A method for the domestication of bacteria that produce glucose via bamboo cellulase membrane coupling

Abstract

This invention relates to the field of bioengineering technology and discloses a method for the domestication of microbial strains for glucose production via bamboo cellulase membrane coupling. The method includes the following steps: S1, selection of initial microbial strains; S2, preparation of domestication culture medium; S3, gradient domestication; S4, enzyme membrane coupling adaptive domestication; and S5, performance testing and screening. This invention, through gradient domestication and enzyme membrane coupling adaptive domestication, allows the microbial strain to gradually adapt to a high-concentration bamboo powder environment. During gradient domestication, as the bamboo powder concentration gradually increases, the microbial strain continuously adjusts its metabolic mechanism to enhance its ability to produce cellulase in order to survive and grow. After this series of domestications, the degradation efficiency of bamboo cellulose by the microbial strain is greatly improved, enabling the conversion of bamboo cellulose into glucose in a shorter time, thereby significantly increasing the glucose yield, reducing production costs, and effectively solving the yield problem caused by the low degradation efficiency of natural microbial strains.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for the domestication of bacterial strains that produce glucose via bamboo cellulase membrane coupling. Background Technology

[0002] With the continuous growth of global energy demand and the increasing emphasis on renewable energy, the development and utilization of biomass energy has become a research hotspot. Bamboo, as an abundant renewable biomass resource, has advantages such as rapid growth, high yield, and wide distribution. Bamboo is rich in cellulose, and converting bamboo cellulose into fermentable sugars such as glucose can be used to produce biofuels (such as ethanol) or other high-value-added chemicals, which has significant economic and environmental implications.

[0003] Currently, the main method for converting bamboo cellulose into glucose is enzymatic hydrolysis, which utilizes cellulase to hydrolyze bamboo cellulose into glucose. However, existing cellulase-producing bacteria have some shortcomings in processing bamboo cellulose. On the one hand, natural cellulase-producing bacteria have low degradation efficiency for bamboo cellulose, requiring long reaction times and high enzyme dosages, which increases production costs. On the other hand, during enzymatic hydrolysis, the glucose produced inhibits cellulase activity, affecting the continuous enzymatic hydrolysis and resulting in low glucose yield. Therefore, a method for the domestication of bacteria for the membrane-coupled production of glucose from bamboo cellulase is proposed. Summary of the Invention

[0004] The purpose of this invention is to provide a method for the domestication of bacteria for producing glucose via bamboo cellulase membrane coupling, in order to address some shortcomings of existing cellulase-producing bacteria in the treatment of bamboo cellulose as mentioned in the background art. On the one hand, natural cellulase-producing bacteria have low degradation efficiency for bamboo cellulose, requiring a long reaction time and a high enzyme dosage, which increases production costs. On the other hand, during enzymatic hydrolysis, the glucose produced inhibits the activity of cellulase, affecting the continuous enzymatic hydrolysis and resulting in low glucose yield.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for the domestication of bacterial strains for glucose production via bamboo cellulase membrane coupling, comprising the following steps: S1. Selection of initial strains: Screening for initial strains with cellulose degradation capabilities from cellulose-rich environments; S2. Preparation of acclimatization culture medium: Bamboo powder is used as the main carbon source. The particle size of bamboo powder is 80-120 mesh, accounting for 2%-5% of the mass of the culture medium. Add appropriate amounts of nitrogen source, inorganic salts and trace elements, adjust the pH value to 4.5-6.0, and sterilize by autoclaving for later use. S3. Gradual acclimatization: The initial strain is inoculated into an acclimatization medium containing a low concentration of bamboo powder, and the concentration of bamboo powder is gradually increased for acclimatization. S4. Enzyme membrane coupling adaptive domestication: Construct an enzyme membrane coupling reaction device, inoculate the gradient-acclimated strains into an acclimation culture medium containing 5% bamboo powder, place them in the enzyme membrane coupling reaction device for reaction, and separate glucose in real time through the membrane separation component. S5. Performance Testing and Screening: After the reaction, cellulase activity and glucose content are tested, and strains with high cellulase activity and high glucose yield are screened out.

[0006] Preferably, in S1, the initial strain is any one of fungi such as Trichoderma, Aspergillus, or Penicillium, or bacteria such as Bacillus or Pseudomonas.

[0007] As a preferred option, in S1, during the initial strain selection process, strains with cellulose degradation capabilities are further subjected to molecular biological identification, and the accurate taxonomic position of the strains is determined by using 16S rDNA or ITS sequence analysis.

[0008] As a preferred option, in S2, the bamboo powder needs to be pretreated by first drying it in an oven at 60-80℃ for 2-4 hours, and then pulverizing it using a ball mill. The nitrogen source can be one or a mixture of sodium nitrate and ammonium sulfate. The amount of nitrogen source added is 0.1%-0.5% of the mass of the culture medium. If a mixed nitrogen source is used, the mass ratio of sodium nitrate to ammonium sulfate is 1:1-3:1. The inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, and calcium chloride. The amount of potassium dihydrogen phosphate added is 0.02%-0.08% of the mass of the culture medium, the amount of magnesium sulfate added is 0.01%-0.05%, the amount of calcium chloride added is 0.005%-0.02%, and the total amount of inorganic salts added is 0.05%-0.2% of the mass of the culture medium. Small amounts of trace elements, including any one of iron, zinc, and manganese, are added. Iron is added in the form of ferrous sulfate at a rate of 0.001%-0.005%, zinc in the form of zinc sulfate at a rate of 0.0005%-0.002%, and manganese in the form of manganese sulfate at a rate of 0.0005%-0.002%, to meet the needs of the strain for growth. Adjust the pH of the culture medium using either a 1 mol / L sodium hydroxide solution or a 1 mol / L hydrochloric acid solution. After adding water to the required volume, pour the solution into a suitable container and sterilize at 121°C for 20-30 minutes.

[0009] Preferably, in S2, a small amount of surfactant can be added to the culture medium during the preparation of the acclimatization culture medium, with an addition amount of 0.01%-0.05%.

[0010] As a preferred option, in S3, the initial bamboo powder concentration is 1%, the inoculation amount is 3%-8%, and the inoculated culture medium is placed in a constant temperature shaking incubator and shaken for 2-3 days at a temperature of 25-35℃ and a rotation speed of 150-200r / min. During the culture process, samples are taken every 12-24 hours, and the cellulose degradation of the strain is observed by the Congo red staining method. Once the bacterial strain has stabilized, observe it under a microscope to ensure that the strain is morphologically normal and growing vigorously. Also, determine the cellulase activity in the culture medium using the DNS method, which shows an enzyme activity ≥5 U / mL. Then, transfer the bacterial solution to an acclimatization medium with a bamboo powder concentration of 2% at an inoculation rate of 5%-10%, and continue to culture it under the same conditions for 2-3 days. Repeat the above steps, gradually increasing the concentration of bamboo powder in the acclimatization culture medium by 1% each time, until the bamboo powder concentration reaches 5%.

[0011] As a preferred option, in S3, during the gradient acclimatization process, a small amount of lignin degradation products, such as vanillin or syringic acid, can be added to the acclimatization culture medium at a concentration of 0.001%-0.005%, so that the strain can gradually adapt to the bamboo powder environment containing lignin.

[0012] Preferably, in S4, the enzyme membrane coupling reaction device includes a reactor and a membrane separation component. The reactor is a closed container with a stirring device and a temperature control system. The membrane separation component uses an ultrafiltration membrane with a molecular weight cutoff of 10,000-50,000 Da. The ultrafiltration membrane is made of any one of polysulfone, polyethersulfone, or polyvinylidene fluoride. The reaction system was placed in the enzyme membrane coupling reaction device, and the stirring device was turned on to ensure that the reaction system was fully mixed. The stirring speed was 100-150 r / min, and the reaction temperature was controlled at 30-35℃ through the temperature control system. This temperature range is conducive to the growth of the strain and the expression of enzyme activity. During the reaction, glucose separation was performed using cross-flow filtration, with the transmembrane pressure difference controlled at 0.05-0.2 MPa, maintaining the glucose concentration in the reaction system at <10 g / L. Samples were taken every 2-4 hours to determine the glucose concentration and cellulase activity in the reaction system.

[0013] Preferably, in S4, during the enzyme membrane coupling adaptation process, a small amount of antifouling agent, such as polyethylene glycol, can be added to the reaction system at an addition amount of 0.005%-0.02%. The antifouling agent can form a protective film on the surface of the ultrafiltration membrane.

[0014] As a preferred option, in S5, after the reaction is completed, the reaction solution is taken for cellulase activity detection. The activity of cellulase is determined by the DNS method. The standard for high cellulase activity is enzyme activity ≥15U / mL. The glucose content in the reaction solution was determined by high performance liquid chromatography. The standard for high glucose yield was glucose yield ≥ 0.3 g / g bamboo powder. The culture is carried out in a fermenter with a liquid volume of 60%-70%, a culture temperature of 30-35℃, a stirring speed of 150-200 r / min, a ventilation rate to culture medium volume ratio of 0.5:1-1:1, and a culture time of 3-5 days.

[0015] Compared with the prior art, the present invention, employing the above technical solution, has the following technical effects: 1. This invention allows the microorganisms to gradually adapt to a high-concentration bamboo powder environment through gradient domestication and enzyme membrane coupling adaptive domestication. During the gradient domestication process, as the bamboo powder concentration gradually increases, the microorganisms will continuously adjust their metabolic mechanisms to enhance their ability to produce cellulase in order to survive and grow. After this series of domestications, the degradation efficiency of bamboo cellulose by the microorganisms is greatly improved, and they can convert bamboo cellulose into glucose in a shorter time, thereby significantly increasing the glucose yield, reducing production costs, and effectively solving the yield problem caused by the low degradation efficiency of natural microorganisms.

[0016] 2. This invention employs a specialized enzyme membrane coupling adaptive acclimatization method, enabling the strain to grow stably and produce enzymes in the enzyme membrane coupling reaction device. The membrane separation component in the enzyme membrane coupling reaction device can separate the produced glucose in real time, keeping the glucose concentration in the reaction system at a consistently low level. This significantly reduces the inhibitory effect of the product glucose on the strain and enzyme, allowing the enzymatic hydrolysis reaction to proceed continuously and stably, improving the continuity and stability of the enzymatic hydrolysis reaction, and solving the key problem of product inhibition. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the method of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 Please see Figure 1 This invention provides a technical solution: a method for the domestication of bacterial strains for glucose production via bamboo cellulase membrane coupling, comprising the following steps: S1. Selection of initial strains: Initial strains with cellulose degradation capabilities were screened from cellulose-rich environments. The initial strains were Trichoderma fungi. During the initial strain selection process, the strains with cellulose degradation capabilities were further identified by molecular biology. The ITS sequence analysis method was used to determine the accurate taxonomic position of the strains.

[0021] S2. Preparation of acclimatization culture medium: Bamboo powder is used as the main carbon source. The bamboo powder particle size is 100 mesh and accounts for 3% of the culture medium mass. Add appropriate amounts of nitrogen source, inorganic salt and trace elements, adjust the pH value to 5, and sterilize by high pressure steam for later use. Bamboo powder needs to be pretreated. First, it is dried in an oven at 70℃ for 3 hours, and then pulverized using a ball mill. The nitrogen source is one or a mixture of sodium nitrate and ammonium sulfate, with the amount of nitrogen source added being 0.3% of the mass of the culture medium. If a mixed nitrogen source is used, the mass ratio of sodium nitrate to ammonium sulfate is 2:1. Inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, and calcium chloride. The amount of potassium dihydrogen phosphate added is 0.05% of the mass of the culture medium, the amount of magnesium sulfate added is 0.03%, and the amount of calcium chloride added is 0.01%. The total amount of inorganic salts added is 0.09% of the mass of the culture medium. A small amount of trace element is iron, which is added in the form of ferrous sulfate at a rate of 0.003% to meet the needs of bacterial growth. The pH of the culture medium is adjusted with 1 mol / L hydrochloric acid solution, and after adding water to the required volume, it is poured into a suitable container and sterilized at 121℃ for 25 minutes. During the preparation of the acclimatization culture medium, a small amount of surfactant can be added to the culture medium, with an addition amount of 0.03%, to improve the utilization rate of bamboo powder. The surfactant can reduce the surface tension of bamboo powder and promote the contact and degradation of bamboo powder by the strain.

[0022] S3. Gradual acclimatization: The initial strain is inoculated into an acclimatization medium containing a low concentration of bamboo powder, and the concentration of bamboo powder is gradually increased for acclimatization. The initial bamboo powder concentration was 1%, and the inoculum size was 5%. The inoculated culture medium was placed in a constant temperature shaking incubator and shaken for 2.5 days at 30℃ and 180 r / min. During the culture, samples were taken every 18 hours, and the cellulose degradation of the strain was observed using the Congo red staining method. After the strain growth stabilized, the strain was observed under a microscope to ensure normal morphology and vigorous growth. The cellulase activity in the culture medium was measured using the DNS method, and the enzyme activity was ≥5 U / mL. The bacterial culture was then transferred to an acclimatization culture medium with a bamboo powder concentration of 2% at a 7% inoculum size, and cultured under the same conditions for another 2.5 days. The above steps were repeated, gradually increasing the bamboo powder concentration in the acclimatization culture medium by 1% each time, until the bamboo powder concentration reached 5%. After each increase in bamboo powder concentration, the adaptation of the strain was observed. If slow growth or decreased cellulase activity occurred, the increase rate could be appropriately reduced or the culture time extended. In order to improve the strain's tolerance to lignin in bamboo powder during the gradient acclimatization process, a small amount of lignin degradation products, such as vanillin, can be added to the acclimatization culture medium at a concentration of 0.003%, so that the strain can gradually adapt to the bamboo powder environment containing lignin.

[0023] S4. Enzyme membrane coupling adaptive domestication: Construct an enzyme membrane coupling reaction device, inoculate the gradient-acclimated strains into an acclimation culture medium containing 5% bamboo powder, place them in the enzyme membrane coupling reaction device for reaction, and separate glucose in real time through the membrane separation component. The enzyme membrane coupling reaction device includes a reactor and a membrane separation component. The reactor is a sealed container equipped with a stirring device and a temperature control system to ensure uniform temperature in the reaction system. The membrane separation component uses an ultrafiltration membrane with a molecular weight cutoff of 30,000 Da. The ultrafiltration membrane is made of polyethersulfone, which has good chemical stability and antifouling properties. The reaction system is placed in the enzyme membrane coupling reaction device, and the stirring device is turned on to ensure thorough mixing. The stirring speed is 120 r / min, and the reaction temperature is controlled at 32℃ by the temperature control system. This temperature range is conducive to the growth of the strain and the expression of enzyme activity. During the reaction, glucose separation is performed using cross-flow filtration, controlling the transmembrane pressure difference at 0.1 MPa to maintain the glucose concentration in the reaction system <10 g / L, in order to reduce the inhibitory effect of the product on the strain and enzyme. Samples are taken every 3 hours to measure the glucose concentration and cellulase activity in the reaction system. The reaction is continued for 6 days to allow the strain to gradually adapt to the enzyme membrane coupling process environment. During the reaction, appropriate nutrients are periodically added to the reaction system to maintain the growth of the strain and enzyme production. In the process of enzyme membrane coupling adaptation, in order to prevent the ultrafiltration membrane from being contaminated and clogged, a small amount of antifouling agent, such as polyethylene glycol, can be added to the reaction system at a concentration of 0.01%. The antifouling agent can form a protective film on the surface of the ultrafiltration membrane, reducing the adsorption of impurities on the membrane surface.

[0024] S5. Performance testing and screening: After the reaction, cellulase activity and glucose content are tested, and strains with high cellulase activity and high glucose yield are screened out. After the reaction was completed, the reaction solution was taken for cellulase activity detection. The activity of cellulase was determined by the DNS method. The standard for high cellulase activity was ≥15 U / mL. The specific operation was as follows: Take an appropriate amount of reaction solution and add it to the reaction system containing 1% sodium carboxymethyl cellulose solution. React at a certain temperature of 50℃ for a certain time of 30 minutes. Then add DNS reagent, boil and develop color, and measure the absorbance at a wavelength of 540 nm. Calculate the cellulase activity according to the standard curve. The enzyme activity of the strain screened in this example was 16.2 U / mL. The glucose content in the reaction solution was determined by high performance liquid chromatography (HPLC). The standard for high glucose yield was glucose yield ≥ 0.3 g / g bamboo powder. The HPLC instrument was equipped with an amino column, the mobile phase was an acetonitrile-water mixture with a volume ratio of 75:25, the flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detector was a differential refractive index detector. In this example, the glucose yield was 0.34 g / g bamboo powder. Select strains with high cellulase activity and high glucose yield for further expansion and preservation. Fermentation was conducted in a fermenter with a liquid volume of 65%, a culture temperature of 32℃, a stirring speed of 180 r / min, and an aeration-to-culture-volume ratio of 1:0.8. The culture time was 4 days. During the culture process, the culture conditions were adjusted as needed based on the growth of the strain and the generation of metabolites. Qualified strains were then preserved.

[0025] Example 2 The difference from Example 1 is as follows: S1, Selection of initial bacterial strain: Initial bacterial strains with cellulose degradation ability are screened from the humus environment of the bamboo garden. In this example, Bacillus subtilis is selected, which belongs to the genus Bacillus. The molecular biological identification is performed by 16S rDNA analysis to determine the accurate taxonomic position of the bacterial strain.

[0026] S2. Preparation of the acclimatization culture medium: Bamboo powder, with a particle size of 80 mesh, was used as the main carbon source, accounting for 2% of the culture medium mass. The bamboo powder was first dried in an oven at 60℃ for 4 hours, and then pulverized using a ball mill. Sodium nitrate was used as the nitrogen source, with an addition amount of 0.1% of the culture medium mass. Inorganic salts included potassium dihydrogen phosphate, magnesium sulfate, and calcium chloride, with potassium dihydrogen phosphate added at 0.03% of the culture medium mass, magnesium sulfate at 0.015%, and calcium chloride at 0.005%, for a total inorganic salt addition of 0.05%. Zinc in the form of zinc sulfate was used as the trace element, with an addition amount of 0.001%. 0.01% surfactant was added to improve the utilization rate of bamboo powder. The pH of the culture medium was adjusted to 4.5 using a 1 mol / L hydrochloric acid solution, and after adding water to make up the volume, it was poured into containers and sterilized at 121℃ for 20 minutes before use.

[0027] S3. Gradual Acclimation: The initial strain was inoculated into an acclimation medium with a bamboo powder concentration of 1%, at a rate of 3%. The inoculated medium was placed in a constant temperature shaking incubator and cultured for 3 days at 25℃ and 150 rpm. Samples were taken every 12 hours during the culture, and the cellulose degradation of the strain was observed using the Congo red staining method. Once the strain growth was stable, and the strain morphology was normal and the growth was vigorous under a microscope, and the cellulase activity in the culture medium was ≥5 U / mL using the DNS method, the strain was transferred to an acclimation medium with a bamboo powder concentration of 2% at a rate of 5%, and cultured for another 3 days under the same conditions. The above steps were repeated, increasing the bamboo powder concentration by 1% each time, until the bamboo powder concentration reached 5%. During the acclimation process, 0.001% syringic acid was added to improve the strain's tolerance to lignin in the bamboo powder. If slow growth or decreased enzyme activity occurred, the concentration increase was appropriately reduced to 0.5%.

[0028] S4. Enzyme Membrane Coupling Adaptation and Acclimation: An enzyme membrane coupling reaction apparatus was constructed, comprising a closed reactor with a stirring device and a temperature control system, and a polysulfone ultrafiltration membrane separation component with a molecular weight cutoff of 10000 Da. Gradient-acclimated bacterial strains were inoculated into an acclimation medium containing 5% bamboo powder and placed in the reaction apparatus. The stirring device was turned on at a speed of 100 r / min, and the reaction temperature was controlled at 30℃ using the temperature control system. During the reaction, cross-flow filtration was used to separate glucose, controlling the transmembrane pressure difference at 0.05 MPa to maintain a glucose concentration <10 g / L in the reaction system, reducing product inhibition. Samples were taken every 2 hours to measure glucose concentration and cellulase activity. 0.005% polyethylene glycol was added as an antifouling agent during the reaction to reduce impurity adsorption on the ultrafiltration membrane surface. The reaction was continued for 5 days, with nutrients periodically replenished to maintain bacterial growth and enzyme production, allowing the strains to gradually adapt to the enzyme membrane coupling process environment.

[0029] S5. Performance Testing and Screening: After the reaction, cellulase activity was determined using the DNS method. The procedure was as follows: An appropriate amount of reaction solution was added to a reaction system containing 1% sodium carboxymethyl cellulose solution. The reaction was carried out at 50°C for 30 minutes. After adding DNS reagent and boiling for color development, the absorbance was measured at a wavelength of 540 nm. The enzyme activity was calculated based on the standard curve. The enzyme activity of the strain screened in this example was 15.7 U / mL, which meets the high activity standard of ≥15 U / mL. Glucose content was determined using high-performance liquid chromatography (HPLC). An amino column was used, the mobile phase was a 75:25 volume ratio acetonitrile-water mixture, the flow rate was 1.0 mL / min, the column temperature was 30°C, and a differential refractive index detector was used for detection. The glucose yield in this example was 0.31 g / g bamboo powder, which meets the high yield standard of ≥0.3 g / g bamboo powder. The selected strains were expanded for culture. The fermentation tank was filled with 60% liquid, the culture temperature was 30℃, the stirring speed was 150r / min, the ratio of aeration rate per minute to culture medium volume was 1:0.5, and the culture time was 3 days. During the culture process, the culture conditions were adjusted in a timely manner according to the growth and metabolism of the strains. Qualified strains were preserved.

[0030] Example 3 Unlike Examples 1 and 2, in S1, the initial strain was selected by screening initial strains with cellulose degradation capabilities from the bamboo product processing waste accumulation environment. In this example, Aspergillus niger was selected, which belongs to the Aspergillus genus. The molecular biological identification was performed using the ITS sequence analysis method to determine the accurate taxonomic position of the strain.

[0031] S2. Preparation of the acclimatization culture medium: Bamboo powder, with a particle size of 120 mesh, was used as the main carbon source, accounting for 5% of the culture medium mass. The bamboo powder was first dried in an oven at 80℃ for 2 hours, and then pulverized using a ball mill. Ammonium sulfate was used as the nitrogen source, with an addition amount of 0.5% of the culture medium mass. Inorganic salts included potassium dihydrogen phosphate, magnesium sulfate, and calcium chloride, with potassium dihydrogen phosphate added at 0.08% of the culture medium mass, magnesium sulfate at 0.05%, and calcium chloride at 0.02%, for a total inorganic salt addition of 0.15%. Manganese in the form of manganese sulfate was used as the trace element, with an addition amount of 0.002%. 0.05% surfactant was added to improve the utilization rate of bamboo powder. The pH of the culture medium was adjusted to 6.0 using a 1 mol / L sodium hydroxide solution, and after adding water to make up the volume, it was poured into containers and sterilized at 121℃ for 30 minutes before use.

[0032] S3. Gradual Acclimation: The initial strain was inoculated into an acclimation medium with a bamboo powder concentration of 1%, at an inoculation rate of 8%. The inoculated medium was placed in a constant temperature shaking incubator and cultured for 2 days at 35℃ and a shaking speed of 200 rpm. Samples were taken every 24 hours during the culture, and the cellulose degradation of the strain was observed using the Congo red staining method. Once the strain growth was stable, and the strain morphology was normal and the growth was vigorous under a microscope, and the cellulase activity in the culture medium was ≥5 U / mL using the DNS method, the strain was transferred to an acclimation medium with a bamboo powder concentration of 2% at an inoculation rate of 10%, and cultured for another 2 days under the same conditions. The above steps were repeated, increasing the bamboo powder concentration by 1% each time, until the bamboo powder concentration reached 5%. During the acclimation process, 0.005% vanillin was added to improve the strain's tolerance to lignin in the bamboo powder. If slow growth or decreased enzyme activity occurred, the culture time was appropriately extended until the strain adapted.

[0033] S4. Enzyme Membrane Coupling Adaptation and Acclimation: An enzyme membrane coupling reaction apparatus was constructed, comprising a closed reactor with a stirring device and a temperature control system, and a polyvinylidene fluoride (PVDF) ultrafiltration membrane separation component with a molecular weight cutoff of 50,000 Da. Gradient-acclimated bacterial strains were inoculated into an acclimation medium containing 5% bamboo powder and placed in the reaction apparatus. The stirring device was activated at a speed of 150 rpm, and the reaction temperature was controlled at 35°C using the temperature control system. During the reaction, cross-flow filtration was used to separate glucose, controlling the transmembrane pressure difference at 0.2 MPa and maintaining a glucose concentration <10 g / L in the reaction system to reduce product inhibition. Samples were taken every 4 hours to measure glucose concentration and cellulase activity. 0.02% polyethylene glycol was added as an antifouling agent during the reaction to reduce impurity adsorption on the ultrafiltration membrane surface. The reaction was continued for 7 days, with nutrients periodically replenished to maintain bacterial growth and enzyme production, allowing the strains to gradually adapt to the enzyme membrane coupling process environment.

[0034] S5. Performance Testing and Screening: After the reaction, cellulase activity was determined using the DNS method. The procedure was as follows: An appropriate amount of reaction solution was added to a reaction system containing 1% sodium carboxymethyl cellulose solution. The reaction was carried out at 50°C for 30 minutes. After adding DNS reagent and boiling for color development, the absorbance was measured at a wavelength of 540 nm. The enzyme activity was calculated based on the standard curve. The enzyme activity of the strain screened in this example was 17.4 U / mL, which meets the high activity standard of ≥15 U / mL. Glucose content was determined using high-performance liquid chromatography (HPLC). An amino column was used, the mobile phase was a 75:25 volume ratio acetonitrile-water mixture, the flow rate was 1.0 mL / min, the column temperature was 30°C, and a differential refractive index detector was used for detection. The glucose yield in this example was 0.38 g / g bamboo powder, which meets the high yield standard of ≥0.3 g / g bamboo powder. The selected strains were expanded for culture. The fermentation tank was filled with 70% liquid, the culture temperature was 35℃, the stirring speed was 200r / min, the ratio of aeration rate per minute to culture medium volume was 1:1, and the culture time was 5 days. During the culture process, the culture conditions were adjusted in a timely manner according to the growth and metabolism of the strains. Qualified strains were preserved.

[0035] In summary, this invention enables the microorganisms to gradually adapt to a high-concentration bamboo powder environment through gradient domestication and enzyme membrane coupling adaptive domestication. During the gradient domestication process, as the bamboo powder concentration gradually increases, the microorganisms continuously adjust their metabolic mechanisms to enhance their ability to produce cellulase in order to survive and grow. After this series of domestications, the degradation efficiency of bamboo cellulose by the microorganisms is greatly improved, and they can convert bamboo cellulose into glucose in a shorter time, thereby significantly increasing the glucose yield, reducing production costs, and effectively solving the yield problem caused by the low degradation efficiency of natural microorganisms. Furthermore, a specialized enzyme-membrane coupling adaptive acclimatization method is employed, enabling the strain to grow stably and produce enzymes in the enzyme-membrane coupling reaction device. The membrane separation component in the enzyme-membrane coupling reaction device can separate the produced glucose in real time, keeping the glucose concentration in the reaction system at a consistently low level. This significantly reduces the inhibitory effect of the product glucose on the strain and enzyme, allowing the enzymatic hydrolysis reaction to proceed continuously and stably, improving the continuity and stability of the enzymatic hydrolysis reaction, and solving the key problem of product inhibition.

[0036] Those skilled in the art will understand that the features described in the various embodiments and / or claims of the present invention can be combined or combined in various ways, even if such combinations or combinations are not explicitly described in the present invention. In particular, the features described in the various embodiments and / or claims of the present invention can be combined or combined in various ways without departing from the spirit and teachings of the present invention. All such combinations and / or combinations fall within the scope of the present invention.

Claims

1. A method for the domestication of a bacterial strain for glucose production via bamboo cellulase membrane coupling, characterized in that, Includes the following steps: S1. Selection of initial strains: Screening for initial strains with cellulose degradation capabilities from cellulose-rich environments; S2. Preparation of acclimatization culture medium: Bamboo powder is used as the main carbon source. The particle size of bamboo powder is 80-120 mesh, accounting for 2%-5% of the mass of the culture medium. Add appropriate amounts of nitrogen source, inorganic salts and trace elements, adjust the pH value to 4.5-6.0, and sterilize by autoclaving for later use. S3. Gradual acclimatization: The initial strain is inoculated into an acclimatization medium containing a low concentration of bamboo powder, and the concentration of bamboo powder is gradually increased for acclimatization. S4. Enzyme membrane coupling adaptive domestication: Construct an enzyme membrane coupling reaction device, inoculate the gradient-acclimated strains into an acclimation culture medium containing 5% bamboo powder, place them in the enzyme membrane coupling reaction device for reaction, and separate glucose in real time through the membrane separation component. S5. Performance Testing and Screening: After the reaction, cellulase activity and glucose content are tested, and strains with high cellulase activity and high glucose yield are screened out.

2. The method for acclimatizing a bamboo cellulase membrane-coupled glucose production strain according to claim 1, characterized in that, In S1, the initial strain is any one of the fungi such as Trichoderma, Aspergillus, or Penicillium, or any one of the bacteria such as Bacillus or Pseudomonas.

3. The method for acclimatizing a bamboo cellulase membrane-coupled glucose production strain according to claim 1, characterized in that, In S1, during the initial strain selection process, strains with cellulose degradation capabilities are further identified through molecular biological identification, using 16S rDNA or ITS sequence analysis to determine their accurate taxonomic position.

4. The method for acclimatizing a bamboo cellulase membrane-coupled glucose production strain according to claim 1, characterized in that, In S2, bamboo powder needs to be pre-treated by drying it in an oven at 60-80℃ for 2-4 hours, and then pulverizing it using a ball mill. The nitrogen source can be one or a mixture of sodium nitrate and ammonium sulfate. The amount of nitrogen source added is 0.1%-0.5% of the mass of the culture medium. If a mixed nitrogen source is used, the mass ratio of sodium nitrate to ammonium sulfate is 1:1-3:

1. The inorganic salts include potassium dihydrogen phosphate, magnesium sulfate, and calcium chloride. The amount of potassium dihydrogen phosphate added is 0.02%-0.08% of the mass of the culture medium, the amount of magnesium sulfate added is 0.01%-0.05%, the amount of calcium chloride added is 0.005%-0.02%, and the total amount of inorganic salts added is 0.05%-0.2% of the mass of the culture medium. Small amounts of trace elements, including any one of iron, zinc, and manganese, are added. Iron is added in the form of ferrous sulfate at a rate of 0.001%-0.005%, zinc in the form of zinc sulfate at a rate of 0.0005%-0.002%, and manganese in the form of manganese sulfate at a rate of 0.0005%-0.002%, to meet the needs of the strain for growth. Adjust the pH of the culture medium using either a 1 mol / L sodium hydroxide solution or a 1 mol / L hydrochloric acid solution. After adding water to the required volume, pour the solution into a suitable container and sterilize at 121°C for 20-30 minutes.

5. The method for acclimatizing a bamboo cellulase membrane-coupled glucose production strain according to claim 1, characterized in that, In S2, a small amount of surfactant can be added to the culture medium during the preparation of the acclimatization medium, with an addition amount of 0.01%-0.05%.

6. The method for acclimatizing a bamboo cellulase membrane-coupled glucose production strain according to claim 1, characterized in that, In S3, the initial bamboo powder concentration was 1%, and the inoculation amount was 3%-8%. The inoculated culture medium was placed in a constant temperature shaking incubator and shaken for 2-3 days at a temperature of 25-35℃ and a rotation speed of 150-200r / min. During the culture process, samples were taken every 12-24 hours, and the cellulose degradation of the strain was observed using the Congo red staining method. Once the bacterial strain has stabilized, observe it under a microscope to ensure that the strain is morphologically normal and growing vigorously. Also, determine the cellulase activity in the culture medium using the DNS method, which shows an enzyme activity ≥5 U / mL. Then, transfer the bacterial solution to an acclimatization medium with a bamboo powder concentration of 2% at an inoculation rate of 5%-10%, and continue to culture it under the same conditions for 2-3 days. Repeat the above steps, gradually increasing the concentration of bamboo powder in the acclimatization culture medium by 1% each time, until the bamboo powder concentration reaches 5%.

7. The method for acclimatizing a bamboo cellulase membrane-coupled glucose production strain according to claim 1, characterized in that, In S3, during the gradient acclimatization process, a small amount of lignin degradation products, such as vanillin or syringic acid, can be added to the acclimatization medium at a rate of 0.001%-0.005%, so that the strain can gradually adapt to the bamboo powder environment containing lignin.

8. The method for acclimatizing a bamboo cellulase membrane-coupled glucose production strain according to claim 1, characterized in that, In S4, the enzyme membrane coupling reaction device includes a reactor and a membrane separation component. The reactor is a closed container with a stirring device and a temperature control system. The membrane separation component uses an ultrafiltration membrane with a molecular weight cutoff of 10,000-50,000 Da. The ultrafiltration membrane is made of any one of polysulfone, polyethersulfone, or polyvinylidene fluoride. The reaction system was placed in the enzyme membrane coupling reaction device, and the stirring device was turned on to ensure that the reaction system was fully mixed. The stirring speed was 100-150 r / min, and the reaction temperature was controlled at 30-35℃ through the temperature control system. This temperature range is conducive to the growth of the strain and the expression of enzyme activity. During the reaction, glucose separation was performed using cross-flow filtration, with the transmembrane pressure difference controlled at 0.05-0.2 MPa, maintaining the glucose concentration in the reaction system at <10 g / L. Samples were taken every 2-4 hours to determine the glucose concentration and cellulase activity in the reaction system.

9. The method for acclimatizing a bamboo cellulase membrane-coupled glucose production strain according to claim 1, characterized in that, In S4, during the enzyme membrane coupling adaptation process, a small amount of antifouling agent, such as polyethylene glycol, can be added to the reaction system at a concentration of 0.005%-0.02%. The antifouling agent can form a protective film on the surface of the ultrafiltration membrane.

10. The method for acclimatizing a bamboo cellulase membrane-coupled glucose production strain according to claim 1, characterized in that, In S5, after the reaction is completed, the reaction solution is taken for cellulase activity detection. The activity of cellulase is determined by the DNS method. The standard for high cellulase activity is enzyme activity ≥15U / mL. The glucose content in the reaction solution was determined by high performance liquid chromatography. The standard for high glucose yield was glucose yield ≥ 0.3 g / g bamboo powder. The culture is carried out in a fermenter with a liquid volume of 60%-70%, a culture temperature of 30-35℃, a stirring speed of 150-200 r / min, a ventilation rate to culture medium volume ratio of 0.5:1-1:1, and a culture time of 3-5 days.

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