FEMS Microbiology Ecology, 91, 2015, fiv124
doi: 10.1093/femsec/fiv124
Advance Access Publication Date: 15 October 2015
Research Article
RESEARCH ARTICLE
Predation of nitritation–anammox biofilms used
for nitrogen removal from wastewater
Carolina Suarez1 , Frank Persson2 and Malte Hermansson1,∗
1
Department of Chemistry and Molecular Biology/Microbiology, University of Gothenburg, SE-405 30, Sweden;
and 2 Water Environment Technology, Department of Civil and Environmental Engineering, Chalmers
University of Technology, SE-412 96, Sweden
∗
Corresponding author: Department of Chemistry and Molecular Biology/Microbiology, University of Gothenburg, Medicinaregatan 9C, SE-405 30
Gothenburg, Sweden. Tel: +46-31-7862574; Fax: +46-31-7862599; E-mail: [email protected]
One sentence summary: We show predation of anaerobic and aerobic ammonium oxidizing bacteria at different depths in intact biofilms for nitrogen
removal from wastewater, indicating that predation can be an important factor regulating these guilds.
Editor: Tillmann Lueders
ABSTRACT
Predation is assumed to be a major cause of bacterial mortality in wastewater treatment plants (WWTP). Grazing on the
slowly growing autotrophic ammonia oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AMX) may
result in loss of biomass, which could compromise nitrogen removal by the nitritation–anammox process. However,
predation, particularly of anaerobic AMX, is unknown. We investigated the presence of protozoa, AOB and AMX and the
possible predation in nitritation–anammox biofilms from several WWTPs. By fluorescence in situ hybridization (FISH) and
confocal laser scanning microscopy (CLSM), predator and prey were localized in intact biofilm cryosections. Different broad
morphological types of protozoa were found at different biofilm depths. Large variations in abundance of protozoa were
seen. One reactor showed a predation event of amoeba-like protozoa, forming grazing fronts reaching deep biofilm regions
that were dominated by the anaerobic AMX. Both AOB and AMX were grazed by the amoeba, as revealed by FISH–CLSM.
Hence, even AMX, living in the deeper layers of stratified biofilms, are subjected to predation. Interestingly, different
colocalization was observed between the amoeba-like protozoa and two different Ca. Brocadia AMX sublineages, indicating
different grazing patterns. The findings indicate that predation pressure can be an important factor regulating the
abundance of AOB and AMX, with implications for nitrogen removal from wastewater.
Keywords: biofilm; predation; nitritation-anammox; MBBR; FISH–CLSM
INTRODUCTION
The combined nitritation–anammox process in one-stage reactors is becoming a viable alternative to the nitrification/denitrification process for removal of nitrogen from
wastewater (Joss et al. 2009; Lackner et al. 2014). In the
nitritation–anammox process, ammonia oxidizing bacteria
(AOB) oxidize part of the ammonia to nitrite. Nitrite together
with remaining ammonium is used by anaerobic ammonium
oxidizing bacteria (AMX), resulting in production of N2 (Jetten
et al. 2009). Oxygen consumption by AOB and other microorganisms in the outer layers of microbial assemblages allow AMX to
thrive in the deeper anoxic parts (Vlaeminck et al. 2010; Almstrand, Persson and Hermansson 2014). Nitritation–anammox
communities are often cultivated on biofilm carriers in moving
bed biofilm reactors (MBBR) or in granules (free-floating biofilm
aggregates) to retain high concentrations of the slow growing
AOB and AMX in the wastewater treatment reactors (Lackner
et al. 2014; Persson et al. 2014). The nitritation–anammox process can be unstable, and inhibition of AOB, for example by toxic
Received: 21 May 2015; Accepted: 8 October 2015
C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
1
2
FEMS Microbiology Ecology, 2015, Vol. 91, No. 11
substances, would result in increased oxygen penetration in the
biofilm that would in turn inhibit AMX and reduce process performance (Joss et al. 2009). Both AOB and AMX have long generation times (van der Star et al. 2007; Joss et al. 2009), which impacts
the resilience of the nitration-anammox process after a loss of
biomass by for instance detachment or bacterial mortality.
The major causes of bacterial mortality in aquatic ecosystems are virus attacks and grazing by eukaryotic and prokaryotic predators (Fuhrman and Noble 1995; Thingstad 2000;
Pernthaler 2005; Dolinšek et al. 2013). Studies of predation on
bacteria in biofilms have been performed mostly in laboratory
experiments where protozoa have been added to single- or multispecies bacterial biofilms (Huws, McBain and Gilbert 2005;
Matz et al. 2005; Weitere et al. 2005; Böhme, Risse-Buhl and Küsel
2009). Elegant laboratory experiments have shown how bacteria
were protected from protozoan predation by forming biofilms,
aggregates and by production of an antiprotozoa substance by
the biofilm bacteria (Matz et al. 2005; Weitere et al. 2005). However, bacteria in biofilms are not always protected from protozoa predation. Amoeba significantly reduced the thickness of
a laboratory multispecies bacterial biofilm (Huws, McBain and
Gilbert 2005) and protozoa with various feeding strategies grazed
on biofilm bacteria and changed the thickness and structure of
laboratory biofilms (Böhme, Risse-Buhl and Küsel 2009). Thus,
the aggregated growth of both AOB and AMX in nitrificationanammox biofilms (Almstrand, Persson and Hermansson 2014)
may be expected to reduce, but perhaps not prevent predation.
Furthermore, the anoxic conditions in the deeper parts, where
the AMX are located, might help these bacteria to escape predation from aerobic predators.
Compared with predation on suspended bacteria, surprisingly little is known about predation in natural multispecies
biofilms (Parry 2004). In wastewater treatment plants (WWTP),
protozoa have been suggested to act as general process performance indicators in activated sludge systems (Madoni 2011).
Protozoa have also been detected in biofilms on MBBR carriers
(Fried and Lemmer 2003; Canals et al. 2013). Predation resulted
in decreased nitrification rates in an MBBR (Lee and Welander
1994). However, both Lee and Oleszkiewicz (2003) and Yu, Peng
and Ren (2011) showed that no change in nitrification performance occurred with or without predation, even if the latter
study indicated changes in both AOB and nitrite oxidizing bacterial (NOB) community compositions due to predation. Preferential predation of AOB by a wastewater peritrich ciliate has also
been suggested, based on 13 CO2 labeling experiments (Moreno
et al. 2010). Similar labeling experiments and FISH analyses suggested that NOB sublineage I Nitrosospira were attacked by a bacterial predator related to Micavibrio (Dolinšek et al. 2013). To the
best of our knowledge, no studies of predation on AMX have
been reported. In fact, it is not clear if and how AOB or AMX
bacteria are grazed upon inside nitritation–anammox biofilms
or how common it is. In order to utilize the complex nitritation–
anammox process in wastewater treatment more efficiently, a
better understanding is required, not only of the growth of AOB
and AMX, but also of the mortality caused by predation on these
bacteria.
Predation depends on the actual encounter between predator
and prey. Therefore colocation of predator and prey should indicate a potential predation pressure. AOB and AMX are typically
found at different depths in a stratified nitritation–anammox
biofilm (Almstrand, Persson and Hermansson 2014) where AMX
might be protected from predation in the deeper, anoxic parts of
the biofilm. Here, we test the hypothesis that AOB and AMX are
exposed to different predation pressures. We studied biofilms
from nitrogen removal systems of three large-scale one-stage
nitritation–anammox pilot MBBRs and one full-scale Integrated
Fixed Film Activated Sludge (IFAS) reactor. In contrast to the
other MBBRs, the IFAS reactor is designed to have AOB mainly
in the activated sludge phase and AMX mainly in the biofilm.
We used fluorescence in situ hybridization (FISH), with probes
directed against prokaryotic 16S rRNA and eukaryotic 18S rRNA
targets, and confocal laser scanning microscopy (CLSM) to analyze cryosectioned biofilms. This allowed, for the first time, the
simultaneous identification and physical locations of key bacterial groups (in this case AOB and AMX) and broad morphological groups of eukaryotic predators at different depths in intact
biofilm structures. A detailed colocalization and identification
of prey inside predators also indicated predation of AOB and
AMX. In one of the nitritation–anammox MBBRs, a reduction in
AOB abundance was noticed earlier by quantitative PCR during
a down-shift temperature experiment (Persson et al. 2014), and
here, we studied the possible role of grazing during this AOB
reduction.
MATERIALS AND METHODS
Reactors
We choose reactors with different characters, fed with different
water (reject water from anaerobic sludge digestion and mainstream wastewater) and operated at different temperatures and
ammonium loads. Characteristics of the different reactors are
summarized in Table 1. The KTH pilot MBBR was situated at
the Centre for municipal wastewater purification (Hammarby
Sjöstadsverk), Stockholm, Sweden and received reject water
from anaerobic sludge digestion. It was filled to 40% with biofilm
R
carriers (K1
, AnoxKaldnes AB, Lund, Sweden, specific surface
area 500 m2 m−3 ). The reactor is described in detail elsewhere
(Persson et al. 2014).
A pilot MBBR plant was situated at Sjölunda WWTP in
Malmö, Sweden. One MBBR received reject water from anaerobic sludge digestion and two consecutive MBBRs received main
stream wastewater from a high-loaded activated sludge plant
for COD removal. The first of the main stream MBBRs (Sjmanammox) and the reject water MBBR (Sj-reject) were investigated here. The reactors were filled to 40% with biofilm carriers
(K1). The operational strategies of the pilot plant included a periodical transfer of carriers between the mainstream and reject
stream MBBRs. The pilot plant is described in detail elsewhere
(Gustavsson, Persson and la Cour Jansen 2014).
The full-scale IFAS reactor is located at the Sjölunda WWTP,
Malmö, and operated by AnoxKaldnes AB, Lund, Sweden (Veuillet et al. 2014). The reactor was filled to 50% with biofilm carriR
ers (K3
, AnoxKaldnes AB, Lund, Sweden, specific surface area
800 m2 m−3 ) and received reject water from anaerobic sludge digestion. The IFAS reactor is described in detail elsewhere (Veuillet et al. 2014).
Fixation and cryosectioning of biofilms
Biofilm cryosectioning was performed as described in (Lydmark
et al. 2006), with minor modifications for sampling of K1 and
K3 carriers, see also Fig. S1, Supporting Information. Samples
for cryosectioning were fixed by submerging the biofilm carriers in 4% paraformaldehyde for 8 h at 4◦ C, followed by rinsing
twice for 30 min with PBS. After fixation, the entire carriers were
embedded in O.C.T. compound (VWR, USA) in individual plastic containers. The containers with the embedded carriers were
Suarez et al.
3
Table 1. Operational conditions and process performance of the pilot reactors at KTH and Sjölunda WWTP (Sj) and the full-scale IFAS reactor,
Anox Kaldnes AB (see text for details)∗ .
Reactor
Ammonium concentration
effluent (mgN L−1 )
Sludge reject liquor feed
KTH1a
KTH2a
KTH3a
Sj-rejecta
IFASb
108
43
301
57
109
Main stream wastewater feed
Sj-manammoxa
9.0 ± 3.0
±
±
±
±
±
96
56
143
28
46
Nitrogen loading rate
(gN m−2 d−1 )
2.25
1.05
1.02
2.4
4.71
±
±
±
±
±
0.36
0.07
0.05
0.45
0.60
Nitrogen removal rate
(gN m−2 d−1 ) (N removal %)
1.67
0.81
0.55
1.8
3.66
0.96 ± 0.19
±
±
±
±
±
0.4 (74 ± 11%)
0.12 (77 ± 9%)
0.13 (54 ± 13%)
0.39 (74 ± 4.1%)
0.53 (78 ± 5%)
0.39 ± 0.18 (40 ± 8.3%)
DO (mg L−1 )
T (◦ C)
±
±
±
±
±
0.2
0.4
0.1
0.70
0.2
19
16
13
25 ± 1.2
30 ± 1
2.4 ± 0.26
16 ± 0.9
1.3
1.1
1.2
1.1
0.4
∗
Mean values +/- S.D. during at least two months prior to sampling. Data on KTH reactor from Persson et al. 2014, data on Sjölunda reactors from (Gustavsson et al.
2013; Gustavsson, Persson and la Cour Jansen 2014) and data on IFAS from AnoxKaldnes AB, Lund, Sweden.
a
One-stage nitritation anammox MBBR with K1 carriers.
b
Integrated IFAS reactor with K3 carriers
incubated overnight at 4◦ C. They were thereafter placed in a liquid nitrogen fume chamber until the O.C.T. compound was completely frozen. The plastic container was removed and the intact
biofilm from one of the compartments in each carrier was taken
out using a forceps and a scalpel, by gently removing the excess
O.C.T. compound surrounding the carrier and cutting the carrier
open with the scalpel to sample the biofilm. The intact frozen
biofilm from one compartment was again embedded in O.C.T. in
another plastic container and frozen solid in a block in the liquid
nitrogen fume chamber. The blocks were stored at −70◦ C until
use. They were sectioned in 20–25-μm-thick slices with a HM550
microtome cryostat (MICROM International GmbH, Germany) at
−20◦ C. The slices were collected on SuperFrost Plus Gold microscope slides (Menzel GmbH, Braunschweig, Germany). After dehydration in an ethanol series (50, 80 and 96% v/v), the cryosectioned biofilm slices were stored on the microscope slides at
−20◦ C until use.
Biofilm suspensions were used to quantify the relative abundance of AOB and AMX in the entire biofilms. For this, the
fixed biofilm was brushed off the carriers and homogenized in
PBS. The biofilm suspensions were stored in PBS-ethanol (1:1)
at −20◦ C until use. Prior to microscopy, 2–4 μl aliquots of the
fixed biofilm suspensions were spotted onto the wells in microscopic diagnostic slides (8 × 6 mm diameter wells, Menzel
GmbH, Braunschweig, Germany).
Fluorescence in situ hybridization
The cryosectioned biofilm samples were fixed again directly on
the microscope slides at room temperature for 20 min. with
4% paraformaldehyde to ensure that all cells in the biofilm
cryosections were fixed. Fixation was followed by submersion
in PBS for 30 min. Prior to FISH a hydrophobic barrier frame
was applied to the glass slides around the regions containing
the biofilm cryosections by using a Liquid Blocker Mini Pap Pen
(Life Technologies, Carlsbad, CA, USA). FISH was performed at
46◦ C for 4 h for the cryosectioned biofilms and 2 h for the
biofilm suspensions (Manz et al. 1992). When probes with different hybridization stringency optima were applied to the same
sample, consecutive hybridizations were performed, beginning
with the probe(s) requiring the most stringent conditions (Manz
et al. 1992). Slides were counterstained with DAPI (3 μg/ml for
10 min) and washed with water. After FISH and counterstaining,
the slides were mounted in the antifadant Citifluor AF1 (Citifluor
Ltd., UK).
Table 2. FISH oligonucleotides used in this study. Sequences and references are shown in Tables S1 and S2 (Supporting Information).
Probe
EUB338 (I-IV)
EUK-mix
AMX368
AMX820
Ban162b
Bfu613c
Nse1472d
NEUd
Cluster6a192
NmV
Target organisms
FAa (%)
Most bacteria
Most eukaryotes
AMX
Ca. Brocadia and Ca. Kuenenia
Ca. Brocadia sp.40
Ca. Brocadia KTH11-AMX-C10
N. europaea/eutropha
N. europaea/eutropha/halophila
N. oligotropha
Nitrosococcus mobilis
10–50
20–50
15
40
40
30
50
40
35
35
FA = Formamide concentration in hybridization buffer.
Probe applied with unlabeled competitor probe cBan162 designed in this study
(see Table S1, Supporting Information). Ban162 is intended to target Ca. Brocadia
anammoxidans, but in fact also detects Ca. Brocadia sp. 40.
c
Probe applied with unlabeled competitor probe cBfu613 (Persson et al. 2014).
Although the Bfu613 probe is intended to target Ca. Brocadia fulgida, it was used
to detect the Ca. Brocadia C10 populations in the KTH reactor.
d
Both Nse1472 and NEU target AOB in N. europaea/eutropha, N. halophila. However,
neither Nse1472 nor NEU target all of these. Hence, both probes were used in the
study.
a
b
Probe specificity at domain- and genus levels was determined using the TestProbe web tool (http://www.arb-silva.de/
search/testprobe). Probe specificity within the Nitrosomonas
genus, was determined with ARB 6.0.2 (Ludwig et al. 2004) using the SILVA NR 119 SSU Nitrosomonas sequences (Quast et al.
2013). Probes BFU613 and Ban162 were used to target anammox
subpopulations in the KTH reactor; these targets were selected
according to 16SrRNA sequence information of the AMX community in the reactor (Persson et al. 2014) and differ from the
intended target described in literature. For FISH probes and hybridization conditions, see Table 2. A new AMX probe competitor (cBan162) was designed, which allowed us to differentiate
between Ca. Brocadia sp. 40 and other AMX populations in the
KTH reactor (Tables S1 and S2, Supporting Information).
Microscopy and image analyses
Images were acquired using a Zeiss LSM700 (Carl Zeiss, Germany) with laser lines of 405, 488, 555 and 639 nm. Larger images covering several millimeters were acquired with a 10×/0.45
4
FEMS Microbiology Ecology, 2015, Vol. 91, No. 11
objective. High-resolution images were obtained with a 40×/1.3
plan-apochromat oil objective. When using the 555 nm laser for
the Cy3 probes, autofluorescence in the near IR was sometimes
noticed in the biofilm. A 600 nm short pass filter was used to reduce this background fluorescence. To create composite images
of large size from the 160 × 160 μm images and for 3D reconstruction, the tile and Z functions of the Zeiss ZEN2010 software
were used, respectively.
For screening of the location of eukaryotes in the biofilms,
160 × 480 μm non-overlapping random composite images were
taken near the biofilm–water interface. These were divided into
50 μm sections, (perpendicular to the biofilm–water interface)
starting from the biofilm–water interface up to 300 μm depth in
the biofilms, using the Slicer tool (Almstrand et al. 2013) in the
image analysis software daime (Daims, Lücker and Wagner 2006).
For comparing protozoa and AMX abundance between samples,
30 images from two different carriers (15 each) were taken for
each sample. For comparisons of protozoa abundance between
the outer and the inner biofilm in a carrier from KTH2, where
a grazing event was observed, 13 pictures were taken in each
location. To study biovolume fractions of different AOB populations in the reactors, biofilm suspensions were used; 10 random pictures were taken for each population in every sample.
The same method was used to distinguish between AMX in suspended phase and in the biofilm in the IFAS reactor. 3D visualizations of protozoa grazing in the biofilms were done with the
software daime, using z-stacks.
Differential interference contrast (DIC) microscopy was used
to study protozoan morphology.
Statistics
Statistical analyses were done with R 3.2.2 (R Core Team 2015).
Due to deviations from normality and variance homogeneity, as
observed by Shapiro–Wilks test and Cochran’s C test, respec-
tively, implemented in the R package GAD (Sandrini-Neto and
Camargo 2015), differences in protozoa abundances between
samples were analyzed non-parametrically using Kruskal–
Wallis test (Neuhäuser 2012).
RESULTS
Bacterial biofilm structure
A screening with FISH probes showed that AOB in all the reactors were dominated by Nitrosomonas europaea/eutropha/halophila
within cluster 7, targeted by the probes NEU and Nse1472 (Fig. S2,
Supporting Information). The anammox bacteria in all reactors
were targeted with the probe AMX820, and cohybridization with
AMX368 (data not shown), showed that all anammox bacteria
were Ca. Kuenenia sp./Brocadia sp. (see below for more details
on different AMX populations). Hence, for showing localization
of AOB and AMX in the intact cryosections from all reactors, the
probes NEU, Nse1472 and AMX820 were used (Fig. 1). The carriers had a more or less pronounced channel in the middle of the
biofilm (Fig. 1G). The general biofilm structure, with AOB close
to the biofilm–water interface and AMX located deeper into the
biofilm, was seen in all the nitritation–anammox MBBR reactors
(Fig. 1A–E). This pattern was seen both in the outer biofilms facing the bulk water phase (Fig. 1A–E) and in the inner biofilms
inside the channel of the carriers (Fig. 1G). Micrographs in Fig. 1
are selected to show the generally layered AOB and AMX structure. Random images also show how the distribution of AMX increases with biofilm depth from the surface in all reactors except
the IFAS reactor (Fig. 2B). In the IFAS reactor, most of the AOB
were found in the activated sludge (biovolume fraction as % of
EUB signal of 4.2% in the biofilm and 17.8% in the water phase)
(Fig. S2, Supporting Information), while the AMX were located
in the biofilm (biovolume fractions as % of EUB signal of 20.8% ±
6.4 in the biofilm and 0.6% ± 0.9 in the water phase).
Figure 1. Lengthwise FISH–CLSM composite images from cryosections of different biofilms: (A–C) KTH1-3; (D) Sj-reject; (E) Sj-manammox; (F) IFAS; (G) larger composite
image, showing the structure of the biofilm including the channel from KTH2. The water-biofilm interface is oriented to the left in all images. Blue: all bacteria;
green–cyan: AMX (Ca. Brocadia sp. / Kuenenia sp.); red–magenta: AOB (N. europaea/eutropha/halophila). Scale bars A–F: 50 μm, G: 500 μm.
Suarez et al.
5
Figure 2. (A) Lengthwise FISH–CLSM composite images from cryosections of different biofilms. The water-biofilm interface is oriented to the left in all images. Yellow:
protozoa; blue: bacteria; green–cyan: AMX (Ca. Brocadia sp. / Kuenenia sp.). The arrow points toward the grazing fronts. Scale bars: 50 μm. (B) Average AMX biovolume
fractions at different biofilm depths. (C) Average number of protozoa observed at different biofilm depths. For B and C, error bars show confidence interval (95%); each
observation area corresponds to a 50 × 160 μm section; n = 30 for each biofilm.
Eukaryotic predators in the biofilms
Use of different FISH probes allowed for the detection and localization of eukaryotic predators together with the different
bacterial groups (Table 2). By combining FISH with other staining methods, classification of protozoa into broad morphological groups was also possible. Stalked sessile protozoa (Fig. S3A,
Supporting Information) were present in all reactors, attached
to the outer biofilm bulk water interface and also to the inner,
water filled biofilm channel (e.g. Fig. 1G and Fig. S8, Supporting Information). Stalked structures extending from the biofilm
were preserved in the cryosections, indicating that biofilms were
kept intact during preparation. Surface dwelling protozoa were
present at the biofilm surface and in small channels, close to
the surface. These protozoa did not seem to penetrate to more
than about 150 μm into the biofilm. The protozoa in the outer
parts of the biofilms were of different types, including some that
were identified as small flagellated protozoa, resembling Kinetoplastids, by staining of nucleus and kinetoplast DNA and by
the presences of flagella, as seen by DIC (Fig. S3C, Supporting Information). Amoeba-type protozoa were detected by combining
DAPI and FISH, which allowed for detection of a DAPI stained
ring-like nucleus (Fig. S3B, Supporting Information). There were
large variations in the abundance of protozoa in all biofilm
6
FEMS Microbiology Ecology, 2015, Vol. 91, No. 11
samples (Fig. 2 and Fig. S7, Supporting Information). However,
they were only found in large numbers in grazing fronts (Kalmbach, Manz and Szewzyk 1997) deep inside the biofilms in the
KTH1 and KTH2 samples (Fig. 2A, B and Fig. S7, Supporting Information). Judging from the overview composite images no grazing fronts were seen in the biofilm channels, and here, no protozoa were penetrating deep into the biofilm (Fig. 5).
Predation in the biofilms
Both AMX and AOB cells were detected inside the amoeba
(Fig. 3). The absence of overlapping eukaryotic FISH probe signals
at the same position as the bacterial probe inside the eukaryotes, indicate that bacteria were seen in food vacuoles and not
in the cytoplasm. In fact, in one composite image of the KTH2
biofilm, as many as 71% of the protozoa in the grazing front had
AMX cells inside. Occasionally, single AMX cells were also observed inside the surface dwelling protozoa (data not shown).
AMX predation was seen in all reactors (Fig. S7B, Supporting Information). Predation of AMX by the stalked protozoa was, however, never observed.
In the KTH reactor, we followed a dynamic grazing event for
nine months during a temperature decrease experiment (Persson et al. 2014). In contrast to the other protozoan types, amoeba
penetrated deep into the biofilms where they could form grazing fronts in KTH1 and KTH2 (Figs 2A, C and 4). The depth of the
grazing fronts generally changed from 4–160 to 4–250 μm during
the two months between sample KTH1 and KTH2 and reached
into the AMX layers (Fig. 2B and C). They penetrated into large
AMX and AOB aggregates (Fig. 3C, D and Fig. S6, Supporting Information). No grazing fronts were detected in KTH3 six months
after the appearance of major grazing fronts, indicating that the
amoeba grazing was a dynamic event (Fig. 2A, C). As mentioned
above, amoeba grazing fronts were never seen in the KTH1 and
KTH2 biofilm channels (Fig. S8, Supporting Information), neither
did the few protozoa in the channels penetrate deep into the
biofilm (Fig. 5)
Two AMX Ca. Brocadia populations, sp. 40 and C10, were detected in the KTH1 and KTH2 biofilms (see Tables 2, S1 and
S2, Supporting Information for probe specificities). Hence, we
investigated if there was a possible difference in predation of
these populations. Ca. Brocadia C10 was present close to the
biofilm–water interface, while Ca. Brocadia sp. 40 was growing
at all depths (Fig. S4, Supporting Information). Both populations
were observed within the amoeba (data not shown). However,
the amoeba only formed grazing fronts in the Ca. Brocadia sp.
40 aggregates, but never in the C10 aggregates (Fig. 4 and Fig. S5,
Supporting Information). Instead, single amoeba was seen inside C10 aggregates (Fig. S5B, Supporting Information).
DISCUSSION
We surveyed the AOB and AMX communities and studied eukaryotic predation on these bacteria in intact biofilms from four
different nitritation–anammox reactors. Our working hypothesis was that AMX were protected from predation in the deeper,
anaerobic part of biofilms and that AOB in the biofilm close to
the water interface were more vulnerable to predation. Predation of AMX has not been investigated before and only very few
studies have detected mechanisms of nitrifier mortality (Moreno
et al. 2010; Vlaeminck et al. 2010; Dolinšek et al. 2013).
Despite the different conditions and water composition of
the four reactors (Table 1), the major AOB populations in all reactors were affiliated to N. europaea/eutropha /halophila within clus-
Figure 3. Predation of AMX and AOB in the KTH2 reactor. Yellow: protozoa; green:
AMX (Ca. Brocadia sp./ Kuenenia sp.); red: AOB (N. europaea/eutropha/halophila).
(A) Grazing of AMX within the biofilm, the water-biofilm interface is 60 μm to
the left, AMX are seen inside protozoa. (B) Grazing of AOB at the water-biofilm
interface, AOB are seen inside protozoa. (C) 3D visualization of a grazing front in
an anammox aggregate; protozoa are rendered semitransparent to show AMX
inside. (D) 3D visualization of grazing on both AOB and AMX. Scale bars (A–B):
10 μm.
ter 7 and the major AMX populations to the Ca. Brocadia sp./
Ca. Kuenenia sp. These AOB and AMX phylogenetic clusters have
also been found before in a number of different nitritation–
anammox reactors at different conditions (e.g. Park et al. 2010;
Vlaeminck et al. 2010; Gilbert et al. 2014), suggesting a broad ecophysiology.
Suarez et al.
Figure 4. Predation patterns for different Ca. Brocadia populations in the KTH2
biofilm. The water-biofilm interface is oriented to the left. Red–magenta: Ca. Brocadia C10; blue: Ca. Brocadia sp. 40; yellow: protozoa. Scale bar: 50 μm.
Figure 5. Depth distribution of total numbers of protozoa in different parts of
the KTH2 biofilm. Patterned bars: Protozoa at different depths from the outer
biofilm–water interface. White bars: protozoa at different depths from the inner
channel biofilm–water interface. See Fig. 1G for locations. Each observation area
corresponds to a 160 × 50 μm section. Error bars: confidence interval 95%. n =
13 (for each location). Significant differences (P < 0.05, Kruskal–Wallis) between
inner and outer biofilms at specific depths are indicated by asterisks (∗ ) above
the bars.
Stalked protozoa were attached to the biofilm at the outer
water interface as well as in the channel. Since they feed mainly
on suspended bacteria (Parry 2004), they were not directly affecting the AOB or AMX in the MBBR biofilms. In the IFAS reactor,
however, a large fraction of the AOB community is present in the
bulk water (Fig. S2, Supporting Information) and might hence
be affected by these predators. Small protozoa, including flagellates (Fig. S3C, Supporting Information), located in the outer
layers (about 150 μm) of the biofilms were detected in all reactors. They occasionally had AMX cells inside but did not seem
to graze the AOB. The small size of flagellates might have prevented grazing of AOB aggregates, as suggested before (Weitere
et al. 2005; Wey et al. 2008). Amoeba in the KTH biofilms were
shown to engulf AOB and were able to penetrate into the AOB
aggregates (Fig 3). However, no or very few amoeba were seen in
the biofilm channels, indicating that the channels were microhabitats that might function as a general refuge. Interestingly,
the presence of the amoeba grazing front in KTH2 and a signif-
7
icantly higher abundance of protozoa, compared with the other
reactors (Fig. 2C and Fig. S7B, Supporting Information), coincided
with a significant reduction in the AOB abundance, as previously
measured by qPCR (Persson et al. 2014). Even though a drop in
temperature at the same time period may have influenced the
AOB activity, it is tempting to suggest that the reduction in AOB
abundance might have been, at least partially, caused by grazing.
The nitritation–anammox system is sensitive to disturbances
and AOB carry out the rate-limiting step. Hence, predation of
AOB may explain unforeseen changes in ecosystem functions.
Amoeba was shown to engulf AMX cells (Fig. 3), and predation was seen in all reactors although at very different extent
(Fig. S7B, Supporting Information). Thus, AMX are not always
protected from grazing by their aggregated growth in biofilms, or
by their anaerobic microhabitat. Also, not all protozoa are limited by anoxic conditions, for example the archamoeba (comprising the entamoeba group) are anaerobic/microaerophilic
(Ptáčková et al. 2013) and control of chemolithoautotrophic bacterial growth by protozoa grazing in oxic–anoxic interfaces has
been reported (Anderson et al. 2013).
Interestingly, we detected a clear difference in grazing of the
two Ca. Brocadia AMX populations in the KTH biofilms. While
both Ca. Brocadia C10 and Ca. Brocadia sp. 40 were seen inside
food vacuoles in amoeba, the grazing fronts were only seen associated with Ca. Brocadia sp. 40 aggregates (Fig. 4 and Fig. S5,
Supporting Information). On the other hand, single amoeba was
seen inside Ca. Brocadia C10 aggregates. We can only speculate
about possible differences in the predation defense mechanisms
of the two AMX populations. A different grazing pattern may be
a selective force that can favor one AMX over another and thus
influences the structure of the AMX guild and therefore the function of nitritation–anammox reactors.
In summary, the techniques we used allowed the localization
of predators and prey in intact biofilm and the detection of bacteria inside predators, indicating a predation pressure on the different populations. Stalked protozoa and surface dwelling protozoa were found in all reactors, while amoeba forming grazing
fronts deep into the biofilms was only found in the KTH biofilms,
where the grazing fronts seemed to reduce the AOB population.
No grazing fronts were seen in the biofilm channels and channels may have acted as a repository for bacteria. Amoeba grazing
fronts could also reach down into the biofilm to graze on AMX,
showing that neither AOB nor AMX were protected from predation. There was also a clear difference in the grazing pattern of
two different AMX populations. Predation of these slow growing, autotrophic bacteria should not be overlooked as a potential explanation for variations in process function in wastewater nitritation–anammox reactors. Further, research to clarify
the mechanisms and extent of predation could include identification of active predators in these biofilms, by developing specific eukaryotic FISH probes from 18S rRNA sequence information and analyses by CLSM and FISH. Other lines of investigation
may include the use of laboratory reactors to study the extent of
predation and its effect on prey communities and reactor performance under different conditions.
SUPPLEMENTARY DATA
Supplementary data are available at FEMSEC online.
ACKNOWLEDGEMENTS
We acknowledge Elzbieta Plaza at the Royal Institute of Technology (KTH); AnoxKaldnes AB, Lund, Sweden; and VA SYD for
8
FEMS Microbiology Ecology, 2015, Vol. 91, No. 11
allowing us access to their reactors and for providing data on
the reactors. We also acknowledge David Gustavsson at VA SYD
for help with sampling and for discussions and Simon Piveteau
at AnoxKaldnes AB for help with sampling from the IFAS reactor. We thank two anonymous referees for valuable comments
on the manuscript. The authors would like to acknowledge the
Centre for Cellular Imaging (CCI) at the University of Gothenburg
for support and use of their CLSM.
FUNDING
This work was supported by the Swedish research council FORMAS (Contract no. 243-2010-2259 and 245-2014-1528) and Adlerbertska forskningsstiftelsen.
Conflict of interest. None declared.
REFERENCES
Almstrand R, Daims H, Persson F, et al. New methods for
analysis of spatial distribution and coaggregation of microbial populations in complex biofilms. Appl Environ Microbiol
2013;79:5978–87.
Almstrand R, Persson F, Hermansson M. Biofilms in nitrogen
removal: population dynamics and spatial distribution of
nitrifying- and anammox bacteria. In: Marco D (ed.). Metagenomics of the Microbial Nitrogen Cycle: Theory, Methods and Applications. Portland, USA: Caister Academic Press, 2014, 227–60.
Anderson R, Wylezich C, Glaubitz S, et al. Impact of protist grazing on a key bacterial group for biogeochemical cycling in
Baltic Sea pelagic oxic/anoxic interfaces. Environ Microbiol
2013;15:1580–94.
Böhme A, Risse-Buhl U, Küsel K. Protists with different feeding modes change biofilm morphology. FEMS Microbiol Ecol
2009;69:158–69.
Canals O, Salvadó H, Auset M, et al. Microfauna communities as performance indicators for an a/o shortcut biological nitrogen removal moving-bed biofilm reactor. Water Res
2013;47:3141–50.
Daims H, Lücker S, Wagner M. Daime, a novel image analysis
program for microbial ecology and biofilm research. Environ
Microbiol 2006;8:200–13.
Dolinšek J, Lagkouvardos I, Wanek W, et al. Interactions of
nitrifying bacteria and heterotrophs: identification of a
micavibrio-like putative predator of nitrospira spp. Appl Environ Microbiol 2013;79:2027–37.
Fried J, Lemmer H. On the dynamics and function of ciliates in sequencing batch biofilm reactors. Water Sci Technol
2003;47:189–96.
Fuhrman JA, Noble RT. Viruses and protists cause similar
bacterial mortality in coastal seawater. Limnol Oceanogr
1995;40:1236–42.
Gilbert EM, Agrawal S, Karst SM, et al. Low temperature partial nitritation/anammox in a moving bed biofilm reactor treating
low strength wastewater. Environ Sci Technol 2014;48:8784–92.
Gustavsson DJ, Aspegren H, Stålhanske L, et al. Mainstream
anammox at Sjölunda Wastewater Treatment Plant—pilot
operation of sludge liquor treatment and start-up of the full
process. 13th Nordic Wastewater Conference, NordIWA, Malmö,
Sweden, The Swedish Water & Wastewater Association,
2013.
Gustavsson DJI, Persson F, la Cour Jansen J. Manammox mainstream anammox at Sjölunda WWTP. IWA World Water
Congress and Exhibition, Lisbon, Portugal, 2014.
Huws SA, McBain AJ, Gilbert P. Protozoan grazing and its impact
upon population dynamics in biofilm communities. J Appl Microbiol 2005;98:238–44.
Jetten MSM, Niftrik van L, Strous M, et al. Biochemistry and
molecular biology of anammox bacteria. Crit Rev Biochem Mol
Biol 2009;44:65–84.
Joss A, Salzgeber D, Eugster J, et al. Full-scale nitrogen removal
from digester liquid with partial nitritation and anammox in
one sbr. Environ Sci Technol 2009;43:5301–6.
Kalmbach S, Manz W, Szewzyk U. Dynamics of biofilm formation
in drinking water: phylogenetic affiliation and metabolic potential of single cells assessed by formazan reduction and in
situ hybridization. FEMS Microbiol Ecol 1997;22:265–79.
Lackner S, Gilbert EM, Vlaeminck SE, et al. Full-scale partial nitritation/anammox experiences - an application survey. Water
Res 2014;55:292–303.
Lee NM, Welander T. Influence of predators on nitrification
in aerobic biofilm processes. Water Sci Technol 1994;29:
355–63.
Lee Y, Oleszkiewicz JA. Effects of predation and ORP conditions
on the performance of nitrifiers in activated sludge systems.
Water Res 2003;37:4202–10.
Ludwig W, Strunk O, Westram R, et al. ARB: a software environment for sequence data. Nucleic Acids Res 2004;32:1363–71.
Lydmark P, Lind M, Sorensson F, et al. Vertical distribution of nitrifying populations in bacterial biofilms from a full-scale nitrifying trickling filter. Environ Microbiol 2006;8:2036–49.
Madoni P. Protozoa in wastewater treatment processes: a minireview. Ital J Zool 2011;78:3–11.
Manz W, Amann R, Ludwig W, et al. Phylogenetic oligodeoxynucleotide probes for the major subclasses of proteobacteria:
problems and solutions. Syst Appl Microbiol 1992;15:593–600.
Matz C, McDougald D, Moreno AM, et al. Biofilm formation and
phenotypic variation enhance predation-driven persistence
of Vibrio cholerae. P Natl Acad Sci USA 2005;102:16819–24.
Moreno AM, Matz C, Kjelleberg S, et al. Identification of ciliate
grazers of autotrophic bacteria in ammonia-oxidizing activated sludge by rna stable isotope probing. Appl Environ Microbiol 2010;76:2203–11.
Neuhäuser M. Nonparametric Statistical Tests: a Computational Approach. Boca Raton, Florida, USA: CRC Press, 2012.
Park H, Rosenthal A, Jezek R, et al. Impact of inocula and growth
mode on the molecular microbial ecology of anaerobic ammonia oxidation (anammox) bioreactor communities. Water
Res 2010;44:5005–13.
Parry JD. Protozoan grazing of freshwater biofilms. Adv Appl Microbiol 2004;54:167–96.
Pernthaler J. Predation on prokaryotes in the water column
and its ecological implications. Nat Rev Microbiol 2005;3:
537–46.
Persson F, Sultana R, Suarez M, et al. Structure and composition
of biofilm communities in a moving bed biofilm reactor for
nitritation–anammox at low temperatures. Bioresource Technol 2014;154:267–73.
Ptáčková E, Kostygov AY, Chistyakova LV, et al. Evolution of archamoebae: morphological and molecular evidence for pelobionts including rhizomastix, entamoeba, iodamoeba, and
endolimax. Protist 2013;164:380–410.
Quast C, Pruesse E, Yilmaz P, et al. The SILVA ribosomal RNA gene
database project: improved data processing and web-based
tools. Nucleic Acids Res 2013;41:D590–6.
R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing,
2015.
Suarez et al.
Sandrini-Neto L, Camargo MG. GAD: General ANOVA Designs.
Brazil: Centro de Estudos do Mar da Universidade Federal do
Parana, 2015.
Thingstad TF. Elements of a theory for the mechanisms controlling abundance, diversity, and biogeochemical role of
lytic bacterial viruses in aquatic systems. Limnol Oceanogr
2000;45:1320–8.
van der Star WRL, Abma WR, Blommers D, et al. Startup of reactors for anoxic ammonium oxidation: experiences from
the first full-scale anammox reactor in Rotterdam. Water Res
2007;41:4149–63.
Veuillet F, Lacroix S, Bausseron A, et al. Integrated fixed-film activated sludge ANITATM Mox process—a new perspective for
advanced nitrogen removal. Water Sci Technol 2014;69:915–22.
9
Vlaeminck SE, Terada A, Smets B, et al. Aggregate size and
architecture determine microbial activity balance for onestage partial nitritation and anammox. Appl Environ Microbiol
2010;76:900–9.
Weitere M, Bergfeld T, Rice S, et al. Grazing resistance of Pseudomonas aeruginosa biofilms depends on type of protective
mechanism, developmental stage and protozoan feeding
mode. Environ Microbiol 2005;7:1593–601.
Wey JK, Scherwass A, Norf H, et al. Effects of protozoan grazing
within river biofilms under semi-natural conditions. Aquat
Microb Ecol 2008;52:283–96.
Yu L, Peng D, Ren Y. Protozoan predation on nitrification performance and microbial community during bioaugmentation.
Bioresource Technol 2011;102:10855–60.