Klebsiella strain with electroactivity and application of Klebsiella strain in plastic degradation

Abstract

The invention discloses a Klebsiella strain with electroactivity and application of the Klebsiella strain in degradation of plastics. The Klebsiella strain is Klebsiella sp. X11, and the preservation number of the Klebsiella strain is CGMCC (China General Microbiological Culture Collection Center) 1.62470. The degradation strain X11 is obtained through separation and screening, belongs to gram-negative bacteria, and can realize efficient degradation of the polyvinyl chloride film, so that the polyvinyl chloride film reaches a relatively high weight loss ratio. Meanwhile, it is found that the strain X11 has electrochemical activity, can degrade polyvinyl chloride (PVC) and generate electricity by means of a microbial fuel cell, and has wide application prospects in the fields of plastic pollution bioremediation, plastic solid waste resourceful treatment and utilization and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a Klebsiella strain with electrical activity and its application in the degradation and resource utilization of polyvinyl chloride (PVC) plastics. Background Art

[0002] PVC is an important material with multiple uses. With the rapid development of social economy, its production is also increasing. In 2017, the global plastic production reached 345 million tons, and it will increase to about 400 million tons in 2022. PVC production accounts for about 12.7%, and a large amount of waste PVC cannot be recycled. These waste PVC are mainly converted into microplastics and distributed in nature. Their stability, adsorption, cytotoxicity and reproductive toxicity in the environment pose a great threat to the ecosystem. Therefore, finding suitable PVC degradation or recycling technology is a major demand for social sustainable development.

[0003] At present, the commonly used plastic degradation technologies are mainly chemical degradation and thermal degradation, but they have many disadvantages such as high cost, low efficiency and easy to produce toxic by-products. Biodegradation is a process in which microorganisms such as bacteria, fungi and algae produce enzymes that cut plastic polymer chains, thereby breaking polymers into smaller molecules (such as oligomers, dimers and monomers), and finally converting them into CO2 and H2O. In addition, some microorganisms can achieve high-value conversion of degraded monomers while degrading plastics. Therefore, compared with physical and chemical degradation methods, biodegradation has the advantages of low cost, environmental protection and sustainability.

[0004] Microbial fuel cells (MFCs) are a new electrochemical device that generates electricity based on electroactive microbial metabolism and extracellular electron transfer processes, and can sustainably combine pollutant treatment and energy recovery. Microbial metabolic activity is a key factor in determining the use of MFCs to degrade PVC. However, the existing technology lacks electroactive microorganisms that can efficiently degrade PVC. Summary of the invention

[0005] In view of the shortcomings and deficiencies of the prior art, the object of the present invention is to provide an electroactive Klebsiella strain X11 and its application in degrading plastics, which can provide a new method for solving polyvinyl chloride plastic pollution in the environment and realizing waste resource utilization.

[0006] The present invention first provides a Klebsiella Klebsiella sp. X11 strain, its deposit number is CGMCC1.62470, the depository unit is the General Microbiology Center of China Culture Collection Administration, and the deposit date is October 26, 2024.

[0007] The strain in this study is of novel origin, isolated from the laboratory MFC anode liquid, and verified through research that the strain can be used to degrade PVC and has electrochemical activity. It is the first Klebsiella member found to be able to degrade PVC and have electrochemical activity.

[0008] The strain is a bacterium-oxytocin Klebsiella ( Klebsiella oxytoca ), the bacteria were first reported to degrade pollutants such as phenanthrene, pyrene and cyanide. The bacteria can secrete specific degradation enzymes to degrade pollutants. It is a Gram-negative bacteria. There are also reports that the bacteria can be iron-reducing bacteria. So far, there has been no report on the bacteria degrading plastic pollutants and converting them into electrical energy at the same time.

[0009] The second object of the present invention is to provide the use of the above-mentioned strain in degrading PVC.

[0010] Preferably, the application comprises: inoculating the strain in a degradation medium, and then adding PVC as a carbon source for cultivation.

[0011] Preferably, the degradation culture medium is a liquid carbon-free inorganic salt culture medium (LCFBM) culture medium.

[0012] Preferably, the culture temperature is 28-32°C.

[0013] Preferably, the culture rotation speed is 140~160 rpm.

[0014] Preferably, the application comprises: taking the strain and inoculating it into a liquid carbon-free inorganic salt culture medium so that the initial OD600 is 0.2, then adding PVC as a carbon source, and continuously culturing at 28-32° C. and 140-160 rpm.

[0015] The third object of the present invention is to provide a method for degrading polyvinyl chloride and generating electricity using a microbial fuel cell, wherein the anode liquid of the microbial fuel cell is inoculated with the strain.

[0016] Preferably, the method comprises: inoculating the strain in the anode liquid of a dual-chamber microbial fuel cell, using polyvinyl chloride as an anode electron donor and trivalent iron ions as a cathode electron acceptor, and acclimating and operating the dual-chamber microbial fuel cell.

[0017] The present invention has the following advantages and beneficial effects: The strain described in the present invention can significantly degrade polyvinyl chloride film under liquid culture conditions for a short period of time (15 days). Even for the more difficult to degrade PVC film without additives, X11 can still reduce the weight of the PVC film by nearly 11.1%; the weight of the PVC film can be reduced by 24.7% in 60 days; the PVC film turns yellow, and obvious cracks and pits appear on the surface; the infrared spectrum changes, and new hydroxyl and carbonyl functional groups appear; the X-ray photoelectron energy spectrum changes, and the chlorine content decreases significantly. This effect is better than other PVC degradation strains reported. In addition, the present invention also found that the strain has electrochemical activity, and constructed an MFC based on it. After the constructed dual-chamber MFC runs stably, the maximum voltage is measured to be 401 mV, and the power density is 206 mW / m -2 . BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration: Figure 1 This is a colony morphology characteristic diagram of Klebsiella X11 provided by the present invention.

[0019] Figure 2 The Gram staining diagram of Klebsiella X11 provided by the present invention.

[0020] Figure 3 The present invention provides a phylogenetic tree of Klebsiella X11 based on 16S rDNA gene sequence analysis.

[0021] Figure 4 This is the weight loss rate of PVC film after culturing Klebsiella X11 with PVC film for 15 days, 30 days and 60 days.

[0022] Figure 5 The cracks and holes produced by degradation on the surface of the clean PVC film observed by SEM when Klebsiella X11 was cultured with PVC film for 60 days.

[0023] Figure 6 These are the infrared spectrum test results of PVC film after Klebsiella X11 was cultured with the PVC film for 60 days.

[0024] Figure 7 These are the X-ray photoelectron spectroscopy test results of PVC film during the 60-day culture period of Klebsiella X11.

[0025] Figure 8 The results of the electroactivity detection of Klebsiella X11.

[0026] Description of biological deposit: The biological material of the present invention is classified and named as: Klebsiella ( Klebsiella sp.) X11, depository unit: General Microbiology Center of China Culture Collection Administration, address: Datun Road, Chaoyang District, Beijing, deposit date: October 26, 2024, deposit number: CGMCC 1.62470. DETAILED DESCRIPTION

[0027] Example 1 Klebsiella oxytoca ( Klebsiella oxytoca )Enrichment, separation and purification of strains: (1) Micro carbon source culture medium enrichment: Using the laboratory microbial fuel cell anode liquid as the bacterial source, 0.35% (m / V) sterile PVC film (without additives) was added to the flask. The anode liquid was inoculated into the flask at 10% of the inoculum volume and cultured at 30°C and 150 rpm for 10 days to enrich the micro carbon source.

[0028] (2) Enrichment in carbon-free medium: After 10 days of micro-carbon source enrichment culture, the bacterial solution was centrifuged to collect the precipitate, which was resuspended in sterile phosphate buffer (PBS) at pH 7.2 and concentration of 0.1 mol / L and then centrifuged to collect the precipitate. After repeating this process twice, the collected precipitate was resuspended in LCFBM and 0.35% (m / V) sterile PVC film (without additives) was added. The culture was then kept at 30°C and 150 rpm for 4 weeks. After the enrichment was completed, 10% of the enrichment solution was transferred to fresh LCFBM for carbon-free medium enrichment and subculture, and the transfer was repeated for 5 generations.

[0029] (3) Isolation of PVC-degrading bacteria: Take 6 10 ml sterilized centrifuge tubes and label them 1-6, which represent different dilution multiples. Take 1 ml of the culture enriched in carbon-free medium and add it to tube 1 and mix it with 9 ml of sterile water. Dilute the bacterial solution to 10 −1 Then take 1 ml of the bacterial solution from tube 1 and add it to tube 2 and mix it with 9 ml of sterile water to dilute it to 10 -2 By analogy, the initial bacterial solution is diluted to 10 -6 Take 100 μL of 10 -5 , 10 -6 The gradient dilutions were evenly spread on LB solid medium and cultured for 36 hours. After that, different single colonies were selected and separated and purified by streaking method to obtain PVC-degrading bacteria, which were numbered and stored. The obtained PVC-degrading bacteria were cultured in liquid carbon-free medium with sterile PVC film (without additives) as the only carbon source, and finally the strain X11 capable of efficiently degrading PVC was obtained, which was again streaked and purified on a sterile LB plate. Figure 1 shown.

[0030] The trace carbon source culture medium used in the present invention is composed of: 0.5 g of Yeast extract, 2.0 g of (NH4)2SO4, 100 mL of trace element mother solution, and 900 mL of distilled water. The trace element mother solution is composed of: 1.0 g of FeSO4·7H2O, 1.0 g of MgSO4·7H2O, 0.1 g of CuSO4·5H2O, 0.1 g of MnSO4·H2O, 0.1 g of ZnSO4·7H2O, and 1000 mL of distilled water, pH 7.0. The components of the carbon-free medium are: K2HPO4·3H2O 0.92 g, KH2PO4 0.92 g, MgSO4·7H2O 0.7 g, (NH4)2SO4 1.0 g, NaCl 0.005 g, FeSO4·7H2O 0.002 g, ZnSO4·7H2O 0.002 g, MnSO4·H2O 0.001 g, 1000 mL distilled water. The components of the LB medium are: Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, 1000 mL distilled water, and LB solid medium is supplemented with 18-20 g of agar powder.

[0031] Example 2 Identification and preservation of strain X11: (1) Morphological identification: Use oil immersion microscope to identify the morphology of strain X11 after Gram staining. Figure 2 As shown, strain X11 is a Gram-negative bacterium, which appears as a short and thick pink rod under an oil microscope.

[0032] (2) Gene sequencing: strain X11 was inoculated in LB medium. After culturing for 36 hours, 2 mL of bacterial solution was collected by centrifugation at 8000 rpm, and the genomic DNA of the strain was extracted using a kit. The extracted total DNA of the strain was used as a template and amplified using bacterial universal primers. The PCR product was detected and sequenced by 1% agarose gel electrophoresis. The 16S rDNA sequence of strain X11 was obtained as shown in SEQ ID No. 1.

[0033] (3) Construction of phylogenetic tree: BLAST comparison was performed on NCBI. Based on the principle of selecting reference sequences, some homologous sequences with similarities between 98% and 100% were selected. The phylogenetic tree was established using MEGA 11 software. The phylogenetic tree was constructed using the Neighbor joining method in the distance method. The bootstrap parameter was set to 1000. Figure 3 From the clustering diagram, we can see that strain X11 and Klebsiella oxytoca Therefore, it can be determined that strain X11 belongs to the genus Klebsiella oxytoca ( Klebsiella oxytoca ).

[0034] (4) Strain preservation: The strain is preserved under the name: Klebsiella sp. X11, depository unit: General Microbiology Center of China Microbiological Culture Collection Administration, deposit date: October 26, 2024, deposit number: CGMCC 1.62470.

[0035] Example 3 Degradation of PVC film by strain X11 Degradation system: Take Klebsiella ( Klebsiella sp.) X11 strain, activated and inoculated into LB liquid medium, 36 hours after inoculation, centrifuged at 10000 rpm for 10 minutes, discarded the supernatant, washed twice with sterile PBS, and finally resuspended in sterile PBS. Take an appropriate amount of the resuspended bacterial solution and inoculate it into 50 mL LCFBM medium, so that the initial OD600 = 0.2, then add 0.1 g sterile PVC film (without additives) and culture continuously in an incubator at 30°C and 150 rpm for 60 days, and at the same time add a group of 0.1 g sterile commercial PVC film (with additives) as a control group.

[0036] The experiment was divided into three groups: (1) liquid carbon-free inorganic salt medium (LCBFM) supplemented with strain X11 and washed PVC film (without additives), (2) liquid carbon-free inorganic salt medium (LCBFM) supplemented with strain X11 and washed commercial PVC film (with additives), and (3) liquid carbon-free inorganic salt medium (LCBFM) supplemented with washed PVC film (without additives) as the control group. The cells were cultured at 30°C in a temperature-controlled shaker at 150 rpm for 60 days, and tests were performed at set time points during the period.

[0037] Degradation: The PVC films after 15, 30 and 60 days of culture were taken out and rinsed with phosphate buffer (PH=7). After removing the excess culture medium and bacteria on the surface, they were soaked in 2% sodium dodecyl sulfate (SDS) solution for 2 hours, rinsed with distilled water to remove the excess SDS, added with 75% ethanol and allowed to stand overnight, ultrasonicated with anhydrous ethanol for 1 hour, and rinsed with sterile water after the ultrasonication. The washed PVC films were placed in a 60℃ oven for 24 hours to dry, and finally weighed with a precision balance. The PVC weight loss rate was calculated according to the following formula: PVC weight loss rate (%) = (PVC original weight - PVC weight after degradation) / PVC original weight * 100%. The results are as follows: Figure 4As shown. Within 60 days of culturing the PVC film with strain X11, the weight loss rate of the PVC film (without additives) reached 24.7%, which is better than other PVC-degrading strains reported so far. In addition, the weight loss rate of the PVC film (without additives) after 15 days of degradation by strain X11 (11.1%) was less than the weight loss rate of the commercial PVC film (with additives) control group (23.6%). This shows that PVC film (without additives) is more difficult to degrade than ordinary commercial PVC film (with additives) because commercial PVC film contains organic additives such as plasticizers that are relatively easy to be used by bacteria. When strain X11 degraded PVC film (without additives) for 60 days, as shown Figure 5 As shown in the figure, by comparing the film surface treated with strain X11 for 0 days and 60 days under a scanning electron microscope, it can be observed that obvious cracks and pits appeared on the film surface after X11 treatment. At the same time, Fourier-infrared spectroscopy was used to detect the changes of PVC film (without additives) before and after, and new hydroxyl and carbonyl groups appeared on the film surface, such as Figure 6 This indicates that an oxidation reaction occurred during the degradation of PVC. The X-ray photoelectron spectroscopy of the X11 strain and PVC film (without additives) for 60 days showed that the chlorine content on the film surface was significantly reduced compared with the control group, as shown in Figure 2. Figure 7 As shown, dechlorination is the key step in PVC degradation.

[0038] Example 4 Detection and verification of the electrical activity of strain X11 The electroactivity of strain X11 was first detected in a three-electrode system: the carbon felt electrode with attached bacteria was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the platinum wire electrode was used as the counter electrode. The CV scan was performed using an electrochemical workstation, with the voltage range from -800 mV to 800 mV and the scan rate of 10 mV·s -1In order to explore the electron transfer mode of strain X11, three experimental groups were set up: (1) carbon felt with bacteria + sterile LB culture medium (as electrolyte), (2) sterilized carbon felt + sterile supernatant (X11 culture medium was filtered out of bacteria and used as electrolyte), and (3) carbon felt with bacteria + sterile supernatant (X11 culture medium was filtered out of bacteria and used as electrolyte). The electroactivity of strain X11 was further verified, and the carbon felt with bacteria was used as the anode of the double-chamber MFC. The double-chamber MFC uses a proton exchange membrane to separate the cathode and anode chambers. The cathode liquid is a phosphate buffer containing 50 mM potassium ferrocyanide, and the anode liquid is a sterile LB culture medium. It is operated in a batch incubator at a constant temperature of 30°C. The cathode and anode liquids are replaced every 3 days, so that X11 is enriched on the anode electrode, so that it forms an electroactive biofilm for extracellular electron transfer while degrading PVC. MFC anolyte: Tryptone 10 g, Yeast extract 5 g, NaCl 10 g, 1000 mL distilled water. MFC cathode solution: NaH2PO4 2.132 g, Na2HPO4 4.576 g, KCl 0.13 g, potassium ferrocyanide 13.1696 g, 1000 mL distilled water.

[0039] The results are as follows Figure 8 As shown in the figure, there is no obvious redox peak in the group of carbon felt with bacteria + sterile LB medium, while similar redox peaks appear in the group of sterilized carbon felt + sterile supernatant and the group of carbon felt with bacteria + sterile supernatant, indicating that the electron transfer mode of strain X11 is indirect electron transfer. The maximum voltage of the double-chamber MFC reached 401 mV and the power density reached 206 mW / m -2 .

Claims

1. A Klebsiella species ( Klebsiella sp.) strain X11, whose deposit number is CGMCC 1.62470.

2. Use of the strain according to claim 1 in degrading polyvinyl chloride.

3. The use according to claim 2, characterized in that: include: The strain was inoculated into a degradation medium, and then polyvinyl chloride was added as a carbon source for cultivation.

4. The use according to claim 3, characterized in that: The degradation culture medium is a liquid carbon-free inorganic salt culture medium.

5. The use according to claim 3, characterized in that: The culture temperature is 28-32°C.

6. The use according to claim 3, characterized in that The culture rotation speed is 140~160 rpm.

7. The use according to any one of claims 3 to 6, characterized in that: The application comprises: taking the strain and inoculating it in a liquid carbon-free inorganic salt culture medium to make the initial OD600=0.2, then adding polyvinyl chloride as a carbon source, and continuously culturing under the conditions of 28-32°C and 140-160 rpm.

8. A method for degrading polyvinyl chloride and generating electricity using a microbial fuel cell, characterized in that: The anode liquid of the microbial fuel cell is inoculated with the strain.

9. The method according to claim 8, characterized in that include: The strain is inoculated into the anode liquid of a double-chamber microbial fuel cell, and polyvinyl chloride is used as an anode electron donor and trivalent iron ions are used as a cathode electron acceptor to domesticate and operate the microbial fuel cell.

10. The method according to claim 9, characterized in that The anode is a carbon felt with attached bacteria, and the anode is obtained by co-culturing the carbon felt and the strain in a shaking flask; or by domestication in a double-chamber microbial fuel cell.

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