Predominant Methanogens And Methanogenic Pathway In The Rhizosphere Soil Of Reed In Saline-alkaline Zhalong Wetland

The total saline-alkaline land area is more than 500 million mu.As a unique wetland ecosystem,the inland saline-alkaline wetland is the main source of greenhouse gas methane.Under saline-alkaline soil,stable halophyte community structure is an important reason to maintain wetland ecosystem.However,wetland ecosystem is vulnerable to human disturbance and global climate change.At present,researches on the environment of inland saline-alkaline wetlands mainly include field greenhouse gas monitoring and related microbial analysis,etc.However,the metabolic pathways of different dominant methanogens in reed rhizosphere soil and their contributions to the greenhouse effect remain unclear.In this study,the rhizosphere soil of phragmites australis in Zhalong wetland,the most representative inland saline-alkali wetland in the lower reaches of Wuyu River in western Heilongjiang Province,was selected as the research subjects.Based on 16S r RNA gene high-throughput sequencing analysis,we investigated the dominant methane-producing bacteria in the rhizosphere soil of Zhalong wetland,namely the composition of methanogens.The phylogeny and saline-alkaline adaptation mechanism of dominant methanogenic archaea in Zhalong wetland were explored by combining the clone library construction and sequencing analysis and metagenomic analysis.In order to reveal the dominant methanogenic archaea and their interacting bacteria in the Zhalong wetland,we used 16S r RNA high-throughput sequencing and metagenomics to analyze the composition of dominant methanogenic archaea and their interacting bacteria in the Zhalong wetland microcosmic enrichment experiments.The study had the following key findings:（1）Reed has a high methane emission flux during the growing season.The study showed that the dominant methanogenic archaea in the high-water plot included the CO₂reducing Methanobacterium（36.42%）and Rice Cluster II（11.55%）,the methane anaerobic oxidizer Candidatus Methanoperedens（35.06%）,acetotrophic Methanosaeta（11.29%）and methylotrophic Methanosarcina（6.53%）were the main contributors of methane greenhouse gas in the Zhalong saline-alkaline wetland.The dominant bacteria included Chloroflexi（21.55%）,Proteobacteria（16.88%）,Actinobacteria（13.37%）,and Acidobacteria（10.0%）.Bacteroidota（7.42%）,Desulfobacterota（7.77%）,and Firmicutes（5.18%）,respectively.（2）The RCII group of uncultured methane archaea in the saline-alkaline Zhalong wetland accounts for about 13%of the abundance of methane archaea in the topsoil,and its contribution to methane production may have been underestimated in previous studies.Phylogenetic analysis showed that there were three branches and one new group of RCII in saline-alkaline Zhalong wetland.Metagenomic analysis assembled two dominant uncultured RCII strain genomes,The average nucleotide identity（g ANI）of Candidatus’Methanoflorens stordalenmirensis’,the only strain found in the permafrost of northern Sweden with a near-complete genome,is 63%and 64%,respectively.Our results indicated that RCII in the surface soil of Zhalong wetland was a new species,which was different from the Arctic Circle.The genomes of ZL＿bin.22 and ZL＿bin.25 strains contained complete genes of hydrogen-reduced carbon dioxide methanogenesis pathway.Metagenomic analysis suggested that salt tolerance was related to the transport and synthesis of trehalose.（3）The highest methane-producing rate was observed in the media with the addition of TMA and betaine.The dominant bacteria reducing betaine to TMA included Sporomusaceae,Sedimentibacteraceae,Hungateiclostridiaceae,and Clostridiaceae and the dominant archaea producing CH₄from TMA included Methanosarcina（methylotroph）and Methanomassiliicoccus（H₂-reducing methyl substance）.Metagenome sequencing analysis of heirloom betaine enrichment obtained to two assembled genomes of Methanosarcina and two assembled genomes of Methanomassiliicoccus with more complete functional genes for metabolizing methyl substances such as methanol and methylamine,as well as the genome of a bacterial strain of the Sporomusaceae with the functional gene grd H for metabolizing betaine.GBT＿2nd＿bin.2 and GBT＿2nd＿bin.8 had an average nucleotide identity（g ANI）of 63.57%and 74.51%,respectively,compared with reported pure cultures of methanogenic Methanomassiliicoccus＿luminyensis derived from the human intestine,and may belong to new taxa.