Method for three-dimensionally culturing magnetotactic bacteria and application of magnetotactic bacteria in synthesis of magnetosomes

Abstract

The invention belongs to the technical field of nano biology, and particularly discloses a method for three-dimensional culture of magnetotactic bacteria and application of the magnetotactic bacteria to synthesis of magnetosome.The method for three-dimensional culture of the magnetotactic bacteria comprises the following steps that alginate gel beads are prepared, specifically, AMB-1 bacteria liquid and filtered and sterilized Alg-Na solution are evenly mixed, and alginate gel beads are prepared; dropwise adding the alginate gel beads into a filtered and sterilized FeCl3 solution through a high-voltage electrostatic encapsulation device to form alginate gel beads embedded with AMB-1; pMCG film forming reaction: soaking the prepared alginate gel beads in a PMCG solution to carry out film forming reaction, so that a layer of PMCG film is formed on the surfaces of the alginate gel beads; microcapsule membrane surface treatment: soaking the treated alginate gel beads in a solution to carry out microcapsule surface modification; and microcapsule core liquefaction: soaking the treated alginate gel beads in a sodium citrate solution for reaction to liquefy the core of the gel beads, and finally washing with ultrapure water to obtain the bacterium-loaded microcapsule. The three-dimensional culture of the magnetotactic bacteria is realized, and the magnetic production period of the magnetotactic bacteria is prolonged by 4-6 hours.

Description

Technical Field

[0001] The invention belongs to the field of nano-biotechnology, and specifically relates to a method for three-dimensionally culturing magnetotactic bacteria and an application thereof in synthesizing magnetosomes. Background Art

[0002] Magnetotactic bacteria (MTB) is a general term for a class of Gram-negative bacteria that can be arranged and directed along the magnetic field lines of the Earth's magnetic field. It was first discovered by Bellini in 1963 and can synthesize magnetic nanoparticles - magnetosomes. Magnetosomes are the core structure of magnetotactic bacteria, composed of an external lipid bilayer and an internal mineral crystal (Fe3O4 or Fe3S4), and are arranged in chains within the cell. Magnetosomes have become a classic model for studying the formation of prokaryotic organelles. Its formation mechanism involves a variety of genes and proteins and has developed into a unique field of multidisciplinary intersection. Compared with artificially synthesized magnetic nanoparticles, magnetosomes synthesized in bacteria have uniform morphology, narrow particle size distribution, biofilm wrapping and good biocompatibility, which make them have great application value in biomedicine, biomaterials and other fields. However, there are currently few reports on the application of magnetosomes, mainly due to the low yield of magnetosomes. Therefore, how to improve the density of bacteria and the yield of magnetosomes is a key issue that needs to be solved urgently.

[0003] At present, the main methods for culturing magnetotactic bacteria are liquid submerged culture in culture bottles and fermentation tanks. The former has the advantage of simple operation, but the disadvantages are low bacterial density (generally, the bacterial OD value is only about 0.2) and low magnetosome production. The latter has the advantage of high bacterial density and large magnetosome production. Liu et al. used a 42L fermentation tank and a constant culture method to culture MSR-1. The bacterial OD value was 0.2. 565 The value reached 12, and the magnetosome yield was 83.23±5.36 mg / L (dry weight); however, this method also has disadvantages, namely, it has high requirements for instruments, high cost, and the experimenters need to perform continuous operations for a long time.

[0004] Chang first reported the microencapsulation of bioactive substances in 1957. Bioactive substances such as enzymes, proteins and hormones were encapsulated in a selective permeable membrane to form spherical microcapsules. Through the selective permeation of the microcapsule membrane, substances with a molecular weight greater than a certain value outside the capsule cannot diffuse into it, while nutrients in the biological environment and bioactive substances or small molecular products secreted by cells in the capsule can freely enter and exit the microcapsule. At present, the research on microencapsulated bacteria is mainly focused on the use of probiotics, such as lactic acid bacteria and bifidobacteria; or using bacteria to obtain some useful metabolites, including biomaterials, such as polyhydroxybutyrate (PHB). Commonly used methods for preparing microcapsules of bacteria or cells include spray drying, spray freeze drying and high-voltage electrostatic method. Spray drying is the most commonly used method for preparing microcapsules in the food industry. It is both economical and effective. Yu used spray drying to prepare biological microcapsules for encapsulating Gram-negative bacteria. After optimizing the conditions, the bacterial survival rate reached 84.57%, and then after a period of anaerobic culture, the number of bacteria increased by 148%. However, the disadvantage of this method is that the simultaneous dehydration, heat treatment and oxygen pressure during the drying process cause a high bacterial mortality rate. Semyonov used spray freeze drying to prepare microcapsules and successfully encapsulated lactic acid bacteria, and the bacterial activity exceeded 60%. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide a method for three-dimensionally culturing magnetotactic bacteria and its application in synthesizing magnetosomes. By culturing magnetotactic bacteria in microencapsulation, the growth law of bacteria in a microenvironment is mastered, the bacterial density and the number of magnetosomes are increased, and the optimal conditions for preparing microcapsules carrying magnetotactic bacteria are obtained, including relevant information such as materials, voltage, liquid surface distance, propulsion speed, etc.

[0006] The present invention explores the interaction between the culture system and magnetotactic bacteria, and promotes the use and development of new microcapsule materials. Through this culture method, a relatively stable microenvironment is provided for magnetotactic bacteria, the bacteria are less affected by external factors in the microcapsule, and nutrients can freely enter the microcapsule; another significant advantage is that iron ions are used as microcapsule crosslinking agents, which overcomes the problem of low concentration of dissolved iron ions in conventional culture, realizes the enrichment culture of iron ions in microcapsules, and is conducive to the synthesis of magnetosomes. At the same time, microencapsulation culture has the advantages of both suspension culture and adherent culture, which can increase the yield of magnetotactic bacteria.

[0007] In order to achieve the above object, one of the technical solutions of the present invention is: a method for three-dimensionally culturing magnetotactic bacteria, comprising the following steps:

[0008] (1) Preparation of alginate beads: AMB-1 bacterial liquid and a filtered and sterilized sodium alginate (Alg-Na) solution were mixed evenly, and then added dropwise to a filtered and sterilized FeCl3 solution using a high-voltage electrostatic encapsulation device to form alginate beads encapsulating AMB-1;

[0009] (2) poly(methylenec-co-quanidine) (PMCG) film-forming reaction: immersing the alginate beads prepared in step (1) in a PMCG solution for film-forming reaction, so that a layer of PMCG film is formed on the surface of the alginate beads;

[0010] (3) Surface treatment of microcapsule membrane: immersing the alginate beads treated in step (2) in an Alg-Na solution to repair the surface of the microcapsule;

[0011] (4) Liquefaction of microcapsule core: The alginate beads treated in step (3) are immersed in a sodium citrate solution for reaction for min to liquefy the microcapsule core, and finally washed with ultrapure water to obtain bacteria-loaded microcapsules.

[0012] In a preferred embodiment of the present invention, the preparation method of AMB-1 bacterial solution in step (1) comprises the following steps: placing the magnetotactic bacteria ABM-1 at -4°C for 1 to 60 minutes, and then placing it at room temperature for 1 to 20 minutes, and after the strain is thawed, immediately transferring it to 10 mL of sterilized culture medium with an inoculum of 400-1200 μL and 100-400 μL, respectively, and culturing it at 25-27°C anaerobic environment for 3 to 20 days, and observing the growth status under an inverted microscope; inspecting the bacterial solution under an inverted microscope to observe the growth status of the strain and whether it is contaminated; selecting the bacterial solution with better growth status, and using a pipette to transfer 400-1200 μL and 100-400 μL of the bacterial solution into 10 mL of sterilized culture medium, respectively; Sterilized culture medium, incubate at 25-27℃ anaerobic environment; examine the bacterial solution under a microscope, and select the bacterial solution with good growth for experiment; add about 800μL culture medium to the capillary, transfer enough bacterial solution to fill the sample chamber, place the magnet 1-10cm away from the tip of the capillary, and magnetic separation for 10-60min; add 800μL culture medium; break the capillary at 1-10cm from the tip of the capillary, transfer the bacterial solution into the above centrifuge tube, and incubate at 25-27℃; after 2-8d, examine the growth status of the bacterial solution and whether it is contaminated, and transfer all the bacterial solution into 10ml culture medium, incubate at 25-27℃, examine under a microscope and measure the OD value and magnetic sensitivity coefficient (coefficient ofmagnetically induced differential lightscattering, Cmag); examine the bacterial solution under a microscope and select the bacterial solution with good growth condition for preservation; add about 50-500 μL of glycerol to the preservation tube, transfer about 500-1500 μL of bacterial solution and glycerol to mix thoroughly, mark the number and quickly store in a -80℃ refrigerator.

[0013] In a preferred embodiment of the present invention, in step (1), the volume ratio of the AMB-1 bacterial solution to the Alg-Na solution is 1:(8-14), the concentration of the Alg-Na solution is 0.5-5.0% (w / v), the volume ratio of the AMB-1 bacterial solution to the FeCl3 solution is 1:(25-30), and the concentration of the FeCl3 solution is 10-300 mM.

[0014] In a preferred embodiment of the present invention, in step (2), the volume ratio of alginate beads to PMCG solution is 1:(1-10), and the concentration of PMCG solution is 1.0-3.5% (w / v).

[0015] In a preferred embodiment of the present invention, the film-forming reaction time in step (2) is 5-100 min.

[0016] In a preferred embodiment of the present invention, in step (3), the volume ratio of alginate beads to Alg-Na solution is 1:(3-6), the concentration of Alg-Na solution is 0.1-1.0% (w / v), and the modification time is 1-10 min.

[0017] In a preferred embodiment of the present invention, in step (4), the volume ratio of alginate beads to sodium citrate solution is 1:(1-10), the concentration of sodium citrate solution is 0.1-1.0% (w / v), and the reaction time is 1-30 min.

[0018] In order to achieve the above objectives, the second technical solution of the present invention is: a bacteria-loaded microcapsule obtained by a method for three-dimensionally culturing magnetotactic bacteria.

[0019] In order to achieve the above objectives, the third technical solution of the present invention is: application of a method for three-dimensionally culturing magnetotactic bacteria in synthesizing magnetosomes.

[0020] In a preferred embodiment of the present invention, the method for synthesizing magnetosomes comprises placing the bacteria-loaded microcapsules in a fresh culture medium, culturing them at 20-40°C for 24-72 hours, and after the membrane is broken, taking the broken membrane bacterial solution and transferring it to the culture medium for further cultivation, and measuring the OD every 12 hours. 600 and Cmag value, draw the growth curve, extract and purify the magnetosomes.

[0021] In a preferred embodiment of the present invention, the volume ratio of the membrane-breaking bacterial solution to the culture medium is 1:(40-60).

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The method of the present invention realizes the three-dimensional culture of magnetotactic bacteria and prolongs the magnetization period of magnetotactic bacteria by 4-6 hours; a new method of culturing magnetotactic bacteria is successfully created;

[0024] 2. The present invention adopts an electrostatic encapsulation device, takes bacteria as the capsule core, and iron ions as the cross-linking agent, and optimizes the preparation of an embedded magnetotactic bacteria culture unit with uniform particle size, good permeability, and the advantages of both adherent culture and suspended culture; while overcoming the disadvantage of low iron ion concentration in traditional culture methods, the three-dimensional culture of magnetotactic bacteria provides a good microaerobic environment for magnetotactic bacteria to synthesize magnetosomes, thereby realizing the large-scale synthesis of magnetosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The growth curve and magnetic sensitivity curve of the magnetotactic bacteria AMB-1 in the present invention;

[0026] Figure 2 is the density curve of the magnetotactic bacteria AMB-1 in the present invention;

[0027] Figure 3 The growth curve and magnetic sensitivity curve of the magnetotactic bacteria AMB-1 under different culture conditions in the present invention;

[0028] Figure 4 The microscopic examination results of the effect of the microencapsulated reagent of the present invention on the growth of magnetotactic bacteria AMB-1;

[0029] Figure 5 The microscopic examination results of the effect of high voltage static electricity on the growth of magnetotactic bacteria AMB-1 in the present invention;

[0030] Figure 6 Optical microscope photos of iron alginate gel beads and iron alginate-PMCG / sodium alginate microcapsules in the present invention;

[0031] Figure 7 The effect of PMCG concentration in the film-forming solution on the biomass of microencapsulated AMB-1 in the present invention;

[0032] Figure 8 The growth curve of AMB-1 under different iron source culture conditions in the present invention;

[0033] Fig. 9 The figure is a transmission electron microscope photograph of the magnetic body prepared in the present invention. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in more detail below with reference to the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited to these embodiments.

[0035] The magnetotactic bacteria AMB-1 used in the following examples were cultured in the following manner:

[0036] (1) Activation of bacteria: The stored magnetotactic bacteria ABM-1 were taken out from a -80°C refrigerator, placed in a -4°C refrigerator for 10 min, and then placed at room temperature for 3 min. After the strain was thawed, it was immediately transferred to a screw-capped test tube containing 10 mL of sterilized culture medium with an inoculum of 800 μL and 200 μL, respectively. The tubes were placed in an anaerobic incubator at 26°C for 7 days, and the growth status was observed under an inverted microscope.

[0037] (2) Transfer of bacteria: Use an inverted microscope to examine the bacterial solution to observe the growth of the bacteria and whether it is contaminated; select the bacterial solution with better growth, use a pipette to transfer 800 μL and 200 μL of the bacterial solution into screw-capped test tubes containing 10 mL of sterilized culture medium, mark the date, and culture in an anaerobic incubator at 26°C;

[0038] (3) Optimization of magnetic separation of bacteria: Examine the bacterial solution under a microscope and select the bacterial solution with good growth condition for the experiment; add about 800 μL of culture medium to the capillary, transfer enough bacterial solution to fill the sample chamber, place a magnet (25.4×12.7×5.35 mm) 2 cm away from the tip of the capillary, and perform magnetic separation for 20 min; add 800 μL of culture medium to the centrifuge tube; break the capillary 2 cm from the tip, transfer the bacterial solution into the above centrifuge tube, and incubate at a constant temperature of 26°C; after 2 days, examine the growth condition of the bacterial solution and whether it is contaminated, and transfer all the bacterial solution into a 10 ml screw-capped test tube filled with culture medium, incubate at a constant temperature of 26°C, examine under a microscope and measure the OD value and the coefficient of magnetically induced differential lightscattering (Cmag);

[0039] (4) Preservation of bacteria: Examine the bacterial solution under a microscope and select the bacteria with good growth conditions for preservation; add 150 μL of glycerol to the preservation tube, transfer 850 μL of the bacterial solution and glycerol and mix thoroughly, mark the tube with the number, and quickly store it in a -80°C refrigerator.

[0040] Figure 1 The growth curve and magnetosusceptibility curve of magnetotactic bacteria AMB-1; Figure 2 This is the density curve of magnetotactic bacteria AMB-1.

[0041] Example 1

[0042] A bacteria-carrying microcapsule is prepared by a method of three-dimensionally culturing magnetotactic bacteria, which specifically comprises the following steps:

[0043] 1) Preparation of alginate beads: 1.75% (w / v) sodium alginate (Alg-Na) solution and 100 mM FeCl3 solution were prepared and sterilized by three-stage filtration through a microporous filter membrane; the above-mentioned AMB-1 bacterial solution was mixed with the 1.75% (w / v) Alg-Na solution that had been sterilized by filtration at a volume ratio of 1:12 (10 ml of AMB-1 bacterial solution and 120 ml of Alg-Na solution), and the mixture was evenly mixed by pipetting, and the above-mentioned mixture was added dropwise to 300 ml of 10 mM FeCl3 solution through a high-voltage electrostatic encapsulation device to form alginate beads encapsulating AMB-1. After the reaction was complete, the mixture was filtered through a tea filter and rinsed with ultrapure water to remove the residual solution. Figure 4 The microscopic examination results of the effect of microencapsulation reagent on the growth of magnetotactic bacteria AMB-1. Figure 5 The microscopic examination results of the effect of high voltage static electricity on the growth of magnetotactic bacteria AMB-1. Figure 8 The growth curve of AMB-1 under different iron source culture conditions;

[0044] 2) Poly(methylenec-co-quanidine) (PMCG) film-forming reaction: 2 ml of the alginate beads prepared in the previous step were immersed in 10 mL of PMCG solution for 10 min to form a layer of PMCG film on the surface of the alginate beads, which was then filtered and washed with a tea filter; Figure 7 The effect of PMCG concentration in the film-forming solution on the biomass of microencapsulated AMB-1 in the present invention;

[0045] 3) Microcapsule membrane surface treatment: The film-forming beads prepared in the previous step were immersed in 10 mL of 0.15% (w / v) Alg-Na solution to treat the microcapsule surface for 5 min, filtered and washed with a tea filter;

[0046] 4) Liquefaction of microcapsule core: The microcapsules were immersed in 15 mL of 0.16% (w / v) sodium citrate solution for 3 min to liquefy the microcapsule core, and then washed with ultrapure water for 5 times, filtered through a tea filter, and the AMB-1-loaded sodium alginate / polymethylene biguanide microcapsules were placed in a 50 mL centrifuge tube for later use. Figure 6 Optical microscope photos of iron alginate gel beads and iron alginate-PMCG / sodium alginate microcapsules.

[0047] Example 2

[0048] The AMB-1-loaded sodium alginate / polymethylene biguanide microcapsules prepared in Example 1 were placed in fresh culture medium and cultured at 26°C for 48 h. After the membrane was broken, 1 mL of the broken bacterial solution was transferred to 50 mL of culture medium and cultured continuously. The OD was measured every 12 h. 600 and Cmag value, draw the growth curve, extract and purify the magnetosomes. Figure 3 The growth curve and magnetosensitivity curve of magnetotactic bacteria AMB-1 under different culture conditions. Fig. 9 This is a transmission electron microscope photo of the prepared magnetic body.

[0049] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for three-dimensionally culturing magnetotactic bacteria, characterized in that: The steps include: (1) Preparation of alginate beads: AMB-1 bacterial solution and filtered sterilized Alg-Na solution were mixed evenly, and then added dropwise to filtered sterilized FeCl3 solution through a high-voltage electrostatic encapsulation device to form alginate beads encapsulating AMB-1; (2) PMCG film-forming reaction: immersing the alginate beads obtained in step (1) in a PMCG solution for film-forming reaction, so that a layer of PMCG film is formed on the surface of the alginate beads; (3) Surface treatment of microcapsule membrane: immersing the alginate beads treated in step (2) in a solution to modify the surface of the microcapsule; (4) Liquefaction of microcapsule core: The alginate beads treated in step (3) are immersed in a sodium citrate solution to react and liquefy the microcapsule core, and finally washed with ultrapure water to obtain bacteria-loaded microcapsules.

2. The method for three-dimensionally culturing magnetotactic bacteria as claimed in claim 1, characterized in that: In the step (1), the volume ratio of the AMB-1 bacterial solution to the Alg-Na solution is 1:(8-14), the concentration of the Alg-Na solution is 0.5-5.0% (w / v), the volume ratio of the AMB-1 bacterial solution to the FeCl3 solution is 1:(25-30), and the concentration of the FeCl3 solution is 10-300 mM.

3. The method for three-dimensionally culturing magnetotactic bacteria as claimed in claim 1, characterized in that: In the step (2), the volume ratio of alginate beads to PMCG solution is 1:(1-10), and the concentration of PMCG solution is 1.0-3.5% (w / v).

4. The method for three-dimensionally culturing magnetotactic bacteria as claimed in claim 1, characterized in that: The film-forming reaction time in step (2) is 5-100 minutes.

5. The method for three-dimensionally culturing magnetotactic bacteria according to claim 1, characterized in that: In the step (3), the volume ratio of alginate beads to Alg-Na solution is 1:(3-6), the concentration of Alg-Na solution is 0.1-1.0% (w / v), and the modification time is 1-10 min.

6. The method for three-dimensionally culturing magnetotactic bacteria according to claim 1, characterized in that: In the step (4), the volume ratio of alginate beads to sodium citrate solution is 1:(1-10), the concentration of sodium citrate solution is 0.1-1.0% (w / v), and the reaction time is 1-30 min.

7. A bacteria-loaded microcapsule obtained by the method for three-dimensionally culturing magnetotactic bacteria as described in any one of claims 1 to 6.

8. Use of the method for three-dimensionally culturing magnetotactic bacteria as described in any one of claims 1 to 6 in synthesizing magnetosomes.

9. Application of the method for three-dimensionally culturing magnetotactic bacteria as claimed in claim 8 in synthesizing magnetosomes, characterized in that: The method for synthesizing magnetosomes comprises placing the bacteria-carrying microcapsules in a fresh culture medium, culturing them at 20-40° C. for 24-72 hours, taking the membrane-breaking bacterial solution and transferring it to the culture medium for further culturing, and measuring the OD every 12 hours. 600 and Cmag value, draw the growth curve, and extract and purify the magnetosomes.

10. Application of the method for three-dimensionally culturing magnetotactic bacteria according to claim 9 in synthesizing magnetosomes, characterized in that: The volume ratio of the membrane-breaking bacterial solution to the culture medium is 1:(40-60).

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