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Chemostatic high density culture method of magnetotactic bacteria high yield magnetosome

A technology for high-density culture and magnetotactic bacteria, which is applied in the field of nanobiotechnology research, can solve the problems of non-commercial application of magnetosomes, difficulty in culturing magnetotactic bacteria, and low yield of magnetosomes, so as to avoid cell autolysis or Effects of growth stop, shorter fermentation period, and simplified operation

Inactive Publication Date: 2009-03-04
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, due to the difficulty in cultivating magnetotactic bacteria, it is difficult to obtain enough magnetosomes for basic and applied research by cultivating magnetotactic bacteria in large quantities. Magnetosomes have not been commercially applied so far.
The problem of large-scale cultivation of magnetotactic bacteria is mainly reflected in the following three aspects: (1) The cultivation period is long. According to the literature report, the shortest cultivation time is 50 hours, and the longest is as long as 10 days; The limitation of the concentration of carbon and nitrogen sources in the middle causes the culture density to be low and the yield of magnetosomes is very low; (3) the relationship between cell density and magnetosome yield needs to be further coordinated, and the cell density has been obtained under certain conditions. Significantly increased, but the yield of magnetosomes did not increase

Method used

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  • Chemostatic high density culture method of magnetotactic bacteria high yield magnetosome
  • Chemostatic high density culture method of magnetotactic bacteria high yield magnetosome
  • Chemostatic high density culture method of magnetotactic bacteria high yield magnetosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Activation of magnetotactic bacteria

[0043] The original bacterial strain MSR-1 (purchased from the German Culture Collection of Microorganisms (DSMZ)) was activated, and the composition of the sodium lactate liquid medium used was as follows:

[0044] Mineral Element Mixture 5ml

[0045] Ferric citrate (0.01mol / L) 6ml

[0046] K 2 HPO 40.5g

[0047] Yeast powder (Oxoid) 0.1g

[0048] MgSO 4 ·7H 2 O 0.1g

[0049] NH 4 Cl 0.6g

[0050] Sodium Thioglycolate 0.05g

[0051] 80% sodium lactate 2.25g

[0052] Distilled water 1000ml

[0053] pH 7.0

[0054] Sodium lactate solid medium: Add agar powder to sodium lactate liquid medium at a ratio of 0.75% to make sodium lactate solid medium.

[0055] The activation and cultivation methods are as follows:

[0056] The original strain was used to streak on a sodium lactate solid medium plate, the petri dish was sealed with parafilm, and cultured at 30° C. for 5 days.

[0057] Pick a single colony, inocu...

Embodiment 2

[0059] Example 2 Effects of carbon and nitrogen source concentration on shake flask and submerged culture of Magnetospira

[0060] Take the seed solution for the following experiments:

[0061] 1) Shake flask carbon source concentration test

[0062] Add 90ml of sodium lactate medium containing 10, 20, 30, 40, and 50mmol / L to a 250ml serum bottle respectively, insert the activated seed solution, and culture in a shaker at 30°C at 100 rpm for 24 hours, and measure the OD 565nm , the result is as figure 1 shown. Obviously, the medium containing 1-30mmol / L sodium lactate has the effect of promoting the growth of Magnetospira. The most suitable concentration of sodium lactate is 20mmol / L, and when the concentration exceeds 30mmol / L, it can obviously inhibit the growth of bacteria.

[0063] 2) Shake flask nitrogen source test

[0064] Add 90ml of sodium lactate liquid culture medium to two 250ml serum bottles respectively. The medium composition of one group is as above, and th...

Embodiment 3

[0067] Example 3 Relationship Between Dissolved Oxygen (DO) and Magnetospira Growth and Magnetosome Synthesis

[0068] In a 42L automatic fermenter, 27L of fermentation medium is loaded, and the composition of the medium is as follows:

[0069] Mineral Element Mixture 5ml

[0070] Ferric citrate (0.01mol / L) 6ml

[0071] NH 4 Cl 0.6g

[0072] 80% sodium lactate 2.25g

[0073] K 2 HPO4 0.5g

[0074] Yeast powder 0.5g

[0075] MgSO 4 ·7H 2 O 0.2g

[0076] Sodium Thioglycolate 0.05g

[0077] Distilled water 1000ml

[0078] Inoculate 10%, culture with air, set the initial ventilation rate of 0.3L / min and the speed of 100 rpm to maintain the dissolved oxygen concentration at a low level, and set the dissolved oxygen (DO) at this time to 100% (500ppb). Under this culture condition, dissolved oxygen drops to 0% after 4 hours of fermentation, and the cell density at this time is 0.26OD 565nm , in order to promote cell growth, the rotation speed was increased by 40 rpm at ...

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Abstract

The invention provides a method for culturing magnetotactic bacterium high-yield magnetic particles chemostatically with high density; magnetotactic bacterium is cultured through a submerged fermentation culture method; the fermentation culture medium contains a carbon source, a nitrogen source and an iron source; dissolved oxygen is controlled in the fermentation process by stages, and materials are supplemented by regulating the pH value. The method realizes the chemostatical high-density culture of the magnetotactic bacterium and the mass production of the magnetic particles; the density of the cells cultured for 36h to 48h is 12OD565nm, the dry weight of the magnetic particles is 80mg / L to 100mg / L, and the fermentation cycle is shortened. The method lays solid foundation for the industrialized production of bacterium magnetic particles.

Description

technical field [0001] The invention relates to the field of nanobiological technology research, in particular to a method for cultivating magnetotactic bacteria with high density and high yield of magnetosomes. Background technique [0002] Magnetotactic bacteria are a kind of special bacteria that form nano-magnetosomes in the cells. The magnetosomes formed by them have the characteristics of stable crystal form, small size, outer membrane coating and not easy to agglomerate. , Nucleic acid separation and purification, etc. have shown advantages over other materials, especially in tumor targeting and gene therapy, and have important application prospects. [0003] However, due to the difficulty in culturing magnetotactic bacteria, it is difficult to obtain enough magnetosomes for basic and applied research by cultivating magnetotactic bacteria in large quantities, and magnetosomes have not been commercially applied so far. The problem of large-scale cultivation of magneto...

Claims

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Application Information

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IPC IPC(8): C12P9/00C12N1/20C12R1/01
Inventor 姜伟刘阳张扬李颖关国华田杰生李季伦
Owner CHINA AGRI UNIV
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