High-throughput Sequencing Analysis Of The Ecological Distribution And Autotrophic Carbon Fixation Potential Of Ammonia-oxidizing Archaea(AOA) In The Deep Waters Of The South China Sea

The discovery of ammonium-oxidizing archaea（AOA）challenged the traditional understanding that the ammonium oxidation was uniquely performend by ammonium-oxidizing bacteria（AOB）,which is the new content of the microbial nitrogen cycle.AOA was known as one of the most abundant prokaryote in meso-and bathypelagic realm,it is pervasive and plays dominant role in ammonium oxidation process in areas of ocean.Recent studies showed that AOA grows autotrophically using a variant of the 3-hydroxypropionate/4-hydroxybutryrate pathway for carbon assimilation,making great contributions to marine primary productivity（especially in meso-and bathy-pelagic）.AOA contributes significantly to the global nitrogen and carbon cycle.In this study,we investigated the abundance,diversity,and distribution of AOA in water samples at difference depths of four stations in the South China Sea by analysis of ammonia monooxygenase gene amoA involved in ammonia oxidation,and biotin carboxylase gene accA putatively involved in archaeal CO₂ fixation.Quantitative PCR and amplicon high-throughput sequencing technique were applied to this study,the primary objectives of the current study were to 1)explore the abundance,diversity,distribution pattern and microbial community of AOA in the South China Sea based on amoA gene.2)explore the diversity of accA,the carbon fixation potential of AOA,and the spatial distribution pattern of AOA carbon fixation process in the South China Sea with accA gene as the molecular marker.The major results are as follows:1)amoA gene sequences of 34 AOA strains from pure or enrichment culture were used as seed sequences.Uncultured amoA gene sequences from environmental samples were downloaded from functional gene database.All sequences were used to construct taxonomic database for amoA gene sequences.By pairwise comparison of 16S rRNA gene and archaeal amoA identities of all recognized species of AOA,we determined the cut-off value for OTU clustering at species-level.We constructed a reference taxonomic database containing 26091 amoA gene sequences and determined 89%sequence identity as cut-off value for OTU clustering at species-level.We developed a method for ammonia-oxidation archaea diversity analysis based on amoA gene amplicons with high-throughput sequencing.This method can effectively analyze the diversity of AOA in environmental samples.2)We examined the community diversity and composition of AOA in the water column of four stations by Quantitative PCR and MiSeq High-throughput sequencing of amoA gene.The analysis results showed that the abundance of AOA was much higher than that of AOB in water deeper than 75m,and the average abudance of AOA was 1-2 order of magnitude more than that of AOB in the South China Sea,indicating that AOA was the main driver of ammonia oxidation in the South China Sea.The results of diversity analysis showed that AOA in the South China sea belonged to Group I.1a cluster.Nitrosopelagicus was the most abundant group at genus level in shallow waters,and the main AOA of the deeper sea was dominated by uncultured genus without defined taxonomic information.The hightest diversity of AOA appeared in 150-200m water layer,there were niche differentiation of AOA in the water column.The amoA genes were divided into two groups,the relative abundance of the“Shallow”group and the“Deep”group changed with depth,resulting in significant vertical distribution differences of AOA in the South China Sea.3)We investigated the abundance,genetic diversity and community structure of AOA accA gene for exploring autotrophic carbon fixation potential and spatial distribution characteristics of ammonia oxidation autotrophic carbon sequestration in the South China Sea by using Q-PCR-based assay and amplicon high-throughput sequencing technology.Quantitative analysis showed that there were high abudance of accA in water deeper than 75m,and the peak value of abudance appeared in 200m water layer;besides,accA gene abundance was correlated with AOA amoA gene abundance and the ratio of accA/amoA gene abundance increased with depth,it indicated that AOA make a significant contribution to primary prodution in the the South China Sea（especially in dark ocean）.The result of diversity analysis showed that the hightest diversity of AOA appeared in 100-150m water layer,and accA gene was divided into two groups,the surface group was mainly distributed in 75-150m while the deep group was mainly distributed in 100-1500m.There were significant differences of vertical distribution of AOA accA gene and evident spatial distribution of chemoautotrophy carbon fixation by AOA,the deep group of AOA may have a higher chemoautotrophy carbon fixation capacity than AOA in the surface water.The results of comprehensive study showed that AOA was the dominant player in the process of ammonia oxidation in the South China Sea,and the autotrophic AOA was an important contributor to primary productivity in the South China Sea（especially in the dark ocean）.AOA amoA gene and accA gene showed both“Shallow”and“Deep”groups,they presented distinct differences in vertical distribution.There showed an obvious spatial distribution pattern of the biogeochemical process of autotrophic production by ammonia oxidation of AOA.