阅读说明：本技术 一种电子传递介体强化拜氏梭菌发酵制备氢气和丁醇的阴极电发酵方法 (Cathode electric fermentation method for preparing hydrogen and butanol by using electron transfer mediator to strengthen fermentation of clostridium beijerinckii ) 是由 李建政 张亚非 孟佳 王鑫 于 2020-06-23 设计创作，主要内容包括：本发明公开了一种电子传递介体强化拜氏梭菌发酵制备氢气和丁醇的阴极电发酵方法,属于微生物发酵生产清洁能源技术领域。本发明解决了现有拜氏梭菌在丁醇发酵过程中,丁醇产量、产率低,产酸相向产溶剂相转变失败、溶剂中丙酮占比较高的问题。本发明在阴极电发酵系统中以拜氏梭菌发酵制备丁醇,通过引入电极和外源电子传递介体中性红(NR)的添加,可以控制和优化发酵环境,改变细胞原有的NAD~+/NADH的平衡,显著优化微生物代谢中不同途径的分流,诱导微生物代谢路径中更多的碳和电子流向丁醇合成路径,提高生物丁醇的产量和产率,同时产生更多的氢气。(The invention discloses a cathode electric fermentation method for preparing hydrogen and butanol by strengthening fermentation of clostridium beijerinckii by an electron transfer mediator, belonging to the technical field of producing clean energy by microbial fermentation. The invention solves the problems that the prior clostridium beijerinckii has low butanol yield and productivity, failed phase transformation from an acid-producing phase to a solvent-producing phase and higher acetone ratio in the solvent in the butanol fermentation process. The invention prepares butanol by fermentation of clostridium beijerinckii in a cathode electric fermentation system, can control and optimize the fermentation environment by introducing electrodes and adding Neutral Red (NR) of an exogenous electron transfer mediator, and changes the original NAD of cells + NADH balance, significantly optimizing different pathways in microbial metabolismShunting, inducing more carbon and electrons in the microbial metabolic pathway to flow to a butanol synthesis pathway, improving the yield and productivity of biological butanol, and simultaneously generating more hydrogen.)

1. A cathodic electrofermentation method for preparing hydrogen and butanol by fermentation of clostridium beijerinckii with an electron transfer mediator, which is characterized by comprising the following steps:

(1) adding an anaerobic fermentation culture medium into a cathode chamber of a double-chamber H-shaped reactor, inoculating clostridium beijerinckii seed liquid into the anaerobic fermentation culture medium added with a fermentation substrate and an electron transfer mediator, and stirring and culturing at 37 ℃ at the rotating speed of 120 r/min;

(2) connecting three electrodes of the double-chamber H-shaped reactor with a constant potential while starting the culture in the step (1), and starting the double-chamber H-shaped reactor;

(3) sampling from the cathode chamber at intervals of 24h, and measuring the biomass, the pH value and the liquid phase product components of the fermentation liquid suspension until the liquid phase product components in the fermentation liquid suspension do not change any more, and ending the fermentation process.

2. The cathodic electrofermentation method for the enhanced fermentation of clostridium beijerinckii for the production of hydrogen and butanol according to claim 1, wherein the anaerobic fermentation medium is prepared by: mixing yeast powder and K₂HPO₄、KH₂PO₄、MgSO₄·7H₂O、MnSO₄·H₂O、FeSO₄·7H₂O, NaCl, mixing p-aminobenzoic acid, thiamine and biotin, introducing nitrogen to aerate and remove oxygen, and then sterilizing at 121 ℃ for 20min to obtain the anaerobic fermentation medium.

3. The cathodic electrofermentation method for the enhanced fermentation of Clostridium beijerinckii to produce hydrogen and butanol according to claim 2, wherein 1L of the anaerobic fermentation medium contains 3g of yeast powder, K₂HPO₄ 0.5g，KH₂PO₄0.5g，MgSO₄·7H₂O 0.2g，MnSO₄·H₂O 0.01g，FeSO₄·7H₂0.01g of O, 0.01g of NaCl, 0.001g of p-aminobenzoic acid, 0.001g of thiamine and 0.0001g of biotin.

4. The cathodic electrofermentation method for the fermentative production of hydrogen and butanol with the aid of clostridium beijerinckii according to claim 1, characterized in that the fermentation substrate is glucose, and the mass of the fermentation substrate added to 1L of the anaerobic fermentation medium is 20 g.

5. The cathodic electrofermentation method for the fermentative production of hydrogen and butanol using clostridium beijerinckii according to claim 1, wherein the electron transfer mediator is neutral red, and the amount of the substance added with the electron transfer mediator is 0.5mmol in 1L of the anaerobic fermentation medium.

6. The cathodic electrofermentation process for the fermentative production of hydrogen and butanol, according to claim 1, using an electron transfer mediator enhanced clostridium beijerinckii, characterized in that the seed liquid of clostridium beijerinckii is inoculated, in a volume ratio of 5%, into an anaerobic fermentation medium to which a fermentation substrate and an electron transfer mediator are added.

7. The cathodic electrofermentation process for the fermentative production of hydrogen and butanol with the electron transfer mediator enhanced Clostridium beijerinckii according to claim 1, characterized in that the constant potential applied by the double-chamber H-type reactor is-0.75V.

8. The cathodic electrofermentation method for the fermentation preparation of hydrogen and butanol by the electron transfer mediator enhanced clostridium beijerinckii according to claim 1, characterized in that the double-chamber H-type reactor uses a titanium wire fixed graphite felt as a cathode and an anode, and an Ag/AgCl electrode as a reference electrode; loading equal volume of K in anode chamber and cathode chamber₄(Fe(CN)₆) The solution acts as an electron donor.

9. The cathodic electrofermentation method for enhanced fermentation of clostridium beijerinckii to produce hydrogen and butanol with an electron transfer mediator according to claim 8, wherein K is₄(Fe(CN)₆) The solution concentration was 200 mmol/L.

10. The cathodic electrofermentation method for enhanced fermentation of clostridium beijerinckii for the production of hydrogen and butanol according to claim 8, wherein the clostridium beijerinckii seed solution is purchased from the china general microbiological culture collection center CGMCC.

Technical Field

The invention relates to a cathode electric fermentation method for preparing hydrogen and butanol by strengthening fermentation of clostridium beijerinckii by an electron transfer mediator, belonging to the technical field of producing clean energy by microbial fermentation.

Background

Renewable biomass has huge reserves, can be converted into various high-efficiency energy sources such as bioethanol, biodiesel, biobutanol, biogas and the like through different ways, and has important effects on the aspects of coping with global climate change, contradiction between energy supply and demand, protecting ecological environment and the like. Under the promotion of national energy strategy, the bioethanol is popularized and applied. Compared with ethanol, the biological butanol has the advantages of high energy, good miscibility, low volatility, small corrosivity and the like, and is a novel biofuel with great potential. And the butanol has high energy density, lower saturated vapor pressure and heat value close to that of gasoline, and can be used as fuel to be added into the gasoline, even to completely replace the gasoline. The existing production methods of butanol mainly comprise a synthesis method and a fermentation method, and along with the rising of price and the increasing severity of environmental problems in recent years, the butanol is produced by adopting cheap renewable biomass (cellulose raw materials such as plant straws, agricultural residues, agricultural byproduct wastes and the like) as a fermentation substrate. However, the microbial fermentation method for preparing butanol has slow industrial application process due to the limitation of low yield and productivity and difficult phase transition regulation. Therefore, it is desirable to provide a method for increasing the yield and productivity of butanol, increasing the butanol fraction in the solvent, and increasing the hydrogen production during fermentation.

Disclosure of Invention

The invention provides a cathode electric fermentation method for preparing hydrogen and butanol by strengthening fermentation of clostridium beijerinckii by an electron transfer mediator, aiming at solving the problems that the yield and the yield of butanol are low, the phase conversion of an acid-producing phase to a solvent-producing phase fails and the acetone in the solvent accounts for a relatively high proportion in the conventional fermentation process of the clostridium beijerinckii.

The technical scheme of the invention is as follows:

a cathodic electrofermentation method for preparing hydrogen and butanol by fermentation of clostridium beijerinckii with an electron transfer mediator, which comprises the following steps:

(1) adding an anaerobic fermentation culture medium into a cathode chamber of a double-chamber H-shaped reactor, inoculating clostridium beijerinckii seed liquid into the anaerobic fermentation culture medium added with a fermentation substrate and an electron transfer mediator, and stirring and culturing at 37 ℃ at the rotating speed of 120 r/min;

(2) connecting three electrodes of the double-chamber H-shaped reactor with a constant potential and starting the double-chamber H-shaped reactor while starting the culture in the step (1);

(3) sampling from the cathode chamber at intervals of 24h, and measuring the biomass, the pH value and the liquid phase product components of the fermentation liquid suspension until the liquid phase product components in the fermentation liquid suspension do not change any more, and ending the fermentation process.

Further, the preparation process of the anaerobic fermentation culture medium comprises the following steps: mixing yeast powder and K₂HPO₄、KH₂PO₄、MgSO₄·7H₂O、MnSO₄·H₂O、FeSO₄·7H₂O, NaCl, mixing p-aminobenzoic acid, thiamine and biotin, introducing nitrogen to aerate and remove oxygen, and then sterilizing at 121 ℃ for 20min to obtain the anaerobic fermentation medium.

Further, 3g of yeast powder and K in 1L of the anaerobic fermentation medium₂HPO₄ 0.5g，KH₂PO₄ 0.5g，MgSO₄·7H₂O 0.2g，MnSO₄·H₂O 0.01g，FeSO₄·7H₂0.01g of O, 0.01g of NaCl, 0.001g of p-aminobenzoic acid, 0.001g of thiamine and 0.0001g of biotin.

Further, the fermentation substrate was glucose, and the mass of the fermentation substrate added to 1L of the anaerobic fermentation medium was 20 g.

Further, the electron mediator was neutral red, and the amount of the substance to which the electron mediator was added was 0.5mmol in 1L of the anaerobic fermentation medium.

Further, the clostridium beijerinckii seed liquid was inoculated in an amount of 5% by volume to an anaerobic fermentation medium to which a fermentation substrate and an electron transfer mediator were added.

Further, the constant potential applied by the double-chamber H-type reactor was-0.75V.

Furthermore, the double-chamber H-shaped reactor is fixed by titanium wiresThe graphite felt is used as a cathode and an anode, and the Ag/AgCl electrode is used as a reference electrode; loading equal volume of K in anode chamber and cathode chamber₄(Fe(CN)₆) The solution acts as an electron donor.

Further, K₄(Fe(CN)₆) The solution concentration was 200 mmol/L.

Further, the clostridium beijerinckii seed solution is purchased from China general microbiological culture collection center (CGMCC).

The invention has the following beneficial effects: the invention prepares butanol by fermentation of clostridium beijerinckii in a cathode electric fermentation system, can control and optimize fermentation environment by introducing electrodes and adding Neutral Red (NR), and changes the original NAD of cells⁺The balance of NADH influences the fermentation process of the microorganism so as to obtain a product with higher purity, is beneficial to the growth of microbial cells and biomass accumulation to realize the extension of carbon chains and realize sustainable butanol preparation. The method has the following advantages:

(1) the cathodic electrofermentation has low energy consumption, can achieve remarkable effect only by applying extremely small current stimulation or potential, can regulate and control biological systems and intracellular redox levels through external potential, controls the metabolic balance of fermentation microorganisms, increases ATP and cell substance synthesis, adjusts metabolic pathways, and improves the yield and the yield of target products.

(2) According to the invention, by adding the exogenous electron transfer mediator, on one hand, the generation of cell metabolites can be changed by direct contact with cell membranes, and on the other hand, the exogenous electron transfer mediator can mediate electron transfer between the fermenting microorganism and the electrode, so that the regulation and control of reducing power in the fermentation process are realized, more carbon and electrons in the microorganism metabolic pathway are induced to flow to a butanol synthesis pathway, the yield and the productivity of biological butanol are improved, and more hydrogen is generated at the same time.

(3) The fermentation system of the invention can remarkably optimize the shunting of different ways in microbial metabolism, improve the composition of fermentation products and is beneficial to the synthesis of reducing products by applying a certain potential and introducing external current into the fermentation system. The final appearance is as follows: in the fermentation with glucose as a single substrate, the acetone (non-target product) produced by the system is reduced, so that the percentage of butanol in the solvent is increased, and further the utilization rate of the substrate (glucose), the butanol yield and the butanol yield are greatly improved.

Drawings

FIG. 1 is a schematic view of a structure of a double-chamber H-type reactor of the present invention;

FIG. 2 shows the change of fermentation index in example 1;

FIG. 3 shows the change in fermentation index of comparative example 1.

Detailed Description

The experimental procedures used in the following examples are conventional unless otherwise specified. The materials, reagents, methods and apparatus used, unless otherwise specified, are conventional in the art and are commercially available to those skilled in the art.

Example 1:

the process of producing butyric acid by fermenting straws by anaerobic mixed flora is carried out in a cathodic electrotransformation system (CEF + NR) added with an exogenous electron transfer mediator. The specific operation process is as follows:

(1) a double-chamber H-shaped reactor with the effective volume of 100mL is used as a fermentation reactor, wherein the fermentation is carried out in a cathode chamber, and an equal volume of 200mmol/L potassium ferrocyanide is contained in an anode chamber and is used as an electron donor in an electric fermentation system. And fixing equal-volume graphite felts serving as a cathode and an anode by using a titanium wire with the diameter of 3mm, and enabling the cathode and the anode fixed on the titanium wire to penetrate through a rubber plug at the top of the polar chamber to finish fixing. The anode reference electrode was Ag/AgCl (196mV vs SHE) and was held in place by a side sample port in the cathode chamber and the front end of the electrode was placed about 2cm below the surface of the fermentation broth as shown in FIG. 1.

(2) Adding the prepared anaerobic fermentation culture medium added with fermentation substrate and electron transfer mediator into a cathode chamber, inoculating Clostridium beijerinckii NCIMB 8052 (purchased from China general microbiological culture Collection center CGMCC) into a fresh anaerobic fermentation culture medium according to the proportion of 5% (v/v), and culturing at 37 ℃ at the rotating speed of 120 r/min.

The anaerobic fermentation culture medium contains 3g/L yeast powder and 0.5g/L K g/L yeast powder₂HPO₄，0.5g/L KH₂PO₄，0.2g/L MgSO₄·7H₂O，0.01g/L MnSO₄·H₂O，0.01g/L FeSO₄·7H₂O, 0.01g/L NaCl, 0.001g/L p-aminobenzoic acid, 0.001g/L thiamine and 0.0001g/L biotin.

20g of fermentation substrate glucose was added to 1L of the anaerobic fermentation medium. The amount of the substance having neutral red as an electron transfer mediator added to 1L of the anaerobic fermentation medium was 0.5 mmol.

The preparation process of the anaerobic fermentation culture medium added with the fermentation substrate and the electron transfer mediator comprises the following steps: mixing yeast powder and K₂HPO₄、KH₂PO₄、MgSO₄·7H₂O、MnSO₄·H₂O、FeSO₄·7H₂O, NaCl, mixing p-aminobenzoic acid, thiamine and biotin, introducing nitrogen to aerate and remove oxygen, sterilizing at 121 ℃ for 20min, cooling to room temperature, adding glucose as a fermentation substrate into a culture medium in a sterile operating platform, adding NR as an electron transfer mediator, and adding an NR concentrated solution by using an injector to enable the NR concentration of the fermentation liquid in a cathode chamber to be about 0.5 mmol.

(3) And (3) connecting three electrodes of the double-chamber H-shaped reactor with a constant potential while starting the culture in the step (2), and starting the double-chamber H-shaped reactor after setting the applied potential of the working electrode to-0.75V (vs Ag/AgCl).

(4) During the electric fermentation, samples were taken from the system every 24h, and key fermentation indicators were determined, and the results are shown in FIG. 2.

(5) After the whole fermentation process lasts for 5 days, the liquid phase product components in the fermentation liquid do not change any more, and the fermentation is finished.

Comparative example 1:

the process of producing butyric acid by fermenting straws by anaerobic mixed flora is carried out in a system (OC) without applied potential. The OC group was not additionally added with neutral red, no constant potential was applied, no wire was connected between the cathode and the anode, the rest of the operation was the same as in example 1, and the test results of the key fermentation indexes are shown in fig. 3.

Results of the Key fermentation index test of comparative example 1 and comparative example 1, i.e., comparisonAs can be seen from FIGS. 2 and 3, the OC system consumed only 7.34g/L glucose after the end of fermentation and accumulated the resulting biomass (OD)₆₀₀) 2.23 with 0.82g/L butanol and 0.53g/L acetone and 0.59g/L and 0.81g/L acetic and butyric acid. Furthermore, 0.88L/L hydrogen was produced after the fermentation in the OC group was completed. It can be seen that at the end of the OC group fermentation, only a small fraction of the consumed glucose is used for the production of solvent and volatile acids, in addition to the synthetic biomass.

In the CEF + NR system, the amount of glucose consumed after the end of the fermentation reached 18.09g/L, whereas the biomass accumulated was only 1.73. The fermentation produced 5.49g/L butanol and 1.17g/L acetone, as well as 1.1g/L acetic acid and 1.48g/L butyric acid. At the same time, 3.74L/L hydrogen generation was also accompanied. The glucose substrate consumed in the fermentation is used for biomass accumulation, and most is used for solvent, volatile acid and hydrogen generation.

By comparison, in the CEF system after adding NR, the combined action of CEF and NR can greatly improve the substrate (glucose) utilization rate, butanol yield and butanol yield of Clostridium beijerinckii NCIMB 8052 in fermentation, which are respectively 2.47 times, 6.70 times and 2.73 times of those of the OC system. In the aspect of hydrogen generation, the yield of the CEF + NR system is 4.25 times that of the OC system, and the improvement effect is very obvious. Meanwhile, the acetone (non-target product) generated in the fermentation can be obviously reduced, so that the percentage of the butanol in the solvent is increased from 60.74% to 82.43% of the OC system, and the strengthening effect is very obvious.

Example 2:

(1) the process of producing butyric acid by fermenting straws by anaerobic mixed flora is carried out in a cathodic electrotransformation system (CEF + NR) added with an exogenous electron transfer mediator. The difference from example 1 is that glucose and butyric acid are used as fermentation substrates, 30g of glucose is added as a fermentation substrate to 1L of anaerobic fermentation medium, and 3g of butyric acid is added as a fermentation substrate to 1L of anaerobic fermentation medium, and the rest of the operation is the same as example 1.

(2) The process of producing butyric acid by fermenting straws by anaerobic mixed flora is carried out in a system (OC) without applied potential. The difference from comparative example 1 is that fermentation substrates are glucose and butyric acid, 30g of fermentation substrate glucose is added to 1L of anaerobic fermentation medium, 3g of fermentation substrate butyric acid is added to 1L of anaerobic fermentation medium, and the rest of the operation is the same as comparative example 1.

The fermentation index data obtained in (1) and (2) above are shown in Table 1 below.

TABLE 1 Butanol fermentation Performance of OC group and CEF + NR group with glucose and butyric acid cosubstrates

When glucose and butyric acid were co-substrates, the amount of glucose consumed at the end of fermentation in the OC system was 22.51g/L, and the resulting biomass (OD) was accumulated₆₀₀) 3.32 with formation of 8.87g/L butanol and 2.73g/L acetone, and 2.37g/L butyric acid consumed. Furthermore, 2.34L/L hydrogen was produced after the fermentation.

When glucose and butyric acid were used as cosubstrates, the amount of glucose consumed in the CEF + NR group after the end of fermentation was increased to 26.35g/L, and the biomass (OD) obtained was accumulated₆₀₀) Only 2.15. The fermentation produced 10.10g/L butanol and 2.54g/L acetone, with 1.81g/L butyric acid consumed. At the same time, 4.29L/L of hydrogen was also produced.

In comparison, in the CEF system after NR addition, the accumulation of c.beijerinckii NCIMB 8052 biomass was less, and more substrate was used for solvent and hydrogen generation. Compared with the OC system, the butanol yield of the CEF + NR system is improved by 13.87%, and meanwhile, the content of butanol in the solvent is improved from 76.47% to 79.91%. The yield of the CEF + NR system was increased by 83.33% compared to the OC group in terms of hydrogen generation. For butanol fermentation of butyrate and glucose co-substrates, the combined application of CEF and NR was still very significant for the c.beijerinckii NCIMB 8052 strain fortification effect.