Method for synthesizing hexanoic acid by C6-HSL and C8-HSL synergistic reinforced carbon chain prolonged microbial electrochemical system

Abstract

The invention discloses a method for synthesizing hexanoic acid by using a C6-HSL and C8-HSL synergistic reinforced carbon chain extension microbial electrochemical system, which comprises the following steps of: inoculating domesticated carbon chain extension function microorganisms into a culture medium by using ethanol and acetic acid as substrates, and adding two specific signal molecules of C6-HSL and C8-HSL to construct a carbon chain extension reaction system; and then reacting under a sealed anaerobic condition. According to the method, C6-HSL and C8-HSL signal molecules are added into a carbon chain extension reaction system at the same time to regulate and control key functional microorganisms, biological membrane thickness and live / dead ratio of caproic acid synthesis through carbon chain extension, caproic acid synthesis through carbon chain extension can be effectively promoted through the method, meanwhile, the method is easy to operate and has high application value, and further, the method has good application prospects. The caproic acid produced by the method has great environmental benefits and economic benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial electrosynthesis, and in particular to a method for synthesizing caproic acid by using a C6-HSL and C8-HSL synergistically enhanced carbon chain extension microbial electrochemical system. Background Art

[0002] In microbial electrosynthesis systems, electroactive biofilms have attracted widespread attention for their unique ability to transfer electrons between solid materials, such as electrodes. Specifically, electroactive biofilms are composed of electroactive microorganisms. Because these microorganisms can exchange electrons with electrodes through various electron transfer mechanisms, electroactive biofilms possess this ability to transfer electrons.

[0003] The biosynthesis of medium-chain fatty acids (C6-C8) is a carbon chain elongation process, primarily through the reverse cycle of β-oxidation, where microorganisms extend the carbon chains of short-chain fatty acids (such as acetic acid and butyric acid) to synthesize medium-chain fatty acids with 6 to 8 carbon atoms. Hexanoic acid is a very important medium-chain fatty acid and a multifunctional chemical that can be used as a food additive, aviation fuel, and chemical reagent. In recent years, research on improving the efficiency of hexanoic acid production in microbial electrosynthesis systems has focused on the development of new cathode materials, the screening of cathode microorganisms, and the online separation of products.

[0004] The cathode microorganisms are mainly composed of Gram-negative bacteria and Gram-positive bacteria. How to regulate the behavior of Gram-negative bacteria and Gram-positive bacteria and thus promote the formation of cathode microorganisms is the current research focus. Chinese invention patent CN 108277239A discloses a method for promoting microbial electrosynthesis of organic acids. This method adds penicillin salts with a final concentration of 5 to 30 mg / L to the culture medium of the microbial electrosynthesis system, which reduces the difficulty of electrons crossing the cell wall of Gram-positive bacteria and increases the speed of electrons and organic acids crossing the cell wall of Gram-positive bacteria to promote the speed and yield of electrosynthetic acid production. Although this method reduces the difficulty of electrons crossing the Gram-positive cell wall, it does not regulate the key functional microorganisms of the biofilm related to the synthesis of organic acids, the thickness of the biofilm, and the live / dead cell ratio. Summary of the Invention

[0005] Quorum sensing signaling molecules are an effective way for bacteria to communicate between cells using chemical signals. C6-HSL and C8-HSL are two N-acylhomoserine lactone (AHLs) signaling molecules, standing for N-hexanoyl-L-homoserine lactone and N-octanoyl-L-homoserine lactone, respectively. They are typical signaling molecules in the quorum sensing system of Gram-negative bacteria, regulating intraspecific communication within Gram-negative bacteria. By secreting these molecules, bacteria can communicate with each other and coordinate group behavior.

[0006] The object of the present invention is to provide a method for synergistically enhancing the carbon chain extension biosynthesis of caproic acid by C6-HSL and C8-HSL. The method of the present invention utilizes two signal molecules to jointly enhance the key functional microorganisms and biofilm thickness and live / dead cell ratio of carbon chain extension biosynthesis of caproic acid, thereby increasing the production of caproic acid and shortening the caproic acid production cycle.

[0007] A method for synthesizing caproic acid by a microbial electrochemical system using C6-HSL and C8-HSL to synergistically enhance carbon chain extension. The method comprises synthesizing caproic acid in a microbial electrosynthesis system, wherein the cathode and anode electrodes of the microbial electrosynthesis system are both unmodified carbon felt electrodes, and the reference electrode is a silver-silver chloride electrode. A titanium wire and a 10-ohm resistor are used to connect the cathode and anode to form a closed circuit. A phosphate buffer solution is used as the anolyte, and a culture medium is used as the catholyte.

[0008] Ethanol and acetic acid are added to the cathode liquid, and carbon chain extension functional microorganisms are inoculated. Then, C6-HSL and C8-HSL signal molecules are added to the cathode liquid, and the cathode liquid of the bioelectrosynthesis system is aerated with nitrogen by aeration. Finally, a non-lethal external voltage is applied by a DC power supply, and the reaction device is operated under sealed anaerobic conditions at 30-36°C.

[0009] The carbon chain extension functional microorganisms include one or more of Clostridium_sensu_stricto_12, Thioclava, and Desulfovibrio. The abundance of the functional microorganisms can be synergistically regulated by two signal molecules, C6-HSL and C8-HSL, to enhance the carbon chain extension and synthesis of hexanoic acid.

[0010] In one embodiment, the reaction temperature may be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C or 36°C, or any value between 30-36°C.

[0011] In one embodiment, the microbial electrochemical synthesis system uses a double-chamber H-type reactor, with the two poles separated by a proton exchange membrane.

[0012] In one embodiment, the concentration of ethanol is 2-10 g / L, and the concentration of acetic acid is 1-5 g / L.

[0013] In one embodiment, the concentration of ethanol can be 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L or 10 g / L, or any value between 2 and 10 g / L; the concentration of acetic acid can be 1 g / L, 2 g / L, 3 g / L, 4 g / L or 5 g / L, or any value between 1 and 5 g / L.

[0014] In one embodiment, the pH of the reaction system is 6.6-8.

[0015] In one embodiment, the total concentration of C6-HSL and C8-HSL added is 5-12 μmol / L.

[0016] In one embodiment, the added concentration of the C6-HSL signal molecule is 1-6 μmol / L, and the added concentration of the C8-HSL signal molecule is 1-6 μmol / L.

[0017] In one embodiment, the added concentrations of C6-HSL and C8-HSL signaling molecules can be 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L or 6 μmol / L, respectively, or any value between 1-6 μmol / L, as long as the total concentration is between 5-12 μmol / L.

[0018] In one embodiment, the nitrogen aeration treatment time is 10-50 minutes, and the applied voltage is 0.6-1.5 V. In one embodiment, the nitrogen aeration treatment time is 10 minutes, 20 minutes, 30 minutes, 40 minutes, or 50 minutes, or any value between 10-50 minutes.

[0019] In one embodiment, the inoculation volume ratio of the bacterial population is 5%-12%.

[0020] In one embodiment, the composition of the culture medium is 0.31 g / L K2HPO4, 0.23 g / L KH2PO4, 0.25 g / L NH4Cl, 0.20 g / LMgSO4·7H2O, 1.0 g / L yeast extract, 2.5 g / L NaHCO3, 0.25 g / L Na2S·9H2O, 0.25 g / L L-cysteine ​​hydrochloride monohydrate, 2.0 g / L sodium 2-bromoethylsulfonate, 1 mL / L trace elements, 1 mL / L selenite tungstate, 0.5 mL / L resazurin indicator, and 1 mL / L vitamin solution.

[0021] In one embodiment, the construction of the microbial electrochemical synthesis system:

[0022] A double-chamber H-type reactor was used. The effective volume of the anode chamber and the cathode chamber was 175 mL. The two poles were separated by a proton exchange membrane. The cathode and anode electrodes were unmodified carbon felt electrodes. The reference electrode was a silver / silver chloride electrode placed in the cathode chamber. The cathode and anode were connected with titanium wire and a 10 ohm resistor to form a closed path. The anolyte was phosphate buffer and the catholyte was culture medium.

[0023] Beneficial effects:

[0024] (1) The present invention simultaneously adds C6-HSL and C8-HSL signal molecules to the carbon chain extension reaction system. These two signal molecules are irreplaceable in regulating the functional microorganisms of biofilms, and can regulate the key functional microorganisms that extend the carbon chain to synthesize hexanoic acid. The method of the present invention can effectively promote the extension of the carbon chain to synthesize hexanoic acid.

[0025] (2) The method of the present invention is simple to operate and has high application value. Furthermore, the production of caproic acid by this method has great environmental and economic benefits.

[0026] (3) In the present invention, the C6-HSL+C8-HSL group synergistically regulates the microorganisms Thioclava and Desulfovibrio, and the total relative abundance of Thioclava+Desulfovibrio increases by 10.00% to 90.00% compared with the group without adding any signal molecules, the C6-HSL group and the C8-HSL group; at the same time, the biofilm thickness of the C6-HSL and C8-HSL groups synergistically increases by 8.00% to 80.00% compared with the group without adding any signal molecules, the C6-HSL group and the C8-HSL group; the live / dead cell ratio of the biofilm of the C6-HSL and C8-HSL groups synergistically increases by 11.00% to 80.00% compared with the group without adding any signal molecules, the C6-HSL group and the C8-HSL group. The C6-HSL and C8-HSL groups synergistically produced caproic acid, increasing caproic acid concentrations by 10.00% to 85.00% compared to the groups without any signal molecule addition, the C6-HSL group, and the C8-HSL group. The order of caproic acid concentration was C6-HSL + C8-HSL group > C6-HSL group > C8-HSL group > without any signal molecule addition. The synergistic effect of the C6-HSL + C8-HSL group significantly enhanced the efficiency of carbon chain extension in the production of caproic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 This is a diagram of the structure, composition and mechanism of the bioelectrochemical system of the present invention. DETAILED DESCRIPTION

[0029] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0030] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0031] The detection method is as follows:

[0032] (1) Hexanoic acid organic acid gas chromatography

[0033] The reaction solution collected was centrifuged at 8000 rpm for 10 minutes, and the supernatant was mixed with an equal volume of 3 mM phosphoric acid. After filtering with a 0.22 μm water filter membrane, the supernatant was stored in a gas chromatograph vial and stored at -20°C. Gas chromatograph (2010 type, Shimadzu Corporation) was used to measure the concentrations of caproic acid, butyric acid, acetic acid, and ethanol in the reaction solution. The gas chromatograph carrier gas was nitrogen, and the FID detector and injection port temperature were 250°C, with a sample size of 1 μL. Standard curves were first made using above-mentioned organic chromatography-grade standard substances (caproic acid, butyric acid, acetic acid, and ethanol), and the corresponding substance content in the reaction solution was calculated based on elution time and peak area.

[0034] (2) Microbial community determination

[0035] After the microbial enrichment culture stabilized, 100 mL was retained and centrifuged at 8000 rpm for 8 minutes to collect the bacteria at the bottom of the centrifuge tube. Subsequently, to extract total DNA from the bacteria, the MoBio Powersoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA) was selected as a DNA test kit. During the experiment, the operating procedures of the kit were strictly followed to ensure that the extracted DNA quality and purity met the requirements. The extracted total DNA samples were subjected to subsequent PCR experiments and sequencing analysis.

[0036] The experiment used the Illumina MiSeq high-throughput sequencing platform (Majorbio Biotechnology Co., Ltd., Shanghai, China). The V3-V4 region of the 16S rRNA was amplified using universal bacterial primers (338F: 5′-ACTCCTACGGGAGGCAGCAG-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′). Sequencing results were analyzed on the Illumina MiSeq Majorbio Cloud Platform.

[0037] (3) Determination of biofilm thickness and live / dead cell ratio

[0038] After the reactor operation was completed, the live / dead cell characterization of the biofilm was performed using a confocal laser scanning microscope (CLSM) (Carl Zeiss LSM880, Germany). The selected fluorescent dye was a live / dead cell fluorescent dye kit (Shanghai Qiyuan Biotechnology Co., Ltd., China). According to the ratio of SYTO 9:PI:distilled water = 1.5μL:1.5μL:1mL, the three reagents were added to the same centrifuge tube and mixed evenly. The dye preparation process should be protected from light and used immediately after preparation. The dye fluorescence excitation / emission wavelengths were selected as follows: 480 / 500nm (SYTO 9, green) and 490 / 635nm (PI, red). The activity of microorganisms in each biofilm layer was calculated using the ratio of live cells to total biofilm cells (obj. / total) using Image-Pro Plus 6.0 software.

[0039] Construction of microbial electrochemical synthesis system:

[0040] A dual-chamber H-type reactor was used, with the effective volumes of the anode and cathode chambers both being 175 mL, and the two electrodes being separated by a proton exchange membrane. The proton exchange membrane used in the embodiment of the present invention was Nafion 117 from DuPont, USA, and other types of proton exchange membranes with the same function could also be selected. The cathode and anode electrodes were both unmodified carbon felt electrodes (3 cm × 5 cm × 0.2 cm, purchased from Tokai Carbon Co., Ltd., Japan), and a reference electrode was a silver / silver chloride electrode (purchased from Shanghai Leici) placed in the cathode chamber. The cathode and anode were connected with a titanium wire and a 10 ohm resistor to form a closed path.

[0041] The anolyte used phosphate buffer (50 mM), and the catholyte used culture medium, specifically: 0.31 g / LK2HPO4, 0.23 g / LKH2PO4, 0.25 g / LNH4Cl, 0.20 g / LMgSO4·7H2O, 1.0 g / L yeast extract, 2.5 g / LNaHCO3, 0.25 g / LNa2S·9H2O, 0.25 g / L L-cysteine ​​monohydrate-hydrochloric acid, 2.0 g / L sodium 2-bromoethylsulfonate, 1 mL / L trace elements, 1 mL / L selenite tungstate, 0.5 mL / L resazurin indicator, 1 mL / L vitamin solution, and the pH was adjusted to 7.0.

[0042] Example 1

[0043] (1) Adding ethanol and acetic acid to the cathode liquid: 6 g / L ethanol and 2 g / L acetic acid were used as substrates.

[0044] (2) Carbon chain extension functional microorganisms were inoculated into the cathode liquid of the microbial electrochemical synthesis system at an inoculation ratio of 10% (v / v).

[0045] (3) Add 10 μmol / L C6-HSL and C8-HSL (5 μmol / L each of C6-HSL and C8-HSL) of two signal molecules to the cathode liquid to the carbon chain extension reaction system.

[0046] (4) Construction of the carbon chain extension reaction system: The anaerobic bottle was aerated with N2 for 30 min at 0.7 V to ensure that the reaction system fully reached anaerobic conditions. After sealing with a silicone stopper, it was connected to a chemical workstation and operated continuously for 15 days in a constant temperature chamber at 35 °C.

[0047] (5) Regularly draw 1 mL of cathode liquid from the sampling port of the reactor and detect and analyze the product.

[0048] (6) After the reactor operation is completed, the biofilm thickness and live / dead cell ratio are measured.

[0049] Example 2

[0050] (1) Adding ethanol and acetic acid as substrates to the cathode liquid: 6 g / L ethanol and 2 g / L acetic acid as substrates.

[0051] (2) Carbon chain extension functional microorganisms were inoculated into the cathode liquid of the microbial electrochemical synthesis system at an inoculation ratio of 10% (v / v).

[0052] (3) Add 10 μmol / L C6-HSL and C8-HSL (C6-HSL 6 μmol / L, C8-HSL 4 μmol / L) of two signal molecules to the cathode liquid to the carbon chain extension reaction system.

[0053] (4) Construction of carbon chain extension reaction system: The effective volume of the reactor was 175 mL. The anaerobic bottle was aerated with N2 for 30 min. An external voltage of 0.7 V was applied to ensure that the reaction system fully reached anaerobic conditions. After covering with a silicone plug and sealing, it was connected to a chemical workstation and operated continuously for 15 days in a constant temperature chamber at 35 °C.

[0054] (5) Regularly draw 1 mL of cathode liquid from the sampling port of the reactor and detect and analyze the product.

[0055] (6) After the reactor operation is completed, the biofilm thickness and live / dead cell ratio are measured.

[0056] Comparative Example 1

[0057] The difference from Example 1 is that no signal molecule is added.

[0058] Comparative Example 2

[0059] The difference from Example 1 is that in step (3), only 10 μmol / L C6-HSL signal molecule is added as the signal molecule.

[0060] Comparative Example 3

[0061] The difference from Example 1 is that in step (3), only 10 μmol / L C8-HSL signal molecules are added.

[0062] Comparative Example 4

[0063] The difference from Example 2 is that no signal molecule is added.

[0064] Comparative Example 5

[0065] The difference from Example 2 is that in step (3), only 10 μmol / L C6-HSL signal molecules are added.

[0066] Comparative Example 6

[0067] The difference from Example 2 is that in step (3), only 10 μmol / L C8-HSL signal molecule is added as the signal molecule.

[0068] Test example

[0069] The reaction systems after the reaction of Examples 1 and 2 and Comparative Examples 1-6 were tested.

[0070] Table 1 shows the caproic acid concentration, microbial community changes, biomass thickness and live / dead cell ratio of Example 1 of the present invention and Comparative Examples 1-3.

[0071]

[0072] Table 2 shows the caproic acid concentration, microbial community changes, biomass thickness and live / dead cell ratio of Example 2 of the present invention and Comparative Examples 4-6.

[0073]

[0074] The experimental results are shown in Table 1, which shows that after adding C6-HSL and C8-HSL signal molecules, the hexanoic acid concentration in Example 1 increased by 80.71%, 35.88% and 18.27% compared with Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively; the relative abundance of the microorganisms Thioclavava and Desulfovibrio in Example 1 increased by 83.65%, 39.08 and 23.25% compared with Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively; the biofilm thickness in Example 1 increased by 75.00%, 27.27% and 16.67% compared with Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively; the live / dead cell ratio of the biofilm in Example 1 increased by 78.57%, 38.89% and 19.05% compared with Comparative Example 1, Comparative Example 2 and Comparative Example 3, respectively.

[0075] The experimental results are shown in Table 2, which shows that after adding C6-HSL and C8-HSL signal molecules, the hexanoic acid concentration in Example 2 increased by 67.19%, 27.38% and 10.31% compared with Comparative Example 4, Comparative Example 5 and Comparative Example 6, respectively; the relative abundance of the microorganisms Thioclavava and Desulfovibrio in Example 2 increased by 69.85%, 27.63% and 10.49% compared with Comparative Example 4, Comparative Example 5 and Comparative Example 6, respectively; the biofilm thickness in Example 2 increased by 50.00%, 20.00% and 9.09% compared with Comparative Example 4, Comparative Example 5 and Comparative Example 6, respectively; the live / dead cell ratio of the biofilm in Example 2 increased by 66.67%, 25.00% and 11.11% compared with Comparative Example 4, Comparative Example 5 and Comparative Example 6, respectively.

[0076] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for synthesizing caproic acid by synergistically enhancing the carbon chain extension microbial electrochemical system using C6-HSL and C8-HSL, characterized in that: Hexanoic acid is synthesized based on a microbial electrochemical synthesis system, wherein the cathode and anode electrodes of the microbial electrochemical synthesis system are both unmodified carbon felt electrodes, and the reference electrode is a silver-silver chloride electrode; the cathode and anode are connected by a titanium wire and a 10 ohm resistor to form a closed circuit; the anolyte is a phosphate buffer solution, and the catholyte is a culture medium; Ethanol and acetic acid are added to the cathode liquid, and carbon chain extension functional microorganisms are inoculated. Then, two signal molecules, C6-HSL and C8-HSL, are added to the cathode liquid. The cathode liquid of the bioelectrochemical synthesis system is aerated with nitrogen by aeration. Finally, a voltage of 0 to 1.5 V is applied through a DC power supply, and the reaction device is operated under sealed anaerobic conditions at 30-36°C. The carbon chain extension functional microorganisms include one or more of Clostridium_sensu_stricto_12, Thioclava, and Desulfovibrio.

2. The method for synthesizing caproic acid by synergistically enhancing the carbon chain extension microbial electrochemical system of C6-HSL and C8-HSL according to claim 1, characterized in that: The microbial electrochemical synthesis system adopts a double-chamber H-type reactor, with the two poles separated by a proton exchange membrane.

3. The method for synthesizing caproic acid by synergistically enhancing the carbon chain extension microbial electrochemical system of C6-HSL and C8-HSL according to claim 1, characterized in that: The concentration of the ethanol is 2-10 g / L, and the concentration of the acetic acid is 1-5 g / L.

4. The method for synthesizing caproic acid by synergistically enhancing the carbon chain extension microbial electrochemical system of C6-HSL and C8-HSL according to claim 1, characterized in that: The pH of the reaction system is 6.6-8.

5. The method for synthesizing caproic acid by synergistically enhancing the carbon chain extension microbial electrochemical system of C6-HSL and C8-HSL according to claim 1, characterized in that: The total added concentration of the C6-HSL and C8-HSL is 5-12 μmol / L.

6. The method for synthesizing caproic acid by synergistically enhancing the carbon chain extension microbial electrochemical system by using C6-HSL and C8-HSL according to claim 1 or 5, characterized in that: The added concentration of the C6-HSL signal molecule is 1-6 μmol / L, and the added concentration of the C8-HSL signal molecule is 1-6 μmol / L.

7. The method for synthesizing caproic acid by synergistically enhancing the carbon chain extension microbial electrochemical system using C6-HSL and C8-HSL according to claim 1, characterized in that: The nitrogen aeration treatment time is 10-50 minutes; the applied voltage is 0.6-1.5V.

8. The method for synthesizing caproic acid by synergistically enhancing the carbon chain extension microbial electrochemical system using C6-HSL and C8-HSL according to claim 1, characterized in that: The inoculation volume ratio of the bacterial community is 5%-12%.

9. The method for synthesizing caproic acid by synergistically enhancing the carbon chain extension microbial electrochemical system using C6-HSL and C8-HSL according to claim 1, characterized in that: The composition of the culture medium is 0.31g / L K2HPO4, 0.23g / LKH2PO4, 0.25g / LNH4Cl, 0.20g / LMgSO4·7H2O, 1.0g / L yeast extract, 2.5g / L NaHCO3, 0.25g / LNa2S·9H2O, 0.25g / L L-cysteine ​​monohydrate-hydrochloric acid, 2.0g / L sodium 2-bromoethylsulfonate, 1mL / L trace elements, 1mL / L selenite tungstate, 0.5mL / L resazurin indicator, and 1mL / L vitamin solution.

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