A method for constructing a multi-layer biofilm electrode and the multi-layer biofilm electrode

Abstract

The present application discloses a method for constructing a multi-layer biofilm electrode and the multi-layer biofilm electrode. The method comprises the following steps: providing a reaction system including an H-type electrolytic cell composed of a cathode chamber and an anode chamber, a working electrode, a counter electrode, a reference electrode, and a culture medium, and adding the culture medium to the cathode or anode chamber; adding a first microbial strain to the cathode and / or anode chamber, and then applying an electric potential to enable the first microbial strain to grow on the outer surface of at least one of the working electrode and the counter electrode to form a first biofilm; then inoculating a second microbial strain into the system to enable the second microbial strain to utilize the metabolites of the first microbial strain to form a second biofilm outside the first biofilm; introducing a gas into the cathode or anode chamber, setting a constant electric potential, culturing and domesticating the biofilm, and performing microbial electrosynthesis; adjusting the pH and temperature of the system according to the optimum growth pH and temperature of the microbial strain, and monitoring the pH.

Description

Technical Field

[0001] The present invention relates to the field of microbial electrochemical technology, and particularly to a method for constructing a multi-layer biofilm electrode and a multi-layer biofilm electrode. Background Art

[0002] Environmental pollution and greenhouse gas emissions have become the main problems in the environmental field and have attracted extensive attention from all sectors of society in recent years. As a new energy production and waste and pollutant treatment technology, microbial electrochemistry can utilize waste resources through microorganisms. For example, in the cathode, carbon dioxide can be converted into organic substances such as acetic acid through microbial electrosynthesis, or nitrate in wastewater can be reduced; in the anode, organic substances in wastewater can be degraded, thereby reducing the effluent COD, etc.

[0003] Biofilm is an important part of a microbial electrolytic cell. However, under natural conditions, the formation rate of biofilm is slow, and the bacterial species are complex, and the metabolic products cannot be regulated directionally. Therefore, it is necessary to develop a method for constructing a multi-layer biofilm electrode. Summary of the Invention

[0004] The following is an overview of the subject matter described in detail in this article. This overview is not intended to limit the protection scope of this application.

[0005] This application provides a method for constructing a multi-layer biofilm electrode and a multi-layer biofilm electrode. The method of this application uses porous corrosion-resistant conductive materials such as foam carbon, nanoporous carbon, and carbon fiber hollow fiber membranes as the electrode biofilm substrate. By controlling the strains, anoxic and anaerobic conditions are created in the reactor, and different bacterial species are used for aerobic conditions and their electroactivity to construct a multi-layer biofilm electrode, promoting interspecies electron transfer. At the same time, problems such as insufficient gas utilization can be avoided, resulting in increased costs and waste of resources. As a key process in the microbial electrosynthesis process, the multi-layer biofilm electrode formed by the present invention can significantly improve the product yield and electrotransformation efficiency, and realize the controllability of microbial electrosynthesis.

[0006] In one aspect, this application provides a method for constructing a multi-layer biofilm electrode, including the following steps:

[0007] 1) Provide a reaction system including an H-type electrolytic cell composed of a cathode chamber and an anode chamber, a working electrode, a counter electrode, a reference electrode, and a culture medium. Add the culture medium to the cathode chamber or the anode chamber, and purge with nitrogen for 30 min - 120 min;

[0008] 2) Add the first electroactive microbial strain to the cathode chamber and / or anode chamber, and then apply an electric potential to the cathode chamber and / or anode chamber to allow the first microbial strain to grow on the outer surface of at least one of the working electrode and the counter electrode to form an electroactive first biofilm; then inoculate the second microbial strain into the reaction system to allow the second microbial strain to utilize the metabolites of the first microbial strain to form a second biofilm outside the electroactive first biofilm;

[0009] 3) Introduce gas into the cathode chamber or anode chamber, set a constant electric potential, cultivate and domesticate the biofilm obtained in step 2), and perform microbial electrosynthesis;

[0010] 4) Adjust the pH and temperature of the reaction system according to the optimal growth pH and temperature of the microbial strains used, and monitor the pH in the reaction system in real time.

[0011] In the embodiments of the present application, in step 1), the working electrode and the counter electrode are made of a carbon-based material selected from carbon foam, nanoporous carbon, carbon fiber hollow fiber membrane or graphite sheet, and the reference electrode is selected from Ag / AgCl electrode, calomel electrode or hydrogen electrode.

[0012] In the embodiments of the present application, in step 2), if the cathode is the working electrode, the applied electric potential is -0.5V to -1.5V (relative to Ag / AgCl), and if the anode is the working electrode, the applied electric potential is 0.5V to 1.5V (relative to Ag / AgCl).

[0013] In the embodiments of the present application, in step 2), after the formation of the second biofilm, a third microbial strain that utilizes the metabolites of the second microbial strain as its own reaction substrate is inoculated into the reaction system to form a third biofilm.

[0014] In the embodiments of the present application, in step 2), an anode multi-layer biofilm or a cathode multi-layer biofilm is constructed according to whether the working electrode is an anode electrode or a cathode electrode.

[0015] In the embodiments of the present application, in step 2), if a cathode multi-layer biofilm is constructed, the first microbial strain is selected from acid-producing bacteria, alcohol-producing bacteria, Pseudomonas, such as Sporomusa, Clostridium, Geobacter or Shewallena; and the second microbial strain is selected from butyric acid-producing bacteria or nitrite-reducing bacteria.

[0016] In an embodiment of the present application, in step 2), if an anode multi-layer biofilm is formed, the first microbial strain is an aerobic fungus selected from the genus Trichoderma, Penicillium, Myrothecium or Chaetomium, such as Trichoderma reesei, Trichoderma viride or Trichoderma harzianum; and the second microbial strain is a lactic acid bacterium selected from the genus Lactobacillus, Pediococcus or Streptococcus, such as Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus bulgaricus or Lactobacillus johnsonii.

[0017] In an embodiment of the present application, if a cathode three-layer biofilm is constructed, the third microbial strain is selected from chain-elongating bacteria, such as the genus Clostridium, Clostridium bacterium or Desulfovibrio; if an anode three-layer biofilm is constructed, the third-layer microbial strain is an anaerobic acid-producing bacterium selected from the genus Clostridium, Bacteroides, Butyrivibrio or Bifidobacterium, such as Clostridium ljungdahlii, Butyrivibrio fibrisolvens, Bifidobacterium or Clostridium tyrobutyricum.

[0018] In an embodiment of the present application, in step 1), the culture medium in the cathode chamber is composed of 2 - 6 g / L KH2PO4, 4 - 8 g / L K2HPO4, 0.2 - 2 g / L NH4Cl, 0 - 0.5 g / L KCl, 0 - 1.0 g / L NaCl, 0 - 0.5 g / L MgCl2·6H2O, 0 - 0.1 g / L CaCl2, 1 - 10 mL / L Wolin's vitamin solution and 1 - 10 mL / L Wolin's mineral solution; the culture medium in the anode chamber is composed of 2 - 6 g / L KH2PO4, 4 - 8 g / L K2HPO4, 0.2 - 2 g / L NH4Cl, 1 - 10 mL / L Wolin's vitamin solution and 1 - 10 mL / L Wolin's mineral solution.

[0019] Optionally, in step 1), a buffer pair, such as dipotassium hydrogen phosphate / monopotassium phosphate, is added to the culture medium.

[0020] In the examples of the present application, in step 1), the working electrode is disposed in the cathode chamber or the anode chamber.

[0021] In the examples of the present application, in step 3), the gas is CO2, CO in the cathode chamber and O2 in the anode chamber.

[0022] In the examples of the present application, in step 1), according to the chamber where the working electrode is located, the culture medium is divided into a cathode culture medium or an anode culture medium.

[0023] In the examples of the present application, an appropriate reducing substance is added to the cathode culture medium to consume the remaining trace oxygen in the cathode culture medium, such as cysteine hydrochloride (0.1 - 0.4 g / L).

[0024] In the examples of the present application, in step 2), after adding the first microbial strain or after adding the second microbial strain, glucose / yeast powder is immediately added as a carbon source to accelerate the growth of the first microbial strain and the second microbial strain subsequently;

[0025] Optionally, in step 2), after adding the first microbial strain or after adding the second microbial strain, 10 - 100 μg / L of a signal molecule, such as N-butyryl-L-homoserine lactone or N-hexanoyl-L-homoserine lactone, is immediately added to the reaction system to promote the growth of the first biofilm and the second biofilm subsequently.

[0026] In the examples of the present application, in step 2), after adding the third microbial strain, 10 - 100 μg / L of a signal molecule, such as N-butyryl-L-homoserine lactone or N-hexanoyl-L-homoserine lactone, is immediately added to the reaction system to promote the growth of the third biofilm subsequently.

[0027] In the examples of the present application, in step 2), the inoculation amounts of the first microbial strain and the second microbial strain are 1 - 20% (v / v), preferably 5 - 10% (v / v).

[0028] In the examples of the present application, in step 2), the inoculation amount of the third microbial strain is 1 - 20% (v / v), preferably 5 - 10% (v / v).

[0029] In the examples of the present application, in step 3), the aeration rate of the gas is 10 - 100 mL / min.

[0030] In an embodiment of the present application, in step 4), the pH of the bacterial strain used is 5-8;

[0031] Optionally, in step 4), during the reaction process, an alkali such as NaOH is used to adjust the pH of the reaction system to 5-8 according to the optimal pH of the strain to avoid anodic acidification, and an acid such as H3PO4 is used to adjust the pH of the reaction system to 5-8 according to the optimal pH of the strain to avoid cathodic alkalization;

[0032] Optionally, in step 4), the temperature of the reaction system is controlled between 25-60 °C.

[0033] On the other hand, the present application provides a multi-layer biofilm electrode manufactured by the above method, and the multi-layer biofilm electrode includes:

[0034] An electroactive first biofilm, which is disposed on the outer surface of at least one of the working electrode and the counter electrode;

[0035] A second biofilm, which is disposed on the first biofilm.

[0036] In an embodiment of the present application, the biofilm electrode further includes a third biofilm disposed on the second biofilm.

[0037] The advantage of the multi-layer biofilm is that the reactions of 2-3 different bioreactors are coupled in one reactor, which can accelerate interspecies electron transfer and metabolite transfer, thereby improving the reaction efficiency and economic benefits; it can also break through the problem that it is difficult for heterogeneous microorganisms to coexist due to different oxygen demands and growth conditions.

[0038] Other features and advantages of the present application will be described in the subsequent specification, and, in part, will become apparent from the specification, or will be understood by implementing the present application. Other advantages of the present application can be achieved and obtained through the solutions described in the specification. Description of the Drawings

[0039] Figure 1 is a schematic diagram of a reactor for constructing a two-layer biofilm on a cathode hollow fiber membrane electrode according to Embodiment 1 of the present application.

[0040] Figure 2 is a schematic diagram of constructing a three-layer biofilm on a cathode hollow fiber membrane electrode according to Embodiment 4 of the present application. Detailed Description of the Embodiments

[0041] To make the objectives, technical solutions, and advantages of this application more clear and understandable, the embodiments of this application will be described in detail below. It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined arbitrarily with each other.

[0042] An embodiment of this application provides a method for constructing a multi-layer biofilm electrode, including the following steps:

[0043] 1) Provide a reaction system including an H-type electrolytic cell composed of a cathode chamber and an anode chamber, a working electrode, a counter electrode, a reference electrode, and a culture medium. Add the culture medium to the cathode chamber or the anode chamber, and purge with nitrogen for 30 min - 120 min;

[0044] 2) Add an electroactive first microbial strain to the cathode chamber and / or the anode chamber, and then apply an electric potential to the cathode chamber and / or the anode chamber to allow the first microbial strain to grow on the outer surface of at least one of the working electrode and the counter electrode to form an electroactive first biofilm; then inoculate a second microbial strain into the reaction system to allow the second microbial strain to utilize the metabolites of the first microbial strain to form a second biofilm outside the electroactive first biofilm;

[0045] 3) Pass a gas into the cathode chamber or the anode chamber, set a constant electric potential, and culture and domesticate the biofilm obtained in step 2);

[0046] 4) Adjust the pH and temperature of the reaction system according to the optimal growth pH and temperature of the microbial strains used, and monitor the pH in the reaction system in real time.

[0047] In the embodiment of this application, in step 1), the working electrode and the counter electrode are made of a carbon-based material selected from carbon foam, nanoporous carbon, carbon fiber hollow fiber membrane, or graphite sheet, and the reference electrode is selected from an Ag / AgCl electrode, a calomel electrode, or a hydrogen electrode.

[0048] In the embodiment of this application, in step 2), if the cathode is the working electrode, the applied electric potential is -0.5 V to -1.5 V (relative to Ag / AgCl), and if the anode is the working electrode, the applied electric potential is 0.5 V to 1.5 V (relative to Ag / AgCl).

[0049] In the embodiment of this application, in step 2), after forming the second biofilm, it further includes inoculating a third microbial strain that uses the metabolites of the second microbial strain as its own reaction substrate into the reaction system to form a third biofilm.

[0050] In an embodiment of the present application, in step 2), an anodic multi-layer biofilm or a cathodic multi-layer biofilm is constructed according to whether the working electrode is an anode electrode or a cathode electrode.

[0051] In an embodiment of the present application, in step 2), if a cathodic multi-layer biofilm is constructed, the first microbial strain is selected from acid-producing bacteria, alcohol-producing bacteria, Pseudomonas, such as Sporomusa, Clostridium, Geobacter or Shewallena; and the second microbial strain is selected from butyric acid-producing bacteria or nitrite-reducing bacteria.

[0052] In an embodiment of the present application, in step 2), if an anodic multi-layer biofilm is constructed, the first microbial strain is an aerobic fungus, which is selected from the genus Trichoderma, Penicillium, Myrothecium or Chaetomium, such as Trichoderma reesei, Trichoderma viride or Trichoderma harzianum; and the second microbial strain is a lactic acid bacterium, which is selected from the genus Lactobacillus, Pediococcus or Streptococcus, such as Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus bulgaricus or Lactobacillus johnsonii.

[0053] In an embodiment of the present application, if a cathodic three-layer biofilm is constructed, the third microbial strain is selected from chain elongation bacteria, such as the genus Clostridium, Clostridium bacterium or Desulfovibrio; if an anodic three-layer biofilm is constructed, the third-layer microbial strain is an anaerobic acid-producing bacterium, which is selected from the genus Clostridium, Bacteroides, Butyrivibrio or Bifidobacterium, such as Clostridium ljungdahlii, Butyrivibrio fibrisolvens, Bifidobacterium or Clostridium tyrobutyricum.

[0054] In an embodiment of the present application, in step 1), the culture medium in the cathode chamber consists of 2 - 6 g / L KH₂PO₄, 4 - 8 g / L K₂HPO₄, 0.2 - 2 g / L NH₄Cl, 0 - 0.5 g / L KCl, 0 - 1.0 g / L NaCl, 0 - 0.5 g / L MgCl₂·6H₂O, 0 - 0.1 g / L CaCl₂, 1 - 10 mL / L of Wolin's vitamin solution, and 1 - 10 mL / L of Wolin's mineral solution; the culture medium in the anode chamber consists of 2 - 6 g / L KH₂PO₄, 4 - 8 g / L K₂HPO₄, 0.2 - 2 g / L NH₄Cl, 1 - 10 mL / L of Wolin's vitamin solution, and 1 - 10 mL / L of Wolin's mineral solution.

[0055] Optionally in step 1), a buffer pair, such as dipotassium hydrogen phosphate / potassium dihydrogen phosphate, is added to the culture medium.

[0056] In an embodiment of the present application, in step 1), the working electrode is disposed in the cathode chamber or the anode chamber.

[0057] In an embodiment of the present application, in step 3), the gas is CO₂ and CO in the cathode chamber, and O₂ in the anode chamber.

[0058] In an embodiment of the present application, in step 1), according to the chamber where the working electrode is located, the culture medium is divided into cathode culture medium or anode culture medium.

[0059] In an embodiment of the present application, an appropriate reducing substance is added to the cathode culture medium to consume the remaining trace oxygen in the cathode culture medium, such as cysteine hydrochloride (0.1 - 0.4 g / L).

[0060] In an embodiment of the present application, in step 2), after adding the first microbial strain or after adding the second microbial strain, glucose / yeast powder is immediately added as a carbon source to subsequently accelerate the growth of the first microbial strain and the second microbial strain.

[0061] Optionally in step 2), after adding the first microbial strain or after adding the second microbial strain, 10 - 100 μg / L of a signal molecule, such as N-butyryl-L-homoserine lactone or N-hexanoyl-L-homoserine lactone, is immediately added to the reaction system to subsequently promote the growth of the first biofilm and the second biofilm.

[0062] In the embodiments of the present application, in step 2), after adding the third microbial strain, 10 - 100 μg / L of a signal molecule, such as N-butyryl-L-homoserine lactone or N-hexanoyl-L-homoserine lactone, is immediately added to the reaction system to promote the growth of the third biofilm subsequently.

[0063] In the embodiments of the present application, in step 2), the inoculation amounts of the first microbial strain and the second microbial strain are respectively 1 - 20% (v / v), preferably 5 - 10% (v / v).

[0064] In the embodiments of the present application, in step 2), the inoculation amount of the third microbial strain is 1 - 20% (v / v), preferably 5 - 10% (v / v).

[0065] In the embodiments of the present application, in step 3), the aeration rate of the gas is 10 - 100 mL / min.

[0066] In the embodiments of the present application, in step 4), the pH of the microbial strain used is 5 - 8;

[0067] Optionally in step 4), during the reaction process, an alkali such as NaOH is used to adjust the pH of the reaction system to 5 - 8 according to the optimal pH of the strain to avoid anodic acidification, and an acid such as H3PO4 is used to adjust the pH of the reaction system to 5 - 8 according to the optimal pH of the strain to avoid cathodic alkalization;

[0068] Optionally in step 4), the temperature of the reaction system is controlled between 25 - 60 °C.

[0069] The embodiments of the present application provide a multi-layer biofilm electrode manufactured by the above method, and the multi-layer biofilm electrode includes:

[0070] An electroactive first biofilm, which is disposed on the outer surface of at least one of the working electrode and the counter electrode;

[0071] A second biofilm, which is disposed on the first biofilm.

[0072] In the embodiments of the present application, the multi-layer biofilm electrode further includes a third biofilm disposed on the second biofilm.

[0073] To further illustrate the content of the present invention, some embodiments will be further enumerated below. It should be noted that in the present application, biofilms can be constructed not only on the cathode but also on the anode; the reactor is not limited to the H-type electrolytic cell used, the electroactive microorganisms are not limited to the strains used, the working electrode is not limited to the carbon-based material used, the counter electrode is not limited to the graphite sheet electrode used, and the present invention is not limited to the enumerated embodiments.

[0074] For the experiments involved in the following examples, if not specified explicitly, they can be carried out according to the conventional conditions or methods in the art. The strains used in the examples and comparative examples of this application are from the German Collection of Microorganisms and Cell Cultures (DSMZ) and the American Type Culture Collection (ATCC). The electrodes used are from Shanghai Chenhua. The reagents used are all purchased from Shanghai Merck Biochemical Technology Co., Ltd. Wolin's vitamin solution and Wolin's mineral solution are both purchased from Shangdong Top Biological Engineering Co., Ltd.

[0075] Example 1

[0076] This example provides a method for constructing a cathode multi-layer biofilm electrode

[0077] (1) The reactor is an H-type electrolytic cell composed of a cathode chamber and an anode chamber. The volumes of both the cathode chamber and the anode chamber are 100 mL. The cathode is the working electrode, and the electrode material is a carbon fiber hollow fiber membrane; the anode is the counter electrode, and the electrode material is a graphite sheet; the reference electrode (Ag / AgCl) is set in the cathode chamber. First, 80 mL of culture medium is added to the cathode chamber. The culture medium is composed of 3 g / L KH2PO4, 5.8 g / L K2HPO4, 0.5 g / L NH4Cl, 0.09 g / L MgCl2·6H2O, 0.02 g / L CaCl2, 10 mL / L Wolin's vitamin solution, and 10 mL / L Wolin's mineral solution; then the culture medium is purged with nitrogen for 60 min to remove the dissolved oxygen in the culture medium; 80 mL of electrolyte is added to the anode chamber. The electrolyte is composed of 3 g / L KH2PO4 and 5.8 g / L K2HPO4, and the pH is adjusted to 5.0 with 1 M phosphoric acid.

[0078] (2) The first microbial strain Sporomusa ovata DSM2662 is added to the cathode chamber, and the inoculation concentration is 10% (v / v) (that is, 10 mL of OD is added to 100 mL of the culture medium) 600Sporomusa ovata bacterial solution with a concentration of 0.5) and 0.5 g / L of yeast powder was added to promote the growth and biofilm formation of Sporomusa ovata. Then, a potential of -0.6 V (versus Ag / AgCl) was applied to the reaction system, and after 3 days of cultivation, Sporomusa ovata DSM2662 grew on the cathode hollow fiber membrane electrode to form an electroactive first biofilm. Then, the second microbial strain, Clostridium kluyveri, which is a chain extender, was inoculated into the cathode chamber at an inoculation concentration of 10% (v / v), and 0.5 g / L of yeast powder and 10 μg / L of N-butyryl-L-homoserine lactone as a signaling molecule were added to promote the growth and biofilm formation of Clostridium kluyveri. Clostridium kluyveri can utilize acetic acid to synthesize organic acids such as butyric acid and caproic acid. After 3 days of cultivation, Clostridium kluyveri grew outside the first biofilm to form a second biofilm.

[0079] (3) Only CO2 gas was introduced into the cathode chamber at an aeration rate of 50 mL / min, and the potential was gradually adjusted from -0.6 V to -1.2 V for microbial electroreduction to reduce CO2 to butyric acid, caproic acid, etc., and the anode underwent a water electrolysis reaction.

[0080] (4) The reaction temperature was controlled at 35 °C, and the optimal pH of the bacterial strain was about 7.0. However, alkalization easily occurred during the electro-synthesis process in the cathode chamber. Therefore, every day, according to the pH of the cathode medium, 1 M phosphoric acid solution was used to adjust the pH to 6.8.

[0081] In this example, the CO2 reduction efficiency at the cathode reached more than 90%, the acetic acid concentration reached 3000 mg / L, the butyric acid concentration reached 1800 mg / L, and the caproic acid concentration reached 500 mg / L.

[0082] Example 2

[0083] This example provides a method for constructing a cathode multi-layer biofilm electrode.

[0084] (1) The reactor is an H-type electrolytic cell composed of a cathode chamber and an anode chamber. The volumes of both the cathode chamber and the anode chamber are 100 mL. The cathode is the working electrode, and the electrode material is a carbon fiber hollow fiber membrane; the anode is the counter electrode, and the electrode material is a graphite sheet; the reference electrode (Ag / AgCl) is set in the cathode chamber. First, 80 mL of a culture medium is added to the cathode chamber. The culture medium consists of 3 g / L KH2PO4, 5.8 g / L K2HPO4, 1 g / L NH4Cl, 0.1 g / L KCl, 0.8 g / L NaCl, 0.09 g / L MgCl2·6H2O, 0.02 g / L CaCl2, 10 mL / L Wolin's vitamin solution, and 10 mL / L Wolin's mineral solution. Then, the culture medium is purged with nitrogen for 60 min to remove the dissolved oxygen in the culture medium. 80 mL of an electrolyte solution is added to the anode chamber. The electrolyte solution consists of 3 g / L KH2PO4 and 5.8 g / L K2HPO4, and the pH is adjusted to 5.0 using 1 M phosphoric acid.

[0085] (2) The first microbial strain, Clostridium ljungdahlii, is added to the cathode chamber at an inoculation concentration of 10% (v / v) (i.e., 10 mL of a Clostridium ljungdahlii bacterial solution with an OD 600 of 0.5 is added to 100 mL of the culture medium), and 0.5 g / L of yeast powder and 10 μg / L of N-butyryl-L-homoserine lactone as a signaling molecule are added to promote the growth and biofilm formation of Clostridium ljungdahlii. Then, a potential of -0.6 V (relative to Ag / AgCl) is applied to the reaction system. After 3 days of cultivation, Clostridium ljungdahlii grows on the cathode hollow fiber membrane electrode to form an electroactive first biofilm. Then, the second microbial strain, the chain elongation bacterium Clostridium kluyveri, is inoculated into the cathode chamber at an inoculation concentration of 10% (v / v), and 0.5 g / L of yeast powder and 10 μg / L of N-butyryl-L-homoserine lactone as a signaling molecule are added to promote the growth and biofilm formation of Clostridium kluyveri. Clostridium kluyveri can utilize acetic acid and ethanol to produce organic acids such as butyric acid and caproic acid. After 3 days of cultivation, Clostridium kluyveri grows outside the first biofilm to form a second biofilm.

[0086] (3) Only CO2 gas is introduced into the cathode chamber at an aeration rate of 50 mL / min, and the potential is gradually adjusted from -0.6 V to -1.2 V for microbial electroreduction to reduce CO2 to ethanol, acetic acid, butyric acid, caproic acid, etc. The anode undergoes a water electrolysis reaction.

[0087] (4) The reaction temperature is controlled at 35 °C. The optimum pH of the bacterial strain is about 6.0. However, alkalization easily occurs during the electro-synthesis process in the cathode chamber. Therefore, every day, according to the pH of the cathode culture medium, 1 M phosphoric acid solution is used to adjust the pH to 6.0.

[0088] In this example, the cathode CO2 reduction efficiency reaches more than 90%, the ethanol concentration reaches 500 mg / L, the acetic acid concentration reaches 2500 mg / L, the butyric acid concentration reaches 1800 mg / L, and the caproic acid concentration reaches 500 mg / L.

[0089] Example 3

[0090] This example provides a method for constructing an anode double-layer biofilm electrode.

[0091] (1) The reactor is an H-type electrolytic cell composed of a cathode chamber and an anode chamber. The volumes of both the cathode chamber and the anode chamber are 100 mL. The anode is the working electrode, and the electrode material is a carbon fiber hollow fiber membrane; the cathode is the counter electrode, and the electrode material is a graphite sheet; the reference electrode (Ag / AgCl) is set in the anode chamber. First, 80 mL of culture medium is added to the anode chamber. The culture medium consists of 3 g / L KH2PO4, 5.8 g / L K2HPO4, 0.5 g / L NH4Cl, 10 mL / L Wolin's vitamin solution, and 10 mL / L Wolin's mineral solution; 80 mL of electrolyte is added to the cathode chamber. The electrolyte consists of 3 g / L KH2PO4 and 5.8 g / L K2HPO4, and the pH is adjusted to 7.0 using 1 M NaOH.

[0092] (2) Add the first microbial strain Trichoderma reesei to the anode chamber, and the inoculation concentration is 1% (v / v) (that is, 1 mL OD is added to 100 mL of the culture medium) 600Trichoderma reesei bacterial solution with a concentration of 1), the reaction substrate was microcrystalline cellulose at 5 g / L, and 0.5 g / L of yeast powder was added to promote the growth of Trichoderma reesei and biofilm formation. Then, a potential of 0.5 V (relative to Ag / AgCl) was applied to the reaction system, and it was cultured for 48 h to allow Trichoderma reesei to grow on the anode hollow fiber membrane electrode to form an electroactive first biofilm; then, the second microbial strain Lactiplantibacillus pentosus was inoculated into the anode chamber at an inoculation concentration of 1% (v / v), and 0.5 g / L of yeast powder was added to promote the growth of Lactiplantibacillus pentosus and biofilm formation. Lactiplantibacillus pentosus can utilize glucose to convert it into lactic acid, and it was cultured for 48 h to allow Lactiplantibacillus pentosus to grow outside the first biofilm to form a second biofilm.

[0093] (3) Slowly introduce O2 gas (aeration rate: 5 mL / min) into the anode carbon fiber hollow fiber membrane electrode, and gradually adjust the potential from 0.5 V to 1 V for microbial electrofermentation to convert cellulose into lactic acid, and the cathode undergoes an electrolytic water reaction.

[0094] (4) The reaction temperature was controlled at 25 °C, and the optimal pH of the bacterial strain was about 6.0. However, acidification easily occurred during the electrofermentation process in the anode chamber. Therefore, according to the pH of the cathode medium every day, 1 M NaOH solution was used to adjust the pH to 6.0.

[0095] In this example, the anode cellulose degradation rate reached over 80%, and the lactic acid concentration reached 3000 mg / L.

[0096] Example 4

[0097] This example provides a method for constructing an anode three-layer biofilm electrode

[0098] (1) The reactor is an H-type electrolytic cell composed of a cathode chamber and an anode chamber. The volumes of both the cathode chamber and the anode chamber are 100 mL. The anode is the working electrode, and the electrode material is a carbon fiber hollow fiber membrane; the cathode is the counter electrode, and the electrode material is a graphite sheet; the reference electrode (Ag / AgCl) is in the anode chamber. First, 80 mL of a culture medium is added to the anode chamber. The culture medium consists of 3 g / L KH2PO4, 5.8 g / L K2HPO4, 0.5 g / L NH4Cl, 10 mL / L of Wolin's vitamin solution, and 10 mL / L of Wolin's mineral solution; 80 mL of an electrolyte is added to the cathode chamber. The electrolyte consists of 3 g / L KH2PO4 and 5.8 g / L K2HPO4, and the pH is adjusted to 7.0 using 1 M NaOH.

[0099] (2) Add the first microbial strain Trichoderma reesei to the anode chamber, and the inoculation concentration is 1% (v / v) (that is, 1 mL OD is added to 100 mL of the culture medium) 600A Trichoderma reesei bacterial solution with a concentration of 1 was used. The reaction substrate was microcrystalline cellulose at a concentration of 5 g / L, and 0.5 g / L of yeast powder was added to promote the growth of Trichoderma reesei and the formation of biofilms. Then, a potential of 0.5 V (versus Ag / AgCl) was applied to the reaction system. After culturing for 48 h, Trichoderma reesei grew on the anode hollow fiber membrane electrode to form an electroactive first biofilm. Then, a second microbial strain, Lactiplantibacillus pentosus, was inoculated into the anode chamber at an inoculation concentration of 1% (v / v), and 0.5 g / L of yeast powder was added to promote the growth of Lactiplantibacillus pentosus and the formation of biofilms. Lactiplantibacillus pentosus can utilize glucose and convert it into lactic acid. After culturing for 48 h, Lactiplantibacillus pentosus grew outside the first biofilm to form a second biofilm. Finally, a third microorganism, Clostridium tyrobutyricum, was inoculated into the anode chamber at an inoculation concentration of 1% (v / v), and 0.5 g / L of yeast powder and 10 μg / L of N-butyryl-L-homoserine lactone as a signal molecule were added to promote the growth of Clostridium tyrobutyricum and the formation of biofilms. Clostridium tyrobutyricum converts lactic acid into butyric acid under anaerobic conditions. After 48 h of culturing, Clostridium tyrobutyricum grew outside the second biofilm to form a third biofilm. Trichoderma reesei, Lactiplantibacillus pentosus, and Clostridium tyrobutyricum have different sensitivities to oxygen, and the products of the former are the substrates of the latter. Therefore, they can form three-layer biofilms in sequence.

[0100] (3) Slowly introduce O2 gas (aeration rate: 5 mL / min) into the anode carbon fiber hollow fiber membrane electrode, and gradually adjust the potential from 0.5 V to 1 V for microbial electrolysis fermentation to convert cellulose into butyric acid. The cathode undergoes a water electrolysis reaction.

[0101] (4) The reaction temperature is controlled at 24 °C. The optimal pH of the bacterial strains is around 6.0. However, acidification easily occurs during the electrolysis fermentation process in the anode chamber. Therefore, every day, according to the pH of the cathode culture medium, 1 M NaOH solution is used to adjust the pH to 6.0.

[0102] In this example, the cellulose degradation rate in the anode reached over 80%, the lactic acid concentration reached 3000 mg / L, and the butyric acid concentration reached 1500 mg / L.

[0103] Comparative Example 1 constructed a Sporomusa ovata biofilm electrode to reduce CO2 to acetic acid

[0104] (1) The reactor was an H-type electrolytic cell. The volumes of both the cathode chamber and the anode chamber were 100 mL. The cathode was the working electrode, and the electrode material was a carbon fiber hollow fiber membrane; the anode was the counter electrode, and the electrode material was carbon cloth; the reference electrode (Ag / AgCl) was in the cathode chamber. First, 80 mL of medium was added to the cathode chamber, and the medium (medium 311, DSMZ) was purged with nitrogen for 60 min; 80 mL of electrolyte was added to the cathode chamber. The electrolyte was composed of 3 g / L KH2PO4 and 5.8 g / L K2HPO4, and the pH was adjusted to 7.0 using 1M NaOH.

[0105] (2) 5% (v / v) Sporomusa ovata DSM2662 was added to the cathode chamber. CO2:H2 = 1:4 gas was introduced into the cathode carbon fiber hollow fiber membrane (aeration rate was 50 mL / min), and a potential of -0.6 V was applied for 48 h to enable Sporomusa ovata DSM 2662 attached to the fiber membrane to directly utilize the gas overflowing from the fiber membrane to generate small molecule organic substances such as acetic acid.

[0106] (3) Only CO2 gas was introduced into the cathode chamber (aeration rate was 50 mL / min), and the potential was gradually adjusted to -0.6 V for microbial electroreduction.

[0107] In this comparative example, the CO2 reduction efficiency of the cathode could reach 75%, and the acetic acid production rate could reach 145 nM / m 2 ·d -1 。

[0108] In Example 1 of the present application, a double-layer biofilm composed of Sporomusa ovata and Clostridium kluyveri was formed, and the product was further converted from acetic acid to more valuable butyric acid and caproic acid. Moreover, acetic acid was not prone to acid accumulation in this system, so the CO2 conversion efficiency reached 90%.

[0109] Although the disclosed embodiments of the present application are as above, the described content is only the embodiments adopted for facilitating the understanding of the present application and is not used to limit the present application. Any person skilled in the art within the scope of the present application can make any modifications and changes in the form and details of the implementation without departing from the spirit and scope disclosed by the present application. However, the patent protection scope of the present application shall still be subject to the scope defined by the appended claims.

Claims

1. A method for constructing a multi-layer biofilm electrode, characterized in that, Comprising the following steps: 1) Provide a reaction system including an H-type electrolytic cell composed of a cathode chamber and an anode chamber, a working electrode, a counter electrode, a reference electrode, and a culture medium. Add the culture medium to the cathode chamber or the anode chamber, and purge nitrogen for 30 min - 120 min; 2) Add electroactive first microbial strains to the cathode chamber and / or the anode chamber, and then apply an electric potential to the cathode chamber and / or the anode chamber to allow the first microbial strains to grow on the outer surface of at least one of the working electrode and the counter electrode to form an electroactive first biofilm; then inoculate the reaction system with second microbial strains, and allow the second microbial strains to utilize the metabolites of the first microbial strains to form a second biofilm outside the electroactive first biofilm; 3) Pass a gas into the cathode chamber or the anode chamber, set a constant electric potential, culture and acclimatize the biofilm obtained in step 2), and perform microbial electrosynthesis; 4) Adjust the pH and temperature of the reaction system according to the optimal growth pH and temperature of the microbial strains used, and monitor the pH in the reaction system in real time; In step 2), construct an anode multi-layer biofilm or a cathode multi-layer biofilm according to whether the working electrode is an anode electrode or a cathode electrode; In step 2), if a cathode multi-layer biofilm is constructed, the first microbial strains are selected from acid-producing bacteria, alcohol-producing bacteria, and Pseudomonas; and the second microbial strains are selected from butyric acid-producing bacteria or nitrite-reducing bacteria; In step 2), if an anode multi-layer biofilm is constructed, the first microbial strains are aerobic fungi; and the second microbial strains are lactic acid bacteria.

2. The method according to claim 1, wherein in step 1), the working electrode and the counter electrode are composed of a carbon-based material selected from carbon foam, nanoporous carbon, carbon fiber hollow fiber membrane, or graphite sheet, and the reference electrode is selected from an Ag / AgCl electrode, a calomel electrode, or a hydrogen electrode.

3. The method according to claim 1, wherein in step 2), the electric potential applied to the system, if the cathode is the working electrode, the applied electric potential is -0.5 V to -1.5 V relative to Ag / AgCl, and if the anode is the working electrode, the applied electric potential is 0.5 V to 1.5 V relative to Ag / AgCl.

4. The method according to claim 1, wherein in step 2), after forming the second biofilm, it further includes inoculating the reaction system with third microbial strains that utilize the metabolites of the second microbial strains as their own reaction substrates to form a third biofilm.

5. The method according to any one of claims 1 to 4, wherein in step 2), if a cathode multi-layer biofilm is constructed, the first microbial strain is Sporomusa , Clostridium, Geobacter or Shewallena .

6. The method according to any one of claims 1 to 4, wherein in step 2), if an anode multi-layer biofilm is formed, the aerobic fungi are selected from the genus Trichoderma, Penicillium, Myrothecium or Chaetomium ； and the lactic acid bacteria are selected from the genus Lactobacillus, Pediococcus or Streptococcus.

7. The method according to claim 6, wherein the aerobic fungus is Trichoderma reesei , Trichoderma viride or Trichoderma harzianum; and the lactic acid bacteria are Lactobacillus acidophilus, Lactobacillus rhamnosus , Lactobacillus casei , Lactobacillus paracasei , Lactobacillus plantarum , Lactobacillus gasseri , Lactobacillus reuteri , Lactobacillus bulgaricus or Lactobacillus johnsonii .

8. According to the method described in claim 4, if a three - layer biofilm is constructed on the cathode, the third microbial strain is selected from chain - elongating bacteria; if a three - layer biofilm is constructed on the anode, the third microbial strain is an anaerobic acid - producing bacterium.

9. The method according to claim 8, wherein, The chain elongation bacterium is Clostridium genus 、Clostridium bacterium or Desulfovibrio ; The anaerobic acid-producing bacteria are selected from the genus Clostridium, Bacteroides, Butyrivibrio or Bifidobacterium.

10. The method according to claim 9, wherein The anaerobic acidogenic bacteria are Clostridium ljungdahlii , Butyrivibrio fibrisolvens , Bifidobacterium or Clostridium tyrobutyricum .

11. According to the method described in any one of claims 1 to 4, wherein in step 1), the culture medium in the cathode chamber consists of 2 - 6 g / L KH2PO4, 4 - 8 g / L K2HPO4, 0.2 - 2 g / L NH4Cl, 0 - 0.5 g / L KCl, 0 - 1.0 g / L NaCl, 0 - 0.5 g / L MgCl2·6H2O, 0 - 0.1 g / L CaCl2, 1 - 10 mL / L Wolin's vitamin solution and 1 - 10 mL / L Wolin's mineral solution; the culture medium in the anode chamber consists of 2 - 6 g / L KH2PO4, 4 - 8 g / L K2HPO4, 0.2 - 2 g / L NH4Cl, 1 - 10 mL / L Wolin's vitamin solution and 1 - 10 mL / L Wolin's mineral solution.

12. The method according to claim 11, wherein In step 1), a buffer pair is added to the culture medium; the buffer pair is dipotassium hydrogen phosphate / potassium dihydrogen phosphate.

13. According to the method described in any one of claims 1 to 4, wherein in step 1), the working electrode is disposed in the cathode chamber or the anode chamber.

14. According to the method described in any one of claims 1 to 4, wherein in step 3), the gas in the cathode chamber is CO2, CO, and in the anode chamber is O2.

15. According to the method described in claim 13, wherein in step 1), according to the chamber where the working electrode is located, the culture medium is divided into cathode culture medium or anode culture medium.

16. According to the method described in claim 15, wherein an appropriate reducing substance is added to the cathode culture medium to consume the remaining trace oxygen in the cathode culture medium; the reducing substance is 0.1 - 0.4 g / L cysteine hydrochloride.

17. The method according to claim 1, wherein in step 2), after adding the first microbial strain or after adding the second microbial strain, glucose / yeast powder is added immediately as a carbon source for subsequently accelerating the growth of the first microbial strain and the second microbial strain.

18. The method according to claim 17, wherein in step 2), after adding the first microbial strain or after adding the second microbial strain, 10 - 100 μg / L of a signal molecule is added immediately to the reaction system for subsequently promoting the growth of the first biofilm and the second biofilm; the signal molecule is N-butyryl-L-homoserine lactone or N-hexanoyl-L-homoserine lactone.

19. The method according to claim 4, wherein in step 2), after adding the third microbial strain, 10 - 100 μg / L of a signal molecule is added immediately to the reaction system for subsequently promoting the growth of the third biofilm; the signal molecule is N-butyryl-L-homoserine lactone or N-hexanoyl-L-homoserine lactone.

20. The method according to claim 1, wherein in step 2), the volume ratios of the inoculation amounts of the first microbial strain and the second microbial strain are 1 - 20% respectively.

21. The method according to claim 20, wherein in step 2), the volume ratios of the inoculation amounts of the first microbial strain and the second microbial strain are 5 - 10% respectively.

22. The method according to claim 4, wherein in step 2), the volume ratio of the inoculation amount of the third microbial strain is 1 - 20%.

23. The method according to claim 22, wherein in step 2), the volume ratio of the inoculation amount of the third microbial strain is 5 - 10%.

24. The method according to any one of claims 1 to 4, wherein in step 3), the aeration rate of the gas is 10 - 100 mL / min.

25. The method according to any one of claims 1 to 4, wherein in step 4), the pH of the microbial strains used is 5 - 8.

26. The method according to claim 25, wherein in step 4), during the reaction process, the pH of the reaction system is adjusted to 5 - 8 according to the optimal pH of the strain using an alkali to avoid anodic acidification, and the pH of the reaction system is adjusted to 5 - 8 according to the optimal pH of the strain using an acid to avoid cathodic alkalization; the alkali is NaOH; the acid is H3PO4.

27. The method according to claim 26, wherein in step 4), the temperature of the reaction system is controlled between 25 - 60°C.

28. A multi-layer biofilm electrode manufactured by the method according to any one of claims 1-27, characterized in that, The multi-layer biofilm electrode comprises: An electroactive first biofilm, which is disposed on the outer surface of at least one of the working electrode and the counter electrode; A second biofilm, which is disposed on the first biofilm.

29. The multi-layer biofilm electrode according to claim 28, further comprising a third biofilm disposed on the second biofilm.

Images