Effect of Ammonia on Ammonia-oxidizing Microbes Figure 4. Population shifts of AOA in different layers. (sA0-1 represents the first stage in the surface layer; sA0-2, the first stage in the mid layer; sA0-3, the first stage in the deep layer; sA18-1, the last stage in the surface layer; sA18-2, the last stage in the mid layer; and sA18-3, the last stage in the deep layer). doi:10.1371/journal.pone.0044122.g004 Figure 5. Population shifts of AOB 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.g005 25.06 mg?N?L21?g21?h21, while those in the surface and deep layers were 16.13 mg?N?L21?g21?h21 and 9.97 mg?N?L21?g21?h21, respectively. Therefore, ammonia-oxi- dizing microorganisms in the mid layer exhibited the highest nitrification activity among the three layers of the soil column. Discussion Community Succession of AOA and AOB It was found that 69.2% sequences derived in our soil column were related to Nitrosotalea devanaterra-the only ammonia-oxidizing archaea from acid soil [6]. This was in accordance with the high-throughput sequencing results reported by Gubry-Rangina [44], which demonstrated that Nitrosotalea devanaterra-related sequences may be predominant in some agricultural soils. pH in our soil column were neutral or slightly alkaline and was not in accordance with the acid environment where Nitrosotalea devanaterra was found. The contradictory could be explained by that there may be existed AOA belonging to Nitrosotalea devanaterra cluster which could survive and oxidize ammonia to nitrite in neutral or slightly alkaline environment. The community composition of the AOA in the different layers of the soil column underwent great changes during the cultivation process. In the surface layer, the sequences belonging to Nitrososphaera sister cluster disappeared after 18-month cultiva- tion. Since there was no isolated or enrichment strain of this AOA cluster [37], the physiochemical characteristics of Nitroso- sphaera sister cluster was unknown. Combined with the increased Nitrososphaera-related sequences in the surface and the decreased Nitrososphaera sister-related sequences in the deep layer, we deduced that the AOA affiliated with Nitrososphaera sister cluster had relative high affinity for ammonia and prefer to live in the environment with relative low ammonia concentration. Nitroso- sphaera viennensis [7] could grow well in the media containing ammonia concentration as high as 15 mM, indicating a low affinity of this strain of AOA. So, it was reasonable that the proportion Nitrososphaera-related sequences occupied increased in the last stage compared with the first stage in the surface layer, since it preferred conditions with relatively higher ammonia concentration. Correspondingly, in the deep layer, the propor- tion Nitrososphaera-related sequences occupied decreased from 59.1% to 16.7%. Nitrosopumilus maritimus showed extremely high affinity for ammonia and the ammonia inhibition concentration was as low as 2 mM to 3 mM [5], so it was not strange that the Nitrosopumilus-related sequences disappeared in the deep layer after exogenous ammonia was added during the cultivation process. No Nitrosopumilus-related sequence was detected in the surface and mid layer during the whole 18 months. Nitrosotalea- related sequences were the dominant species in our soil column throughout the cultivation process. In the surface layer, the proportion that the Nitrosotalea-related sequences occupied decreased from 66.7% to 52.9%; on the contrary, the proportion in the deep layer increased from 27.3% to 72.2%. It was reported that the acid AOA could grow well with the total NH3& NH4+ concentration ranged from 10 mM to 10 mM (equivalent to 0.18 nM to 0.18 mM of NH3) at pH 4.5 and no growth was detected at 50 mM and 100 mM (equivalent to 9 mM to 18 mM NH3, pKa for NH3: NH4+, 9.25) [6]. The available NH3 concentration in the medium was 44mM (pH, 7.24; 30uC), which was much higher than the ammonia Figure 6. Archaeal and bacterial amoA gene copy numbers in different layers. (s0-1 represents the first stage in the surface layer; s0-2, the first stage in the mid layer; s0-3, the first stage in the deep layer; s18-1, the last stage in the surface layer; s18-2, the last stage in the mid layer; and s18-3, the last stage in the deep layer). doi:10.1371/journal.pone.0044122.g006 PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e44122