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Method for collecting nitrosobacteria

A nitrosative bacteria, cryopreservation technology, applied in the direction of preservation of microorganisms, bacteria, etc., can solve the problems of uncontrollable two-way exchange of extracellular and extracellular substances, increase of van der Waals force between molecules, increase of cell membrane permeability, etc., to achieve excellent performance retention and flow improvement The effect of sex and stability of membrane protein structure

Active Publication Date: 2018-09-04
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When lyophilized and dehydrated, the polar ends of phospholipids remove the hydration hydrogen bonds, forcing the acyl groups to gather together, the density of the polar ends increases, the van der Waals force between molecules increases, and the liquid crystal phase lipid membrane changes to a gel phase. Increased permeability makes cells unable to control the two-way exchange of extracellular and extracellular substances, thus causing metabolic damage to cells

Method used

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  • Method for collecting nitrosobacteria
  • Method for collecting nitrosobacteria

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Preservation method of nitrosative bacteria:

[0029] 1) Prepare the protective agent: add 3 parts of sodium glutamate, 20 parts of tripotassium phosphate and 2 parts of cysteine ​​to 100 parts of sterile water, mix well to obtain the protective agent;

[0030] 2) Adding bacterial cells: Add 160 parts of nitrosative bacterial cells to 110 parts of the protective agent obtained in step 1), shake and resuspend to obtain a mixed solution;

[0031] 3) Cold and heat stress treatment: heat-treat the obtained mixture at 35°C for 12 minutes, and then at 6°C for 1.7 hours;

[0032] 4) Cryopreservation: freeze-dry the mixed solution obtained in step 3), freeze-preserve at -80° C., and perform activation transfer every 8-12 months. The freeze-drying process is: pre-freezing: -30°C for 4 hours; freeze-drying: -60°C, 40Pa freeze-drying for 6 hours, -20°C, 70Pa freeze-drying for 2.2 hours; sublimation drying: 10°C, 150Pa sublimation drying for 3.5 hours; Drying: 16°C, 4Pa analytical ...

Embodiment 2

[0034] Preservation method of nitrosative bacteria:

[0035] 1) Prepare the protective agent: add 3.5 parts of sodium glutamate, 22 parts of tripotassium phosphate and 2 parts of cysteine ​​to 110 parts of sterile water, mix well to obtain the protective agent;

[0036] 2) Add bacterial cells: add 140 parts of nitrosative bacterial cells to 100 parts of the protective agent obtained in step 1), shake and resuspend to obtain a mixed solution;

[0037] 3) Cold and heat stress treatment: heat-treat the obtained mixture at 37°C for 10 minutes, and then at 7°C for 1.2 hours;

[0038] 4) Cryopreservation: freeze-dry the mixed liquid obtained in step 3), freeze-preserve at -85°C, and perform activation transfer every 8-12 months. The freeze-drying process is as follows: pre-freezing: -30°C for 4 hours; freeze-drying: -58°C, 40Pa freeze-drying for 6 hours, -20°C, 70Pa freeze-drying for 2.2 hours; sublimation drying: 10°C, 150Pa sublimation drying for 3.5 hours; Drying: 16°C, 4Pa ana...

Embodiment 3

[0040] Preservation method of nitrosative bacteria:

[0041] 1) Prepare the protective agent: add 2.8 parts of sodium glutamate, 20 parts of tripotassium phosphate and 2 parts of cysteine ​​to 100 parts of sterile water, mix well to obtain the protective agent;

[0042] 2) Adding cells: Add 150 parts of nitrosative bacteria cells to 100 parts of the protective agent obtained in step 1), shake and resuspend to obtain a mixture;

[0043] 3) Cold and heat stress treatment: heat-treat the obtained mixture at 37°C for 10 minutes, and then at 7°C for 1.2 hours;

[0044] 4) Cryopreservation: freeze-dry the mixed solution obtained in step 3), freeze-preserve at -80° C., and perform activation transfer every 8-12 months. The freeze-drying process is as follows: pre-freezing: -35°C for 4 hours; freeze-drying: -60°C, 40Pa freeze-drying for 6 hours, -20°C, 70Pa freeze-drying for 2.2 hours; sublimation drying: 10°C, 140Pa sublimation drying for 3.5 hours; Drying: 16°C, 4Pa analytical dry...

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Abstract

The invention discloses a method for collecting nitrosobacteria. The method comprises the following steps: 1) preparing a protective agent, namely adding 2-4 parts of sodium glutamate, 18-25 parts oftripotassium phosphate and 1-3 parts of cysteine into 90-120 parts of sterile water, and uniformly mixing to obtain the protective agent; 2) adding thalli, namely 0.8-1.2 times that of the volume of nitrosobacteria thalli into the protective agent obtained in the step 1), and performing shock re-suspending so as to obtain a mixed solution; and 3) freezing and preserving, namely performing freeze drying on the mixed solution obtained in the step 2), freezing and preserving at a temperature of 70-90 DEG C, and performing activation transfer every 2-3 months. The method disclosed by the inventionhas the beneficial effects that damage of freeze drying on the nitrosobacteria thalli can be greatly reduced, and the nitrosobacteria survival rate and activities of ATP synthase and ammonia monooxygenase (AMO) in the preservation process are improved.

Description

technical field [0001] The invention relates to the technical field of strain preservation, in particular to a method for preserving nitrosating bacteria. Background technique [0002] Ammonia nitrogen is first converted into nitrite under the action of nitrosifying bacteria, and then nitrite is further converted into nitrate under the action of nitrifying bacteria. The overall reaction formula of nitration reaction is: NH 4 + +1.815O 2 +0.1304CO 2 →0.0261C 5 h 7 o 2 +0.973NO 3 - -N+0.921H 2 O+1.973H + . The oxidative metabolic pathway of the nitrogen cycle of nitrosative bacteria mainly includes ammonia (NH 3 ) is oxidized to hydroxylamine (NH 2 OH), hydroxylamine is oxidized to nitrite under the catalysis of hydroxylamine reductase (HAO). Nitrite bacteria are widely distributed in nature. Nitrite bacteria exist in soil, ocean and fresh water, and they only account for a very small proportion of the total number of bacteria. Different species of nitrous bacte...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/04
CPCC12N1/04C12N1/20
Inventor 汤江武孙宏李园成吴逸飞姚晓红王新沈琦
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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