Klebsiella and application thereof
By screening and optimizing fermentation conditions, the yield of Klebsiella extracellular polysaccharides was increased, solving the problems of high production costs and low efficiency, broadening its application range in the petroleum industry, and providing efficient and environmentally friendly biomaterials.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-25
- Publication Date
- 2026-03-27
AI Technical Summary
Existing technologies for the industrial application of Klebsiella extracellular polysaccharides suffer from high production costs, low efficiency, and environmental pollution, which limit their widespread use.
Klebsiella strains with high yield of novel extracellular polysaccharides were screened. By optimizing the fermentation medium and fermentation conditions, the yield of extracellular polysaccharides was significantly increased. The physicochemical properties of the polysaccharides were comprehensively investigated to reveal their potential application value in industry, especially in the petroleum industry.
It significantly increased the yield of extracellular polysaccharides, broadened their application scope, and provided efficient and environmentally friendly biomaterials for related industrial fields.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial technology, and in particular relates to a type of Klebsiella pneumoniae and its applications. Background Technology
[0002] Klebsiella belongs to the genus Klebsiella and is a group of Gram-negative, facultative anaerobic bacilli belonging to the family Enterobacteriaceae. Klebsiella is widely distributed in nature, including in soil, water bodies, and within plants and animals. Klebsiella also has certain industrial applications, such as in biodiesel, biofertilizers, bioplastics, enzyme production, and wastewater treatment. Overall, the industrial applications of Klebsiella are constantly expanding and being researched, showing broad development prospects.
[0003] In recent years, Klebsiella spp. has gradually become a research hotspot in the field of biotechnology due to its unique production capacity of exopolysaccharides (EPS). The EPS produced by this genus of bacteria has attracted attention due to its abundant capsules, high yield, and unique properties. These polysaccharides not only exhibit significant activity in immunomodulation and potential protective effects against bacterial, fungal, and viral infections, but also demonstrate excellent flocculation properties. Of particular note is the excellent adsorption capacity of Klebsiella polysaccharides for heavy metal ions, which makes them promising for broad applications in wastewater treatment. Compared with traditional organic and inorganic flocculants, microbial-derived polysaccharides have a significantly reduced impact on human health and environmental safety due to their biodegradability.
[0004] Although the applications of Klebsiella pneumoniae extracellular polysaccharides in the biopharmaceutical industry have been extensively explored, their potential applications in other industrial sectors have not yet been fully realized. Currently, research on the physicochemical properties of Klebsiella pneumoniae extracellular polysaccharides is relatively limited, which to some extent restricts their widespread industrial application.
[0005] This invention aims to address the limitations of existing polymer production processes, such as high production costs, low efficiency, and environmental pollution, by utilizing microbial fermentation technology to achieve the production and application of this polymer. Summary of the Invention
[0006] To address the shortcomings of existing technologies, the present invention aims to provide a Klebsiella pneumoniae strain and its applications. This invention seeks to screen Klebsiella pneumoniae strains from nature that produce novel extracellular polysaccharides, and to significantly increase the yield of extracellular polysaccharides by optimizing the fermentation medium and conditions. Simultaneously, this invention will comprehensively investigate the basic physicochemical properties of the produced polysaccharides to reveal their potential industrial applications, particularly in the petroleum industry. Through these studies, the present invention will not only broaden the application scope of Klebsiella pneumoniae extracellular polysaccharides but also provide a highly efficient and environmentally friendly biomaterial for related industrial fields.
[0007] To achieve this objective, the present invention adopts the following technical solution:
[0008] On one hand, the present invention provides a Klebsiella strain with accession number CGMCC No. 29581, accession date January 8, 2024, and depositary institution China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The proposed classification name for this strain is Klebsiella oxytoca.
[0009] This invention provides a novel microbial strain of Klebsiella pneumoniae that can efficiently produce polymers and has potential economic and environmental advantages in industrial applications.
[0010] The microbial strain screening sample of this invention is the root soil of tomatoes. The screening process includes strain isolation, preliminary screening, and re-screening to obtain the strain.
[0011] The rRNA gene sequence of this strain includes the sequence shown in SEQ ID NO.1.
[0012] On the other hand, the present invention provides a fermentation broth of Klebsiella pneumoniae, which is obtained by fermentation of Klebsiella pneumoniae as described above.
[0013] On the other hand, the present invention provides a method for preparing the fermentation broth as described above, the method comprising the following steps:
[0014] The Klebsiella strain described above was cultured in LB medium to obtain a seed culture, which was then inoculated into a shake-flask fermentation medium for further culture to obtain the fermentation broth.
[0015] Preferably, the LB medium consists of 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and a pH of 6.0-8.0 (e.g., 6.0, 6.3, 6.5, 6.8, 7.0, 7.4, 7.8 or 8.0).
[0016] Preferably, the culture time in LB medium is 1 day before transfer.
[0017] Preferably, the shake-flask fermentation medium comprises: 40-60 g / L sucrose (e.g., 40 g / L, 43 g / L, 45 g / L, 48 g / L, 50 g / L, 55 g / L, 58 g / L, or 60 g / L), 2-4 g / L yeast extract (e.g., 2 g / L, 2.3 g / L, 2.5 g / L, 2.8 g / L, 3 g / L, 3.5 g / L, 3.8 g / L, or 4 g / L), 2-3 g / L dimethyl hydrogen phosphate (e.g., 2 g / L, 2.3 g / L, 2.5 g / L, 2.8 g / L, or 3 g / L), 2-3 g / L potassium dihydrogen phosphate (e.g., 2 g / L, 2.3 g / L, 2.5 g / L, 2.8 g / L, or 3 g / L), and 0 g / L magnesium sulfate. 0.1-0.2 g / L (e.g., 0.1 g / L, 0.15 g / L, 0.18 g / L or 0.2 g / L), ferrous sulfate 0.2-0.3 g / L (e.g., 0.2 g / L, 0.23 g / L, 0.25 g / L, 0.28 g / L or 0.3 g / L), calcium carbonate 0.5-2 g / L (e.g., 0.5 g / L, 0.8 g / L, 1 g / L, 1.3 g / L, 1.5 g / L, 1.8 g / L or 2 g / L), potassium chloride 0.5-1 g / L (e.g., 0.5 g / L, 0.8 g / L, 0.9 g / L or 1 g / L), pH 6.0-7.0 (e.g., 6.0, 6.2, 6.4, 6.6, 6.8 or 7.0).
[0018] In this invention, in the shake-flask fermentation medium, sucrose serves as the main carbon source, yeast powder supplies growth factors and vitamins, dimethyl hydrogen phosphate and potassium dihydrogen phosphate maintain phosphate levels, magnesium sulfate and ferrous sulfate provide trace elements, calcium carbonate regulates ionic strength and provides calcium ions, potassium chloride supplements potassium ions, and the pH is adjusted to 6.0-7.0 to provide a suitable growth environment.
[0019] Preferably, the inoculation amount of the seed solution is 3-7%, for example 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5% or 7%.
[0020] Preferably, the fermentation medium is cultured at a temperature of 28-32°C, for example, 28°C, 29°C, 30°C, 31°C or 32°C.
[0021] Preferably, the fermentation medium is cultured for 3-4 days, for example, 3 days, 3.2 days, 3.4 days, 3.5 days, 3.7 days, 3.9 days or 4 days.
[0022] Preferably, the shaking speed during cultivation in the fermentation medium is 200-250 rpm, for example, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm or 250 rpm.
[0023] In this invention, through optimization of the fermentation process, it was determined that the viscosity of the culture medium at the end of fermentation, as described above, can reach a maximum of 5000 cP or more.
[0024] This invention does not use fermentation tanks, but uses a shake flask system (the system used is a 250ml shake flask containing 100ml of liquid). The seed culture is LB medium, and the seed culture time is 1 day before transfer. The inoculum size of the fermentation medium is 3-7%, the culture temperature is 28-32℃, the shaker speed is 200-250rpm, and the fermentation culture time is 3-4 days.
[0025] In this invention, the salt tolerance of the polysaccharide solution produced by fermentation is evaluated. Specifically, the fermentation broth after culture is diluted to a certain ratio, divided into multiple bottles, and then different concentrations of salt are added. The salt ratio is as follows: msodium chloride: mcalcium chloride: mmagnesium chloride = 17.5:1.5:1, ultimately obtaining polymer solutions with different salinities: 0 g / L, 50 g / L, 100 g / L, 150 g / L, 200 g / L, and 250 g / L. Viscosity is then measured using a BROOKFIELD DV-Ⅲ ULTRA viscometer with a 34-piston rotor at 26 rpm (measurement temperature: 25°C; viscosity measurement shear rate: 7.3 s). -1 ).
[0026] The acid and alkali resistance of the polysaccharide solution produced during fermentation was evaluated. Specifically, the fermentation broth was diluted to a certain ratio after the culture was completed, dispensed into multiple bottles, and the pH was adjusted for each bottle to obtain polymer solutions with different pH values: 1, 3, 5, 7, 9, 11, and 13. Viscosity was then measured using a Brookfield DV-Ⅲ ULTRA viscometer with a 34-piston rotor at 26 rpm (measurement temperature: 25°C; viscosity measurement shear rate: 7.3 s). -1 ).
[0027] The temperature resistance of the polysaccharide solution produced during fermentation was evaluated. Specifically, the fermentation broth was diluted to a certain factor after the culture was completed. Then, a Brookfield DV-Ⅲ ULTRA viscometer with a No. 34 rotor and 26 rpm was used to measure the viscosity at different temperatures: 25℃, 35℃, 45℃, 55℃, 65℃, 75℃, and 85℃. (The viscosity was measured at a shear rate of 7.3 s). -1 ).
[0028] On the other hand, the present invention provides a microbial inoculant, which includes Klebsiella pneumoniae as described above or fermentation broth as described above.
[0029] On the other hand, the present invention provides the application of Klebsiella pneumoniae, fermentation broth, or microbial agents as described above in microbial enhanced oil recovery in the petroleum industry.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] The Klebsiella pneumoniae strain of this invention can significantly increase the yield of extracellular polysaccharides, and the viscosity of the fermentation broth can reach more than 5000 cP. The salt tolerance, acid and alkali tolerance and temperature tolerance of the fermentation broth produced were investigated to reveal its potential application value in industry, especially in the petroleum industry. This invention can not only broaden the application range of Klebsiella pneumoniae extracellular polysaccharides, but also provide a high-efficiency and environmentally friendly biomaterial for related industrial fields. Attached Figure Description
[0032] Figure 1 This is a diagram showing the results of the phylogenetic tree comparison of Klebsiella pneumoniae.
[0033] Figure 2 The graph shows the measurement results of the viscosity of the fermentation broth after adding different concentrations of salt.
[0034] Figure 3 The graph shows the measurement results of the effect of different pH values on the viscosity of the fermentation broth.
[0035] Figure 4 The graph shows the measurement results of the effect of different temperatures on the viscosity of the fermentation broth. Detailed Implementation
[0036] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.
[0037] The Klebsiella involved in the following examples is classified and named Klebsiella oxytoca, with accession number CGMCC No. 29581, accession date of January 8, 2024, and deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0038] Example 1
[0039] Screening methods for Klebsiella pneumoniae:
[0040] (1) Soil samples were collected from the roots of tomatoes and safely brought back to the laboratory under low temperature conditions. The samples were then appropriately diluted with 0.9% sterile saline to facilitate subsequent strain screening. After a series of serial dilutions, the samples were plated on LB agar plates and cultured at a constant temperature of 30°C for 36 to 72 hours to ensure microbial growth and reproduction. During LB plate culture, single colonies with distinct morphological characteristics and good growth were selected and further activated and purified to ensure the purity and lack of contamination of the obtained strains. Through a series of screening and purification steps, multiple single colonies were successfully isolated.
[0041] (2) The initial screening process was as follows: After the initial isolation of the bacterial strains, the obtained single colonies were inoculated into screening media specifically designed for further screening. These media were carefully prepared according to the growth requirements of the target microorganisms, aiming to provide a suitable growth environment for the strains to observe and evaluate their growth characteristics and biological activities. The samples were cultured at a constant temperature of 30°C for 36 to 72 hours to ensure the growth and reproduction of the microorganisms. During the culture process, single colonies exhibiting large colony volume and viscous texture were selected and picked up using an inoculation loop. If the colonies exhibited stringy characteristics, they could also be considered as candidate strains.
[0042] (3) The secondary screening process for the strains was as follows: The strains screened initially were inoculated into LB liquid medium and cultured for 24 hours. Afterward, they were used as seed culture and inoculated into the initial fermentation medium at an inoculation rate of 5%. The culture conditions were 30℃, 220 r / min, and fermentation for 72 hours. All culture systems were carried out in 250 ml Erlenmeyer flasks containing 100 ml of liquid. The viscosity of the fermentation broth was measured at the end of fermentation. Viscosity was determined at 25℃ with a shear rate of 7.3 s⁻¹. -1 ).
[0043] LB agar medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L, agar powder 20 g / L, pH 7.0.
[0044] Initial screening culture medium: sucrose 50 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 1 g / L, disodium hydrogen phosphate dodecahydrate 3 g / L, ammonium sulfate 1 g / L, agar powder 20 g / L, pH 7.0.
[0045] Initial fermentation medium: sucrose 60 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 4 g / L, magnesium sulfate 1 g / L, calcium chloride 1 g / L, ferrous sulfate 0.5 g / L, calcium carbonate 1 g / L, pH 7.0.
[0046] As mentioned above, the culture medium can achieve sterilization by using high temperature and high pressure moist heat sterilization method, which can be achieved by treating it at 121℃ for 20 minutes.
[0047] Multiple strains with a viscosity exceeding 100 cP after fermentation were selected as target strains. Biolog analysis was used to detect these strains, and it was preliminarily determined that one Klebsiella strain achieved a fermentation broth viscosity exceeding 2000 cP after fermentation. The results were obtained using Biolog's A and B detection solutions, as shown in Table 1. Solutions A and B are two identification solutions in the Biolog system, with similar functions, differing only in the types of detectable bacteria they contain. Using two solutions ensured the rigor of the experiment. As shown in Table 1, in the Biolog system, a higher similarity indicates a higher degree of matching between the unknown sample and one or more known microorganisms in the database. A lower difference value in the Biolog system indicates a closer similarity between the unknown sample and a known microorganism in the database, thus increasing the reliability of the identification results. Biotype is a parameter describing the type or category of the identified microorganism; here, it is divided into Gram-positive and Gram-negative bacteria.
[0048] Table 1
[0049]
[0050]
[0051]
[0052] Sequencing results, analyzed using 16S RNA in the BLAST database, identified the bacteria as Klebsiella pneumoniae, and phylogenetic alignment results were as follows: Figure 1 As shown.
[0053] Example 2
[0054] The preparation method of the fermentation broth for extracellular capsular polysaccharides is as follows:
[0055] (1) Preparation of single colonies on solid culture medium
[0056] The preserved strain of Klebsiella was inoculated onto slant agar and cultured at 30°C for 24 hours to obtain colonies on a solid culture medium. The slant agar medium formula was as follows: 20 g / L agar powder, 20 g / L glucose, 5 g / L yeast extract, 3 g / L peptone, 3 g / L sodium chloride, 1.5 g / L disodium hydrogen phosphate, and the pH was adjusted to 7.0.
[0057] (2) Microbial enrichment
[0058] Under aseptic conditions, colonies were scraped from the solid culture medium and placed in an Erlenmeyer flask containing 50 mL of sterile liquid LB medium. The flask was incubated at 30°C for 48 h to obtain the enriched bacterial solution. The liquid LB medium consisted of 5 g / L yeast extract, 10 g / L peptone, and 10 g / L NaCl, and was sterilized at 121°C for 20 min.
[0059] (3) The optimized fermentation medium in this example
[0060] Through preliminary optimization of the fermentation process, the polymer yield of the strain at the end of fermentation was successfully increased. The composition of the strain was as follows: sucrose 50 g / L, yeast powder 3 g / L, dimethyl hydrogen phosphate 1.5 g / L and potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate 0.2 g / L and ferrous sulfate 0.25 g / L, calcium carbonate 1.0 g / L, potassium chloride 0.5 g / L, pH adjusted to 7.0, and the culture medium was sterilized at 115°C for 30 minutes. This step not only ensured the sterility of the culture medium but also helped to destroy harmful substances that might inhibit the growth of the strain. The study also listed several media for comparison during the optimization process, such as the initial unoptimized fermentation medium: sucrose 60 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 4 g / L, magnesium sulfate 1 g / L, calcium chloride 1 g / L, ferrous sulfate 0.5 g / L, calcium carbonate 1 g / L, pH 7.0; and starch-based medium: starch: 40 g / L, yeast extract: 2.5 g / L, dimethyl hydrogen phosphate: 1 g / L, potassium dihydrogen phosphate: 1 g / L, magnesium sulfate: 0.15 g / L, ferrous sulfate: 0.2 g / L, calcium carbonate: 0.75 g / L, potassium chloride: 0.4 g / L, pH adjusted to 6.8. Glucosyl medium: Glucose: 35 g / L, yeast extract: 3 g / L, dimethyl hydrogen phosphate: 1.2 g / L, magnesium sulfate: 0.25 g / L, ferrous sulfate: 0.3 g / L, calcium carbonate: 0.5 g / L, potassium chloride: 0.6 g / L, pH adjusted to 7.2. Xylose and glycerol medium: Xylose: 25 g / L, glycerol: 15 g / L, yeast extract: 2.5 g / L, dimethyl hydrogen phosphate: 1.2 g / L, potassium dihydrogen phosphate: 1 g / L, magnesium sulfate: 0.2 g / L, ferrous sulfate: 0.2 g / L, calcium carbonate: 0.6 g / L, potassium chloride: 0.5 g / L, pH adjusted to 7.0.
[0061] Fermentation culture
[0062] The enriched strains were inoculated into optimized fermentation media and cultured for 72 hours. Inoculation was performed at a rate of 5% in 250 mL Erlenmeyer flasks, with a total volume of 100 mL. The fermentation process lasted for 3 days. Results for each medium were presented in duplicate.
[0063] The biopolymer product obtained after the above fermentation process has a viscosity of 4800 cP. Viscosity of the fermentation broth was measured at 25℃ using a BROOKFIELD DV-Ⅲ ULTRA viscometer with a No. 34 rotor at 5 rpm.
[0064] Fermentation batch Culture medium type After fermentation, the viscosity of the fermentation broth is reduced. 1 Initial unoptimized fermentation medium 2120cP, 2430cP 2 Glucosyl fermentation medium 3330cP, 2570cP 3 Xylose and glycerol medium 2840cP, 2560cP 4 Starch-based fermentation medium 1500cP, 1870cP 5 This invention optimizes the post-fermentation culture medium. 5120cP, 5060cP
[0065] Note: The table shows two sets of fermentation broth viscosities after fermentation for each fermentation medium. These figures represent data obtained from two parallel experiments.
[0066] Example 3
[0067] The salt tolerance test of the fermentation broth for extracellular capsular polysaccharides follows these steps:
[0068] A batch of fermentation broth from the optimized fermentation medium was taken and diluted to obtain a fermentation broth of a certain viscosity. The diluted fermentation broth was then divided into several beakers, and different amounts of salt were added to study the salt tolerance of the polysaccharide.
[0069] The ratio of added salt is: sodium chloride: calcium chloride: magnesium chloride = 17.5:1.5:1. Different amounts of the above ratio of salt are added to make the final salt concentrations 0 g / L, 50 g / L, 100 g / L, 150 g / L, 200 g / L, and 250 g / L, respectively.
[0070] After adding salt to the fermentation broth and letting it stand at room temperature overnight to allow the solution to stabilize, the viscosity was measured using a BROOKFIELD DV-Ⅲ ULTRA viscometer with a No. 34 rotor at 26 rpm (measurement temperature was 25℃, and the viscosity was measured at a shear rate of 7.3 s). -1 ).
[0071] The viscosity measurements after adding different concentrations of salt are as follows: Figure 2 As shown.
[0072] Depend on Figure 2 It can be seen that the viscosity of the solution exhibits a certain regularity under different salinity conditions. Starting from a viscosity of 42 cP at 0 g / L salinity, the viscosity decreases slightly with increasing salinity, and this decreasing trend continues until around 150 g / L, indicating that salinity has little effect on viscosity. At 200 g / L salinity, the viscosity increases slightly. When the salinity reaches 250 g / L, an anomaly occurs, with the viscosity rising to nearly 50 cP, indicating that the viscosity of the solution increases significantly under high salinity conditions.
[0073] The acid and alkali resistance test of the fermentation broth of extracellular capsular polysaccharides is conducted using the following steps:
[0074] A batch of fermentation broth from the optimized fermentation medium was taken and diluted to obtain a fermentation broth of a certain viscosity. The diluted fermentation broth was then divided into several beakers, and different amounts of salt were added to study the acid and alkali resistance of the polysaccharide.
[0075] The pH of the diluted fermentation broth was adjusted using 2 mol / L sulfuric acid and 4 mol / L sodium hydroxide solutions to different pH values of 1, 3, 5, 7, 9, 11, and 13.
[0076] After adjusting the pH, the fermentation broth was left at room temperature overnight to allow the solution to reach a stable state. Then, the viscosity was measured using a BROOKFIELD DV-Ⅲ ULTRA viscometer with a No. 34 rotor at 26 rpm (the measurement temperature was 25℃, and the viscosity was measured at a shear rate of 7.3 s⁻¹).
[0077] The effect of different pH values on the fermentation broth, and the viscosity measurement results are as follows: Figure 3 As shown:
[0078] Depend on Figure 3 It can be seen that the viscosity of the solution exhibits a certain regularity under different pH conditions. The viscosity reaches its maximum of approximately 40 cP at around pH 5, and gradually decreases with increasing or decreasing pH. The viscosity decrease is more pronounced under strongly alkaline conditions, indicating that the polysaccharide fermentation broth is more acid-resistant than salt-resistant. The viscosity retention rate can reach over 80% at pH 1, while it is only about 60% at pH 13.
[0079] Example 4:
[0080] The instantaneous temperature tolerance test of the fermentation broth for extracellular capsular polysaccharides is conducted using the following steps:
[0081] A batch of fermentation broth from the optimized fermentation medium was taken and diluted to obtain a fermentation broth of a certain viscosity. The temperature resistance test was carried out using a Thermo Scientific-Haake MARS60 rheometer. The temperature gradients were set as follows: 25℃, 35℃, 45℃, 55℃, 65℃, 75℃, and 85℃. The dwell time at each temperature was 5 minutes, and the average value of each temperature measurement stage was taken.
[0082] The effect of different temperatures on the fermentation broth; viscosity measurement results are as follows: Figure 4 As shown.
[0083] Depend on Figure 4 It can be seen that the viscosity of the solution exhibits a significant temperature-dependent change under different temperature conditions. Starting at 25℃, the viscosity is 43 cP, and it gradually decreases with increasing temperature. When the temperature increases to 55℃, the viscosity drops to 27 cP. Further increasing the temperature to 70℃, the viscosity decreases further to 20 cP, demonstrating the significant effect of temperature on viscosity, indicating that the viscosity of the solution decreases significantly with increasing temperature. Overall, the gradually decreasing viscosity trend with increasing temperature suggests that this polysaccharide fermentation broth cannot withstand high temperatures. In applications requiring a thickening solution, this fermentation broth is only suitable for operation at room temperature or slightly above room temperature.
[0084] The applicant declares that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above process steps, that is, it does not mean that the present invention must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A type of Klebsiella pneumoniae, characterized in that, The Klebsiella pneumoniae has the accession number CGMCCNo.29581, the accession date is January 8, 2024, and the classification name is Klebsiella oxytoca.
2. The Klebsiella pneumoniae according to claim 1, characterized in that, The rRNA gene sequence of the Klebsiella pneumoniae includes the sequence shown in SEQ ID NO.
1.
3. A fermentation broth of Klebsiella pneumoniae, characterized in that, The fermentation broth is obtained by fermentation of Klebsiella pneumoniae as described in claim 1 or 2.
4. The method for preparing fermentation broth according to claim 3, characterized in that, The preparation method includes the following steps: The strain of Klebsiella pneumoniae as described in claim 1 or 2 is cultured in LB medium to obtain a seed culture, which is then inoculated into a shake flask fermentation medium for further culture to obtain the fermentation broth.
5. The preparation method according to claim 4, characterized in that, The LB medium consisted of 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, 20 g / L agar powder, and a pH of 6.0-8.
0.
6. The preparation method according to claim 4 or 5, characterized in that, The culture time in LB medium is 1 day.
7. The preparation method according to any one of claims 4-6, characterized in that, The shake-flask fermentation medium consists of: 40-60 g / L sucrose, 2-4 g / L yeast extract, 2-3 g / L dimethyl hydrogen phosphate, 2-3 g / L potassium dihydrogen phosphate, 0.1-0.2 g / L magnesium sulfate, 0.2-0.3 g / L ferrous sulfate, 0.5-2 g / L calcium carbonate, 0.5-1 g / L potassium chloride, and a pH of 6.0-7.
0.
8. The preparation method according to any one of claims 4-7, characterized in that, The inoculation amount of the seed solution is 3-7%; Preferably, the fermentation medium is cultured at a temperature of 28-32°C; Preferably, the fermentation medium is used for 3-4 days; Preferably, the shaking speed is 200-250 rpm when the fermentation medium is used for cultivation.
9. A microbial inoculant, said microbial inoculant comprising Klebsiella pneumoniae as described in claim 1 or 2 or fermentation broth as described in claim 3.
10. The application of Klebsiella pneumoniae according to claim 1 or 2, or the fermentation broth according to claim 3, or the microbial agent according to claim 9 in microbial enhanced oil recovery in the petroleum industry.