Diversity Of Nitrogen Cycling Bacteria In Kitezh Lake Basin,Fildes Peninsula,Antarctica

Kitezh Lake basin is a typical microenvironment formed by the stream of meltedice and snowin the interior of Fildes Peninsula,Antarctica.This basin is located in the hills,far from the coast,with few animals and almost no human activities,and a few mosses be scattered on sides of the stream.In summer,ice and snowon the upstream mountains melt into Kitezh Lake.In this study,four soil samples collected from the upper,middle and lower reaches of Kitezh Lake basin were selected as objects.Using three technological approaches including culturable method,high-throughput sequencing method and metagenomic sequencing method,the diversity characteristics,functional activities,enzyme-producing strains and main nitrogenmetabolic pathways of nitrifying bacteria,denitrifying bacteria,nitrogen-fixing bacteria and ammoniating bacteria were systematically studied and analyzed.The results are as follows:1.The results of culturable nitrogen cycling bacteria showed as follows:The number of nitrogen cycling bacteria in the soil samples of Kitezh Lake basin were ammonifying bacteria>nitrifying bacteria>denitrifying bacteria>nitrogen fixing bacteria.The total number of its ranged were from 1.1×10~5?2.5×10~8 MPN/g or CFU/g,and between each other differed by about one order of magnitude.The morphology of bacteria were mainly rod-shaped and short rod-shaped,and a few were spherical(but ammonifying bacteria are mainly spherical).The shape and size of bacterial colonywere different,and its colour were mainly white,milky white,yellow,dark yellow and so on.On the whole,the morphological diversities of ammoniating bacteria and nitrifying bacteria were higher than that of denitrifying bacteria and nitrogen-fixing bacteria.in this study,we found that 4 ammonifying bacteria had strong ammonia oxidation function,3 nitrifying bacteria had strong nitrification function,and 4 denitrifying bacteria had strong denitrification activity.In addition,a group of bacteria with high enzyme producing activity were also found,including 6 strains producing catalase,2 strains producing oxidase,1 strain producing DNA enzyme,2 strains producing casein enzyme,3strains producing aescinase,1 strain producing tween60 decomposition enzyme and 2strains producing alginase.Phylogenetic identification results of 16S showed that,ammonifying bacteria of soils samples were distributed in three classes(Gammaproteobacteria?Actinomycetia?Alphaproteobacteria)and four genera(Psychrobacter?Pseudomonas?Pseudarthrobacter?Caulobacter),nitrogen-fixing bacteria were also distributed in three classes(Gammaproteobacteria?Bacilli?Actinomycetia)and four genera(Pseudomonas?Bacillus?Pseudarthrobacter?Rhodococcus),nitrifying bacteria were distributed in two classes(Gammaproteobacteria?Alphaproteobacteria)and three genera(Pseudomonas?Pantoea?Agrobacterium),denitrifying bacteria were also distributed in two classes(Gammaproteobacteria?Alphaproteobacteria)and three genera(Pseudomonas?Acinetobacter?Agrobacterium).The dominant bacteria of the four types of nitrogen-cycling bacteria were all Pseudomonas of Gammaproteobacteria.In addition,the16S r DNA sequence similarities between isolated bacteria and model bacteria were99.05-100%,so no potential new species were found.2.High throughput sequencing analysis results of nitrogen cycling genes(nxr A?nir S?nif H?amo A)showed as follows:In the soil samples of Kitezh Lake basin,there were 5 classes and 6 genera of nitrifying bacteria(the dominant bacteria was unclassified?d??Bacteria),7 classes and14 genera of denitrifying bacteria(the dominant bacteria were Rhodanobacter of Gammaproteobacteria and unclassified?k??norank?d??Bacteria),6 classes and 8 genera of nitrogen-fixing bacteria(the dominant bacteria was unclassified?k??norank?d??Bacteria),3 classes and 3 genera of ammonia oxidizing bacteria(the dominant bacteria was Nitrosospira of Betaproteobacteria).On the whole,in soils of Kitezh Lake basin,the diversities of denitrifying bacteria and nitrogen-fixing bacteria were higher than that of nitrifying bacteria and ammonia oxidizing bacteria,and perhaps many uncultured nitrogen cycling bacteria presented in these soils.According to the composition and distribution of nitrogen cycling bacteria among samples,the composition of nitrifying bacteria in sample JCB-3 was special,and sample JCB-2 was close to sample JCB-4.The composition of denitrifying bacteria in sample JCB-1 and JCB-2 were close,and sample JCB-3 was close to JCB-4.Sample JCB-2 and JCB-3 had similar nitrogen-fixing bacteria composition,but JCB-4 had little similarity with the other three samples.Sample JCB-1 and JCB-2 have similar compositions of ammonia oxidizing bacteria,but JCB-3 had very low similarity with the other three samples.These results showed that the composition of nitrifying bacteria and ammonia oxidizing bacteria in sample JCB-3 from the middle reaches of Kitezh Lake basin was special compared with the other three samples.The analysis results of the influence of soil physical and chemical factors on the composition of microbial community showed that,the soils of Kitezh Lake basin were neutral and acidic,high water content,low organic C and organic N contents,and low other nutrient salt contents,which belongs to the extremely poor nutrition soils.Relatively,organic C had a higher influence on the composition of nitrifying bacteria,denitrifying bacteria and nitrogen-fixing bacteria,but had a lower influence on ammoniaoxidizing bacteria.Organic N had a higher influence on the composition of nitrogen-fixing bacteria,but little influence on that of nitrifying bacteria,denitrifying bacteria and ammonia oxidizing bacteria.NO₂^--N?NO₃^--N?NH₄⁺-N had higher influence on the composition of nitrifying bacteria,denitrifying bacteria and ammonia oxidizing bacteria,but little on that of nitrogen fixing bacteria.Si had a higher influence on the composition of nitrifying bacteria and ammoniaoxidizing bacteria,but little on that of denitrifying bacteria and nitrogen-fixing bacteria.In addition,PO₄^3--P and p H hadhigh influence on all of four nitrogen cycling bacteria in soil samples.3.The results of metagenomic sequencing analysis showedas follows:Six metabolic pathways of nitrogen cycle were observed in soil samples of Kitezh Lake basin,including nitrification,denitrification,nitrogen fixation,anammox,dissimilatory nitrate reduction,and assimilatory nitrate reduction.Among them,the enzymes and genes involved in nitrification pathway were ammonia monooxygenases(Amo CAB),hydroxylamine dehydrogenase(Hao)and nitrite oxidoreductases(Nxr AB).The enzymes and genes involved in the denitrification pathway were nitrate reductases(Nar GHI,Nap AB),nitrite reductases(Nir KS),nitric oxide reductases(Nor BC)and nitrous-oxide reductase(Nos Z).The main enzymes involved in nitrogen fixation pathway were nitrogenases(Nif DKH).The enzymes involved in the ammonia oxidation pathway were ammonia monooxygenases(Amo CAB),hydroxylamine dehydrogenase(Hao),but the anammox pathway would not exist due to the lack of Hzs and Hdh gene annotations.The enzymes involved in the dissimilatory nitrate reduction pathway were nitrate reductases(Nar GHI,Nap AB)and nitrite reductases(Nir BD,Nrf AH).In addition,MFS transporter protein,nitratetransport system substrate-binding protein,permease protein and ATP-binding protein(related genes of NRT and Nrt ABCD)related to extracellular nitrate transport were found.In addition,carbon fixation pathways in prokaryotes,cyanoamino acid metabolism,methane metabolism and glyoxylate metabolism and other metabolic pathways related to nitrogen metabolism were all found.According to the number of genes related to nitrogen metabolism in samples,sample JCB-4 would hada stronger nitrogen metabolism activity,and sample JCB-3 hadaweaker activity,which would be related to the geographical microenvironment of samples.Similarity analysis of nitrogen metabolism pathways in samples showed that,sample JCB-2 and JCB-4 had similar nitrogen metabolism pathways,and sample JCB-1 also had similarities with them.While sample JCB-3 had little similarity with the other three samples,which would be caused by the relatively low amount of soil nutrients in this microenvironment.In addition,the analysis of soil physical and chemical factors on nitrogen metabolic pathways showed that organic N had litter influence,but organic C,p H,NO₂^--N,NO₃^--N,NH₄⁺-N,Si and PO₄^3--P had great influence.In this paper,the diversities,functional activities,enzyme-producing strains,and the main metabolic pathways of nitrifying,denitrifying,nitrogen-fixing and ammonifying bacteria in soils of the Kitezh Lake basin,Fildes Peninsula of Antarctica,were comprehensively and systematically studied.These results not only make up for the shortcomings of previous studies on nitrogen cycling microorganisms of Fildes Peninsula,but also may provide valuable reference for similar studies ofthe Antarctic,Arctic,snow mountainsand plateaus environment.