KR20190044954A - Biomass pre-treatment and saccharification method for the production of biofuel or bioplastic - Google Patents

Abstract

The present invention relates to a biomass preprocessing and saccharification method for producing biofuel or bioplastic. When biomass is cultured with KPYBE10 which is new cellulose decomposing mold before physical and chemical preprocessing, it is possible to manufacture a saccharification liquid having the same glucose concentration even with low sulfuric acid concentration, a low temperature, a short time, and a small amount of saccharification enzymes. Physical and chemical conditions reduce generation of fermentation inhibiting materials such as furfural, hydromethyl furfural, etc. such that a decontamination process is not needed. A preprocessing method using acid hydrolysis remarkably reduces preprocessing and saccharification costs and high-pressure steam and electricity costs which occupy more than a half (53%) of direct producing costs of conventional second generation bioethanol. A low-cost saccharification liquid manufactured by the method can be used as a microbial culture medium for industrial fermentation to produce second generation biofuel or bioplastic. Therefore, the present invention is an inexpensive and eco-friendly new method for rapidly realizing commercial scale production.

Description

TECHNICAL FIELD The present invention relates to a biomass pre-treatment and saccharification method for producing biofuels or bioplastics,

The present invention relates to a simple and cost-effective biomass pretreatment for the production of biofuels or bioplastics using lignocellulosic biomass and novel fibrinolytic fungi, which are second generation biofuels, It is about narration.

Currently, bioethanol and biodiesel for transport are being commercialized in the US and Brazil, and gasoline (BE10) containing 10% bioethanol is also readily available in European gas stations. However, the current commercialized way of producing first-generation bio-ethanol under the support of governments by introducing food resources such as corn, wheat, sugarcane, etc. has a problem of deepening the food shortage problem, and the problem of cornstalks, sugarcane remnants economical production of cellulosic bioethanol using non-food resources such as inexpensive agricultural fuels byproducts such as bagasse, straw, straw, reed, switchgrass, fruit husks, beet residue, Commercialization is urgent.

The agrochemical by-products consist of 38-45% cellulose, 22-40% hemicellulose, and 5-25% lignin. Fibers, which are the most abundant organic matter on the planet, are made up of thousands of glucose molecules, which are converted into bioethanol by glycosylation and fermentation. However, despite the existence of various technologies for glycosylation, commercial pretreatment processes have not yet been proved and commercial production is not realized. In the US, Canada, Europe, Japan, and China, the 2nd generation bioethanol production test plant is operating by pretreatment of acid hydrolysis using agricultural by-products such as cornstalks and straw as raw materials, but the time spent in pretreatment / The development of the pretreatment process which can reduce the cost considerably has progressed and it is not yet commercialized.

When comparing the second-generation bio-ethanol production process with the first-generation bio-ethanol production process, the second-generation bio-ethanol production process includes the purchase and handling of inexpensive agricultural by-products and the pre-treatment and saccharification processes, Etc.) is the same as the first generation bioethanol production process. Therefore, the cost of direct production of second-generation bio-ethanol is relatively low using cheap cornstalks or sugarcane remnants instead of expensive food resources. However, pretreatment and saccharification processes are costly factors, It is time to develop a new concept of pre-treatment and saccharification that shortens time.

International Publication No. 2017010789 (published on Jan. 19, 2017)

It is an object of the present invention to provide a simple and cost-effective biomass preprocessing and a method for enhancing saccharification efficiency for the production of biofuels or bioplastics using the novel fibrinolytic fungus and the novel fibrinolytic fungus.

In order to achieve the above object, the present invention provides a novel fibrinolytic fungus, which is a penicillium species microorganism collected from soil, and is viable under severe conditions.

The present invention also provides a method for producing a biomass comprising: (a) pulverizing a biomass; And (b) a step of spraying and pulverizing the mixed solution obtained by suspending the fibrinolytic fungus in water or a sulfuric acid solution to the pulverized biomass, and culturing the biomass for biomass production. .

According to the present invention, the biomass is cultivated with KPYBE10, a novel fibrinolytic fungus, prior to physico-chemical pretreatment, so that even with a low concentration of sulfuric acid, low temperature, short time, and a small amount of saccharifying enzyme, The physicochemical condition of the present invention has the additional effect of reducing the generation of fermentation inhibiting substances such as furfural, hydro-methylfurfural and the like, and thus eliminating the need for a detoxification process.

The pretreatment method using the acid hydrolysis of the present invention can significantly reduce the cost of pretreatment / saccharification, high-pressure steam, and electricity, which accounts for more than half of the conventional second-generation bioethanol direct production cost at 53% The produced low cost saccharified liquid can be used as an industrial fermentation microorganism medium for the production of second generation biofuels and bioplastics, and is a cheap and environmentally friendly new method for realizing commercial scale production at an early stage.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual diagram of a method for improving biomass pretreatment and saccharification efficiency using the woody biomass of the present invention and the novel fibrin degrading fungus KPYBE10.
FIG. 2 is a step-by-step representation of the manufacturing process of A) second generation bio-ethanol and B) bio-plastic using biomass cultured with KPYBE10.
Fig. 3 is a microscopic image of the conidia and conidia of KPYBE10.
FIG. 4 shows the results of the cultivation of KPYBE10 after sterilization of cornstarch powder, inoculation with KPYBE10 and incubation at various culture temperatures (28 ° C, 21 ° C, 18 ° C and 15 ° C) for one week or B) .
Figure 5 shows the growth activity of KPYBE10 after sterilization of sawdust medium, inoculation with KPYBE10, and subsequent incubation at various incubation temperatures (28 ° C, 21 ° C, 18 ° C, 15 ° C) Respectively.
6 shows the inhibition of the growth of the resistant strain existing in the biomass after incubation for one week at various temperatures (28 DEG C, 35 DEG C, 40 DEG C) after inoculating KPYBE10 without sterilization of the medium containing the cornstarch powder .
FIG. 7 shows the inhibition of the growth of germs after adding 0.1% or 0.2% of sulfuric acid solution to the medium to which cornstarch powder without sterilization was added, inoculating KPYBE10 and culturing at 28 DEG C for 7 days.

The inventors of the present invention have found that a novel cellulolytic fungus, KPYBE10, is directly cultured in a biomass to significantly reduce the total pretreatment cost, the time required and the cost of purchasing the saccharification enzyme, and significantly increase the concentration of glucose produced as a result of saccharification, The present invention can be applied to the industrialization of second-generation bio-ethanol, bio-butanol, bio-plastic, etc. by providing a biomass pretreatment and saccharification method having an effect of increasing the biomass.

The present invention provides a fungi degrading fungus characterized by being a Penicillium species microorganism collected from soil and capable of surviving under severe conditions.

This harsh condition may be, but is not limited to, treating at a temperature of 40 to 50 占 폚.

The fibrinolytic fungus is deposited with KCTC 13356BP, which can improve the saccharification efficiency of biomass.

The present invention also provides a method for producing a biomass comprising: (a) pulverizing a biomass; And (b) a step of spraying and pulverizing the mixed solution obtained by suspending the fibrinolytic fungus in water or a sulfuric acid solution to the pulverized biomass, and culturing the biomass for biomass production. .

The water, sulfuric acid solution or biomass of step (b) can be used without sterilization treatment, and KPYBE10, a novel fibrinolytic fungus, can be cultured in the biomass without the addition of additional nutrients.

The biofuel may be, but is not limited to, bioethanol or biobutanol.

The method for enhancing the pretreatment and saccharification efficiency of the biomass for producing biofuels or bioplastics is the same as the first step (Steps a to e).

The method for improving the pretreatment and saccharification efficiency of the biomass may include: (a) pulverizing the biomass; (b) inoculating and pulverizing the pulverized biomass with a mixture solution in which the fibrin degrading fungus is suspended in water or a sulfuric acid solution; (c) treating the cultured culture with an acid to hydrolyze it; (d) separating the resultant product after the hydrolysis into a solid phase and a liquid phase; (e) treating the solid phase with a cellulose degrading enzyme to obtain a saccharified saccharide per hexane or treating the liquid phase with a neutralizing agent to obtain a pentasaccharide solution; (f) preparing a biofuel fermentation broth by inoculating and culturing the biofuel fermentation microorganism in the saccharified saccharide solution, the pentasaccharide solution or the mixed saccharification solution thereof; And (g) distilling the fermentation broth to produce a concentrated biofuel.

More specifically, in step (f), the saccharified saccharide solution is subjected to ethanol fermentation using a hexose fermentation strain (such as yeast), and the pentose saccharified solution is fermented with a pentose fermentation strain (Pichia stipitis) (Pachysolen tannophilus, Candida shehata, etc.) or fermented with ethanol or a mixture of the saccharified saccharide solution and the pentasaccharide solution simultaneously (recombinant Zymomonas mobilis , Recombinant E. coli, etc.) can be used for simultaneous fermentation. In the case of biobutanol, the mixture can be fermented by inoculating Clostridium to a mixture of the saccharified saccharide and pentose saccharide.

The method for improving the pretreatment and saccharification efficiency of the biomass may include: (a) pulverizing the biomass; (b) inoculating and pulverizing the pulverized biomass with a mixture solution in which the fibrin degrading fungus is suspended in water or a sulfuric acid solution; (c) treating the cultured culture with an acid to hydrolyze it; (d) separating the resultant product after the hydrolysis into a solid phase and a liquid phase; (e) treating the solid phase with a cellulolytic enzyme to obtain a saccharide-saccharide solution, and treating the liquid phase with a neutralizing agent to obtain a saccharide-saccharide solution; And (f) preparing a bioplastic by inoculating and culturing a strain producing the bioplastic synthetic material in the hexagonal and pentagonal mixed saccharified liquid, and a method for enhancing the saccharification efficiency of the biomass.

Specifically, in the step (f), the saccharified saccharified liquid and the pentasaccharide mixed saccharified liquid are mixed with an alkali to synthesize a hydroxyvalerate, hydroxy butyrate, A strain such as Alcaligenes eutrophus can be inoculated and cultured, and the culture can be used as industrial fermentation medium.

The fibrinolytic fungus is deposited with KCTC 13356BP and is a microorganism of the genus Penicillium species.

The biomass may be selected from the group consisting of corn stover, corn cobs, bagasse, beet residue, straw, chaff, straw, reed, sunflower stem, switchgrass, rapeseed, , Sawdust, grass, cotten, flax, and fruit peel, but is not limited thereto.

After the step (a), the pulverized biomass may be passed through a mesh having a length of 0.25 to 10 mm on one side.

In the step (b), the fibrinolytic fungus may be suspended in water, inoculated into the pulverized biomass, and cultured for 3 to 14 days at a temperature of 40 to 50 ° C. However, the present invention is not limited thereto.

In the step (b), the fibrinolytic fungus may be suspended in a 0.1 to 0.2% (w / v) sulfuric acid solution to inoculate the pulverized biomass and cultured for 3 to 14 days, But is not limited thereto. Thereafter, in the pretreatment reaction, sulfuric acid is used in an amount corresponding to the amount of sulfuric acid added in advance.

In the step (b), the inoculum of the fibrinolytic fungus is sprayed with a sufficient amount of spores to actively grow in the entire biomass within 5 days. (5 to 10 stages) culture method according to the scale of biomass to be used for pretreatment. The amount of biomass to be used in the next inoculation is sequentially increased by 10 to 50 times, and the seed culture is cultured to obtain a sufficient number After forming the spore bacterium, the spore-forming biomass is suspended in water (industrial water, no sterilized water is needed when cultivation is carried out at 40 ° C) and is sprayed on the biomass yard.

The hydrolysis in step (c) may be carried out in an acidic solution of 0.1 to 10% (w / v) at a temperature of 130 to 300 ° C for 30 to 90 minutes, but it is not limited thereto.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a conceptual diagram of a method for improving the pretreatment and saccharification efficiency of biomass using the woody biomass of the present invention and the novel fiber decomposing fungus KPYBE10.

The method of enhancing the biomass pretreatment and saccharification efficiency of the present invention can be carried out by inoculating KPYBE10 spore bacterium into woody biomass in a biomass yard without the need for installation of additional equipment and then cultivating it for 5-7 days at 40 ° C, , And then the cellulase treatment can increase the glucose concentration by 40% or more. This means that even milder physico-chemical conditions (lower pretreatment pressure, lower temperature, shorter time, lower sulfuric acid concentration) and lesser amounts of cellulase can be used to obtain the same amount of sugars from woody biomass, It provides a way to accelerate the realization of inexpensive mass production and commercialization of second generation biofuels and bioplastics.

The present invention also relates to a process for the preparation of all woody biomass physical, chemical and biological pretreatment methods (acid hydrolysis, steam explosion, organosolv, sulfite pretreatment, SO ₂ - ethanol - water (AVAP®) fraction _{(SO 2 -ethanol-water (AVAP®} ) fractionation), alkaline wet oxidation and ozone pretreatment (alkaline wet oxidation and ozone pretreatment) , pokswae ammonia (ammonia fiber explosion; APEX), ammonia recycle Prior to the introduction of ammonia recycle percolation (ARP), lime, sonication, irradiation, high temperature / high pressure method, biological pretreatment method, etc.), the saccharification efficiency By providing improved methods, the cost of producing second-generation bio-ethanol, second-generation bio-butanol and bio-plastics (lignocellulosic biodegradable bioplastic) can be drastically reduced, enabling an empirical realization of these industries It provides a way to.

FIG. 2 is a step-by-step representation of the manufacturing process of A) second generation bio-ethanol and B) bio-plastic using biomass cultured with KPYBE10.

S100 is a biomass feeding step. The biomass used as the feedstock of the present invention includes various agricultural by-products, and preferably includes corn stover, corn cobs, bagasse, Straw, reeds, switchgrass, rapeseed, waste wood, sawdust, grass, cotten, flax, or fruit husks, more preferably cornstalks, agar Straw, reed, switchgrass, wood, sawdust, or fruit husks, and most preferably cornstalks, cornstarch, sugarcane remnants, sugar beet removers, straw straw, straw, reed straw, sugar cane residue, sugar cane residue, straw straw, , Switchgrass, sawdust, but not limited thereto.

S110 is a biomass grinding step. In the present invention, the step of grinding the biomass includes a physical shredding method such as shearing, milling or grinding in various ways known in the art, , A knife cutter or a blender may be used to pulverize the biomass, but is not limited thereto.

According to a preferred embodiment of the present invention, the method of the present invention may further comprise the step of passing the pulverized biomass after the above-mentioned step into a net having a length of 0.25 to 10 mm on one side. More preferably, the length of one side of the network may be 0.25-3 mm, and most preferably 0.25-1 mm, but is not limited thereto.

S120 is a step of suspending a fibrinolytic microorganism strain with a sparger, wherein the fibrinolytic microorganism strain includes all microorganisms that secrete a cellulase (fungi: Aspergillus, Fusarium, Humicola ), Melanocarpus, Penicillium, Trichoderma; Bacteria: Acidothermus, Alcaligenes, Anoxybacillus, Bacillus, Cellulomonas, Cellvibrio, Clostridium, Saito The use of Cytophaga, Microbispora, Nocardiopsis, Pseudomonas, Rhodothermus, Streptomyces, Thermomonospora, etc.) However, when the novel fibrinolytic fungal KPYBE10 strain of the present invention is used, it is possible to provide an eco-friendly method for improving the saccharification efficiency of woody biomass at low cost.

S130 is a step of mixing cultivated biomass and KPYBE10 in a yard. If necessary, water is sprayed on the upper part to maintain moisture, and on top of it, a vinyl insulating waterproofing film is covered to prevent evaporation of water and to help maintain the temperature. In addition, considering the seasonal factors such as the winter season, some heat is supplied to the bottom of the yard in the form of heat pipes.

Reference numeral 10 denotes a continuous mixed culture of biomass and fungi. The apparatus is designed to be used in a case where the temperature of the field is maintained at 15 ° C or less due to seasonal factors or the like, so that the proper growth rate of the fungus is lowered, KPYBE10 This is a continuous mixing chamber with a simple structure that is kept warm with an insulating material that can be used when there is a need to increase the cultivation processing speed. After crushing from the top, the biomass sieved with a 0.25-2 mm mesh is continuously introduced and spore suspension suspended in water is cultured at 40 ° C using a sprinkler (S120) on the ceiling, and cultured at 25-35 ° C In order to inhibit the growth of germs, spores were suspended in a 0.1 to 0.2% sulfuric acid solution and mixed periodically with an agitator installed in the culture room. After 5-7 days of storage, KPYBE10 The cultured biomass is continuously sampled and used for the pretreatment / saccharification process as a subsequent process. When seed spores are suspended in a 0.1 to 0.2% sulfuric acid solution, use less sulfuric acid by the amount of sulfuric acid added beforehand in the pretreatment reaction.

S140 is an acid hydrolysis step of the biomass. The acid used for hydrolyzing the pulverized biomass includes various acids known in the art, but preferably sulfuric acid, hydrochloric acid, nitric acid, acetic acid, formic acid or phosphoric acid, More preferably sulfuric acid, hydrochloric acid or nitric acid, most preferably sulfuric acid, but is not limited thereto.

According to another preferred embodiment of the present invention, the hydrolysis is carried out at a concentration of sulfuric acid of 0.1-80% (w / v) at a temperature of 60-250 ° C for 20-240 min, more preferably of 0.1-5 % (w / v), at a temperature of 100-200 ° C for 30-120 minutes, most preferably at a concentration of 0.5% (w / v) of sulfuric acid, at a temperature of 140-160 ° C for 20-60 minutes But is not limited to,

When the low-concentration sulfuric acid (H ₂ SO ₄ ) used in the above process is neutralized with calcium carbonate (CaCO ₃ ) or calcium hydroxide (Ca (OH) ₂ ], gypsum (CaSO ₄ ) is formed and used as a raw material for making an environment- have. In addition, relatively low temperatures and short processing times can reduce the total energy costs required for the pretreatment, enabling economical bio-ethanol production.

Step S150 is a step of separating the pretreatment product into a liquid phase and a solid phase, and separates the solid (S) and the liquid (L) as a solid-liquid separation filter. The solids are mainly composed of hexose (glucose), and the liquid is mainly composed of xylose.

S160 is a step of treating the cellulase in the solid phase in the saccharification tank to obtain a saccharified saccharide solution of the saccharified saccharide, whereby saccharification of cellulose is caused by the added cellulase and glucose is formed.

In step S170, the saccharified liquid of the saccharified saccharide is fermented with ethanol by the ethanol-producing fermentation strain in the fermentation tank of the granulocyte.

S180 is a step of separating solid matter such as fibrin lignin and the like, which is saccharified in the centrifuge, from the liquid containing ethanol. Solid materials are used in livestock feed or in the factory for power generation or high pressure steam production, and the remaining materials are sold to nearby plants.

S190 is a step in which an osmopolitan saccharified liquid produced by neutralizing the liquid phase of S150 with calcium carbonate (CaCO3) or calcium hydroxide (Ca (OH) 2] in a fermentation tank per ounce is fermented with ethanol by an ethanol producing fermentation strain.

S200 is a step of separating gypsum (CaSO ₄ , gypsum) and other solid substances produced after the neutralization process from a fermentation broth with a centrifuge. The gypsum can be separated and used for manufacturing an environmentally friendly gypsum board.

S210 is a distillation step using a distillation apparatus to produce 99.9% of bioethanol through conventional distillation and molecular sieves.

Reference numeral 20 denotes a bioethanol storage tank of 99.9%.

S220 is a mixed fermentation tank of hexane saccharified liquid and pentane saccharified liquid, which is a synthetic material of bioplastics. Alcaligenes eutrophus, which synthesizes hydroxy valerate, hydroxy butyrate and the like, ), And the cultured product can be used as industrial fermentation medium.

Among the conventional methods, biomass is used as a wood waste fungus (Phanerochaete chrysosporium, Dichomitus squalens, etc.) for use as a biomass for bioethanol production, ), Shiitake mushroom, mushroom mushroom, oyster mushroom, etc.). (Korean Patent No. 10-1055623), a method of using panergotectosifolia and decomitus squalens (Korean Patent No. 10-1055623) is a method in which the above-mentioned woody dead fungi are mixed with high nutrients (glucose 3-5 g / L, (10 g / L, KH ₂ PO ₄ 2.25 to 11 g / L) and a saccharification enzyme (cellulase 5% (v / v) -30 days), followed by partial saccharification. Then, the saccharified solution is further saccharified by sending the beta-glucosidase to an enzyme saccharification tank fixed to the carrier, and the resulting saccharified solution is used as a bioethanol fermentation medium .

This method is characterized in that biomass is charged into a packed column together with cellulase and various nutrients without a conventional physico-chemical pretreatment process, and is dependent on the biological saccharification process of white rot fungi which occurs slowly when white rot fungi are grown. Considering the industrial scale of producing at least several tens to millions of liters of second generation bio-ethanol per day, (1) a very long pretreatment period of up to 30 days is required, (2) pretreatment of a packed tower type with a capacity of hundreds to thousands of liters It is necessary to maintain a high concentration of nutrients such as glucose, yeast extract, phosphoric acid, and vitamins and cellulase enzymes at a high concentration in a glucose and enzyme saccharification tank, and (3) when the nutrients are consumed due to contamination of germs It is more costly to prevent pollution and to maintain the concentration of nutrients. Therefore, the second generation Production of bioethanol is not only low in production but also costly for rising costs, and may not be suitable for industrial scale commercialization considering economies of scale.

Meanwhile, it is sufficient that the KPYBE10 spore bacterium easily grown in cornstalks, rice straw, sawdust, etc. is suspended in water, sprinkled on the biomass pulverized in the yard, and covered with the vinyl thermal insulation tarpaulin for 5-7 days. According to the present invention, (1) no additional nutrients need to be added, (2) an additional small amount of water is required to maintain a minimum amount of moisture necessary for growth of the mold, and (3) (4) It is necessary to cultivate KPYBE10 at 40 占 폚 or less to inhibit the growth of germs, when the temperature is kept at 40 占 폚 or less, by providing a heat pipe at the bottom of the yard and maintaining the culture temperature at 40 占 폚 , The spore suspension is suspended and cultured in a 0.1 to 0.2% (w / v) sulfuric acid solution, and in the pretreatment reaction, the biomass is used in an amount as small as that of sulfuric acid.

Further, in addition to the practice in the yard, a biomass and a fungi mixed continuous culture room having a simple structure, such as 10 in Fig. 2, can be used. After the crushing from the upper part of the culture room, the biomass sifted with a 0.25-2 mm mesh is continuously introduced, and the spore bacterium suspended in water or a 0.1 to 0.2% sulfuric acid solution is sprayed using a sprinkler (S120) The cells were cultured for 5-7 days at 40 ° C (suspended in water) or 25-35 ° C (suspended in 0.1-0.2% sulfuric acid solution), periodically mixed with the agitator installed in the culture chamber, and cultured from the bottom to KPYBE10 The processed biomass is continuously sampled to continuously perform a pretreatment and saccharification process as a subsequent process. This is because when the temperature of the field is kept below 15 ° C due to seasonal factors, the growth rate of the fungus is lowered, or when there is a need to increase the speed of the fungal culture while using a minimum yard area, It is a cheap mixed culture room.

As described above, the present invention is very cost-effective to cultivate molds in biomass, and has the advantage that several to several tens of tons can be rapidly treated in a yard or mixed continuous culture room at a time. This means simplicity, convenience, speed and economy, and enables the rapid realization of commercialization of second-generation biofuels.

In addition, the present invention provides a method of increasing the concentration of glucose by more than 40%, even though using a lower concentration of sulfuric acid, a lower temperature, a shorter time, and a smaller amount of cellulase than the conventional acid hydrolysis pretreatment method, It provides a new way to treat biomass at once.

Of the conventional pretreatment methods, a method widely used in test plants in the United States, Canada, Europe, and China is acid hydrolysis pretreatment using sulfuric acid. In this case, the conditions of high temperature (200 ° C or higher) and high pressure (10 or higher) are used in many cases. Further, there is a double pretreatment method (Korean Patent Laid-open No. 10-2016-01211991) in which preliminary pretreatment is performed at 150 ° C with 1% sulfuric acid and then second pretreatment at 190 ° C. (International Patent Application No. WO 2011/016591 A1), which requires special high-temperature and high-pressure reactors under the conditions of high-temperature and high-pressure reactor, and is difficult to mass-produce due to high energy cost and high facility cost. In addition, there are various methods such as an acid hydrolysis pretreatment method (U.S. Patent No. US 8,278,080 B2) which requires expensive equipment such as an ultrasonic generator, but the use of expensive equipment, high pretreatment cost, long treatment time, , Low saccharification efficiency, and so on.

According to the conventional literature (Bioresource Technology. 2005. 96: 2019-2025), in the case of pretreatment using acid hydrolysis, the second-generation bioethanol direct production cost (biomass purchase and handling, pretreatment, saccharification, fermentation, The total amount of the above two items corresponds to 53%, and the total amount of the above two items is 53% It has been reported that it accounts for more than half of the production cost. On the other hand, the present invention provides a method for reducing the above-mentioned costs, which accounts for more than half of the total direct production cost.

The present invention also relates to a method for producing KPYBE10, which comprises culturing KPYBE10 in a biomass yard, pretreating KPYBE10 at a low sulfuric acid concentration (0.5%) and a low temperature (150 DEG C) It is a simple method to obtain the concentration, it is a new method characterized by the reduction of sulfuric acid usage, the reduction of the total energy cost of the pretreatment, and the cost of the purchase of the celluler can be drastically reduced. In addition, the present invention does not require a detoxification process since a fermentation inhibitory substance such as furfural and hydromethyl furfural (HMF) is used at a low pretreatment temperature at which the inhibitory concentration is not exceeded.

Therefore, the present invention can greatly reduce the costs of pretreatment, saccharification, high-pressure steam and electricity, and is used in a second-generation bioethanol test plant using acid hydrolysis pretreatment methods in various places in the US, Canada, Europe and China City, which can dramatically reduce direct production costs, enabling the transition to commercial production.

Further, gypsum (CaSO ₄ ) produced by neutralizing the sulfuric acid used in the present invention with calcium carbonate (CaCO ₃ ) or calcium hydroxide (Ca (OH) ₂ ] can be used as an eco-friendly gypsum board raw material. As a result of reduction of coal power generation to prevent global warming, production of flue gas desulfurization gypsum board from thermal power generation coal ash production is shrunk, phosphorus gypsum board produced from fertilizer by processing phosphorus stone is high in radon release, There is a concern. However, the gypsum produced as a by-product of the present invention can be recycled at low cost as a raw material for environmentally friendly gypsum board without such a problem. In addition, the solid residues remaining after ethanol fermentation can be used as livestock feeds or used for electricity generation and steam generation, and the remaining ones can be sold.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1: Isolation of novel cellulose degrading microorganisms

In the present invention, soil samples were collected from riversides and tidal flats in Kyungbuk, Gyeongnam, and Jeolla provinces in order to purify microorganisms which can increase the glycosylation efficiency by decomposing the cellulose in plant cell walls.

1 g of each soil sample is weighed, placed in 100 ml of sterilized water, thoroughly suspended, diluted to 10 ² and 10 ³ , and then immersed in CMC (carboxymethyl cellulose) agar medium [5 g of CMC per liter of distilled water, 5 g of peptone , 5 g of KH ₂ PO ₄ , 0.5 g of yeast extract, 0.5 g of MgSO ₄ .7H ₂ O, 15 g of agar, pH 7.0] and then cultured at 28 ° C for 3 days. After culturing, each colony was plated on a fresh CMC agar medium, and then pure culture was performed at 28 ° C for 3 days. 1 ml of a 0.5% congo red reagent was added, and the mixture was allowed to stand for 30 minutes, discarded, After incubation for 5 minutes with NaCl added, the strains showing a relatively large yellow halo zone were selected.

Example 2: Pure culture and identification of a novel fibrinolytic fungal KPYBE10

In order to isolate the microorganism strains that grow well in the biomass without adding any special nutrients from the microorganisms isolated from the CMC agar medium, the naturally dried cornstalks were sieved with a blender and sieved with a 1 mm mesh, Was placed in a 150 x 25 mm glass petri dish and 40 ml of distilled water was added. Thereafter, the microorganism strains were inoculated and cultured at 48 DEG C for one week. The selected strain was selected by sterilization. The selected strain was collected by sterilized needle, and 30 g of cornstarch powder and 20 ml of distilled water After incubation in an Erlenmeyer flask, the cells were incubated with a sili stopper to maintain aerobic conditions at 48 ° C for one week. After that, it was stored at 4 캜 and used as a preservation strain. The strain was named KPYBE10 and deposited with the Korean Institute of Bioscience and Biotechnology under accession number KCTC13356BP.

A novel fibrinolytic fungal KPYBE10 strain was disrupted in the same manner as described above to form a greenish green colonies in cornstaff powder, sawdust and rice straw sieve sieved with a 1 mm mesh, and cultured in CMC agar medium at 48 ° C for 7 days On the day of culture, a cotton-like flat white colony was formed. As shown in Fig. 3, Penicillium sp. (Sp. Penicillium sp.) Was formed on the basis of characteristics such as a circumferential conidium observed with a microscope and a smooth- .).

Example 3: Optimal culture temperature of the novel fibrinolytic fungus KPYBE10

To determine the optimal temperature for mycelial growth of KPYBE10, KPYBE10 was inoculated with sterilized needles in the center of a CMC agar medium made of 100-mm Petri dishes and then incubated at various temperatures (15 ° C, 20 ° C, 25 ° C, 30 ° C, 40 ° C , 45 占 폚, 48 占 폚, 50 占 폚), and the growth time of KPYBE10 was analyzed by measuring the time taken to completely cover the medium.

As a result, it took 6 days at 20 ~ 25 ℃, 3-4 days at 30 ~ 45 ℃, 5 days at 48 ℃ and 7 days at 50 ℃, and no rapid growth was observed at 15 ℃. From the above results, it was confirmed that KPYBE10 exhibited optimal growth at temperatures ranging from 30 to 45 ° C.

In order to confirm the change of the growth rate of KPYBE10 according to the temperature change in the biomass, 30 g of cornstarch powder, sawdust and rice straw sifted with a 1 mm mesh were pulverized in the same manner as above, ml Erlenmeyer flasks and sterilized. KPYBE10 was inoculated with sterilized needles at the center of the culture medium and cultured at 28 ° C, 21 ° C, 18 ° C and 15 ° C, which are the expected temperatures of the yard for one week and one month.

As a result, referring to Fig. 4A (cornstarch powder) and Fig. 5A (sawdust), when cultured for one week at 28 DEG C, KPYBE10 mycelium grown vigorously all over the surface of cornstarch powder and sawdust biomass, Respectively. At 21 ℃, KPYBE10 mycelium was observed at about 60% of the whole biomass surface. At 18 ℃, KPYBE10 mycelium was observed at about 5% of the entire biomass surface. At 15 ℃, the growth of KPYBE10 Was not observed at all. (Rice straw is not shown as a similar result)

As shown in FIG. 4B (cornstalks powder) and FIG. 5B (sawdust), when cornstarch powder and sawdust biomass were heated at 28 ° C and 21 ° C, KPYBE10 mycelium was observed at 18 ℃, and KPYBE10 mycelium was observed at an area of about 70-80% of the whole biomass surface. At 15 ℃, the area of about 50-60% of the entire biomass surface KPYBE10 mycelium was observed. (Rice straw is not shown as a similar result)

The results show that cultivation of biomass in KPYBE10 in a yard requires cultivation for one week at 28 ° C. For the above temperature conditions, considering the seasonal factors such as covering with vinyl thermal insulation tarpaulins or winter season, It may require a method of supplying a small amount of heat in the form of a pipe. In addition, when there is a need to increase the mold processing rate while using a minimum yard area, a low-cost mixed continuous culture chamber of simple structure, which is kept warm with an insulating material, as shown in Fig.

In the present invention, the biomass is pulverized without being subjected to the sterilization treatment, and then KPYBE10 is inoculated by suspending it in unsterilized water (industrial water) and spraying it. Therefore, to determine the temperature at which the growth of endogenous fungi in biomass is inhibited, 3 g of unsterilized cornstarch powder is added to 100 ml Erlenmeyer flask, 15 ml of untreated tap water is added, and then 28 Lt; 0 > C, 35 [deg.] C and 40 [deg.] C for 10 days.

As a result, referring to FIG. 6, when cultured at 28 ° C and 35 ° C, black fungi and white fungi which were endogenously present in the biomass were grown, whereas when cultured at 40 ° C, The growth of pollutant fungi was not observed. Therefore, it was confirmed that the temperature of 40 ℃ inhibited the growth of endogenous pollutant fungi.

The possibility of the use of sulfuric acid was investigated in order to inhibit the growth of germs and to improve the growth efficiency of KPYBE10 only by observing the growth of germs when cultured at 25 ° C -35 ° C. 10 g of cornstarch powder not sterilized was added to a 150 x 25 mm glass petri dish and 40 ml of a 0.1% or 0.2% sulfuric acid solution was added thereto, followed by inoculation with KPYBE10 and incubation at 28 ° C.

As a result, referring to FIG. 7, it took 7 days to cover the entire surface when suspended and cultured in water, and it took 9 days to cultivate with sulfuric acid solution. In addition, it was confirmed that the 0.1% and 0.2% sulfuric acid solution effectively inhibited the growth of the fungus, and did not significantly affect the growth of the KPYBE10 strain.

Example 4: Culture of KPYBE10 in biomass

3 g of cornstal powder was added to a 100 ml Erlenmeyer flask, and the nutrient solution (Na ₂ HPO ₄ .12H ₂ O 4 g / L, MgSO ₄ .7H ₂ O 1 g / L of CaCl ₂ , 0.5 g / L of NaNO _3, 1 g / L of NH ₄ Cl, 1 g / L of KCl, 0.3 g / L of FeSO ₄ .4H ₂ O, 0.1 g / L of ZnSO ₄ .8H ₂ O, L, MnSO ₄揃 H ₂ O 0.02 g / L, CuCl ₂揃 2H ₂ O 0.01 g / L, Na ₂ MoO ₄揃 2H ₂ O 0.01 g / L, yeast extract 1 g / , 1 g / L of peptone) was diluted 20 times with distilled water. Then, KPYBE10 was inoculated with sterilized needles and cultured at 28 ° C for 3-4 days. Then, the whole culture was suspended in 5 L of water, and 60 ml of each of 150 x 25 mm glass petri dishes containing 10 g of cornstalks were added, and cultured at 40 캜 for one week. Biomass and suspended water were not sterilized and were cultured at 40 ° C to inhibit the growth of various bacteria.

Example 5: Pretreatment and saccharification of biomass

As described above, cornstarch powder biomass cultured in KPYBE10 was added to a 4 L flask for acid hydrolysis pretreatment and diluted with various concentrations of sulfuric acid dilution (0.2% (w / v), 0.5% (w / v) ), 1.0% (w / v)] were added to the mixture, and the mixture was pre-treated at 140 ° C or 150 ° C for 1 hour. A vacuum filter was used to separate the pretreated biomass into a solid and a liquid. The water content of the solid was measured, and water and NaOH were added to prepare a slurry having a total solid content of 7% (pH 5.0). 142.8 g of the slurry was quantitatively added to a 250 ml Erlenmeyer flask.

The cellulase for saccharification was purchased from Enzym Tech, Inc., Cellic® CTec2 and Cellic® HTec2 enzyme from Novozyme (Bagsvard, Denmark). Add 1 ml of Cellic® CTec2 or 1 ml of Cellic® CTec2 and Cellic® HTec2, respectively, to the Erlenmeyer flask and perform the glycation reaction at 50 ° C with slow stirring at 100 rpm.

Each Erlenmeyer flask was given an AABB number, and only 1 ml of Cellic® CTec2 was added when AA was odd (01, 03, 05, 07, 09, 11) , 06, 08, 10, 12), Cellic® CTec2 and Cellic® HTec2 were added in the same volume of 1 ml each. BB represents saccharification time (3, 5, 7, 9 days).

After the saccharification was performed for 3 to 9 days, 5 ml of each sample was collected and centrifuged to separate into a solid phase and a liquid phase. The liquid phase containing glucose was filtered with a 0.25 μm pore size membrane filter for HPLC, Lt; / RTI > (300 mm x 7.8 mm, BIO-RAD, USA) in HPLC (Waters 2690, USA) to quantify glucose, ethanol, furfural and xylose 5 mmol sulfuric acid at 65 ° C was measured with a RI detector (refractive index detector) at a flow rate of 0.5 ml / min.

For ethanol fermentation, 1 kg of the enzyme-treated sample was separated into a solid phase and a liquid phase by spinning at 8,000 rpm for 15 minutes using a high-speed centrifuge (Supra 22K, Korea) and an A2505-6N rotor (Korea) The liquid phase was filtered using a 142 mm pressure filter holder equipped with a filter having a pore size of 0.2 mu m, and the glucose concentration was measured using a glucose assay kit (GAHK-20, Sigma-Aldrich, USA) And stored in the refrigerator until fermentation.

a: pretreatment sulfuric acid concentration

b: No KPYBE10 incubation treatment

c: Cultivation treatment of KPYBE10

d: Pretreatment temperature

e: Glucose concentration after saccharification

As shown in Table 1, when the saccharification time was influenced on the concentration of glucose, the concentration of glucose was increased until 5 days when Cellic® CTec2 alone was used, but the rate of increase was insignificant thereafter I could confirm. This means that the optimal number of saccharification days is 5 days, and when using Cellic® CTec2 and Cellic® HTec2 in the same way, the optimal saccharification days were 7 days.

To investigate the effect of sulfuric acid concentration on the glycation in the pretreatment, 16.98 g / L (0105), 25.10 g / L (0305), 33.35 g / L (0505) and 17.91 g / L (0705) (W / v) sulfuric acid, the concentration of glucose obtained by pretreatment with 1.0% (w / v) sulfuric acid was 2 (w / v) Times higher. From the above results, it was confirmed that the saccharification efficiency was improved as the sulfuric acid concentration increased in the pretreatment.

When biomass was cultured in KPYBE10, pretreatment was carried out at two concentrations of sulfuric acid (0.5%, 1%) and two temperatures (140 ° C and 150 ° C), and the effect of CTec2 alone on glucose concentration Respectively.

When the biomass that was not cultured with KPYBE10 was subjected to pretreatment / saccharification under the condition of 0.5% sulfuric acid / 140 ° C / 1 hour / CTec2, the glucose concentration was 25.10 g / L (0305) and the biomass cultured with KPYBE10 was used , The glucose concentration was increased by 25% to 31.40 g / L (0905) under the same conditions as above. When the pretreatment temperature was raised to 150 ° C by 10 ° C under the same conditions, 34.11 g / L (1305 ), It was confirmed that the glucose concentration was greatly increased by 36%.

When the biomass that was not cultured with KPYBE10 was subjected to pretreatment / saccharification under the conditions of 1% sulfuric acid / 140 ° C / 1 hour / CTec2, the glucose concentration was 33.35 g / L (0505), and the biomass cultured with KPYBE10 The glucose concentration was increased by 8.2% at 36.09 g / L (1105), and 39.02 g / L (1505) when the pretreatment temperature was elevated by 10 ° C. at only 150 ° C., The glucose concentration was increased by 17%. Also, it was confirmed that the glucose concentration was increased when the biomass which was not cultured with KPYBE10 was used even when it was pretreated with 0.2% sulfuric acid.

Using the biomass cultured with KPYBE10, the effect of increasing the glucose concentration by up to 36% was observed under the condition of 0.5% sulfuric acid. That is, the present invention provides a method of greatly increasing the saccharification efficiency by using biomass cultured with KPYBE10 using the same saccharification enzyme.

When 1% sulfuric acid was used, the glucose concentration was 33.35 g / L (0505). When 0.5% sulfuric acid was used, the glucose concentration was 34.11 g / L (1305) and the glucose concentration was similar to that of KPYBE10 Using treated biomass means that a similar glucose concentration can be obtained even with a small amount of sulfuric acid. That is, the present invention provides a method for maintaining the saccharification efficiency at a similar level while drastically reducing the amount of sulfuric acid used by using biomass cultured with KPYBE10.

Next, we analyzed the effect of CTec2 alone and CTec2 & HTec2 combination on glucose concentration in two concentrations of sulfuric acid (0.5%, 1.0%) when cultured with KPYBE10 in biomass.

When the biomass that was not cultured with KPYBE10 was pretreated and saccharified under conditions of 0.5% sulfuric acid / 140 ° C / 1 hour / CTec2 & HTec2, the glucose concentration was 29.08 g / L (0407), and the biomass cultured with KPYBE10 (0905) in the case of using CTec2 alone, the glucose concentration was 8% higher than that in the case of using CTec2 alone. In addition, when the pretreatment temperature was raised to 150 캜 by 10 캜, the glucose concentration was increased by 17.3% to 34.11 g / L (1305).

From the above results, it was confirmed that using the biomass cultured with KPYBE10, CTec2 and HTec2 were not used together but CTec2 alone could obtain higher saccharification efficiency.

When the biomass that was not cultured with KPYBE10 was subjected to pretreatment / saccharification under the conditions of 1.0% sulfuric acid / 140 ° C / 1 hour / CTec2 & HTec2, the glucose concentration was 36.63 g / L (0607), and cultured with KPYBE10 When biomass was pretreated and saccharified under the same conditions as above, CTec2 alone was similar to 36.42 g / L (1107). In addition, when the pretreatment temperature was increased to 150 ° C by 10 ° C, the glucose concentration was increased by 8.4% to 39.72 g / L (1507) when CTec2 alone was used.

From the above results, it can be seen that the use of the biomass cultured with KPYBE10, which is not cultured with KPYBE10 and used in combination with CTec2 & HTec2, results in higher glycation efficiency even when CTec2 alone is used, Respectively. That is, the present invention provides a method of obtaining a similar or higher saccharification efficiency by using only relatively inexpensive CTec2 alone, without using HTec2, which is 2.5 times more expensive than CTec2, Or more.

Next, we analyzed the effect of biomass cultured with KPYBE10 on glucose concentration in combination with CTec2 and HTec2.

When the biomass that was not cultured with KPYBE10 was pretreated and saccharified under conditions of 0.5% sulfuric acid / 140 ° C / 1 hour / CTec2 & HTec2, the glucose concentration was 29.08 g / L (0407), and the biomass cultured with KPYBE10 , The glucose concentration was increased by 42% at 41.29 g / L (1407) when pretreated at 150 ° C.

From the above results, it was confirmed that using the biomass cultured with KPYBE10 can greatly increase the saccharification efficiency and still obtain a similar saccharification efficiency even if the amount of the two enzymes is greatly reduced. That is, the present invention provides a method for obtaining a similar saccharification efficiency while simultaneously reducing the amount of the two saccharifying enzymes.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Korea Biotechnology Research Institute KCTC13356BP 20170920

Claims (16)

Penicillium species microorganism collected from soil, characterized by surviving in harsh conditions. The fibrin degrading fungus according to claim 1, wherein said harsh condition is treated at a temperature of 40 to 50 ° C. The fibrinolytic fungus according to claim 1, wherein the fibrin degrading fungus is deposited with KCTC 13356BP. The fibrin degrading fungus according to claim 1, wherein the fibrin degrading fungus improves the saccharification efficiency of the biomass. (a) pulverizing the biomass; And
(b) a step of spraying and pulverizing the pulverized biomass with a mixture solution obtained by suspending the fibrinolytic fungus according to any one of claims 1 to 4 in water or a sulfuric acid solution;
And a method for improving the pretreatment and saccharification efficiency of biomass for producing biofuels or bioplastics. The method according to claim 5, wherein the water, the sulfuric acid solution or the biomass of the step (b) can be used without sterilization treatment and is cultured without the addition of additional nutrients. And a method for improving the saccharification efficiency. 6. The method of claim 5, wherein the biofuel is bioethanol or biobutanol. 5. The method of claim 1, wherein the biofuel is bioethanol or biobutanol. 6. The method of claim 5, wherein the pretreatment and saccharification efficiency of the biomass is improved,
(a) pulverizing the biomass;
(b) inoculating and pulverizing the pulverized biomass with a mixture solution obtained by suspending the fibrinolytic fungus according to any one of claims 1 to 4 in water or a sulfuric acid solution;
(c) treating the cultured culture with an acid to hydrolyze it;
(d) separating the resultant product after the hydrolysis into a solid phase and a liquid phase;
(e) treating the solid phase with a cellulose degrading enzyme to obtain a saccharified saccharide per hexane or treating the liquid phase with a neutralizing agent to obtain a pentasaccharide solution;
(f) preparing a biofuel fermentation broth by inoculating and culturing the biofuel fermentation microorganism in the saccharified saccharide solution, the pentasaccharide solution or the mixed saccharification solution thereof; And
(g) distilling the fermentation broth to produce a concentrated biofuel;
And a method for improving the pre-treatment and saccharification efficiency of the biomass. 6. The method of claim 5, wherein the pretreatment and saccharification efficiency of the biomass is improved,
(a) pulverizing the biomass;
(b) inoculating and pulverizing the pulverized biomass with a mixture solution obtained by suspending the fibrinolytic fungus according to any one of claims 1 to 4 in water or a sulfuric acid solution;
(c) treating the cultured culture with an acid to hydrolyze it;
(d) separating the resultant product after the hydrolysis into a solid phase and a liquid phase;
(e) treating the solid phase with a cellulolytic enzyme to obtain a saccharide-saccharide solution, and treating the liquid phase with a neutralizing agent to obtain a saccharide-saccharide solution; And
(f) preparing a bioplastic by inoculating and culturing a strain producing the bioplastic synthetic material in the mixed sugar solution of hexane and pentane;
And a method for improving the pre-treatment and saccharification efficiency of the biomass. 10. The method of claim 8 or 9, wherein the fibrinolytic fungus is deposited with KCTC 13356BP to improve the pretreatment and saccharification efficiency of the biomass. The method of claim 8 or 9, wherein the fibrin degrading fungus is a penicillium species microorganism. 10. The method of claim 8 or 9, wherein the biomass is selected from the group consisting of corn stover, corn cobs, bagasse, sugar beet residue, rice straw, chaff, straw, reed, sunflower stem, Wherein the plant is at least one selected from the group consisting of switchgrass, oilseed rape, waste wood, sawdust, grass, cotten, flax and fruit peel. The method according to claim 8 or 9, further comprising: after the step (a), passing the pulverized biomass through a mesh having a length of one side of 0.25 to 10 mm. A method for improving saccharification efficiency. [9] The method according to claim 8 or 9, wherein the step (b) is characterized by suspending the fibrinolytic fungus in water at a temperature of 40 to 50 DEG C, inoculating the pulverized biomass, and culturing for 3 to 14 days A method for improving the pretreatment and saccharification efficiency of biomass. The method according to claim 8 or 9, wherein the step (b) comprises suspending the fibrinolytic fungus in a 0.1 to 0.2% (w / v) sulfuric acid solution at a temperature of less than 40 캜, inoculating the pulverized biomass, And culturing the cells for 3 to 14 days. The method for improving the pretreatment and saccharification efficiency of biomass. The method according to claim 8 or 9, wherein the hydrolysis in step (c) is performed in an acidic solution of 0.1 to 10% (w / v) at a temperature of 130 to 300 ° C for 30 to 90 minutes A method for improving the pretreatment and saccharification efficiency of biomass.

Images