# Effects of Simulated Nitrogen Deposition on the Bacterial Community of Urban Green Spaces
## 3 Results
### Effects of N Deposition on Soil Bacterial Community Composition  
Upon reads processing ad filtering the actual sequences that were obtained in the bacterial community analysis of three sampling times were 530,447; 960,673; and 522,682; respectively. Classified OTUs belonged to 51 phyla among all samples. As common, due to our still partial knowledge of global microbial diversity, available reference databases do not allow an unambiguous assignment up to the genus or species level for a substantial number of the obtained sequence reads. Therefore, the most reliable annotation is to date prudentially assumed at relatively high taxonomy ranks. For this reason, we will present to the majority of results sticking to the phylum level, and we will later comment the data at finer resolution (from order to genus level) for those cases in which the assignment was backed up by a robust degree of sequence identity.  
The bacterial community composition at phylum level (relative abundance \(>\)1%) is shown in Figure 3. The dominant phyla (i.e., the ones showing highest values in relative sequence abundance) in all samples were Proteobacteria and Acidobacteria. Besides, the subdominant phyla in all samples were Chloroflexi, Bacteroidetes, Nitrospirae, Acidobacteria, Gemmatimonadetes, Verrucomicrobia, Planctomycetes, Firmicutes, Actinobacteria, Parcubacteria, and Latesobacteria. These phyla represented more than 88% of the sequences in all the samples. After 12 months of N treatment, the relative abundance of Proteobacteria significantly differed among different treatments (\(p<0.01\), Figure S1 in the Supplementary Material), specifically increasing following N enrichment. The percentage of Acidobacteria was low in the N3 treatment but did not differ significantly among the treatments (Figure S1).  
Figure 2: Effects of N deposition on soil bacterial chao1 index. Different letters indicate significant differences among treatments within the same sampling time (\(p<0.05\), ANOVA).  
The variations in soil bacterial communities caused by N deposition were evaluated by PERMDISP2. Differences in the overall bacterial composition were expressed based on the Bray-Curtis distance. Results are shown in Figure 4. After 3 months (July 2016) of N deposition, different N treatments did not separate, which indicated that at such early time, the bacterial community composition was still similar among all treatments (Figure 4). Subsequently, starting from the second time point (October 2016), a significant difference arose between communities in relation to the N level received. After 12 months (May 2017) of N deposition, the communities separate clearly between N3 and N0 treatments (Figure 4), and the bacterial community composition of N3 treatment resulted profoundly changed. Such significance attains its highest value in the last sampling at 12 months (May 2017). An N deposition rate of N3 or higher was required for a significant shift to occur.  
Figure 3: Effects of N deposition on soil bacterial relative abundance. The groups accounting for \(\geq\)1% are shown while those >1% are integrated into “other”.