| Nitrification is a central process in nitrogen cycle in soil and is important for soil nutrition and nitrate pollution. Ammonia-oxidation is the key step of nitrification which is driven by ammonia-oxidizing microorganisms, mainly including ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). However, very little is known about their distinct physiological characteristics. So the research for the influence of environmental factors on the abundance and community composition of AOA and AOB are of great significance for understanding the soil nitrification process in Inner Mongolia.In this study, soil samples were collected from the Inner Mongolia Grassland Ecosystem Research Station (IMGERS) of Chinese Academy of Sciences. The abundance and community structure of soil ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) in soils with long-term different N addition rates (0, 1.75,5.25,10.5,17.5, and 28 g N m-2 yr-1) and vitriol-loading rates for pH adjustment were investigated using quantitative real-time PCR, cloning and sequencing by targeting on amoA gene. In addition, soil potential ammonia oxidation rate was analyzed and short-term lab incubation experiment was completed. We analyzed the central factors which affected the soil ammonia-oxidizing microorganisms and ammonia activity, and discussed the influence and mechanism of ammonia oxidation resulted from N accumulation in the soil of Inner Mongolia Grassland. The results are as follow.With the increase of nitrogen added, soil pH declined significantly from 6.6 without N addition to 4.9 with the highest N addition rate, and potential ammonia oxidation rate also declined which was positively correlated with soil pH (p<0.01). Contrary to the variation of pH and potential ammonia oxidation rate, the copy number of bacterial amoA gene increased with nitrogen addition rates and it was positively (p<0.01) correlated with ammonia concentrations in soil while it was negatively correlated with PNR and pH. The archaeal amoA gene copy number did not change a lot with the increase of N addition, but decreased significantly under high N concentration. Sequencing of clone libraries revealed that all bacterial amoA sequences belong to genus of Nitrosospira. In the treatment without N addition, middle-level N addition, and high N addition, AOB was dominated by Cluster 3al,Cluster 3a2, Cluster 2, respectively., The profile of T-RFLP showed that no significant variation on community structure of AOA was found among all the treatments.With the increase of vitriol added, soil pH and PNR declined significantly and positively correlation was found between them. The results were similarly to N addition experiments. Differently, with the decreased of soil pH, the copy number of bacterial amoA gene declined, while the amoA gene copies of archaea increase significantly, though the copies declined under the lowest pH. The profile of T-RFLP showed that the composition of AOA change little among all the treatments. From the results of N addition experiment and vitriol addition experiment, we conclude that soil PNR may be determined by pH, and the change on abundance of AOB and AOA may be resulted from the influence of soil ammonia concentration.To further validate the effect of ammonia concentration and pH which acted on the compositions and activity of soil ammonia-oxidizing microorganisms, we made a cultivation of two soil sample(NO treatment without N addition, N28 treatment with addition of 28g N/m2) under acid and neutral condition respectively. Before cultivating, AOB has been dominated by Cluster 3a1 for N0 and Cluster 2 for N28. Through adding N, for N0, all the sequences we got grouped into Cluster 3a2 under neutral medium while about 56% sequences grouped into Cluster 2 under acid medium; for N28, near 56% for Cluster 3a2 under neutral medium while near 87% for Cluster 2 under acid medium. This result just give the evidence of the result before that composition of AOB can be changed with N adding. From this we can conclude that the AOB which can be grouped into Cluster 3a2 may adjust to the neutral environment of high N concentration and AOB which can be grouped into Cluster 2 may adjust to the acid environment.
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