World J Microbiol Biotechnol (2010) 26:19811990

DOI 10.1007/s11274-010-0382-y

ORIGINAL PAPER

Characterization of microbial community in an aerobic moving

bed biofilm reactor applied for simultaneous nitrification
and denitrification
Bo Fu Xiaoyi Liao Lili Ding Hongqiang Ren

Received: 4 January 2010 / Accepted: 2 March 2010 / Published online: 13 March 2010
Springer Science+Business Media B.V. 2010

Abstract A continuous-flow moving bed biofilm reactor

(MBBR) under aerobic conditions was established for
simultaneous nitrification and denitrification (SND), and
microbial communities were investigated by a combination
of denaturing gel gradient electrophoresis (DGGE) and
fluorescence in situ hybridization (FISH). DGGE analysis
has revealed more similar microbial community structures
formed in the biofilms with more similar carbon nitrogen
(C/N) ratios. FISH analysis shows that the dominance of
both Betaproteobacteria ammonia-oxidizing bacteria and
Nitrospira-like nitrite-oxidizing bacteria were negatively
correlated to C/N ratios. Sequence analysis of DGGE bands
has indicated the presence of anoxic denitrifying bacteria
Agrobacterium tumefaciens and Rhizobium sp., suggesting
that the oxygen gradient inside the biofilm may be responsible for the mechanism of SND in aerobic MBBRs. The
study confirms that appropriate control of microbial community structure resulting from optimal C/N ratio is beneficial in improving SND, thus optimizing nitrogen removal
in aerobic MBBR. The established SND-based MBBR can
save operation space and time in comparison to the traditional nitrogen removal process, and might be very attractive
for future practical applications.
Keywords Biofilm  Microbial community 
Simultaneous nitrification and denitrification (SND) 
C/N ratio

B. Fu  X. Liao  L. Ding  H. Ren (&)

State Key Laboratory of Pollution Control and Resource Reuse,
School of the Environment, Nanjing University,
210093 Nanjing, China
e-mail: hqren@nju.edu.cn

Introduction
Nitrogen removal from wastewaters has attracted increasing attention due to strict environmental regulations on
nitrogen discharge. Biological treatment is one of the most
economical processes for nitrogen removal. While conventional biological nitrogen removal processes usually
employ at least two separate reactors for aerobic nitrification and anoxic denitrification, biofilm systems do reveal
some advantages on eliminating nitrogen in a single bioreactor (Helmer and Kunst 1998; Luostarinen et al. 2006).
Moving bed biofilm reactors (MBBRs) exposed to alternating anoxic and aerobic conditions have been proved to
be feasible for nitrogen removal (Rusten et al. 1994; Pastorelli et al. 1997; Luostarinen et al. 2006), whereas
nitrogen removal based on simultaneous nitrification and
denitrification (SND) in single aerobic MBBR with continuous operation has not been well studied.
A better understanding of microbial ecology in the biofilm
community is essential for improving reactor performance
and control. The available literature shows that changing the
C/N ratio of wastewater can affect nitrogen removal in the
bioreactors, such as bench-scale biological aerated filter
(BAF; Ohashi et al. 1995), fluidized-bed bioreactor (Xing
et al. 2000) and SND-based sequencing batch reactor (SBR;
Chiu et al. 2007). However, little investigation has been
carried out to characterize the microbial community in aerobic MBBR applied for SND. One of the difficulties in
exploring microbial ecology in complex biofilms is that pure
cultures of many important microorganisms cannot be
obtained by conventional microbiological techniques. Fortunately, the development of the molecular biological tools
has made it possible to characterize the microbial communities involved in the wastewater. In addition, denaturing
gradient gel electrophoresis (DGGE) and fluorescent in situ

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1982

hybridization (FISH) techniques are powerful tools for

comparison and quantification of the changes in the microbial composition under different operation conditions (Sanz
and Kochling 2007). The objectives of the present study
were: (1) to investigate the microbial communities in aerobic
MBBR applied for SND at different C/N ratios by a combination of DGGE and FISH, and (2) to provide experimental
evidence on the microbial diversity and the nitrifying bacteria involved in nitrogen removal for reactor performance
optimization. The mechanisms of SND in aerobic MBBR
have also been discussed.

World J Microbiol Biotechnol (2010) 26:19811990

analysed immediately after filtering through 0.45 lm filter

paper. Soluble COD, ammonium (NH4N), nitrate (NO2
N) and nitrite (NO3N) concentrations were measured
according to the Standard Method (APHA 1998).
Biofilm sampling
Carriers taken from the reactor were kept in 1.0 mM phosphate buffer solution (PBS). The biofilm was detached from
carriers and homogenized by ultrasonic for 10 min. The
carriers were subsequently washed in PBS for three times to
collect cells by centrifuging at 12,000g for 10 min at 4C.

Materials and methods

DNA extraction, PCR and DGGE
MBBRs operation
Three identical plexiglass columns (10 cm in diameter and
40 cm in height), each with a working volume of 3.1 l, were
used as MBBRs for biofilm formation. The reactors were
inoculated with activated sludge from Suojincun municipal
wastewater treatment plant (Nanjing, China). Polyethylene
carriers (Jiangsu Yulong E. P. Co., Ltd., China) used in this
study had a density of 0.940.98 kg/m3, a specific surface
area of 500 m2/m3 and void volume of 0.95 m3/m3, occupying 30% of the reactor volume. They were shaped like
small cylinders, about 25 mm in diameter and 10 mm in
height, with longitudinal fins on the outside. The pH was
automatically maintained between 7.0 and 8.0 by addition of
a NaOH-NaHCO3 solution. MBBRs were operated at a
constant temperature of 24 1C with dissolved oxygen
(DO) concentrations ranging from 3.0 to 4.0 mg/l. Prepared
wastewater was continuously pumped into the lab-scale
MBBRs with the flow rate of 7.44 l/day, resulting in the
theoretical hydraulic retention time (HRT) of 10 h.
Synthetic wastewater containing different glucose concentrations, i.e. 500, 1,000 and 1,500 mg COD/l, were fed
continuously to the three MBBRs (M1, M2 and M3). Prior
to the experimental phase, the MBBRs were operated for at
least 35 days with approximate initial COD:N:P ratio of
100:5:1 (nitrogen as NH4Cl and phosphorus as KH2PO4),
allowing time for biofilm formation. The ammonia concentrations in the three bioreactors gradually increased to
the same final concentration of 100 mg/l, resulting in five
different C/N ratios of 4.5 (M1), 6.0 (M1), 8.9 (M2), 11.9
(M2) and 13.4 (M3). Trace elements including Ca, Mg, Cu,
Fe, Co, Mn and Zn were added to the influent.
Chemical analysis methods
During the study, manual grab samples were collected from
the effluent of each reactor every day. The samples were

123

The wet samples were then freeze-dried, ground with liquid

nitrogen, and stored at -20C. The genomic DNA in dry
samples was extracted following the protocol described by
Miller et al. (1999). For DGGE analysis, the V3 region of
the bacterial 16S rRNA gene was amplified using Bac341F
with a GC clamp at its 50 end and Bac518R (Muyzer et al.
1993; Liang et al. 2007) with the following protocol: 94C
for 5 min, 30 cycles of 94C for 30 s, 65C for 30 s, 72C
for 1 min, and a final elongation cycle at 72C for 10 min.
The size and amount of PCR products were estimated by
1.2% agarose gel (w/v) electrophoresis and ethidium bromide staining.
Denaturing gel gradient electrophoresis was performed
using the DCodeTM universal mutation detection system
(BioRad, Hercules, CA, USA). PCR products were separated on 810% (wt/vol) gradient polyacrylamide gels,
containing a 4570% denaturant gradient (100% denaturant is 7 M urea and 40% (v/v) deionized formamide).
Electrophoresis was performed at 80 V for 15 h at a constant temperature of 60C. The gel was stained with ethidium bromide for 20 min and visualized with ultraviolet
transillumination. Specific bands were manually excised
from the gel, and used as a template in a re-amplification
using the primers Bac341F and Univ518R for sequencing.
Gel images were analysed to determine the microbial
diversity using Quantity One Software Vers. 4.1 (Bio-Rad,
Hercules, CA, USA). The Shannon-Wiener index of species diversity (H) was calculated by the following equation:
H

s
X

pi loge pi

i1

where pi represents the intensity proportion of band i in the

DGGE profile and s is the total number of bands. Community similarities in band patterns were calculated using
the Dice coefficients and displayed graphically in a form
of UPGMA dendrogram. The sequences obtained were

World J Microbiol Biotechnol (2010) 26:19811990

compared with 16S rRNA gene sequences in the NCBI

database using the BLASTn search program.

1983

Results and discussion

MBBR performance

FISH analysis
Wet biofilm biomass were fixed in freshly prepared 4%
paraformaldehyde at 4C for 12 h immediately after sampling, washed twice in PBS, and subsequently stored in
PBS-ethanol (1:1) at -20C until further analysis. After the
fixation and washing steps, samples were spotted onto
gelatin-coated microscopic slides in duplicate, followed by
air-drying and dehydration in each 50, 80 and 96% of
ethanol solutions for 3 min, and finally dried in the air.
Details on the oligonucleotide probes are available at
probeBase (Loy et al. 2003) and are listed in Table 1. A
specific amount of formamide and NaCl concentration
depending on the probe is shown in Table 1 (Daims et al.
2001; Mobarry et al. 1996). The probes were labeled with
cyanine 3 (Cy3) or carboxyfluorescein (FAM) at the 50 end
(Genscript Co., Ltd, Nanjing, China).
A mixture of probe and hybridization buffer [0.9 M
NaCl, 20 mM TrisHCl, 0.01% SDS, and a specific
amount of formamide depending on the probe used in
Table 1 (pH 7.2)], was applied to each slide, and followed
by incubation for 1.52 h in a humid chamber at 46C. The
final probe concentration was approximately 2.5 ng ml-1.
The slides were washed in pre-warmed washing buffer
[20 mM TrisHCl (pH 7.2), 5 mM EDTA, 0.01% SDS,
and NaCl concentrations as shown in Table 1] for 20 min
at 48C, then rinsed with deionized water and air-dried.
An AXIO IMAGER A II fluorescent microscope
equipped with FITC/DAPI specific filters (Zeiss, Oberkochen, Germany) was used for microscopic observation. The
number of probe-hybridized cells was determined by direct
counting the minimum of ten randomly selected fields
covering 36,600 lm2 in sample covering area of 400 mm2
of slide and the counting was repeated three times. The
hybridization experiments were carried out in duplicate,
and the number was averaged.

The MBBRs showed high COD removal efficiencies irrespective of C/N ratios with stable operation throughout the
study period (Fig. 1). For the influent CODs of 500 mg/l (M1),
1,000 mg/l (M2) and 1,500 mg/l (M3), the effluent COD
concentrations ranged from 10.5 to 93.3 mg/l with the average
of 41.6 mg/l, from 23.3 to 123.0 mg/l with the average of
77.6 mg/l and from 53.3 to 75.0 mg/l with the average of
64.0 mg/l for M1, M2 and M3, respectively (Fig. 1). The
average COD removal efficiencies increased from 91.0, 92.8
to 95.7% as C/N ratios increased from 4.5, 8.9 to 13.4.
The effluent ammonia, nitrite, nitrate, ammonia
removal, as well as SND efficiencies under the different C/
N ratios are illustrated in Fig. 2. The maximum average
ammonia removal efficiency (72.4%) was achieved at C/N
ratio of 13.4, and those decreased in the following order: C/
N ratios of 11.9 (60.3%), 8.9 (49.6%), 6.0 (43.3%) and 4.5
(41.1%) (Fig. 2). The ammonia removal efficiency
decreased with either increasing ammonium loading rates
at a constant COD loading or decreasing COD loading
rates at a constant ammonium loading.
The percentage of SND efficiency was calculated from
the sum of the nitrite and nitrate presented and the amount
of ammonium oxidized during the operation period (Third
et al. 2003). The MBBRs exhibited excellent capability of
denitrification as average SND efficiencies reached 97.6,
98.5 and 98.4% at C/N ratios of 4.5, 8.9 and 13.4,
respectively. Moreover, the moving bed biofilm with C/N
ratios of 11.9 and 6.0 also exhibited high SND efficiencies,
which averaged 99.2 and 92.0%, respectively (Fig. 2). The
nitrogen removal performance of the aerobic MBBR with
C/N ratios of 4.513.4 resulted in nearly complete denitrification yet relatively low nitrification (41.172.4%)
without nitrite accumulation.
The results showed an optimum C/N ratio of 13.4 for
maximum nitrogen and carbon removals in aerobic MBBR,

Table 1 FISH oligonucleotide probes in this study

Probe

Sequence (50 -30 )

Specificity

% FA/NaCl concen (mM)a,

EUB338

GCTGCCTCCCGTAGGAGT

Most Bacteria

20/225

ALF1b

CGTTCGYTCTGAGCCAG

Most Alphaproteobacteria

20/60

BET42a

GCCTTCCCACTTCGTTT

Most Betaproteobacteria

35/80

GAM42a

GCCTTCCCACATCGTTT

Most Gammaproteobacteria

35/60

Nso190

CGATCCCCTGCTTTTCTCC

Ammonia-oxidizing

55/20

Betaproteobacteria
Ntspa662

GGAATTCCGCGCTCCTCT

FA, formamide concentration in the hybridization buffer

NaCl concen, NaCl concentration in the wash buffer

Genus Nitrospira

35/80

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World J Microbiol Biotechnol (2010) 26:19811990

Fig. 1 Profiles of COD concentrations in MBBRs at different C/N ratios filled sqaureinfluent open sqaureeffluent timesremoval
efficiency

which is generally in accordance with the results of former

researchers. Chiu et al. (2007) reported that SND-based
SBR achieved complete removal of NH4?N and COD

123

without leaving any NO2-N in the effluent when the

initial COD/NH4?N ratio was adjusted to 11.1, and the
nitrogen removal efficiency decreased gradually with

World J Microbiol Biotechnol (2010) 26:19811990

Fig. 2 Profiles of NH4?-N, NO2--N and NO3--N concentrations in

MBBRs at different C/N ratios filled squareinfluent NH4N open
squareeffluent NH4N filled triangleeffluent NO2N inverted

1985

filled triangle effluent NO3N open squareNH4N removal

efficiency times SND efficiency

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1986

World J Microbiol Biotechnol (2010) 26:19811990

(a)

2
8
9
10

1
5

6
7

3
4

(b)

M1-0

M1-1

M1-2

M2-0

M2-1

M2-2

M3-0

M3-1

Fig. 3 DGGE pattern (a) and cluster analysis in the form of UPGMA
dendrograms (b) of the bacterial communities from three MBBRs
with different C/N ratios. M1-0, M1-1, M1-2: samples collected from
M1 with C/N ratio of 20, 6.0, 4.5, respectively; M2-0, M2-1, M2-2:

samples collected from M2 with C/N ratio of 20, 11.9, 8.9,

respectively; M3-0, M3-1: samples collected from M3 with C/N
ratio of 20, 13.4, respectively

increasing ammonium-loading rate to the SNDSBR system. Another investigation of SND-based suspended carrier biofilm reactor (SCBR) exhibited good capability of
both nitrogen and organic carbon removal at C/N ratio of
10:1 (Xia et al. 2008). Study of shrimp aquaculture
wastewater treatment by SBR by Fontenot et al. (2007) also
showed an optimum C:N ratio of 10:1 for the best removal
of both nitrogen and organic carbon. Therefore, the
established SND-based MBBR with optimal C/N ratio
can save operation space and time in comparison to the
traditional nitrogen removal process, and might be very
attractive for future practical applications.

with different C/N ratios. The results reveal that the bacterial
communities from more similar C/N ratios were more
associated to each other (Fig. 3b), indicating that similar C/N
ratios resulted in similar microbial population structures in
the biofilm. For example, the similarity coefficient of C/N of
4.5 with C/N of either 8.9 or 6.0 was approximately 0.50, and
that with C/N of 13.4 or 11.9 was 0.49 or 0.45, respectively.
However, compared with all the communities mentioned
above, the correlation coefficient of C/N ratio 4.5 with the
communities of C/N ratio 20.0 was the lowest at 0.330.38.
The ShannonWiener index H is used as a measurement
of the microbial diversity, which is influenced by both the
richness and abundance of species in the population.
DGGE-based H index increases with increasing number of
DGGE bands or the even distribution of individual band.
The diversity index for the bacterial communities varied in
the three reactors fed with different C/N ratios (Fig. 4). The
H value for the C/N ratio of 20.0 was 2.6332.861, which
increased to 2.926, 2.922, and 2.990 for C/N ratio of 13.4,

Microbial community structure analysis

All the biofilm mass for community analysis samples were
taken after the effluent of the system had been stable for at
least a week. Figure 3 shows the DGGE pattern and cluster
analysis of the bacterial community obtained from MBBRs

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1987

Fig. 4 The band number

(a) and Shannon-Wiener index
(b) of the microbial
communities from MBBRs with
different C/N ratios

11.9, and 8.9, respectively. The diversity index H for the

biofilm with influent C/N ratios of 6.0 and 4.5 were 2.732
and 2.741, respectively. It is apparent that the reactor with
the C/N ratios of 8.913.4 had the highest species richness
and diversity, while those with the C/N ratios of 4.5 and 6.0
had lower diversity. The majority of bacterial population,
heterotrophic bacteria, may be restricted due to carbon
resource shortage at low C/N ratios, which may explain the
lower species richness and diversity at low C/N ratios. In
this study, the C/N ratio was responsible for the species
selection and dominance of the microbial communities in
the moving bed biofilm.

Sequence analysis of DGGE bands

The highest similarity results based on the partial 16S
rRNA gene sequences of ten DGGE bands (Fig. 3) are
summarized in Table 2. The majority of the bacterial 16S
rDNA sequences were grouped with members of Proteobacteria, with six in the b subdivision and two in the a
subdivision. The remaining two sequences were related to
members of the Bacteroidetes.
Band 1 and Band 5 possibly belong to Zoogloea oryzae,
and sequences of band 6 and 7 were closely related to
Zoogloea resiniphila. Zoogloea is known to form
Table 2 Percentage similarity
based on the partial 16S rRNA
gene sequences of DGGE bands
to the closest relatives in the
NCBI nucleotide sequence
database

characteristic cell aggregates embedded in gelatinous

matrices and is commonly found in activated sludge. They
are aerobic, but can grow anaerobically in the presence of
nitrate (Mohn et al. 1999; Xie and Yokota 2006). Zoogloea
sp. dominated in all the biofilms with different C/N ratios,
indicating its important role in the biofilm formation and
denitrification performance.
Band 2 and Band 10 were identified as Acidovorax
konjaci and Acidovorax delafieldii, respectively. Acidovorax sp. was observed in activated sludge processes (Snaidr
et al. 1997; You et al. 2002) and was well known for the
utilization of a wide spectrum of carbon sources. They were
both capable of reducing nitrate (Willems et al. 1992), and
Acidovorax defluvii could also produce polyhydroxybutyrate (PHB), which might be denitrifying polyphosphateaccumulating organisms (DPAO; Willems et al. 1990).
Band 8 and 9 were identified to be close relatives of
Runella limosa, which was isolated from activated sludge
performing enhanced biological phosphorus removal
(EBPR). The strain produced acids from carbohydrates
including D-glucose and could not reduce nitrate to nitrite
(Ryu et al. 2006).
Two clones affiliated with AlphaProteobacteria had
highest sequence similarities to Agrobacterium tumefaciens
(Band 3) and Rhizobium sp. (Band 4). They were both able
to reduce nitrate under anaerobic conditions (Daniel et al.

Band no.

Closest relative in NCBI database

Identity (%)

Phylogenetic division

Zoogloea oryzae strain A-4

98

Betaproteobacteria

Acidovorax konjaci

96

Betaproteobacteria

Agrobacterium tumefaciens strain LRC6

100

Alphaproteobacteria

Rhizobium sp. Mp12

100

Alphaproteobacteria

Zoogloea oryzae strain A-4

93

Betaproteobacteria

Zoogloea resiniphila strain DhA-35

98

Betaproteobacteria

Zoogloea resiniphila strain DhA-35

92

Betaproteobacteria

Runella limosa EMB111

96

Bacteroidetes

Runella limosa EMB111

94

Bacteroidetes

10

Acidovorax delafieldii strain PCWCS4

100

Betaproteobacteria

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World J Microbiol Biotechnol (2010) 26:19811990

Table 3 Bacterial community analysis of the biofilms in the three MBBRs with different C/N ratios as determined by FISH cell counts
C/N ratio

ALF1b (%)

BET42a (%)

GAM42a (%)

NSO190 (%)

Ntspa662 (%)

4.5

13.03 2.78

66.54 9.92

13.11 3.78

4.51 0.20

4.35 0.45

6.0

20.04 1.64

55.43 0.57

10.21 2.78

4.32 0.87

4.33 0.57

8.9

11.51 4.77

66.60 7.78

11.38 7.78

3.78 1.57

2.64 0.22

11.9

16.73 1.45

56.04 7.14

15.17 1.48

4.06 1.25

2.77 0.84

13.4

16.74 3.50

60.42 1.64

12.25 6.80

3.29 1.55

2.48 0.25

The values obtained with the probes ALF1b, BET42a, GAM42a, NSO190 and Ntspa662 were expressed as percentages of the number of cells
detected with the probe EUB338

1982; Baek et al. 2003). They should be responsible to the

denitrification performance in moving bed biofilm. The
existence of the above bacteria supports the hypothesis that
the anoxic/anaerobic environments may concurrently
appear in the deeper layers of the biofilm under aerobically
operated conditions.
The explanations given for the phenomenon of SND are
either physical or biological in nature. SND occurs naturally as a consequence of the DO concentration gradients
within microbial biofilms due to diffusion limitations
(Munch et al. 1996). Nitrifiers are active in areas of high
DO concentration, whereas denitrifiers exist in regions with
very low DO concentration. On the other hand, many
heterotrophic bacteria such as Thiosphaera pantotropha are
also capable of performing SND by converting ammonium
aerobically into nitrogen gas (Robertson and Kuenen
1988). Some denitrifiers are able to denitrify under aerobic
conditions (Hellinga et al. 1998). Pathways of heterotrophic SND metabolism are different from those of aerobic
autotrophic nitrifiers and anoxic heterotrophic denitrifiers.
Consequently, the presence of DO micro-gradients resulting from oxygen diffusion limitation within the biofilm
might be responsible for SND in the aerobic MBBR. Pochana and Keller (1999) observed that the lower DO concentration inhibited the aerobic nitrification process
whereas the anoxic denitrification process was enhanced in
an SND-based SBR. Thus, we speculate that the higher
efficiency for denitrification than that for nitrification in the
aerobic MBBR might be partially due to the low DO level
in the bulk liquid.
Quantitative analysis of specific groups by FISH
Two groups of organisms are involved in nitrification:
AOB and NOB. With the exception of two marine species
of the genus Nitrosococcus, all ammonia-oxidizers belong
to the Betaproteobacteria. Recent studies indicated a more
general importance of Nitrospira spp., not Nitrobacter spp.,
as the dominant nitrite-oxidizing bacteria in wastewater
treatment systems (Wagner et al. 1996; Okabe et al. 1999).
Nitrospira adapts to low nitrite concentrations, while

123

Nitrobacter thrives in higher nitrite concentrations (Bartosch et al. 2002). Consequently, Betaproteobacteria AOB
and Nitrospira-like NOB were selected to quantify the
nitrifiers in the biofilm by FISH with oligonucleotide
probes Nso190 and Ntspa662.
FISH cell counts of specific groups are given in Table 3
as percentages of the number of cells detected with the
probe EUB338. FISH analysis reveals that the moving bed
biofilm was dominated by a large number of Proteobacteria consisting of alpha, beta and gamma subclasses. The
bacteria affiliated with beta-subclass Proteobacteria were
the most numerically important populations (55.466.6%),
which are in accordance with the identification of DGGE
band sequences (Table 2).
The relative abundances of Betaproteobacteria AOB
were 3.29, 4.06, 3.78, 4.32 and 4.51% for C/N ratio of 13.4,
11.9, 8.9, 6.0 and 4.5, respectively, and those of Nitrospiralike NOB were 2.48, 2.77, 2.64, 4.33 and 4.35% (Table 3),
which indicates the increasing proportion of Betaproteobacteria AOB and Nitrospira-like NOB with decreasing C/
N ratios. The increases in nitrifier population due to C/N
ratio are consistent with the results obtained by Ohashi
et al. (1995) and Xia et al. (2008).
Decreases in COD loading rates at a constant ammonium loading cause less supply of carbon sources, and
consequently the heterotrophic bacterial population
decreased, while nitrifying bacteria become a more
important component in the biofilm. The relationship
between C/N ratio and nitrifier population suggests larger
relative abundance of nitrifiers in biofilm reactors at low C/
N ratio. However, the competition for DO between heterotrophic bacteria and nitrifiers in biofilm systems is well
known, and it results in a stratified biofilm structure, the
fast growing heterotrophic bacteria being placed in the
outer layers, while the slow growing nitrifiers locate deeper
inside the biofilm. Thus oxygen limitation resulting from
consumption and resistance to mass transfer within the
heterotrophic layer affects the nitrifying bacteria negatively
in the presence of organic substrates when the bulk liquid
oxygen concentration is low (Ohashi et al. 1995; Satoh
et al. 2000). Meanwhile, the activity of both AOB and

World J Microbiol Biotechnol (2010) 26:19811990

NOB was inhibited by free ammonia when ammonium

loading rate increases at a constant COD loading (Kim
et al. 2006). Therefore, the inhibition of nitrifying bacteria
by free ammonia and interspecies competition for oxygen
leads to the deterioration of nitrification performance. This
may be responsible for the increase in the proportion of
nitrifying bacteria but the decrease in the nitrification
efficiency with decreasing C/N ratios.

Conclusions
In continuous-flow MBBRs under constantly aerobic conditions, appropriate control of microbial community
structure resulting from optimal C/N ratio is beneficial to
improve SND, thus maximizing nitrogen removal. The
results show that the aerobic MBBRs were efficient for
SND and COD removal with respective efficiencies of
92.098.5% and over 90.0%. The nitrogen removal performance of the aerobic MBBRs at C/N ratios of 4.513.4
resulted in nearly complete denitrification, yet relatively
low nitrification (41.172.4%) without nitrite accumulation. DGGE results indicate that the microbial community
structures based on more similar C/N ratios were more
similar in terms of highest species richness and diversity at
C/N ratios of 8.913.4. FISH analysis has confirmed that
the dominance of both Betaproteobacteria AOB and
Nitrospira-like NOB negatively correlated with C/N ratios.
Acknowledgments This research is supported by Natural Science
Foundation of Jiangsu Province of China (No. BK2009252) and the
Key Project of Chinese Ministry of Education (No. 108150).

References
American Public Health Association (APHA) (1998) Standard
methods for the examination of water and wastewater, 17th ed.
American public health association, New York
Baek SH, Kim KH, Yin CR, Jeon CO, Im WT, Kim KK, Lee ST
(2003) Isolation and characterization of bacteria capable of
degrading phenol and reducing nitrate under low-oxygen conditions. Curr Microbiol 47:462466
Bartosch S, Harttwig C, Spieck E, Bock E (2002) Immunological
detection of Nitrospira-like bacteria in various soils. Microbiol
Ecol 43:2633
Bjorn Rusten, Siljudalen JG, Nordeidet B (1994) Upgrading to
nitrogen removal with the KMT moving bed biofilm process.
Water Sci Technol 29(12):185195
Chiu YC, Lee LL, Chang CN, Chao AC (2007) Control of carbon and
ammonium ratio for simultaneous nitrification and denitrification
in a sequencing batch bioreactor. Int Biodeter Biodegr 59:17
Daims H, Nielsen JL, Nielsen PH, Schleifer KH, Wagner M (2001) In
situ characterization of nitrospira-Like nitrite-oxidizing bacteria
active in wastewater treatment plants. Appl Environ Microbiol
67(11):52735284

1989
Daniel RM, Limmer AW, Steele KW, Smith IM (1982) Anaerobic
growth, nitrate reduction and denitrification in 46 Rhizobium
strains. J Gen Microbiol 128:18111815
Fontenot Q, Bonvillain C, Kilgen M, Boopathy R (2007) Effects of
temperature, salinity, and carbon: nitrogen ratio on sequencing
batch reactor treating shrimp aquaculture wastewater. Bioresour
Technol 98:17001703
Hellinga C, Schellen AAJC, Mulder JW, van Loosdrecht MCM,
Heijnen JJ (1998) The Sharon process: an innovative method for
nitrogen removal from ammonium-rich waste water. Water Sci
Tech 37(9):135142
Helmer C, Kunst S (1998) Simultaneous nitrification/denitrification in
an aerobic biofilm system. Water Sci Tech 37:183187
Kim DJ, Lee DI, Keller J (2006) Effect of temperature and free
ammonia on nitrification and nitrite accumulation in landfill
leachate and analysis of its nitrifying bacterial community by
FISH. Bioresour Technol 97:459468
Liang DW, Zhang T, Fang HerbertHP (2007) Anaerobic degradation
of dimethyl phthalate in wastewater in a UASB reactor. Water
Res 41:28792884
Loy A, Horn M, Wagner M (2003) probeBase: an online resource for
rRNA-targeted oligonucleotide probes. Nucleic Acids Res
31:514516
Luostarinen S, Luste S, Lara V, Jukka R (2006) Nitrogen removal
from on-site treated anaerobic effluents using intermittently
aerated moving bed biofilm reactors at low temperatures. Water
Res 40:16071615
Miller DN, Bryant JE, Madsen EL et al (1999) Evaluation and
optimization of DNA extraction and purification proced ures for
soil and sodiment samples. Appl Environ Microbiol 65(11):
47154724
Mobarry BK, Wagner M, Urbain V, Rittmann BE, Stahl DA (1996)
Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl Environ Microbiol 62:2156
2162
Mohn WW, Wilson AE, Bicho P, Moore ERB (1999) Physiological
and phylogenetic diversity of bacteria growing on resin acids.
Syst Appl Microbiol 22:6878
Munch EV, Lant P, Keller J (1996) Simultaneous nitrification and
denitrification in bench-scale sequencing batch reactors. Water
Res 30:277284
Muyzer G, Waal EC, Uitterlinden AG (1993) Profiling of complex
microbial populations by denaturing gradient gel electrophoresis
analysis of polymerase chain reaction-amplified genes coding for
16S rRNA. Appl Environ Microbiol 59:695700
Ohashi A, Silva DG, Rittmann BE (1995) Influence of substrate C/N
ratio on biofilms consisting of nitrifiers and heterotrophs. Water
Sci Technol 32(8):7584
Okabe S, Satoh H, Watanabe Y (1999) In situ analysis of nitrifying
biofilms as determined by in situ hybridization and the use of
microelectrodes. Appl Environ Microbiol 65:31823191
Pastorelli G, Andreottola G, Canziani R, Darriulat C, De fraja
frangipane E, Rozzi A (1997) Organic carbon and nitrogen
removal in moving-bed biofilm reactors. Wat Sci Tech 35(6):
9199
Pochana K, Keller J (1999) Study of factors affecting simultaneous
nitrification and denitrification (SND). Water Sci Technol
39(6):6168
Robertson LA, Kuenen JG (1988) Heterotrophic nitrification on
Thiosphaera pantotropha: oxygen uptake and enzyme studies.
General Microbiology 134:857863
Ryu SH, Nguyen TTH, Park W, Kim CJ, Jeon CO (2006) Runella
limosa sp. nov., isolated from activated sludge. Int J Syst Evol
Microbiol 56:27572760

123

1990
Sanz JL, Kochling T (2007) Molecular biology techniques used in
wastewater treatment: An overview. Process Biochem 42:
119133
Satoh H, Okabe S, Norimatsu N, Watanabe Y (2000) Significance of
substrate C/N ratio on structure and activity of nitrifying biofilms
determined by in situ hybridization and the use of microelectrodes. Water Sci Technol 41(45):317321
Snaidr J, Amman R, Huber I, Ludwig W, Schleifer KH (1997)
Phylogenetic analysis and in situ identification of bacteria in
activated sludge. Appl Environ Microbiol 63:28442896
Third KA, Burnett N, Cord-Ruwisch R (2003) Simultaneous nitrification and denitrification using stored substrate (PHB) as the
electron donor in an SBR. Biotechnol Bioeng 83(6):706720
Wagner M, Rath G, Koops HP, Flood J, Amann R (1996) In situ
analysis of nitrifying bacteria in sewage treatment plants. Water
Sci Technol 34:237244
Willems A, Falsen E, Pot B, Jantaen E, Hoste B, Vandamme P, Gillis
M, Kersters K, Deley J (1990) Acidovorax, a New Genus for
Pseudomonas facilis, Pseudomonas delaJieldii, E. Falsen (EF)
Group 13, EF Group 16, and Several Clinical Isolates, with the
Species Acidovorax facilis comb. nov., Acidovorax delaJieldii
comb. nov., and Acidovorax temperans sp. nov. Int J Syst Bac
40(4):107119

123

World J Microbiol Biotechnol (2010) 26:19811990

Willems A, Goor M, Thieiemans S, Gillis M, Kersters K, Deley J
(1992) Transfer of several phytopathogenic Pseudomonas species to Acidovorax as Acidovorax avenae subsp. avenae subsp.
nov., comb. nov., Acidovorax avenae subsp. citrulli, Acidovorax
avenae subsp. cattleyae, and Acidovorax konjaci. Int J Syst Bac
42(1):107119
Xia SQ, Li JY, Wang RC (2008) Nitrogen removal performance and
microbial community structure dynamics response to carbon
nitrogen ratio in a compact suspended carrier biofilm reactor.
Ecol Eng 32:256262
Xie CH, Yokota A (2006) Zoogloea oryzae sp. nov., a nitrogen-fixing
bacterium isolated from rice paddy soil, and reclassification of
the strain ATCC 19623 as Crabtreella saccharophila gen. nov.,
sp. nov. Int J Syst Evol Microbiol 56:619624
Xing XH, Jun BH, Yanagida M, Tanji Y, Unno H (2000) Effect of C/
N values on microbial simultaneous removal of carbonaceous
and nitrogenous substances in wastewater by single continuousflow fluidized-bed bioreactor containing porous carrier particles.
Biochem Eng J 5:2937
You SJ, Hsu CL, Ouyang CF (2002) Identification of the microbial
diversity of wastewater nutrient removal processes using
molecular biotechnology. Biotechnol Lett 24:13611366