Research On The Community Structure Characteristics Of Denitrifying Anaerobic Methanotrophic Bacteria In The Permafrost Region Of Daxing'an Mountains

Global warming will accelerate the degradation of permafrost and release more greenhouse gases(CO₂ and CH₄),further aggravating the greenhouse effect.Denitrification-type anaerobic methane oxidation(nitrate/nitrite-dependent anaerobic methane oxidation,n-damo)is a key link between biogeochemical carbon and nitrogen cycles,and plays an important role in alleviating global greenhouse gas emissions.However,studies on the community structure and reaction rate of n-damo bacteria in high-latitude permafrost regions are rarely reported.In this study,the soil of three typical forests(Larix gmelinii,Betula platyphylla,and Pinus sylvestris var.mongolica)in the permafrost region of the Daxing'an Mountains was used as the research object.The phylogeny,community composition,abundance,diversity and reaction rate of bacteria,revealing the key environmental factors that control the n-damo process and their impact mechanisms,and exploring the carbon biogeochemistry of n-damo flora in high-latitude permafrost regions importance in the cycle.The research results can provide a theoretical basis for in-depth understanding of the methane cycle process in permafrost regions and accurate evaluation of the contribution of permafrost ecosystems to the global carbon cycle.The results of the study are as follows:(1)Based on the 16S rRNA gene clone library,a total of 638 valid sequences were obtained and divided into 11 OTUs.Based on the pmoA gene clone library,a total of579 valid sequences were obtained and divided into 8 OTUs.Phylogenetic analysis showed that the 16S rRNA gene sequence was 93.8%-97.5%close to the bacterium"Candidatus Methylomirabilis oxyfera",and the pmoA gene sequence was 86.1%-96.3%close to the bacterium"Candidatus Methylomirabilis sinic".The diversity index analysis of n-damo bacteria based on 16S rRNA and pmoA genes showed that the diversity index in the three forest types were all higher in the middle soil layer(20-40 cm and 40-60 cm)than in the upper layer(0-20 cm)and deep layers(60-80 cm and 80-100 cm).Correlation analysis showed that the environmental factors regulating the diversity of n-damo bacteria were mainly total nitrogen(TN),nitrate(NO₃^--N)and temperature(T).(2)Principal coordinate(PCoA)analysis results based on 16S rRNA and pmoA genes showed that the composition of n-damo bacterial community was significantly affected by soil depth,but less correlated with forest type.Redundancy analysis(RDA)indicated that NO₃^--N and nitrite(NO₂^--N)were the key drivers affecting the community structure of n-damo bacteria.(3)The abundances of n-damo bacteria(16S rRNA and pmoA genes)were1.55×10~4-1.47×10~5 copies·g^-1 and 1.31×10~3-3.11×10~4 copies·g^-1,respectively.Among them,the abundance of 16S rRNA gene was the highest in birch forest,the abundance of pmoA gene was the highest in Pinus sylvestris forest,and the abundance of n-damo bacteria in the three forest types increased first and then decreased with soil depth.Correlation analysis showed that the abundance of n-damo bacteria was significantly positively correlated with soil water content(SWC),NO₂^--N and TN(P<0.05).(4)The results of ¹³C stable isotope tracer measurement showed that the reaction rate of n-damo bacteria was 0-1.26·g^-1·d^-1.The n-damo reaction rate in the middle layers(20-40 cm and 40-60 cm)of the three forest types were significantly higher than that in the upper layer(0-20 cm)and deep layers(60-80 cm and 80-100 cm)(P<0.05),and the three forest types showed a trend of increasing first and then decreasing with the increase of soil depth.