Culture method for prolonging ammonia-oxidizing archaea (AOA) passage time

A technology of ammonia oxidizing archaea and culture method, which is applied in the field of culture to prolong the passage time of ammonia oxidizing archaea, which can solve the problems of increasing the risk of AOA contamination and increasing workload, so as to prolong the culture time, ensure activity, prolong Effect of passaging time

Active Publication Date: 2016-08-31
INST OF URBAN ENVIRONMENT CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Frequent passage not only increases the workload, but also increases the risk of AOA contamination

Method used

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  • Culture method for prolonging ammonia-oxidizing archaea (AOA) passage time

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] AOA (Nitrosotalea devanaterraNd2) was isolated from acidic soil in China. Chemoautotrophic NOB pure bacteria (ATCC 51922) were purchased and streptomycin medium containing 50 μM was prepared. Can not grow, but on the plate without streptomycin, NOB grows well, it is clear that streptomycin has a good inhibitory effect on NOB. And it was verified that the NOB pure bacteria can grow well under the environment of pH 5.5 and culture temperature 30°C.

[0020] Prepare archaeal medium: 1L contains NaCl 1.0g, MgCl 2 0.4g, CaCl 2 0.1g, KH 2 PO 4 0.2g, KCl 0.5g, trace elements 1mL, 7.5mM NaFeEDTA1mL, 2mM NaHCO 3 2ml, 500μM NH 4 Cl 1ml, after autoclaving, adjust the pH to 5.5.

[0021] Take Nitrosotalea devanaterra Nd2 and NOB in the logarithmic growth phase, respectively 1ml to 50ml of culture medium, and culture at 30°C.

[0022] Determination of NO in the medium after 3d, 14d, 42d of culture 2 - and NO 3 - concentration:

[0023] NO 2 - Determination: Pipette ...

Embodiment 2

[0029] In the process of Example 1, when it is necessary to regain the pure culture of Nitrosotaleadevanaterra Nd2, 50 μM streptomycin is added to the medium to inhibit the growth of NOB; once every 2 weeks, after 3-4 passages, Nd2 is detected by 16S rDNA PCR purity.

[0030] Detection method:

[0031] Primer: 27f-1492r

[0032] PCR system: DNA Taq enzyme 12.5 μl, primer (27f) 1 μl, primer (1492r) 1 μl, culture medium 1 μl, sterilized water 9.5 μl.

[0033] PCR amplification program: 94°C for 5min, (94°C, 60S, 53°C, 60S, 72°C, 90S) X 30cycles, 72°C, 10min.

[0034] Electrophoresis results: the 2nd lane is DNA Mark, the 3rd lane is the negative control, the 4th lane is the medium of Nitrosotalea devanaterra Nd2, and the 5th and 6th lanes are the positive control. After 16S rDNA PCR amplification, no electrophoresis band was found, which proved that there was no bacterial contamination in the culture medium, and the pure culture of Nitrosotalea devanaterra Nd2 was obtained. ...

Embodiment 3

[0036] Isolate AOA (Nitrosotalea devanaterraNd1) from Scottish soil, buy autotrophic NOB pure bacterium (ATCC 25381), clear that streptomycin has a good inhibitory effect on NOB by the method as described in Example 1, and verify that The NOB pure bacteria can grow well in the environment of pH 5.5 and culture temperature 25°C.

[0037] Archaeal media were prepared as described in Example 1.

[0038] Take Nitrosotalea devanaterra Nd1 and NOB in the logarithmic growth phase, respectively 0.5ml to 50ml of culture medium, and culture at 25°C.

[0039] Determination of NO in the medium after 7d, 14d, 42d of culture 2 - and NO 3 - Concentration, assay method is the same as in embodiment 1, repeats no more here, and result is as shown in table 2. Table 2. NO in the culture medium of Nitrosotalea devanaterra Nd1 after 7d, 14d, 42d culture 2 - and NO 3 - concentration

[0040] Training days

[0041] The bacterium liquid after 42 days of culture was absorbed and sub...

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Abstract

The invention relates to a culture method for prolonging ammonia-oxidizing archaea (AOA) passage time and belongs to the technical field of bioengineering. The culture method comprises synchronously inoculating a medium with nitrobacteria (NOB) and AOA, carrying out culture at a temperature of 25-37 DEG C for 6 weeks, carrying out subculture, adding an antibiotic into the medium with NOB and AOA and carrying out passage 3-4 times to obtain pure AOA culture. Through coculture of NOB and AOA, AOA passage time is prolonged, passage number is reduced and AOA activity is guaranteed. Through use of the antibiotic, NOB growth is inhibited and pure AOA is obtained. The culture method is very simple and convenient.

Description

technical field [0001] The invention relates to a culture method for prolonging the passage time of ammonia oxidizing archaea, belonging to the technical field of bioengineering. Background technique [0002] Ammonia oxidation, as the first step in the nitrification process, is a key step in the nitrogen biogeochemical cycle. Ammonia-oxidizing bacteria in the β and γ subgroups of the Proteobacteria have long been considered to be the main undertakers of ammonia oxidation. However, in recent years, studies have found that ammonia-oxidizing archaea (AOA) are similar to ammonia-oxidizing bacteria (AOB) and have ammonia oxidation. From the perspective of the entire ecological function, the intensity and total amount of ammonia oxidation are in Ammonia oxidizing bacteria are widely found in various ecosystems and play a very important role in the nitrogen biogeochemical cycle. However, AOA can only be cultured in liquid, ammonium chloride (NH 4 Cl) as a substrate, the reaction...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/04C12R1/01
CPCC12N1/04C12N1/20
Inventor 李雅颖姚槐应
Owner INST OF URBAN ENVIRONMENT CHINESE ACAD OF SCI
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