Method for producing butanol by pretreating straw through deep eutectic solvent combined with biocatalysis

Abstract

The present invention provides a method for pretreating straw by using a deep eutectic solvent in combination with a biocatalytic enzymatic method to produce butanol, belonging to the technical field of straw treatment. The present invention provides a method for enzymatic hydrolysis and saccharification of straw, comprising: pretreating the straw with a deep eutectic solvent, and then mixing the pretreated straw and sugar solution, inoculating an enzyme-producing microorganism, and successively performing microbial culture to produce enzymes and enzymatic hydrolysis to obtain a high-concentration sugar solution. In the present invention, DES is used to pretreat the straw to break the natural anti-degradation barrier of lignin and improve the accessibility of cellulose; the enzyme-producing microorganism is inoculated on the DES-pretreated straw to efficiently prepare cellulase, and then enzymatic hydrolysis of the straw is carried out. After DES pretreatment, the degradation rate of lignin in the straw is as high as 23.52%. After enzymatic hydrolysis is completed, the yield of glucose is as high as 88.23%. The obtained high-concentration sugar solution does not contain furan toxic substances and can be used for the preparation of butanol, and will not have an adverse effect on the growth and metabolism of the butanol-producing microbial preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of straw treatment, and specifically relates to a method for pretreating straw by eutectic solvent combined with enzymatic hydrolysis to produce butanol. Background Art

[0002] A large amount of crop straw is generated every year. If these straws are not reasonably utilized, it will cause huge resource waste and environmental pollution. Cellulase has broad application prospects and markets in the biomass energy industry. Using cellulase to treat lignocellulosic biomass raw materials, enzymatically hydrolyzing to produce sugars and then fermenting to produce green renewable and pollution-free liquid biofuels such as ethanol and butanol is one of the important ways to solve the energy crisis and environmental pollution.

[0003] Butanol has more advantages as a biofuel than ethanol: (1) Butanol has better miscibility with diesel and does not require any co-solvent, and can be miscible with diesel in any proportion. Its kinematic viscosity is roughly the same as that of diesel, and it is more adaptable to the existing engine structure. Due to its higher heat of vaporization and lower combustion temperature, it can also reduce NOx emissions. (2) Butanol has a very low vapor pressure point and a high flash point, so it is safer to use at high temperatures, and the possibility of cavitation and vapor lock problems is smaller. Engines burning butanol are easier to start in cold weather. (3) Butanol has less corrosion and is more suitable for distribution through existing pipelines without the need to modify the existing pipelines for transportation and distribution, while ethanol must be transported by rail, barge or truck. Butanol is the most promising biofuel to replace ethanol and bio-oil. Using cellulase to degrade corn straw raw materials to produce bio-butanol is of epoch-making significance for ultimately solving the energy shortage problem faced by mankind.

[0004] Currently, the methods for preparing butanol from straw mostly use a combination of various chemical agents and cellulase to further prepare butanol from straw. The related methods for preparing butanol have disadvantages such as a long production chain and many uncertain factors. At the same time, the high-concentration sugar solution prepared after straw degradation will also have an adverse effect on Clostridium butylicum due to the chemical agents contained, which is not conducive to the improvement of butanol production. Summary of the Invention

[0005] Aiming at the defects in the prior art, the purpose of the present invention is to provide a method for enzymatic hydrolysis and saccharification of straw. The high-concentration sugar solution prepared by this method does not contain chemical substances that are unfavorable to the growth of microorganisms, has low toxicity to Clostridium butylicum, is conducive to increasing butanol production, and has a simple production chain and easy control of the whole operation process, etc.

[0006] The purpose of the present invention is achieved by the following technical solutions:

[0007] The present invention provides a method for enzymatic hydrolysis and saccharification of straw, comprising the following steps:

[0008] Mix straw with a deep eutectic solvent for straw pretreatment to obtain pretreated straw; the mass-volume ratio of the straw to the deep eutectic solvent is 1 g:10 mL;

[0009] Mix the pretreated straw with a sugar solution, inoculate with an enzyme-producing microbial agent, and sequentially carry out microbial culture for enzyme production and enzymatic hydrolysis to obtain a high-concentration sugar solution; the solid content after mixing the pretreated straw with the sugar solution is 25% - 30%; the mass concentration of glucose in the sugar solution is 5% - 8%; the enzyme-producing microbial agent includes Trichoderma reesei and Aspergillus niger; the Trichoderma reesei includes Trichoderma reesei CICC13052; the Aspergillus niger includes Aspergillus niger CMCC(F)98003.

[0010] Preferably, the hydrogen bond acceptor of the deep eutectic solvent includes choline chloride; the hydrogen bond donor of the deep eutectic solvent includes glycerol and / or triethanolamine; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor in the deep eutectic solvent is 1:(1 - 2);

[0011] The pretreatment time is 0.5 - 1 h.

[0012] Preferably, the ratio of the viable cell count of Trichoderma reesei to the viable cell count of Aspergillus niger in the enzyme-producing microbial agent is 5:1; the total viable cell count in the enzyme-producing microbial agent is 1×10 8 ~5×10 8 CFU / mL.

[0013] Preferably, the temperature for microbial culture for enzyme production is 37°C; the time for microbial culture for enzyme production is 3 - 5 d; the temperature for enzymatic hydrolysis is 50°C; the time for enzymatic hydrolysis is 4 - 5 d.

[0014] Preferably, the straw includes any one or more of corn straw, wheat straw, and rice straw.

[0015] The present invention provides a method for producing butanol from a high-concentration sugar solution prepared by the method according to the above technical solution, including the following steps:

[0016] Inoculate Clostridium acetobutylicum into the high-concentration sugar solution for anaerobic fermentation to produce butanol.

[0017] Preferably, the Clostridium acetobutylicum includes Clostridium acetobutylicum CICC8020.

[0018] Preferably, the temperature for anaerobic fermentation is 37°C; the time for anaerobic fermentation is ≥3 d.

[0019] Preferably, the method for butanol extraction includes: extraction by stripping.

[0020] Preferably, feeding is also included during the anaerobic fermentation process; the feeding includes supplementing high-concentration sugar solution and Clostridium acetobutylicum.

[0021] Advantages of the present invention

[0022] The present invention provides a method for enzymatic hydrolysis and saccharification of straw, comprising the following steps: mixing straw and a deep eutectic solvent for pretreatment of the straw to obtain pretreated straw; the mass-volume ratio of the straw to the deep eutectic solvent is 1 g:10 mL; mixing the pretreated straw with a sugar solution and then inoculating an enzyme-producing microbial agent to sequentially perform microbial culture for enzyme production and enzymatic hydrolysis to obtain a high-concentration sugar solution; the solid content after mixing the pretreated straw with the sugar solution is 25% - 30%; the mass concentration of glucose in the sugar solution is 5% - 8%; the enzyme-producing microbial agent includes Trichoderma reesei and Aspergillus niger; the Trichoderma reesei includes Trichoderma reesei CICC13052; the Aspergillus niger includes Aspergillus niger CMCC(F)98003. A deep eutectic solvent (DES) is a kind of ionic liquid-like solvent, which is composed of a binary or multi-component mixture of a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA). After mixing, due to the charge delocalization of the HBD hydrogen bond, the melting point is reduced, making the DES present a liquid state at room temperature. The present invention first pretreats the straw with DES to break the natural anti-degradation barrier of lignin and improve the accessibility of cellulose; then inoculates enzyme-producing microorganisms such as Trichoderma reesei and Aspergillus niger on the straw pretreated with DES, uses Trichoderma reesei, Aspergillus niger and other microorganisms to efficiently prepare cellulase, and then performs enzymatic hydrolysis of the straw, that is, uses DES reagent combined with biocatalysis method to pretreat the straw and perform dynamic enzymatic hydrolysis and saccharification of the straw. The degradation rate of lignin in the straw is as high as 23.52% after DES pretreatment. After enzymatic hydrolysis is completed, the yield of glucose is as high as 88.23%, and the yield of reducing sugar is as high as 90.05%. At the same time, the high-concentration sugar solution obtained by enzymatic hydrolysis does not contain furan toxic substances, and further prepares butanol from the obtained sugar solution, which will not have an adverse effect on the growth and metabolism of the butanol-producing microbial preparation, and is beneficial to increasing the yield of butanol. Description of the drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the following-described drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 It is a flow chart of the enzymatic hydrolysis and saccharification of straw and the production of butanol from high-concentration sugar solution in the present invention;

[0025] Figure 2 It is a schematic diagram of the device for the enzymatic hydrolysis and saccharification of straw to produce butanol. Specific implementation mode

[0026] The present invention provides a method for the enzymatic hydrolysis and saccharification of straw, comprising the following steps:

[0027] Mix straw and a deep eutectic solvent for the pretreatment of straw to obtain pretreated straw; the mass-volume ratio of the straw to the deep eutectic solvent is 1 g:10 mL;

[0028] Mix the pretreated straw with a sugar solution, inoculate with an enzyme-producing microbial inoculum, and successively carry out microbial culture for enzyme production and enzymatic hydrolysis to obtain a high-concentration sugar solution; the solid content after mixing the pretreated straw with the sugar solution is 25% - 30%; the mass concentration of glucose in the sugar solution is 5% - 8%; the enzyme-producing microbial inoculum includes Trichoderma reesei and Aspergillus niger; the Trichoderma reesei includes Trichoderma reesei CICC13052; the Aspergillus niger includes Aspergillus niger CMCC(F)98003.

[0029] In the present invention, straw and a deep eutectic solvent are mixed for pretreatment of the straw to obtain pretreated straw. In the present invention, the straw may include any one or more of corn straw, wheat straw, and rice straw. In the embodiments of the present invention, corn straw is taken as an example to illustrate the specific method steps of enzymatic hydrolysis and saccharification of the straw. Based on the fact that corn straw can be efficiently enzymatically hydrolyzed and saccharified by a corresponding method in the present invention, it can be further determined that wheat straw and rice straw, which are more easily enzymatically hydrolyzed than corn straw, can also be enzymatically hydrolyzed and saccharified by this method. As an optional implementation manner of the present invention, the straw may be fresh straw or dried straw. Theoretically, using fresh straw may also have better effects. In the specific embodiments of the present invention, in order to facilitate verification of the effect of the test scheme, dried straw is used to specifically illustrate the method of enzymatic hydrolysis and saccharification of the straw. As an optional implementation manner of the present invention, the dried straw is preferably straw with a water content ≤ 5%. The present invention has no special limitation on the drying method of the straw, and any conventional drying method in the art can be used. The present invention has no special limitation on the composition of the deep eutectic solvent, and any deep eutectic solvent with a conventional composition in the art can be used. As an optional implementation manner of the present invention, the hydrogen bond acceptor of the deep eutectic solvent may include choline chloride; the hydrogen bond donor of the deep eutectic solvent may include glycerol and / or triethanolamine; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor in the deep eutectic solvent may be 1:(1 - 2). In the present invention, the mass-volume ratio of the straw to the deep eutectic solvent may be 1 g:10 mL. In the present invention, the pretreatment time may be 0.5 - 1 h. The present invention uses a deep eutectic solvent to pretreat the straw. The protons dissociated from the organic acid group of the deep eutectic solvent strongly impact corn straw by promoting the cleavage of unstable ether bonds in the lignin phenylpropane unit, have a strong depolymerization effect, can destroy the natural anti-degradation barrier of lignin, improve the accessibility of cellulose, and thus facilitate the degradation of the straw. In the present invention, the degradation rate of lignin in the straw after DES pretreatment is as high as 23.52%.

[0030] After obtaining the pretreated straw, the present invention mixes the pretreated straw with a sugar solution, inoculates an enzyme-producing microbial inoculum, and sequentially performs microbial culture for enzyme production and enzymatic hydrolysis to obtain a high-concentration sugar solution.

[0031] In the present invention, the mass concentration of glucose in the sugar solution can be 5% to 8%. As an alternative embodiment of the present invention, the mass concentration of glucose in the sugar solution can also be 5%, 6%, 7%, or 8%. In the present invention, the solid content after mixing the pretreated straw with the sugar solution can be 25% to 30%, or can also be 25%, 26%, 27%, 28%, 29%, or 30%. When calculating the solid content in the present invention, the influence of the deep eutectic solvent on the solid content is ignored, that is, the solid content = mass of dry matter of straw / (mass of straw + mass of sugar solution). In the present invention, the glucose solution mainly serves as a carbon source for the growth of molds and an inducer for enzyme production, promoting the growth and enzyme production of molds.

[0032] The present invention has no special limitation on the mixing method of the pretreated straw and the sugar solution, and any conventional mixing method in the art can be used. The present invention obtains a mixed system of the pretreated straw and the sugar solution by mixing the pretreated straw and the sugar solution, or it can be called a straw-sugar solution mixed substrate.

[0033] After obtaining the mixed system of the pretreated straw and the sugar solution, the present invention inoculates the enzyme-producing microbial inoculant into the mixed system of the pretreated straw and the sugar solution, and sequentially performs microbial culture for enzyme production and enzymatic hydrolysis to obtain a high-concentration sugar solution.

[0034] In the present invention, the enzyme-producing microbial inoculant can include Trichoderma reesei and Aspergillus niger; the Trichoderma reesei can include Trichoderma reesei CICC13052; the Aspergillus niger can include Aspergillus niger CMCC(F)98003. As an alternative embodiment of the present invention, the ratio of the viable count of Trichoderma reesei to the viable count of Aspergillus niger in the enzyme-producing microbial inoculant can be 5:1; the total viable count in the enzyme-producing microbial inoculant can be 1×10 8 ~5×10 8 CFU / mL, or can also be 1.45×10 8CFU / mL. Before preparing the enzyme-producing microbial agent of the present invention, Trichoderma reesei CICC13052 and Aspergillus niger CMCC(F)98003 are preferably subjected to activation culture and seed culture respectively to obtain a Trichoderma reesei CICC13052 seed liquid and an Aspergillus niger CMCC(F)98003 seed liquid. Then, the Trichoderma reesei CICC13052 seed liquid and the Aspergillus niger CMCC(F)98003 seed liquid are compounded according to the viable count ratio to obtain the enzyme-producing microbial agent. The method for activating culture and seed culture of Trichoderma reesei CICC13052 and Aspergillus niger CMCC(F)98003 of the present invention is not particularly limited, and any conventional method for culturing corresponding microbial agents in the art can be used. As an optional implementation mode of the present invention, Trichoderma reesei CICC13052 and Aspergillus niger CMCC(F)98003 can be subjected to activation culture and seed culture by using the same culture method. The cultivation of Trichoderma reesei CICC13052 and Aspergillus niger CMCC(F)98003 of the present invention can be carried out respectively by using a comprehensive potato medium; the activation culture can be carried out in a sterile solid medium; the temperature of the activation culture can be 28°C; the time of the activation culture can be 5-7d; the activation culture is preferably carried out continuously for 2 times. After the activation culture is completed, the present invention preferably inoculates the obtained activated microbial agent into a sterile liquid medium for seed culture; the temperature of the seed culture can be 28°C; the time of the seed culture can be 24-48h; the seed culture is preferably accompanied by rotation; the rotation speed can be 180rpm. After the seed culture is completed, a seed culture solution of the microbial agent is obtained. As an optional implementation mode of the present invention, the viable count of the obtained Trichoderma reesei CICC13052 seed culture solution can be 1×10 8 CFU / mL; the viable count of the Aspergillus niger CMCC(F)98003 seed culture solution can be 1×10 8 CFU / mL. After the Trichoderma reesei CICC13052 seed culture solution and the Aspergillus niger CMCC(F)98003 seed culture solution are obtained respectively, the present invention preferably mixes the Trichoderma reesei CICC13052 seed culture solution and the Aspergillus niger CMCC(F)98003 seed culture solution according to the above-mentioned viable count ratio to obtain the enzyme-producing microbial agent. The viable count of the enzyme-producing microbial agent obtained in the present invention can be 1×10 8 ~5×10 8 CFU / mL, or can also be 1.45×10 8 CFU / mL. In the present invention, the Trichoderma reesei can produce a cellulase complex; Aspergillus niger CMCC(F)98003 can increase the proportion of cellobiase in the cellulase complex, thereby being conducive to efficiently promoting the enzymatic hydrolysis and saccharification of straw.

[0035] After obtaining the enzyme-producing microbial agent, the present invention can inoculate the enzyme-producing microbial agent into the mixed system of the pretreated straw and sugar solution according to a volume ratio of 1% to 10% and sequentially carry out microbial culture for enzyme production and enzymatic hydrolysis. In the present invention, the temperature of the microbial culture for enzyme production can be 37°C; the time of the microbial culture for enzyme production can be 3 to 5 days, or can also be 4 days; the process of the microbial culture for enzyme production is preferably accompanied by rotation; the rotation speed can be 110 rpm. In the present invention, there is also an enzymatic hydrolysis reaction during the process of microbial growth and enzyme production, in which Trichoderma reesei and Aspergillus niger mainly grow by utilizing the substances in the straw, and then the process of producing cellulase occurs. After the microbial culture for enzyme production is cultured until the enzyme production of the microorganism enters the vigorous period, the present invention preferably adjusts the solid content of the enzyme production system. The present invention can adjust the solid content by introducing pure water or a liquid for enzymatic hydrolysis to produce sugar with a low concentration into the enzyme production system. In the present invention, the liquid for enzymatic hydrolysis to produce sugar with a low concentration can be the low-concentration sugar solution produced by enzymatic hydrolysis of sugar in other equipment in the factory. When the present invention adjusts the solid content, it is preferably to use the dry matter in the straw as the solid matter to adjust the solid content; and use the sugar solution and the added water or the liquid for enzymatic hydrolysis to produce sugar with a low concentration and the sugar solution as the liquid system to adjust the solid content. When the present invention adjusts the solid content, if the straw used is the dried straw with a water content ≤ 5%, it is preferably to calculate and determine the addition amount of water or the liquid for enzymatic hydrolysis to produce sugar with a low concentration based on the solid content of the straw being 95%; when the present invention adjusts the solid content, if the straw used is fresh straw, it is preferably to calculate and determine the addition amount of water or the liquid for enzymatic hydrolysis to produce sugar with a low concentration based on the solid content in the fresh straw. In the present invention, the method for calculating the solid content can be solid content = dry matter content in the straw / (mass of the straw + mass of water or the liquid for enzymatic hydrolysis to produce sugar with a low concentration + mass of the sugar solution). The present invention preferably adjusts the solid content of the enzyme production system to 25% to obtain a high-solid hydrolysis system. After obtaining the high-solid hydrolysis system, the present invention preferably carries out enzymatic hydrolysis. In the present invention, the temperature of the enzymatic hydrolysis can be 50°C; the time of the enzymatic hydrolysis can be 4 to 5 days; the process of the enzymatic hydrolysis is preferably accompanied by rotation; the rotation speed can be 150 rpm. In the present invention, the enzymatic hydrolysis mainly involves the hydrolysis of the straw by the cellulase complex to produce glucose and obtain a high-concentration sugar solution. After the enzymatic hydrolysis of the present invention is completed, a high-concentration sugar solution is obtained. After the enzymatic hydrolysis of the present invention is completed, it is preferably to stop rotation and carry out static settlement to make the prepared high-concentration sugar solution and the remaining hydrolysis substances in the enzymatic hydrolysis system be stratified. In the present invention, the obtained high-concentration sugar solution is located below the remaining hydrolysis substances due to the gravity sedimentation effect. After obtaining the high-concentration sugar solution, the present invention preferably measures the glucose yield or the degradation rate of the straw. After obtaining the high-concentration sugar solution, the present invention can separate a part of the high-concentration sugar solution from the remaining hydrolysis substances in the hydrolysis system through the gravity sedimentation effect to obtain the high-concentration sugar solution. In the present invention, the steps of enzymatic hydrolysis and saccharification of the straw to prepare a high-concentration sugar solution can be carried out in a hydrolysis tank.

[0036] After obtaining the high-concentration sugar solution, the present invention preferably filters the obtained high-concentration sugar solution. The present invention has no special limitation on the filtering method, and any conventional filtering method in the art can be used. As an optional implementation manner of the present invention, the filtering method can be drip filtration.

[0037] After obtaining the filtered high-concentration sugar solution, the obtained high-concentration sugar solution can be directly added to an anaerobic fermentation tank for the preparation of butanol. After the present invention obtains the high-concentration sugar solution, DES-treated straw can be continuously added to the remaining hydrolysis substances in the enzymatic hydrolysis system, and at the same time, the enzyme-producing microbial inoculant is inoculated, and the straw enzymatic hydrolysis and saccharification are continued. When the present invention continues the straw enzymatic hydrolysis and saccharification, the remaining hydrolysis substances are located at the bottom layer. As the pore carbon at the bottom layer also has a microbial community and cellulase, it can hydrolyze the straw to produce sugar solution more quickly. Cellulase is an inducible enzyme, and glucose is both a decomposition product and a carbon source required for growth and also an inducer for enzyme production. Then, the method for preparing the high-concentration sugar solution is the same as described above, and the high-concentration sugar solution is prepared in such a cycle.

[0038] The method for straw enzymatic hydrolysis and saccharification provided by the present invention uses the DES combined with cellulase method to treat straw to produce sugar solution. The enzyme-producing microbial inoculant is inoculated on the straw pretreated with a deep eutectic solvent, that is, the straw is first pretreated with DES to break the natural anti-degradation barrier of lignin and improve the accessibility of cellulose; then, enzyme-producing microbial inoculants such as Trichoderma reesei and Aspergillus niger are inoculated on the straw pretreated with DES, and cellulase is efficiently prepared by using microorganisms such as Trichoderma reesei and Aspergillus niger, and then the enzymatic hydrolysis of the straw is carried out, that is, the straw is pretreated by using a DES reagent combined with a bioenzymatic method, and the straw is dynamically enzymatically hydrolyzed and saccharified. In the present invention, the degradation rate of lignin in the straw is as high as 23.52% after DES pretreatment, and the cellulose is slightly degraded; in the enzymatic hydrolysis stage, the degradation of cellulose is mainly carried out. After the enzymatic hydrolysis is completed, the glucose yield in the high-concentration glucose solution is as high as 88.23%, and the yield of reducing sugar is as high as 90.05%. In the present invention, the obtained high-concentration sugar solution does not contain furan-like toxic substances, and further preparing butanol from the obtained sugar solution will not have an adverse effect on the growth and metabolism of the butanol-producing microbial preparation.

[0039] The present invention also provides a method for producing butanol from a high-concentration sugar solution prepared by the method according to the above technical solution, including the following steps:

[0040] Clostridium acetobutylicum is inoculated into the high-concentration sugar solution for anaerobic fermentation to produce butanol.

[0041] In the present invention, the Clostridium acetobutylicum includes Clostridium acetobutylicum CICC8020. Preferably, after activating and expanding the culture of Clostridium acetobutylicum to obtain an expanded culture solution of Clostridium acetobutylicum, the expanded culture solution of Clostridium acetobutylicum is inoculated into a high-concentration sugar solution. The present invention has no special limitation on the method for activating and expanding the culture of Clostridium acetobutylicum, and any conventional method in the art can be used. In the present invention, the method for activating and expanding the culture of Clostridium acetobutylicum is preferably carried out according to the method described in the inoculum instruction manual to obtain an expanded culture solution of Clostridium acetobutylicum. In the present invention, the OD 600 value of the expanded culture solution of Clostridium acetobutylicum is 2.3, indicating that the bacteria grow well and can be used for subsequent inoculation. After obtaining the expanded culture solution of Clostridium acetobutylicum, the present invention preferably inoculates the expanded Clostridium acetobutylicum CICC8020 into a high-concentration sugar solution at a mass concentration of 2% - 10% for anaerobic fermentation. In the present invention, the temperature of the anaerobic fermentation can be 37°C; the time of the anaerobic fermentation can be ≥3 days. In the present invention, butanol begins to be produced in the fermentation system after 3 days of anaerobic fermentation. In the present invention, feeding is also included during the anaerobic fermentation process; the feeding includes supplementing a high-concentration sugar solution and Clostridium acetobutylicum. In the present invention, the high-concentration sugar solution can be fed at regular intervals; the timing of the regular feeding is to feed once every 11 days. In the process of producing butanol in the present invention, Clostridium acetobutylicum is also supplemented. The present invention has no special limitation on the timing of supplementing Clostridium acetobutylicum. During the anaerobic fermentation process, the addition of Clostridium acetobutylicum can be determined by observing the production rate of butanol. When the butanol production rate slows down and / or the gas production is insufficient, Clostridium acetobutylicum can be supplemented; when supplementing Clostridium acetobutylicum in the present invention, a low-concentration Clostridium acetobutylicum should be supplemented to ensure the coordination between the production rate of the high-concentration sugar solution and the utilization of the high-concentration sugar solution. In the present invention, butanol is produced during the anaerobic fermentation process; the butanol is preferably extracted by a stripping method. The present invention has no special limitation on the stripping method, and any conventional stripping method in the art can be used. In the present invention, ethanol and acetone can also be produced during the anaerobic fermentation process. In the present invention, the method for producing butanol from the high-concentration sugar solution can be carried out in an anaerobic fermentation tank.

[0042] In the present invention, the entire process of preparing a high-concentration sugar solution by enzymatic hydrolysis and saccharification of straw, filtering the high-concentration sugar solution, and producing butanol using the high-concentration sugar solution can be carried out in one device. Among them, the hydrolysis tank for performing the step of preparing a high-concentration sugar solution by enzymatic hydrolysis and saccharification of straw can be located above the high-concentration sugar solution filtration device; the device for producing butanol using the high-concentration sugar solution can be located below the high-concentration sugar solution filtration device. By using the above device to produce butanol from straw in the present invention, during the entire production process, the high-concentration sugar solution can be separated, filtered, and used as a fermentation substrate by the action of gravitational sedimentation. Technologically, through wet-dry coupled continuous saccharification, and physically isolating saccharification and butanol fermentation, it enables the conversion from the hydrolysis of lignocellulose to the synthesis of butanol to be completed in one process.

[0043] In the present invention, when the entire process of the enzymatic hydrolysis and saccharification of straw and the production of butanol using the high-concentration sugar solution are carried out in one device, that is, the enzymatic hydrolysis and saccharification process of straw is carried out in the hydrolysis tank, and the process of producing butanol from the high-concentration sugar solution is carried out in the anaerobic fermentation tank. During the anaerobic fermentation process when feeding regularly, only need to open the valve to let the hydrolyzed high-concentration sugar solution drip in after filtration; at the same time, add DES-pretreated straw and sugar solution to the hydrolysis tank for feeding the hydrolysis tank.

[0044] The present invention uses the high-concentration sugar solution as a fermentation substrate for anaerobic fermentation of Clostridium acetobutylicum. The high-concentration sugar solution does not contain furan toxic substances, will not have an adverse effect on the growth of Clostridium acetobutylicum, and can promote the rapid growth of Clostridium acetobutylicum in the fermentation substrate, thereby increasing the butanol yield.

[0045] As an implementable mode, the present invention also provides a device for producing butanol by enzymatic hydrolysis and saccharification of straw, specifically as Figure 2As shown in the figure, it includes a hydrolysis tank, a filtration device, and an anaerobic fermentation tank; the filtration device is located at the lower part of the hydrolysis tank; the anaerobic fermentation tank is located at the lower part of the filtration device; the lower opening of the hydrolysis tank is communicated with the filtration device; the lower opening of the filtration device is connected to the anaerobic fermentation tank. As an optional implementation manner of the present invention, the hydrolysis tank is located at the upper part of the filtration device. In the hydrolysis tank, enzymatic hydrolysis and saccharification of straw are carried out to prepare a high-concentration sugar solution. There is a stirring device in the hydrolysis tank to facilitate stirring during the enzymatic hydrolysis and saccharification process. The lower opening of the hydrolysis tank is communicated with the filtration device. After the high-concentration sugar solution produced by the enzymatic hydrolysis and saccharification of straw in the hydrolysis tank is allowed to stand and precipitate, it directly enters the filtration device through the lower opening for filtration of the high-concentration sugar solution. The filtration device is connected to the anaerobic fermentation tank, and the filtered high-concentration sugar solution can directly enter the anaerobic fermentation tank for anaerobic fermentation to produce butanol. In the present invention, the anaerobic fermentation tank is preferably located at the lower part of the filtration device, and the filtered high-concentration sugar solution can directly drip into the lower anaerobic fermentation tank due to gravity for anaerobic fermentation to produce butanol. The device provided by the present invention can directly carry out the processes of enzymatic hydrolysis and saccharification of straw and production of butanol from high-concentration sugar solution. The device, through wet-dry coupling continuous saccharification in terms of process and physically isolating saccharification and butanol fermentation, enables the completion of the synthesis of butanol from lignocellulose hydrolysis in one process.

[0046] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0047] The flow chart of the enzymatic hydrolysis and saccharification of straw and production of butanol from high-concentration sugar solution in the present invention is as Figure 1 shown.

[0048] Example 1

[0049] A method for enzymatic hydrolysis and saccharification of straw comprises the following steps:

[0050] 1. Preparation of enzyme-producing microbial inoculum

[0051] The source of the enzyme-producing microbial inoculum is Trichoderma reesei ( Trichoderma reesei ) CICC13052 freeze-dried bacterial powder and Aspergillus niger CMCC(F)98003 freeze-dried bacterial powder.

[0052] 1) Activation of strains and preparation of seed liquid

[0053] (1) Activation of Trichoderma reesei CICC13052 and preparation of seed liquid: Add 1.1 mL of anaerobic water to the freeze-dried powder of the microbial agent, and vortex for 5 - 10 s to completely dissolve it. Pipette 1 mL of the completely dissolved bacterial liquid, add it to 9 mL of sterile physiological saline, mix well, and perform serial 10-fold dilutions step by step until the bacterial count is approximately 500 - 1000 CFU / mL, obtaining the Trichoderma reesei CICC13052 bacterial liquid. Take 100 μL of the Trichoderma reesei CICC13052 bacterial liquid (with a bacterial content of 50 - 100 CFU), add it to the solidified sterile culture medium plate, and then use a sterile L-shaped spreader to spread the bacterial liquid evenly on the plate in the same direction. Wait until the bacterial liquid penetrates into the culture medium, invert the plate, and place it at 28 °C for 5 - 7 d until spores grow, obtaining the activated Trichoderma reesei CICC13052. The activated Trichoderma reesei CICC13052 is subcultured and activated using the same method as above to obtain the subcultured and activated Trichoderma reesei CICC13052. Add an appropriate amount of sterile physiological saline to the culture medium of the subcultured and activated Trichoderma reesei CICC13052, mix well for 2 min, and prepare a spore suspension with a concentration of 1×10 6 ~1×10 7 cells / mL. Inoculate the spore suspension into the seed medium at an inoculation amount of 5% (v / v). Shake-culture at 28 °C and 180 rpm for 24 - 48 h to obtain the seed culture solution of Trichoderma reesei CICC13052, and adjust the viable bacterial count of the obtained seed culture solution of Trichoderma reesei CICC13052 to 1×10 8 CFU / mL.

[0054] The culture medium used in the above cultivation is a comprehensive potato medium (PDA), and its composition is: 1000 mL of potato extract; 20 g of glucose; pH is natural. 15 g of agar needs to be added to the solid medium, and no agar needs to be added to the liquid medium. The preparation method of the potato extract is: Take 200 g of peeled potatoes, cut them into small pieces, add 1000 mL of water, boil for 30 min, filter out the potato pieces, and make up the filtrate to 1000 mL.

[0055] (2) Activation of Aspergillus niger CMCC(F)98003 and preparation of seed liquid are the same as in step (1), obtaining the seed culture solution of Aspergillus niger CMCC(F)98003, and adjusting the viable bacterial count of the obtained seed culture solution of Aspergillus niger CMCC(F)98003 to 1×10 8 CFU / mL.

[0056] 2) Preparation of enzyme-producing microbial agent

[0057] Trichoderma reesei CICC13052 and Aspergillus niger CMCC(F)98003 were used to prepare an enzyme-producing microbial inoculant according to the ratio of the viable cell count of Trichoderma reesei CICC13052 to the viable cell count of Aspergillus niger CMCC(F)98003 being 5:1. The viable cell count of the obtained enzyme-producing microbial inoculant was 1.45×10 8 CFU / mL.

[0058] 2. Prepare the DES reagent

[0059] Choline chloride and glycerol were mixed according to a molar ratio of 1:2, that is, 139.62 g of choline chloride powder was dissolved in 184.18 g of glycerol to obtain the DES reagent.

[0060] 3. Corn straw with a moisture content ≤ 5% after drying was mixed with the DES reagent according to a mass-to-volume ratio of 1 g:10 mL for straw pretreatment. The pretreatment time was 1 h to obtain pretreated straw. The obtained pretreated straw was mixed with a sugar solution with a glucose mass concentration of 8% according to a mass-to-volume ratio of 75 g:175 mL to obtain a straw-sugar solution mixed substrate. The enzyme-producing microbial inoculant was inoculated into the straw-sugar solution mixed substrate according to a volume ratio of 5%, and microbial culture for enzyme production was carried out at 37 °C and 110 rpm. Microbial culture for enzyme production was mainly the process of microbial growth and enzyme production. After 3 d of microbial culture for enzyme production and waiting for the microorganisms to produce enzymes vigorously, pure water was pumped in. Using the sugar solution and pure water as the liquid system and the straw dry matter as the solid system, the system was adjusted to a high-solid hydrolysis system with a solid content of 25% (calculated based on dry matter). The addition amount of pure water was determined based on a straw solid content of 95%. Enzymatic hydrolysis was carried out on the high-solid hydrolysis system at 50 °C and 150 rpm for 4 - 5 d. After the hydrolysis was completed, the stirring device was turned off, and after precipitation, a high-concentration sugar solution was obtained in the lower layer. The process of step 3 was carried out in a hydrolysis tank.

[0061] The obtained high-concentration sugar solution in the lower layer could directly flow out through the interface and then enter an anaerobic fermentation tank after filtration for the preparation of butanol.

[0062] After the high-concentration sugar solution in the lower layer flowed out and immediately after transferring away the first batch of sugar solution in the hydrolysis tank, a mixture of DES-pretreated straw and sugar solution and the prepared microbial flora, that is, the enzyme-producing microbial inoculant, could be directly added to the remaining system to continue straw enzymatic hydrolysis and saccharification. At this time, there were also microbial communities and cellulase in the pore carbon at the bottom layer of the remaining system, which could hydrolyze straw to produce sugar solution more quickly. Cellulase is an inducible enzyme, and glucose is both a decomposition product, a carbon source required for growth, and an inducer for enzyme production. After that, the method for preparing the high-concentration sugar solution was the same as described above, and the high-concentration sugar solution was prepared in such a cycle.

[0063] After pretreating the straw with DES solvent for one hour, the degradation rate of lignin in the straw was measured to be as high as 23.53%. After enzymatic hydrolysis was completed, the glucose content in the high-concentration sugar solution was measured, and the results showed that the glucose yield was as high as 88.23%, and the reducing sugar yield was as high as 90.05%.

[0064] Example 2

[0065] A method for producing butanol using the high-concentration sugar solution prepared in Example 1 is as follows:

[0066] 1. Activation and scale-up culture of Clostridium acetobutylicum CICC8020: (1) Add 50 g of corn flour to a small amount of distilled water to make a paste, and then add it to boiling water. Continuously stir, add distilled water during the stirring process, boil for 1 h, and finally make up the volume to 1 L to obtain a corn flour medium with a mass concentration of 5%. Aliquot the corn flour medium into test tubes and sterilize at 121 °C for 15 min. (2) Inoculate Clostridium acetobutylicum CICC8020 after cooling. (3) After inoculation, place the test tubes in a boiling water bath for heat treatment for 1 - 2 min. (4) Immediately cool the test tubes in cold water and wipe dry the test tubes. (5) Anaerobically culture at 37 °C for 7 - 10 days. When the gelatinized liquid is completely liquefied, the top cover floats on the surface, and there is a cloudy band at the bottom, indicating that Clostridium acetobutylicum CICC8020 grows well and can be used as the seed liquid. Expand the culture of the seed liquid in the above corn paste medium at 37 °C for 7 - 10 days according to the above steps. Obtain the expanded culture solution of Clostridium acetobutylicum CICC8020, and through detection, the OD 600 value of the expanded culture solution of Clostridium acetobutylicum CICC8020 is 2.3, indicating that the bacteria grow well and can be used for subsequent inoculation.

[0067] 2. Filter the high-concentration sugar solution prepared in Example 1 and use it as the fermentation substrate. Inoculate the expanded Clostridium acetobutylicum CICC8020 into the filtered high-concentration sugar solution at a mass concentration of 5% for anaerobic fermentation. The temperature of anaerobic fermentation is 37 °C, and the time of anaerobic fermentation is 11 d. Detect the butanol yield of the anaerobic fermentation system, and regularly supplement the high-concentration sugar solution every 11 d during the anaerobic fermentation process. During the anaerobic fermentation process, the produced butanol can be extracted by the stripping method.

[0068] By calculation, when anaerobic fermentation culture is carried out for 11 d, the butanol concentration is detected by GC-MS, and the butanol yield is 8.65 g / L.

[0069] Example 3

[0070] A method for enzymatic hydrolysis and saccharification of straw is as follows:

[0071] 1. The preparation of the enzyme-producing microbial inoculum is the same as in Example 1.

[0072] 2. Preparation of DES reagent

[0073] Mix choline chloride and triethanolamine (TEA) in a molar ratio of 1:1, that is, weigh 139.62 g of choline chloride and dissolve it in 149.19 g of triethanolamine to obtain the DES reagent.

[0074] 3. Pretreat the corn stover with a moisture content ≤ 5% after drying by mixing it with the DES reagent at a mass-to-volume ratio of 1 g:10 mL for 1 h to obtain the pretreated stover. Mix the obtained pretreated stover with a sugar solution with a glucose mass concentration of 5% at a mass-to-volume ratio of 75 g:175 mL to obtain a stover-sugar solution mixed substrate. Inoculate the enzyme-producing microbial inoculum into the stover-sugar solution mixed substrate at a volume ratio of 5%, and carry out microbial culture for enzyme production at 37 °C and 110 rpm. Microbial culture for enzyme production is mainly the process of microbial growth and enzyme production. After 4 d of microbial culture for enzyme production and waiting for the microorganisms to produce enzymes vigorously, pump in pure water. Using pure water and the sugar solution as the liquid system and the stover as the solid system, adjust the system to a high-solid hydrolysis system with a solid content of 25% (dry matter basis). Calculate and determine the addition amount of pure water based on a stover solid content of 95%. Carry out enzymatic hydrolysis on the high-solid hydrolysis system at 50 °C and 150 rpm for 4 - 5 d. After the hydrolysis is completed, turn off the stirring device. After precipitation, a high-concentration sugar solution is obtained at the lower layer. The process of step 3 is carried out in a hydrolysis tank.

[0075] After the obtained high-concentration sugar solution at the lower layer can directly flow out through the interface, it enters the anaerobic fermentation tank for the preparation of butanol after filtration.

[0076] After the high-concentration sugar solution at the lower layer flows out and immediately after transferring away the first batch of sugar solution in the hydrolysis tank, a mixture of DES-pretreated stover and sugar solution and the prepared microbial community, that is, the enzyme-producing microbial inoculum, can be directly added to the remaining system to continue the enzymatic hydrolysis and saccharification of the stover. At this time, there are also microbial communities and cellulase in the pore carbon at the bottom layer of the remaining system, which can hydrolyze the stover to produce sugar solution more quickly. Cellulase is an inducible enzyme, and glucose is both a decomposition product, a carbon source required for growth, and an inducer for enzyme production. Then, the method for preparing the high-concentration sugar solution is carried out in a cycle as described above to prepare the high-concentration sugar solution.

[0077] One hour after the pretreatment of the stover with the DES solvent, the degradation rate of lignin in the stover is measured to be as high as 16.18%. After the enzymatic hydrolysis is completed, the glucose content in the high-concentration sugar solution is measured, and the results show that the glucose yield is as high as 85.66%, and the reducing sugar yield is as high as 89.56%.

[0078] Example 4

[0079] The method for producing butanol using the high-concentration sugar solution prepared in Example 3 is as follows:

[0080] The activation and scale-up culture method of Clostridium acetobutylicum CICC8020 was the same as that in Example 2.

[0081] 2. After filtering the high-concentration sugar solution prepared in Example 3, it was used as the fermentation substrate. The scale-up cultured Clostridium acetobutylicum CICC8020 was inoculated into the filtered high-concentration sugar solution at a mass concentration of 2% for anaerobic fermentation. The temperature of anaerobic fermentation was 37 °C. When the anaerobic fermentation time reached 11 d, the butanol yield of the anaerobic fermentation system was detected. During the anaerobic fermentation process, the high-concentration sugar solution was regularly supplemented every 11 d. During the anaerobic fermentation process, the produced butanol was extracted by the air stripping method.

[0082] By calculation, when the anaerobic fermentation culture time reached 11 d, the butanol yield was 8.81 g / L.

[0083] Comparative Example 1

[0084] The corn straw was mixed with 60 wt% sulfuric acid solution according to a liquid-solid ratio of 10 mL:1 g for hydrolysis of corn straw. The hydrolysis process was carried out at room temperature of 25 °C for 2 h to obtain a high-concentration sugar solution. The glucose yield in the high-concentration sugar solution was 70.43%.

[0085] Ca(OH) was added to the obtained high-concentration sugar solution 2 The pH was adjusted to 6.5, and Clostridium acetobutylicum CICC8020 was inoculated for anaerobic fermentation according to the method of Example 4. The butanol yield obtained after 11 d of anaerobic fermentation was 0.29 g / L.

[0086] Comparative Example 2

[0087] It was the same as the method of enzymatic hydrolysis and saccharification of straw in Example 1. The only difference was that the straw was not mixed with the DES reagent, that is, the enzyme-producing microbial inoculant was directly inoculated into the corn straw aqueous system with a solid content of 25% and cultured at 37 °C and 110 rpm for 3 d, and then the reaction system was carried out at 50 °C and 150 rpm for 4 d.

[0088] During the above enzymatic hydrolysis and saccharification process of straw, the enzyme-producing microbial inoculant would gradually die, and the produced enzyme was not enough to break the natural anti-degradation barrier composed of lignin in the biomass raw material. After soaking for one week, there were only some molds on the straw, and no saccharification and liquefaction occurred.

[0089] Comparative Example 3

[0090] The method is the same as that of Example 1 for enzymatic hydrolysis and saccharification of straw. The only difference is that instead of mixing the straw with the DES reagent, the straw is directly mixed with a glucose solution with a glucose mass concentration of 5% at a mass-to-volume ratio of 75 g:175 mL. After that, an enzyme-producing microbial inoculum is inoculated into the mixed system, and then the mixture is cultured at 37 °C and 110 rpm for 3 days. After the culture is completed, the solid content of the high-solid hydrolysis system is adjusted to 25% according to the method of Example 1, and then the reaction system is carried out at 50 °C and 150 rpm for 4 days. Under this condition, the glucose yield is also zero, and the straw cannot be hydrolyzed at all. If the time is extended to one month, molds will appear but the straw cannot be degraded.

[0091] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for producing butanol by enzymatic saccharification of straw, characterized in that: The following steps are involved: The straw and the low eutectic solvent are mixed to pretreat the straw to obtain pretreated straw; the mass volume ratio of the straw to the low eutectic solvent is 1 g:10 mL; the hydrogen bond acceptor of the low eutectic solvent comprises choline chloride; the hydrogen bond donor of the low eutectic solvent comprises glycerol and / or triethanolamine; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor in the low eutectic solvent is 1:(1-2); the pretreatment time is 0.5-1 h; The pretreated straw is mixed with the sugar solution, and then an enzyme-producing microbial agent is inoculated to perform microbial culture, enzyme production and enzymolysis in sequence to obtain a high-concentration sugar solution; the solid content of the pretreated straw and the sugar solution after mixing is 25%-30%; the mass concentration of glucose in the sugar solution is 5%-8%; the enzyme-producing microbial agent includes Trichoderma reesei and Aspergillus niger; the Trichoderma reesei includes Trichoderma reesei CICC13052; the Aspergillus niger includes Aspergillus niger CMCC (F) 98003; the ratio of the number of live bacteria of Trichoderma reesei to the number of live bacteria of Aspergillus niger in the enzyme-producing microbial agent is 5:1; the total number of live bacteria in the enzyme-producing microbial agent is 1×10 8 ~5×10 8 CFU / mL; The acetobutylic acid Clostridium is inoculated into a high-concentration sugar solution for anaerobic fermentation to produce butanol; the acetobutylic acid Clostridium includes acetobutylic acid Clostridium CICC8020.

2. The method according to claim 1, characterized in that: The temperature for culturing the microorganisms to produce enzymes is 37° C.; the time for culturing the microorganisms to produce enzymes is 3 to 5 days; the temperature for the enzymatic hydrolysis is 50° C.; and the time for the enzymatic hydrolysis is 4 to 5 days.

3. The method according to claim 1, characterized in that: The straw includes any one or two or more of corn straw, wheat straw and rice straw.

4. The method according to claim 1, characterized in that: The temperature of the anaerobic fermentation is 37° C.; the time of the anaerobic fermentation is ≥ 3 days.

5. The method according to claim 1, characterized in that: The method for extracting butanol includes: extracting it by gas stripping.

6. The method according to claim 1, characterized in that: The anaerobic fermentation process also includes feeding; the feeding includes supplementing high-concentration sugar solution and Clostridium acetobutylicum.

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