CN103484446A - Chemically modified fiber immobilized carrier and preparation method and application thereof - Google Patents
Chemically modified fiber immobilized carrier and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to a chemically modified fiber immobilized carrier, a preparation method and application thereof. The invention provides a method for preparing a chemically modified fiber immobilized carrier, which comprises the following steps: (1) soaking a fiber-based immobilized carrier in a polyethyleneimine solution so as to obtain a polyethyleneimine-modified carrier, wherein polyethyleneimine molecules are formed on the surface of the polyethyleneimine-modified carrier; and (2) soaking the polyethyleneimine modified carrier in a glutaraldehyde solution so as to react amino groups in polyethyleneimine molecules with aldehyde groups of glutaraldehyde, thereby obtaining the chemically modified fiber-based immobilized carrier. The fiber immobilized carrier obtained by the embodiment of the invention can be used as a cell immobilized carrier to be filled into an immobilized fiber bed and applied to single-batch fermentation and repeated-batch fermentation of biological butanol.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a chemically modified fiber immobilized carrier, a preparation method and application thereof, and more particularly relates to a method for preparing the chemically modified fiber immobilized carrier, equipment for preparing butanol and a method for preparing butanol by using the equipment.
Background
Under the influence of world petroleum resources, price, environmental protection and global climate change, the development of biofuel has become an important measure for improving energy safety, reducing emission of greenhouse gases and coping with climate change in many countries. The biological butanol is mainly generated by clostridium through anaerobic fermentation, the method can effectively relieve two outstanding problems of energy shortage and environmental pollution, and the biological butanol serving as a novel liquid fuel has the advantages of high energy density, low corrosivity, direct application to an internal combustion engine, convenient transportation and the like. Biobutanol, like ethanol, can be blended with gasoline, but has many advantages over ethanol, for example, butanol has a higher energy density, similar to gasoline, whereas ethanol has an energy density 35% lower than gasoline; because of its hydrocarbon-like structure, butanol has much less solubility for water than ethanol and can be blended at refineries and piped, while ethanol must be blended at distribution terminals; biobutanol may utilize the original infrastructure of the refinery, whereas ethanol does not. Therefore, research and development of biobutanol are increasingly receiving attention from many countries.
Butanol, also known as n-butanol, is widely regarded as a good chemical raw material and a novel liquid fuel, can be used as paint and surface coating, can also be used for producing plasticizers such as dibutyl phthalate (DBP) and Butyl Benzyl Phthalate (BBP), can also be used for producing butyl acetate, butyl methacrylate, butyl acrylate and glycol ether, and can be used in the aspects of surface coating, leather treatment, spice, rubber processing aid, agricultural chemicals and the like. Different from the traditional chemical method, the biological butanol is mainly produced by clostridium through anaerobic fermentation, and the method can effectively relieve two prominent problems of energy shortage and environmental pollution, and is a research hotspot at present. With the continuous development of chemical industry and medical industry in China, the demand of butanol will increase to a certain extent.
At present, butanol is mainly synthesized by a chemical method, and with the accelerated exhaustion of petroleum resources and the rising price, the preparation of butanol by a biological fermentation method is also widely regarded. However, the biological method for preparing butanol has the bottlenecks of long fermentation period, low production rate, low conversion rate, low product concentration and the like.
The current approach to butanol production remains to be improved.
Disclosure of Invention
The present invention aims to solve at least one of the above technical problems to at least some extent or to at least provide a useful commercial choice.
According to an embodiment of the present invention, there is provided a method for preparing a chemically modified fiber-based immobilization support, including:
(1) soaking a fiber-based immobilization support in a polyethyleneimine solution to obtain a polyethyleneimine-modified support, wherein polyethyleneimine molecules are formed on the surface of the polyethyleneimine-modified support.
(2) And soaking the polyethyleneimine modified carrier in a glutaraldehyde solution so as to enable amino groups in polyethyleneimine molecules to react with aldehyde groups of glutaraldehyde, thereby obtaining the chemically modified fiber immobilized carrier.
The fiber immobilized carrier obtained by the embodiment of the invention can be used as a cell immobilized carrier to be filled into an immobilized fiber bed and applied to single-batch fermentation and repeated-batch fermentation of biological butanol. According to the embodiment of the invention, amino groups in the polyethyleneimine react with hydroxyl groups in a fiber structure to form chemical bonds by soaking in the polyethyleneimine solution, a large number of polyethyleneimine molecules are formed on the surface of the fiber immobilized carrier, amino groups in the polyethyleneimine molecules react with aldehyde groups of glutaraldehyde by soaking in the glutaraldehyde solution to complete crosslinking of the polyethyleneimine molecules, and a molecular membrane with strong chemical adsorption force is formed on the surface of the fiber immobilized carrier after crosslinking is completed. Furthermore, the surface roughness, positive charge quantity and active group number of the fiber material can be improved, so that the immobilization efficiency of the fiber immobilized material on the clostridium acetobutylicum is improved, the fermentation period is shortened, and the production strength is improved.
According to an embodiment of the present invention, the method for preparing the chemically modified fiber-based immobilization support described above may further have the following additional technical features:
according to an embodiment of the present invention, the fiber-based immobilization carrier may be plant straw, preferably bagasse, rice straw, and corn straw. These materials contain a cellulosic component with a large number of hydroxyl groups on the surface. Therefore, the efficiency of the fiber immobilized carrier which is chemically modified and is used as a cell immobilized carrier to be filled into an immobilized fiber bed and applied to single-batch fermentation and repeated-batch fermentation of the biological butanol can be further improved.
According to the embodiment of the invention, the concentration of the polyethyleneimine solution is 4g/L, the pH is 7.0-8.0, and according to the embodiment of the invention, the concentration of the glutaraldehyde solution is 1 g/L. According to an embodiment of the present invention, the polyethyleneimine solution and the glutaraldehyde solution may be prepared as follows, respectively. Adding a proper amount of polyethyleneimine into pure water in a ventilation kitchen, stirring for 10-30 minutes, adjusting the pH after dissolution to be stable to 7.0-8.0, and sealing and storing the obtained polyethyleneimine solution at 4 ℃. Since glutaraldehyde has a strong bactericidal effect, according to an embodiment of the present invention, the concentration of the glutaraldehyde crosslinking agent solution is 1 g/L, but the concentration can be increased appropriately according to the tolerance of the cells to be immobilized to glutaraldehyde, so as to enhance the crosslinking effect and increase the immobilization efficiency. The glutaraldehyde solution of 25% used in the present invention was diluted to 1 g/L by adding it to a phosphate buffer solution (PH = 7.0), and the resulting glutaraldehyde solution was stored under sealed conditions at 4 ℃.
According to one embodiment of the present invention, in the step (1), the fiber-based immobilization support is previously washed and dried before being soaked in the polyethyleneimine solution. This can further improve the efficiency of immobilization of the chemically modified fiber-based immobilization support.
According to an embodiment of the present invention, in the step (1), the fiber-based immobilization support is soaked in the polyethyleneimine solution for 2 to 6 hours. This can further improve the efficiency of the method for chemically modifying the fiber-based immobilization support.
According to one embodiment of the present invention, it is characterized in that, in the step (2), the polyethyleneimine modified carrier is washed and dried in advance before being soaked in a glutaraldehyde solution. This can further improve the efficiency of the method for chemically modifying the fiber-based immobilization support.
According to one embodiment of the invention, the polyethyleneimine modified carrier is soaked in a glutaraldehyde solution for 2-4 hours. This can further improve the efficiency of the method for chemically modifying the fiber-based immobilization support.
In yet another aspect of the present invention, the present invention provides a chemically modified fiber-based immobilization support prepared according to the method described above. The fiber immobilized carrier subjected to chemical modification according to the embodiment of the invention can be used as a cell immobilized carrier to be filled into an immobilized fiber bed and applied to single-batch fermentation and repeated-batch fermentation of biological butanol. According to the embodiment of the invention, amino groups in the polyethyleneimine react with hydroxyl groups in a fiber structure to form chemical bonds by soaking in the polyethyleneimine solution, a large number of polyethyleneimine molecules are formed on the surface of the fiber immobilized carrier, amino groups in the polyethyleneimine molecules react with aldehyde groups of glutaraldehyde by soaking in the glutaraldehyde solution to complete crosslinking of the polyethyleneimine molecules, and a molecular membrane with strong chemical adsorption force is formed on the surface of the fiber immobilized carrier after crosslinking is completed. Furthermore, the surface roughness, positive charge quantity and active group number of the fiber material can be improved, so that the immobilization efficiency of the fiber immobilized material on the clostridium acetobutylicum is improved, the fermentation period is shortened, and the production strength is improved. Thus, in a further aspect of the invention, the invention proposes the use of the chemically modified fiber-based immobilization support described above for the preparation of butanol. As described above, the chemically modified fiber-based immobilization carrier according to the embodiment of the invention can be used as a cell immobilization carrier to be packed into an immobilized fiber bed, and can be applied to single-batch fermentation and repeated-batch fermentation of bio-butanol. According to the embodiment of the invention, amino groups in the polyethyleneimine react with hydroxyl groups in a fiber structure to form chemical bonds by soaking in the polyethyleneimine solution, a large number of polyethyleneimine molecules are formed on the surface of the fiber immobilized carrier, amino groups in the polyethyleneimine molecules react with aldehyde groups of glutaraldehyde by soaking in the glutaraldehyde solution to complete crosslinking of the polyethyleneimine molecules, and a molecular membrane with strong chemical adsorption force is formed on the surface of the fiber immobilized carrier after crosslinking is completed. Furthermore, the surface roughness, positive charge quantity and active group number of the fiber material can be improved, so that the immobilization efficiency of the fiber immobilized material on the clostridium acetobutylicum is improved, the fermentation period is shortened, and the production strength is improved. It should be noted that other features and advantages described above with respect to the preparation method are also applicable to this use and will not be described in further detail here.
Further, in still another aspect of the present invention, the present invention is an apparatus for producing butanol, comprising: the immobilized fibrous bed reactor is internally provided with the chemical modified fiber immobilized carrier; and a fermentor connected to the immobilized fiber bed reactor and containing a culture medium suitable for clostridium acetobutylicum, wherein the fermentor is in fluid communication with the immobilized fiber bed reactor via a peristaltic pump. According to the embodiment of the invention, the fermentation tank and the immobilized chemical fiber minimally invasive reactor can be connected through the silicone tube, and the peristaltic pump is arranged on the silicone tube, so that the fluid communication between the fermentation tank and the immobilized chemical fiber bed reactor is realized. Therefore, the fiber-based immobilized carrier chemically modified according to the embodiment of the invention can be used as a cell immobilized carrier to be filled into an immobilized fiber bed, and can be applied to single-batch fermentation and repeated-batch fermentation of biological butanol. According to the embodiment of the invention, amino groups in the polyethyleneimine react with hydroxyl groups in a fiber structure to form chemical bonds by soaking in the polyethyleneimine solution, a large number of polyethyleneimine molecules are formed on the surface of the fiber immobilized carrier, amino groups in the polyethyleneimine molecules react with aldehyde groups of glutaraldehyde by soaking in the glutaraldehyde solution to complete crosslinking of the polyethyleneimine molecules, and a molecular membrane with strong chemical adsorption force is formed on the surface of the fiber immobilized carrier after crosslinking is completed. Furthermore, the surface roughness, positive charge quantity and active group number of the fiber material can be improved, so that the immobilization efficiency of the fiber immobilized material on the clostridium acetobutylicum is improved, the fermentation period is shortened, and the production strength is improved. It should be noted that other features and advantages described above with respect to the preparation method are equally applicable to the device and will not be described in further detail here.
In still another aspect of the present invention, the present invention also provides a method for preparing butanol using the aforementioned apparatus, comprising: (1) performing activation culture on clostridium acetobutylicum by using a seed culture medium, transferring a seed solution obtained after the activation culture into a fermentation tank in an inoculation amount of 5-10%, wherein the fermentation tank is filled with the seed culture medium, performing closed culture on the clostridium acetobutylicum at the temperature of 35-40 ℃, and maintaining an anaerobic environment by using nitrogen or carbon dioxide in the culture process; (2) after the closed culture is carried out for 8-12 hours, starting the peristaltic pump so as to pump the fermentation liquor generated in the fermentation tank into the immobilized fiber bed, and carrying out immobilization treatment on the clostridium acetobutylicum cells by using the chemically modified fiber immobilized carrier; and (3) after the immobilization treatment is carried out for 12-24 hours, replacing the fermentation broth in the fermentation tank with a fermentation medium (P2), pumping the fermentation medium into the immobilized fiber bed reactor, so as to carry out fermentation culture by using the clostridium acetobutylicum to produce the biological butanol, wherein the fermentation culture utilizes nitrogen or carbon dioxide to maintain an anaerobic environment. Therefore, the fiber-based immobilized carrier chemically modified according to the embodiment of the invention can be used as a cell immobilized carrier to be filled into an immobilized fiber bed, and can be applied to single-batch fermentation and repeated-batch fermentation of biological butanol. According to the embodiment of the invention, amino groups in the polyethyleneimine react with hydroxyl groups in a fiber structure to form chemical bonds by soaking in the polyethyleneimine solution, a large number of polyethyleneimine molecules are formed on the surface of the fiber immobilized carrier, amino groups in the polyethyleneimine molecules react with aldehyde groups of glutaraldehyde by soaking in the glutaraldehyde solution to complete crosslinking of the polyethyleneimine molecules, and a molecular membrane with strong chemical adsorption force is formed on the surface of the fiber immobilized carrier after crosslinking is completed. It should be noted that other features and advantages described above with respect to the preparation method are equally applicable to the device and will not be described in further detail here.
According to an embodiment of the present invention, the acetone butanol shuttleThe bacterium isClostridium acetobutylicum XY16、 Clostridium acetobutylicum AS1.135 andClostridium beijerinckii 8052.
According to an embodiment of the invention, in step (3), the fermentation medium is replaced when the sugar concentration in the fermentation broth is below 20 g/L. Thus, the strain viability can be maintained, and the efficiency of butanol production by biological fermentation can be further improved.
According to the embodiment of the invention, the rotation speed of the peristaltic pump is 100-200 rpm, and preferably 150 rpm. Thus, the efficiency of butanol production by biological fermentation can be further improved.
According to the embodiment of the invention, chemical reagents of polyethyleneimine and glutaraldehyde are used for carrying out chemical modification on the fiber immobilized carrier, so that the cell immobilization efficiency is improved, and the acting force between cells and the carrier is enhanced, thereby improving the production rate and saving the production cost. The positive charge quantity, the surface roughness, the active group number and the like of the surface of the carrier material after chemical modification are all improved, the adsorption capacity to cells can be greatly enhanced, the production efficiency of immobilized cells in repeated batch fermentation processes is improved, and the carrier material plays a vital role in long-term stable and efficient operation of an immobilized cell system.
According to one embodiment of the invention, the fiber immobilized carrier is chemically modified by using 4g/L polyethyleneimine and 1 g/L glutaraldehyde, the chemically modified carrier is used as a cell immobilized carrier and is filled into an immobilized fiber bed to be applied to single batch fermentation of biological butanol, and the concentrations of butanol and total solvent are highest after 36 hours of fermentation, and respectively reach 12.24 g/L and 21.67 g/L. Compared with free cell fermentation, the concentration of butanol is increased by 11%, the total solvent is increased by 26%, the production rate of the solvent reaches 0.60 g/(L.h), and the production rate is increased by 130.8% and 66.7% respectively compared with batch fermentation of free cells and unmodified bagasse immobilized cells. The modified carrier-immobilized chemical cells are used for 6 times of repeated batch fermentation, the yield of butanol and total solvent is stable, the solvent production rate is gradually increased to 0.83 g/(L.h), and the conversion rate is also increased to 0.42 g/g. Therefore, the acting force between the cells and the carrier is enhanced by forming chemical bonds between the cells and the carrier, and the cell immobilization efficiency can be effectively improved, so that the production rate of the biological fermentation process is improved, and the production cost is saved.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
fig. 1 shows a schematic configuration of an apparatus for preparing butanol according to an embodiment of the present invention.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as described in the claims.
General procedure
In the following examples, unless otherwise specified, the chemical modification, reactor assembly and butanol fermentation were carried out according to the following steps:
a) preparing a polyethyleneimine solution: adding a proper amount of polyethyleneimine into pure water in a ventilation kitchen, stirring for 10-30 minutes, adjusting the pH after dissolution to be stable to 7.0-8.0, and sealing and storing at 4 ℃.
b) Preparing a glutaraldehyde solution: the concentration of the glutaraldehyde crosslinking agent solution is set to be 1 g/L because glutaraldehyde has strong bactericidal effect, but the concentration can be properly increased according to the tolerance condition of the cells needing to be immobilized to the glutaraldehyde, so that the crosslinking effect is enhanced, and the immobilization efficiency is improved. The present invention uses a 25% glutaraldehyde solution, which is diluted to 1 g/L by adding it to a phosphate buffer solution (PH = 7.0), and stored sealed at 4 ℃.
c) Chemical modification of fiber-based immobilization carrier: cleaning a fiber immobilized carrier, drying, soaking in a polyethyleneimine solution for 2-6 hours to enable amino in polyethyleneimine to react with hydroxyl in a fiber structure to form chemical bonds, forming a large amount of polyethyleneimine molecules on the surface of the fiber immobilized carrier, and drying after cleaning with pure water; and soaking the dried modified carrier in a glutaraldehyde solution for 2-4 h to enable amino groups in polyethyleneimine molecules to react with aldehyde groups of glutaraldehyde, so that crosslinking of the polyethyleneimine molecules is completed, and a layer of molecular membrane with strong chemical adsorption force is formed on the surface of the fiber immobilized carrier after crosslinking is completed. And (3) cleaning and drying the modified and crosslinked fiber immobilized carrier, and storing at 4 ℃ for later use.
d) Building an immobilized fiber bed: adding the chemically modified fiber immobilized carrier into a self-made glass chromatographic column (the length of the column is 30cm, the inner diameter is 3 cm), compacting to form a fiber bed, introducing nitrogen for 30 minutes to 1 hour to remove air, sealing, placing into a sterilization pot at 121 ℃, sealing for 15min, and sterilizing for later use.
e) And connecting the built immobilized fiber bed and the fermentation tank together through a peristaltic pump to obtain the equipment for preparing the butanol. As shown in FIG. 1, the apparatus for preparing butanol includes an apparatus for preparing butanol including an immobilized fiber bed reactor, a fermenter, wherein the fermenter and the immobilized fiber bed reactor are in fluid communication by a peristaltic pump, and the immobilized fiber bed reactor is provided with the chemically modified fiber-based immobilized carrier obtained above, and the fermenter is filled with a culture medium suitable for Clostridium acetobutylicum. The medium herein may be selected to be of an appropriate type and composition as required for the subsequent steps. The fermentation tank and the fixed chemical fiber minimally invasive reactor can be connected through a silicone tube, and the peristaltic pump is arranged on the silicone tube.
f) And (3) regulating and controlling the fermentation process: will be provided withActivating and culturing the clostridium acetobutylicum by using a seed culture medium, transferring the seed solution subjected to the activation culture into a fermentation tank (seed culture medium) according to the inoculation amount of 5-10%, connecting a glass chromatographic column filled with a chemically modified fiber immobilized carrier with the fermentation tank, introducing N2Or CO2 Maintaining anaerobic environment for 30min, performing closed culture at 35-40 ℃ for 8-12 h, then opening a peristaltic pump, pumping the fermentation broth into an immobilized fiber bed, circulating the fermentation broth between the fiber bed and the fermentation tank at a circulation speed of 150rpm, completing immobilization of the fiber carrier on the clostridium butyricum cells, removing seed liquid after circulating immobilization for 12-24 h, pumping fresh fermentation medium, introducing N2Or CO2 Fermenting for 30min to prepare butanol. During repeated fermentation batches, the broth was removed and fresh fermentation medium was pumped in each case as described above, when the glucose concentration during the fermentation was below 20 g/L.
The seed activation is to inoculate the strain into a conventional liquid culture medium which contains starch and has the pH of 6.0-7.0 and can provide a carbon source, a nitrogen source and inorganic salt, wherein the conventional liquid culture medium is prepared by freezing the strain in a glycerol tube and introducing N2Or CO2Culturing in a constant-temperature incubator at 30-40 ℃ for 12-24 h by activation culture, and inoculating in a seed culture medium and preserving strains.
The seed culture step of the invention can adopt the seed culture method adopted by the conventional clostridium acetobutylicum. In the invention, the culture medium for seed culture is a conventional liquid culture medium which contains saccharides and can provide a carbon source, a nitrogen source and inorganic salts and has pH of 6.0-7.0, 100-400 mL of the seed culture medium is added into a 500 mL serum bottle during culture, and N is introduced2Or CO2Sterilizing at 115-121 ℃ for 15-30 min, cooling, inoculating activated seed liquid, culturing at 30-40 ℃ in a constant-temperature incubator for 8-16 h, and inoculating in a fermentation tank.
In the step of producing butanol by anaerobic fermentation, the culture medium for fermentation culture can be a conventional liquid culture medium which contains saccharides and can provide a carbon source, a nitrogen source and inorganic salts, and the pH value of the conventional liquid culture medium is 6.0-7.0The volume of the fermentation medium in the 5L fermentation tank is 1.5L; the operation and fermentation conditions of the anaerobic fermentation are conventional methods for producing butanol by using clostridium acetobutylicum anaerobic fermentation, in the invention, the inoculation amount is 5-10% by volume, the temperature is 35-40 ℃, and N is introduced into a fermentation tank2Or CO2Keeping the anaerobic environment of the fermentation system, and the stirring speed is 100-200 rpm.
Example 1
This example is intended to demonstrate the use of modified bagasse as an immobilized carrier in a single fermentation batch of butanol.
Strain: clostridium acetobutylicumClostridium acetobutylicum XY16（CCTCC NO：M 2010011）
Seed culture medium: seed culture medium: 3 g/L yeast powder, 5 g/L peptone, 10 g/L soluble starch, 2 g/L ammonium acetate, 2 g/L NaCl, MgSO4 3 g/L，KH2PO4 1 g/L，K2HPO4 1 g/L，FeSO4·7H2O 0.1 g/L，pH 6.0；
Fermentation medium (P2): carbon source (glucose 60g/L digested), K2HPO4 0.5 g/L，KH2PO4 0.5 g/L，CH3COONH4 2.2 g/L，MgSO4·7H2O 0.2 g/L，MnSO4·H2O 0.01 g/L，NaCl 0.01 g/L，FeSO4·7H2O0.01 g/L; corn steep liquor 1 g/L.
In this experiment, bagasse is taken as an example to illustrate the specific steps of selecting the modification amount of polyethyleneimine.
The addition amount of the bagasse is 10 g/L (the bagasse is modified by 4g/L polyethyleneimine and 1 g/L glutaraldehyde, the ratio of the bagasse to the modifier is 1: 20), after 36 h of fermentation, the glucose (60 g/L) is completely consumed, the butanol and the total solvent respectively reach 12.24 g/L and 21.67 g/L, compared with batch fermentation of unmodified bagasse immobilized cells, the butanol concentration is increased by 11 percent,the total solvent is improved by 26 percent. OD of free cells600The immobilization efficiency is reduced from 6.54 to 0.57, the immobilization efficiency is as high as 91.3 percent, and the immobilization efficiency of bagasse modified by polyethyleneimine is greatly improved and is 2.75 times that of unmodified bagasse. When the concentration of polyethyleneimine is higher than 6 g/L, the total solvent concentration begins to decrease, possibly due to contact of polyethyleneimine with proteins on the cell membrane surface, affecting cell growth and metabolism. As glutaraldehyde has strong inhibition effect on cells, the addition amount of the glutaraldehyde is set to be 1 g/L in the experiment.
Example 2
This example is to demonstrate the use of modified corn stover as an immobilized carrier in butanol single batch fermentation.
Strain: clostridium acetobutylicumClostridium acetobutylicum XY16（CCTCC NO：M 2010011）
Seed culture medium: seed culture medium: 3 g/L yeast powder, 5 g/L peptone, 10 g/L soluble starch, 2 g/L ammonium acetate, 2 g/L NaCl, MgSO4 3 g/L，KH2PO4 1 g/L，K2HPO4 1 g/L，FeSO4·7H2O 0.1 g/L，pH 6.0；
Fermentation medium (P2): carbon source (glucose 60g/L digested), K2HPO4 0.5 g/L，KH2PO4 0.5 g/L，CH3COONH4 2.2 g/L，MgSO4·7H2O 0.2 g/L，MnSO4·H2O 0.01 g/L，NaCl 0.01 g/L，FeSO4·7H2O0.01 g/L; corn steep liquor 1 g/L.
In the experiment, corn straws are taken as an example to illustrate the specific steps of selecting the modification amount of the polyethyleneimine.
The adding amount of the corn straws is 10 g/L (the corn straws are modified by 4g/L polyethyleneimine and 1 g/L glutaraldehyde, the ratio of the corn straws to the modifier is 1: 20), and after 36 hours of fermentation, butanol and total solvent are addedThe preparation respectively reaches 12.00 g/L and 21.27 g/L, and compared with batch fermentation of unmodified bagasse immobilized cells, the concentration of butanol is improved by 8.9%, and the total solvent is improved by 23.8%. OD of free cells600The immobilization efficiency is reduced from 6.43 to 0.77, and is as high as 88.0%.
Example 3
This example is to demonstrate the application of modified rice straw as immobilized carrier in butanol fermentation.
Strain: clostridium acetobutylicumClostridium acetobutylicum AS1.135（CGMCC 1.135）
Seed culture medium: seed culture medium: 3 g/L yeast powder, 5 g/L peptone, 10 g/L soluble starch, 2 g/L ammonium acetate, 2 g/L NaCl, MgSO4 3 g/L，KH2PO4 1 g/L，K2HPO4 1 g/L，FeSO4·7H2O 0.1 g/L，pH 6.0；
Fermentation medium (P2): carbon source (glucose 60g/L digested), K2HPO4 0.5 g/L，KH2PO4 0.5 g/L，CH3COONH4 2.2 g/L，MgSO4·7H2O 0.2 g/L，MnSO4·H2O 0.01 g/L，NaCl 0.01 g/L，FeSO4·7H2O0.01 g/L; corn steep liquor 1 g/L.
The experiment takes straw stalks as an example to illustrate the specific steps of selecting the modification dosage of the polyethyleneimine.
The addition amount of the straw stalks is 10 g/L (the corn stalks are modified by 4g/L of polyethyleneimine and 1 g/L of glutaraldehyde, the ratio of the corn stalks to the modifier is 1: 20), after 36 hours of fermentation, the butanol and the total solvent respectively reach 11.89 g/L and 21.53 g/L, and compared with batch fermentation of unmodified bagasse immobilized cells, the concentration of the butanol is improved by 7.8 percent, and the total solvent is improved by 25.4 percent. OD of free cells600The immobilization efficiency is reduced from 6.68 to 0.97, and the immobilization efficiency is as high as 85.5 percent.
Example 4
This example is intended to demonstrate the effect of modification of fiber-based immobilization supports on the rate of cell sugar consumption and the rate of solvent production.
Strain: clostridium acetobutylicumClostridium acetobutylicum XY16（CCTCC NO：M 2010011）
Seed culture medium: seed culture medium: 3 g/L yeast powder, 5 g/L peptone, 10 g/L soluble starch, 2 g/L ammonium acetate, 2 g/L NaCl, MgSO4 3 g/L，KH2PO4 1 g/L，K2HPO4 1 g/L，FeSO4·7H2O 0.1 g/L，pH 6.0；
Fermentation medium (P2): carbon source (glucose 60g/L digested), K2HPO4 0.5 g/L，KH2PO4 0.5 g/L，CH3COONH4 2.2 g/L，MgSO4·7H2O 0.2 g/L，MnSO4·H2O 0.01 g/L，NaCl 0.01 g/L，FeSO4·7H2O0.01 g/L; corn steep liquor 1 g/L.
The surface modification of the fiber immobilized carrier has obvious promotion effect on the sugar consumption rate and the solvent production rate, the sugar consumption rate is increased to 1.66 g/(L.h) and the solvent production rate is increased to 0.60 g/(L.h) in single batch fermentation of the modified carrier, compared with batch fermentation using unmodified bagasse as the immobilized carrier, the sugar consumption rate and the solvent production rate are respectively increased by 48.2 percent and 66.7 percent, which indicates that a large amount of high-activity cells are loaded on the modified bagasse.
Example 5
This example is to demonstrate that modified bagasse as an immobilized carrier can be applied in the production of butanol by repeated fermentation batches.
Strain: clostridium acetobutylicumClostridium acetobutylicum AS1.135（CGMCC 1.135）
Seed culture medium: seed culture medium: 3 g/L yeast powder, 5 g/L peptone, 10 g/L soluble starch, 2 g/L ammonium acetate, 2 g/L NaCl, MgSO4 3 g/L，KH2PO4 1 g/L，K2HPO4 1 g/L，FeSO4·7H2O 0.1 g/L，pH 6.0；
Fermentation medium (P2): carbon source (glucose 60g/L digested), K2HPO4 0.5 g/L，KH2PO4 0.5 g/L，CH3COONH4 2.2 g/L，MgSO4·7H2O 0.2 g/L，MnSO4·H2O 0.01 g/L，NaCl 0.01 g/L，FeSO4·7H2O0.01 g/L; corn steep liquor 1 g/L.
The immobilized cells are used for repeated batch fermentation, so that the fermentation period can be shortened, and the production rate and the conversion rate are improved. The P2 fermentation medium containing 60g/L glucose is adopted to perform repeated batch fermentation on the clostridium acetobutylicum XY16 immobilized by the chemically modified bagasse, and the stability of the immobilization effect of the chemically modified bagasse is examined. After 6 times of repeated batch fermentation, the adsorption capacity of the modified bagasse on cells is kept stable. Through 6 times of repeated batch fermentation, the concentration of butanol can be maintained at 9-10 g/L, and the total solvent can be maintained at about 16 g/L. The production intensity of the solvent is gradually improved to 0.83 g/(L.h), the glucose conversion rate reaches 0.42 g/g, and the glucose conversion rate is respectively improved by 130.6 percent and 31.2 percent compared with batch fermentation using unmodified bagasse as an immobilized carrier.
Claims (10)
1. A method for preparing a chemically modified fiber-based immobilization support, comprising:
(1) soaking a fiber-based immobilized carrier in a polyethyleneimine solution so as to obtain a polyethyleneimine-modified carrier, wherein polyethyleneimine molecules are formed on the surface of the polyethyleneimine-modified carrier; and
(2) soaking the polyethyleneimine modified carrier in a glutaraldehyde solution so as to enable amino groups in polyethyleneimine molecules to react with aldehyde groups of glutaraldehyde, thereby obtaining the chemically modified fiber immobilized carrier,
optionally, the fiber-based immobilization carrier is plant straw, preferably bagasse, rice straw, and corn straw.
2. The method according to claim 1, wherein the concentration of the polyethyleneimine solution is 4g/L, the pH is 7.0 to 8.0,
optionally, the concentration of the glutaraldehyde solution is 1 g/L.
3. The method according to claim 1, wherein in the step (1), the fiber-based immobilization support is previously washed and dried before being soaked in the polyethyleneimine solution.
4. The method according to claim 1, wherein in the step (1), the fiber-based immobilization support is immersed in the polyethyleneimine solution for 2 to 6 hours.
5. The method according to claim 1, wherein in step (2), the polyethyleneimine modified carrier is washed and dried in advance before being soaked in a glutaraldehyde solution,
optionally, soaking the polyethyleneimine modified carrier in a glutaraldehyde solution for 2-4 hours.
6. A chemically modified fiber-based immobilization support produced by the method according to any one of claims 1 to 5.
7. Use of the chemically modified fiber-based immobilization support according to claim 6 for producing butanol.
8. An apparatus for producing butanol, comprising:
an immobilized fiber bed reactor in which the chemically modified fiber-based immobilized carrier of claim 6 is disposed; and
a fermenter connected to the immobilized fiber bed reactor and containing a culture medium suitable for Clostridium acetobutylicum,
wherein the fermentor is in fluid communication with the immobilized-fiber bed reactor via a peristaltic pump.
9. A method for producing butanol using the apparatus of claim 8, comprising:
(1) performing activation culture on clostridium acetobutylicum by using a seed culture medium, transferring a seed solution after the activation culture into the fermentation tank in an inoculation amount of 5-10%, wherein the fermentation tank is filled with the seed culture medium, so that the clostridium acetobutylicum is subjected to closed culture at the temperature of 35-40 ℃ in the fermentation tank, and an anaerobic environment is maintained by using nitrogen or carbon dioxide in the culture process;
(2) after the closed culture is carried out for 8-12 hours, starting the peristaltic pump so as to pump the fermentation liquor generated in the fermentation tank into the immobilized fiber bed, so that the clostridium acetobutylicum cells are immobilized by using the chemically modified fiber immobilized carrier;
(3) after the immobilization treatment is carried out for 12-24 hours, replacing the fermentation broth in the fermentation tank with a fermentation medium, pumping the fermentation medium into the immobilized fibrous bed reactor, so as to carry out fermentation culture by using the clostridium acetobutylicum, thereby obtaining butanol, wherein the fermentation culture utilizes nitrogen or carbon dioxide to maintain an anaerobic environment,
wherein,
preferably, the clostridium acetobutylicum isClostridium acetobutylicum XY16、 Clostridium acetobutylicumAS1.135 andClostridium beijerinckii 8052.
10. The method according to claim 9, wherein in step (3), the fermentation medium is replaced when the sugar concentration in the fermentation broth is less than 20 g/L,
optionally, the rotation speed of the peristaltic pump is 100-200 rpm, preferably 150 rpm.
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