CN115627277A

CN115627277A - Method for performing solid state fermentation on 2, 3-butanediol by high-solid-state enzymolysis of straws

Info

Publication number: CN115627277A
Application number: CN202211442594.2A
Authority: CN
Inventors: 王岚; 姚长洪; 陈洪章
Original assignee: Institute of Process Engineering of CAS
Current assignee: Institute of Process Engineering of CAS
Priority date: 2022-11-18
Filing date: 2022-11-18
Publication date: 2023-01-20

Abstract

The invention provides a method for straw high-solid enzymolysis solid state fermentation of 2, 3-butanediol, which comprises the following steps: (1) Mixing the pretreated straw raw material with a buffer solution to obtain a mixture, carrying out disc grinding treatment on the mixture, and adding cellulase for enzymolysis to obtain an enzymolysis material; (2) Detoxifying the enzymolysis material, preparing a fermentation culture medium, adding an adsorption carrier, and performing solid fermentation to obtain the 2, 3-butanediol. The method realizes the improvement of the enzymolysis efficiency under a high solid system, effectively improves the production efficiency of the 2, 3-butanediol and solves the problem of high-valued conversion utilization of straw resources.

Description

Method for performing solid state fermentation on 2, 3-butanediol by high-solid-state enzymolysis of straws

Technical Field

The invention belongs to the field of microbial fermentation, relates to a method for fermenting 2, 3-butanediol, and particularly relates to a method for performing solid state fermentation on 2, 3-butanediol by high-solid enzymolysis of straws.

Background

The importance of renewable bio-based chemicals is increasing with the consumption of fossil energy and the negative impact that its excessive use has on the environment. Of these, 2, 3-butanediol is recognized as an important platform chemical. It is not only a biofuel with excellent performance, but also its derivatives are widely used in many fields such as food, medicine, synthetic rubber, etc. Biosynthesis is a major research direction for scaling up the production of 2, 3-butanediol. The energy consumption of the production process is lower compared with that of a chemical method, and meanwhile, the 2, 3-butanediol prepared by the biological method can be produced by utilizing lignocellulose and other renewable resources, so that the problems of abuse of non-renewable resources and the production cost of the 2, 3-butanediol are effectively relieved. The production efficiency is one of the key problems of preparing the 2, 3-butanediol by using a biological method, and the enhancement of the metabolic rate of a 2, 3-butanediol fermentation strain by proper strengthening measures is an important way for improving the production efficiency of the 2, 3-butanediol. By means of the characteristic of high air permeability of the adsorption carrier, the adsorption carrier is utilized to carry out solid state fermentation, so that a fermentation system can be ensured to have higher oxygen content, and thus the thallus density and the fermentation yield are improved.

The carbon source is another key problem of preparing the 2, 3-butanediol by using a biological method, and the grain carbon sources such as glucose, sucrose, starch and the like in the existing fermentation process are still the main raw materials for producing the 2, 3-butanediol. However, the food of the carbon source of the grain inevitably has the problem of competing with people for the grain. China is a big agricultural country, non-grain resources are abundant, and only straw resources are stabilized at about 8 hundred million tons per year every year, which is far greater than the annual corn yield in China. Therefore, the application of straw resources to the production of 2,3 butanediol is a very potential industry development direction and is also the key point of the industry development in the future. The preparation of cellulose sugar by degrading straw raw materials is the first link of high-value utilization of straws. The preparation of cellulose sugar by using straws under the prior art system is realized, but the concentration of the obtained cellulose sugar is generally lower. This increases the difficulty and cost of subsequent separation of the product, making it economically unattractive and difficult to continue to develop the cellulosic sugar industry. The preparation of high-concentration cellulose sugar is the inevitable direction of high-value utilization of straws in the future and is also the premise of industrial production of bio-based chemicals. The existing low concentration of cellulose sugar is mainly caused by low content of cellulose in straw raw materials and overlarge mass transfer resistance in a high-solid enzymolysis system. Firstly, the cellulose content in the straws is only 30-40%, and the solid content of the straw sugar solution with the concentration of more than 100 g/L is required to be more than 30%. And secondly, under a high-solid system, the excessive mass transfer resistance limits the reaction process, so that the enzymolysis efficiency is greatly reduced. The preparation of high concentration cellulosic sugars must therefore overcome both of the above problems. Xylose and furfural are important bio-based chemicals, and the production process mainly processes pentose separated from straws, while other raw materials are mainly treated as waste. Due to the removal of pentose in the straw, the cellulose content in the straw processing by-products reaches a very high level compared with the straw raw material. Meanwhile, the original structure of the straw is damaged by severe treatment conditions in the preparation process, so that the enzymolysis performance of the straw raw material is greatly improved. Therefore, the method has great potential as a preparation raw material of high-concentration cellulose sugar. In addition, water is an important transfer medium in the lignocellulose high-solid enzymolysis process, and the mass transfer medium is continuously reduced along with the increase of the solid content in a reaction system. The viscosity in the system is increased, and the traditional strengthening modes such as stirring and the like are gradually ineffective. The lack of effective mass transfer enhancement means results in mass transfer resistance becoming a major limiting factor for high solids enzymatic hydrolysis. The mass transfer resistance in a high-solid system is overcome by reasonable strengthening measures, and the improvement of the straw enzymolysis efficiency is the key point for realizing the high-solid enzymolysis and is the premise for realizing the lignocellulose biotransformation technology.

CN201110321670.X discloses a method for co-producing furfural, ethanol and lignin from corncobs, wherein the content of solid matters is low in the enzymolysis process, and the concentration of cellulose sugar is below 50 g/L; CN200810141424.4 discloses a method for producing furfural coupled co-production of acetone and butanol by using straws, and the highest concentration of cellulose sugar obtained by performing membrane separation on the product after enzymolysis is only 52.3 g/L; CN201210332435.7 discloses a furfural residue pretreatment efficient saccharification method, and the sugar concentration in the disclosed embodiment is about 50 g/L. The concentration of cellulose sugar obtained by enzymolysis in the method is generally lower, and is only about 50 g/L at most. Lower sugar concentrations mean lower product concentrations which undoubtedly increase the cost of subsequent separations. The existing methods can not realize the production of high-concentration cellulose sugar, mainly because the enzymolysis process is carried out in a low-solid state, and how to overcome the mass transfer resistance in a high-solid system is the key for producing the high-concentration cellulose sugar.

CN202011555799.2 discloses a method for improving the enzymatic hydrolysis and saccharification efficiency of cellulase in a high-solid enzymatic hydrolysis system, and an amino acid type surfactant is used as a mixing agent and added into the enzymatic hydrolysis system to reduce the viscosity of the high-solid system so as to improve the enzymatic hydrolysis efficiency. However, the addition amount is high, which increases the production cost. And the amino acid type surfactant has a certain bacteriostatic effect, and a certain inhibiting effect may exist on the fermentation effect of part of industrial strains, so that the further application of the method is limited. CN201910443752.8 discloses a method and a system for continuous enzymolysis of biomass raw material with high solid content, wherein a large number of machines are combined to form a set of complete production flow applied to high solid system enzymolysis. However, a large number of mechanical devices are used in this system, such as a screw heating conveyor, a two-way material-separating screw system, a mixing conveyor, a pre-enzymolysis screw combination system, an aggregate screw conveyor, an intermediate tank, a screening machine, a temporary storage tank, and the like. This sharply increases the number of unit operations of the entire production flow, greatly increasing the production cost and the production difficulty. CN201910451353.6 discloses a pretreatment method for preparing fermentable sugar by improving the biological enzymolysis conversion rate of fiber raw materials, which is to add a large amount of hydrogen peroxide, sodium hydroxide, sodium silicate, DTPA and other medicines after the raw materials are disintegrated by other mechanical equipment and a disc mill to pretreat the raw materials to obtain a better treatment effect. On one hand, the addition of the medicine possibly influences the utilization of the straw sugar, and on the other hand, the disc mill only serves as a crushing means to pretreat the raw material and cannot promote mass transfer under a high-solid system. How to utilize the disc grinding function to the maximum extent and effectively promote the enzymolysis efficiency of the lignocellulose in a high solid state becomes a problem to be solved urgently.

CN200910012176.8 discloses a clean technology for producing 2, 3-butanediol by using corn straw resources, and CN200910012166.4 provides a method for producing clean fuel by using 2, 3-butanediol by using straw resource solid state fermentation, wherein the fermentation of 2, 3-butanediol is carried out by using straw cellulose raw materials, however, the concentration of enzymolysis sugar is low, which results in the low concentration of 2, 3-butanediol products, and the problems of long fermentation period of 2, 3-butanediol, low product conversion efficiency and the like exist in the second place. In the prior art, the fermentation process of the 2, 3-butanediol only has two modes of liquid fermentation and solid fermentation of a nutrient carrier, and the production efficiency of the 2, 3-butanediol by utilizing the two production modes is low and difficult to improve, and the process is difficult to meet the requirement of the 2, 3-butanediol.

In order to solve the problems, how to provide a method for preparing high-concentration enzymatic hydrolysis sugar and efficiently converting the enzymatic hydrolysis sugar into high-value chemicals is an urgent problem to be solved.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a method for performing solid-state fermentation on 2, 3-butanediol by straw high-solid enzymolysis, which realizes the improvement of the enzymolysis efficiency under a high-solid system, effectively improves the production efficiency of the 2, 3-butanediol and solves the problem of high-valued conversion and utilization of straw resources.

In order to achieve the technical effect, the invention adopts the following technical scheme:

the invention provides a method for straw high-solid enzymolysis solid state fermentation of 2, 3-butanediol, which comprises the following steps:

(1) Mixing the pretreated straw raw material with a buffer solution to obtain a mixture, carrying out disc grinding treatment on the mixture, and adding cellulase for enzymolysis to obtain an enzymolysis material;

(2) Detoxifying the enzymolysis material, preparing a fermentation culture medium, adding an adsorption carrier, and performing solid state fermentation to obtain the 2, 3-butanediol.

In the invention, the disc grinding treatment is a strengthening measure suitable for industrial scale production, the improvement of the straw enzymolysis efficiency is realized by adding a single-stage unit operation, and the possibility that new substances are introduced into an enzymolysis system to interfere enzymolysis and a subsequent production process is avoided. The disc mill does not influence the use of other mass transfer strengthening modes, and can be strengthened by adopting additional strengthening measures in the enzymolysis process, so that the enzymolysis efficiency is promoted. The disc mill is used as an enzymolysis strengthening measure, and the application requirements of various production scales can be met by replacing disc mill equipment with different specifications.

According to the invention, the robustness and the production efficiency of the thallus are effectively improved by adding a detoxification process and solid fermentation of an adsorption carrier, and the inhibition effect of an inhibitor on the thallus is overcome. In addition, solid state fermentation is adopted, so that the separation difficulty of the product is effectively reduced, the energy consumption in the separation process and the consumption of a separation reagent are saved, and the production cost of the whole process is reduced.

As the preferable technical scheme of the invention, the straw raw material comprises straw or straw industrial waste residue.

Preferably, the straw comprises any one of corn stover, rice stover, or wheat straw, or a combination of at least two of these, typical but non-limiting examples of which include: the combination of corn stalks and rice stalks, the combination of rice stalks and wheat stalks, the combination of wheat stalks and corn stalks or the combination of corn stalks, rice stalks and wheat stalks, etc.

Preferably, the straw industrial residue comprises any one of furfural residue, xylose residue or a byproduct generated in a straw processing process or a combination of at least two of the furfural residue, the xylose residue and the byproduct.

In the invention, the high-concentration cellulose sugar is prepared by high-solid enzymolysis of the straw and the straw industrial waste residue, so that the problem of waste generated in the process of processing and preparing xylose, furfural and other products is effectively solved, and the application value of the straw is improved. In addition, the cellulose sugar is prepared by using the straw processing by-products, so that the problem of straw raw material collection is solved, and the problem of high cost in the cellulose sugar production process is solved. The removal of pentose sugars increases the cellulose content of the feedstock during straw processing, so higher cellulose sugar concentrations can be achieved with straw processing by-products than with straw feedstock at the same solids content. The treatment conditions of acidolysis, high temperature, high pressure and the like are commonly used in the straw processing process, and the severe treatment conditions in the straw processing process can be utilized to effectively pretreat the straw raw material. Effectively reduces the recalcitrance of the straw raw material. The pretreatment difficulty before enzymolysis is reduced, and the pretreatment cost in the preparation process of the cellulose sugar is saved.

As a preferred technical solution of the present invention, the pretreatment method in step (1) includes any one common treatment method or a combination of at least two of steam explosion, hydrothermal pretreatment, and acid-base pretreatment, and typical but non-limiting examples of the combination include: a combination of steam explosion and hydrothermal pretreatment, a combination of hydrothermal pretreatment and acid-base pretreatment, a combination of steam explosion and acid-base pretreatment, or a combination of steam explosion, hydrothermal pretreatment and acid-base pretreatment, and the like.

In the present invention, the steam explosion, hydrothermal pretreatment or acid-base pretreatment included in the pretreatment method is similar to the pretreatment method commonly used in the art, and the specific conditions thereof can be appropriately adjusted according to the different pretreatment objects, which is not specifically limited herein.

As a preferred technical scheme of the invention, the buffer solution in the step (1) comprises a citric acid buffer solution.

Preferably, the mass concentration of the buffer solution is 0.01 to 0.1M, such as 0.02M, 0.03M, 0.04M, 0.05M, 0.06M, 0.07M, 0.08M or 0.09M; the pH of the buffer solution is 4.5 to 5.0, such as 4.6, 4.7, 4.8 or 4.9, but the buffer solution is not limited to the values listed, and other values not listed in the above numerical ranges are also applicable.

Preferably, the solid content of the mixture is 15 to 25%, such as 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, or 24%, but is not limited to the recited values, and other unrecited values within the range are also applicable.

In the invention, the specific conditions of the disc grinding treatment can be that the distance between the grinding discs is 0.6-3.0 mm, and the disc grinding times are 3-10.

As a preferred technical scheme of the invention, the dosage of the cellulase in the step (1) is 5 to 20FPU/g DM, such as 6 FPU/g DM, 7 FPU/g DM, 8 FPU/g DM, 9 FPU/g DM, 10 FPU/g DM, 11 FPU/g DM, 12 FPU/g DM, 13 FPU/g DM, 14 FPU/g DM, 15 FPU/g DM, 16 FPU/g DM, 17 FPU/g DM, 18 FPU/g DM or 19 FPU/g DM, and the like, but the dosage is not limited to the enumerated values, and other unrecited values in the numerical range are also applicable.

In the invention, the reaction device for enzymolysis comprises a shaking table, a stirring tank and other devices commonly applied to enzymolysis reaction.

In the present invention, the conditions of the enzymatic hydrolysis, such as temperature and time, can be adjusted according to the selection of the cellulase, and are not specifically limited herein.

As a preferable technical scheme of the invention, the detoxification treatment method in the step (2) comprises activated carbon detoxification and/or air stripping detoxification.

As a preferred embodiment of the present invention, the method for detoxifying activated carbon comprises: and (3) placing the enzymolysis material in a shaking table, adding active carbon, stirring, performing solid-liquid separation, and collecting a liquid phase.

In the invention, partial insoluble substances exist in the straws after enzymolysis and need to be removed, and the concentration of solid matters is too high, so that the subsequent treatment cannot be carried out, and therefore, the solid-liquid separation treatment is needed before the detoxification treatment. The solid-liquid separation method is usually performed by centrifugation, filtration or the like.

Preferably, the temperature of the shaker is from 30 ℃ to 50 ℃, such as 32 ℃, 35 ℃, 38 ℃, 40 ℃, 42 ℃, 45 ℃ or 48 ℃, but is not limited to the recited values, and other values not recited in the range of values are equally applicable.

Preferably, the stirring time is 3 to 12 hours, such as 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours or 11 hours, but the stirring time is not limited to the enumerated values, and other unrecited values in the numerical range are also applicable.

Preferably, the activated carbon is added in an amount of 1 to 10 wt%, such as 2 wt%, 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%, 8 wt%, or 9 wt%, but is not limited to the recited values, and other values not recited in the range of the values are also applicable.

As a preferred technical solution of the present invention, the adsorption carrier of step (2) comprises any one of polyurethane sponge, pulp or zeolite or a combination of at least two of them, and typical but non-limiting examples of the combination include: a combination of polyurethane sponge and pulp, a combination of pulp and zeolite, a combination of zeolite and polyurethane sponge or a combination of polyurethane sponge, pulp and zeolite, and the like.

As a preferable technical scheme of the invention, the solid state fermentation method in the step (2) comprises the step of absorbing inoculated culture medium into an adsorption carrier for fermentation.

In a preferred embodiment of the present invention, the inoculation amount is 1 to 10%, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%, but is not limited to the above-mentioned values, and other values not shown in the above-mentioned range are also applicable.

Compared with the prior art, the invention has at least the following beneficial effects:

(1) The invention provides a method for performing solid state fermentation on 2, 3-butanediol by straw high-solid enzymolysis, which realizes the improvement of the enzymolysis efficiency under a high-solid system, effectively improves the production efficiency of the 2, 3-butanediol and solves the problem of high-valued conversion utilization of straw resources;

(2) The invention provides a method for straw high-solid enzymolysis solid state fermentation of 2, 3-butanediol, which utilizes a cheap carbon source to prepare the 2, 3-butanediol, solves the problem of overhigh cost of the carbon source in the production process and effectively reduces the production cost;

(3) The invention provides a method for straw high-solid enzymolysis solid state fermentation of 2, 3-butanediol, which effectively prolongs the industrial chain of straw processing by using straw raw materials to produce the 2, 3-butanediol and realizes high-valued utilization of straw resources.

Drawings

FIG. 1 is a graph showing the distribution of water in a pond with a corn stover content of 20% solids in example 1 and comparative example 1 according to the present invention;

FIG. 2a is a graph showing the change of glucose concentration during the straw enzymolysis process in example 1 and comparative example 1 of the present invention;

FIG. 2b is a graph showing the change of xylose concentration during the straw enzymolysis process in example 1 and comparative example 1 of the present invention.

The present invention is described in further detail below. The following examples are merely illustrative of the present invention and do not represent or limit the scope of the claims, which are defined by the claims.

Detailed Description

To better illustrate the invention and to facilitate the understanding of the technical solutions thereof, typical but non-limiting examples of the invention are as follows:

example 1

The embodiment provides a method for straw high-solid enzymolysis solid state fermentation of 2, 3-butanediol, which comprises the following steps:

(1) Cleaning impurities such as dust on the surface of corn straw, cutting into 3-5 cm segments, placing in a steam explosion reactor after rehydration, performing steam explosion under the conditions of 0.8 MPa and 10 min, collecting the straw raw material after steam explosion, cleaning with clear water of 3 times of volume, and drying in the air for later use;

(2) Adding a citric acid buffer solution (0.05M and pH 4.8) into the treated straws to adjust the solid content to be 20 percent, fully mixing the materials, putting the mixed materials into a disc mill device for disc milling for 5 times, continuously adding purified water to supplement the water lost due to heat in the disc mill process, adding cellulase (20 FPU/g DM) into the straws after the disc milling is finished, fully mixing the materials and putting the straws into an enzymolysis reactor for enzymolysis;

(3) Placing the straw enzymolysis liquid in a shaking table at 30 ℃, adding 10 percent of active carbon (w/v), stirring for 12 hours, filtering to remove the active carbon, and collecting filtrate. Adjusting the sugar content in the straw enzymatic hydrolysate to 30 g/L, adding 13.33 g/L peptone, 13.33 g/L yeast extract powder, 0.67 g/L sodium acetate and 2 mL/L microelement mother liquor (weighing 0.68 g magnesium sulfate heptahydrate, 0.372 g manganese sulfate monohydrate, 0.3325 g sodium chloride and 0.608 g ferrous sulfate heptahydrate, dissolving in 50 mL distilled water), adjusting the pH value to 6.0, and preparing a corresponding fermentation culture medium; adding polyurethane sponge (with a charging coefficient of 0.8) as an adsorption carrier into a fermentation device, inoculating according to an inoculation amount of 10%, and sufficiently sucking the polyurethane sponge into an inert carrier for fermentation.

Example 2

The embodiment provides a method for performing solid state fermentation on 2, 3-butanediol by straw high-solid enzymolysis, which comprises the following steps:

(1) Preparing an enzymolysis liquid by adopting a residual furfural residue raw material for preparing furfural, and adjusting the pH of the enzymolysis liquid to be about 4.8 by using an alkaline solution;

(2) Adding a citric acid buffer solution (0.05M and pH 4.8) into the treated furfural residues to adjust the solid content to be 20 percent, fully mixing the residues, putting the mixed materials into a disc mill device for disc milling for 5 times, continuously adding purified water to supplement the water lost due to heat in the disc mill process, adding cellulase (20 FPU/g DM) into the straws after the disc milling is finished, fully mixing the materials and putting the mixture into an enzymolysis reactor for enzymolysis;

(3) Placing the straw enzymatic hydrolysate in a shaking table at 30 ℃, adding 10% active carbon (w/v), stirring for 12 hours, performing suction filtration to remove the active carbon, and collecting filtrate; adjusting the sugar content in the straw enzymolysis liquid to 90 g/L, adding 13.33 g/L peptone, 13.33 g/L yeast extract powder, 0.67 g/L sodium acetate and 2 mL/L microelement mother liquor (0.68 g magnesium sulfate heptahydrate, 0.372 g manganese sulfate monohydrate, 0.3325 g sodium chloride and 0.608 g ferrous sulfate heptahydrate are weighed and dissolved in 50 mL distilled water), adjusting the pH value to 6.0, and preparing a corresponding fermentation culture medium. The adsorption carrier polyurethane sponge (with a charging coefficient of 0.8) added to the fermentation device was inoculated at an inoculum size of 10% and sufficiently absorbed into the inert carrier for fermentation.

Example 3

(1) Cleaning impurities such as dust on the surface of rice straws, cutting into small sections of 3-5 cm, placing in a steam explosion reactor after rehydration, performing steam explosion under the conditions of 0.8 MPa and 10 min, collecting the straw raw materials after steam explosion, cleaning with clear water of 3 times of volume, and airing for later use;

(2) Adding a citric acid buffer solution (0.05M, pH 4.8) into the treated straws to adjust the solid content to 25%, fully and uniformly mixing, putting the uniformly mixed materials into a disc mill device for disc milling for 5 times, continuously adding purified water in the disc mill process to supplement moisture lost due to heat, adding cellulase (15 FPU/g DM) into the straws after the disc milling is finished, fully mixing, and putting the straws into an enzymolysis reactor for enzymolysis;

(3) Placing the straw enzymolysis liquid in a shaking table at 50 ℃, adding 10% of activated carbon (w/v), stirring for 3 hours, then carrying out suction filtration to remove the activated carbon, collecting filtrate, adjusting the sugar content in the straw enzymolysis liquid to 30 g/L, adding 13.33 g/L of peptone, 13.33 g/L of yeast extract powder, 0.67 g/L of sodium acetate and 2 mL/L of microelement mother liquid (weighing 0.68 g of magnesium sulfate heptahydrate, 0.372 g of manganese sulfate monohydrate, 0.3325 g of sodium chloride and 0.608 g of ferrous sulfate heptahydrate, dissolving in 50 mL of distilled water), adjusting the pH to 6.0, and preparing a corresponding fermentation culture medium; pulp (with a loading factor of 0.8) as an adsorption carrier was added to the fermentation apparatus and inoculated in an amount of 5% and sufficiently absorbed into an inert carrier for fermentation.

Example 4

(1) Cleaning impurities such as dust on the surface of wheat straw, cutting into 3-5 cm segments, placing in a steam explosion reactor after rehydration, performing steam explosion under the conditions of 0.8 MPa and 10 min, collecting the straw raw material after steam explosion, cleaning with clear water of 3 times of volume, and air-drying for later use;

(2) Adding a citric acid buffer solution (0.05M and pH 4.8) into the treated straws to adjust the solid content to be 15 percent, fully mixing the materials, putting the mixed materials into a disc mill device for disc milling for 5 times, continuously adding purified water to supplement the water lost due to heat in the disc mill process, adding cellulase (5 FPU/g DM) into the straws after the disc milling is finished, fully mixing the materials and putting the straws into an enzymolysis reactor for enzymolysis;

(3) Placing the straw enzymolysis liquid in a shaking table at 40 ℃, adding 1% of activated carbon (w/v), stirring for 12 hours, then carrying out suction filtration to remove the activated carbon, collecting filtrate, adjusting the sugar content in the straw enzymolysis liquid to 30 g/L, adding 13.33 g/L of peptone, 13.33 g/L of yeast extract powder, 0.67 g/L of sodium acetate and 2 mL/L of microelement mother liquid (weighing 0.68 g of magnesium sulfate heptahydrate, 0.372 g of manganese sulfate monohydrate, 0.3325 g of sodium chloride and 0.608 g of ferrous sulfate heptahydrate, dissolving in 50 mL of distilled water), adjusting the pH to 6.0, and preparing a corresponding fermentation culture medium; the fermentation apparatus was inoculated with zeolite (loading factor 0.8) as an adsorption carrier in an amount of 1% and sufficiently adsorbed into an inert carrier to carry out fermentation.

Comparative example 1

The comparative example provides a method for straw high-solid enzymolysis solid state fermentation of 2, 3-butanediol, comprising the steps of:

(1) Cleaning impurities such as dust on the surface of the straw, cutting or crushing into small sections of 3-5 cm, and performing steam explosion pretreatment under the steam explosion condition of 0.8 MPa for 10 min; washing the treated straws by using 5 times of clear water to remove inhibitors generated in the pretreatment process, airing and crushing the washed straws through a 40-mesh screen, adding a citric acid buffer solution (0.05M, PH 4.8) to adjust the solid content to be 20%, adding cellulase (20 FPU/g DM) to fully mix uniformly, and then putting the mixture into an enzymolysis reactor for enzymolysis to prepare a straw enzymolysis solution;

(2) Placing the straw enzymatic hydrolysate in a shaking table at 30 ℃, adding 10% active carbon (w/v), stirring for 12 hours, performing suction filtration to remove the active carbon, and collecting filtrate; adjusting the sugar content in the straw enzymatic hydrolysate to 30 g/L, adding 13.33 g/L peptone, 13.33 g/L yeast extract powder, 0.67 g/L sodium acetate and 2 mL/L microelement mother liquor (weighing 0.68 g magnesium sulfate heptahydrate, 0.372 g manganese sulfate monohydrate, 0.3325 g sodium chloride and 0.608 g ferrous sulfate heptahydrate, dissolving in 50 mL distilled water), adjusting the pH value to 6.0, and preparing a corresponding fermentation culture medium. After sterilization, inoculating bacillus licheniformis seed solution according to the inoculation amount of 10% for fermentation.

Comparative example 2

The comparative example provides a method for straw high-solid enzymolysis solid state fermentation of 2, 3-butanediol, the method comprising:

fermentation of 2, 3-butanediol with glucose: the concentration of sugar in the culture medium is adjusted to 90 g/L, and 2 mL/L of peptone 13.33 g/L, yeast extract powder 13.33 g/L, sodium acetate 0.67 g/L and microelement mother liquor (0.68 g of magnesium sulfate heptahydrate, 0.372 g of manganese sulfate monohydrate, 0.3325 g of sodium chloride and 0.608 g of ferrous sulfate heptahydrate are weighed and dissolved in 50 mL of distilled water) are added. Adjusting the pH value to 6.0, and preparing a corresponding fermentation culture medium. After sterilization, inoculating bacillus licheniformis seed liquid according to the inoculation amount of 10 percent for fermentation.

The glucose concentration and xylose concentration of the enzymatic hydrolysates obtained in examples 1 to 4 and comparative example 1 were measured, and the results are shown in table 1.

The sugar concentration and the product concentration were determined by HPLC. The concrete conditions are as follows: diluting the enzymolysis solution, centrifuging at 8000 rpm for 10 min, collecting supernatant, passing through 0.45 um filter membrane, and measuring glucose and xylose concentration in the enzymolysis solution by HPLC, wherein the chromatographic column is ion chromatographic column (Aminex HPX-87H), the detector is differential Refractometer (RID), the mobile phase is 5 mmol/l sulfuric acid water solution, the flow rate is 0.6 ml/min, and the column temperature is 65 deg.C.

TABLE 1

As can be seen from the test results in Table 1, the comparison between example 1 and comparative example 1 shows that the disc grinding effectively enhances the enzymolysis process of the straws. Not only enhances the enzymolysis speed, but also increases the final enzymolysis rate. The comparative example 1 and the example 2 show that the disc mill can effectively solve the mass transfer problem of the cellulose enzymatic hydrolysis produced by the high-solid enzymatic hydrolysis of the xylose residues, and the concentration of the cellulose is 2-3 times of that of the straw sugar under the same condition.

FIG. 1 is a graph of the change in pond distribution at 20% solids in corn stover for example 1 and comparative example 1, with longer straw relaxation time before the pan grinding process, which shows that the water in the feedstock has a higher degree of freedom and is less bound to the substrate. Although the higher degree of freedom of water is beneficial to enhancing the mobility of water and strengthening the mass transfer effect, the larger relaxation time width indicates that the moisture state is not uniform, even free water appears and is far lower than the water saturation point of the straws under the solid content of 20 percent, which indicates that the distribution of the moisture in the straws is extremely non-uniform, and most of the moisture is in weak contact with the straws, thus the distribution of cellulase is influenced. The water distribution of the straw subjected to disc grinding is extremely concentrated, although the mass transfer difficulty is higher due to the reduction of the degree of freedom, the improvement of the peak height and the distribution of the relaxation time are concentrated, so that the mixing is extremely uniform, and the uniform mixing process of a high-solid system is extremely enhanced in the disc grinding process. The water is mainly in the form of capillary water, and cellulase is distributed in the substrate uniformly, so that the liquefaction time is shortened.

FIGS. 2a and 2b show the sugar concentration change during the straw enzymolysis process in example 1 and comparative example 1, wherein it can be seen that the straw after disc grinding can obtain higher sugar concentration and enzymolysis rate.

The fermentation of 2, 3-butanediol in examples 1 to 4 and comparative examples 1 and 2 was tested, and the results are shown in Table 2.

The substrate and metabolite concentrations in the fermentation broth were determined by HPLC. The specific measurement conditions are as follows: the detector is a differential refraction detector; the chromatographic column is an AminexHpx-87H ion exchange chromatographic column; the column temperature is 65 ℃; the temperature of the detector is 50 ℃; the mobile phase is 5 mmol/L sulfuric acid, and the sample amount is 10 mu L.

TABLE 2

As can be seen from the test results of Table 2, the adsorption carrier significantly improves the fermentation efficiency of 2, 3-butanediol. Particularly, the inhibition effect of the inhibitor in the straw enzymolysis liquid on the fermentation is relieved, and the comparison of example 1 and comparative example 2 shows that the production efficiency of the adsorption carrier fermentation even exceeds the production efficiency of using glucose as a carbon source.

The applicant states that the present invention is described by the above embodiments to explain the detailed structural features of the present invention, but the present invention is not limited to the above detailed structural features, that is, it is not meant to imply that the present invention must be implemented by relying on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims

1. A method for straw high-solid enzymolysis solid state fermentation of 2, 3-butanediol is characterized by comprising the following steps:

(2) Detoxifying the enzymolysis material, preparing a fermentation culture medium, adding an adsorption carrier, and performing solid fermentation to obtain the 2, 3-butanediol.

2. The method of claim 1, wherein the straw feedstock comprises straw or straw industrial residue;

the straws comprise any one or the combination of at least two of corn straws, rice straws or wheat straws;

the straw industrial waste residue comprises any one or combination of at least two of furfural residue, xylose residue or byproducts generated in the straw processing process.

3. The method according to claim 1, wherein the pretreatment method in step (1) comprises any one of the conventional methods of steam explosion, hydrothermal pretreatment and acid-base pretreatment, or a combination of at least two of the conventional methods.

4. The method according to claim 1, wherein the buffer solution of step (1) comprises a citric acid buffer;

the mass concentration of the buffer solution is 0.01-0.1M, and the pH value of the buffer solution is 4.5-5.0;

the solid content of the mixture is 15-25%.

5. The method as claimed in claim 1, wherein the cellulase in step (1) is used in an amount of 5 to 20FPU/g DM.

6. The method of claim 1, wherein the detoxification treatment of step (2) comprises activated carbon detoxification and/or air-assisted detoxification.

7. The method of claim 6, wherein the method of detoxification of activated carbon comprises: placing the enzymolysis material in a shaking table, adding active carbon, stirring, performing solid-liquid separation, and collecting a liquid phase;

the temperature of the shaking table is 30 to 50 ℃;

the stirring time is 3 to 12 hours;

the adding amount of the activated carbon is 1 to 10 wt%.

8. The method of claim 1, wherein the adsorbent carrier of step (2) comprises any one of or a combination of at least two of polyurethane sponge, pulp, or zeolite.

9. The method according to claim 1, wherein the solid state fermentation in step (2) comprises fermenting the inoculated culture medium by imbibing it into an adsorbent carrier.

10. The method of claim 9, wherein the amount of inoculation is 1-10%.