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Pure culture method of anaerobic microorganisms

An anaerobic microorganism, pure culture technology, applied in the field of bioengineering, can solve the problems of culture waste liquid pollution, high solubility, excessive reducing dose, etc., to achieve the effect of simplified separation, simple and convenient operation

Pending Publication Date: 2021-07-23
DONGGUAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in this method, the added reducing dose is too much, the solubility is large, and it is eaten by microorganisms, which affects the growth of anaerobic microorganisms; at the same time, the reducing agent is not easy to separate and recover from microorganisms, which affects the purity of the final microbial liquid, resulting in waste cultivation liquid contamination

Method used

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Experimental program
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Effect test

Embodiment 1

[0029] Implementation 1: Taking the cultivation of Geobacter sulfur-reducing as an example

[0030] Liquid culture solution composition: the following components are included in each liter of liquid culture solution: 1.5g NH 4 Cl, 0.6gNa 2 HPO 4 , 0.1g KCl, 0.82g CH 3 COONa, 2.5g NaHCO 3 , 8.0g disodium fumarate, 10mL trace element stock solution; 10mL vitamin solution; the following components are included in each liter of trace element stock solution: 1.5g nitrilotriacetic acid, 3.0g MgSO 4 ·7H 2 O, 0.5gMnSO 4 ·H 2 O, 1.0g NaCl, 0.1g FeSO 4 ·7H 2 O, 0.1g CoCl 2 ·6H 2 O, 0.76g CaCl 2 , 0.1gZnSO 4 ·7H 2 O, 0.01g CuSO 4 ·5H 2 O, 0.02g KAl(SO 4 ) 2 ·12H 2O, 0.01g H 3 BO 3 , 0.01g Na 2 MoO 4 ·12H 2 O; each liter of vitamin liquid includes the following components: 2mg biotin, 2mg folic acid, 10mg vitamin B 6 Hydrochloride, Thiamine Hydrochloride 5mg, Riboflavin 5mg, Vitamin B 5mg 3 , 5mg D-calcium pantothenate, 0.1mg vitamin B 12 , 5mg p-aminobenzoic aci...

Embodiment 2～6

[0034] Examples 2 to 6: all take the cultivation of P. Acetatigenes as an example.

[0035] The components of the liquid culture solution are: each liter of the liquid culture solution includes the following components: 5g polypeptone, 5g tryptone, 10g yeast extract, 10g glucose, 0.96L distilled water, and 40mL salt solution; the salt solution per liter Contains the following ingredients: 0.2g CaCl 2 , 0.4g MgSO 4 ·7H 2 O, 1.0g K 2 HPO 4 , 1.0g KH 2 PO 4 , 10.0g NaHCO 3 , 2.0g NaCl, 1L distilled water.

Embodiment 2

[0037] Step 1: prepare a liquid culture solution, heat it in a water bath, add dropwise 1 mol / L NaOH to adjust pH=7.2; boil, dropwise add resazurin to a concentration of 0.1 mg / L, and dispense into a thin tube to obtain a culture tube; step 2: Insert the aeration needle into the culture tube with a mass ratio of 80% N 2 / 20%H 2 The mixed gas is aerated for 20 minutes, and the rubber stopper is quickly closed;

[0038] Step 3: Place the culture tube in an autoclave, sterilize it at high temperature, set the temperature to 121°C for 20 minutes; transfer it to an ultra-clean bench and cool it to room temperature, put it in a constant temperature incubator for 48 hours, and sterilize it with UV light 25 minutes.

[0039] Step 4: Use a syringe to inject 3% anaerobic microorganism seed liquid into the culture tube, and put it into a 37°C constant temperature incubator for 6 days to obtain anaerobic microorganisms.

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Abstract

The invention discloses a pure culture method of anaerobic microorganisms. The pure culture method comprises the following steps of: step 1, preparing a liquid culture solution and boiling; dropwise adding resazurin; sub-packaging into a thin-opening pipe to obtain a culture pipe; step 2, introducing N2 / H2 mixed gas and aerating; rapidly covering a rubber plug tightly; step 3, putting the culture pipe into a high-pressure sterilization pot and sterilizing at high temperature; transferring into a super-clean platform and carrying out ultraviolet sterilization; and step 4, injecting a magnetic antioxidant into the culture pipe by utilizing an injector and uniformly mixing; injecting an anaerobic microorganism seed solution into the culture pipe by utilizing the injector; putting the culture pipe into a constant-temperature culture box and culturing to obtain an anaerobic microorganism group. The pure culture method of the anaerobic microorganisms has the beneficial effects that (1) an anaerobic culture box does not need to be utilized and the operation is simple and convenient; and (2) the magnetic antioxidant is not dissolved into the liquid culture solution and is not easily absorbed by the anaerobic microorganisms, so that the growth is not influenced; the separation of the anaerobic microorganisms can be simplified through magnetic separation and the relatively pure anaerobic microorganism group is obtained.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for pure cultivation of anaerobic microorganisms. Background technique [0002] In the common anaerobic microbial culture process, there are fermentation methods and multi-microbial co-culture methods, but there will be separation problems. In the pure culture method, anaerobic incubators or anaerobic culture tanks are generally used to cultivate anaerobic microorganisms, but this method is more complicated to operate and is more suitable for solid medium culture, not liquid medium culture. At present, reducing agents such as cysteine ​​and ascorbic acid are pre-added in common liquid culture media to reduce the oxidation-reduction potential and isolate oxygen, thereby forming an anaerobic environment for culturing microorganisms. However, in this method, the added reducing dose is too much, the solubility is large, and it is eaten by microorganisms, which affect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20
CPCC12N1/20
Inventor 郭膘虎刘倩林辉吕斯濠文震
Owner DONGGUAN UNIV OF TECH
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