Method for determining Tetrasphaera subgroup intracellular metabolites by combining flow cytometry sorting and Raman technology

Abstract

The invention discloses a method for determining Tetrasphaera subgroup intracellular metabolites by combining flow cytometry sorting and a Raman technology, and belongs to the technical field of biological sewage treatment. According to the method, the flow cytometry sorting and the Raman technology are combined, all subgroups of Tetrasphaera phosphorus-accumulating bacteria are accurately sorted out by utilizing the sensitivity and the high accuracy of flow cytometry sorting determination samples, and then intracellular metabolites of the subgroups are determined through a Raman spectrometer. The invention provides a method for determining intracellular metabolites of specific functional florae in different stages in a period in a complex environment of a sewage treatment system, and a theoretical basis is provided for biological phosphorus removal of novel fermentation type phosphorus-accumulating bacteria Tetrasphaera in an actual sewage plant. And meanwhile, a new insight is provided for the determination of intracellular metabolites of other special functional flora in a complex environment.

Description

technical field [0001] The invention relates to a method for measuring intracellular metabolites of fermented phosphorus-accumulating bacteria Tetrasphaera subgroup by combining flow cytometry sorting and Raman technology, which belongs to the technical field of sewage biological treatment and is used for the determination of Tetrasphaera genus aggregates in sewage biological treatment systems. Studies on intracellular metabolites of phosphorus bacteria. Background technique [0002] The eutrophication of water body has become one of the important environmental problems restricting the sustainable development of our country. Excessive entry of nutrients such as phosphorus and nitrogen into the receiving water will cause algae growth, which will lead to eutrophication of the water body. Therefore, before the wastewater is discharged into the environment Nitrogen and phosphorus need to be removed to reduce the occurrence of eutrophication. The current relatively efficient, ch...

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Problems solved by technology

[0008] (1) Use DAPI staining to relatively quantify the intracellular polyphosphorus content, perform PHA quantification by gas chromatography, use gas chromatography-mass spectrometry to measure intracellular amino acids, and use anthrone colorimetric method to analyze sugar principle, but due to mud Due to the complexity of the sample, the chemical analysis method can only measure the intracellular polymers of all bacterial groups, and cannot accurately determine the metabolism of specific bacterial groups, and the intracellular phosphorus accumulation determined by DAPI staining may also be caused by the selection of DAPI concentration. error;

[0009] (2) Raman technology alone can monitor the intracellular polymers of all phosphorus-accumulating bacteria such as polyphosphate, glycogen, PHA, etc., but it cannot distinguish whether these cells belong to Candidatus Accumulibacter, Tetrasphaera, Dechloromonas-type phosphorus-accumulating bacteria or polysaccharide bacteria , In addition, the researchers determined that Candidatus Accumulibacter and Tetrasphaera contributed equally to biological phosphorus removal in some sewage treatment plants through the combination of fluorescence in situ hybridization and Raman technology, but did not perform metabolic analysis on the Tetrasphaera subgroup. In addition, the traditional The probes required for fluorescence in situ hybridization generally have strong quenching properties and need to be strictly protected from light, and the fluorescent dyes of the probes will interfere with the determination of Raman spectroscopy;

[0012] Therefore, the present invention takes the microorganisms in the activated sludge of the sewage treatment system, and uses flow cytometry and Raman technology in combination to measure the intracellular metabolism of the Tetrasphaera type phosphorus accumulating bacteria subgroup, and there is no relevant research report

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