International Biodeterioration & Biodegradation 71 (2012) 72e79

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International Biodeterioration & Biodegradation

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Enhanced biological nitrogen removal via dissolved oxygen partitioning and step
feeding in a simulated river bioreactor for contaminated source water
remediation
Li-Juan Feng b, Jian Xu b, Xiang-Yang Xu b, c, Liang Zhu a, b, *, Jing Xu b, Wei Ding b, Jing Luan b
a
Zhejiang Provincial Key Laboratory of Environmental Pollution Control Technology, No.16 Haishu Road, Hangzhou 310007, PR China
b
Department of Environmental Engineering, Zhejiang University, No.866 Yuhangtang Road, Hangzhou 310058, PR China
c
ZJU-UWA Joint Centre in Integrated Water Management and Protection, No. 866 Yuhangtang Road, Hangzhou 310058, PR China

a r t i c l e i n f o a b s t r a c t

Article history: In recent years, nitrogen pollution has been increasingly serious in natural waters including drinking
Received 6 May 2011 source water. A simulated river biofilm reactor fed with contaminated drinking source water was
Received in revised form established to evaluate the effects of dissolved oxygen (DO) partitioning and step feeding on the nitrogen
20 December 2011
removal performance and biofilm microbial community. Results showed that after the hydraulic
Accepted 29 December 2011
Available online 15 May 2012
retention time of anoxic zone extending and step feeding, the effluent concentration of ammonia was
below 0.2 mg L
1, and the removal efficiency of total nitrogen increased from 12.02%  4.59% to
34.98%  2.65%, which indicated the occurrence of simultaneous nitrification and denitrification. The
Keywords:
Contaminated source water
results of denaturing gradient gel electrophoresis showed that the microbial community of biofilm
Biofilm obviously shifted via DO controlling and step feeding. Low DO concentration favored the enrichment of
Microbial community denitrifying bacteria and coexistence of algae and bacteria, and the pattern of step feeding could increase
Simultaneous nitrification and the community abundance. The dominant heterotrophic bacteria species of biofilm in oligotrophic niche
denitrification (SND) belonged to Hyphomicrobium sp., Pseudomonas sp., Chloroflexi sp., Enterobacter sp., Pantoea sp., and
Oligotrophic niche Synechococcus sp., which were mostly associated with denitrification and refractory organics utilization.
It was worth noting that the ammonia-oxidizing bacteria (AOB) community of biofilm was stable
throughout the whole experiment, and Nitrosomonas sp. was the predominant AOB in the oligotrophic
niche.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Biological nitrogen removal involves a combination of nitrifi-

In recent years, nitrogen pollution in natural waters has been
causing a series of problems such as eutrophication, odor, and
microcystin pollution, damaging the ecosystem and threatening
human health (Camargo and Alonso, 2006). Therefore, researchers
have been paying attention to the remediation process in order to
guarantee the safety of drinking water. Biological denitrification
appears to be an efficient, economical, and environmentally
friendly approach, which can simultaneously remove organics and
nitrogen (Vidali, 2001; Lynch and Moffat, 2005; Farhadian et al.,
2008).
* Corresponding author. Department of Environmental Engineering, Zhejiang
University, No. 866 Yuhangtang Road, Hangzhou 310058, PR China. Tel./fax: þ86
571 88982343.
E-mail address: felix79cn@hotmail.com (L. Zhu).
cation of ammonia to nitrite and nitrate, and later denitrification of
the oxidized N-species to N2 (Sun et al., 2010). Thus, ammonia is
oxidized via two pathways: Firstly, ammonia is oxidized to
hydroxylamine and then to nitrite by low-growing autotrophic
ammonia-oxidizing bacteria (AOB) or heterotrophic AOB (Hu and
Kung, 2000); Secondly, ammonia and nitrite are anaerobically
converted to dinitrogen gas by novel autotrophic anammox
bacteria (Rick and Stuart, 2001). Denitrification is the conversion of
nitrate and nitrite to nitric or nitrous oxide, which is carried out by
anoxic heterotrophic denitrifiers or aerobic heterotrophic denitri-
fiers. Along with research into the mechanism of the nitrogen cycle,
lots of novel nitrogen removal processes have been developed for
biological wastewater treatment, including the simultaneous
nitrification and denitrification (SND), single reactor high activity
ammonia removal over nitrite (SHARON), oxygen-limited autotro-
phic nitrification-denitrification (OLAND), anaerobic ammonium
oxidation (ANAMMOX), and the completely autotrophic nitrogen

0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ibiod.2011.12.016
 L.-J. Feng et al. / International Biodeterioration & Biodegradation 71 (2012) 72e79
removal over nitrite (CANON) process (Xia et al., 2010). However,
there is little research on these novel processes applied in micro-
polluted water bioremediation. In view of the low C/N ratio of the
micro-polluted water, the SND process could achieve nitrification
and denitrification synchronously with high-utilization efficiency
of the carbon source (Zhang et al., 2009).
Up to date, there are two mechanisms for SND: One occurs
inside the microbial biofilm and flocs due to dissolved oxygen (DO)
concentration gradient, which favors the coexistence of nitrifying
and denitrifying microorganisms; and the other is conducted in
a single reactor under aerobic condition by heterotrophic nitrifiers
and aerobic denitrifiers. Factors such as DO, pH, temperature,
organic carbon source, and ammonia concentration have signifi-
cant influence on the SND process, which has been paid more
attention in wastewater treatment than that in drinking source
water remediation (Chiu et al., 2007; Meng et al., 2008; Zhang et al.,
2009). Studies have shown that nitrification is enhanced and
denitrification is inhibited at high DO concentration, while deni-
trification is well conducted at low DO concentration. When DO is
controlled at 0.5 mg L
1, nitrification and denitrification usually
have similar rates in the system (Munch et al., 1996; Peng et al.,
2001). Meanwhile, an organic carbon source is also important to
the SND because low organic concentration leads to a deficiency of
electron donors for denitrifiers, whereas high organic concentra-
tion causes the inhibition of autotrophic nitrifiers (Tam et al., 1992).
Hence, micro-polluted water is at a disadvantage for denitrification
because of a carbon deficiency (Chang and Ouyang, 2001), and an
attractive approach such as a step-feeding process to enhance
denitrification in environmental waters needs to be developed (Lu
et al., 2009; Zhu et al., 2009a, 2009b).
In recent years, lots of nitrifying and denitrifying microorgan-
isms have been found in natural water and micro-polluted water
bioremediation systems with the development of molecular
biotechnology. There are usually two kinds of microorganisms
responsible for SND process; one is autotrophic nitrifiers, belonging
to a relatively few groups. Previous studies have shown that
Nitrosomonas and Nitrosospira-like ammonia-oxidizing bacteria
(AOB) co-existed in the freshwater and estuarine systems (Regan
Fig. 1. Schematic diagram of the simulated river biofilm reactor (A-Anoxic zone, O-oxic zone).
73
et al., 2002, 2003; de Vet et al., 2009), and the predominance of
Nitrosomonas-type AOB was also found in drinking water biofilms
because of its strong competitive advantage (Lipponen et al., 2004;
Qin et al., 2007). The other type of bacteria responsible for SND is
heterotrophic denitrifiers; there are many clusters, such as Pseu-
domonas, Hyphomicrobia, Azoarcus, Rhodocyclus, and Thauera,
found in biological treatment processes (Wagner and Loy, 2002;
Ginige et al., 2004, 2005; Thomsen et al., 2007; Xia et al., 2010).
However, there is little information on denitrifying microorganism
enrichment in contaminated source water bioremediation system
(Warneke et al., 2011).
The simulated river biofilm reactor was established for
contaminated drinking source water bioremediation in this study,
and two key factors of DO concentration and influent pattern were
evaluated for biological nitrogen removal performance of the bio-
film system. The objectives of this study are as follows: (1) opti-
mizing operating parameters to enhance biological nitrogen
removal in contaminated source water bioremediation; and (2)
investigating the microbial community succession of biofilms in the
oligotrophic niche.
2. Materials and methods
2.1. Running of the simulated river biofilm reactor
The simulated river biofilm reactor (SRBR) was made of plex-
iglass with trapezoidal cross sections (the width of the top and
bottom, and the height of the trapezoidal cross section were 30 cm,
16 cm, and 30 cm, respectively), and the working volume was 100 L.
A schematic illustration of the reactor is provided in Fig. 1. For this
study, TA-II elastic filler used as the biofilm carrier was purchased
from Hangzhou Tianyu Environmental Protection Engineering Co.
Ltd. in China, with a diameter and surface area of 200 mm and
200e300 m2 m
3, respectively. According to previous investiga-
tions of polluted source water in Eastern China, CODMn and total
nitrogen (TN) were two of the main pollutants. The influent of the
reactor was from Huajia Pond in Zhejiang University. The water
characteristics are shown in Table 2. The air was pumped from the
74 L.-J. Feng et al. / International Biodeterioration & Biodegradation 71 (2012) 72e79

Table 1
Pollutants removal performance of the biofilm remediation process.

Pollutants Index Phase I Phase II Phase III Phase IV

CODMn Influent (mg/L) 13.61  1.39 11.26  0.23 8.79  0.84 8.56  0.97
Effluent (mg/L) 9.37  0.65 7.06  0.37 5.58  0.47 5.47  0.40
Removal rate (%) 30.83  2.82 37.30  2.69 36.39  3.36 35.80  3.20
NHþ
4 eN Influent (mg/L) 0.40  0.13 0.54  0.19 0.66  0.14 0.31  0.09
Effluent (mg/L) 0.20  0.03 0.09  0.05 0.12  0.02 0.13  0.01
Removal rate (%) 43.40  12.62 84.10  4.23 80.39  5.48 52.56  4.93
TN Influent (mg/L) 2.29  0.18 2.35  0.14 1.98  0.06 1.86  0.14
Effluent (mg/L) 2.01  0.11 1.79  0.14 1.39  0.04 1.21  0.07
Removal rate (%) 12.02  4.59 24.11  2.45 29.52  2.20 34.98  2.65

bottom of the reactor. The bioreactor system was seeded with river
sediment from different water sources in China’s Hang-Jia-Hu area.
Sediment was screened with a 0.45-mm sieve to remove debris,
snails, sand, and so on. Then, all sediment samples were mixed and
laid evenly on the bottom of the reactor.
The reactor was run continuously at the following operating
parameters: a carrier concentration of 2.33% (volume ratio of
cumulated carriers to the reactor), hydraulic retention time (HRT)
of 24 h, temperature of (25  2) C, pH of 7.0e8.0, DO concentration
of 1.73e4.39 mg L
1 with aeration and 0.06e0.66 mg L
1 without
aeration in the bottom of the reactor. The condition of four phases is
shown in Table 1 and Fig. 1; HRT(A)/HRT(O) referred to the ratio of
HRT of anoxic zone to oxic zone in the reactor. During the experi-
ment, the anoxic zone was gradually extended from phase I to
phase III, and the step-feeding process was operated at the last
phase (phase IV). A one-step feeding process was applied in phase
III with the influent velocity of 48 ml min
1; a two-step feeding
process was applied in phase IV with each step’s influent velocity of
24 ml min
1. Each phase was monitored for more than one month
(40 w 50 days) to ensure that stable performance was achieved. All
of the influent and effluent were sampled on alternate days, and
CODMn, NHþ 4 eN, and TN were analyzed according to standard
analytical methods (State Environmental Protection
Administration, 2002). The removal rates of CODMn, NHþ 4 eN, and
TN in the four phases were calculated from the residual concen-
tration at the end of the reactor (C1) and those pollutants in influent
(C0): %removal rate ¼ 100  (1
C1/C0). The pH was determined
with a digital pH meter (Mettler Toledo 320, Switzerland), and DO
was measured with a DO meter (YSI Model 52, USA).
2.2. DNA extraction, PCR, and DGGE
The biofilm samples were obtained from two clusters of elastic
filler in the front (B1), middle (B2), and back sections (B3) of the
SRBR at the end of each operational phase. Genomic DNA of bio-
films was extracted using a soil DNA kit (OMEGA). The extracted
DNA was checked by 0.8% agarose electrophoresis using Golden
view as the staining dye, and quantified spectrophotometrically at
260 nm and 280 nm.
The PCR was performed with universal bacterial primers (P338f
with GC-clamp and P518r) (Lapara et al., 2002) for DGGE analysis of
the total bacteria community. To analyze the ammonia-oxidizing
Table 2
Operational condition of the simulated river biofilm reactor.
bacteria (AOB) community, 16S rRNA genes were amplified by nes-
ted PCR with the CTO189feCTO654r primer set (Kowalchuk et al.,
1997) and reamplified with the eubacterial 338f-GC-518r primers.
The DGGE was performed using the DCode system (Bio-Rad,
Hercules, CA, USA). PCR products were loaded onto polyacrylamide
gels (10% (w/v), 37.5:1 acrylamide/bisacrylamide) in 1x TAE (20 mM
Tris, 10 mM acetate, and 0.5 mM EDTA at pH 7.4). Denaturing
acrylamide (100%) was defined as 7 M urea with 40% formamide.
Two 8% polyacrylamide gels were prepared with a vertical gradient
of 30e60% of the full strength denaturant (i.e., 100%) for total
bacteria amplicons and 40e60% for AOB amplicons. Electrophoresis
was performed at 30 V and 60  C for 30 min, and then 5.5 h at 155 V
for bacteria and 6.5 h for AOB. DGGE profiles were obtained by
staining gels with SYBR Green for 30 min and capturing images using
a Gel Documentation System (BIO- Rad Laboratories, Segrate, Italy).
Quantity One software (Version 4.62) was used for band anal-
ysis. Microbial diversity was calculated by using the Shannon
diversity index (H) (Eichner et al., 1999). The analysis of DGGE
community profile similarity used pair-wise similarity coefficients
(Cs) of unweighted pair group method with arithmetic mean
(UPGMA) cluster analysis (Fromin et al., 2002) and principal
component analysis (PCA) using SPSS software (Kjellerup et al.,
2005). Selected DNA bands were aseptically excised and ream-
plified by PCR as described previously (Chen et al., 2002). Excised
PCR products were sent to TAKaRa Bio-technology Co. Ltd., (Dalian,
China) for sequencing. The National Center for Biotechnology
Information BLAST program (http://www.ncbi.nlm.nih.gov) was
used for sequence identification.
3. Results
3.1. Pollutants removal performance of the SRBR
The removal efficiencies of NHþ 4 eN, CODMn, and TN at different
phases in the SRBR are shown in Table 2. From phase I to phase III,
TN removal efficiency was increased from 12.02%  4.59% to
29.52%  2.20% because of the anoxic zone extension. In the step-
feeding process (phase IV), it was found that TN removal was
further enhanced to 34.98%  2.65%. Additionally, the NHþ 4 eN
removal efficiency was over 70% when aeration was stopped, but
significantly decreased from 80.38%  5.48% to 52.56%  4.93% due
to the step feeding in phase IV. During the four phases, the removal
efficiency of CODMn increased from 30.83%  2.82% to
37.30%  2.69% by extending the anoxic zone, and there was no
significant change in phases III and IV.

Parameters HRT(A)/HRT(O) Step-feeding 3.2. Bacterial community analysis of the biofilms in different stages
Phase I 12/12 One step
Phase II 16/8 One step The bacterial communities of biofilms in different stages were
Phase III 24/0 One step
Phase IV 24/0 Two steps
analyzed using 16S rRNA gene PCR-DGGE and sequencing analysis.
DGGE profiles are shown in Fig. 2(A and C). In the DGGE gel, the
 L.-J. Feng et al. / International Biodeterioration & Biodegradation 71 (2012) 72e79
Fig. 2. DGGE profiles of the total bacteria and AOB for different HRT(A)/HRT(O)(A and B) ratio and feeding pattern (C and D). The bands indicated by the numbers (T1eT13 for
bacteria and A1eA17 for AOB) represent excised and sequenced bands. B1, B2, B3 refer to the front, middle, end of the reactor, and I, II, III, IV mean different phases.
number, position, and intensity of the bands in each lane gave an
estimate of the number and relative abundance of numerically
dominant species in the samples (Muyzer et al., 1993).
From phase I to phase III, only the biofilms from the end part of
the reactor (B3) were influenced consistently by the change of the
HRT(A)/HRT(O) ratio, and the relative biofilms were prepared for
PCR-DGGE analysis. Therefore, B3-I and B3-II were from the oxic
zone (DO of 1.73e4.39 mg L
1) and B3-III was from the anoxic zone
(DO of 0.06e0.66 mg L
1). It was found that the bacteria commu-
nity shifted a little when the ratio of HRT(A)/HRT(O) changed
(Fig. 2A). Twelve excised bands shown in the DGGE bands were
compared with the GenBank database, and results were shown in
Table 3. In this study, all the sequences showed 98% similarity to
the GenBank data, and most of them were homologous with a-
proteobacteria and g-proteobacteria. Furthermore, some bands
appeared when the environment shifted from oxic to anoxic, such
as bands T4, T5, and T6, which were associated with Synechococcus
sp., Pantoea sp., and Chloroflexi bacterium, respectively.
At the same time, step feeding in the middle of the reactor at
phase IV supplied the nutrients for corresponding biofilms, and
resulted in the Shannon index of B2 (2.29) and B3 (2.29) increasing
to the same level as B1 (Fig. 3). The results showed that some bands,
such as T7, T10, and T11, recovered their intensity in B2 and B3,
because of the step-feeding process (Fig. 2C). The sequences rep-
resented by bands T7, T10, and T11, had more than 99% sequence
similarity to uncultured Hyphomicrobium sp., Pseudomonas putida,
and Hyphomicrobium sp., respectively.
3.3. AOB community analysis of the biofilms in different stages
A nested PCR approach was used to investigate the AOB
community of the biofilms in different stages. This approach can
75
enhance sensitivity to allow detection of less abundant species
(Boon et al., 2002), especially for AOB in the oligotrophic environ-
ment. The DGGE profiles of AOB are shown in Fig. 2(B and D). All
analyzed biofilms produced rich DGGE bands, indicating the pres-
ence of different species of AOB. Seventeen excised and sequenced
bands shown in the DGGE profile bands were compared with the
GenBank databases (Table 4). All excised and sequenced bands
were found to be related to the genus Nitrosomonas sp., except that
band A11 was Nitrosospira sp.
4. Discussion
In this paper, the average removal efficiencies of NHþ 4 eN and TN
reached above 35% and 50% via extending the anoxic zone’s HRT
and step feeding, and simultaneous nitrification and denitrification
finally occurred. Previous studies have proved that DO concentra-
tion is a key factor for the biological nitrogen removal process, and
high DO concentration suppresses denitrification while low DO
concentration inhibits nitrification (Pochana and Keller, 1999).
Munch et al. (1996) reported that nitrification and denitrification
rates were equal at DO concentration of 0.5 mg L
1. The nitrification
and denitrification rates were both improved when DO concen-
tration increased from 0.5 to 1.0 mg L
1, and nitrification could
supply sufficient electronic acceptors for denitrification. However,
the TN removal rate decreased when DO further increased to
1.5 mg L
1 (Meng et al., 2008). In this study, the average concen-
tration of DO stayed at about 0.5 mg L
1 after aeration stopped, and
the TN removal efficiency increased from 12.02%  5.43% to
29.52%  2.20% because of the HRT(A) extending. However, the

1
effluent NHþ 4 eN was still maintained below 0.5 mg L , indicating
that low influent concentration of NHþ 4 eN could be effectively
removed at a DO concentration of 0.5 mg L
1. But the ammonia
76 L.-J. Feng et al. / International Biodeterioration & Biodegradation 71 (2012) 72e79

Table 3
Bacteria DGGE bands with corresponding closest matches (BLASTn).

Band Closet match Phylogenetic % Similarity Origin Accession

group number
T1 Pseudomonas putida strain1389 [GU726875.1] g- 98 Japan Sea and South China Sea JF506010
proteobacteria
T2 Uncultured Pseudomonas sp. [AM711579.1] g- 100 Pristine extreme Andean wetlands JF506011
proteobacteria
T3 Uncultured Pseudomonas sp. [AM711878.1] g- 100 Pristine extreme Andean wetlands JF506012
proteobacteria
T4 Synechococcus sp. ACT9807 [GQ422959.1] Cyanobacteria 100 Shallow lake (Lake Balaton, Hungary) JF506013
T5 Pantoea sp. I-Bh20-21 [FN555402.1] g- 100 Denitrifying soil JF506014
proteobacteria
T6 Uncultured Chloroflexi bacterium [FJ916310.1] Chloroflexi 100 Temperate lake JF506015
T7 Uncultured Hyphomicrobium sp. [GU257860.1] a- 100 Submerged novel polyvinyl chloride membrane bioreactor JF506016
proteobacteria
T8 Enterobacter sp. [HM461132.1] g- 100 Growing on N-acyl homoserine lactones JF506017
proteobacteria
T9 Enterobacter sp. [GQ891865.1] g- 100 Degrading polycyclic aromatic hydrocarbons JF506018
proteobacteria
T10 Pseudomonas putida strain d92 [FJ950674.1] g- 99 Oxytetracycline production wastewater treatment JF506019
proteobacteria plant and the receiving river
T11 Hyphomicrobium sp. [AM502926.1] a- 100 Long-term linuron-treated agricultural soils JF506020
proteobacteria
T12 Uncultured Pantoea sp. [GU569157.1] g- 100 Various geographic populations JF506021
proteobacteria
T13 Hyphomicrobium facile [AB222020.1] a- 100 Aromatic hydrocarbons degrading bacteria JF506022
proteobacteria

removal not only contributed to the oxidization by autotrophic
AOB, but also to assimilation by heterotrophic bacteria, the mech-
anism of ammonia removal in oligotrophic environment needs
further research.
In view of carbon utilization, the CODMn concentrations of the
influent, front, middle, and back parts of the reactor were also
examined in phase III; the results (not shown in the table) were
8.79  0.84(mg L
1), 6.08  0.43(mg L
1), 5.75  0.47(mg L
1), and
5.58  0.47(mg L
1), respectively. It was demonstrated that the
available carbon source was used mainly in the front part of the
reactor, and this resulted in a carbon deficiency for denitrification
in the middle and back parts. The increase of available carbon
source in the back end by step feeding favored nitrogen removal
enhancement (the removal efficiency of TN increased to
34.98  2.65% after the step-feeding process was applied). Previous
studies have focused on the addition of solid carbon sources,
including agricultural byproducts (wheat straw, rice husk, etc.), and
Fig. 3. Shannon index of biofilm samples in phase III and phase IV.
plant carbohydrate from wetlands (giant reed, Typha latifolia,
Elodea canadensis, etc.) (Bastviken et al., 2005; Ovez et al., 2006;
Shao et al., 2009), and high nitrogen removal efficiency was
obtained. However, the COD and suspended solid concentrations in
the effluent were always increased. It was demonstrated that
optimizing the C/N ratio via step-feeding and solid carbon source
addition favored nitrogen removal in contaminated environmental
water, but more research is needed.
DO concentration had a significant influence on the biofilm
microbial community in biological treatment systems (Tan and Ng,
2008; Guo et al., 2009). For the bacterial community analysis in
Fig. 2A, bands T4 to T6 were dominant in the anoxic environment.
Band T4 was associated with Synechococcus sp., which was an
autotrophic bacterium. Thus, the coexistence of the bacteria and
algae occurred at the low DO niche. Band T5 was associated with
Pantoea sp. I-Bh20-21, which could convert nitrate to N2 (Braker
et al., 2010). Band T6 had 100% sequence similarity to Chloroflexi
bacterium, which Miura et al. (2007) found to be closely related to
the accumulation of soluble carbohydrates. Yamada et al. (2005)
found that Chloroflexi sp. was a predominant bacterium in acti-
vated sludge treating high-strength organic wastewater. Therefore,
low DO concentration was not only helpful for the growth of
denitrifying bacteria, but also favored the coexistence of the
bacteria and algae. However, some bands (T8, T9, T10, and T11)
related to refractory organics degradation weakened in the anoxic
zone. Research in industrial wastewater treatment has shown that
a high removal rate of refractory organics was obtained in an oxic
environment. Therefore, a low DO concentration of about 0.5 mg/L
was helpful for some denitrifying bacteria to be dominant in the
oligotrophic environment, but it was not suitable for the growth of
other refractory organics-degrading bacteria. However, bands T10
and T11 maintained their intensity in the step-feeding process in
phase IV. Due to the variety of refractory organics in the source
water (Kolpin et al., 2002; Kasprzyk et al., 2008), it’s necessary to
enrich refractory organics-degrading bacteria in polluted water
bioremediation. Hence, the combination of an anoxic and step-
feeding process was efficient in simultaneous removal of the
refractory organics and nitrogen in this study.
 L.-J. Feng et al. / International Biodeterioration & Biodegradation 71 (2012) 72e79 77

Table 4
AOB DGGE bands with corresponding closest matches (BLASTn).

Band Closet match % Similarity Origin Accession number

A1 Uncultured betaproteobacterium [EU373137.1] 95 Eutrophic freshwater lake JF506023
A2 Uncultured Nitrosomonas sp. [FM997814.1] 97 ANAMMOX bioreactor JF506024
A3 Uncultured betaproteobacterium [FJ916428.1] 97 Temperate lakes JF506025
A4 Uncultured Nitrosomonas sp. [AF527015.1] 94 Wastewater treatment reactors JF506026
A5 Uncultured betaproteobacterium [GQ245689.1] 94 Landfill leachate treatment Plant JF506027
A6 Uncultured Nitrosomonas sp. [FM997833.1] 94 ANAMMOX bioreactor JF506028
A7 Uncultured Nitrosomonas sp. [FM997811.1] 95 ANAMMOX bioreactor JF506029
A8 Uncultured AOB [DQ196231.1] 99 Wastewater treatment process JF506030
A9 Uncultured betaproteobacterium [AF289172.1] 100 Lakes and rivers JF506031
A10 Uncultured betaproteobacterium [EU169043.1]. 96 Landfill cover soils JF506032
A11 Uncultured Nitrosospira sp.[HQ727018.1] 97 South Chile Sea JF506033
A12 Uncultured Nitrosomonas sp. [GU073375.1] 98 Contact oxidation system JF506034
A13 Uncultured Nitrosomonas sp. [AY648575.1] 97 Rivers JF506035
Nitrosomonas sp. [AY123811.1] 95
A14 Uncultured betaproteobacterium [FJ916650.1] 97 Temperate lakes JF506036
A15 Uncultured Nitrosomonas sp. [EU155071.1] 96 Prawn farm sediment JF506037
Uncultured Nitrosomonas sp. [FN429865.1] 96 ANAMMOX bioreactor
A16 Uncultured Nitrosomonas sp. [GQ183204.1] 94 Surface-flow wetland mesocosms JF506038
A17 Uncultured Nitrosomonas sp. [DQ857301.1] 98 Activated sludge JF506039

Previous studies have shown that the changes of pollutant
removal efficiencies were closely related to the microbial commu-
nity in the biological treatment system (Zhang et al., 2010). In phase
IV, it was interesting to note that some bands, such as T1 to T6, and
T12, appeared to be dominant in B1 to B3. Bands T1 and T3 had
more than 98% sequence similarity to Pseudomonas sp. and band
T12 was associated with Hyphomicrobium sp. (Table 2). Pseudo-
monas sp. and Hyphomicrobium sp. were the most well-known
denitrifying bacteria (Timmermans and Vanhaute, 1983; Bedzyk
et al., 1999; Kariminiaae et al., 2004; Labbe et al., 2007; Nakano
et al., 2008; Rajakumar et al., 2008; Green et al., 2010). A strain of
Pseudomonas sp. isolated by Su et al. (2006) and Hyphomicrobium
sp. isolated by Meiberg et al. (1980) were also capable of hetero-
trophic nitrification and denitrification, which might be related to
the occurrence of the SND.
The effluent ammonia concentrations in the four phases were
below 0.2 mg L
1, and there were two possible reasons for the
abnormal increase in ammonia removal in phase II: (1) Ammonia-
oxidizing bacteria (AOB) are chemolitho-autotrophs and exist
widely in most environments. Previous studies have shown that
higher concentrations of NHþ 4 eN favor AOB growth and nitrogen
removal in the oligotrophic environment (Koops and
Pommerening, 2001). In this study, the influent NHþ 4 eN concen-
trations in phases I and II were 0.40  0.13 mg L
1 and
0.54  0.19 mg L
1, and the NHþ 4 eN removal efficiency increased
from 43% in phase I to 84% in phase II; (2) The extension of the
anoxic zone may favor the occurrence of the anammox process, in
which nitrite and ammonia are converted directly into nitrogen gas
in the absence of oxygen.
Regarding the AOB community in this study, most of the excised
bands belonged to Nitrosomonas sp., which showed that Nitro-
somonas lineages could grow in oligotrophic niches (Regan et al.,
2002, 2003; Cebron et al., 2003; Lipponen et al., 2004; Qin et al.,
2007; de Vet et al., 2009; Yapsakli et al., 2010). Koops and
Pommerening (2001) found that N. lineages had low Ks value of
1.9e4.2 mM NHþ
4 eN, which made N. lineages a strong competitor in
low NHþ 4 eN concentration (Qin et al., 2007). The results in this
study showed that the AOB population had high similarity in both
oxic and anoxic environments, except for bands A4 and A5, which
disappeared without aeration. The results were in accordance with
those of Park et al. (2008). Furthermore, it was interesting that the
DGGE profiles of B2 (the biofilm from the middle of the reactor) and
B3 (the biofilm from the end of the reactor) in phase IV had higher
similarity to B1 than that in phase III. Therefore, the DO and influent
substrate concentration had little influence on the AOB community
in oligotrophic niche with low NHþ 4 eN concentration, and the AOB
community seemed to be more stable than the bacterial commu-
nity in different conditions. Limpiyakorn et al. (2005) also found
that no obvious shift of AOB community occurred until there was
the ammonia concentration over 5 mmol L
1 and nitrite concen-
tration over 2 mmol L
1.
5. Conclusions
The simultaneous nitrification and denitrification in contami-
nated source water bioremediation was achieved via DO controlling
step feeding. Low DO concentration at about 0.5 mg L
l favored the
enrichment of denitrifiers and the coexistence of functional
bacteria and algae, and the step-feeding process could increase the
community abundance in the end of biofilm system. The dominant
microorganisms such as Hyphomicrobium sp., Pseudomonas sp.,
Chloroflexi sp., Enterobacter sp., Pantoea sp., and Synechococcus sp.,
associated with denitrification and refractory organics utilizing,
were successfully enriched in the biofilms under low DO concen-
tration with step-feeding. AOB community of biofilms were stable
under different conditions and Nitrosomonas sp. was the predom-
inant AOB in the oligotrophic niche.
Acknowledgments
This work was supported by the National Key Technology R&D
Program (No. 2006BAJ08B01/2012BAJ25B07), Open Project of the
Zhejiang Provincial Key Laboratory of Environmental Pollution
Control Technology (No. 2010-12), and Research Projects of the
Department of Education of Zhejiang Province (No. Y200909172).
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