Method for in-situ revealing anaerobic iron ammonia oxidizing bacteria in river and lake sediment by DNA stable isotope probe

An anaerobic iron ammonium oxidation and isotope technology, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of small bacterial population ratio and difficult analysis, and achieve the effect of improving the identification rate

Pending Publication Date: 2020-09-25
GUANGDONG UNIV OF TECH
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Problems solved by technology

Therefore, the proportion of bacterial flora reflected by traditional high-throughput sequencing is also small, and analysis is difficult

Method used

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  • Method for in-situ revealing anaerobic iron ammonia oxidizing bacteria in river and lake sediment by DNA stable isotope probe
  • Method for in-situ revealing anaerobic iron ammonia oxidizing bacteria in river and lake sediment by DNA stable isotope probe
  • Method for in-situ revealing anaerobic iron ammonia oxidizing bacteria in river and lake sediment by DNA stable isotope probe

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Embodiment 1

[0044] A method for in situ revealing of anaerobic iron ammonia oxidizing bacteria in sediments of rivers and lakes by DNA stable isotope probes, the process operation is as follows figure 1 As shown, it specifically includes the following steps:

[0045] 1, 13 C, 15 N-SIP incubation and DNA extraction

[0046] (1) On-site sampling: According to the investigation purpose and sampling plan, collect about 2L of biological samples from river (lake) sediment at a depth of 0-20cm at each sampling point, and divide them into two equal parts and place them in sterile plastic sampling bags. Empty the air on the upper part of the sampling bag and seal it, and transport it to the laboratory through a sampling box with built-in ice cubes;

[0047] (2) For each sampling point sample, one of the samples was analyzed according to the method of "Physical and Chemical Analysis of Soil" (China Forestry Press, 2017) to measure water content, pH, particle size distribution, dissolved nitrogen...

Embodiment 2

[0079] A DNA stable isotope probe in situ reveals the method for anaerobic iron ammonia oxidizing bacteria in river and lake sediments, the specific steps are basically the same as in Example 1, the difference is that in the formal cultivation of step 1-(6), Fe (III) The ion concentration is 20mg-Fe / L, 12 C-NaHCO 3 with 14 N-NH 4 The amount of Cl added is 30 mg-C / L and 15 mg-N / L respectively; step 3-(2) ultra-high speed centrifugation, the temperature is 18°C, 180000×g, ultra-high speed centrifugation for 44 hours.

Embodiment 3

[0081] A DNA stable isotope probe in situ reveals the method for anaerobic iron ammonia oxidizing bacteria in river and lake sediments, the specific steps are basically the same as in Example 1, the difference is that in the formal cultivation of step 1-(6), Fe (III) The ion concentration is 23mg-Fe / L, 12 C-NaHCO 3 with 14 N-NH 4 The amount of Cl added is 33 mg-C / L and 17 mg-N / L respectively; step 3-(2) ultra-high speed centrifugation, the temperature is 22° C., 200,000×g, ultra-high speed centrifugation for 46 hours.

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Abstract

The invention discloses a method for in-situ revealing anaerobic iron ammonia oxidizing bacteria in river and lake sediment by a DNA stable isotope probe. The method comprises the following steps of (1) collecting a nitrogen-polluted river and lake sediment sample; (2) carrying out <13>C- and <15>N- isotope incubation experiments; (3) separating heavy-layer DNA by adopting a cesium chloride density gradient solution ultra-high-speed centrifugation method; and (4) carrying out DNA marking degree evaluation and flora structure analysis. The invention provides a method for analyzing and identifying the anaerobic iron ammonia oxidizing bacteria in a non-culture mixed flora in a natural water body environment, which is of great significance for researching and understanding a nitrogen circulation process in which iron reduction participates and developing an autotrophic nitrogen transformation technology.

Description

technical field [0001] The invention relates to the technical field of microbial ecology, and more specifically relates to a method for in-situ revealing of anaerobic iron ammonia oxidizing bacteria in river and lake bottom mud by DNA stable isotope probes. Background technique [0002] Nitrogen pollution in rivers (lakes) is a wide-ranging and persistent environmental pollution problem. and urban life and other non-point source pollution. In order to improve the restoration ability of the ecosystem and seek new biological denitrification methods, people have been continuously exploring and understanding the nitrogen cycle path in nature. Anaerobic iron ammonium oxidation (also known as Feammox) is a newly discovered nitrogen conversion pathway in aquatic and terrestrial ecosystems. Together with heterotrophic denitrification, co-denitrification and anammox pathways, it promotes nitrogen conversion in polluted waters. Feammox has been reported in forest soils, swamps, wetl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/04
CPCC12Q1/6869C12Q2535/122C12Q2563/101C12Q2531/113
Inventor 李宁江进许燕滨潘汉平王桢胡颖斌曹节
Owner GUANGDONG UNIV OF TECH
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