A method for in situ revealing of formic acid-utilizing methanogenic archaea in paddy fields using DNA stable isotope probes

Abstract

The method of using DNA stable isotope probes to in situ reveal and identify formic acid-utilizing methanogenic archaea in paddy fields includes the following steps: (1) Collecting paddy soil samples; (2) Conducting 13C-formic acid microcosm cultivation experiments; (3) Use an ultra-high-speed centrifuge to centrifuge and stratify the total DNA of microorganisms in the soil cultivated with 13C-formic acid; (4) conduct real-time quantitative PCR analysis and fingerprint analysis on the methanogenic archaeal genes in multiple buoyant densities after stratification, and determine the Does paddy soil contain metabolically active formate-utilizing methanogenic archaea. In summary, the present invention can sensitively and in situ reveal formic acid-utilizing methanogenic archaea in paddy fields through DNA-based stable isotope probes (DNA-based? Stable? Isotope? Probing), which is useful for understanding the nutrient cycle process driven by microorganisms in paddy fields It is of great significance to recognize the ecological function of functional groups of methanogenic archaea in paddy fields.

Description

technical field [0001] The invention relates to a method for using a DNA-based Stable Isotope Probing (DNA-based Stable Isotope Probing) in situ to reveal and distinguish formic acid-utilizing methanogenic archaea in paddy fields, and belongs to the field of soil microbial ecology. Background technique [0002] Of the 150 million hectares of rice fields in the world, 75% are flooded. The anaerobic environment makes the paddy soil rich in small molecule organic acids. By metabolizing these substances, the microbes drive the biogeochemical cycles of the paddy ecosystem. Formic acid is one of the important intermediate products in the degradation process of organic matter in paddy fields, and its concentration in paddy soil can be as high as 150 μM. A variety of microorganisms in paddy soil can metabolize formic acid. For example, a variety of anaerobic bacteria in the Clostridia class can use formic acid as an electron donor to perform sulfate reduction and iron reduction pr...

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Problems solved by technology

However, formic acid-utilizing methanogenic archaea have not been reported in paddy soils

The reason for this is firstly that the currently cultivable microorganisms only account for 1% of all microorganisms, and a large number of microorganisms can only survive and perform ecological functions in specific habitats, and cannot be purely cultivated; secondly, methanogenic archaea are strictly anaerobic microorganisms , which have high requirements on the growth environment, and it is difficult to screen them by traditional methods; third, although some methanogenic archaea can use formic acid to grow under pure culture conditions, pure culture conditions cannot represent the in situ habitat in paddy soil, So the obtained strains cannot reflect the real situation in the paddy soil

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