Method for revealing and distinguishing paddy field formic acid utilization type methanogenic archaea in situ by adopting DNA-based stable isotope probing technology

Abstract

A method for revealing and distinguishing paddy field formic acid utilization type methanogenic archaea in situ by adopting DNA-based stable isotope probing technology comprises the followings steps: (1), collecting a paddy soil sample; (2), performing a micro universe cultivation experiment of <13>C-formic acid; (3), performing centrifugation layering on the microbe total DNA of <13>C-formic acid cultivation soil by using an ultracentrifuge; (4), performing real-time quantitative PCR analysis and finger-print analysis on methanogenic archaea genes in layers of multiple buoyant densities after layering to judge whether the paddy soil contains the formic acid utilization type methanogenic archaea with metabolic activity. Therefore, the method can be used for keenly revealing the paddy field formic acid utilization type methanogenic archaea in situ based on the DNA-based stable isotope probing technology, and has great significance on the understanding of the paddy field nutrient cycling process driven by microbes and the cognition of the ecological functions of paddy field methanogenic archaea functional groups.

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 ...

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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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