3.3. ANAMMOX bacteria and phylogenetic analysis
The nested PCR process seemed to be more sensitive for amplifying
the 16S rDNA fragments of the ANAMMOX bacteria. Some
available sequences and their relatives from the GenBank are
shown in Table 4. The clones from R5PLA1 to R5PLA14 are the
sequencing results of the PCR products for Planctomycete, while
clones R5Amx2 and R5Amx3 are the specific amplification results
for the ANAMMOX bacteria. Candidatus Kuenenia and Candidatus
Brocadia are the commonly known ANAMMOX bacteria genera
(Bae et al., 2010), and some bacteria affiliated with Planctomycete
had a similarity of 95% with the uncultured Candidatus Kuenenia
sp. (R5PLA10 and R5PLA11). While the clones from R5PLA12 to
R5PLA14 also seemed to be related to the ANAMMOX bacteria,
even they shared a similarity of less than 90% with some known
ANAMMOX bacteria, as is shown in Table 4. The specific amplification
using Amx368f/Amx820r as primers was more accessible for
identifying ANAMMOX bacteria, since R5Amx2 and R5Amx3
shared a higher similarity with Candidatus Kuenenia stuttgartiensis
and anaerobic ammonium-oxidizing Planctomycete, respectively;
and they could be clustered into a group after the phylogenetic tree
analysis (see supplementary material, Fig. S3). It was demonstrated
that the ANAMMOX reaction indeed occurred in the biofilter.
The existence of the ANAMMOX bacteria that occurred in the
aerobic biofilter was interesting but also confusing. The diffusion
of DO into the inner layer of the biofilm could be hindered by
the resistance of the bacteria in the outer layer of the biofilm,
and therefore a suitable anaerobic environment in the inner layer
for the ANAMMOX bacteria had to be created.