Primers sets Arch-amoAF, Arch-amoAR [34] and amoA-1F, amoA-2R [35] were used for the amplification of archaeal and bacterial amoA genes, respectively. The PCR thermal cycling programs employed were described previously [36]. The amplified products were examined in a 1.0% agarose gel by electrophoresis. Cloning and Sequencing The obtained PCR products were cloned using the pMD19-T vector (TaKaRa, Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Plasmid DNA was isolated using the Gene JETTM Plasmid Miniprep kit (Fermentas Life Sciences, Burlington, Canada) and was digested with 5 U of EcoRI enzyme in EcoRI buffer for 1.5 h at 37uC. The digestion products were examined for an insert of the expected size using agarose (1.0%) gel electrophoresis. At least 20 positive clones from each library were randomly selected for sequencing using an ABI3100 automated sequencer (Applied Biosystems, California, USA). Phylogenetic Analyses All of the amoA gene sequences obtained were imported into MEGA4.1 to construct alignment files in combination with the most similar environmental sequences and sequences of known strains of AOA and AOB. Phylogenetic trees were constructed using the neighbour-joining method. Bootstrap analysis with 1000 replicates was applied to estimate the confidence values for the tree nodes. Statistical Analyses Operational taxonomic unit (OTUs) was used to determine the archaeal and bacterial amoA gene diversity in different soil samples. For both AOA and AOB, researchers found that 1%–3% 16S rRNA distance equate to a 15% amoA gene distance [37,38]. 85% sequence identity at the amoA gene level could be regarded as an approximate threshold below which ammonia-oxidizing microor- ganisms can be assigned to different species. Hence, OTUs were defined by 15% differences for both archaeal amoA gene and bacterial amoA gene in nucleotide sequences. OTU numbers were determined by the farthest neighbour algorithm in the DOTUR program [39]. The coverage of the clone libraries was calculated as C = [1 - (n1/N)] 6100, where n1 is the number of unique OTUs and N is the total number of clones in a library. Real-time Quantitative PCR (qPCR) The primer used to quantify the copy numbers of amoA genes of AOA and AOB were the same with those used in the clone library construction. Primer pair P128r, P365r designed by Dang [40] was used to quantify the copy numbers of Nitrosomonas amoA gene. qPCR was performed using an iCycler iQ5 thermocycler and a real-time detection system (Bio-Rad, California, USA) according to the method described by Francis et al. [34], Mc Tavish et al. [41] and Dang [40]. Quantification standards consisted of 102–108, 101–107, 102–108 copy number dilution series of plasmid containing inserted amoA genes of AOA, AOB and Nitrosomonas, respectively. R2 and the amplification efficiencies of the AOA, AOB and Nitrosomonas standard curves were 0.996, 0.998 and 0.996 (as shown in Figure S3, S4 and S5) and 96%, 89% and 95%, respectively. Analytical Methods Measurements of NO3–N, NO2–N and NH4+-N in the experimental medium were performed according to standard methods [42]. The total solid (TS) content was measured by the weighing method after drying at 105uC. The inorganic nitrogen Effect of Ammonia on Ammonia-oxidizing Microbes contents in the soils were determined colorimetrically via flow injection analysis of soil-KCl extracts as previously described [43]. GenBank under accession Accession Numbers Sequences were deposited in numbers JQ320498 to JQ320554. Results Diversity and Phylogenetic Analysis of AOA and AOB 185 derived archaeal amoA genes belonged to Nitrososphaera cluster, Nitrososphaera sister cluster, Nitrosotalea cluster and Nitroso- pumilus cluster according to a new nomenclature system for archaeal amoA genes reported by Pester [37] (Figure 2), but no Nitrosocaldus-related sequences was detected. The sequences affiliated with Nitrosotalea cluster were the most and accounted for 69.2% of all the archaeal amoA genes obtained. Only two sequences were detected belonging to Nitrosopumilus cluster and the rest sequences belonged to Nitrososphaera cluster and Nitrososphaera sister cluster. A total of 9 OTUs were obtained using the DUTOR program based on a 15% amoA gene sequence distance cutoff [37] for all 185 AOA amoA sequences detected in this study. Nitrosopumilus cluster contained only one OTU and the other three clusters contained at least two OTUs. Sequences belonging to OTU1 were found in all three soil layers during the entire cultivation process, totalling 121 sequences, which accounted for 65.4% of the detected AOA amoA gene sequences. As to AOB, all 201 sequences obtained for the AOB amoA gene in the clone libraries belonged to b-proteobacteria, as shown in Figure 3. Among these sequences, 91.0% were grouped into the Nitrosospira genus, and the remainders were grouped into Nitrosomonas. A total of 8 OTUs were obtained for the bacterial amoA genes using the DUTOR program based on a 15% amoA gene sequence distance cutoff [38]. Nitrosomonas cluster contained 2 OTUs, and the remainders were grouped into Nitrosospira cluster. Community Succession of AOA and AOB The community structure of the AOA and AOB in the soil column changed during the cultivation process and was closely related to soil depth. An obvious population shift was found for AOA in different layers when the proportions that each cluster occupied in the first stage and the last stage were compared. In the surface layer, the proportion of Nitrosotalea cluster decreased from 66.7% to 52.9% (Figure 4). Accordingly, an increase from 20% to 47.1% was found for Nitrososphaera cluster. The sequences affiliated with Nitrososphaera sister cluster disappeared during the 18-month cultivation process. Interestingly, in the mid layer, we found that cluster Nitrosotalea-related sequences represented more than 94.4% of the sequences detected, and this proportion did not change during the cultivation process. Accordingly, the proportion of Nitrososphaera-related sequences was less than 5.6% throughout the cultivation process. In the deep layer, the observed changes were dramatic. In the first stage, the diversity of the population was high and all the four AOA amoA clusters were found, the Nitrosopumilus- related sequences were only detected in the first stage of the deep layer and represented 9.1% of the sequences detected. However, 18 months later, the sequences belonging to Nitrosopumilus disappeared and the proportion that Nitrosotalea cluster occupied increased from 27.3% to 72.2%. In the 18-month cultivation process, the number of OTUs in all three layers decreased first and then increased. The number of OTUs in the deep layer was greater than in the other two layers in the first stage (Figure S1). In the second stage, the numbers of OTUs in all three layers were reduced dramatically. In the surface layer, the number of OTUs PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e44122