阅读说明：本技术 一种采用电发酵强化短链挥发性脂肪酸加链产己酸的方法 (Method for producing caproic acid by adopting electric fermentation to strengthen short-chain volatile fatty acid addition ) 是由 张宇 孙睿 刘德丽 高秀君 于 2021-03-05 设计创作，主要内容包括：本发明公开了一种采用电发酵强化短链挥发性脂肪酸加链产己酸的方法。本方法要解决目前采用的短链挥发性脂肪酸加链产己酸方法的成本高,需外加电子供体,己酸转化率低等突出问题,旨在通过利用微生物电解池提高己酸产率并连续回收己酸。微生物电解池为己酸生产提供了一条新途径。方法如下：一、组装并连接数据记录仪；二、电极材料预处理；三、组装反应器；四、MEC的接种启动与功能微生物的驯化；五、在厌氧条件下进行正式电发酵实验,完成电发酵强化短链脂肪酸加链产己酸方法。本发明以附着在电极上的微生物作为自再生催化剂,以原位产生的电极电子和氢气为还原力促进己酸合成,是一种快速、高效的产己酸方法。(The invention discloses a method for producing hexanoic acid by reinforcing short-chain volatile fatty acid addition through electric fermentation. The method aims to solve the outstanding problems of high cost, additional electron donor, low conversion rate of the caproic acid and the like of the conventional method for producing the caproic acid by adding the short-chain volatile fatty acid, and aims to improve the yield of the caproic acid and continuously recover the caproic acid by using a microbial electrolytic cell. The microbial electrolytic cell provides a new way for producing the caproic acid. The method comprises the following steps: firstly, assembling and connecting a data recorder; secondly, pretreating electrode materials; thirdly, assembling a reactor; fourth, inoculating and starting MEC and domesticating functional microorganisms; and fifthly, performing formal electric fermentation experiments under anaerobic conditions to finish the method for producing the caproic acid by reinforcing the short-chain fatty acid by electric fermentation. The invention takes the microorganisms attached to the electrode as the self-regeneration catalyst, and takes the electrode electrons and hydrogen generated in situ as the reducing power to promote the synthesis of the caproic acid, thereby being a quick and efficient method for producing the caproic acid.)

1. A method for producing hexanoic acid by adopting electric fermentation to strengthen short-chain volatile fatty acid addition is characterized by comprising the following steps:

firstly, assembling and connecting a data recorder: the data recorder is used for recording the current data change condition of the reactor in real time;

secondly, pretreatment of electrode materials: the anode material is a carbon brush, firstly deionized water is used for washing away impurities on the surface of the carbon brush, then acetone is used for soaking for 24 hours, then the carbon brush is placed into a muffle furnace and is sintered for 30min at the temperature of 400-600 ℃, and finally the carbon brush is placed into deionized water for later use; soaking the cation exchange membrane in a saturated sodium chloride solution for 24 hours, and then rinsing the cation exchange membrane with deionized water for later use; the cathode material is carbon cloth, according to the calculation that each square centimeter of cathode surface needs 0.5mg of platinum carbon, 90mg of 10% platinum carbon is weighed firstly, then 75 mu L of deionized water is added, 600 mu L of 5% Nafion membrane solution and 300 mu L of isopropanol are added after vortex oscillation, the vortex oscillation is carried out, then the platinum carbon dissolved into paste is evenly brushed on the carbon cloth, and the carbon cloth is dried for 24 hours for standby;

thirdly, assembling a single-chamber MEC reactor: adopting a single-pole-chamber microbial electrolytic cell, wherein the anode is a carbon brush, the cathode is carbon cloth, the volume of the electrolytic cell is 100-250 mL, the material of the electrolytic cell is glass, and the whole device is in a sealed anaerobic environment;

fourth, inoculation starting of MEC and domestication of functional microorganisms: starting a single-chamber MEC reactor at room temperature, applying a voltage of 0.8-1.2V, and simultaneously connecting a 10 omega resistor to measure the current of the reactor; mixing the domesticated caproic acid-producing bacteria in an anaerobic bottle with a culture medium in a ratio of 1: 10, adding the mixture into a single-chamber MEC reactor for starting, wherein the concentration of sodium acetate is 1-3 g/L, and the concentration of ethanol is 0-12 mL/L; 5d is a period, and inoculation starting of the MEC reactor and domestication of functional microorganisms are completed when the current periodic change is stable and the power generation peak value is stable;

fifthly, performing formal electric fermentation experiments under anaerobic conditions: adjusting the concentration of sodium acetate to be 1.5g/L and the concentration of ethanol to be 0-6 mL/L, running for 5-7 days at room temperature, and measuring the concentration change of the substrate and the product by using a gas chromatograph.

2. The method for producing hexanoic acid by using electric fermentation to strengthen short-chain volatile fatty acid addition according to claim 1, wherein in the second step, the carbon brush is soaked in acetone for 24 hours and then is fired at 450 ℃ of a muffle furnace for 30 minutes.

3. The method for producing hexanoic acid by using electric fermentation to strengthen short-chain volatile fatty acid chain addition is characterized in that in the fourth step, each liter of culture medium base solution contains 3.6 g/L ammonium dihydrogen phosphate, 0.33 g/L magnesium chloride hexahydrate, 0.2 g/L magnesium sulfate heptahydrate, 0.5 g/L calcium chloride dihydrate, 0.15 g/L potassium chloride, 4 g/L potassium carbonate, 3.7 g/L sodium hydroxide, 10mL/L minerals and 10mL/L vitamins.

4. The method for producing hexanoic acid by using electric fermentation to strengthen short-chain volatile fatty acid addition according to claim 1, wherein the electrolyte is added in step four under the protection of nitrogen.

5. The method for producing hexanoic acid by using electric fermentation to strengthen short-chain volatile fatty acid chain addition according to claim 1, wherein the sodium acetate is added in the fourth step at a concentration of 2g/L and the ethanol is added at a concentration of 0-8 mL/L.

6. The method for producing hexanoic acid by using electric fermentation to strengthen short-chain volatile fatty acid chain addition according to claim 1, wherein the sodium acetate is added in the fourth step at a concentration of 1.5g/L and the ethanol is added at a concentration of 0-6 mL/L.

Technical Field

The method relates to a method for producing hexanoic acid by adopting electric fermentation to strengthen short-chain volatile fatty acid addition.

Background

As social production develops, the demand for fuels and chemicals continues to increase resulting in an increasing amount of organic waste. Currently used fuels and chemicals are mainly derived from fossil resources, and the production of grain crops such as corn, sugarcane and palm is expanding. However, environmental pollution and global warming are aggravated by the large consumption of fossil resources, and the use of food crops for fuel and chemical production may compete with human food production. The production of high value additional products from mixed organic waste, such as anaerobic fermentation to produce biogas and composting anaerobic digestion to produce small molecular fatty acids, but the water content of the fermented substrate is high, and a huge cost factor exists in extracting miscible fermented products from the fermentation broth. Hexanoic acid, an emerging microbial fuel, can be produced from low-grade mixed organic waste. Hexanoic acid has low solubility and high energy density and is considered a very potential biofuel. Furthermore, caproic acid has wide application, can be directly used as feed additive, antibiotic and plant growth promoter, and can also be used as precursor of various commodities such as lubricating oil, perfume, paint additive and medicine.

The method for producing the caproic acid by anaerobic fermentation is a widely researched caproic acid production method at present, and the synthesis of the caproic acid is carried out by taking reductive chemicals such as ethanol, methanol, lactic acid and the like as external electron donors and taking sodium acetate as a substrate through reverse beta oxidation circulation. However, the addition of an electron donor during the synthesis results in a less economical synthesis system. The Microbial Electrolysis Cell (MEC) is one of the important means for recycling waste due to wide substrate range, short reaction period and high energy recovery rate, and has wide application in recycling high-value fermentation products. MEC technology is also considered to be the most efficient method for sustainable energy and biochemical production today.

Disclosure of Invention

The invention aims to improve the conversion rate of short-chain volatile fatty acid to caproic acid by using a microbial electrolytic cell and overcome the defects of large floor area, poor continuous operation effect and the like in the traditional anaerobic fermentation process.

The method for producing the caproic acid by strengthening the short-chain volatile fatty acid by microbial electrocatalysis specifically comprises the following steps:

firstly, assembling and connecting a data recorder: the data recorder is used for recording the current data change condition of the reactor in real time;

secondly, pretreatment of electrode materials: the anode material is a carbon brush, firstly deionized water is used for washing away impurities on the surface of the carbon brush, then the carbon brush is soaked in acetone for 24 hours and then is placed into a muffle furnace, the carbon brush is burnt for 30 minutes at the temperature of 400-600 ℃, and finally the carbon brush is placed into deionized water for standby (the purpose is to remove organic substances on the carbon brush); the cathode material is carbon cloth, according to the calculation that each square centimeter of cathode surface needs 0.5mg of platinum carbon, 90mg of 10% (referring to the mass percentage content of platinum in the platinum carbon) of platinum carbon is firstly weighed, 75 muL of deionized water is then added, 600 muL of 5% Nafion membrane solution (5% referring to the solid content of resin in the Nafion membrane solution) and 300 muL of isopropanol are added after vortex oscillation, vortex oscillation is carried out, then the platinum carbon dissolved into paste is uniformly brushed on the carbon cloth, and drying is carried out for 24h for standby application (the membrane solution is used as an adhesive, the platinum carbon can be successfully attached to the surface of the carbon cloth, the platinum carbon is used as a catalyst, and the platinum carbon catalyst is loaded on the carbon cloth, so that the electrocatalytic performance can be improved);

thirdly, assembling a single-chamber MEC reactor: adopting a single-pole-chamber microbial electrolytic cell, wherein the anode is a carbon brush, the cathode is carbon cloth, the volume of the electrolytic cell is 100-250 mL, the material of the electrolytic cell is glass, and the whole device is in a sealed anaerobic environment;

fourth, inoculation starting of MEC and domestication of functional microorganisms: starting a single-chamber MEC reactor at room temperature, applying a voltage of 0.8-1.2V, and simultaneously connecting a 10 omega resistor to measure the current of the reactor; mixing the domesticated caproic acid-producing bacteria in an anaerobic bottle with a culture medium in a ratio of 1: 10, adding the mixture into a single-chamber MEC reactor for starting, wherein the concentration of sodium acetate is 1-3 g/L, and the concentration of ethanol is 0-12 mL/L (sodium acetate/ethanol with different proportions affects the yield of hexanoic acid); 5d is a period, and inoculation starting of the MEC reactor and domestication of functional microorganisms are completed when the current periodic change is stable and the power generation peak value is stable;

fifthly, performing formal electric fermentation experiments under anaerobic conditions: adjusting the concentration of sodium acetate to be 1.5g/L and the concentration of ethanol to be 0-6 mL/L, running for 5-7 days at room temperature, and measuring the concentration change of the substrate and the product by using a gas chromatograph.

The acetate can be anodized and the proton at the cathode can be reduced to hydrogen biologically by using the MEC treatment system, the hydrogen production in the system is proved to be high-energy-efficiency and high-selectivity, the hydrogen can also be used as an electron donor to participate in the hexanoic acid synthesis process in situ, and the electrode can also provide part of the electron donor to realize the hexanoic acid synthesis without an external electron donor.

In the fourth step, each liter of culture medium base solution comprises 3.6 g/L of ammonium dihydrogen phosphate, 0.33 g/L of magnesium chloride hexahydrate, 0.2 g/L of magnesium sulfate heptahydrate, 0.5 g/L of calcium chloride dihydrate, 0.15 g/L of potassium chloride, 4 g/L of potassium carbonate, 3.7 g/L of sodium hydroxide, 10mL/L of mineral substances and 10mL/L of vitamin. The culture solution can ensure that the domestication of the functional microorganisms meets the requirements of electric fermentation.

The invention has the following beneficial results: the invention adopts anaerobic fermentation assisted with electrical stimulation to promote the conversion of short-chain fatty acids to caproic acid. The reactor can stably run after the caproic acid-producing microorganism is inoculated for multiple times, the microorganism attached to the electrode can carry out self-regeneration and is used as a self-regeneration catalyst to promote the conversion of short-chain fatty acid to caproic acid, and meanwhile, the electrode can provide part of electron donors for the reaction in the electrocatalysis process.

Drawings

FIG. 1 is a graph of volatile acid concentration versus treatment time during a run for examples and comparative experiments.

FIG. 2 is an electron distribution diagram before and after the run of the example and comparative experiment I.

Detailed Description

The technical solution of the present invention is not limited to the specific embodiments listed below, and includes any combination of the specific embodiments.

Firstly, assembling and connecting a data recorder: selecting a keithley2700 data recorder for recording the current data change condition of the reactor in real time;

secondly, pretreatment of electrode materials: the anode material is a carbon brush, firstly deionized water is used for washing away impurities on the surface of the carbon brush, then acetone is used for soaking for 24 hours, then the carbon brush is placed into a muffle furnace and is sintered for 30min at 450 ℃, and finally the carbon brush is placed into deionized water for later use; soaking the cation exchange membrane in a saturated sodium chloride solution for 24 hours, and then rinsing the cation exchange membrane with deionized water for later use; the cathode material is carbon cloth, according to the calculation that every square centimeter of cathode surface needs 0.5g of platinum carbon, 90mg of 10% of platinum carbon is weighed firstly, then 75 muL of deionized water is added, 600 muL of 5% Nafion membrane solution and 300 muL of isopropanol are added after vortex oscillation, the vortex oscillation is carried out, then the platinum carbon dissolved into paste is evenly brushed on the carbon cloth, and the drying is carried out for 24 hours for standby;

thirdly, assembling a single-chamber MEC reactor: adopting a single-pole chamber microbial electrolytic cell, wherein the anode is a carbon brush, the cathode is carbon cloth, the volume of the electrolytic cell is 200mL, the material of the electrolytic cell is glass, and the whole device is in a sealed anaerobic environment;

fourth, inoculation starting of MEC and domestication of functional microorganisms: starting the single-chamber MEC reactor at room temperature, applying a voltage of 0.8V-0.9V, and connecting a 10 omega resistor to measure the current of the reactor. Mixing the domesticated caproic acid-producing bacteria in an anaerobic bottle with a culture medium in a ratio of 1: 10, adding the mixture into a single-chamber MEC reactor for starting, wherein the concentration of sodium acetate is 2g/L, and the concentration of ethanol is 0-8 mL/L; 5d is a period, and inoculation starting of the MEC reactor and domestication of functional microorganisms are completed when the current periodic change is stable and the power generation peak value is stable;

fifthly, performing formal electric fermentation experiments under anaerobic conditions: adjusting the concentration of sodium acetate to 1.5g/L, running for 5-7 days at room temperature, and measuring the concentration change of the substrate and the product by using a gas chromatograph.

The second embodiment is as follows: the first difference between the present embodiment and the specific embodiment is: any data control instrument capable of recording data in real time can be adopted in the first step. The rest is the same as the first embodiment.

The third concrete implementation mode: the present embodiment is different from the first to the second embodiments in that: in the second step, the cathode material can adopt carbon brushes, carbon cloth or carbon felt and other materials. The rest is the same as the first embodiment.

The fourth concrete implementation mode: the difference between this embodiment mode and one of the first to third embodiment modes is: and in the second step, the carbon brush can be burnt for 30min at 400-600 ℃ after being soaked in acetone to remove impurities. The rest is the same as the first embodiment.

The fifth concrete implementation mode: the difference between this embodiment and one of the first to fourth embodiments is: in the third step, an MEC reactor with a volume of 100-250 mL can be used. The rest is the same as the first embodiment.

The sixth specific implementation mode: the difference between this embodiment and one of the first to fifth embodiments is: in the fourth step, the applied voltage may be 0.8-1.2V, and the other steps are the same as those of the first embodiment.

The seventh embodiment: the difference between this embodiment and one of the first to sixth embodiments is: in the fourth step and the fifth step, the adding amount of the sodium acetate can be 1-3 g/L, and the rest is the same as that of the first embodiment.

The specific implementation mode is eight: the present embodiment differs from one of the first to seventh embodiments in that: the amount of ethanol added in the fourth and fifth steps can be 0-12 mL/L, and the rest is the same as that in the first embodiment.

The following examples and comparative experiments were used to verify the beneficial effects of the present invention:

the first embodiment is as follows:

the method for producing the caproic acid by reinforcing the short-chain volatile fatty acid by the electrocatalysis of the microorganism specifically comprises the following steps:

firstly, assembling and connecting a data recorder: selecting a keithley2700 data recorder for recording the current data change condition of the reactor in real time;

secondly, pretreatment of electrode materials: the anode material is a carbon brush, firstly deionized water is used for washing away impurities on the surface of the carbon brush, then acetone is used for soaking for 24 hours, then the carbon brush is placed into a muffle furnace and is sintered for 30min at 450 ℃, and finally the carbon brush is placed into deionized water for later use; soaking the cation exchange membrane in a saturated sodium chloride solution for 24 hours, and then rinsing the cation exchange membrane with deionized water for later use; the cathode material is carbon cloth, according to the calculation that every square centimeter of cathode surface needs 0.5g of platinum carbon, 90mg of 10% of platinum carbon is weighed firstly, then 75 muL of deionized water is added, 600 muL of 5% Nafion membrane solution and 300 muL of isopropanol are added after vortex oscillation, the vortex oscillation is carried out, then the platinum carbon dissolved into paste is evenly brushed on the carbon cloth, and the drying is carried out for 24 hours for standby;

thirdly, assembling a single-chamber MEC reactor: adopting a single-pole chamber microbial electrolytic cell, wherein the anode is a carbon brush, the cathode is carbon cloth, the volume of the electrolytic cell is 200mL, the material of the electrolytic cell is glass, and the whole device is in a sealed anaerobic environment;

fourth, inoculation starting of MEC and domestication of functional microorganisms: the single-chamber MEC reactor was started at room temperature, and a voltage of 0.8V was applied while a 10 Ω resistor was switched in to measure the reactor current. Mixing the domesticated caproic acid-producing bacteria in an anaerobic bottle with a culture medium in a ratio of 1: 10 volume ratio is added into a single-chamber MEC reactor for starting, wherein the concentration of sodium acetate is 2g/L, and the concentration of ethanol is 8 mL/L; 5d is a period, and inoculation starting of the MEC reactor and domestication of functional microorganisms are completed when the current periodic change is stable and the power generation peak value is stable;

fifthly, performing formal electric fermentation experiments under anaerobic conditions: adjusting the concentration of sodium acetate to be 1.5g/L and the concentration of ethanol to be 6mL/L, running for 5-7 days at room temperature, and measuring the concentration change of the substrate and the product by using a gas chromatograph.

Comparison experiment one:

the method for producing the caproic acid by adding the short-chain volatile fatty acid without carrying out microbial electrocatalysis strengthening is specifically completed according to the following steps:

firstly, pretreatment of electrode materials: the anode material is a carbon brush, firstly deionized water is used for washing away impurities on the surface of the carbon brush, then acetone is used for soaking for 24 hours, then the carbon brush is placed into a muffle furnace and is sintered for 30min at 450 ℃, and finally the carbon brush is placed into deionized water for later use; soaking the cation exchange membrane in a saturated sodium chloride solution for 24 hours, and then rinsing the cation exchange membrane with deionized water for later use; the cathode material is carbon cloth, according to the calculation that every square centimeter of cathode surface needs 0.5g of platinum carbon, 90mg of 10% of platinum carbon is weighed firstly, then 75 muL of deionized water is added, 600 muL of 5% Nafion membrane solution and 300 muL of isopropanol are added after vortex oscillation, the vortex oscillation is carried out, then the platinum carbon dissolved into paste is evenly brushed on the carbon cloth, and the drying is carried out for 24 hours for standby;

secondly, assembling a single-chamber MEC reactor: adopting a single-pole chamber microbial electrolytic cell, wherein the anode is a carbon brush, the cathode is carbon cloth, the volume of the electrolytic cell is 200mL, the material of the electrolytic cell is glass, and the whole device is in a sealed anaerobic environment;

thirdly, starting inoculation of anaerobic fermentation and domesticating functional microorganisms: starting the single-chamber anaerobic reactor at room temperature. Mixing the domesticated caproic acid-producing bacteria in an anaerobic bottle with a culture medium in a ratio of 1: 10, adding the mixture into a single-chamber anaerobic reactor for starting, wherein the concentration of sodium acetate is 2 g/L; 5d is a period for waiting for substrates and products;

fourthly, performing formal electric fermentation experiments under anaerobic conditions: adjusting the concentration of sodium acetate to be 1.5g/L, operating at room temperature for 5-7 days, adjusting the concentration of ethanol to be 6mL/L, and measuring the concentration change of a substrate and a product by using a gas chromatograph.

FIG. 1 is a graph of volatile acid concentration versus treatment time during a run for examples and comparative experiments. FIG. 1A shows the change in the concentration of substrate and product in the reactor under open circuit conditions, and FIG. 1B shows the change in the concentration of substrate and product in the reactor under closed circuit conditions. It can be seen from the figure that the content of caproic acid in the applied voltage experiment group is obviously increased, and finally reaches 8722.4920 mg COD/L, which is 1.28 times of that in the simple anaerobic fermentation process. The consumption of sodium acetate and ethanol in the applied voltage group and the pure anaerobic fermentation group are basically consistent, but the yield of the caproic acid is larger and the yield of the butyric acid is less in the applied voltage group, which shows that the applied voltage can promote the conversion process of the butyric acid to the caproic acid.

FIG. 2 is an electron distribution diagram before and after the run of the example and comparative experiment I. It can be seen from the figure that the electron donor of the applied voltage group increases an electron donor-current compared with the pure anaerobic fermentation test group, the electron recovery rates of the two groups after the reaction are 93.05% and 94.60%, respectively, and the electron recovery rate of the applied voltage group is slightly improved, but the electron recovery rate of the applied voltage group in the caproic acid is 75.5% which is 1.32 times that of the pure anaerobic fermentation group, which indicates that the applied voltage can promote the electron recovery of the caproic acid in the product.