Key Processes Of Nitrogen Transformation And Their Microbial Driving Mechanisms In Antarctic Tundra

Nitrogen is one of the most basic elements for all living organisms and the main limited nutrient element of the ecosystem.The nitrogen biogeochemical cycle is mediated by microbial communities,forming a complex nitrogen cycle network.Generally,the nitrogen cycle includes nitrogen fixation,nitrification,denitrification,anaerobic ammonia oxidation,organic nitrogen decomposition,and dissimilation nitrate reduction to ammonium.With the development of molecular biology technology,genomic data collected during the past decade has revealed that microorganisms involved in nitrogen transformation have complex and diverse metabolic functions:such as phototrophic nitrite oxidation,complete ammonia oxidation,ammonia-oxidizing archaea,and denitrifying eukaryotic foraminifera.These findings have updated people’s understanding of the nitrogen cycle driven by microorganisms.Therefore,the mechanism of the nitrogen cycle mediated by organisms has become a research hotspot in geoscience.At present,extensive studies have been carried out on the nitrogen cycle and the mechanism of functional microorganisms in terrestrial and aquatic ecosystems.However,studies on the nitrogen transformation process and functional microbial community diversity are still limited in Antarctic ecosystems.In coastal Antarctica,the ice-free tundra areas are often colonized by sea animals,such as penguins and seals.Marine animals,acting as biological pumps connecting the ocean and land,transfer large amounts of nutrients such as nitrogen and phosphorus from the ocean to the land.The nitrogen provided by marine animal excrement transfers and transforms in the soil,vegetation,waterbody,and the surrounding atmosphere through various forms or pathways,which controls and affects the nitrogen biogeochemical cycle processes in the tundra ecosystem.Therefore,it is of great significance to study the environmental nitrogen transformation process and the response of functional microbial community diversity to marine animal activities in Antarctica.In this paper,the Fildes Peninsula and adjacent Ardley Island in western Antarctic were selected as the study area.Different types of tundra soils from the Fildes Peninsula and Ardley Island were collected,including seal colony soils,penguin colony soils,and adjacent seal-lacking tundra soils,penguin-lacking tundra soils,and the background upland tundra soil.Lake sediments of G Lake,Y2 Lake,Y3 Lake,and Y4 Lake were also collected.The15N isotope tracer method and real-time quantitative PCR,high-throughput sequencing,and metagenomics were used to(1)systematically study the rates and their influence factors of nitrification,denitrification,DNRA,and anammox of tundra soils and lake sediments in Antarctica;(2)analyze the microbial abundance,diversity and community structure of nitrogen fixation,denitrification,and DNRA microorganisms and their influence factors;(3)discuss the composition and diversity of soil microbial communities;(4)explore the relative abundance of functional genes in the main nitrogen transformation process and construct a metabolic network of soil nitrogen transformation in Antarctic tundra soil.The main contents and results are as follows:1.Characteristics of nitrogen fixation microbial community structure in Antarctic tundra soils.High-throughput sequencing and quantitative PCR were used to study the abundance,community composition,and diversity of nifH-encoding nitrogen fixation microorganisms in Antarctic tundra soil.The results showed that the nifH gene abundances of seal colony soil and penguin-lacking soil ranged from 1.0×105-3.9×105 copies g-1,significantly higher than those of penguin colony soil(950-1297 copies g-1).The abundances of nifH genes in sea animal colony soil and penguin-lacking soil were significantly higher than those of background soil(13-582 copies g-1),indicating that sea animal activities increased the abundance of nifH genes.However,the continuous input of excessive organic matter by penguin activities reduced the abundance of nifH gene.Seal activity significantly reduced nifH gene diversity,while penguin activity had no significant effect on nifH gene diversity.The results of classification showed that the seal colony soil and background soil had similar nitrogen fixation microbial communities.The dominant bacteria were Methanothrix,Methylobacter,and Desulfocapsa in seal colony soil and background soil.The dominant genus of the penguin colony soil and penguin-lacking soil was Bradyrhizobium.The cluster analysis and CCA results showed that penguin activities significantly changed the community compositions of soil nitrogen fixation microorganisms.Compared with background soil,penguin activities increased soil TC and OM content,providing more carbon sources for nitrogen fixation microorganisms,and increased soil moisture and ammonia nitrogen content,changing the activity of nitrogen fixation microorganisms.In general,sea animal activities had significant effect on the abundance,diversity,and community composition of nifH nitrogen fixation microorganisms in Antarctic tundra soils.2.Characteristics of denitrifier microbial community diversity in the Antarctic tundra soils.The 15N isotope tracer method and molecular biology methods were used to investigate the effects of sea animal activities on the activity,abundance,and community composition diversity of nirS-encoding and nirK-encoding denitrifiers.The results showed that the denitrification rates of background tundra soils were extremely low(0.01 μmol N kg-1 h-1),and the nirS and nirK genes could not be amplified.Both nirS and nirK genes were successfully amplified in sea animal colony soils and the animal-lacking soils.The abundances of nirS gene range were 5.9×103-2.5×107 copies g-1,which were significantly higher than the abundances of nirK gene abundance(7.4×102-6.2×105 copies g-1).The denitrification rates ranged from 0.04-59.49 μmol N kg-1 h-1.Compared with the animal-lacking soils,sea animal activity significantly increased the abundances of nirS and nirK genes and the denitrification rates(p<0.05).The denitrification rates were significantly positively correlated(p<0.05)with nirS gene abundances instead of nirK gene abundances,indicating that nirS-encoding denitrifiers dominated the denitrification in tundra soils.The diversity of nirS-encoding denitrifiers was higher in animal colonies than in animal-lacking tundra,but the diversity of nirK-encoding denitrifiers was lower.Canonical correspondence analysis indicated that the community structures of nirS-encoding and nirK-encoding denitrifiers were closely related to tundra soil biogeochemical processes associated with penguin or seal activities:the supply of nitrate and ammonium from penguin guano or seal excreta,and low C:N ratios.Ammonium nitrogen generated nitrate nitrogen through nitrification to provide the reaction substrate for denitrification.The lower C:N ratio was conducive to the denitrification process in the sea animal colonies.Therefore,the sea animal activities significantly improved the activity and abundance and changed the community composition of denitrifying microorganisms in Antarctic tundra soil by changing the physicochemical properties of tundra soil.3.Characteristics of DNRA microbial community diversity in the Antarctic tundra soils.The 15N isotope tracer method and molecular biology methods were used to study the activity,abundance,community composition diversity,and influencing factors of DNRA bacteria.The results showed that the DNRA rates of the seal-lacking soil and the background tundra soils were 0-0.09 μmol N kg-1 h-1,neither of which could amplify the nrfA gene.The abundances of nrfA gene in animal colonies soils and the penguin-lacking soils were from 4.6 × 103-6.1 × 104 copies g-1,and the DNRA rates were in the range of 0.35-3.79 μmol N kg-1 h-1.The DNRA rates were significantly positively correlated with nrfA gene abundances.Among them,the nrfA gene abundances and DNRA rate of the penguin colony soils were the highest,higher than those of the seal colony soils,and both were higher than that of the penguin-lacking soils,indicating that sea animal activities promoted the DNRA process.The diversity of nrfA genes in the sea animal colonies soils was lower than that in penguin-lacking soils,and the Chao 1 index of the nrfA gene was significantly negatively correlated with C:N.The CCA results showed that the composition of the DNRA bacterial community was mainly affected by ammonia and nitrate in tundra soil.Soil C:N ratio and ammonia nitrogen and nitrate nitrogen are closely related to animal activities.Therefore,sea animal activities had significant effect on the diversity and composition of the DNRA bacterial community.4.Characteristics of soil microbial community structure and nitrogen transformation function gene in the Antarctic tundra soils.Metagenomics approach was used to reveal the effects of sea animal activities on tundra soil microbial community and function,and their potential involvement in the overall N cycle.Sea animal activities reduced alpha diversity of bacteria and archaea.At the taxonomic level,sea animal activities increased the relative abundance of Proteobacteria,Bacteroidetes,and Euryarchaeota.The relative abundances of the phyla of archaea and bacteria were closely related to soil pH,C/N,and NH4+-N.Analysis of function reads showed that sea animal activities increased the relative abundance of nitrification,denitrification,DNRA,and nitrogen fixation genes.While organic N metabolism and assimilatory nitrate reduction did not change significantly under sea animal activities.The soil nitrogen metabolic process of the Antarctic tundra was driven by a series of highly diverse microorganisms.Actinobacteria,Betaproteobacteria,and Gammaproteobacteria in bacteria participated dominantly and widely in soil N metabolic process.5.Characteristics of nitrogen transformation rates of tundra soils and lake sediments in Antarctic.The rates of nitrification,denitrification,DNRA,and anammox of Antarctic tundra soils ranged from 0.06-9.91 μmol N kg-1 h-1,0.01-59.49 μmol N kg-1 h-1,0-3.79 μmol N kg-1 h-1,and 0-2.55 μmol N kg-1 h-1,respectively.The nitrogen transformation rates were higher in sea animal colonies than in background tundra.The statistical analysis showed that ammonium was the key factor controlling nitrogen transformation rates in tundra soils(p<0.05).The potential rates of nitrification,denitrification,DNRA,and anammox of Antarctic lake sediments ranged from 0.15-0.62 μmol N kg-1 h-1,0.01-3.11 μmol N kg-1 h-1,0.31-0.83 μmol N kg-1 h-1,and 0.05-0.25 μmol N kg-1 h-1,respectively.The highest rates of nitrification,denitrification,and DNRA occurred in G lake.The lowest rates of nitrification,denitrification,and anammox occurred in Y2 lake.The statistical analysis showed that ammonium was the key factor controlling nitrification and anammox rates in lake sediments(p<0.05).The rate of DNRA was mainly affected by TP(p<0.05).In sea animal colonies,denitrification contributed 49.3%-96.4%to total nitrate reduction,while in the non-animal activity area with high C:N,the DNRA process contributed 8.1%-92.5%to total nitrate reduction.In Antarctic lake sediments,anammox contributed 5.9%-35.5%to total nitrate reduction,which was lower than the contribution of the denitrification and DNRA processes.In G Lake and Y3 Lake,denitrification was the dominant nitrate reduction process,while in Y2 Lake and Y4 lake,the DNRA process was the dominant nitrate reduction process.