CN112501213B - Cathode electrofermentation method for preparing hydrogen and butanol by enhancing clostridium beijerinckii fermentation by using electron transfer mediator - Google Patents

Cathode electrofermentation method for preparing hydrogen and butanol by enhancing clostridium beijerinckii fermentation by using electron transfer mediator Download PDF

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CN112501213B
CN112501213B CN202010581805.5A CN202010581805A CN112501213B CN 112501213 B CN112501213 B CN 112501213B CN 202010581805 A CN202010581805 A CN 202010581805A CN 112501213 B CN112501213 B CN 112501213B
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李建政
张亚非
孟佳
王鑫
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Harbin Institute of Technology
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Abstract

The invention discloses a cathode electric fermentation method for preparing hydrogen and butanol by enhancing clostridium beijerinckii fermentation by an electron transfer mediator, belonging to the technical field of clean energy production by microbial fermentation. The invention solves the problems of low butanol yield and low yield, failure of phase transition of acid production phase to solvent production phase and higher acetone occupation ratio in the solvent in the butanol fermentation process of the traditional clostridium beijerinckii. The invention prepares butanol by clostridium beijerinckii fermentation in a cathode electrofermentation system, and can control and optimize the fermentation environment and change the original NAD of cells by introducing electrodes and adding Neutral Red (NR) of an exogenous electron transfer mediator + Balance of NADH, significant advantageAnd (3) shunting different paths in microbial metabolism, inducing more carbon and electrons in the microbial metabolism path to flow to a butanol synthesis path, improving the yield and the yield of biological butanol, and simultaneously generating more hydrogen.

Description

Cathode electrofermentation method for preparing hydrogen and butanol by enhancing clostridium beijerinckii fermentation by using electron transfer mediator
Technical Field
The invention relates to a cathode electric fermentation method for preparing hydrogen and butanol by enhancing clostridium beijerinckii fermentation by an electron transfer mediator, belonging to the technical field of clean energy production by microbial fermentation.
Background
Renewable biomass has huge reserves, can be converted into various high-energy sources such as bioethanol, biodiesel, biobutanol, biogas and the like through different ways, and has important effects in coping with global climate change, energy supply and demand contradiction, ecological environment protection and the like. Under the promotion of national energy strategy, bioethanol has been popularized and applied. Compared with ethanol, the biological butanol has the advantages of high energy, good miscibility, low volatility, low corrosiveness and the like, and is a novel biological fuel with great potential. The butanol has high energy density, low saturated vapor pressure and heat value close to that of gasoline, and can be used as fuel to be added into the gasoline or even completely replace the gasoline. The existing butanol production method mainly comprises a synthesis method and a fermentation method, and in recent years, along with the rising price and increasing environmental problems, inexpensive renewable biomass (plant straws, agricultural residues, agricultural and sideline product waste materials and other cellulosic raw materials) is adopted as a fermentation substrate to produce butanol. However, the microbial fermentation method for preparing butanol is limited by low yield and productivity, and phase transition is difficult to regulate and control, so that the industrial application process is slow. Therefore, it is desirable to provide a process that increases butanol yield, increases butanol fraction in the solvent, and increases hydrogen production during fermentation.
Disclosure of Invention
The invention provides a cathode electrofermentation method for preparing hydrogen and butanol by using an electron transfer mediator to strengthen clostridium beijerinckii fermentation, which aims at solving the problems that the existing clostridium beijerinckii has low butanol yield and low yield, and the phase transition of an acid-producing phase to a solvent-producing phase fails and the acetone in the solvent is relatively high.
The technical scheme of the invention is as follows:
a cathodic electrofermentation method for preparing hydrogen and butanol by enhancing clostridium beijerinckii fermentation by an electron transfer mediator, comprising the following steps:
(1) Adding an anaerobic fermentation culture medium into a cathode chamber of a double-chamber H-type 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 a temperature of 37 ℃ and a rotating speed of 120 r/min;
(2) The three electrodes of the double-chamber H-type reactor are connected with constant potential and the double-chamber H-type reactor is started while the culture is started in the step (1);
(3) Sampling from the cathode chamber every 24h, and measuring the biomass, pH value and liquid phase product component of the fermentation liquid suspension until the liquid phase product component in the fermentation liquid suspension is not changed continuously, and ending the fermentation process.
Further, the anaerobic fermentation medium is prepared by the following steps: mixing yeast powder, K 2 HPO 4 、KH 2 PO 4 、MgSO 4 ·7H 2 O、MnSO 4 ·H 2 O、FeSO 4 ·7H 2 O, naCl mixing para aminobenzoic acid, thiamine and biotin, introducing nitrogen, aerating to remove oxygen, and sterilizing at 121 ℃ for 20min to obtain the anaerobic fermentation culture medium.
Further, 3g of yeast powder and 3g of K in 1L of anaerobic fermentation medium 2 HPO 4 0.5g，KH 2 PO 4 0.5g，MgSO 4 ·7H 2 O 0.2g，MnSO 4 ·H 2 O 0.01g，FeSO 4 ·7H 2 O0.01 g, naCl 0.01g, p-aminobenzoic acid 0.001g, thiamine 0.001g, biotin 0.0001g.
Further, the fermentation substrate was glucose, and the mass of the fermentation substrate added to 1L of anaerobic fermentation medium was 20g.
Further, the electron transfer mediator was neutral red, and the amount of the electron transfer mediator-added substance in 1L of anaerobic fermentation medium was 0.5mmol.
Further, clostridium beijerinckii seed solution is inoculated into an anaerobic fermentation medium added with a fermentation substrate and an electron transfer mediator according to the volume ratio of 5 percent.
Further, the constant potential applied by the dual-chamber H-type reactor was-0.75V.
Further, the double-chamber H-type reactor takes a titanium wire fixed graphite felt as a cathode and an anode, and an Ag/AgCl electrode as a reference electrode; the anode chamber is loaded with K equal to the volume of the cathode chamber loading material 4 (Fe(CN) 6 ) The solution acts as an electron donor.
Further, K 4 (Fe(CN) 6 ) The concentration of the solution was 200mmol/L.
Further, clostridium beijerinckii seed liquid is purchased from China general microbiological culture collection center CGMCC.
The invention has the following beneficial effects: the invention prepares butanol by clostridium beijerinckii fermentation in a cathode electric fermentation system, and can control and optimize the fermentation environment and change the original NAD of cells by introducing electrodes and adding Neutral Red (NR) + The balance of NADH affects the fermentation process of microorganisms to obtain a product with higher purity, and facilitates growth of microbial cells and biomass accumulation to achieve elongation of carbon chains and sustainable butanol production. The method has the following advantages:
(1) The cathodic electrofermentation has lower energy consumption, can play a remarkable role by only needing extremely small current stimulation or potential application, can control the metabolic balance of fermenting microorganisms by regulating and controlling a biological system and intracellular oxidation-reduction level through externally applied potential, increases synthesis of ATP and cellular substances, adjusts metabolic pathways, and improves the yield and the productivity of target products.
(2) According to the invention, through adding the exogenous electron transfer medium, on one hand, the generation of metabolic products of cells can be changed through direct contact with cell membranes, and on the other hand, electron transfer between fermenting microorganisms and electrodes can be mediated, so that the regulation of reducing force in the fermentation process is realized, more carbon and electrons in a microbial metabolic pathway are induced to flow to a butanol synthesis pathway, the yield and the yield of biological butanol are improved, and more hydrogen is generated.
(3) The fermentation system of the invention can obviously optimize the diversion of different approaches in microbial metabolism by applying a certain potential and introducing external current into the fermentation system, improve the composition of fermentation products and facilitate the synthesis of reductive products. The final appearance is: in the fermentation with glucose as a single substrate, acetone (non-target product) produced by the system is reduced, so that the percentage of butanol in a solvent is increased, and the substrate (glucose) utilization rate, butanol yield and butanol yield are greatly improved.
Drawings
FIG. 1 is a schematic structural view of a double-chamber H-type reactor according to the present invention;
FIG. 2 shows the fermentation index change of example 1;
FIG. 3 shows the fermentation index change of comparative example 1.
Detailed Description
The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods and apparatus used, without any particular description, are those conventional in the art and are commercially available to those skilled in the art.
Example 1:
and (3) carrying out an anaerobic mixed flora fermentation straw butyric acid production process in a cathodic electrofermentation system (CEF+NR) added with an exogenous electron transfer mediator. The specific operation process is as follows:
(1) A double-chamber H-type reactor with an effective volume of 100mL is used as a fermentation reactor, wherein fermentation is carried out in a cathode chamber, and 200mmol/L potassium ferrocyanide with an equal volume is filled in an anode chamber and used as an electron donor in an electric fermentation system. And (3) fixing graphite felt with the same volume by using a titanium wire with the diameter of 3mm as a cathode and an anode, wherein the cathode and the anode fixed on the titanium wire penetrate through a rubber plug at the top of the polar chamber to complete fixation. The anode reference electrode was Ag/AgCl (196 mV vs SHE) which was fixed through the cathode chamber side sampling port and the front end of the electrode was placed at a depth of about 2cm below the fermentation liquid surface as shown in FIG. 1.
(2) Adding the prepared anaerobic fermentation medium added with the fermentation substrate and the electron transfer medium into a cathode chamber, inoculating clostridium beijerinckii (Clostridium beijerinckii NCIMB 8052) (purchased in China general microbiological culture collection center (CGMCC)) into a fresh anaerobic fermentation medium according to the proportion of 5% (v/v), and culturing at 37 ℃ and the rotating speed of 120 r/min.
The anaerobic fermentation medium contains 3g/L yeast powder and 0.5g/L K 2 HPO 4 ，0.5g/L KH 2 PO 4 ，0.2g/L MgSO 4 ·7H 2 O，0.01g/L MnSO 4 ·H 2 O，0.01g/L FeSO 4 ·7H 2 O,0.01g/L NaCl,0.001g/L p-aminobenzoic acid, 0.001g/L thiamine and 0.0001g/L biotin.
20g of fermentation substrate glucose is added into 1L of anaerobic fermentation medium. The amount of neutral red as an electron transfer mediator added to 1L anaerobic fermentation medium was 0.5mmol.
The anaerobic fermentation medium added with the fermentation substrate and the electron transfer medium is prepared by the following steps: mixing yeast powder, K 2 HPO 4 、KH 2 PO 4 、MgSO 4 ·7H 2 O、MnSO 4 ·H 2 O、FeSO 4 ·7H 2 O, naCl mixing para aminobenzoic acid, thiamine and biotin, introducing nitrogen, aerating, deoxidizing, sterilizing at 121 ℃ for 20min, cooling to normal temperature, adding glucose as a fermentation substrate into a sterile operation table, adding NR as an electron transfer mediator, and adding NR concentrate into a cathode chamber by using a syringe to ensure that the NR concentration of the fermentation liquid in the cathode chamber is about 0.5mmol.
(3) And (3) starting the culture in the step (2), connecting three electrodes of the double-chamber H-type reactor with constant potential, setting the applied potential of the working electrode to be-0.75V (vs Ag/AgCl), and starting the double-chamber H-type reactor.
(4) During the electrofermentation, samples were taken from the system every 24 hours, and key fermentation indicators were measured, 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 are not changed any more, and the fermentation is finished.
Comparative example 1:
and (3) carrying out the process of producing butyric acid by fermenting the straw by anaerobic mixed flora in a system (OC) without applying electric potential. The OC group was not additionally added with neutral red, no constant potential was applied, no wire connection between the cathode and the anode, and the rest of the operations were the same as in example 1, and the test results of the key fermentation index are shown in fig. 3.
As can be seen from the key fermentation index test results of comparative example 1 and comparative example 1, i.e., comparing FIGS. 2 and 3, the OC system only consumed 7.34g/L glucose consumption after the fermentation was completed, and the resulting biomass (OD 600 ) 2.23, accompanied by 0.82g/L butanol and 0.53g/L acetone formation, and 0.59g/L and 0.81g/L acetic acid and butyric acid formation. In addition, the OC group fermentation was completed to yield 0.88L/L of hydrogen gas. It can be seen that at the end of the OC group fermentation, only a small fraction of the glucose consumed, apart from the synthetic biomass, was used for the production of solvent and volatile acids.
In the CEF+NR system, the amount of glucose consumed after the end of 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, and 1.1g/L acetic acid and 1.48g/L butyric acid. At the same time, 3.74L/L of hydrogen gas is generated. The glucose substrates consumed in fermentation are used for biomass accumulation, mostly for solvent, volatile acid and hydrogen generation.
In comparison, the combined action of CEF and NR in the CEF system after NR addition can greatly improve the substrate (glucose) utilization, butanol yield and butanol yield of Clostridium beijerinckii NCIMB 8052 in fermentation by 2.47 times, 6.70 times and 2.73 times respectively compared with 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, acetone (non-target product) generated in fermentation can be obviously reduced, so that the percentage of butanol in a solvent is increased from 60.74% of an OC system to 82.43%, and the strengthening effect is very obvious.
Example 2:
(1) And (3) carrying out an anaerobic mixed flora fermentation straw butyric acid production process in a cathodic electrofermentation system (CEF+NR) added with an exogenous electron transfer mediator. The procedure of example 1 was repeated except that glucose and butyric acid were used as fermentation substrates, 30g of glucose was used as a fermentation substrate in 1L of anaerobic fermentation medium, 3g of butyric acid was used as a fermentation substrate in 1L of anaerobic fermentation medium.
(2) And (3) carrying out the process of producing butyric acid by fermenting the straw by anaerobic mixed flora in a system (OC) without applying electric potential. The procedure was the same as in comparative example 1 except that the fermentation substrates were glucose and butyric acid, 30g of glucose as a fermentation substrate was added to 1L of anaerobic fermentation medium, 3g of butyric acid as a fermentation substrate was added to 1L of anaerobic fermentation medium.
The fermentation index data obtained in the above (1) and (2) are shown in the following Table 1.
TABLE 1 Butanol fermentation efficacy for OC and CEF+NR groups with glucose and butyric acid co-substrates
Figure BDA0002553456280000051
When glucose and butyric acid are co-substrates, the glucose consumed at the end of fermentation in the OC system is 22.51g/L, and the biomass (OD) thus obtained is accumulated 600 ) 3.32, accompanied by the formation of 8.87g/L butanol and 2.73g/L acetone, and the consumption of 2.37g/L butyric acid. In addition, after the fermentation was completed, 2.34L/L of hydrogen gas was produced.
When glucose and butyric acid are co-substrates, CEF+NR group, the amount of glucose consumed after fermentation is increased to 26.35g/L, and the biomass (OD) 600 ) 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 hydrogen generation was accompanied.
In contrast, in the CEF system after NR addition, the accumulation of C.beijerinckii NCIMB 8052 biomass was less, and the substrate was more 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 butanol content in the solvent is improved from 76.47% to 79.91%. In terms of hydrogen generation, the yield of the CEF+NR system was increased by 83.33% compared to the OC group. For butanol fermentation of butyrate and glucose co-substrates, the combined application of CEF and NR was still very pronounced for strain c.beijerinckii NCIMB 8052.

Claims (6)

1. A cathodic electrofermentation method for preparing hydrogen and butanol by fermenting 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-type 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 a temperature of 37 ℃ and a rotating speed of 120 r/min;
the fermentation substrate is glucose, and the mass of the fermentation substrate added into 1L anaerobic fermentation medium is 20g;
the electron transfer medium is neutral red, and the amount of substances added with the electron transfer medium in 1L anaerobic fermentation medium is 0.5mmol;
(2) The three electrodes of the double-chamber H-type reactor are connected with constant potential while the culture is started in the step (1), and the double-chamber H-type reactor is started;
the double-chamber H-type reactor takes a titanium wire fixed graphite felt as a cathode and an anode, and an Ag/AgCl electrode as a reference electrode; the anode chamber is loaded with K equal to the volume of the cathode chamber loading material 4 (Fe(CN) 6 ) The solution serves as an electron donor;
the constant potential applied by the double-chamber H-type reactor is-0.75V;
(3) Sampling from the cathode chamber every 24 hours, 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 are not changed continuously, and ending the fermentation process;
the clostridium beijerinckii strain seed liquid is clostridium beijerinckii NCIMB 8052.
2. The cathodic electrofermentation method for preparing hydrogen and butanol by fermenting clostridium beijerinckii reinforced with an electron transfer mediator according to claim 1, wherein the anaerobic fermentation medium is prepared by the following steps: mixing yeast powder, K 2 HPO 4 、KH 2 PO 4 、MgSO 4 ·7H 2 O、MnSO 4 ·H 2 O、FeSO 4 ·7H 2 O, naCl mixing para aminobenzoic acid, thiamine and biotin, introducing nitrogen, aerating to remove oxygen, and sterilizing at 121 ℃ for 20min to obtain the anaerobic fermentation culture medium.
3. The cathodic electrofermentation method for preparing hydrogen and butanol by fermenting clostridium beijerinckii reinforced with electron transfer mediator according to claim 2, wherein 1L of yeast powder 3g, K in the anaerobic fermentation medium 2 HPO 4 0.5g，KH 2 PO 4 0.5g，MgSO 4 ·7H 2 O 0.2g，MnSO 4 ·H 2 O 0.01g，FeSO 4 ·7H 2 O0.01 g, naCl 0.01g, p-aminobenzoic acid 0.001g, thiamine 0.001g, biotin 0.0001g.
4. The cathodic electrofermentation process for preparing hydrogen and butanol by enhanced fermentation of clostridium beijerinckii with an electron transfer mediator according to claim 1, wherein the clostridium beijerinckii seed solution is inoculated into the anaerobic fermentation medium to which the fermentation substrate and the electron transfer mediator are added in an amount of 5% by volume.
5. The cathodic electrofermentation process for producing hydrogen and butanol by fermentation of clostridium beijerinckii enhanced by an electron transfer mediator of claim 1, wherein said K is 4 (Fe(CN) 6 ) The concentration of the solution was 200mmol/L.
6. The cathodic electrofermentation method for preparing hydrogen and butanol by fermenting clostridium beijerinckii reinforced with an electron transfer mediator as claimed in claim 1, wherein said clostridium beijerinckii seed solution is purchased from China general microbiological culture collection center (CGMCC).
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