| The Fildes Peninsula is located in the southwest of King George Island,Antarctica,and is the largest summer ice-free area on the edge of the southwest pole.Due to its diverse geographical,geological,and ecological environments,many distinct microecological areas within the island have been formed,such as animal activity zones,moss-covered zones,snow and ice melt flow areas,rare animal and plant areas,ice cover front areas,and areas with frequent human activities,etc.Therefore,it is a relatively concentrated area for Antarctic scientific research in various countries.In this study,soil samples from 10 characteristic microenvironment of the Fildes Peninsula were taken as the objects,the culturable pathway,high-throughput sequencing pathway of nitrogen-cycling functional genes（nxr A,nir S,amo A,and nif H）,and metagenomic sequencing pathway of nitrogen-cycling metabolism,the diversity composition and distribution characteristics of nitrogen-cycling bacteria（including nitrifying bacteria,denitrifying bacteria,ammoniating bacteria and nitrogen-fixing bacteria）,enzyme production status,difference in nitrogen metabolism pathways and related enzymes or genes,as well as the effects of soil physicochemical factors were comprehensively compared and analyzed.The aims are to comprehensively and systematically understand the status of soil nitrogen cycling bacteria in Fildes Peninsula,and to provide reference for related studies in other similar environments.At the same time,the strains with strong nitrogen cycling function or high extracellular enzyme capacity would be excavated,which could provide good strain resources for further research on the properties of cryogenic enzymes and development and utilization of cryogenic enzyme producing microorganisms.The research results are as follows:1.The analysis of cultivable pathways showed:（1）The content order of nitrogen cycling bacteria in the 10 characteristic microenvironment was:Biobay basin>Gold Bay land area>southeast side of Penguin Island>front of the Green Ice Sheet>Kitake Lake area>yangjiao Mountain area>Pingdingyan land area>northwest side of Penguin Island>Biyutan area>Nelson Island.According to the total amounts of nitrogen cycling bacteria,the ammoniating bacteria（1.38×106～1.82×109MPN/g）>nitrifying bacteria（2.0×104～1.6×108MPN/g）>denitrifying bacteria（1.20×105～9.00×107MPN/g）>nitrogen fixing bacteria（1.00×103～1.1×107CFU/g）.Among them,the highest and lowest concents of ammoniating bacteria were found on the land side of Biobay and Biyutan land area,respectively.The most and least contents of nitrifying bacteria and nitrogen fixing bacteria were found in the Golden Bay and the northwest side of Penguin Island,respectively;The most and the least contents of denitrifying bacteria were in the southeast and northwest of Penguin Island,respectively.（2）It was found that 53 strains had strong ammonification function and 14 strains had strong denitrification function.In addition,87 isolated bacteria were found with strong abilities to produce amylase,protease（casein hydrolase）,cellulase,ester hydrolase（Tween 60,Tween 80）,alginate enzyme,esculin hydrolase,DNAase,oxidase,and catalase,and belonging to 7 classes and 17 genera（including 6 potential new species of bacteria）.（3）The 16S rDNA phylogenetic diversity order of isolated nitrogen cycling bacteria was:ammonifying bacteria（7 classes,19 genera）>nitrogen fixing bacteria（6 classes,12genera）>nitrifying bacteria（5 classes,9 genera）>denitrifying bacteria（3 classes,7genera）,and among which the dominant classes and genera of four nitrogen cycling bacteria were Pseudomonas of Gammaproteobacteria.From the distribution of the 10characteristic microenvironment,the phylogenetic diversities of the northwest side of Penguin Island（8 classes and 12 genera）were>the southeast side of Penguin Island（6classes and 9 genera）>Pingdingyan area（4 classes and 8 genera）>Yangjiao Mountain area（4 classes and 7 genera）>Biobay basin（3 classes and 9 genera）>Kitake Lake area（3 classes and 7 genera）>front of the Green Ice Sheet（3 classes and 3 genera）>Golden Bay（2 classes and 4 genera）=Biyutan area（2 classes and 4 genera）>Nelson Island area（2 classes and 3 genera）.Additionally,18 strains of isolated bacteria would be the potential new species within the genera Flavobacterium,Pedobacterium,Shinella,Dyadobacterium,and Mucilaginibacter.2.High-throughput sequencing analysis of nitrogen cycling functional genes（nxr A,nir S,amo A,nif H）showed:The species diversities of nitrogen cycling bacteria in the four microenvironment with obvious characteristics at the class level were:Biobay basin（19 classes）>northwest side of Penguin Island（13 classes）>southeast side of Penguin Island（11classes）>Kitake Lake basin（10 classes）.Among them,the southeast side of Penguin Island and the Kitake Lake were showed high similarity in the community composition of nitrifying bacteria and ammoniating bacteria.However,there were particularities in the composition of denitrifying bacteria in Biobay basin and the Kitake Lake area,and in the composition of nitrogen fixing bacteria in the east and west sides of Penguin Island.In addition,organic C,organic N,PO43--P,and p H had a greater impact on the composition and abundance of nitrogen cycle bacteria in four characteristic microenvironments,especially the p H had the most obvious impact on nitrifying bacteria,denitrifying bacteria,and ammoniating bacteria,but the smallest impact on nitrogen fixing bacteria.3.The results of metagenomic sequencing analysis indicated:Six nitrogen metabolism pathways were annotated in six representative microenvironment soil samples,including assimilative nitrate reduction,dissimilating nitrate reduction,nitrogen fixation,denitrification,nitrification,and anaerobic ammoxidation.There were also other related metabolic pathways involved,such as cyano amino acid metabolism,glutamic acid metabolism,arginine biosynthesis,glyoxalic acid metabolism and acetaldehyde metabolism.The comparative analysis of nitrogen metabolism pathways and related enzymes or genes among six characteristic microenvironment samples showed that the southeast side of Penguin Island lacked nitrogen fixing enzymes and nif DKH,anf G genes,nitroalkane oxidase and NAO genes required for the transformation of nitro compounds into nitrite;The east and west sides of Penguin Island and Yangjiao Mountain area lacked the glutamate synthase and GLT1genes required for the conversion of glutamine to glutamate;The Kitake Lake area,Great Wall Station area and Yangjiao Mountain area lacked hydroxylamine reductase,Hcp and Nir S genes required in the process of converting hydroxylamine into ammonia,as well as Nir S genes required for denitrification and anaerobic ammoxidation.According to the functional pathway of nitrogen metabolism,the samples of six characteristic microenvironments had been annotated with 7 classes and 36 genera,which were rich in diversity.Among them,the similarities of nitrogen metabolism pathways were high between the Yangjiao Mountain area and Kitake Lake area,the southeast side of Penguin Island and the Biobay basin,and the northwest side of Penguin Island and the Great Wall Station,which was consistent with the sampling environment.In addition,the organic N,NH4+-N,water content,NO2--N,p H,Si O4--Si,organic C,and NO3--N had a greater impact on the nitrogen metabolism pathways of the six characteristic microenvironment,but PO43--P had the smallest impact.This study has not only comprehensively and systematically understood and compared the status of soil nitrogen cycle bacteria in various microenvironment of the Fildes Peninsula,but also found many strains with strong nitrogen cycle function and high enzyme producing capacity,which can provide reference for related basic research and provide a good strain resource for the development and utilization of low-temperature strain. |
