w a t e r r e s e a r c h 4 9 ( 2 0 1 4 ) 2 1 5 e2 2 4

Available online at www.sciencedirect.com

ScienceDirect
journal homepage: www.elsevier.com/locate/watres

Nitrifying moving bed biofilm reactor (MBBR)

biofilm and biomass response to long term
exposure to 1
C
V. Hoang a, R. Delatolla a,*, T. Abujamel b, W. Mottawea b, A. Gadbois c,
E. Laflamme c, A. Stintzi b
a

Department of Civil Engineering, University of Ottawa, 161 Louis Pasteur, Ottawa, Ontario K1N 6N5, Canada
Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology, and Immunology, University of
Ottawa, Ontario K1H 8M5, Canada
c
John Meunier Inc., Montreal, Quebec H4S 2B3, Canada
b

article info

abstract

Article history:

This study aims to investigate moving bed biofilm reactor (MBBR) nitrification rates, ni-

Received 1 August 2013

trifying biofilm morphology, biomass viability as well as bacterial community shifts during

Received in revised form

long-term exposure to 1
C. Long-term exposure to 1
C is the key operational condition for

4 November 2013

potential ammonia removal upgrade units to numerous northern region treatment sys-

Accepted 12 November 2013

tems. The average laboratory MBBR ammonia removal rate after long-term exposure to 1
C

Available online 21 November 2013

was measured to be 18  5.1% as compared to the average removal rate at 20
C. Biofilm

morphology and specifically the thickness along with biomass viability at various depths in

Keywords:

the biofilm were investigated using variable pressure electron scanning microscope

Nitrification

(VPSEM) imaging and confocal laser scanning microscope (CLSM) imaging in combination

Cold temperature

with viability live/dead staining. The biofilm thickness along with the number of viable

MBBR

cells showed significant increases after long-term exposure to 1
C. Hence, this study

Biofilm morphology

observed nitrifying bacteria with higher activities at warm temperatures and a slightly

Biomass viability

greater quantity of nitrifying bacteria with lower activities at cold temperatures in nitri-

Bacterial population analysis

fying MBBR biofilms. Using DNA sequencing analysis, Nitrosomonas and Nitrosospira
(ammonia oxidizers) as well as Nitrospira (nitrite oxidizer) were identified and no population shift was observed between 20
C and after long-term exposure to 1
C.
2013 Elsevier Ltd. All rights reserved.

1.

Introduction

Ammonia discharged from wastewater effluent is responsible

for the toxicity of natural water around the world and
particularly in northern regions (Canada Gazette, 2012). The
process of nitrification is the most economical means of
ammonia removal (Metcalf and Eddy, 2003), and as such

ammonia removal in cold climates often becomes extensively

limited or non-existent due to the temperature sensitivity of
nitrifiers (Sharma and Ahlert, 1977; Painter and Loveless,
1983). Contrary to conventional suspended growth processes, attached growth processes and specifically the moving
bed biofilm reactor (MBBR) have shown an ability to achieve
and maintain lower temperature nitrification (Wessman and
Johnson, 2006; Houweling et al., 2007; Delatolla et al., 2011;

* Corresponding author. Tel.: 1 613 562 5800x2677.

E-mail address: robert.delatolla@uottawa.ca (R. Delatolla).
0043-1354/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.watres.2013.11.018

216

w a t e r r e s e a r c h 4 9 ( 2 0 1 4 ) 2 1 5 e2 2 4

Di Trapani et al., 2013; Zhang et al., 2013). Hence, MBBR systems have demonstrated promise as a potential upgrade or
replacement technology for low temperature ammonia
removal.
Passive wastewater treatment systems such as wetlands
and lagoons are common in northern regions due to availability of land. For example, there are currently over 1000
lagoon systems operating as wastewater treatment facilities
in Canada alone and due to recent ammonia effluent
discharge regulations many of these existing treatment lagoons will require upgrading in the near future (Canada
Gazette, 2012). To date, MBBR upgrade units that have
demonstrated the ability to nitrify at low temperatures have
been investigated and implemented to treat the effluent from
the first or middle lagoon of a multiple lagoon treatment
systems; where temperature drops are limited and the carbon/nitrogen (C/N) ratio is higher than the downstream ponds
(Delatolla et al., 2011). Higher C/N ratios, specifically the
presence of influent readily degradable carbon, increases the
quantity of heterotrophic growth in the system. The heterotrophs outcompete the nitrifiers for oxygen and may significantly alter the activity of the nitrifying communities and the
ability of the system to remove ammonia. The optimum C/N
ratio for nitrification exists at the end of the carbon treatment
train, or in the last lagoon, where temperatures can drop as
low as 1
C for extended periods of time. Installing the nitrifying MBBR unit after the last pond will allow the upgrade
system to benefit from low C/N ratios, which will reduce the
size and operational costs of the upgrade unit and prevent
carbon removal redundancy in the downstream ponds. Longterm effects of exposure to 1
C on MBBR nitrifying biofilm,
however, have yet to be investigated or quantified.
Up to as recent as 20 years ago, autotrophic bacteria
responsible for nitrification were not believed to experience a
population shift as temperatures decrease, where the same
bacterial population was believed to be responsible for nitrification at warm and cold temperatures (Wijffels et al., 1995).
More recently however, it has been observed that the
ammonia oxidizing bacteria (AOB) populations, which coexist in wastewater, demonstrate shifts in their community
populations with a change in temperature (Hallin et al., 2005;
Layton et al., 2005; Siripong and Rittmann, 2007). Nitrite
oxidizing bacteria (NOB), however, have been shown to not
experience a population shift during temporal changes but
rather during substrate scarcity (Gieseke et al., 2003; Haseborg
et al., 2010; Huang et al., 2010). Nitrosomonas, co-existing with
Nitrosospira, are considered to be the dominant AOB population in wastewaters with the two common genera of NOB in
wastewater being Nitrobacter and Nitrospira (Siripong and
Rittmann, 2007; Ducey et al., 2010; Park et al., 2008;
Rodriguez-Caballero et al., 2012; Daims et al., 1999; Wagner
et al., 2002).
The aim of this work was to investigate the response of
nitrifying biofilm, the embedded biomass and population
shifts of the embedded bacterial communities as laboratory
MBBR treatment systems are exposed to cold temperatures.
Particularly, this study aims to characterize the effects of
long-term exposure to 1
C on the thickness and morphology
of MBBR nitrifying biofilms, the viability of cells in the biofilm
and bacterial community shifts.

2.

Methods and materials

2.1.

Biofilm samples

The nitrifying biofilm analyzed in this study was sampled

from two laboratory scale MBBRs operating under continuous
flow conditions without recycle. Both reactors were operated
under the same loading and operational conditions
throughout the study. The two laboratory MBBR reactors were
cylindrical in shape with a liquid volume of 2 L. The reactors
were fed from the same feed tank and were operated with a
hydraulic retention time (HRT) of 4 h during 20
C experiments
and during the first month of operation at 1
C. The HRT was
subsequently increased to 6 h for the remainder of experiments at 1
C to conserve the feed while maintaining a
traditional HRT at lower kinetics. The pH of both reactors was
maintained between 7 and 8 and the dissolved oxygen (DO)
concentration was maintained between 7 and 8 mg L1, with
the slightly lower DO concentrations occurring at warmer
temperatures. Both reactors were operated at 20
C for six
weeks and then acclimatized to 1
C and operated at 1
C for
four months. Synthetic wastewater (SWW) with an ammonia
concentration of 22 mg-N L1 was used to feed the reactors
(modified version of Delatolla et al., 2009): (NH4)2SO4
(104 mg L1), NaHCO3 (286 mg L1), FeSO4$7H2O (5 mg L1),
KH2PO4 (80 mg L1), CaCl2$2H2O (29 mg L1), and MgSO4$7H2O
(71 mg L1). Trace nutrients included molybdenum, copper,
cobalt and zinc.
No organic carbon sources were added as the motivation of
this work is to investigate the response of MBBR nitrifying
biofilm and biomass under temperature conditions expected
in upgrading units installed after passive treatment systems
in northern countries where the readily degradable carbon
concentrations are expected to be low. Furthermore, the
influent carbon sources was set to zero to best investigate
attached growth kinetics of nitrification in MBBR systems with
limited effects due to heterotrophic growth. It should be noted
that nitrifying MBBR units installed after MBBR carbon
removal or conventional carbon removal treatment units will
traditionally not reach temperatures as low as 1
C in Canada,
nor will integrated treatment systems that combine conventional treatment and MBBR carriers in a single treatment
system reach as low as 1
C in Canada.
K3 anoxkaldnes carriers, with specific surface areas of
500 m2/m3 and a fill percentage of 35e40% by volume, were
used as substratum for biofilm growth (Rusten et al., 2006). All
carriers were inoculated at an MBBR nitrifying pilot plant that
was operated at the Vaudreuil, QC, Canada lagoon wastewater
treatment plant. The pilot plant was fed with lagoon effluent
and operated as a nitrifying unit prior to harvesting of carriers
for this study. The biofilm in reactor 1 (R1) was slightly more
mature than reactor 2 (R2) at the start of the study due to
differences in operation times in the laboratory reactors prior
to the start of the study. R1 was operated for approximately six
months longer than R2 prior to the 20
C experimental phase.
The biofilm of reactor R2 was thus slightly less developed and
exhibited a thinner biofilm at the start of the study with the
biofilm developing throughout the study. The initial biofilm on
the R1 carriers was darker in color and covered more carrier

w a t e r r e s e a r c h 4 9 ( 2 0 1 4 ) 2 1 5 e2 2 4

surface area than R2. The end of the start-up phase was
defined by steady ammonia removal rates in the laboratory
reactors at 20
C.

2.2.

Nitrification kinetics

Nitrification rates were measured at 20
C and during expo

sure time to 1
C. Ammonia (NH
4 -N), nitrite (NO2 -N), and ni-N)
concentrations
were
measured
using
standard
trate (NO
3
methods (APHA, 1995). Ammonia removal rates of the laboratory MBBRs are expressed in this work relative to the surface
area of the carriers (Zhang et al., 2013) and the average
removal rates at 1
C are presented as the percent removal
relative to the removal rate at 20
C. The average removal rate
values presented in this study are based on a minimum of four
influent and effluent ammonia concentrations measured over
a 48 h period. All nitrogen constituents were measured in
triplicate.

2.3.

VPSEM image acquisition & analysis

Variable pressure electron scanning microscope (VPSEM) imaging was used for direct imaging of biofilm specimens
without pre-treatment; thus eliminating the destructive effects of traditional SEM pre-treatment. Samples were
analyzed at a pressure of 40 Pa using a Vega II-XMU SEM
(Tescan USA Inc., Cranberry, PA). Images were captured at
magnifications between 20 and 2000. Biofilm thicknesses
were directly measured using Altas image processing software (Tescan USA Inc., Cranberry, PA). Twenty thickness
measurements along with morphological observational images were acquired at random locations on four carriers for
each reactor at each experimental phase.

biofilm detachment protocols. The FilmTracer LIVE/DEAD

biofilm viability kit (Life Technologies, Burlington, Canada)
was used to analyze the viability of the samples and specifically quantify the live and dead cells in the biofilm. The K3
carriers were cut at various sections to enable biofilm attached
to the inner portion of the carrier to be visualized using the
CLSM. Random locations of the biofilm attached to the carrier
were analyzed with the biofilm remaining attached to the
substratum.

2.5.

CLSM image acquisition & analysis

Fluorescent stained biofilm samples were analyzed using an

LSM 5 Pa confocal microscope (Carl Zeiss Canada Ltd., Toronto, Canada) equipped with an Argon laser (488 nm, 514 nm)
and a HeNe laser (543 nm). A 36 oil immersion lens was used
to capture 1.0 mm thick, 214  143 mm optical sections. Four
image stacks, 20 total images, per reactor were acquired and
analyzed during each experimental phase. Stacks of images
were taken at random locations on the biofilm, where each
stack contained five images with maximum vertical depths of
80 mm to ensure that stain penetration did not limit the
identification of the cells and to capture the upper portion of
the biofilm that is most exposed to substrate and nutrients
(degaard, 2006). The biofilm area of each image was quantified as the portion of the image excluding the pore spaces and
hence the percent coverage of the illuminated cells in the
biofilm was calculated relative to the biofilm area of each
image (Delatolla et al., 2012). NI Vision Assistant 7.1 (LabView
8.0, National Instruments Canada, Vaudreuil-Dorion, Canada)
was used to quantify the percent coverage of live and dead
cells in the nitrifying biofilm.

2.6.
2.4.

217

DNA extraction & amplification

CLSM sample preparation

Confocal laser scanning microscope (CLSM) imaging was used

for this work to capture high-resolution optical images of the
biomass at selective depths in the biofilm. CLSM configuration
did not require the biofilm to be detached from the substratum, hence minimizing loss of biofilm mass due to traditional

DNA was extracted from the biofilm using a FastDNA spin kit
(MP Biomedicals, Santa Ana, CA) in which two mechanical
lysis cycles were performed with a FastPrep Instrument (MP
Biomedicals, Santa Ana, CA) set at speed 6.0 for 40 s. Extracted
DNA was stored at 20
C until it was used for library
construction.

Table 1 e Library construction primers: Paired-end sequencing adapter (green nucleotides); barcodes (underlined
nucleotides); V6 universal primers (red nucleotides, Sundquist et al., 2007); Illumina flow-cell adapters sequence (blue
nucleotides).

218

w a t e r r e s e a r c h 4 9 ( 2 0 1 4 ) 2 1 5 e2 2 4

2.7.

DNA sequencing analysis

The paired-end reads were assembled using the fast length

adjustment of short reads (FLASH) software (Majoc and
Salzberg, 2011) and quality filtered using the fastq_quality_filter command from the Fastx toolkit with a minimum quality
score of 20 over 97% of the sequence. Reads that failed to
overlap or to pass our quality threshold were discarded. Subsequently, the reads were demultiplexed and the barcodes
trimmed using Novobarcode (Goecks et al., 2010). Finally, the
quantitative insights into microbial ecology (QIIME) software
(Majoc and Salzberg, 2011) was used to compute operational
taxonomic units (OTUs) clustering at 97% sequence similarity
by using UCLUST against the Greengenes database (version
4feb2011), the singletons were removed, and the relative
abundance of the bacterial taxa present in the biofilm at 20
C
and 1
C was determined (Caporaso et al., 2010).

2.8.

Statistical analysis

Statistically significant differences in values obtained at 20
C

and 1
C and between the two reactors were tested by
comparing 95% confidence intervals and confirmed using the
t-test and p-values (0.05 signifying statistical relevance).

3.

(82.4  2.5 g N m3 d1 at 20
C and 14.8  3.4 g N m3 d1 at

1
C). The removal rates in R1 were slightly higher than those
in R2, most likely due to natural variances inherent to the
systems, errors inherent to the analytical methods and/or the
biofilm in R1 being more mature than the biofilm in R2. Two
temperature shocks were observed due to malfunctions of the
temperature controlled chamber that housed the MBBR reactors. An increase in removal rate in both reactors was
observed with a rapid increase in temperature.

3.2.

Biofilm morphology

VPSEM imaging was used to measure the thickness of the

biofilm and characterize the morphology of the biofilm
without pretreatment of the samples (Fig. 2). Since conventional MBBR design is based upon the assumption that the
inner surface area of the carrier supports the attachment of
biofilm, only the internal protected surface of the carriers was
investigated for thickness and used to characterize the biofilm
in this study. Images acquired at 90 magnification clearly
showed attached biofilm along the inner surfaces of the carriers, while biofilm attached to the outer surfaces was not
visible at this same magnification due to the very thin nature
of the outer carrier biofilm. The VPSEM images demonstrate
that the biofilm at the corners and ridges of the inner surface
of the carriers, where the biofilm was best protected from
higher intensities of abrasion and erosion, was thicker than
the biofilm along the straight inner surfaces of the carrier
(Fig. 2). The nitrifying biofilm was thus not uniformly thick
and nor was the surface of the biofilm smooth. Throughout
the experiments at 20
C and the long exposure to 1
C, the
morphology of the biofilm qualified from the VPSEM images
did not display any notable changes.
Biofilm thicknesses in R1 were however observed to be
significantly greater than biofilm thicknesses in R2 (Fig. 3).
Again, this was expected since the biofilm in R1 was more
mature at the start of the study as compared to R2. Furthermore, a significant increase in biofilm thickness in both reactors was observed after four months of exposure to 1
C as
compared to 20
C.

Results

R1

R2

Temperature
25

3.1.

Nitrification kinetics

The nitrification kinetics of the laboratory MBBRs during longterm exposure to 1
C are expressed as the average surface
area normalized removal rates compared to 20
C (Fig. 1,
adapted from Hoang et al., in press). Error bars indicating 95%
confidence intervals are too small to be seen in Fig. 1. Nitrogen

mass balances of removed NH
4 -N and produced NO2 -N and




-N
at
20
C
and
1
C
were
consistently
less
than
10%,
with
NO
3


-N
removed
being
slightly
higher
than
NO
-N
and
NO
NH
4
2
3 -N

produced; which was expected since NH4 -N will be preferentially used as a source of nitrogen for cell synthesis (Rusten
et al., 1995). The average removal rates of the two reactors at
1
C after four months of exposure relative to the removal
rates at 20
C were measured to be approximately 18  5.1%

Removal Rate Relative to 20oC (%)

100
90
Steady temperature decline:
acclimatization from 20C to 1C

80

20

70
15

60

50
10

40
30
20

10
0

0
0

50

100

150

Time (d)

Fig. 1 e Average relative ammonia surface area removal

rate of R1 and R2 during long-term 1
C exposure and
during rapid temperature spikes.

Temperature (oC)

DNA amplification was achieved by using a Phusion HighFidelity PCR Master Mix (Thermo Fisher Scientific Inc., Waltham, MA) where a two-step polymerase chain reaction (PCR)
targeting the V6 hyper-variable region of the 16S rRNA was
performed as previously described using a barcoding
approach (Arthur et al., 2012). The primers used in the first and
second PCR reaction are listed in Table 1. Next, the PCR
amplicons were inspected by electrophoresis on a 2% agarose
gel and purified with a Montage PCR96 cleanup kit (EMD Millipore, Billerica, MA). A Quant-iT dsDNA BR Assay Kit (Life
Technologies, Burlington, Canada) was used to quantify the
purified amplicons. Amplicons were then pooled and
sequenced on one lane of an Illumina Hiseq2500 at the center
for applied genomics (TCAG, Toronto, Canada) generating
paired-end reads of 2100 bases (a mean of 901,070  331,232
high quality reads were generated per sample).

w a t e r r e s e a r c h 4 9 ( 2 0 1 4 ) 2 1 5 e2 2 4

219

Fig. 2 e VPSEM images of nitrifying biofilm attached to a single K3 carrier sampled from R1 at 1
C: a) image of biofilm
coverage on the inner portion of the K3 at 316 magnification, b) and c) images displaying biofilm thicknesses on an edge and
on a ridge of the K3 carrier at 390 magnification, respectively, and d) image displaying the morphology of the biofilm at
32000 magnification.

3.3.

Viability of biomass

A CLSM in combination with viability staining was used to

quantify the live and dead cell coverage of biomass at different
depths within the biofilm, where live cells are illuminated
green and dead cells are illuminated red (Fig. 4). No significant
change in biofilm live cell coverage was observed between
experiments at 20
C and after one month of exposure to 1
C.
However, there was a significant increase in live cell coverage
after four months at 1
C compared to 20
C (Fig. 5). Particularly, the live cell coverage was observed to increase by a

R1

R2

250

Thickness (m)

200

150

100

50

0
20oC

1oC (1 month)

1oC (4 months)

Fig. 3 e Average biofilm thickness with 95% confidence

intervals for R1 and R2 throughout the experimental phase.

magnitude of 2.5  1.5% (1.7 times compared to 20
C) in R1

and by 3.9  1.9% (2.1 times compared to 20
C) in R2 after four
months of exposure to 1
C. Dead cell coverage did not show
significant changes throughout the experimental phase, with
average values for both R1 and R2 being 1.4  0.7% at 20
C and
1.0  0.6% at 1
C.

3.4.

Bacterial population

OTUs from all samples identified 25 bacterial phyla. The three

most abundant phyla identified in the two reactors are Proteobacteria, Nitrospirae, and Bacteroidetes at both 20
C and after
four months of exposure to 1
C. The majority of sequences
(43.0e50.6%) in all samples belong to the Proteobacteria
phylum. This phylum includes the class b-Proteobacteria that is
comprised of organic matter decomposing bacteria, AOB,
NOB, and denitrifying bacteria. Interestingly, the phylum
Nitrospirae, which is the phylum of major NOBs, is observed to
be the second major bacterial phylum in the young (R2) biofilm
at both 20
C and 1
C; while in the mature (R1) biofilm,
Nitrospirae is also a major phylum, however it is the fourth
major bacterial phylum at 20
C and 1
C with Bacteroidetes and
Acidobacteria being the second and third most abundant phyla.
In both mature and young biofilms, the percent abundance of
Nitrospirae increases after long-term exposure to 1
C (Table 2).
At the genus level, Nitrosomonas and Nitrosospira (Proteobacteria phylum) are identified as the two AOB genera at 20
C
and after long-term exposure at 1
C, with both reactors

220

w a t e r r e s e a r c h 4 9 ( 2 0 1 4 ) 2 1 5 e2 2 4

Fig. 4 e CLSM images of nitrifying biofilm sampled from R2 at 1
C. Live cells are illuminated green and dead cells are
illuminated red with images being 143 3 143 mm in lateral area: (a) biofilm layer at the top of the biofilm, closest to the
biofilm/liquid interface and (b) biofilm layer 5 mm deeper into the biofilm and hence closer to the carrier. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)

showing a higher relative abundance of Nitrosomonas than

Nitrosospira. Other known major AOB genera, such as Nitrosococcus, are not detected in either reactor. Furthermore,
Nitrospira (Nitrospirae phylum) is identified to be the main NOB
genus at 20
C and after long-term exposure at 1
C in both
reactors. The relative abundance of Nitrospira in the young
biofilm (R2) is higher than R1 at both warm and cold temperatures. At 20
C, Nitrospira accounts for 20% of the total bacteria in R2 where the abundance increased at 1
C to 31%. In
the mature biofilm (R1), Nitrospira abundance is 3.8% at 20
C
and again was observed to increase at 1
C to reach 7.9% (Table
3).
Potential denitrifiers are also detected in all samples where
90% of the genera identified in this group are Nitrosomonas,
Flavobacterium, Pseudomonas, and Rhodoplanes, with Nitrosomonas being a potential AOB or denitrifier that will act as an
AOB in the presence of ammonia (Bock et al., 1995). The denitrifiers account for 5.0% of the bacteria in R1 at 20
C and 10%
at 1
C. Their relative abundance is lower in the young biofilm
(R2) where they show 0.09% and 6.2% of total bacteria at 20
C
and 1
C, respectively.

R1

R2

9.0

Live Cell Coverage (%)

8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0

20oC

1oC (1 month)

1oC (4 months)

Fig. 5 e Average live cell coverage with 95% confidence

intervals for R1 and R2 at each experimental phase.

Anaerobic ammonium oxidation (anammox) bacteria,

mainly represented by the phylum Planctomycetes, were also
among the detected bacteria. Major bacterial genera belong to
the Planctomycetes phylum include Planctomyces, Isosphaera,
Rhodopirellula, and Pirellula. Anammox bacteria represent
0.11e3.8% of the total bacteria. Planctomycetes were detected in
highest relative abundance in R2 at 1
C (3.8%) and was lower
at 20
C (0.83%). The mature biofilm (R1) demonstrated lower
relative abundances of Planctomycetes at both 1
C (0.11%) and
20
C (1.0%).

4.

Discussion

4.1.
Effect of low temperature on MBBR nitrification
kinetics
Previous studies and practical experiences have shown a sharp
decrease in nitrification rates at low temperatures (Sharma and
Ahlert, 1977; Painter and Loveless, 1983; Wessman and
Johnson, 2006; Houweling et al., 2007; Delatolla et al., 2011).
The MBBR ammonia removal rate after long exposure to 1
C in
this study are measured to be 18  5.1% compared to the rate at
20
C. The decrease of ammonia removal rates as temperatures
decrease may be indicative of a loss or death of the nitrifying
bacteria during cold temperature exposure. However, the kinetic data shows that both reactors are able to quickly recover
from the rapid temperature changes and perform at high
removal rates for the remainder of the experiment at 1
C. The
ability of the MBBR reactors to quickly adapt and recover from
two temperature spikes suggests that the nitrifiers are not lost
nor lysed but rather maintained in the biofilm. Both laboratory
MBBRs show an immediate increase in ammonia removal rates
while experiencing two rapid temperature increases. A similar
response was observed during temperature shock experiments
of a nitrifying biological aerated filtration system (Delatolla
et al., 2009). These findings suggest that the nitrifying bacteria
exist at a higher level of activity at higher temperatures and at a

w a t e r r e s e a r c h 4 9 ( 2 0 1 4 ) 2 1 5 e2 2 4

Table 2 e - Percent abundance of the major bacterial

phyla identified.
Bacterial phyla

Proteobacteria
Nitrospirae
Bacteroidetes

Abundance (%)
R1
20
C

R2
20
C

R1
1
C

R2
1
C

51
3.8
25

44
20
10

47
7.9
8.5

43
30
15

lower level of activity at lower temperatures. These results

demonstrate the ability of the MBBR system to perform
consistent nitrification at 1
C over an extended period of time
representative of a northern region winter.

4.2.

Effect of low temperature on nitrifying biofilm

A significant increase in biofilm thickness is observed in both

reactors after four months of exposure to 1
C as compared to
20
C. This observation is consistent with previous studies that
show increases in nitrifying biofilm thicknesses with exposure
to cold temperatures (Delatolla et al., 2009; Bjornberg et al.,
2009). Bjornberg et al. (2009) observed a relatively thin biofilm
during the summer months and an average increase in biofilm
thickness of 40 mm after the winter months in a full-scale MBBR
system. The observed increase in biofilm thickness at colder
temperatures in this previous work may have been due to
increased DO concentrations in the reactors at cold temperatures. Although the concentration of ammonia in this study
was maintained above 5 mg-N L1 in both reactors, which
suggests that DO is the mass transfer limited constituent for
bacterial growth (Gujer and Boller, 1986), the difference in DO
between 20
C and 1
C was relatively small. Hence the significant change in biofilm thickness with exposure to 1
C
observed in both reactors was likely more than an effect of the
slight increase in DO. Particularly, the observed increase in
biofilm thickness is with respect to the overall thickness of the
biofilm and does not imply an increase or change to the depth
of the active biofilm; where the active biofilm depth is restricted
to the volume of the biofilm that is fully penetrated by
ammonia, nitrite and DO and hence participates in nitrification.

4.3.

Effect of low temperature on nitrifying biomass

The increase in viable cells observed in this study occurred in

a biofilm that showed a significant increase in thickness while
the ammonia removal kinetics dropped considerably to an
average of 18  5.1% of the rates observed at 20
C. Therefore,
the observed thicker biofilm and the increase in cell coverage
Table 3 e - Percent abundance of identified AOB and NOB
genera.
Type

AOB
AOB
NOB

Genus

Nitrosomonas
Nitrosospira
Nitrospira

Abundance (%)
R1
20
C

R2
20
C

R1
1
C

R2
1
C

3.7
0.63
3.8

2.5
1.8
20

0.0022
0.00021
7.9

9.4
0.41
31

221

of viable cells after long exposure to cold temperatures demonstrates that a larger quantity of viable cells are promoted at
1
C as compared to 20
C; however based on the observed
decrease in nitrification rates, these cells are significantly less
active with respect to the oxidation of ammonia and nitrite.
Furthermore, the live cell coverage in the younger biofilm
(R2) was similar to that in R1 at 20
C after one month exposure
at 1
C but was significantly greater than R1 after four months
of exposure to 1
C (Fig. 5). It should be noted that throughout
the study and specifically after four months of exposure to
1
C the more mature biofilm in R1 showed significantly
greater biofilm thicknesses than the thicknesses measured in
R2. Nonetheless, both reactors demonstrated similar kinetics
at all temperatures. Hence, the younger and thinner biofilm in
R2 maintained comparable rates of nitrification with respect
to a more mature and thicker biofilm in R1 at all temperatures
in the study by maintaining a larger quantity of viable biofilm
cells per area of biofilm. As such, the operation of the laboratory MBBR reactors demonstrate that a start-up time of 6
weeks at warm temperatures is sufficient to achieve cold
temperature nitrification for a four month period at 1
C.
Furthermore, an extended operational time at warm temperatures, beyond the start-up period of 6 weeks, did not
demonstrate a kinetic advantage during the first four month
period of nitrification at 1
C; nor did the extended operational
time at warm temperatures demonstrate any indications of
the nitrifying biofilm being less susceptible to biofilm loss or
washout. Although viable cell quantities were shown to increase with exposure to cold temperature and differences
were observed between younger and more mature biofilms,
no significant change in dead cell coverage was observed between the two reactors or at the different temperatures of the
study. The ratio of dead cells to viable cells was approximately
0.4  0.3 at 20
C and decreased slightly to 0.2  0.1 at 1
C for
both reactors. The increased biofilm volume and quantity of
viable cells with stagnant percentage coverage of dead cells
suggests evidence of biofilm growth as opposed to cell death
due to cold temperature exposure.
The biofilm and biomass data of this study demonstrate
that the MBBR reactors did not show any loss of biofilm or loss
of viable cell counts as nitrification rates were shown to
decrease during 1
C temperature exposure. The loss of biofilm
and/or loss of active nitrifying cells during periods of lower
kinetics can be indicative that MBBR systems would be at risk
of losing nitrification during long term 1
C exposure.
Contrarily the biofilm and viable cell counts were shown to
increase in the laboratory MBBR reactors, indicating that
MBBR units have shown the potential of maintaining a stable
and robust biofilm with active embedded nitrifying biomass
during long term 1
C exposure. Hence, these findings, based
on controlled laboratory conditions, support subsequent
studies of optimizing these units for cold temperature
nitrification.

4.4.
Nitrification kinetics normalized to biofilm and
biomass at 1
C
The application of VPSEM and CLSM imaging in combination
with viability staining provided valuable information with
respect to the response of the MBBR biofilm and biomass to

222

w a t e r r e s e a r c h 4 9 ( 2 0 1 4 ) 2 1 5 e2 2 4

exposure to cold temperature. The biofilm thickness data

measured in this study were integrated with measured MBBR
ammonia removal rates to calculate nitrification kinetics
normalized to the approximate total volume of biofilm in the
reactor as opposed to the conventional method of normalizing
the kinetics to the total surface area of the carriers in the reactors. Hence, based on the biofilm thickness measurements
acquired in this study, a biofilm volume ammonia removal
rate (BVRRA) was calculated according to the following
equation:
BVRRA CIN  COUT $Q=LAVG $Vb $a

(1)

where COUT and CIN are ammonia concentrations of the outlet

and inlet (mg-N L1), respectively, Q is the flow rate in and out
of the reactors (L/d), LAVG is the average biofilm thickness (m),
Vb is the media bed volume (m3), and a is the specific surface
area of the carrier (m2/m3). Furthermore, using the results
obtained from CLSM analysis, ammonia removal rates were
also established relative to the viable cell coverage (VCRRA)
according to the following equation:
VCRRA CIN  COUT $Q=VC$LAVG $Vb $a

(2)

where VC is the percent cell coverage of viable cells (% of live

cell coverage). The BVRRA and VCRRA of both laboratory
MBBRs after four months exposure to 1
C relative to the
BVRRA and VCRRA at 20
C are compared to the conventional
surface area removal rates in Table 4. A sharp decrease in
BVRRA and VCRRA is observed when both reactors are exposed
to cold temperatures; the BVRRA and VCRRA at 1
C was
approximately 10 times less than the BVRRA and VCRRA at
20
C.
The average BVRRA of the two reactors at 20
C and 1
C
were measured to be approximately 3.1  0.2 kg N m3 d1 and
0.36  0.03 kg N m3 d1, respectively. The average VCRRA of
the two reactors at 20
C and 1
C were measured to be
approximately 94  5 kg N m3 d1 and 7.0  0.4 kg N m3 d1,
respectively. Both the BVRRA and VCRRA at 1
C relative to
20
C were significantly less than the carrier surface area
specific removal rates at 1
C relative to 20
C (Table 4). The
BVRRA at 1
C corresponds to a thicker biofilm and thus higher
biofilm volume along with a much lower removal rate as
compared to the BVRRA calculated at 20
C, which corresponds
to a thinner biofilm and larger removal rate. Thus the larger
biofilm thickness during cold temperatures increased the
difference between the removal rates normalized to the biofilm volume at 20
C and 1
C. Similarly, the VCRRA at 1
C
corresponds to a higher percentage of live cells but a much
lower removal rate as compared to those at 20
C. Hence the
BVRRA and VCRRA values shown in Table 4 confirm that

Table 4 e - Average and standard deviations of relative

ammonia surface area removal rate, BVRRA and VCRRA
after four months exposure time to 1
C.

R1
R2

Removal rates
at 1
C relative
to 20
C (%)

BVRRA at 1
C
relative
to 20
C (%)

VCRRA at 1
C
relative to 20
C (%)

19  5.5
17  4.7

12  6.4
11  7.7

86
75

thinner nitrifying biofilm with fewer cells and higher activity

levels exist at warmer temperatures and thicker nitrifying
biofilm with more cells and lower activity levels exist at cold
temperatures in MBBR systems investigated in this study.

4.5.

Effect of low temperature on bacterial population

The AOB and NOB genera identified in this study are in general
agreement with those found in past studies investigating
microbial communities of nitrifying biofilms (Daims et al.,
1999; Schramm et al., 1999; Gieseke et al., 2001, 2003; Okabe
et al., 2002; Park et al., 2008; Vejmelkova et al., 2012). This
study demonstrates that Nitrosomonas and Nitrosospira coexist at both 20
C and 1
C. The decrease in relative abundance of Nitrosospira observed in both mature (R1) and young
(R2) biofilms after long-term exposure to 1
C in this work is
supported by previous studies that demonstrate that Nitrosospira is more sensitive to temperature than Nitrosomonas
(Park et al., 2008). The observed increase in relative abundance
of Nitrosomonas in the young biofilm and the decrease in
relative abundance in the mature biofilm during exposure to
cold temperatures may be related to differences in bacterial
communities in the young and mature biofilm and their subsequent response to exposure to cold temperatures.
Previous studies have shown that Nitrobacter and Nitrospira
are the two common genera of NOBs in wastewater (Siripong
and Rittmann, 2007; Daims et al., 1999; Wagner et al., 1996),
with recent work demonstrating that Nitrospira, not Nitrobacter, are the dominant nitrite oxidizers in wastewater systems (Daims et al., 1999; Matsumoto et al., 2007; Zeng et al.,
2009). In particular, previous work showed that Nitrobacter
was dominant when the available substrate was abundant
and Nitrospira thrived when substrate concentrations were
scarce or when the system experienced temporary nitrite elevations (Gieseke et al., 2003; Haseborg et al., 2010; Huang
et al., 2010; Schramm et al., 1999; Blackburne et al., 2007). No
NOB population shift was observed in this study with Nitrospira being identified as the genera responsible for nitrite
oxidation at both 20
C and 1
C, while Nitrobacter was not
detected in either reactor throughout the entire experimental
phase. Nitrospira was not only shown to be the only identified
NOB genera in both young and mature biofilms at 20
C and
during long exposures to 1
C, but its relative abundance was
also observed to increase in both reactors during exposure to
cold temperatures. Hence, these observations support the kinetic and microscopic findings that acclimatized nitrifying
MBBRs are capable of maintaining or increasing the relative
abundance of the NOB population during long exposure to
very cold temperatures.
The non-nitrifying bacteria identified in this study coincide
with those identified in past studies investigating activated
sludge and biofilm systems treating wastewater (Okabe et al.,
2002; Benedict and Carlson, 1971; Wagner and Loy, 2002).
Numerous heterotrophic species belonging to the Bacteroidetes, Chloroflexi, Proteobacteria, and Acidobacteria phyla were
identified in this study along with the nitrifying bacteria
belonging to the Proteobacteria (AOB) and Nitrospirae (NOB)
phyla. It has been reported that species of the Proteobacteria
phylum form dense biofilm layers close to the surface of
substrata and provide a growth matrix for nitrifiers (Ducey

w a t e r r e s e a r c h 4 9 ( 2 0 1 4 ) 2 1 5 e2 2 4

et al., 2010; Wagner and Loy, 2002). Numerous bacterial

strands have been identified as cryoprotective exopolysaccharides (EPS) producers that when mixed with Escherichia
coli were shown to increase the survival rate during freeze and
thaw cycles (Sung and Joung, 2007). As such, it has been previously hypothesized that the presence of these protective
non-nitrifiers can actually improve nitrification rates at cold
temperatures (Ducey et al., 2010). Thus, a symbiotic relationship between nitrifiers and the identified non-nitrifiers in this
study could potentially offer cold temperature resistance in
biofilms and be a contributing factor to the maintenance of
nitrification during long exposure to cold temperatures.

5.

Conclusion

The application of VPSEM and CLSM in combination with

viability staining along with DNA sequencing analyses provided valuable information with respect to the response of
MBBR biofilm and biomass to exposure to very cold temperature for long duration. The study shows an increase in biofilm
thickness and live cell coverage after 4 months of exposure to
1
C in MBBR reactors with young and mature biofilm, while
rates of nitrification decreased significantly. The calculated
dead cell coverage on the other hand did not show significant
differences at 20
C and 1
C and thus supports evidence of
biofilm growth as opposed to cell death due to cold temperature exposure. Hence, this study observed a greater quantity
of nitrifying bacteria with lower activities and a greater
quantify of biofilm attached to MBBR carriers at cold temperatures as compared to warm temperatures.. Using DNA
sequencing analysis, Nitrosomonas and Nitrosospira (ammonia
oxidizers) as well as Nitrospira (nitrite oxidizer) were identified
and no population shift was observed between 20
C and after
long-term exposure to 1
C.
The biofilm and biomass data of this study demonstrates
that the laboratory MBBR reactors maintained a stable and
robust biofilm with active embedded nitrifying biomass,
without indication of biofilm loss or washout, during long
term exposure to 1
C. The kinetic data also showed that the
MBBR reactors were responsive to shock temperature increases and that an extended operational time at warm temperatures, beyond the start-up period of 6 weeks, is neither
kinetically advantageous nor advantageous with respect to
producing biofilms less susceptible to biofilm loss or washout.
Hence, this laboratory study demonstrates the potential of
MBBR systems for 1
C treatment and therefore supports
subsequent studies of optimizing these units for cold temperature nitrification using real wastewater.

Acknowledgments
The authors are grateful for financial support from the National Sciences and Engineering Research Council of Canada
(NSERC) and Veolia Water. The authors graciously thank
Jianqun Wang (Carleton University), Raed Hanania and Kim
Wong (University of Ottawa) for their help with VPSEM and
CLSM image acquisitions.

223

references

APHA, A., WEF (Eds.), 1995. Standard Methods for the

Examination of Water and Wastewater, 19 ed.
A.P.H.A.A.W.W.A.W.E. Federation, Washington DC, USA.
Arthur, J.C., Perez-Chanona, E.., Muhkbauer, M., Tomkovich, S.,
Uronis, J.M., Fan, T.J., Campbell, J., Abujamel, T., Dogan, B.,
Rogers, A.B., Rhodes, J.M.., Stintzi, A., Simpson, K.W.,
Hansen, J.J., Keku, T.O., Fodor, A.A., Jobin, C., 2012. Intestinal
inflammation targets cancer-inducing activity of the
microbiota. Science 338, 120e123.
Benedict, D.A., Carlson, D.A., 1971. Aerobic heterotrophic bacteria
in activated sludge. Water Res. 5, 1023e1030.
Bjornberg, C., Lin, W., Zimmerman, R., 2009. Effect of temperature
on biofilm growth dynamics and nitrification kinetics in a fullscale MBBR system. In: Proceedings of the 82nd Annual Water
Environment Federation Technical Exposition and
Conference, Orlando, Florida, Oct. 17e21. Water Environment
Federation, Alexandria, Virginia, pp. 4407e4426.
Blackburne, R., Vadivelu, V.M., Yuan, Z.G., Keller, J., 2007. Kinetic
characterisation of an enriched Nitrospira culture with
comparison to Nitrobacter. Water Res. 41 (14), 3033e3042.
Bock, E., Schmidt, I., Stuven, R., Zart, D., 1995. Nitrogen loss
caused by denitrifying Nitrosomonas cells using ammonium or
hydrogen as electron donors and nitrite as electron acceptor.
Arch. Microbiol. 163, 16e20.
Canada Gazette, July 18 2012. Wastewater Systems Effluent
Regulations. Part II, vol. 146, No. 15.
Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K.,
Bushman, F.D., Costello, E.K., Fierer, N., Pena, A.G.,
Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T.,
Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A.,
McDonald, D., Muegge, B.D., Pirrung, M., Reeder, J.,
Sevinsky, J.R., Turnbaugh, P.J., Walters, W.A., Widmann, J.,
Yatsunenko, T., Zaneveld, J., Knight, R., 2010. QIIME allows
analysis of high-throughput community sequencing data. Nat.
Methods 7, 335e336.
Daims, H., Bruhl, A., Amann, R., Schleifer, K.H., Wagner, M., 1999.
The domain-specific probe EUB338 is insufficient for the
detection of all bacteria: development and evaluation of a
more comprehensive probe set. Syst. Appl. Microbiol. 22,
434e444.
Delatolla, R., Tufenkji, N., Comeau, Y., Gadbois, A., Lamarre, D.,
2009. Kinetic analysis of attached growth nitrification in cold
climates. Water Sci. Technol. 60 (5), 1173e1184.
Delatolla, R., Tufenkji, N., Comeau, Y., Gadbois, A., Lamarre, D.,
2011. Investigation of laboratory-scale and pilot-scale
attached growth ammonia removal kinetics at cold
temperature and low influent carbon. Water Qual. Res. J. Can.
45 (4), 427e436.
Delatolla, R., Tufenkji, N., Comeau, Y., Gadbois, A., Lamarre, D.,
Berk, D., 2012. Effects of long exposure to low temperatures on
nitrifying biofilm and biomass in wastewater treatment.
Water Environ. Res. 84 (4), 328e338.
Di Trapani, D., Christensson, M., Torregrossa, M., Viviani, G.,
Odegaard, H., 2013. Performance of hybrid activated sludge/
biofilm process for wastewater treatment in a cold climate
region: influence of operating conditions. Biochem. Eng. J. 77,
214e219.
Ducey, T.F., Vanotti, M.B., Shriner, A.D., Szogi, A.A., Ellison, A.Q.,
2010. Characterization of a microbial community capable of
nitrification at cold temperature. Bioresour. Technol. 101,
491e500.
Gieseke, A., Purkhold, U., Wagner, M., Amann, R., Schramm, A.,
2001. Community structure and activity dynamics of nitrifying
bacteria in a phosphate-removing biofilm. Appl. Environ.
Microbiol. 67 (3), 1351e1362.

224

w a t e r r e s e a r c h 4 9 ( 2 0 1 4 ) 2 1 5 e2 2 4

Gieseke, A., Bjerrum, L., Wagner, M., Amann, R., 2003. Structure
and activity of multiple nitrifying bacterial populations coexisting in a biofilm. Environ. Microbiol. 5 (5), 355e369.
Goecks, J., Nekrutenko, A., Taylor, J., 2010. Galaxy: a
comprehensive approach for supporting accessible,
reproducible, and transparent computational research in the
life sciences. Genome Biol. 11, R86.
Gujer, W., Boller, M., 1986. Design of nitrifying tertiary trickling
filter based on theoretical concepts. Water Res. 20, 1353e1367.
Hallin, S., Lydmark, P., Kokalj, S., Hermansson, M., Sorensson, F.,
Jarvis, A., Lindgren, P.E., 2005. Community survey of
ammonia-oxidizing bacteria in full-scale activated sludge
processes with different solids retention time. J. Appl.
Microbiol. 99, 629e640.
Haseborg, E., Zamora, T.M., Frohlich, J., Frimmel, F.H., 2010.
Nitrifying microorganisms in fixed-bed biofilm reactors fed
with different nitrite and ammonia concentrations. Bioresour.
Technol. 101 (6), 1701e1706.
Hoang, V., Delatolla, R., Laflamme, E., Gadbois, A., 2013. An
investigation of MBBR nitrification during long term exposure
to cold temperatures. Water Environ. Res. (in press).
Houweling, D., Monette, F., Millette, L., Comeau, Y., 2007.
Modelling nitrification of a lagoon effluent in moving-bed
biofilm reactors. Water Qual. Res. J. Can. 42 (4), 284e294.
Huang, Z., Gedalanga, P.B., Avapathanagul, P., Olsen, B.H., 2010.
Influence of physiochemical and operational parameters on
Nitrobacter and Nitrospira communities in an aerobic
activated sludge bioreactor. Water Res. 44, 4351e4358.
Layton, A.C., Dionisi, H., Kuo, H.W., Robinson, K.G., Garrett, V.M.,
Meyers, A., Sayler, G.S., 2005. Emergence competitive
dominant ammonia-oxidizing bacterial populations in a fullscale industrial wastewater treatment plant. Appl. Environ.
Microbiol. 71, 1105e1108.
Majoc, T., Salzberg, S.L., 2011. FLASH: fast length adjustment of
short reads to improve genome assemblies. Bioinformatics 27,
2957e2963.
Matsumoto, S., Terada, A., Aoi, Y., Tsuneda, S., Alpkvist, E.,
Picioreanu, C., van Loosdrech, M.C.M., 2007. Experimental and
simulation analysis of community structure of nitrifying
bacteria in a membrane-aerated biofilm. Water Sci. Technol.
55, 283e290.
Metcalf and Eddy, 2003. Wastewater Engineering: Treatment and
Reuse, fourth ed. McGraw-Hill, New York.
degaard, H., 2006. Innovations in wastewater treatment: the
moving bed biofilm process. Water Sci. Technol. 9, 17e33.
Okabe, S., Naitoh, H., Satoh, H., Watanabe, Y., 2002. Structure and
function of nitrifying biofilms as determined by molecular
techniques and the use of microelectrodes. Water Sci.
Technol. 46 (1), 233e241.
Painter, H.A., Loveless, J.E., 1983. Effect of temperature and pH
value on the growth-rate constants of nitrifying bacteria in the
activated-sludge process. Water Res. 17, 237e248.
Park, J.J., Byun, I.G., Park, S.R., Park, T.J., 2008. Nitrifying bacterial
communities and its activities in aerobic biofilm reactors
under different temperature conditions. Korean J. Chem. Eng.
25, 1448e1455.
Rodriguez-Caballero, A., Hallin, S., Pahlson, C., Odlare, M.,
Dahlquist, E., 2012. Ammonia oxidizing bacterial community
composition and process performance in wastewater plants

under low temperature conditions. Water Sci. Technol. 65 (2),

197e204.
Rusten, B., Hem, L.J., degaard, H., 1995. Nitrification of municipal
wastewater in moving-bed biofilm reactors. Water Environ.
Res. 67 (1), 75e86.
Rusten, B., Eikebrokk, B., Ulgenes, Y., Lygren, E., 2006. Design and
operations of the kaldnes moving bed biofilm reactors.
Aquacult. Eng. 24, 322e331.
Schramm, A., De Beer, D., Van Den Heuvel, J.C., Ottengraf, S.,
Amann, R., 1999. Microscale distribution of populations and
activities of Nitrosospira and Nitrospira spp. along a
macroscale gradient in a nitrifying bioreactor: quantification
by in situ hybridization and the use of microsensors. Appl.
Environ. Microbiol. 65 (8), 3690e3696.
Sharma, B., Ahlert, R.C., 1977. Nitrification and nitrogen removal.
Water Res. 11, 879e925.
Siripong, S., Rittmann, B.E., 2007. Diversity study of nitrifying
bacteria in full-scale municipal wastewater treatment plants.
Water Res. 41, 1110e1120.
Sundquist, A., Bigdeli, S., Jalili, R., Druzin, M.L., Waller, S.,
Pullen, M.K., El-Sayed, Y., Taslimi, M.M., Batzoglou, S.,
Ronaghi, M., 2007. Bacterial flora-typing with targeted, chipbased Pyrosequencing. BMC Microbiol. 7 (108), 1e11.
Sung, J.K., Joung, H.Y., 2007. Cryoprotective properties of
exopolysaccharide (P-21653) produced by the Antarctic
bacterium, Pseudoalteromonas arctica KOPRI 21653. J.
Microbiol. 45, 510e514.
Vejmelkova, D., Sorokin, D.Y., Abbas, B., Kovaleva, O.L.,
Kleerebezem, R., Kampschreur, M.J., Muyzer, G., van
Loosdrecht, M.C.M., 2012. Analysis of ammonia-oxidizing
bacteria dominating in lab-scale bioreactors with high
ammonium bicarbonate loading. Environ. Biotechnol. 93,
401e410.
Wagner, M., Loy, A., 2002. Bacterial community composition and
function in sewage treatment systems. Biotechnology 13,
218e227.
Wagner, M., Rath, G., Koops, H.P., Flood, J., Amann, R., 1996. Insitu analysis of nitrifying bacteria in sewage treatment plants.
Water Sci. Technol. 34 (1), 237e244.
Wagner, M., Loy, A., Nogueira, R., Purkhold, U., Lee, N., Daims, H.,
2002. Microbial community composition and function in
wastewater treatment plants. Antonie Van Leeuwenhoek 81,
665e680.
Wessman, F.G., Johnson, C.H., 2006. Cold Weather Nitrification of
Lagoon Effluent Using a Moving Bed Biofilm Reactor (MBBR)
Treatment Process. WEFTEC, pp. 4738e4750.
Wijffels, R.H., Englund, G., Hunik, J.H., Leenen, E.J.T.M., Bakketun, A.,
Gunther, A., Obon de Castro, J.M., Tramper, J., 1995. Effects of
diffusion limitation on immobilized nitrifying microorganisms
at low temperatures. Biotechnol. Bioeng. 45, 1e9.
Zeng, W., Zhang, Y., Li, L., Peng, Y.Z., Wang, S.Y., 2009. Control
and optimization of nitrifying communities for nitritation
from domestic wastewater at room temperatures. Enzyme
Microb. Technol. 45 (3), 226e232.
Zhang, S., Wang, Y., He, W., Wu, M., Xing, M., Yang, J., Gao, N.,
Yin, D., 2013. Responses of biofilm characteristics to variations
in temperature and NH
4 -N loading in a moving-bed biofilm
reactor treating micro-polluted raw water. Bioresour. Technol.
131, 365e373.