Bioresource Technology 211 (2016) 273279

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Suspended sludge and biofilm shaped different anammox communities

in two pilot-scale one-stage anammox reactors
Bingyu Zheng a, Liang Zhang b, Jianhua Guo a, Shujun Zhang b, Anming Yang b, Yongzhen Peng a,
a
b

Key Laboratory of Beijing for Water Quality Science and Water Environment Recovery Engineering, Engineering Research Center of Beijing, Beijing, China
Beijing Drainage Group Co. Ltd (BDG), Beijing, China

h i g h l i g h t s

One-stage partial nitritation and anammox was realized in two pilot-scale IFAS.

Anammox abundance reached 87.8% and 73.3% of the total bacteria in biofilm.

Brocadia and Kuenenia tended to live in biofilm and suspended sludge, respectively.

Niche difference is vital to govern the distribution of anammox bacteria.

a r t i c l e

i n f o

Article history:
Received 22 January 2016
Received in revised form 3 March 2016
Accepted 8 March 2016
Available online 12 March 2016
Keywords:
Anammox bacteria
Autotrophic nitrogen removal
Biofilm
Flocculants

a b s t r a c t
The abundance and diversity of anammox bacteria was investigated in two pilot-scale integrated fixedfilm activated sludge (IFAS) reactors treating high ammonium wastewater. Reactor A was inoculated with
nitrifying sludge, while Reactor B was inoculated with suspended anammox sludge with the dominant
anammox bacteria of Candidatus Kuenenia. After 180 days operation, the predominate anammox bacteria was Candidatus Brocadia (65%) in the biofilm, while Candidatus Kuenenia (86%) outcompeted with
other anammox bacteria in suspended sludge in Reactor A. Candidatus Kuenenia were dominated in suspended sludge through the entire experiment in Reactor B. In contrast, the predominated species shifted
from Candidatus Kuenenia (89%) into Candidatus Brocadia (66%) in the biofilm of Reactor B. This study
indicated that Candidatus Brocadia preferred to grow in the biofilm, while Candidatus Kuenenia would
dominant over other anammox bacteria in the suspended sludge. Further studies are required to identify
the internal factors affecting the distribution of anammox bacteria.
2016 Elsevier Ltd. All rights reserved.

1. Introduction
The discovery of anaerobic ammonium oxidation (anammox)
bacteria has not only changed the understanding of the global
nitrogen cycle, but also provided a promising solution to
autotrophically remove nitrogen from industrial and domestic
wastewater (Kartal et al., 2010; Guo et al., 2013). As a sustainable
biological nitrogen removal process, anammox process has exhibited many advantages, including saving aeration consumption, no
requirement of organic carbon and producing much less sludge
(Ma et al., 2016). So far, various configurations have been developed to achieve autotrophic nitrogen removal via anammox, which
can be divided into two categories, two-stage and one-stage anam Corresponding author at: Key Laboratory of Beijing for Water, Quality Science
and Water Environment Recovery Engineering, Engineering, Research Center of
Beijing, Beijing University of Technology, Beijing 100124, China.
E-mail address: pyz@bjut.edu.cn (Y. Peng).
http://dx.doi.org/10.1016/j.biortech.2016.03.049
0960-8524/ 2016 Elsevier Ltd. All rights reserved.

mox. For the two-stage anammox process, partial nitritation is first

achieved by enriching ammonia oxidation bacteria (AOB) in an aerobic reactor. Afterward, anammox bacteria will convert both
ammonium and nitrite from the first stage into dinitrogen gas
(van der Star et al., 2007; Wett, 2007). Differently, for one-stage
anammox process, both partial nitritation and anammox are
simultaneously achieved in the same reactor by controlling low
dissolved oxygen (DO) (Jetten et al., 2001). For one-stage anammox
process, one of the major challenges are to sustain an efficient synergy between AOB and anammox bacteria to enhance total nitrogen (TN) removal rate, because improper DO concentrations
would inhibit the activity of anammox bacteria and favored the
growth of nitrate oxidation bacteria (NOB) (Jetten et al., 2009).
As a novel technology, the integrated fixed-film activated
sludge system (IFAS) has been proposed to incorporate an attached
growth media within the suspended growth reactors (Su and
Ouyang, 1996). IFAS has been applied to achieve one-stage anammox process. Partial nitritation and anammox reactions can

274

B. Zheng et al. / Bioresource Technology 211 (2016) 273279

proceed spatially in the IFAS reactor, in which AOB grow in suspended aggregates to consume DO, while anammox bacteria attach
to the carrier to form biofilm to achieve a high nitrogen removal
rate. Although several studies regarding IFAS process have been
conducted to optimize operational performance (Regmi et al.,
2011; Malovanyy et al., 2015; Zhang et al., 2015a), the anammox
bacteria community structure of different sludge aggregates in
IFAS is still not fully understood. It is assumed that different sludge
aggregates would shape different dominant anammox bacteria.
The investigation of microbial communities and anammox diversity in the IFAS reactor would be beneficial to understand the effect
of environments on the distribution of anammox bacteria.
The objective of this study was to determine if the dominant
anammox bacteria would be affected by sludge aggregates. To this
aim, the abundance and diversity of anammox bacteria in the suspended activated sludge and biofilm collected from two pilot-scale
IFAS reactors were investigated by phylogenetic analysis and realtime quantitative polymerase chain reaction (PCR).

Concentrated sludge was returned to the inlet of the reactor with

a sludge return ratio of 200300%. Cubic sponge polyesters were
used as carriers for the biofilm attachment. The DO of the reactors
was controlled at 0.51.5 mg/L through an on/off aeration controller. Temperature in both reactors were controlled in the range
of 2429 C.
The mixture of synthetic wastewater contained (NH4)2SO4 and
NaHCO3 and actual effluent from primary clarifier of Gaobeidian
wastewater treatment plant (WWTP, Beijing, China) was used as
the influent. The variation range of influent ammonia and COD
concentration were set as 173920 mg/L and 108331 mg/L,
respectively. The influent ammonium loading rate (ALR) was
increased from 0.05 kg N/(m3 d) to 0.7 kg N/(m3 d) and the hydraulic retention time (HRT) was 0.91.0 d in Reactor A. While in Reactor B the ALR and HRT were set as 0.40.55 kg N/(m3 d) and 1.0 d,
respectively.

2.2. Seeding inocula

2. Methods
2.1. Reactor set-up and wastewater
Two pilot-scale IFAS reactors (Reactor A and Reactor B) with the
same working volume (12 m3) were adopted in this study (Fig. 1).
Both bioreactors were made of steel and divided into five equal
compartments (2.4 m3). The first compartment was set as the
anoxic phase, which was continually stirred by a mechanical
mixer. The other four compartments were aerated by an air compressor through fine diffusers installed at the bottom of the bioreactor. The settler was cylindrical with a working volume of 2 m3.

Reactor A was inoculated with returned activated sludge in

Gaobeidian WWTP which contained little anammox bacteria
(Wang et al., 2015). The initial mixed liquor suspended solids
(MLSS) was 3500 mg/L. The carrier possessing no biofilm on their
surface was fixed in the aerobic zones, which accounted for a volume of 15% of the total reactor.
Reactor B was inoculated with the suspended sludge from Reactor A. In addition, the carriers with mature anammox biofilm (Day
180) of Reactor A were collected as inoculation, which accounted
for 3% of the volume of Reactor B. Fresh carriers without any biomass were also added to reach a volume ratio of 15% of Reactor B.

Fig. 1. Schematic diagram of IFAS reactor (A: anoxic zone, O: oxic zone).

275

B. Zheng et al. / Bioresource Technology 211 (2016) 273279

2.3. Chemical analysis methods

Samples were taken from these two reactors at appropriate

intervals (23 times per weeks). NH+4-N, NO
2 -N, NO3 -N, COD, MLSS
and mixed liquor volatile suspended solids (MLVSS) concentrations
were analyzed according to the standard methods (APHA, 2005).
TN was analyzed using a TN/TOC analyzer (MultiN/C 3100, Jena,
Germany). The suspended activated sludge (AS) or biofilm samples
were centrifuged at 3000 rpm for 2 min and the supernatants were
filtered by 0.45 lm acetate cellulose membranes before analysis.
Temperature, DO and pH values were monitored using a WTW
pH/oxi340i meter (WTW Company, Germany).
2.4. Sampling, DNA extraction and PCR amplification
For Reactor A, the biofilm from the carriers which were fixed in
the reactor at the beginning and suspended sludge samples were
collected during the steady period (Day 180) for DNA extractions.
For Reactor B, the biofilm formed on blank carriers and suspended
sludge samples in the successful set-up phase (Day 35), and longterm stable operation phase (Day 110) were collected for DNA
extractions.
Samples collected from the biofilm were immediately centrifuged at 10,000 rpm for 20 min and the supernatants were discarded. Sediments of 250 mg were used for DNA extraction with
the CTAB lytic solution and Proteinase K. DNA solutions with a volume of 100 lL were obtained, from which 2 lL was used for concentration determination by a Nanodrop 1000 Spectrophotometer
(Nanodrop, USA).
The 16S rRNA gene of anammox bacteria (amx) was amplified
by the primer set Amx368f (50 -TTCGCAATGCCCGAAAG-30 ) and
Amx820r (50 -AAAACCCCTCTACTTAGTGCCC-30 ). The PCR assays
were conducted in 25 lL volume reactions using an ABI PCR System 9700 (ABI, USA). The PCR mixture consisted of 1 rTaq buffer,
5 dNTP, 0.625 U rTaq (Takara, Japan), 400 nM each primer, 0.5 mg/
mL BSA, and 1 lL DNA. Temperature was set at 95 C for 5 min, followed by 35 cycles of 30 s at 95 C, 30 s at different annealing temperatures and extension at 72 C for 45 s, and finished with a final
extension step at 72 C for 7 min. The PCR products were checked
by electrophoresis on a 1% (weight/volume) agarose gel in 1 TBE
buffer. The positive amplicons were assured by cloning and
sequencing.
2.5. Cloning and sequencing of 16S rRNA gene and phylogenetic
analysis
Cloning of the 16S rRNA genes of anammox bacteria was realized by utilizing the pGEMT-easy (Promega, Madison, WI). Fragment of 478 bp was obtained for anammox bacteria. The PCR
products purified by the E.Z.N.A Gel Extraction Kit (Omega, USA)
were ligated by plasmid pMD-18T (Takara, Dalian, China) and
transformed into competent Escherichia coli TOP10 (Tiangen,
China). Positive clones of the amx gene were verified by PCR with
the plasmid specific primer set M13-47f/M13-48r, which were
sequenced with an ABI3300 sequencing machine (ABI, USA) for
each sample. Sequences were subjected to homology analysis
using the software Lasergene (DNAStar, USA), and those with identity higher than 97% were considered as one OTU (operational taxonomy unit). One representative sequence from each OTU,
together with the most similar GenBank sequences searched online
using the NCBI BLAST tool were used to construct the neighborjoining phylogenetic trees of anammox bacteria with MEGA 4.1
(http://megasoftware.net/). Bootstrap analysis with 1000 replicates was used to evaluate the significance of the nodes. The
sequences of anammox bacteria-specific 16S rRNA gene obtained

in this study were deposited in the NCBI GenBank under Accession

No. JX548510 to JX548525 and KM008573.1 to KM008601.1.
2.6. Quantification of anammox bacteria abundance with real-time
qPCR
In order to investigate the abundance of anammox bacteria in
different sludge aggregates, the amx and universal bacterial 16S
rRNA genes for the total bacteria were quantified for all collected
samples using SYBR-Green real-time PCR. Standard plasmids carrying the target genes were obtained by TA clone and extracted using
a TIANpure Mini Plasmid kit (Tiangen, China) (Liu et al., 2012).
Concentrations of the standard plasmids (ng/lL) were determined
with the Nanodrop ND-1000 (Nanodrop, USA). Their copy concentrations (copies/lL) were then calculated according to the following equation (Pei et al., 2006):

copy concentrationcopies=lL

DNA mass concentrationng=lL

DNA molecular weightg=mol

 6:02  1023  109

The 25 lL real-time PCR reactions typically contained 1 Sybr
Green I, 1 Dye (Takara), 200 nM each primer, 0.5 mg/mL BSA
and 2 lL DNA templates. Real-time PCR was run on an ABI7300
apparatus (ABI, USA) by the following program: 95 C for 30 s, 40
cycles including: 95 C for 10 s, annealing temperature for 15 s,
72 C for 15 s and 78 C for 26 s to collect the fluorescent signals,
and the melting process automatically generated by the ABI7300
software. Triplicate assays were performed for the decimally
diluted standard plasmids, properly diluted samples and negative
controls. The specificity was assured by the melting curves and
agarose gel electrophoresis. Gene copies were normalized to both
the DNA concentrations and the bacterial 16S rRNA gene copies
to minimize the variance caused by different bacterial abundance,
DNA extraction and quantification efficiencies.
3. Results and discussion
3.1. Performance of two pilot-scale IFAS reactors
The operational performance of the nitritation-anammox process in the IFAS Reactor A has been reported in our previous reports
(Zhang et al., 2015b). The experimental period lasted for about
6 months. The start-up time of Reactor A was relatively short
(about 100 days). The influent TN concentration increased from
300 to 600 mg/L during the start-up phase, while the pH of the system was kept in the range of 8.158.38. The effluent TN decreased
progressively as the experiment went on. After the successful
achievement of nitritation, TN removal efficiency increased continuously to a maximum of 70%, with a nitrogen removal rate of
0.44 kg N/(m3 d).
Reactor B was operated for about 117 days, including phase I
(Day 017) for starting up anammox process and phase II (Day
18117) for stable operation. Due to the inoculation of anammox
activated sludge and mature biofilm, the start-up time of anammox in Reactor B reduced significantly compared to Reactor A. In
phase I, the influent NH+4-N was controlled to be about 420 mg/L.
At the beginning the effluent NH+4-N concentration showed a continuous increase trend, and the nitrogen removal performance was
gradually improved in the next two weeks despite of the increasing
of ammonium loading rate. Till the end of phase I, a nitrogen
removal efficiency of 80% was obtained. In phase II, the influent
TN concentration was further elevated to more than 500 mg/L.
During this phase, although some sharp fluctuations of influent
NH+4-N concentrations caused a transient decrease of nitrogen
removal efficiency, the anammox performance was maintained.

276

B. Zheng et al. / Bioresource Technology 211 (2016) 273279

Effluent TN concentration were lower than 10 mg/L and the average nitrogen removal efficiency was 85%. Furthermore, the TN
removal rate increased progressively and reached 1.2 kg N/(m3 d)
after nearly 3-months operation (Fig. 2).
3.2. Abundance of anammox bacteria
Genetic specific primer targeting anammox bacteria was used
to quantify their abundances in both reactors (Table 1). In Reactor
A, the abundance of anammox bacteria steadily increased, which
reached from 0 to 8.25  105 copies/ng DNA on Day 180 in biofilm.
The surfaces of the biofilm carriers were white at the beginning
and gradually turned to reddish- the typical color of anammox bacteria aggregates (Ma et al., 2011) (Fig. S1), which indirectly indicated the enrichment of quite abundant anammox bacteria in the
biofilm. Meanwhile, qPCR demonstrated that the relative abundance of anammox bacteria reached 87.8% of the total bacteria in
biofilm, implying anammox bacteria to be dominant in the end
of the experiment.
In Reactor B, the abundances of anammox bacteria in the suspended sludge steadily increased throughout the whole experiment, which reached from 1.68  104 copies/ng DNA on Day 1 to
5.91  104 copies/ng DNA on Day 117, accounting for 8.4% of the
total bacteria. The relative abundance of anammox bacteria in biofilm was 73.3%, which was much higher than that in suspended
sludge. In addition, nearly all the carriers turned from white to
brick-red gradually (Fig. S2).
3.3. Community structure and biodiversity of anammox bacteria
The communities of anammox bacteria in suspended sludge
and biofilm in Reactor A were investigated by establishing a gene

library. The sequences of anammox bacteria were clustered into

4 OTUs (Fig. 3), suggesting limited biodiversity of anammox bacteria in this reactor. Intriguing, phylogenetic distributions of anammox bacteria were significantly different between suspended
sludge and biofilm. Candidatus Kuenenia (85.7%) was the predominant anammox bacteria in the suspended sludge compared with
Candidatus Brocadia accounting for 14.3%. In contrast, Candidatus
Brocadia (64.5%) outcompeted with Candidatus Kuenenia
(29.0%) in the biofilm. In addition, other anammox bacteria species,
such as Candidatus Jettenia asiatica, Candidatus Anammoxoglobus
propionicus and Candidatus Scalindua brodae, were not found in
both suspended sludge and biofilm in Reactor A.
In Reactor B, 198 positive clones from the 16S rRNA gene clone
library of the 4 samples in 2 phases were randomly selected and
successfully sequenced. With 99% of the phylogenetic tree branch
in the same sequence as the near point, the statistics of biodiversity index including OTUs, Shannon index, and chao1 in 4 libraries
are shown in Table S1. On Day 117, the OUT number and Shannon
index in biofilm were 7 and 1.33, respectively, which were relatively higher than that in suspended sludge (6 and 0.50). The
results indicated the biofilm could harbor much more diverse of
anammox bacteria compared to suspended sludge.
Neighbor-joining phylogenetic trees of anammox bacteria
based on their 16S rRNA genes were constructed as shown in
Fig. 4. Reactor B was mainly inoculated by suspended activated
sludge abundant with Candidatus Kuenenia. In Reactor B, Candidatus Kuenenia was the dominant species in suspended sludge during the whole experiment. On Day 35, more than 86% of the
anammox 16S rRNA gene sequences had a high similarity with
Candidatus Kuenenia. On Day 117, over 91% of the anammox
16S rRNA gene sequences had a high similarity with Candidatus
Kuenenia while only 9% was similar to Candidatus Brocadia. It

900
Inf NH 3 -N
Eff NO 2 -N

800

600
500
400
300
200
100

100

removal efficiency
Inf TN
Eff TN

1400

90

Total nitrogen(mg/L)

1200

80

1000

70

Phase II

800

60

Phase I

50

600

40
400

30

200
0
0

20
20

40

60

80

10 0

10
117

Time (d)
Fig. 2. Nitrogen compounds in influent and effluent and removal efficiency of IFAS Reactor B.

removal efficiency(%)

Nitrogen Compounds

700

Eff NH 3 -N
Eff NO 3 -N

277

B. Zheng et al. / Bioresource Technology 211 (2016) 273279

Table 1
Abundance of anammox bacteria in Reactors A and B.
Reactor
A

Sludge

Copy number of anammox bacteria [copies ng DNA1]

4

Copy number of total bacteria [copies ng DNA1]

5

Relative abundance [%]

Suspended
Sludge

1.68  10
(2.03  102)a

6.47  10
(7.08  104)

2.59

Biofilm

6.82  105
(1.01  105)

8.25  105
(6.34  104)

87.8

Suspended
Sludge

5.91  104
(6.57  103)

7.02  105
(1.97  104)

8.4

Biofilm

1.23  106
(1.13  105)

1.71  106
(1.29  104)

73.3

Values in parentheses represent standard deviation.

OTU1 (Suspended sludge: 24/28; Biofilm:9/31)

Candidatus Kuenenia stuttgartiensis (AF375995)
100 Candidatus Kuenenia clone (HM769655)
OTU2 (Suspended sludge: 4/28; Biofilm:20/31)
94
66
Candidatus Brocadia fulgida (DQ459989)
87
58
Candidatus Brocadia clone (HM769652)
OTU3 (Biolilm:1/31)
OTU4 (Biofilm:1/31)
76
Uncultured Planctomycetales (HM537218)
Candidatus Jettenia asiatica (DQ301513)
Candidatus Anammoxoglobus propionicus (DQ317601)
94
Candidatus Scalindua wagneri (AY254882)
Candidatus
Scalindua sorokinii(AY257181)
100
100
Candidatus Scalindua brodae (AY254883)
0.01
50

Fig. 3. Neighbor-joining phylogenetic tree of representative amx gene sequences of anammox bacteria in suspended sludge and biofilm in Reactor A.

should be noted that Candidatus Brocadia was not observed at the

beginning but its proportion increased through the experiment
period.
The phylogenetic analysis results of the biofilm samples
showed different communities with the suspended sludge sample
in Reactor B (Table 2). During the experimental period, the predominant anammox bacteria in biofilm were changed from Candidatus Kuenenia to Candidatus Brocadia. On Day 35, 89% of the
clone sequences in this reactor were similar with Candidatus Kuenenia, while the residual 11% of the sequences were more similar
to Candidatus Brocadia. However, the shift of dominant species
obviously occurred in the stable operation phase of the reactor
(Day 117). The results showed that 26% of the clone sequences
were with a high similarity with Candidatus Kuenenia, while
66% of the sequences were more similar to Candidatus Brocadia.
3.4. Factors influencing the distribution of anammox bacteria in the
two reactors
In this study, the one-stage anammox process was successfully
achieved in two pilot-scale IFAS reactors. When seeded with common activated sludge from WWTP, the nitritation-anammox process could be established in 100 days in Reactor A, indicating a
possible solution to establish the anammox process without anammox bacteria-rich inoculums (Wett, 2006). In Reactor B that inoculated by enriched anammox bacteria inoculums, the start-up time
could be reduced to one month (Fig. 2). In addition, the nitrogen
removal efficiency has reached 85% in Reactor B, which was higher
than that obtained in moving bed biofilm reactor (Veuillet et al.,
2014). Overall, IFAS reactor provides an alternative to apply onestage anammox process to treat high ammonium wastewater.
The abundance of anammox bacteria was higher in the biofilm
than that in the suspended sludge in both pilot-scale IFAS reactors,
which was consistent with previous reports (van der Star et al.,

2008). The phylogenetic analysis showed that the dominant species mainly included Candidatus Kuenenia and Candidatus Brocadia in IFAS reactor. However, the anammox bacteria communities
in both suspended sludge and biofilm were obviously different. In
Reactor A and B, Candidatus Kuenenia was always the predominant anammox bacteria in suspended sludge, while Candidatus
Brocadia outcompeted with other anammox bacteria in the biofilm. Moreover, the shift of predominant species in biofilm was
observed in Reactor B, which strongly indicated that Candidatus
Brocadia was more likely to be enriched in the biofilm.
In our experiments there was only one kind of anammox bacteria dominated in one habitat. It was also indicated that niche difference might play an important role on the competition among
anammox species. Tsushima et al. (2007) found that the main functional anammox bacteria were Candidatus Brocadia in a two-stage
biofilm anammox reactor. Egli et al. (2001) used a rotating biological contactor (RBC) treating ammonium-rich leachate resulting in
90% accumulative rate of anammox bacteria, most of which were
Candidatus Kuenenia. However, unlike the Candidatus Scalindua
which can survive in high salinity water (Kindaichi et al., 2011)
and Candidatus Anammoxoglobus propionicus that tends to grow
under relatively high propionate concentration condition (Hu et al.,
2010), there is no obvious environmental characteristics for cultivating Candidatus Brocadia and Candidatus Kuenenia.
The growth rate of anammox bacteria is different in certain conditions, which might determine the predominated genus of anammox bacteria. It has been reported that the doubling time of
Candidatus Brocadia (7 days) was shorter than that of Candidatus
Kuenenia (811 days) (Kartal et al., 2012). In our IFAS, biofilm
provided long sludge retention time and a better protection from
oxygen inhibition, which could enhance the growth of Candidatus
Brocadia. In addition, anammox bacteria often grow in the internal part of biofilm (Tsushima et al., 2007). Nitrite must struggle
through microorganisms in the outer layer. As a result, anammox

278

B. Zheng et al. / Bioresource Technology 211 (2016) 273279

80 Suspended sludge 1-OTU1(7/52)

Uncultured bacterium clone UASB anammox (JQ307178.1)
63
Candidatus Brocadia fulgida (JQ889388.1)
Candidatus Brocadia sinica (AB565477.1)
Candidatus Brocadia anammoxidans (AF375994.1)
100Suspended
sludge 1-OTU2(45/52)
Candidatus Kuenenia stuttgartiensis (AF375995.1)
100
Candidatus Jettenia asiatica (DQ301513.1)
95

0.01

80 Suspended sludge 2-OTU6(1/48)

53 Uncultured bacterium clone UASB anammox (JQ307178.1)
Suspended sludge 2-OTU2(1/48)
85
Candidatus Brocadia fulgida (JQ889388.1)
41
Candidatus Brocadia sinica (AB565477.1)
99 Candidatus Brocadia anammoxidans (AF375994.1)
Suspended sludge 2-OTU5(1/48)
Suspended sludge 2-OTU4(1/48)
70 100 Suspended sludge 2-OTU3(1/48)
32 Candidatus Kuenenia stuttgartiensis (AF375995.1)
32 Suspended sludge 2-OTU1(43/48)
Candidatus Jettenia asiatica (DQ301513.1)
0.01

99 Biofilm1-OTU2(4/48)
68
Uncultured bacterium clone UASB anammox (JQ307178.1)
89
Biofilm 1-OTU3(1/48)
59
Candidatus Brocadia fulgida (JQ889388.1)
99
Candidatus Brocadia sinica (AB565477.1)
Candidatus Brocadia anammoxidans (AF375994.1)
100 Biofilm 1-OTU4(1/48)
Candidatus Kuenenia stuttgartiensis (AF375995.1)
51 Biofilm 1-OTU1(42/48)
Candidatus Jettenia asiatica (DQ301513.1)
0.01

93
57 Biofilm 2-OTU2(2/50)
35 Uncultured bacterium clone UASB anammox (JQ307178.1)
Biofilm 2-OTU1(25/50)
49
Biofilm 2-OTU4(7/50)
54
Biofilm 2-OTU5(1/50)
Candidatus Brocadia fulgida (JQ889388.1)
56 34
Biofilm 2-OTU7(1/50)
Biofilm 2-OTU6(1/50)
Biofilm 2-OTU3(13/50)
69
Candidatus Kuenenia stuttgartiensis (AF375995.1)
100
Candidatus Brocadia sinica (AB565477.1)
99 Candidatus Brocadia anammoxidans (AF375994.1)
Candidatus Jettenia asiatica (DQ301513.1)

0.01

Fig. 4. Phylogenetic relationships of anammox bacteria 16S rRNA gene in Reactor B.

(a) Phylogenetic tree of anammox bacteria in suspended sludge on Day 35. (b)
Phylogenetic tree of anammox bacteria in suspended sludge on Day 117. (c)
Phylogenetic tree of anammox bacteria in biofilm on Day 35. (d) Phylogenetic tree
of anammox bacteria in biofilm on Day 117.
Table 2
Sample description and statistics among the 16S rRNA gene libraries in Reactor B.
Sampling time(d)

35
120

Suspended sludge

Biofilm

Kueneniaa

Brocadiab

Kuenenia

Brocadia

86%
91%

0c
9%

89%
26%

11%
66%d

c,d

The rest percentage include other genus of anammox bacteria and anammox
bacteria that could not be classified.
a
Clones sequence highly similar to Kuenenia/Clones in library.
b
Clones sequence highly similar to Brocadia/Clones in library.

bacteria in aggregates are less susceptible to nitrite, which favors

the enrichment of Candidatus Brocadia. Candidatus Kuenenia
appeared to be dominant in both reactors in suspended sludge,
which could be due to its high tolerance to nitrite inhibition. It
was reported that Candidatus Kuenenia had a better tolerance

on nitrite (180 mg/L) compared to Candidatus Brocadia (70 mg/

L) (Schmidt et al., 2003). In suspended sludge, high nitrite concentrations gradually eliminated Candidatus Brocadia and resulted in
the predomination of Candidatus Kuenenia.
However, according to van der Star et al. (2008), the switch
from a Kuenenia-dominated culture to a Brocadia- dominated
culture in biofilm occurred when the SRT was controlled at a high
level (about 30 days). It is assumed that the affinity for nitrite of
anammox bacteria caused the switch. The half saturation constant
(Ks) for nitrite of Candidatus Kuenenia (0.23 mmol/L) is much
lower than that of Candidatus Brocadia (5 mmol/L). In this case,
Candidatus Kuenenia in biofilm appeared more competitive due
to its lower Ks, while Candidatus Brocadia became the key player
in suspended sludge. In fact, the obtained results in this study were
not consist with the hypothesis mentioned above. It seemed that
the tolerance on nitrite more significantly affected the anammox
bacteria communities, rather than Ks.
Studies have shown that Candidatus Brocadia prefers to live in
aggregated sludge, in which its cells gather together and secrete
extracellular polymeric substances (EPS) (Vlaeminck et al., 2010).
As for Candidatus Kuenenia and Candidatus Brocadia, the ratio
of polysaccharide to cell protein in EPS were 2.1 0.3 and
1.0 0.2, respectively. Correspondingly, in anaerobic granular
sludge, the ratio appeared to be between 0.17 and 0.5 (Cirpus
et al., 2006). This reveals the fact that Candidatus Brocadia, which
has a lower polysaccharide/protein ratio can adapt itself better to
the aggregated sludge systems. The existence of EPS protein and
the hydrophobic amino acids would be beneficial for forming
anammox bacteria aggregate (Yan et al., 2015; Yin et al., 2015).
The EPS composition of autotrophic bacteria might also play an
important role on the sludge property, which also requires further
research.
The relationships among sludge morphology, EPS amount and
composition and the suitable habitat for anammox bacteria still
lack direct experimental evidence. The key factors should be the
interaction of multiple elements including internal and external
causes. To obtain further explanation of our results, some details
could be improved by: (i) increasing detection of OTUs and running
time to research on the symbiotic relationship between anammox
bacteria and other functional bacteria; (ii) developing further laboratory studies to reveal the influence of niche differences within
anammox bacteria species on their distribution in different sludge
aggregates; (iii) Using advanced technology such as metagenomics
and metatranscriptomics to uncover the genomics properties of
Candidatus Kuenenia, Candidatus Brocadia and other anammox
bacteria.
4. Conclusions
One-stage partial nitritation and anammox was successfully
achieved in two pilot-scale IFAS reactors. The abundance of anammox bacteria in biofilm reached 87.8% and 73.3% of the total bacteria, which were higher than that in suspended sludge in both
reactors. Candidatus Brocadia which has a worse tolerance for
nitrite tended to exist in biofilm, while Candidatus Brocadia which
can endure higher nitrite concentration preferred to thrive in suspended sludge. The results supported that the niche difference to
be the vital factor influencing the distribution of different kinds
of anammox bacteria.
Acknowledgements
This research was supported by Natural Science Foundation of
China (51478013 and 51208009) and the Funding Projects of Beijing Municipal Commission of Education. Jianhua Guo acknowl-

B. Zheng et al. / Bioresource Technology 211 (2016) 273279

edges the support from Specialized Research Fund for the Doctoral
Program of Higher Education (20121103120010).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2016.03.
049.
References
APHA, 2005. Standard methods for the examination for water and wastewater
american public health association,
21th Ed. American Public Health
Association, Washington, DC.
Cirpus, I.E.Y., Geerts, W., Hermans, J.H.M., Op, H.J., Strous, M., Kuenen, J.G., Jetten, M.
S.M., 2006. Challenging protein purification from anammox bacteria. Int. J. Biol.
Macromol. 39, 8894.
Egli, K., Fanger, U., Alvarez, P.J., Siegrist, H., van der Meer, J.R., Zehnder, A.J., 2001.
Enrichment and characterization of an anammox bacterium from a rotating
biological contactor treating ammonium-rich leachate. Arch. Microbiol. 175,
198207.
Guo, J.H., Peng, Y.Z., Wang, S.Y., Ma, B., Ge, S.J., Wang, Z.W., Huang, H.J., Zhang, J.R.,
Zhang, L., 2013. Pathways and organisms involved in ammonia oxidation and
nitrous oxide emission. Crit. Rev. Environ. Sci. Technol. 43, 22132296.
Hu, B.L., Zheng, P., Tang, C.J., Chen, J.W., van der Biezen, E., Zhang, L., Ni, B.J., Jetten,
M.S.M., Yan, J., Yu, H.Q., Kartal, B., 2010. Identification and quantification of
anammox bacteria in eight nitrogen removal reactors. Water Res. 44, 5014
5020.
Jetten, M.S.M., Wagner, M., Fuerst, J., van Loosdrecht, M., Kuenen, G., Strous, M.,
2001. Microbiology and application of the anaerobic ammonium oxidation
(anammox) process. Curr. Opin. Biotechnol. 12, 283288.
Jetten, M.S.M., van Niftrik, L., Strous, M., Kartal, B., Keltjens, J.T., Den, O.P., Camp, H.J.,
2009. Biochemistry and molecular biology of anammox bacteria. Crit. Rev.
Biochem. Mol. Biol. 44, 6584.
Kartal, B., Kuenen, J.G., van Loosdrecht, M.C.M., 2010. Sewage treatment with
anammox. Science 328, 702703.
Kartal, B., van Niftrik, L., Keltjens, J.T., Op, H.J., Jetten, M.S., 2012. Anammox growth
physiology, cell biology, and metabolism. Adv. Microb. Physiol. 60, 211262.
Kindaichi, T., Awata, T., Suzuki, Y., Tanabe, K., Hatamoto, M., Ozaki, N., Ohashi, A.,
2011. Enrichment using an up-flow column reactor and community structure of
marine anammox bacteria from coastal sediment. Microbes Environ. 26, 6773.
Liu, M., Zhang, Y., Yang, M., Tian, Z., Ren, L., Zhang, S., 2012. Abundance and
distribution of tetracycline resistance genes and mobile elements in an
oxytetracycline production wastewater treatment system. Environ. Sci.
Technol. 46, 75517557.
Ma, B., Zhang, S.J., Zhang, L., Yi, P., Wang, J.M., Wang, S.Y., Peng, Y.Z., 2011. The
feasibility of using a two-stage autotrophic nitrogen removal process to
treatsewage. Bioresour. Technol. 102, 83318334.
Ma, B., Wang, S.Y., Cao, S.B., Miao, Y.Y., Jia, F.X., Du, R., Peng, Y.Z., 2016. Biological
nitrogen removal from sewage via anammox: recent advances. Bioresour.
Technol. 200, 981990.

279

Malovanyy, A., Trela, J., Plaza, E., 2015. Mainstream wastewater treatment in
integrated fixed film activated sludge (IFAS) reactor by partial nitritation/
anammox process. Bioresour. Technol. 198, 478487.
Pei, R., Kim, S.C., Carlson, K.H., Pruden, A., 2006. Effect of river landscape on the
sediment concentrations of antibiotics and corresponding antibiotic resistance
genes (ARG). Water Res. 40, 24272435.
Regmi, P., Thomas, W., Schafran, G., Bott, C., Rutherford, B., Waltrip, D., 2011.
Nitrogen removal assessment through nitrification rates and media biofilm
accumulation in an IFAS process demonstration study. Water Res. 45, 6699
6708.
Schmidt, I., Sliekers, O., Schmid, M., Bock, E., Fuerst, J., Kuenen, J.G., Jetten, M.S.M.,
Strous, M., 2003. New concepts of microbial treatment processes for the
nitrogen removal in wastewater. Fems Microbiol. Rev. 27, 481492.
Su, J.L., Ouyang, C.F., 1996. Nutrient removal using a combined process with
activated sludge and fixed biofilm. Water Sci. Technol. 34, 477486.
Tsushima, I., Ogasawara, Y., Kindaichi, T., Satoh, H., Okabe, S., 2007. Development of
high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors. Water
Res. 41, 16231634.
Van der Star, W.R.L., Abma, W.R., Blommers, D., Mulder, J.W., Tokutomi, T., Strous,
M., Picioreanu, C., van Loosdrecht, M.C.M., 2007. Startup of reactors for anoxic
ammonium oxidation: experiences from the first full-scale anammox reactor in
Rotterdam. Water Res. 41, 41494163.
Van der Star, W.R.L., Miclea, A.I., van Dongen, U., Muyzer, G., Picioreanu, C., van
Loosdrecht, M.C.M., 2008. The membrane bioreactor: a novel tool to grow
anammox bacteria as free cells. Biotechnol. Bioeng. 101, 286294.
Veuillet, F., Lacroix, S., Bausseron, A., Gonidec, E., Ochoa, J., Christensson, M.,
Lemaire, R., 2014. Integrated fixed-film activated sludge ANITA (TM) Mox
process - a new perspective for advanced nitrogen removal. Water Sci. Technol.
69, 915922.
Vlaeminck, S.E., Terada, A., Smets, B.F., De Clippeleir, H., Schaubroeck, T., Bolca, S.,
Demeestere, L., Mast, J., Boon, N., Carballa, M., Verstraete, W., 2010. Aggregate
size and architecture determine microbial activity balance for one-stage partial
nitritation and anammox. Appl. Environ. Microbiol. 76, 900909.
Wang, S.Y., Peng, Y.Z., Ma, B., Wang, S.Y., Zhu, G.B., 2015. Anaerobic ammonium
oxidation in traditional municipal wastewater treatment plants with lowstrength ammonium loading: widespread but overlooked. Water Res. 84, 66
75.
Wett, B., 2006. Solved upscaling problems for implementing deammonification of
rejection water. Water Sci. Technol. 53, 121128.
Wett, B., 2007. Development and implementation of a robust deammonification
process. Water Sci. Technol. 56, 8188.
Yan, L., Liu, Y., Wen, Y., Ren, Y., Hao, G., Zhang, Y., 2015. Role and significance of
extracellular polymeric substances from granular sludge for simultaneous
removal of organic matter and ammonia nitrogen. Bioresour. Technol. 179,
460466.
Yin, C.Q., Meng, F.G., Chen, G.H., 2015. Spectroscopic characterization of
extracellular polymeric substances from a mixed culture dominated by
ammonia-oxidizing bacteria. Water Res. 68, 740749.
Zhang, L., Liu, M.M., Zhang, S.J., Yang, Y.D., Peng, Y.Z., 2015a. Integrated fixed-biofilm
activated sludge reactor as a powerful tool to enrich anammox biofilm and
granular sludge. Chemosphere 140, 114118.
Zhang, L., Zhang, S.J., Peng, Y.Z., Han, X.Y., Gan, Y.P., 2015b. Nitrogen removal
performance and microbial distribution in pilot- and full-scale integrated fixedbiofilm activated sludge reactors based on nitritation-anammox process.
Bioresour. Technol. 196, 448453.