Studies On The Microbial Community,Enrichment Cultivation And Characteristics Of High Ammonia Tolerant Ammonia-oxidizing Archaea In Agricultural Soil

Ammonia-oxidizing archaea（AOA）plays an important role in the biogeochemical cycle of nitrogen.In agricultural soils,excessive application of chemical fertilizer will increase the concentration of ammonia nitrogen in,which would result in the differentiation of ammonia-oxidizing bacteria（AOB）,ammonia-oxidizing archaea（AOA）,complete ammonia-oxidizing bacteria（Comammox）and other ammonia-oxidizing microorganisms in the soil niche.AOB usually has tolerance to high ammonia,and then is often regarded as the main contributor to ammonia oxidation in farmland soil.However,with the discovery of ammonia-tolerant AOA genus Candidatus Nitrosocosmicus in recent years,the opinions that AOA prefer low ammonia-nitrogen environment and AOB dominant in high ammonia-nitrogen environment have been challenged.Moreover,these opinions underestimate the role of AOA playing in farmland soil.In order to find the AOA groups who have tolerance to high ammonia in agricultural soils,this study studied the microbial community changes of AOA in respond to high ammonia,obtained AOA enrichments in use of different concentrations of ammonia and a previously reported two-step enrichment method,and determined the morphology and physicochemical properties of an AOA enrichment.The main results are as follows:（1）The results of microcosmic experiments under different ammonia nitrogen concentrations showed that:a)The response of AOA in farmland soil to ammonia nitrogen concentration was affected by p H;when cultivated in high ammonia nitrogen condition,the AOA in neutral soil maintained good ammonia oxidation activity and the number of AOA amo A gene increased.b)During the cultivation,the number of AOA amo A gene in soils with no addition of NH₄⁺-N increased from 6.04×10⁷copies g^-1soil_dwto 7.92×10⁷copies g^-1soil_dwafter 28 days,while the number of AOA amo A gene in soils with addition of 300μg NH₄⁺-N g^-1soil_dw,increased from 6.07×10⁷copies g^-1soil_dwto 1.17×10⁸copies g^-1soil_dw.At the same time,AOA also showed high transcriptional activity.After 28 days of cultivation,the transcript of amo A in soils with no addition of NH₄⁺-N was determined to be 1.15×10⁵copies g^-1soil_dw,in contrast to 1.30×10⁵copies g^-1soil_dwin the soils with addition of 300μg NH₄⁺-N g^-1soil_dwand 600μg NH₄⁺-N g^-1soil_dw.c)High-throughput sequencing based on DNA and RNA showed that:The abundance of Nitrosocosmicus in the soil without ammonia nitrogen was 1%,while the abundance of Nitrosocosmicus in the soil with 300μg NH₄⁺-N g^-1soil_dwammonia nitrogen increased to 10%and 16%,respectively.Adding 600μg NH₄⁺-N g^-1soil_dwincreased the abundance of ammonia nitrogen to 16%and 48%,respectively.The potential functional AOA group resistant to high ammonia nitrogen in soil is Nitrosocosmicus,which shows good tolerance to high ammonia nitrogen concentration.Within a certain range,the higher the ammonia nitrogen concentration,the higher the proportion of the number and expression activity of the AOA of Nitrosocosmicus.（2）Four AOA enrichments was obtained after the cultivation using the neutral soil as culture sample and different concentration of ammonia（0.5 m M,1 m M,20 m M or50 m M）as substrate.Among the four enrichments,the number of AOA cells were determined as3.04×10⁷,1.04×10⁸,2.32×10⁹and 6.40×10⁸copies g^-1sand,respectively;the abundance of AOA was determined to be 10%,10%,65%and 30%,respectively.According to the results of high-throughput sequencing of the archaea amo A gene,the main AOA groups in the enrichment cultivated under 0.5 m M ammonia nitrogen was Nitrosotenuis,the Nitrosopumilus,Nitrosotenuis,Nitrosotalea,Nitrososphaera-like and Nitrosocosmicus were the main groups when cultivated under 1.0 m M,while the Nitrosocosmicus was the main group and accounted for 66%and 96%of total AOA cells when cultivated under 20 m M and 50 m M of ammonia In conclusion,the higher the concentration of ammonia nitrogen was used in enrichment culture,the more the AOA population tends to Nitrosocosmicus.（3）The morphology,physiological and biochemical analysis of AOA cells in theenrichment showed that AOA cells were irregular spheres with a diameter of 0.8-1μm,and present extracellular polymers on the surface.The optimum growth temperature wasdetermined as 35-38℃,and the optimum p H was 7.0-8.0.When the initial nitriteconcentration is greater than 5 m M,the growth of AOA is inhibited.The presence of organic matter will inhibit the ammonia oxidation of AOA tolerate to high ammonia nitrogen.AOA can tolerate ammonia oxidation inhibitors of certain concentration,and its tolerance todicyandiamide（DCD）,allylthiourea（ATU）and 3,4-dimethylpyridine phosphate（DMPP）is stronger than that of AOB.