Method for detecting community structures and abundance of nitrite oxidizing bacteria in wastewater

Abstract

The invention discloses a method for detecting the community structures and abundance of nitrite oxidizing bacteria in wastewater. The method comprises the concrete steps as follows: (a) extracting the whole-genome DNA of activated sludge in the wastewater; (b) performing PCR amplification of a gene nxrA and a gene nxrB; (c) sequencing amplification products respectively by a Roche 454 pyrosequencing method; (d) dividing the OTUs (Operational Taxonomic Units) of sequencing results; and (e) selecting OTUs sequences with the similarity of 97%, performing BLASTn comparison between the OTUs sequences, and dividing the phylogenetic trees of the gene nxrA and the gene nxrB respectively by using MEGA software, wherein the phylogenetic trees are the community structures of the nitrite oxidizing bacteria in the wastewater, and the relative abundance of the different OTUs of the gene nxrA and the gene nxrB is the abundance of the nitrite oxidizing bacteria in the wastewater. The method is simple and convenient to operate, has a wide detection range, can be applied to detection and analysis in a laboratory and can also realize real-time detection of the nitrogen removal efficiency of a wastewater treatment plant.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular to a method for detecting the community structure and abundance of nitrite oxidizing bacteria in sewage. Background technique [0002] In recent years, with the increase of nitrogen content in industrial wastewater and domestic sewage, the degree of eutrophication in surface water bodies such as lakes and estuaries has intensified, and the ecological balance of water bodies has been destroyed. In order to alleviate this process, it is imperative to reduce the nitrogen content in sewage Row. [0003] At present, nitrogen in sewage is mainly degraded and removed by denitrification microorganisms, including nitrification and denitrification processes. The nitrification reaction includes ammonia oxidizing bacteria oxidizing ammonia nitrogen to nitrite nitrogen and nitrite oxidizing bacteria oxidizing nitrite to In the process of nitrate, nitrite oxidizing bacteria are the second...

AI Technical Summary

Problems solved by technology

The purification culture in this article only cultivated a certain kind of bacteria, and it is difficult to cover all the nitrite oxidizing bacteria in the actual environment. However, the 16S rDNA sequence fragment amplification detection and analysis process used in this article has a large deviation, and it is difficult to apply to the microorganisms in the actual environment. detection

The current quantitative detection methods for nitrite oxidizing bacteria are mainly FISH technology and Real time Q-PCR technology; Patent Publication No. CN 101348836A, patent name "A Fluorescent In Situ Hybridization Detection Method for Nitrifying Bacteria 16SrDNA" was invented in the article A FISH technique was developed to detect nitrifying bacteria. This technique is complicated to operate, with cumbersome steps, poor probe specificity and high requirements, and cannot fully cover all species of nitrite oxidizing bacteria.

In addition, the patent publication number CN 103555831 A, the patent name "Fluorescent Quantitative PCR Detection Method for Nitrite Oxidizing Bacteria in Sewage Treatment Plants" uses QPCR as a technical means to quantitatively detect and analyze the two main species of nitrite oxidizing bacteria , the production of standard DNA samples in this quantitative technique is difficult, the standard curve is difficult to unify, and the cost of experimental detection is high, the community structure analysis is difficult, and it is difficult to apply the community structure analysis and abundance detection of corresponding microorganisms in actual sewage plants on a large scale

[0005] Franck Poly et al. used the nxrA functional gene to detect Nitrobacter ("First exploration of Nitrobacter diversity in soils by a PCR cloning-sequencing approach targeting functional gene nxrA” , FEMS MICROBIOLOGY ECOLOGY, 2008 (64): 132-140); Michael Pester et al. used nxrB functional gene to detect Nitrospira (" wxya encoding the beta subunit of nitrite oxidoreductase as functional and phylogenetic marker for nitrite-oxidizing Nitrospira ", Environmental Microbiology, (2014) 16 (10), 3055–3071), but the use of nxrA and nxrB functional genes for the detection of nitrite oxidizing bacteria has not yet been reported

Images