Environ Sci Pollut Res
DOI 10.1007/s11356-016-7914-4
SHORT RESEARCH AND DISCUSSION ARTICLE
Detection of comammox bacteria in full-scale wastewater
treatment bioreactors using tag-454-pyrosequencing
Alejandro Gonzalez-Martinez 1 & Alejandro Rodriguez-Sanchez 2 &
M. C. M van Loosdrecht 3 & Jesus Gonzalez-Lopez 2 & Riku Vahala 1
Received: 7 June 2016 / Accepted: 14 October 2016
# Springer-Verlag Berlin Heidelberg 2016
Abstract The nitrogen cycle has been expanded with the re-
cent discovery of Nitrospira strains that can conduct complete
ammonium oxidation (commamox). Their importance in the
nitrogen cycle within engineered ecosystems has not yet been
analyzed. In this research, the community structure of the
Bacteria domain of six full-scale activated sludge systems
and three autotrophic nitrogen removal systems in the
Netherlands and China has been investigated through tag-
454-pyrosequencing. The phylogenetic analyses conducted
in the present study showed that just a few of the Nitrospira
sequences found in the bioreactors were comammox.
Multivariate redundancy analysis of nitrifying genera showed
an outcompetition of Nitrosomonas and non-comammox
Nitrospira. Operational data from the bioreactors suggested
that comammox could be favored at low temperature, low
nitrogen substrate, and high dissolved oxygen. The non-
ubiquity and low relative abundance of comammox in full-
scale bioreactors suggested that this phylotype is not very
relevant in the nitrogen cycle in wastewater treatment plants.
Responsible editor: Gerald Thouand
Electronic supplementary material The online version of this article
(doi:10.1007/s11356-016-7914-4) contains supplementary material,
which is available to authorized users.
* Alejandro Gonzalez-Martinez
Alejandro.gonzalezmartinez@aalto.fi
1
Department of Built Environment, School of engineering, Aalto
University, P.O. Box 15200, Aalto, FI-00076 Espoo, Finland
2
Institute of Water Research, University of Granada, C/Ramón y
Cajal, 4, 18071 Granada, Spain
3
Department of Biotechnology, Technical University of Delft,
Julianalaan 67, 2628 BC Delft, The Netherlands
Keywords Activated sludge . Comammox . Nitrogen .
Phylogenetics . Pyrosequencing
Introduction
Nitrogen is one of the major pollutants found in wastewaters
around the world. Since the introduction of wastewater treat-
ment processes, several technologies have been found to elim-
inate nitrogen from wastewater. To date, these technologies
can be divided into two main categories: complete
nitrification/denitrification processes and autotrophic nitrogen
removal processes (Gonzalez-Martinez et al. 2011). In the
complete nitrification/denitrification process, all ammonium
are oxidized to nitrate by aerobic, autotrophic microorganisms
and then nitrate is reduced to molecular nitrogen by anaerobic,
heterotrophic microorganisms. Complete nitrification/
denitrification has been widely used within activated sludge
systems and has been regarded as the main process for nitro-
gen removal from wastewater around the world (Gajewska
et al. 2015; Tang and Chen 2015). On the other hand, autotro-
phic nitrogen removal consists in a partial oxidation of ammo-
nium to nitrite by aerobic, autotrophic microorganisms and a
simultaneous consumption of nitrite and ammonium to yield
molecular nitrogen by the metabolism of anaerobic
ammonium-oxidizing (anammox) bacteria. Several technolo-
gies have been developed using this metabolic route for nitro-
gen removal such as two-stage partial nitritation/anammox
technology, deammonification (DEMON) technology, or
completely autotrophic nitrogen-removal over nitrite
(CANON) technology (Gonzalez-Martinez et al. 2011;
Lackner et al. 2014).
The technologies for nitrogen removal from wastewater thus
depend on the metabolism of ammonium-oxidizing bacteria
(AOB), nitrite-oxidizing bacteria (NOB), and/or anammox

bacteria. Several recent studies have shown the diversity and
relative abundance of AOB, NOB, and anammox bacteria in
full-scale wastewater treatment systems. The most represented
AOB in full-scale activated sludge systems appeared to be
Nitrosomonas and Nitrosospira, while other species such as
Nitrosococcus, Nitrosolobus, or Nitrosovibrio have also been
found (Ge et al. 2014). In autotrophic nitrogen removal systems,
the dominant AOB found were Nitrosomonas, with the presence
of Nitrosospira and Nitrosococcus (Gonzalez-Martinez et al.
2015a, b). Candidatus Brocadia and Candidatus Kuenenia spe-
cies of anammox bacteria have been found in full-scale activated
sludge systems (Wang et al. 2015). Also, they are one of the
dominant phylotypes in autotrophic nitrogen removal systems,
and several species such as Candidatus Brocadia and
Candidatus Jettenia have been found in full-scale anammox bio-
reactors (Gonzalez-Martinez et al. 2015a, b). With respect to
NOB, the dominant genera found in activated sludge systems
were K-strategist Nitrospira, followed by r-strategist
Nitrobacter (Ge et al. 2014). Nitrospira have been found in some
full-scale anammox bioreactors in relative abundances between
0.1 and 1.0 % (Gonzalez-Martinez et al. 2015b).
Nitrospira has been claimed as a very diverse genus con-
taining many different ecophysiological species with different
biological capabilities (Gruber-Dorninger et al. 2015).
Recently, the capacity of microorganisms to oxidize nitroge-
nous compounds has been expanded with the discovery of
Nitrospira strains capable of both ammonium and nitrite oxi-
dation (Van Kessel et al. 2015; Daims et al. 2015; Santoro
2016). This finding showed the first microorganism with this
singular capacity, which gave it the acronym complete ammo-
nium oxidizer (comammox). Given its metabolic capability, it
is possible that comammox can grow in wastewater treatment
systems in which nitrogen is intended to be removed.
In order to evaluate the presence and relative abundance of
comammox bacteria with respect to the total bacterial com-
munity of wastewater treatment systems, the bacterial diversi-
ty of nine wastewater treatment bioreactors has been evaluated
by the means of tag-454-pyrosequencing technology, which
has proven accurate results for different microbial ecosystems
such as wastewater treatment systems, surface water, or hu-
man gut, among others (Wei et al. 2015; Jordaan and
Bezuidenhout 2015). These bioreactors were activated sludge
systems and autotrophic nitrogen removal systems, which in-
tend to remove nitrogen via ammonium oxidation to nitrite or
ammonium oxidation to nitrate and subsequent denitrification.
Materials and methods
Description of bioreactors
The nine bioreactors sampled in this study were all full-scale
bioprocesses. These were activated sludge (AS) systems (six)
Environ Sci Pollut Res
or autotrophic nitrogen removal (ANR) systems (three). The
six AS sampled were located in the Netherlands. These had
two technological configurations: conventional activated
sludge (CAS) systems with enhanced biological phosphorous
removal (EBPR) (four bioreactors sampled, two of them with
presettling and two without presettling) and A-stage bioreac-
tors of AB-stage AS systems (two bioreactors sampled). The
three samples coming from the ANR bioreactors were collect-
ed from WWTPs in the Netherlands (two) and China (one).
The technological configurations of these were DEMON sys-
tem (1) or CANON systems (2), one of them treating indus-
trial wastewater and the other urban anaerobic digester super-
natant. A description of the bioreactors sampled in the study
and some of their environmental and operational conditions
are given in Table 1.
Sample collection and DNA extraction procedure
For each of the nine bioreactors, 200 mL of mixed liquor was
extracted from five different points distributed among the en-
tire volume of the bioreactor. Collected samples were kept at
4 °C and immediately sent to the laboratory. After reception of
samples, these were subjected to centrifugation at 3500 rpm
during 10 min at ambient temperature to obtain a pellet of
biomass. The supernatant was discarded, and the pelleted bio-
mass was stored at −20 °C until further DNA extraction
procedure.
DNA extraction procedure was done according to the pro-
tocol described in Gonzalez-Martinez et al. 2015c. All
pelleted biomass samples were subjected to a DNA extraction
procedure using the FastDNA SPIN Kit for Soil (MP
Biomedicals, Solon, OH, USA) and the Fast Prep24 apparatus
(MP Biomedicals, Solon, OH, USA) following the instruc-
tions provided by the DNA extraction kit’s manufacturer.
The five DNA extracts coming from the same bioreactor were
merged into the same DNA pool for further tag-454-pyrose-
quencing process. Extracted pools of DNA were then stored at
−20 °C and sent to the Research and Testing Laboratory
(Lubbock, TX, USA) for next-generation sequencing
procedure.
For the AS samples, the amplification of extracted DNA
was done under the following conditions: preheating at 94 °C
for 3 min; then proceeded with 40 cycles of 94 °C for 30 s,
60 °C for 40 s, and 72 °C for 1 min; and amplification ended
with an elongation step at 72 °C for 5 min (Gonzalez-Martinez
et al. 2016a). For the ANR samples, this was done under the
different following conditions: preheating step at 94 °C for
3 min; 32 cycles at 94 °C for 30 s, 45 °C for 40 s, and 72 °C
for 1 min; and elongation at 72 °C for 5 min (Gonzalez-
Martinez et al. 2015a).
For the AS samples, the primer pair 28F (5′-GAGT
TTGATCNTGGCTCAG-3′)-519R (5′-GTNTTACNG
CGGCKGCTG-3′) was used for the amplification of the V1-
Environ Sci Pollut Res

Temperature
V2-V3 hypervariable regions of the 16S ribosomal RNA (rRNA)
gene of the domain Bacteria (Gonzalez-Martinez et al. 2016a).

18.8
(°C)
Likewise, for the ANR samples, the primer pair 530F (5′-GTGC

19
20

30
35
35
18

15
18
CAGCMGCNGCGG-3′)-1100R (5′-GGGTTNCGNTCGTTG-
3′) was used to amplify the hypervariable regions V4-V5-V6 of
Dissolved oxygen

the 16S rRNA gene of the domain Bacteria, as it has been de-
fined as the best primer pair for the purpose of bacterial charac-
terization of ANR samples (Gonzalez-Martinez et al. 2015a). For
(mg O/L)

1.5–2.5
all samples, tag-454-pyrosequencing process was done using the

0–0.3

1.5–2
1.5
1.5

1.5
0.2

Roche 454 GS-FLX+ apparatus.

1

1
TNeff (mg N/L)

tag-454-pyrosequencing postprocess

22.5 The raw data from the next-generation sequencing procedure

1.5
2.6
3.4
10

60
30
12
24

was treated to yield data for an ecological analysis of each

bioreactor. The samples were treated separately using mothur
v1.34.4 software (Schloss et al. 2009) and following the 454
TNinf (mg N/L)

SOP guidelines provided by Schloss et al. 2011. First, se-

quences were extracted from the raw next-generation se-
quencing (NGS) data. These were then subjected to a quality
53

33

200
600
250
44
47

56
47

screening control in order to eliminate poor quality reads from

analysis. The quality screening control criteria selected se-
HRT hydraulic retention time, SRT solids retention time, TNinf influent total nitrogen, TNeff effluent total nitrogen

quences with the following characteristics: (i) sequences

Technical, environmental, and operational parameters of full-scale bioreactors sampled in the study

SRT (day)

with 250-bp length or more, (ii) sequences with a max-

imum of zero ambiguous bases, (iii) sequences with a
0.60

0.27
20
27

14
–

–
–
–

maximum of eight homopolymers, and (iv) sequences

that had 35 average quality score or higher as deter-
mined by quality checking using windows of 50 bp.
HRT (hour)

Sequences that remained were then aligned against the

0.41

SiLVA SEED alignment v119. After this, a screening

6.4
1.7
0.7

69
17

27
20
35

was done to eliminate misaligned sequences.

Misalignment was defined as follows: (a) sequences that
started before the primer position and (b) sequences that
CAS (without presettling)
CAS (without presettling)

ended further than the 95 % of total sequences. Then,

CANON (industrial)

sequences were subjected to a preclustering process

CAS (presettling)

CAS (presettling)

CANON (urban)

(Huse et al. 2010) merging sequences within two mis-

Technology

matches. Finally, chimera detection and elimination were

DEMON
A-stage
A-stage

done using the UCHIME v4.2 (Edgar et al. 2011) im-

plemented in mothur v1.34.4.
After processing of sequences, the number of reads for the
samples was between 9579 and 16,039. To conduct a
The Netherlands
The Netherlands
The Netherlands

The Netherlands
The Netherlands
The Netherlands
The Netherlands

The Netherlands

proper ecological analysis, all samples were rarified and

cut to provide subsamples of 9579 sequences. The num-
Country

bers of sequences on each of the tag-454-pyrosequenc-

China

ing samples at each of the postprocess steps are offered

in Table 2.
All subsamples were then separately subjected to a taxo-
nomic affiliation procedure, which involved measurement be-
Amsterdam West

Harnaschpolder

tween sequences by generation of Phylip distance matrices,

Meihua East
Kortenoord

Apeldoorn
Bioreactor

Dokhaven

clustering of these in a 97 % similarity threshold to form

Olburgen
Table 1

Vianen
Breda

operational taxonomic units (OTUs), and taxonomic affilia-

tion of these OTUs using the SiLVA SEED v119 taxonomy.
 Environ Sci Pollut Res

Table 2 Number of reads in tag-454-pyrosequencing samples during the tag-454-pyrosequencing postprocess

Amsterdam West Breda Dokhaven Harnaschpolder Kortenoord Vianen Apeldoorn Meihua East Olburgen

Data extraction 20,372 11,706 23,501 22,202 13,660 13,411 13,223 17,694 15,058
Quality trimming 17,239 10,274 16,057 17,419 10,766 10,594 10,503 14,122 12,100
Alignment screening 16,205 9660 15,258 16,434 10,141 9929 9814 13,312 11,420
Chimera checking 16,039 9242 14,632 15,733 9760 9672 9579 12,920 11,150
Subsample 9242 9242 9242 9242 9242 9242 9242 9242 9242

Finally, all OTUs were merged into a consensus taxonomy
using an 80 % consensus confidence threshold.
To check the diversity coverage of the NGS subsamples,
complexity curves were developed for all subsamples taking
into account the counts of each consensus taxonomy OTU and
calculated using aRarefactWin software.
Phylogenetic analysis of all OTUs
redundancy analysis was done using the software CANOCO
4.5 for Windows with a 499 unconstrained Monte Carlo sim-
ulation under a full permutation model (Gonzalez-Martinez
et al. 2016b). Also, a correlation between the nitrifying genera
Nitrosomonas, Nitrobacter, Nitrospira, and Comammox and
the different bioreactor technologies sampled in this study was
investigated through a network analysis done by Cytoscape
v3.4.0.

A phylogenetic analysis was done over all OTUs from all

subsamples in order to investigate the presence of Bacteria
members taxonomically affiliated with Nitrospira bacteria ca-
pable of complete ammonium oxidation. First, representative
sequences of all OTUs from all subsamples were blasted
against the Nitrospira sp. ENR4 complete genome
LN885086.1 (Daims et al. 2015). All significant matches were
used to construct phylogenetic trees. The phylogenetic trees
also contained representative sequences of several Nitrospira
lineages and groups as shown in Daims et al. (2015) and van
Kessel et al. (2015).
The sequences of each of the trees were separately aligned
using the MUSCLE algorithm (Edgar 2004) in MEGA6 soft-
ware (Tamura et al. 2013). Then, the aligned sequences
were used to calculate Bayesian interference trees using
the MrBayes v3.2 software (van Kessel et al. 2015)
with a GTR substitution model with gamma-distributed
rate variation until deviation was lower than 0.01. The
resulting Bayesian interference trees were then depicted
using TreeGraph 2.8.0-591 beta software (Stöver and
Müller 2010).
Multivariate redundancy analysis and network analysis
The environmental data collected from the bioreactors ana-
lyzed was linked to the relative abundance of genera
conducting ammonium and/or nitrite oxidation by developing
a multivariate redundancy analysis. The environmental data
used was the hydraulic retention time, the solids retention
time, the influent and effluent total nitrogen concentrations,
the dissolved oxygen concentration, and the temperature of
the bioreactor. The genera used were Nitrosomonas,
Nitrobacter, Nitrospira, and Comammox. The multivariate
Results and discussion
Complexity curves of subsamples
The complexity curves of all tag-454-pyrosequencing sub-
samples are shown in Fig. 1. It can be seen that diversity
was higher for the subsamples coming from AS and lower
for samples from A-stage and ANR systems. In both cases,
the samples reached a plateau with an increasing number of
reads.
Bacterial community structure of the bioreactors analyzed
The bacterial community structure of the bioreactors ana-
lyzed showed significant differences among them. In the
case of the AS, the CAS with presettling presented a high
diversity and shared several genera at >0.5 % relative abun-
dance, such as Candidatus Accumulibacter, Candidatus
Competibacter, Haliscomenobacter, Thiococcus, Saprospira,
or Dokdonella. For the case of the CAS without presettling,
there was a high diversity with Haliscomenobacter,
Saprospira, Thiococcus, Candidatus Accumulibacter,
Rubrivivax, Dechloromonas, or Byssovorax. In this sense,
the bacterial communities of the CAS with and without
presettling shared different genera, such as
Haliscomenobacter, Saprospira, Candidatus Competibacter,
or Thiococcus. The four CAS sampled were operated as en-
hanced biological phosphorous removal systems (EBPR), and
therefore, their bacterial communities shared resemblances in
terms of dominant genera in spite of the differences in the
presettling technology. On the contrary, the bacterial commu-
nities of the A-stage systems were clearly dominated by a few
Environ Sci Pollut Res
Fig. 1 Complexity curves of tag-
454-pyrosequencing subsamples
genera with >10–30 % relative abundance, such as
Hydrogenophaga and two Sphingobacteriales clones in the
Breda A-stage and Flavobacterium and Zoogloea in the
Dokhaven A-stage. These two bioreactors shared some genera
at >0.5 % relative abundance than the CAS systems, such as
Acidovorax, Hydrogenophaga, Zoogloea, Cloacibacterium,
and Bacteroides. In this way, the technological differences
between the A-stage and CAS systems were reflected in their
bacterial community structure. On the other hand, the bacterial
community structure of the ANR was very different than that
of the AS, showing that the shared genera at >0.5 % relative
abundance were related to Nitrosomonas, different members
of the Anaerolineaceae family, and Candidatus Brocadiales
bacteria. The domination of the systems belonged to
Leptolinea.
Phylogenetic analysis of all OTUs
Branches of interest of the Bayesian interference tree clado-
grams of comammox candidate sequences for the AS subsam-
ples and ANR subsamples compared with Daims et al. 2015
and van Kessel et al. 2015 phylogenetics are shown in Figs. 2,
3, 4, and 5. The complete Bayesian interference tree clado-
grams are provided in the supplementary material as Fig. S1,
Fig. S2, Fig. S3, and Fig. S4.
Following the phylogeny provided by Daims et al. 2015,
the majority of OTUs found in the AS systems showed a
phylogenetic affiliation with the HM485591 Nitrospira en-
richment culture clone Ga3a, which is related to the lineage
6 of Nitrospira according to Daims et al. 2015. Four OTUs
were related to FP929003 Candidatus Nitrospira defluvii in
the lineage 1 of Nitrospira; other four were related to
JN868922 Uncultured bacterium clone MC48, which is close-
ly related to Nitrospira lenta (Daims et al. 2015); the OTU
IBTM6NN03HEGXV was the only one related to Y14644
Nitrospira sp. 16S rRNA gene strain GC86, which is very
close to known comammox MBR Nitrospira bin 1.
Following the phylogeny provided by van Kessel et al.
2015, only the OTU IBTM6NN03HEGXV was found in the
Nitrospira sp. 1 group, being related to HM438556
Uncultured Nitrospira sp. clone T502G8. Other four OTUs
were found in the Ntspa476 which were not related to
Nitrospira sp.1 or Nitrospira sp. 2 but to KP054177
Uncultured bacterium clone MN 23. All the other OTUs were
affiliated to other Nitrospira groups.
The OTU IBTM6NN03HEGXV, affiliated to Y14644
Nitrospira sp. 16S rRNA gene strain GC86 in the Nitrospira
lineage 2 and to HM438556 Uncultured Nitrospira sp. clone
T502G8 in the Nitrospira sp.1 group, was found in the Vianen
AS and counted only eight sequences among the 9242 of the
subsample, which represents a 0.08 % of total relative abun-
dance. The relative abundance of other common ammonium-
oxidizing bacteria in AS systems, such as Nitrosomonas, was
o f 0. 40 % , fiv e tim es h igh er t ha n tha t of OTU
IBTM6NN03HEGXV. As well, Nitrosomonas was present in
the Amsterdam West subsample at 1.24 % relative abundance,
at 0.43 % in the Houtrust subsample, and at 0.53 % in the
Kortenoord subsample, where comammox were not found.
Therefore, it is possible that comammox bacteria cannot compete
with Nitrosomonas as ammonium-oxidizing bacteria in full-scale
CAS systems with and without presettling studied. On the other
hand, a consensus taxonomy OTU analysis of the Vianen tag-
454-pyrosequencing subsample showed that the relative abun-
dance of Nitrospira genus was of 0.64 %. Also, Nitrospira were
found at 1.30 % relative abundance at Amsterdam West, at
0.93 % at Houtrust, and at 0.55 % at Kortenoord, but no
comammox were found among Nitrospira OTUs in these sub-
samples. In this sense, comammox could also not compete with
other strains of Nitrospira for nitrite oxidation in the full-scale
CAS with and without presettling analyzed in this study. In the
A-stage subsamples, Nitrosomonas, Nitrospira, and other puta-
tive ammonium oxidizers were not found.

Fig. 2 Bayesian interference tree
cladogram of comammox
candidates in the ANR systems
compared to Daims et al. 2015
phylogeny
The majority of OTUs found in the ANR systems
showed a relation with EU084879 Candidatus Nitrospira
bockiana and thus were located in the lineage 5 of
Nitrospira as following the phylogeny provided by
Daims et al. 2015. Only one of the OTUs found in these
systems was related to FP929003 Candidatus Nitrospira
defluvii in the lineage 1 of Nitrospira but none with the
lineage 2. Following the taxonomy of van Kessel et al.
2015, none of the OTUs were related to either Nitrospira
sp. 1 or Nitrospira sp. 2. On the contrary, a 0.53 % rela-
tive abundance of Nitrosomonas, which has been reported
as the most important ammonium-oxidizing bacteria in
Environ Sci Pollut Res
full-scale ANR systems (Gonzalez-Martinez et al.
2015a), was found at Apeldoorn full-scale bioreactor,
0.30 % at Meihua East, and 0.32 % at Olburgen.
Therefore, results suggested that comammox cannot compete
with ammonium-oxidizing bacteria Nitrosomonas in full-
scale ANR systems.
The presence of comammox bacteria in environmental sys-
tems, such as drinking water system (Pinto et al. 2015),
has been detected by shotgun DNA sequencing includ-
ing 16S rRNA and amoA gene, among others. These
results suggested that a suite of genes of comammox
bacteria was present in a drinking water system and
Environ Sci Pollut Res

Fig. 3 Bayesian interference tree

cladogram of comammox
candidates in the ANR systems
compared to van Kessel et al.
2015 phylogeny

therefore that these microorganisms could play an im-
portant role in the nitrogen cycle in engineered systems.
In the study presented here, environmental samples have
been analyzed for the presence of comammox using
next-generation sequencing tools for the first time. The
results indicate that Nitrospira-like comammox bacteria
are not ubiquitous in wastewater treatment systems and
that when they are present, their relative abundance in
terms of 16S rRNA gene copies are low compared with
other ammonium-oxidizing bacterial genera such as

Fig. 4 Bayesian interference tree
cladogram of comammox
candidates in the ANR systems
compared to Daims et al. 2015
phylogeny
Nitrosomonas. These results are in accordance with
those obtained in other activated sludge systems, show-
ing a very low relative abundance of 16S rRNA genes
of comammox-like Nitrospira with respect to other phy-
lotypes (Chao et al. 2016).
Linkage of ammonium and/or nitrite-oxidizing genera
with environmental conditions and bioreactor technology
in the bioreactors analyzed
The plot of the multivariate redundancy analysis con-
structed is shown in Fig. 6. All environmental parame-
ters were related except for the hydraulic retention time,
which seemed to be independent of all others.
Temperature and total nitrogen concentrations were pos-
itively correlated and related to short solids retention
time and low dissolved oxygen concentrations. In this
sense, these results suggested that the elimination of
nitrogen in the bioreactors did not depend on the
Environ Sci Pollut Res
hydraulic retention time, but on the temperature, dissolved
oxygen, and solids retention time. The main ammonium-
oxidizing bacteria found was Nitrosomonas, which showed a
high correlation with the hydraulic retention time. On the oth-
er hand, the major nitrite-oxidizing genus, Nitrospira, was
positively correlated with the dissolved oxygen concentration
and solids retention time. The presence of comammox only at
the Vianen AS may signify that the conditions of low temper-
ature, low influent nitrogen, and high dissolved oxygen favor
comammox growth. The nearly absence of the genus
Nitrobacter and of comammox bacteria in all samples ana-
lyzed was shown by an independence of these phylotypes
from all of the variables considered.
The network analysis linking bioreactor technology and
presence of nitrifying genera is shown in Fig. 7. It was ob-
served that the A-stage bioreactors have no presence of any of
the nitrifying genera. Also, the network demonstrated that
Nitrosomonas genus was present in all other bioreactors re-
gardless of the bioreactor technology, which suggested its
Environ Sci Pollut Res

Fig. 5 Bayesian interference tree

cladogram of comammox
candidates in the ANR systems
compared to van Kessel et al.
2015 phylogeny

Fig. 6 Multivariate redundancy analysis plot of nitrifying genera and
environmental conditions in the bioreactors analyzed. Samples are
depicted as blue circles, genera as green triangles, and environmental
conditions as arrows
ubiquitousness and its importance as ammonium oxidizer in
wastewater treatment systems (Nielsen et al. 2010; Gonzalez-
Martinez et al. 2015b). All CAS accounted for the presence of
Nitrospira, but only two of the samples showed presence of
Nitrobacter. These results suggested that Nitrospira could be
the dominant nitrite-oxidizing bacteria in EBPR systems, as
reported previously (Nielsen et al. 2010). Among the ANR
bioreactors, only the CANON fed with urban anaerobic diges-
tion supernatant showed presence of the nitrite-oxidizing bac-
teria Nitrospira, showing that nitrite oxidizers do not develop
to high relative abundances in these systems (Gonzalez-
Martinez et al. 2015b). Finally, comammox were only found
in the CAS without presettling, which suggested its low
Fig. 7 Network analysis of
nitrifying bacterial genera and
bioreactor technologies.
Nitrifying bacterial genera are
depicted as yellow circles, and
bioreactor technologies are
depicted as orange circles
Environ Sci Pollut Res
importance with respect to other nitrifying genera in full-
scale wastewater treatment systems. This result was also sup-
ported by analysis of presence of comammox bacteria in
a full-scale activated sludge system in Hong Kong
(Chao et al. 2016).
Conclusions
The phylogenetic analysis of full-scale wastewater treat-
ment bioreactors using tag-454-pyrosequencing tech-
niques showed that Nitrospira-related comammox bacte-
ria were not present in all systems. Only one OTU
phylogenetically affiliated with a comammox bacteria
was found among the nine systems analyzed, which
suggested the non-ubiquitous presence of comammox
bacteria in full-scale wastewater treatment systems. Its
low relative abundance compared to typical
ammonium-oxidizing bacteria Nitrosomonas suggested
that this species may outcompete comammox bacteria
in full-scale wastewater treatment systems. As well, the
relative abundance of non-comammox Nitrospira over
comammox Nitrospira suggested that comammox strains
of Nitrospira could be outcompeted by non-comammox
strains of Nitrospira in terms of nitrite oxidation in full-
scale wastewater treatment systems. A multivariate re-
dundancy analysis showed that the main oxidizing ge-
nus Nitrosomonas was related to the hydraulic relation
time of the bioreactors, and the main nitrite-oxidizing
genus Nitrospira was related to high dissolved oxygen
concentrations and high solids retention time. Future
research should be done in order to investigate the pres-
ence of comammox bacteria in natural and engineered
systems, using identification tools like those presented
in this study and others, such as identification through
amoA gene.
Environ Sci Pollut Res
Acknowledgments The authors would like to acknowledge the support
given by the Department of Civil and Environmental Engineering of the
University of Aalto, Finland; the Institute of Water Research of the
University of Granada, Spain; and the Department of Biotechnology of
the Technical University of Delft, the Netherlands; as well, the authors
would like to acknowledge the support provided by the personnel of the
wastewater treatment plants of Amsterdam West, Breda, Dokhaven,
Harnaschpolder, Kortenoord, Vianen, Apeldoorn, Meihua East, and
Olburgen.
Compliance with ethical standards
Conflicts of interest The authors declare that they have no conflict of
interest.
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