## 3 Result
### Diversity of NirK-Dependent Denitriying Communities  
Partial _nirK_ gene fragments (ca. 500 b.p.) from the three sample libraries and 150 clones were sequenced. A BLAST analysis revealed that only 31 (20%) of the 150 sequences showed homology to known copper-containing nitrite reductase (_nirK_) sequences, with 63-96% nucleotide identity (Figure 4). The deduced amino acid sequences shared 56-99% identity with NCBI NirK sequences. Consistent with a previous classification [24], the phylogenetic tree grouped all the _nirK_ sequences into five clusters, and predominantly (86.67%) within the AOB cluster. Interestingly, only nine clones from sample QL-H fell into _nirK_, and six clones belonged to one unique protein. Some clones retrieved  
Figure 3: Analysis of nitrite reductase (nirS) gene sequences constructed based on protein sequences using the neighbor-joining method. Sequences from this study are shown in red (**A**) sample QL-H; (**B**) sample QL-V; (**C**) sample SR-W. Other representative nirS from the protein database and GenBank accession numbers are OIO76922 (_Hydrogenophilaceae_), WP_011386514 (_Magnetospirillum magneticum_), OFZ68978 (_Betaproteobacteriabacteria_), BAV32586 (_Sulfuricaulis limicola_), WP_011310590 (_Thioochillus denitrificans_), OQY70110 (_Rhodocyclaceae_), OQY57414 (_Beggiatao_ sp.), WP_045480144 (_Thiooloca nigra_), BAE45629 (_Hydrogenobacter thermophilus_), OGW13923 (_Nitrospirine bacterium_), OGP39028 (_Deltaproteobacteriabacteria_), OHB90560 (_Planctomycetes_), WP_054521542 (_Thermaneroflrix daxensis_), KPL78197 (_Bellillinea calidifstulae_), WP_075072401 (_Longilinea arroyzae_), OGP16868 (_Deltaproteobacteriaa_), CBE69462 (_Candidatus Methylomirabilis oxifera_), CAJ74898 (_Candidatus Kuenenia stuttgartiensis_), OGT00430 (_Gallionellales bacterium_), and OHC62023 (_Rhodcyclales bacterium_).  
from QL-V and SR-W were grouped with denitrifying bacterial cluster 2, and a single clone from QL-H was grouped into the ammonia-oxidizing archaea (AOA) cluster. These results indicate that bacterial population structures and diversity based on the copper-containing NirK protein differ according to the nitrogen source, which may reflect the occurrence of different 'ecotypes'. Furthermore, a metagenomic qPCR analysis was used to confirm that the copy numbers of _nirS_ retrieved from QL-H, QL-V, and SR-W were \(1.15\times 10^{5}\), \(1.76\times 10^{5}\), and \(7.15\times 10^{4}\) gene copies per ng of DNA, respectively, and those of _nirK_ were \(3.67\times 10^{3}\), \(8.70\times 10^{3}\), and \(1.04\times 10^{4}\) gene copies per ng of DNA, respectively (Figure 5). The abundances of _nirS_ relative to the overall bacterial population were 0.54%, 0.44%, and 0.13%, respectively (Figure 6), and those of _nirK_ were 0.017%, 0.022%, and 0.018%, respectively. These results indicate that _nirS_ was more abundant than _nirK_ in all three samples, which is similar to the results observed in some wastewaters [12]. However, the relative abundances of _nirK_ and _nirS_ were clearly significantly lower than those in previously reported samples [8]. These differences could be attributable to the wastewater treatment plants having higher carbon and nitrogen concentrations than our three samples and involving treatment processes that benefit denitrifiers, although our samples were also clearly from habitats that benefit denitrifiers.  
Figure 4: Phylogenetic analysis of _NirK_ gene products (partial, 140 amino acids) from sample QL-H (HnirK), sample QL-V (VnirK), and sample SR-W (WnirK) bacteria (in red). The 19 candidate _NirK_ sequences (in green) were used for designing the _NirS_ gene degenerate primer pair. Database sequences are shown in black, with GenBank accession numbers in parentheses. Roman numerals refer to clusters discussed in the text. Bootstrap values (50%) for 1000 replicates are shown at the branch points.