Effect of Ammonia on Ammonia-oxidizing Microbes Figure 2. Neighbour-joining phylogenetic tree showing the relationships of the 185 sequences of archaeal amoA genes detected in the clone libraries constructed from samples from the soil column with their most similar sequences and sequences of known AOA. The numbers at the nodes are percentages that indicate the levels of bootstrap support from 1,000 replicates. The scale bar represents 0.1 nucleic acid substitutions per nucleotide position. doi:10.1371/journal.pone.0044122.g002 dropped from 4 to 1, while it decreased from 2 to 1 and from 7 to 3 in the mid and deep layer, respectively. The only difference was that the number of OTUs in the surface and mid layers recovered more rapidly than was observed for the deep layer. The number of OTUs in the surface and mid layers began to increase in the third stage; however, the number of OTUs in the deep layer did not increase until the last stage. With respect to AOB, increasing sequences belonging to Nitrosomonas were detected in all three layers in the soil column, and the proportions that Nitrosomonas-related sequences occupied were 25%, 15.8% and 18.8% from the surface layer to the deep layer (Figure 5). The numbers of AOB OTUs varied in a similar way as was observed for the AOA OTUs. The AOB OTU numbers in the mid and deep layers decreased in the first three stages and increased in the last stage. However, in the surface layer, the number of OTUs remained stable at three after the third stage (Figure S2). Real-time Quantitative PCR (qPCR) The AOA and AOB amoA genes in the three soil layers were quantified every 6 months. AOA were more abundant than AOB in almost all soil samples, with the exception of the sample from the surface layer in the last stage, in which the AOB/AOA ratio was 16.95. The ratios of AOA/AOB ranged from 1.03–12.96 in the other 11 soil samples (Figure 6). The copy number of Nitrosomonas amoA genes increased substantially in the surface layer during the cultivation process, from 2.26106 to 1.26108. As for the other two layers, the change of was not obvious (Figure 7). Nitrification Activity Nitrification activity was tested after the 18-month cultivation period. Samples were collected from the three different layers of the soil column. Different nitrification activities were observed for the three layers of the soil column. The maximum specific nitrification activity in the mid layer was Figure 3. Neighbour-joining phylogenetic tree showing the relationships of the 201 sequences of bacterial amoA genes detected in the clone libraries constructed from samples from the soil column with their most similar sequences and the sequences of known AOB. The numbers at the nodes are percentages that indicate the levels of bootstrap support from 1000 replicates. The scale bar represents 0.05 nucleic acid substitutions per nucleotide position. doi:10.1371/journal.pone.0044122.g003 PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e44122