fraction through a sieve treatment using an 80 to 600 mesh sieve; a distillation step of separating and recovering a fermentation product from the liquid fraction obtained after the sieve treatment excluding fine fibers, through distillation; a centrifugation step of centrifuging the residual distillate from the distillation step to remove any remaining residue and thereby obtain a liquid fraction containing enzymes and saccharides; and a step of recycling and supplying the liquid fraction from the centrifugation step to the step of treating enzyme saccharification as a solution containing enzyme containing saccharides, without going through the membrane separation step.

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同族专利:

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公开号 | 公开日

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NZ607405A|2013-12-20|

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US20130157318A1|2013-06-20|

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EP2612920A4|2016-08-24|

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US8728770B2|2014-05-20|

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AU2011296986B2|2013-11-21|

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AU2011296986A8|2013-03-21|

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CA2809519A1|2012-03-08|

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CA2809519C|2014-07-29|

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MY160964A|2017-03-31|

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CN103189521A|2013-07-03|

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EP2612920A1|2013-07-10|

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EP2612920B1|2020-10-14|

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AU2011296986A1|2013-03-14|

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BR112013004261A2|2016-08-02|

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WO2012029842A1|2012-03-08|

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CN103189521B|2015-11-25|

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法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-05-28| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2020-04-07| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-12-01| B09W| Decision of grant: rectification|Free format text: ERRO NA DESCRICAO DO QUADRO 1 |

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2021-04-06| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 31/08/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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