Method for producing butanol by pre-treating straw through deep eutectic solvent combined biological enzymolysis method

Abstract

The invention provides a method for producing butanol by pretreating straws through a deep eutectic solvent combined biological enzymolysis method, and belongs to the technical field of straw treatment. The invention provides a straw enzymolysis saccharification method which comprises the following steps: pre-treating straw by adopting a deep eutectic solvent, then mixing the pre-treated straw and a sugar solution, inoculating enzyme-producing microorganisms, and sequentially carrying out microorganism culture enzyme production and enzymolysis to obtain a high-concentration sugar solution. According to the method, DES is used for pretreating the straw, so that the natural anti-degradation barrier of lignin is destroyed, and the accessibility of cellulose is improved; enzyme-producing microorganisms are inoculated on the DES pretreated straw to efficiently prepare cellulase, then straw enzymolysis is performed, the degradation rate of lignin in the straw after DES pretreatment reaches up to 23.52%, and the yield of glucose reaches up to 88.23% after enzymolysis is completed. The obtained high-concentration sugar solution does not contain toxic substances such as furan, can be used for preparing butanol, and does not generate adverse effects on growth and metabolism of a butanol-producing microbial preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of straw processing, and particularly relates to a method for producing butanol by pre-treating straw with a low eutectic solvent combined with a biological enzymatic hydrolysis method. Background Art

[0002] A large amount of crop straw is produced every year. If these straws are not used properly, they will cause huge waste of resources and environmental pollution. Cellulase has broad application prospects and markets in the biomass energy industry. Using cellulase to treat lignocellulosic biomass raw materials, enzymatically hydrolyze sugars, and then ferment 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 than ethanol as a biofuel: (1) Butanol is more miscible with diesel and does not require any co-solvent. It can be miscible with diesel in any proportion. Its kinematic viscosity is roughly the same as that of diesel, which is more suitable for existing engine structures. Since it has a high heat of vaporization and a low 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 is less likely to have cavitation and gas lock problems. Engines burning butanol are easier to start in cold weather. (3) Butanol is less corrosive and more suitable for distribution through existing pipelines. There is no need to modify 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. The use of cellulase to degrade corn straw raw materials to produce biobutanol is of epoch-making significance for ultimately solving the energy shortage problem facing mankind.

[0004] At present, the methods for preparing butanol from straw mostly use a variety of chemical agents combined with cellulase to further prepare butanol from straw. The related methods for preparing butanol have the disadvantages of 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 butanol due to the chemical agents contained in it, which is not conducive to the increase of butanol production. Summary of the invention

[0005] In view of the defects in the prior art, the object of the present invention is to provide a method for enzymatic saccharification of straw, wherein the high-concentration sugar solution prepared by the method does not contain chemical substances that are detrimental to the growth of microorganisms, has low toxicity to Clostridium butanol, is beneficial to increasing the butanol yield, and has a simple production chain and the entire operation process is easy to control.

[0006] The objective of the present invention is achieved through the following technical solutions: The present invention provides a method for enzymatic saccharification of straw, comprising the following steps: The straw is mixed with a low eutectic solvent 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 pretreated straw is mixed with the sugar solution and then inoculated with an enzyme-producing microbial agent to perform microbial culture to produce enzymes and enzymolysis in sequence to obtain a high-concentration sugar solution; the solid content of the pretreated straw after mixing 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.

[0007] Preferably, the hydrogen bond acceptor of the deep eutectic solvent comprises choline chloride; the hydrogen bond donor of the deep eutectic solvent comprises 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); The pretreatment time is 0.5 to 1 h.

[0008] Preferably, the ratio of the number of viable bacteria of Trichoderma reesei to the number of viable bacteria of Aspergillus niger in the enzyme-producing microbial agent is 5:1; the total number of viable bacteria in the enzyme-producing microbial agent is 1×10 8 ~5×10 8 CFU / mL.

[0009] Preferably, 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.

[0010] Preferably, the straw includes any one or more of corn straw, wheat straw and rice straw.

[0011] The present invention provides a method for producing butanol from a high-concentration sugar solution prepared by the method described in the above technical solution, comprising the following steps: Clostridium acetobutylicum was inoculated into high-concentration sugar solution for anaerobic fermentation to produce butanol.

[0012] Preferably, the Clostridium acetobutylicum includes Clostridium acetobutylicum CICC8020.

[0013] Preferably, the temperature of the anaerobic fermentation is 37° C.; and the time of the anaerobic fermentation is ≥ 3 days.

[0014] Preferably, the method for extracting butanol comprises: extracting it by gas stripping.

[0015] Preferably, the anaerobic fermentation process further includes feeding; the feeding includes supplementing high-concentration sugar solution and Clostridium acetobutylicum.

[0016] Beneficial effects of the present invention The invention provides a method for enzymatic saccharification of straw, comprising the following steps: pre-treating the straw by mixing straw and a low eutectic solvent to obtain pre-treated straw; the mass volume ratio of the straw to the low eutectic solvent is 1 g:10 mL; mixing the pre-treated straw with a sugar solution, inoculating an enzyme-producing microbial agent, and sequentially performing microbial culture to produce enzymes and enzymolysis to obtain a high-concentration sugar solution; the solid content of the pre-treated straw after mixing with the sugar solution is 25%-30%; the mass concentration of glucose in the sugar solution is 5%-8%; the enzyme-producing microbial agent comprises Trichoderma reesei and Aspergillus niger; the Trichoderma reesei comprises Trichoderma reesei CICC13052; the Aspergillus niger comprises Aspergillus niger CMCC (F) 98003. A deep eutectic solvent (DES) is a quasi-ionic solvent consisting of a binary or multicomponent mixture of a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA). After mixing, the charge delocalization of the HBD hydrogen bonds lowers the melting point, making the DES liquid at room temperature. The present invention firstly pre-treats the straw with DES to destroy the natural anti-degradation barrier of lignin and improve the accessibility of cellulose; then enzyme-producing microorganisms such as Trichoderma reesei and Aspergillus niger are inoculated on the straw pre-treated with DES, and cellulase is efficiently prepared by using microorganisms such as Trichoderma reesei and Aspergillus niger, and then enzymatic hydrolysis of the straw is performed, that is, the straw is pre-treated with a DES reagent combined with a biological enzymatic hydrolysis method, and the straw is dynamically enzymatically hydrolyzed and saccharified. The degradation rate of lignin in the straw after DES pre-treatment is as high as 23.52%, and 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 the obtained sugar solution is further used to prepare butanol, which will not have an adverse effect on the growth metabolism of butanol-producing microbial preparations, and is conducive to improving the yield of butanol. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 This is a flow chart of the straw enzymatic saccharification and high-concentration sugar solution production of butanol in the present invention; Figure 2 This is a schematic diagram of the device for producing butanol by enzymatic saccharification of straw. DETAILED DESCRIPTION

[0019] The present invention provides a method for enzymatic saccharification of straw, comprising the following steps: The straw is mixed with a low eutectic solvent 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 pretreated straw is mixed with the sugar solution and then inoculated with an enzyme-producing microbial agent to perform microbial culture to produce enzymes and enzymolysis in sequence to obtain a high-concentration sugar solution; the solid content of the pretreated straw after mixing 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.

[0020] The present invention mixes straw and a low eutectic solvent to pretreat 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 embodiment of the present invention, corn straw is taken as an example to illustrate the specific method steps of straw enzymatic saccharification. The present invention takes corn straw as an example. On the basis that corn straw can be efficiently enzymatically saccharified by a corresponding method, it can be further determined that wheat straw and rice straw that are easier to enzymatically hydrolyze than corn straw can also be enzymatically saccharified by this method. As an optional embodiment of the present invention, the straw can be fresh straw or dried straw. In theory, the effect of using fresh straw will also be better. In a specific embodiment of the present invention, in order to facilitate the verification of the effect of the test scheme, the method of enzymatic saccharification of straw is specifically described using dried straw. As an optional embodiment of the present invention, the dried straw is preferably straw with a moisture content of ≤5%. The present invention does not specifically limit the drying method of the straw, and conventional drying methods in the art can be used. The present invention does not specifically limit the composition of the low eutectic solvent, and any low eutectic solvent of conventional composition in the art can be used. As an optional embodiment of the present invention, the hydrogen bond acceptor of the low eutectic solvent may include choline chloride; the hydrogen bond donor of the low eutectic solvent may include glycerol and / or triethanolamine; the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor in the low eutectic solvent may be 1: (1-2). In the present invention, the mass volume ratio of the straw to the low eutectic solvent may be 1g: 10mL. In the present invention, the pretreatment time may be 0.5-1h. The present invention uses a low eutectic solvent to pretreat the straw, and the protons dissociated from the organic acid group of the low eutectic solvent promote the cleavage of the unstable ether bond of the phenylpropane unit of lignin, have a strong impact effect on the corn straw, a strong depolymerization effect, can destroy the natural anti-degradation barrier of lignin, and improve the accessibility of cellulose, thereby promoting the degradation of straw. In the present invention, the degradation rate of lignin in the straw after DES pretreatment is as high as 23.52%.

[0021] After obtaining the pretreated straw, the present invention mixes the pretreated straw with sugar solution, inoculates enzyme-producing microbial agents, and sequentially performs microbial culture to produce enzymes and enzymolysis to obtain high-concentration sugar solution.

[0022] In the present invention, the mass concentration of glucose in the sugar solution can be 5% to 8%. As an optional 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 of the pretreated straw after mixing with the sugar solution can be 25% to 30%, or 25%, 26%, 27%, 28%, 29% or 30%. When calculating the solid content in the present invention, the effect of the low eutectic solvent on the solid content is ignored, that is, the solid content = the mass of the dry matter of the straw / (the mass of the straw + the mass of the sugar solution). In the present invention, the glucose solution is mainly used as a carbon source for mold growth and an inducer for enzyme production to promote the growth and enzyme production of mold.

[0023] The present invention has no particular 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 pretreated straw and sugar solution by mixing the pretreated straw and sugar solution, or can be called a straw sugar solution mixed substrate.

[0024] After obtaining the pretreated straw and sugar solution mixed system, the present invention inoculates the enzyme-producing microbial agent into the pretreated straw and sugar solution mixed system to sequentially perform microbial culture to produce enzymes and enzymolysis to obtain high-concentration sugar solution.

[0025] In the present invention, the enzyme-producing microbial agent may include Trichoderma reesei and Aspergillus niger; the Trichoderma reesei may include Trichoderma reesei CICC13052; the Aspergillus niger may include Aspergillus niger CMCC (F) 98003. As an optional embodiment of the present invention, 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 may be 5:1; the total number of live bacteria in the enzyme-producing microbial agent may be 1×10 8 ~5×10 8 CFU / mL, or 1.45×10 8CFU / mL. Before preparing the enzyme-producing microbial agent, the present invention preferably performs activation culture and seed culture on Trichoderma reesei CICC13052 and Aspergillus niger CMCC (F) 98003, respectively, to obtain Trichoderma reesei CICC13052 seed solution and Aspergillus niger CMCC (F) 98003 seed solution. Then, the Trichoderma reesei CICC13052 seed solution and the Aspergillus niger CMCC (F) 98003 seed solution are compounded according to the ratio of viable bacteria to obtain an enzyme-producing microbial agent. The present invention does not specifically limit the method for activating and seeding Trichoderma reesei CICC13052 and Aspergillus niger CMCC (F) 98003, and the conventional method of the corresponding agent in the field can be used for culturing. As an optional embodiment of the present invention, the Trichoderma reesei CICC13052 and Aspergillus niger CMCC (F) 98003 can be activated and seeded by the same culture method. The present invention can use comprehensive potato culture medium to culture Trichoderma reesei CICC13052 and Aspergillus niger CMCC (F) 98003 respectively; the activation culture can be carried out in a sterile solid culture medium; the temperature of the activation culture can be 28°C; the time of the activation culture can be 5-7 days; the activation culture is preferably carried out twice in a row. After the activation culture is completed, the present invention preferably inoculates the obtained activated bacterial agent into a sterile liquid culture medium for seed culture; the temperature of the seed culture can be 28°C; the time of the seed culture can be 24-48 hours; 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 bacterial agent is obtained. As an optional embodiment of the present invention, the number of viable bacteria in the obtained Trichoderma reesei CICC13052 seed culture solution can be 1×10 8 CFU / mL; the viable count of Aspergillus niger CMCC (F) 98003 seed culture fluid can be 1×10 8 CFU / mL. After obtaining the seed culture solution of Trichoderma reesei CICC13052 and the seed culture solution of Aspergillus niger CMCC (F) 98003 respectively, the seed culture solution of Trichoderma reesei CICC13052 and the seed culture solution of Aspergillus niger CMCC (F) 98003 are preferably mixed according to the above-mentioned viable bacterial count ratio to obtain an enzyme-producing microbial agent. The viable bacterial count of the enzyme-producing microbial agent obtained by the present invention can be 1×10 8 ~5×10 8 CFU / mL, or 1.45×10 8 CFU / mL. In the present invention, the Trichoderma reesei can produce cellulose complex enzymes; Aspergillus niger CMCC (F) 98003 can increase the proportion of cellobiase in the cellulose complex enzymes, thereby facilitating the efficient enzymatic saccharification of straw.

[0026] 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 at a volume ratio of 1% to 10% to perform microbial culture, enzyme production and enzymolysis in sequence. In the present invention, the temperature of the microbial culture and enzyme production can be 37°C; the time of the microbial culture and enzyme production can be 3 to 5 days, or 4 days; the process of the microbial culture and enzyme production is preferably accompanied by rotation; the rotation speed can be 110 rpm. In the present invention, the microbial growth and enzyme production process also has an enzymatic reaction, in which the main process is that Trichoderma reesei and Aspergillus niger grow by utilizing the substances in the straw to produce cellulase. After the microbial culture and enzyme production are cultured until the microbial enzyme production 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 passing pure water or a low-concentration enzymatic sugar production liquid into the enzyme production system. In the present invention, the low-concentration enzymatic sugar production liquid can be a low-concentration sugar solution produced by enzymatic sugar production of other equipment in the factory. When adjusting the solid content, the present invention preferably uses the dry matter in the straw as the solid matter to adjust the solid content; and uses the sugar solution and the added water or the liquid of low-concentration enzymatic sugar production and the sugar solution as the liquid system to adjust the solid content. When adjusting the solid content, if the straw used is a dried straw with a water content of ≤5%, it is preferably determined by calculating the amount of water or the liquid of low-concentration enzymatic sugar production based on the solid content of the straw of 95%; when adjusting the solid content, if the straw used is fresh straw, it is preferably determined by calculating the amount of water or the liquid of low-concentration enzymatic sugar production 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 of low-concentration enzymatic sugar production + mass of the sugar solution). The present invention preferably adjusts the solid content of the enzyme production system to 25%, and obtains a high-solid hydrolysis system. After obtaining the high-solid hydrolysis system, the present invention preferably performs enzymolysis. In the present invention, the temperature of the enzymolysis can be 50°C; the time of the enzymolysis can be 4-5d; the process of the enzymolysis is preferably accompanied by rotation; the rotation speed can be 150rpm. In the present invention, the enzymolysis is mainly carried out by hydrolyzing the straw with cellulose complex enzymes to produce glucose and obtain a high-concentration sugar solution. After the enzymolysis of the present invention is completed, a high-concentration sugar solution is obtained. After the enzymolysis of the present invention is completed, it is preferred to stop the rotation and stand still so that the prepared high-concentration sugar solution and the remaining hydrolyzed substances in the enzymolysis system are separated. In the present invention, the obtained high-concentration sugar solution is located at the bottom of the remaining hydrolyzed substances due to gravity sedimentation. 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 the high-concentration sugar solution part from the remaining hydrolyzed substances in the hydrolysis system by gravity sedimentation to obtain a high-concentration sugar solution. In the present invention, the step of preparing a high-concentration sugar solution by enzymatic saccharification of the straw can be carried out in a hydrolysis tank.

[0027] 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 embodiment of the present invention, the filtering method can be drip filtration.

[0028] After obtaining the filtered high-concentration sugar solution, the obtained high-concentration sugar solution can be directly added to the anaerobic fermentation tank for the preparation of butanol. After the high-concentration sugar solution is obtained in the present invention, the DES-treated straw can continue to be added to the remaining hydrolyzed material in the enzymatic system, and the enzyme-producing microbial agent is inoculated at the same time to continue the enzymatic saccharification of the straw. When the present invention continues to perform enzymatic saccharification of the straw, the remaining hydrolyzed material is located at the bottom layer, and the porous carbon as the bottom layer also has microbial communities and cellulase, which can hydrolyze the straw more quickly to produce sugar solution. Cellulase is an induced enzyme, and glucose is both a decomposition product and a carbon source required for growth and an inducer for enzyme production. The method for preparing the high-concentration sugar solution is as described above, and the high-concentration sugar solution is prepared in a cycle in this way.

[0029] The method for enzymatic saccharification of straw provided by the present invention utilizes DES combined with cellulase to treat straw to produce sugar solution, and inoculates enzyme-producing microbial agents on straw pretreated with a low eutectic solvent, that is, first pretreating the straw with DES to destroy the natural anti-degradation barrier of lignin and improve the accessibility of cellulose; then inoculating enzyme-producing microbial agents such as Trichoderma reesei and Aspergillus niger on the straw pretreated with DES, using microorganisms such as Trichoderma reesei and Aspergillus niger to efficiently prepare cellulase, and then performing enzymatic hydrolysis of the straw, that is, using DES reagent combined with biological enzymatic hydrolysis to pretreat the straw, and performing dynamic enzymatic saccharification of the straw. In the present invention, the degradation rate of lignin in the straw after DES pretreatment is as high as 23.52%, and cellulose is slightly degraded; the enzymatic hydrolysis stage mainly degrades cellulose, and 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.

[0030] The present invention also provides a method for producing butanol based on the high-concentration sugar solution prepared by the method described in the above technical solution, comprising the following steps: Clostridium acetobutylicum was inoculated into high-concentration sugar solution for anaerobic fermentation to produce butanol.

[0031] In the present invention, the Clostridium acetobutylicum includes Clostridium acetobutylicum CICC8020. In the present invention, Clostridium acetobutylicum is preferably activated and expanded to obtain an expanded culture solution of Clostridium acetobutylicum, and then the expanded culture solution of Clostridium acetobutylicum is inoculated into a high-concentration sugar solution. The present invention does not specifically limit the method for activating and expanding the culture of Clostridium acetobutylicum, and any conventional method in the art may be used. In the present invention, the method for activating and expanding the culture of Clostridium acetobutylicum is preferably performed according to the method described in the instruction manual of the bacterial agent to perform activation and expansion culture to obtain an expanded culture solution of Clostridium acetobutylicum. In the present invention, the OD in the expanded culture solution of Clostridium acetobutylicum is 600 The value is 2.3, indicating that the bacteria grow very well and can be used for subsequent inoculation. After obtaining the expanded culture solution of Clostridium acetobutylicum, the present invention preferably inoculates the expanded culture of Clostridium acetobutylicum CICC8020 into a high-concentration sugar solution at a mass concentration of 2% to 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 ≥3d. In the present invention, the fermentation system can start to produce butanol after 3d of anaerobic fermentation. In the present invention, the anaerobic fermentation process also includes feeding; the feeding includes supplementing high-concentration sugar solution and Clostridium acetobutylicum. In the present invention, the high-concentration sugar solution can be fed regularly; the timing of the regular feeding is feeding once every 11 days. In the process of producing butanol, the present invention also includes supplementing Clostridium acetobutylicum. The present invention does not specifically limit the timing of supplementing Clostridium acetobutylicum. The addition of Clostridium acetobutylicum can be determined by observing the output rate of butanol during the anaerobic fermentation process. When the butanol production rate slows down and / or the gas production is insufficient, Clostridium acetobutylicum can be supplemented; when supplementing Clostridium acetobutylicum, the present invention should supplement Clostridium acetobutylicum with a low concentration to ensure that the output rate of the high-concentration sugar solution is coordinated with 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 gas stripping. The present invention does not specifically limit the gas stripping method, and any conventional gas 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 a high-concentration sugar solution can be carried out in an anaerobic fermentation tank.

[0032] In the present invention, the whole process of preparing high-concentration sugar solution by enzymatic saccharification of straw, filtering the high-concentration sugar solution, and producing butanol by using the high-concentration sugar solution can be carried out in one device. Among them, the hydrolysis tank for preparing high-concentration sugar solution by enzymatic saccharification of straw can be located at the upper part of the high-concentration sugar solution filtering device; the device for producing butanol by using the high-concentration sugar solution can be located at the lower part of the high-concentration sugar solution filtering device. The present invention uses the above-mentioned device to produce butanol by using straw, and the high-concentration sugar solution can be separated, filtered and used as a fermentation substrate by the effect of gravity sedimentation during the whole production process. In terms of technology, continuous saccharification is achieved by dry-wet coupling, and saccharification and butanol fermentation are physically isolated, so that the hydrolysis of lignocellulose and the synthesis of butanol can be completed in one process.

[0033] In the present invention, if the method for enzymatic saccharification of straw and the entire process of producing butanol using high-concentration sugar solution are carried out in one device, that is, the process of enzymatic saccharification of straw is carried out in a hydrolysis tank, and the process of producing butanol using high-concentration sugar solution is carried out in an anaerobic fermentation tank, when regular feeding is performed during the anaerobic fermentation process, it is only necessary to open the valve to allow the hydrolyzed high-concentration sugar solution to drip in after filtering; at the same time, DES pretreated straw and sugar solution are added to the hydrolysis tank to feed the hydrolysis tank.

[0034] The present invention uses the high-concentration sugar solution as a fermentation substrate to perform anaerobic fermentation on Clostridium acetobutylicum. The high-concentration sugar solution does not contain furan-like toxic substances, will not have an adverse effect on the growth of Clostridium acetobutylicum, can promote the rapid growth of Clostridium acetobutylicum in the fermentation substrate, and thus improve the yield of butanol.

[0035] As an practicable method, the present invention also provides a device for producing butanol by enzymatic saccharification of straw, specifically as follows Figure 2As shown, it includes: a hydrolysis tank, a filtering device and an anaerobic fermentation tank; the filtering device is located at the lower part of the hydrolysis tank; the anaerobic fermentation tank is located at the lower part of the filtering device; the lower opening of the hydrolysis tank is connected to the filtering device; the lower opening of the filtering device is connected to the anaerobic fermentation tank. As an optional embodiment of the present invention, the hydrolysis tank is located at the upper part of the filtering device, and the enzymatic hydrolysis and saccharification of straw is carried out in the hydrolysis tank to prepare a high-concentration sugar solution. The hydrolysis tank has a stirring device to facilitate stirring during the enzymatic hydrolysis and saccharification process. The lower opening of the hydrolysis tank is connected to the filtering device, and the high-concentration sugar solution produced by the enzymatic hydrolysis and saccharification of straw in the hydrolysis tank is directly entered into the filtering device through the lower opening after static precipitation, and the high-concentration sugar solution is filtered. The filtering device is connected to the anaerobic fermentation tank, and the filtered high-concentration sugar solution can directly enter the anaerobic fermentation tank, and anaerobic fermentation is carried out in the anaerobic fermentation tank to produce butanol. In the present invention, the anaerobic fermentation tank is preferably located at the lower part of the filtering device, and the filtered high-concentration sugar solution can be directly dripped into the lower anaerobic fermentation tank by gravity for anaerobic fermentation to produce butanol. The device provided by the present invention can directly carry out the process of enzymatic saccharification of straw and production of butanol by high-concentration sugar solution. The device physically isolates saccharification and butanol fermentation through dry-wet coupling continuous saccharification in terms of process, so that it can complete the hydrolysis of lignocellulose to the synthesis of butanol in one process.

[0036] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] The flow chart of the invention for producing butanol from straw enzymatic saccharification and high-concentration sugar solution is as follows: Figure 1 shown.

[0038] Example 1 A method for enzymatic saccharification of straw, comprising the following steps: 1. Preparation of enzyme-producing microbial agents The enzyme-producing microbial agent is sourced from Trichoderma reesei ( Trichoderma reesei ) Freeze-dried bacterial powder of CICC13052 and freeze-dried bacterial powder of Aspergillus niger CMCC(F)98003.

[0039] 1) Activation of bacterial strains and preparation of seed solution (1) Activation of Trichoderma reesei CICC13052 strain and preparation of seed solution: Add 1.1 mL of oxygen-free water to the freeze-dried powder of the bacterial agent and vortex for 5 to 10 seconds to completely dissolve it. Take 1 mL of the completely dissolved bacterial solution and add it to 9 mL of sterile saline. Mix well and dilute it 10 times step by step until the bacterial count is about 500 to 1000 CFU / mL to obtain Trichoderma reesei CICC13052 bacterial solution. Take 100 µL of Trichoderma reesei CICC13052 bacterial solution (containing 50 to 100 CFU) and add it to the solidified sterile culture medium plate. Then use a sterile L-shaped coating stick to evenly spread the bacterial solution on the plate in the same direction. After the bacterial solution penetrates into the culture medium, turn the plate upside down and culture it at 28°C for 5 to 7 days until spores grow to obtain activated Trichoderma reesei CICC13052. The activated Trichoderma reesei CICC13052 was subcultured and activated by the same method as above to obtain subcultured activated Trichoderma reesei CICC13052. An appropriate amount of sterile physiological saline was added to the culture medium of subcultured activated Trichoderma reesei CICC13052 and mixed for 2 minutes to prepare a concentration of 1×10 6 ~1×10 7 The spore suspension was inoculated into the seed culture medium at a volume percentage of 5%. The culture was shaken at 28°C and 180 rpm for 24 to 48 hours to obtain the seed culture solution of Trichoderma reesei CICC13052. The viable bacterial count of the obtained seed culture solution of Trichoderma reesei CICC13052 was adjusted to 1×10 8 CFU / mL.

[0040] The culture medium used in the above culture is a comprehensive potato culture medium (PDA), which is composed of: 1000 mL of potato extract; 20 g of glucose; natural pH. 15 g of agar needs to be added to the solid culture medium, and no agar is needed to be added to the liquid culture 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 and boil for 30 min, filter out the potato pieces, and make up the filtrate to 1000 mL.

[0041] (2) Activation of Aspergillus niger CMCC(F)98003 strains and preparation of seed solution The same as step (1) was used to prepare Aspergillus niger CMCC(F)98003 seed culture solution, and the viable bacterial count of the obtained Aspergillus niger CMCC(F)98003 seed culture solution was adjusted to 1×10 8 CFU / mL.

[0042] 2) Preparation of enzyme-producing microbial agents Trichoderma reesei CICC13052 and Aspergillus niger CMCC (F) 98003 were prepared in a ratio of 5:1 between the number of live bacteria of Trichoderma reesei CICC13052 and the number of live bacteria of Aspergillus niger CMCC (F) 98003. The number of live bacteria of the obtained enzyme-producing microbial agent was 1.45×10 8 CFU / mL.

[0043] 2. Prepare DES reagent Choline chloride and glycerol were mixed at a molar ratio of 1:2, that is, 139.62 g of choline chloride powder was weighed and dissolved in 184.18 g of glycerol to obtain a DES reagent.

[0044] 3. The corn straw with a moisture content of ≤5% after drying was mixed with DES reagent at a mass volume ratio of 1g:10mL for straw pretreatment. The pretreatment time was 1h to obtain pretreated straw. The obtained pretreated straw was mixed with a sugar solution with a glucose mass concentration of 8% at a mass volume ratio of 75g:175mL to obtain a straw sugar solution mixed substrate. The enzyme-producing microbial agent was inoculated into the straw sugar solution mixed substrate at a volume ratio of 5%, and the microorganism was cultured and enzyme-produced at 37°C and 110rpm. Microbial culture enzyme production is mainly a microbial growth enzyme production process. After the microbial enzyme production was cultured for 3 days, pure water was pumped in, and the sugar solution and pure water were used as the liquid system, and the straw dry matter was used as the solid system. The system was adjusted to a high solid hydrolysis system with a solid content of 25% (dry matter basis). The amount of pure water added was calculated based on the straw solid content of 95%. The high solid content hydrolysis system is subjected to enzymatic hydrolysis at 50°C and 150rpm for 4-5 days. After the hydrolysis is completed, the stirring device is turned off, and a high-concentration sugar solution is obtained in the lower layer after precipitation. The process of step 3 is carried out in the hydrolysis tank.

[0045] The obtained lower layer high-concentration sugar solution can flow out directly through the interface and enter the anaerobic fermentation tank for butanol preparation after being filtered.

[0046] After the high-concentration sugar solution in the lower layer flows out, that is, after the first batch of sugar solution in the hydrolysis tank is transferred away, the mixture of DES pretreated straw and sugar solution and the prepared bacterial community, i.e., enzyme-producing microbial agent, can be directly added to the remaining system to continue the enzymatic saccharification of the straw. At this time, the bottom layer of the porous carbon in the remaining system also has microbial communities and cellulase, which can hydrolyze the straw more quickly to produce sugar solution. Cellulase is an induced enzyme, and glucose is both a decomposition product and a carbon source required for growth and an inducer for enzyme production. The method for preparing high-concentration sugar solution is the same as described above, and the high-concentration sugar solution is prepared in this way in a cycle.

[0047] After the straw was pretreated with DES solvent for one hour, the degradation rate of lignin in the straw was as high as 23.53%. After the 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 yield of reducing sugar was as high as 90.05%.

[0048] Example 2 The method for producing butanol using the high-concentration sugar solution prepared in Example 1 comprises the following steps: 1. Activation and expansion of Clostridium acetobutylicum CICC8020: (1) Add 50g corn flour to a small amount of distilled water to make a paste, and then add it to boiling water. Stir continuously, add distilled water during stirring, boil for 1h, and finally adjust the volume to 1L to obtain a corn flour medium with a mass concentration of 5%. Dispense the corn flour medium into test tubes and sterilize at 121℃ for 15min. (2) After cooling, inoculate Clostridium acetobutylicum CICC8020. (3) After inoculation, place the test tube in a boiling water bath for heat treatment for 1~2min. (4) Immediately cool the test tube in cold water and wipe dry the test tube. (5) After anaerobic culture at 37℃ for 7~10 days, the gelatinized liquid is completely liquefied, the top cover floats on the surface, and there is a cloud band at the bottom, indicating that Clostridium acetobutylicum CICC8020 grows well and can be used as seed liquid. The seed liquid is then incubated in the above corn paste medium according to the above steps at 37℃ for 7~10 days for expansion culture. The OD value of the expanded culture medium of Clostridium acetobutylicum CICC8020 was obtained by detection. 600 The value is 2.3, which means that the bacteria are growing well and can be used for subsequent inoculation.

[0049] 2. The high-concentration sugar solution prepared in Example 1 was filtered and used as a fermentation matrix. The expanded culture of Clostridium acetobutylicum CICC8020 was inoculated into the filtered high-concentration sugar solution at a mass concentration of 5% for anaerobic fermentation. The temperature of the anaerobic fermentation was 37°C. The anaerobic fermentation time was 11 days. 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 days. The butanol produced during the anaerobic fermentation process can be extracted by gas stripping.

[0050] By calculation, it was found that after 11 days of anaerobic fermentation, the butanol concentration was detected by GC-MS, and the butanol yield was 8.65 g / L.

[0051] Example 3 A method for enzymatic saccharification of straw, comprising the following steps: 1. The preparation of enzyme-producing microbial agent is the same as that in Example 1.

[0052] 2. Prepare DES reagent Choline chloride and triethanolamine (TEA) were mixed at a molar ratio of 1:1, that is, 139.62 g of choline chloride was weighed and dissolved in 149.19 g of triethanolamine to obtain a DES reagent.

[0053] 3. The corn straw with a moisture content of ≤5% after drying was mixed with DES reagent at a mass volume ratio of 1g:10mL for straw pretreatment. The pretreatment time was 1h to obtain pretreated straw. The obtained pretreated straw was mixed with a glucose solution with a mass concentration of 5% at a mass volume ratio of 75g:175mL to obtain a straw sugar solution mixed substrate. The enzyme-producing microbial agent was inoculated into the straw sugar solution mixed substrate at a volume ratio of 5%, and the microorganism was cultured and enzyme-produced at 37°C and 110rpm. Microbial culture enzyme production is mainly a microbial growth enzyme production process. After the microbial culture enzyme production was carried out for 4 days, pure water was pumped in after the microorganisms produced enzymes vigorously. The pure water and sugar solution were used as the liquid system, and the straw was used as the solid system. The system was adjusted to a high solid hydrolysis system with a solid content of 25% (dry matter basis). The amount of pure water added was calculated based on the solid content of the straw being 95%. The high solid content hydrolysis system is subjected to enzymatic hydrolysis at 50°C and 150rpm for 4-5 days. After the hydrolysis is completed, the stirring device is turned off, and a high-concentration sugar solution is obtained in the lower layer after precipitation. The process of step 3 is carried out in the hydrolysis tank.

[0054] The obtained lower layer high-concentration sugar solution can flow out directly through the interface and enter the anaerobic fermentation tank for butanol preparation after being filtered.

[0055] After the high-concentration sugar solution in the lower layer flows out, that is, after the first batch of sugar solution in the hydrolysis tank is transferred away, the mixture of DES-pretreated straw and sugar solution and the prepared bacterial community, i.e., enzyme-producing microbial agent, can be directly added to the remaining system to continue the enzymatic saccharification of the straw. At this time, the bottom layer of the porous carbon in the remaining system also has microbial communities and cellulase, which can hydrolyze the straw more quickly to produce sugar solution. Cellulase is an induced enzyme, and glucose is both a decomposition product and a carbon source required for growth and an inducer for enzyme production. The method for preparing high-concentration sugar solution is then circulated to prepare high-concentration sugar solution as described above.

[0056] After the straw was pretreated with DES solvent for one hour, the degradation rate of lignin in the straw was measured to be as high as 16.18%. After the enzymatic hydrolysis was completed, the glucose content in the high-concentration sugar solution was measured, and the results showed that the yield of glucose was as high as 85.66%, and the yield of reducing sugar was as high as 89.56%.

[0057] Example 4 The method for producing butanol using the high-concentration sugar solution prepared in Example 3 comprises the following steps: 1. The method for activation and expansion culture of Clostridium acetobutylicum CICC8020 is the same as that in Example 2.

[0058] 2. The high-concentration sugar solution prepared in Example 3 was filtered and used as a fermentation matrix. The expanded 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 the anaerobic fermentation was 37°C. The butanol yield of the anaerobic fermentation system was detected when the anaerobic fermentation time was 11 days. During the anaerobic fermentation process, the high-concentration sugar solution was regularly supplemented every 11 days. The butanol produced during the anaerobic fermentation was extracted by gas stripping.

[0059] Calculation showed that the butanol yield was 8.81 g / L after 11 days of anaerobic fermentation.

[0060] Comparative Example 1 A 60wt% sulfuric acid solution was mixed with corn stalks at a liquid-solid ratio of 10mL:1g to hydrolyze the corn stalks. The hydrolysis process was treated at room temperature of 25°C for 2h to obtain a high-concentration sugar solution. The yield of glucose in the high-concentration sugar solution was 70.43%.

[0061] Ca(OH)2 was added to the obtained high-concentration sugar solution to adjust the pH to 6.5, and Clostridium acetobutylicum CICC8020 was inoculated according to the method of Example 4 for anaerobic fermentation. The butanol yield obtained after 11 days of anaerobic fermentation was 0.29 g / L.

[0062] Comparative Example 2 The method of enzymatic saccharification of corn straw was the same as that in Example 1, except that the corn straw was not mixed with the DES reagent, that is, the enzyme-producing microbial agent was directly inoculated into a corn straw water system with a solid content of 25% and cultured at 37°C and 110 rpm for 3 days, and then the reaction system was carried out at 50°C and 150 rpm for 4 days.

[0063] During the above-mentioned enzymatic saccharification of straw, the enzyme-producing microorganisms will gradually die, and the produced enzymes are not sufficient to destroy the natural anti-degradation barrier composed of lignin in the biomass raw materials. After soaking for a week, only some mold will remain on the straw, and saccharification and liquefaction will not occur.

[0064] Comparative Example 3 The method of enzymatic saccharification of straw is the same as that of Example 1, the only difference is that the straw is not mixed with the DES reagent, but the straw is directly mixed with a glucose solution with a glucose mass concentration of 5% at a mass volume ratio of 75g:175mL, and the enzyme-producing microbial agent is inoculated in the mixed system, and then cultured for 3d at 37°C and 110rpm. After the cultivation is completed, the high-solid hydrolysis system with a solid content of 25% is adjusted according to the method of Example 1, and then the reaction system is carried out for 4d at 50°C and 150rpm. The glucose yield under this condition is also zero, and the straw cannot be hydrolyzed at all. If the time is extended to one month, mold will be produced but the straw cannot be degraded.

[0065] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for enzymatic saccharification of straw, characterized in that: The following steps are involved: The straw is mixed with a low eutectic solvent 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 pretreated straw is mixed with the sugar solution and then inoculated with an enzyme-producing microbial agent to perform microbial culture to produce enzymes and enzymolysis in sequence to obtain a high-concentration sugar solution; the solid content of the pretreated straw after mixing 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.

2. The method according to claim 1, characterized in that: The hydrogen bond acceptor of the deep eutectic solvent comprises choline chloride; the hydrogen bond donor of the deep eutectic solvent comprises 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); The pretreatment time is 0.5 to 1 h.

3. The method according to claim 1, characterized in that: 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.

4. 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.

5. 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.

6. A method for producing butanol using a high-concentration sugar solution prepared by the method according to any one of claims 1 to 5, characterized in that: The following steps are involved: Clostridium acetobutylicum was inoculated into high-concentration sugar solution for anaerobic fermentation to produce butanol.

7. The method according to claim 6, characterized in that: The Clostridium acetobutylicum includes Clostridium acetobutylicum CICC8020.

8. The method according to claim 6, characterized in that: The temperature of the anaerobic fermentation is 37° C.; the time of the anaerobic fermentation is ≥ 3 days.

9. The method according to claim 6, characterized in that: The method for extracting butanol includes: extracting it by gas stripping.

10. The method according to claim 6, 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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