阅读说明：本技术 一种基于微生物互养互作与电发酵耦合梯级产氢产乙醇的方法 (A method of step hydrogen-producing and ethanol-producing is coupled with electricity fermentation based on microorganism syntrophism interaction ) 是由 邢德峰 李�真 于 2019-09-24 设计创作，主要内容包括：一种基于微生物互养互作与电发酵耦合梯级产氢产乙醇的方法,属于发酵产氢技术领域。本发明为了解决现有生物发酵制氢和生物发酵产乙醇过程中发酵细菌受代谢产物抑制,产氢效率低、产乙醇纯度低等问题。本发明将发酵产氢细菌哈尔滨产乙醇杆菌YUAN-3和电活性细菌地杆菌PCA在微生物电发酵MEF反应器中进行共培养,通过细菌代谢互作及微生物电发酵系统两步法梯级制取氢气和乙醇。与传统方法相比,本发明大大提高了氢气产率和乙醇纯度,降低了微生物电发酵系统内的细菌代谢产物抑制,提高了产氢效率及产物中氢气和乙醇的比例,同时并具有操作简便的优点。(A method of step hydrogen-producing and ethanol-producing is coupled with electricity fermentation based on microorganism syntrophism interaction, belongs to fermentation and hydrogen production technical field.The problems such as present invention inhibits to solve during existing fermentative hydrogen production and biofermentation producing and ethanol zymogenous bacteria by metabolite, and hydrogen generation efficiency is low, producing and ethanol purity is low.The present invention co-cultures fermentative hydrogen-producing bacteria Harbin producing and ethanol bacillus YUAN-3 and electroactive bacterium ground bacillus PCA in microorganism electricity fermentation MEF reactor, passes through bacterial metabolism interaction and microorganism electricity fermentation system two-step method step hydrogen making and ethyl alcohol.It is compared with the traditional method, the present invention substantially increases hydrogen yield and purity of alcohol, and the opsonigenous substance reduced in microorganism electricity fermentation system inhibits, and improves the ratio of hydrogen and ethyl alcohol in hydrogen generation efficiency and product, while and having the advantages that easy to operate.)

1. a kind of method for coupling step hydrogen-producing and ethanol-producing with electricity fermentation based on microorganism syntrophism interaction, it is characterised in that:

The operating process of this method is as follows:

Step 1 uses liquid EGC culture medium Anaerobic culturel self aggregation Harbin producing and ethanol bacillus YUAN-3 in constant-temperature table To white spherical particle is formed, thalline were collected by centrifugation, obtains the inoculum of Harbin producing and ethanol bacillus YUAN-3；

Step 2 uses liquid EGC culture medium Anaerobic culturel ground bacillus PCA to the muddy bacterium of formation pink in constant-temperature table Liquid, thalline were collected by centrifugation, obtains the inoculum of ground bacillus PCA；

Step 3, assembling microorganism electricity fermentation MEF reactor, first inserts carbon fibre anode and active carbon hot pressing hydrogen reduction cathode Dress in the reactor, then injects liquid EGC culture medium in reactor, the N for being 99.99% through purity₂It is sealed simultaneously after aeration Autoclave sterilization, reactor use disposable venous infusion needle connector gas collection bag, carbon fibre anode and active carbon heat It is connect after outer connecting resistance with DC power supply between pressure hydrogen reduction cathode；

Step 4, by the inoculum of the inoculum and ground bacillus PCA of step 1 and step 2 Harbin producing and ethanol bacillus YUAN-3 In the EGC culture medium in MEF reactor after being seeded to sterilizing, connection circuit, 35 DEG C of constant temperature incubations, and use multichannel magnetic force Blender stir culture cultivates 1 hour~10 hours generation hydrogen and ethyl alcohol.

2. a kind of side for coupling step hydrogen-producing and ethanol-producing with electricity fermentation based on microorganism syntrophism interaction according to claim 1 Method, it is characterised in that: the outer connecting resistance is the Ω of 10 Ω~50, and the voltage of DC power supply is 0.5V~1.0V.

3. a kind of side for coupling step hydrogen-producing and ethanol-producing with electricity fermentation based on microorganism syntrophism interaction according to claim 1 Method, it is characterised in that: the process of methods,anaerobic culture Harbin producing and ethanol bacillus YUAN-3 inoculum in the step one For；First by liquid EGC culture medium through high-temperature boiling, the high-purity N that purity is 99.99%₂Aeration and high temperature and high pressure steam sterilizing, Then it is inoculated with Harbin producing and ethanol bacillus YUAN-3, and is placed on shake culture 12 hours~48 hours in constant-temperature table, is obtained Bottom deposit is the muddy bacterium solution of the spherical agglutinating particle of white, and 10000r/h is centrifuged 2 minutes, abandons supernatant, obtains Harbin production Ethyl alcohol bacillus YUAN-3 inoculum.

4. a kind of side for coupling step hydrogen-producing and ethanol-producing with electricity fermentation based on microorganism syntrophism interaction according to claim 1 Method, it is characterised in that: the process of methods,anaerobic culture ground bacillus PCA inoculum in the step two are as follows: firstly, liquid EGC culture medium is through N₂And CO₂Gaseous mixture aeration and high temperature and high pressure steam sterilizing, are then inoculated with ground bacillus PCA, and be placed on constant temperature Shake culture 6 hours~24 hours in shaking table, obtain pink muddy bacterium solution, and 10000r/h is centrifuged 2 minutes, abandons supernatant, obtain To ground bacillus PCA inoculum.

5. a kind of side for coupling step hydrogen-producing and ethanol-producing with electricity fermentation based on microorganism syntrophism interaction according to claim 1 Method, it is characterised in that: is produced from the Harbin for accessing 0.005g~0.01g in the step four in EGC culture medium described in every 1L The ground bacillus PCA of ethyl alcohol bacillus YUAN-3 and 0.02g~0.04g.

6. according to claim 1, one kind described in any one of 3,4 or 5 is coupled based on microorganism syntrophism interaction with electricity fermentation The method of step hydrogen-producing and ethanol-producing, it is characterised in that: glucose of the EGC culture medium described in every 1L by 10g, the albumen of 1.0g Peptone, the K of the NaCl of 2.0g, 1.8g₂HPO₄, the MgCl of 0.1g₂·6H₂The NaH of the KCl of O, 0.3g, 1.6g₂PO₄, 1.5g's NaHCO₃, the CH of 0.2g₃The resazurin that the half Guang ammonia of L- and 0.3ml volume fraction of COONa, 0.2g~1.0g are 0.1% forms.

7. a kind of side for coupling step hydrogen-producing and ethanol-producing with electricity fermentation based on microorganism syntrophism interaction according to claim 6 Method, it is characterised in that: the configuration method of the liquid EGC culture medium is as follows, and all medicines in addition to L-cysteine are first added Product simultaneously boil 10 minutes, are added and L-cysteine and dissolve after being cooled to room temperature, and continue to boil and are settled to 1L, are then attached to In 250mL anaerobism bottle, every bottle of 150mL.

8. a kind of side for coupling step hydrogen-producing and ethanol-producing with electricity fermentation based on microorganism syntrophism interaction according to claim 1 Method, it is characterised in that: the magnetic stir bar of the multichannel magnetic stirring apparatus is 2cm, revolving speed 100rpm.

Technical field

The present invention relates to a kind of methods for coupling step hydrogen-producing and ethanol-producing with electricity fermentation based on microorganism syntrophism interaction, belong to Fermentation and hydrogen production technical field.

Background technique

Hydrogen Energy is considered as the clean energy resource of most potential, alternative fossil fuel, has calorific value height, flammability Can good, cleanliness without any pollution, it is sustainable many advantages, such as.But the method for traditional industry hydrogen making is at high cost, consumes vast resources And the energy, and generate Heavy environmental pollution.

Fermentative Biohydrogen Production Process generates clear as a kind of sustainable hydrogen producing technology while consuming debirs Clean efficient hydrogen as energy source has compared with traditional process for making hydrogen and produces that hydrogen rate is high, ecology is friendly, low energy consumption, at low cost etc. excellent Point, therefore the very attractive in reality and business application.According to the difference of liquid final product, Hydrogen Bio-production By Anaerobic Fermentation can divide For the types such as ethanol-type fermentation and the fermentation of butyric acid type.Wherein, ethanol-type fermentation production hydrogen acid resistance is stronger, and hydrogen generation efficiency is higher, In While producing hydrogen, main metabolites ethyl alcohol is alternatively arranged as living resources and is recycled.Existing fermentative hydrogen production and life Since zymogenous bacteria is inhibited by metabolite during object fermentation producing and ethanol, that there are hydrogen generation efficiencies is low, producing and ethanol purity is low etc. is asked Topic.

Summary of the invention

The present invention is zymogenous bacteria during the existing fermentative hydrogen production of solution and biofermentation producing and ethanol by metabolite The problems such as inhibition, hydrogen generation efficiency is low, producing and ethanol purity is low, is provided a kind of fermented based on microorganism syntrophism interaction with electricity and couples ladder The method of grade hydrogen-producing and ethanol-producing.

Technical solution of the present invention:

A method of step hydrogen-producing and ethanol-producing, the specific behaviour of this method are coupled with electricity fermentation based on microorganism syntrophism interaction Steps are as follows for work:

Step 1 uses liquid EGC culture medium Anaerobic culturel self aggregation Harbin producing and ethanol bacillus in constant-temperature table For YUAN-3 to white spherical particle is formed, thalline were collected by centrifugation, obtains the inoculum of Harbin producing and ethanol bacillus YUAN-3；

Step 2, it is muddy to pink is formed using liquid EGC culture medium Anaerobic culturel ground bacillus PCA in constant-temperature table Bacterium solution, thalline were collected by centrifugation, obtains the inoculum of ground bacillus PCA；

Step 3, assembling microorganism electricity fermentation MEF reactor, first by carbon fibre anode and active carbon hot pressing hydrogen reduction yin Pole inserts in the reactor, then injects liquid EGC culture medium in reactor, the high-purity N for being 99.99% through purity₂Aeration Afterwards sealing and autoclave sterilization, reactor use disposable venous infusion needle connector gas collection bag, carbon fibre anode and It is connect after outer connecting resistance with DC power supply between active carbon hot pressing hydrogen reduction cathode；

Step 4, by connecing for the inoculum and ground bacillus PCA of step 1 and step 2 Harbin producing and ethanol bacillus YUAN-3 Kind object is seeded in the MEF reactor after sterilizing, connection circuit, 35 DEG C of constant temperature incubations, and is stirred using multichannel magnetic stirring apparatus Culture is mixed, 1 hour~10 hours generation hydrogen and ethyl alcohol are cultivated.

The anode of the microorganism electricity fermentation MEF reactor is carbon fiber, and cathode is active carbon hot pressing hydrogen reduction cathode, Outer connecting resistance is the Ω of 10 Ω~50, and the voltage of DC power supply is 0.1~0.5mV of 0.5V~1.0V or constant cathode current potential.

The process of methods,anaerobic culture Harbin producing and ethanol bacillus YUAN-3 inoculum is in the step one；First By liquid EGC culture medium through high-temperature boiling, the high-purity N that purity is 99.99%₂Aeration and high temperature and high pressure steam sterilizing, then connect Kind Harbin producing and ethanol bacillus YUAN-3, and shake culture 12 hours~48 hours in constant-temperature table are placed on, it is heavy to obtain bottom Muddy bacterium solution of the product for the spherical agglutinating particle of white, 10000r/h centrifugation 2 minutes, abandoning supernatant obtain Harbin producing and ethanol bar Bacterium YUAN-3 inoculum.

The process of methods,anaerobic culture ground bacillus PCA inoculum in the step two are as follows: firstly, liquid EGC is cultivated Base is through N₂And CO₂Gaseous mixture aeration and high temperature and high pressure steam sterilizing, are then inoculated with ground bacillus PCA, and be placed in constant-temperature table and shake Culture 6 hours~24 hours is swung, pink muddy bacterium solution is obtained, 10000r/h is centrifuged 2 minutes, abandons supernatant, obtain ground bacillus PCA inoculum.

The Harbin producing and ethanol bar of 0.005g~0.01g is accessed in the step four in EGC culture medium described in every 1L The ground bacillus PCA of bacterium YUAN-3 and 0.02g~0.04g.

Glucose of the EGC culture medium described in every 1L by 10g, the peptone of 1.0g, the K of the NaCl of 2.0g, 1.8g₂HPO₄, The MgCl of 0.1g₂·6H₂The NaH of the KCl of O, 0.3g, 1.6g₂PO₄, the NaHCO of 1.5g₃, the CH of 0.2g₃COONa, 0.2g~ The resazurin that the half Guang ammonia of L- and 0.3ml volume fraction of 1.0g is 0.1% forms.

The configuration method of the liquid EGC culture medium is as follows, and all drugs in addition to L-cysteine are first added and boil Boiling 10 minutes is added after being cooled to room temperature and L-cysteine and dissolves, and continues to boil and is settled to 1L, is then attached to 250mL anaerobism In bottle, every bottle of 150mL.

The Harbin producing and ethanol bar of 0.005g~0.01g is accessed in the step four in EGC culture medium described in every 1L The ground bacillus PCA of bacterium YUAN-3 and 0.02g~0.04g.The Harbin 0.005g~0.01g is accessed in every 1L liquid EGC culture medium Producing and ethanol bacillus YUAN-3 inoculum and 0.02g~0.04g ground bacillus PCA inoculum connect in goods, and it is most short to start the production hydrogen time；It presses According to accessed in every 1L liquid EGC culture medium the Harbin 0.001g~0.03g producing and ethanol bacillus YUAN-3 inoculum and 0.005g~ 0.05g ground bacillus PCA inoculum is inoculated with, and two kinds of bacterium any valued combinations can carry out producing hydrogen production second within the above range Alcohol can obtain preferable hydrogen-producing and ethanol-producing effect.

It is 2cm, revolving speed 100rpm that the magnetic stirring apparatus, which obtains magnetic stir bar,.

The condition of the high pressure steam sterilization is 120 DEG C, and holding time is 30 minutes.

It is produced the invention has the following advantages: present invention one kind couples step with electricity fermentation based on microorganism syntrophism interaction The method of hydrogen-producing and ethanol, the present invention are total in microorganism electricity fermentation MEF reactor using fermentative hydrogen-producing bacteria and electroactive bacterium Culture, by bacterial metabolism interaction and microorganism electricity fermentation system two-step method step hydrogen making and ethyl alcohol, with conventional method phase Than since the ground bacillus PCA in microorganism electricity fermentation co-culture system can be metabolized using Harbin producing and ethanol bacillus YUAN-3 The acetic acid and CO of generation₂Growth converts electric energy for chemical energy, and produces hydrogen in microorganism electricity fermentation MEF reactor anode；Together When, the consumption of metabolite reduces the metabolic inhibition of fermentation and hydrogen production bacterium, promotes its highly effective hydrogen yield, and make H in product₂And ethyl alcohol Ratio is substantially improved, therefore the hydrogen yield produced is bigger, and hydrogen and purity of alcohol are higher, and have it is easy to operate, have both production The advantage of electricity.Using 10g/L~20g/L glucose as carbon source, microorganism electricity fermentation MEF reactor run 1~10 hour after after Start to generate hydrogen, it is 80mL/L- culture medium h that 60h or so, which reaches maximum hydrogen-producing speed, final obtainable hydrogen yield For 2.1mol- hydrogen/mol- glucose, the final concentration of 2000mg/L of ethyl alcohol accounts for the 72% of liquid fermentation production.

Detailed description of the invention

Fig. 1 is microorganism electricity fermentation MEF reactor of the invention；

1- DC power supply in figure, 2- resistance, 3- gas collection bag, 4- carbon fibre anode, 5- active carbon hot pressing hydrogen reduction yin Pole.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

Harbin producing and ethanol bacillus (Ethanoligenens harbinense) involved in following specific embodiments is The main production hydrogen function bacterium for the ethanol-type fermentation that the separation such as the peak Xing De in 2005 obtain, wherein bacterial strain YUAN-3 is made from biology The highly effective hydrogen yield bacterial strain of one plant of isolated strictly anaerobic in hydrogen reactor, it, which is reported unique one plant, has self-coagulation The hydrogen-producing bacteria of ability, deposit number are CGMCC No.1152.

Ground bacillus (Geobacter sulfurreducens) PCA is to be common in microorganism electrochemical system, is a kind of energy The electroactive bacterium of extracellular electron transmission is enough grown and carried out using acetic acid.Bond DR and Lovley DR et al. is in article Have in Electricity production by Geobacter sulfurreducens attached to electrodes Record about this plant of strain.