Figure 7. Nitrosomonas amoA gene copy numbers in different layers. (sB0-1 represents the first stage in the surface layer; sB0-2, the first stage in the mid layer; sB0-3, the first stage in the deep layer; sB18- 1, the last stage in the surface layer; sB18-2, the last stage in the mid layer; and sB18-3, the last stage in the deep layer). doi:10.1371/journal.pone.0044122.g007 inhibition concentration (9 mM) of Nitrosotalea devanaterra. So, the sequences affiliated with Nitrosotalea cluster decreased in the surface layer where the ammonia concentration was relatively higher during 18 months cultivation process. In our simulated soil column, we found that all three layers contained a certain amount of Nitrosomonas-like sequences after 18 months. In the surface layer the proportion occupied by Nitrosomonas-like sequences was 25.0%, which was higher than in the other two layers (mid layer, 15.8%; deep layer, 18.8%). Quantitative analysis also reached the similar conclusion that the copy number of Nitrosomonas amoA genes increased from 2.26106 to 1.26108 in the surface layer, and the copy numbers were much higher than that in the other two layers. This could be explained by differences in physiological characteristics between Nitrosomonas and Nitrosospira. Schramm et al. [45,46] have reported that the growth of Nitrosomonas species followed an r-strategy. The Nitrosomonas genus had a low ammonia affinity and was adaptable to conditions where the concentration of ammonia was high. In contrast, the growth of Nitrosospira species followed a k-strategy. The Nitrosospira genus had a high ammonia affinity and was adaptable to conditions where the concentration of ammonia was low. The appearance of and increases in Nitrosomonas-like sequences were reflection of the competitive advantage of Nitrosomonas species under higher ammonia concentrations when exogenous ammonia was added. The obtained Nitrosomonas-like sequences were primarily found in the surface layer because of the relatively higher ammonia concentration in this layer compared with the other two layers. Notably, no Nitrosomonas-related sequences was found in the first stage through construction of AOB clone libraries, but we indeed detected Nitrosomonas amoA gene using qPCR, this may be due to the different specificities of the primers used in phylogenetic and quantitative analysis. The richness of AOA and AOB decreased under ammonia stress and then recovered after a period of adaptation, as reflected in the variation in OTU numbers. It seemed that the AOA in the deep layer were less sensitive to exogenous ammonia than those in the other two layers and had a relative longer adaptation period. Another explanation of the OTU numbers variation was the cultivation effect that led to the survival of only a few adapted species which take over the Figure S3 genes. (TIF) Figure S4 genes. (TIF) PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e44122 Effect of Ammonia on Ammonia-oxidizing Microbes community. For instance, in the deep layer of the first stage, AOA population was constituted with OTU1-3 and OTU6-9, and 6 months later just OTU1, OTU7 and OTU9 were left. In the last stage, the population was composed with 4 OTUs and a new OTU was found even though it had never been derived in the first three stages, so finally the population was taken over by the survival species who could adapt to the selective pressure of the environment. Abundance of AOA and AOB and Potential Nitrification Rates (PNR) in Different Layers The ratio of AOA/AOB in soils was first reported by Leininger in 2006 [17]. He found that the ratio of AOA/AOB was correlated with soil depth in long-term fertilized soils and AOA amoA gene copy numbers could be three orders of magnitude greater than AOB amoA gene numbers at a depth of 30–40 cm. In the present study, the highest ratio of AOA/AOB was found to be 12.96 in the second stage in the surface layer. The growth and ammonia oxidation activity of Nitrosopumilus maritimus were inhibited at an ammonia concentration of 28 mg/L [28] which was lower than the inhibitory concentrations (7000–14000 mg/L) of ammonia found for AOB [47]. Therefore, it was expected that the numbers of bacterial amoA gene copies would exceed the archaeal amoA gene copy numbers in the surface layer during the last stage, where the ammonia concentration was the highest. The potential nitrification rates (PNR) were highest in the mid layer and were 1.6 times higher than that in the surface layer and 2.5 times higher than that in the deep layer. The lower potential nitrification rates in the deep layer may be the cause of the change in redox potential along with soil depth, as reported previously [48]. The redox potential in soils is determined by the relative concentrations of substances in an oxidized state versus a reduced state, which are influenced by oxygen concentrations. The deep layer exhibited the lowest redox potential, which resulted from the lowest oxygen concentration. The fact that the lowest pH (7.0960.15) existed in the deep layer compared with the other two layers (surface layer, 7.2460.12; mid layer, 7.4160.1) may also play an important role. Conclusion In general, obvious population dynamic changes were found for both AOA and AOB under the selective pressure of exogenous ammonia and the changes were different in three layers of the soil column. Supporting Information Figure S1 Changes in AOA OTU numbers in different layers during the 18-month cultivation process. (TIF) Figure S2 Changes in AOB OTU numbers in different layers during the 18-month cultivation process. (TIF) The standard curve of qPCR for AOA amoA The standard curve of qPCR for AOB amoA Figure S5 The standard curve of qPCR for Nitrosomo- nas amoA genes. (TIF)