Isolation And Characterization Of Methanotrophs And Their Localization In Root Tissues Of Phragmites Australis

Methane is the second most important greenhouse gas and natural wetland is one of its major sources.Methane oxidizing bacteria(methanotrophs)is a kind of obligate autotrophic microorganisms that utilizes methane as the sole carbon and energy source.In wetland,plant aerenchyma is the main passageway for methane emission,from the anaerobic layer to the atmosphere.During this process,most of the methane is oxidized by the methanotrophs in root zone.However,few is known about root-associated methanotrophic isolates or their distribution from the root of Phragmites australis.It is significative to explore the function status and distribution characteristics of root-associated methanotrophs from Phragmites australis for wetland ecosystem in Inner Mongolia as well as global greenhouse gases reduction.In this study,the root tissues of Phragmites australis were obtained from Hasuhai wetland.Firstly,enrichment method for the cultivation of methanotrophs was established and microbial composition and dynamic changes were analyzed using the second generation high-throughput sequencing,PCR-DGGE and other modern molecular microbial technologies;Secondly,four strains of Type Ⅱmethanotrophs and 17 strains of non-methanotrophic bacteria growing in association with the methanotrophs were isolated.Moreover,phylogenetic trees based on 16S rRNA gene sequence for the isolates were constructed,and the growth and function of represented methanotrophic isolate PRM1 were also performed.Finally,localization of type Ⅱ methanotrophs in root tissues of Phragmites australis was determined using catalyzed reporter deposition fluorescent in situ hybridization(CARD-FISH)technique.The results were showed as follows:(1)Enrichment culture for root-associated methanotrophs of Phragmites australis showed that Methylocystis was the dominant bacteria,followed by methanol oxidation bacteria,Rhizobium sp.,reached up 60.5%,10.4%and 8.8%of relative abundance respectively,as well as other associated heterotrophic bacteria.In the process of diluting subculture of the enrichment,abundance of Methylocystis is further improved with other heterotrophic bacteria gradually eliminated,eventually evolved into the stable mixed bacteria growth system;(2)Four isolates of type Ⅱ methanotrophs strains belongs to the genus of Methylosinus(PRM1,PRM3,PRM4)and Methylocystis(PRM2),respectively.Strain PRM1 was most closely related to Methylosinus sporium NCIMB 11126(Y18946),with 98%identity based on 16S rRNA gene,and its optimum growth conditions were:pH7.0,35°C,initial methane content for 20%.PRM1 got a maximum value of 0.36 for OD600 and 79.9%for CH4 oxidation efficiency at optimum growth conditions.In addition,PRM1 possess nitrogenase gene nifH and cultivation experiment with absent N condition suggested that it was able to fix nitrogen for growth;(3)The result of CARD-FISH showed that type II methanotrophs are distributed in the stele and epidermis of Phragmites australis root tissus,and compared to stele,there were a large number of methanotrophs in the epidermis.That may be related to the demand for CH4 and 02 concentrations of methane oxidizing bacteria.This study for the first time determined the methanotrophs in Phragmites australis root in situ and directly reflected the important role of type II methanotrophs in methane reduction.