Process Biochemistry 43 (2008) 729735

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Nitrogen removal from low carbon-to-nitrogen wastewater in four-stage

biological aerated lter system
Hong-Duck Ryu 1, Daekeun Kim 1, Heun-Eun Lim 1, Sang-Ill Lee *
Department of Environmental Engineering, Chungbuk National University, 361-763 Cheongju, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 6 November 2007
Received in revised form 13 February 2008
Accepted 22 February 2008

In this study, a four-stage biological aerated lter (BAF) system was proposed for the enhancement of
nitrogen removal in the treatment of low carbon-to-nitrogen ratio (C/N ratio) municipal wastewater.
Laboratory experiments were conducted at two different hydraulic retention times (HRTs), i.e., 3 h and
6 h. Results of the long-term operation of the BAF system showed that its overall NH4-N removal
performance was stable with the removal efciency of 9596% on average, regardless of HRT applied. The
effectiveness of the proposed BAF was observed in denitrication performance. At the total chemical
oxygen demand (TCOD) to total kjeldahl nitrogen (TKN) ratio of 3:6, the efuent NO3-N concentration of
denitrication column was 2.7 mg/L. It was concluded that the four-stage BAF system proposed in this
study was excellent in nitrogen removal performance by employing four-column functioning as sorption,
nitrication, denitrication and purication, respectively.
2008 Elsevier Ltd. All rights reserved.

Keywords:
Nitrogen removal
Biological aerated lter
Low carbon-to-nitrogen ratio
Denitrication
Municipal wastewater
Hydraulic retention time

1. Introduction
The biological aerated lter (BAF) system was developed in
Europe and then widely applied all over the world as a novel
wastewater treatment system due to its advantages relative to
other systems [1]. Conventionally, BAF is submerged media
wastewater treatment reactors that combine oxic biological
treatment and biomass separation by depth ltration [2]. It adopts
a granular media as the support for microbial biolms that also
provides the depth ltration action. BAF offers a small footprint
alternative to conventional oxic process and it can be operated at
high loadings of biochemical oxygen demand (BOD) as compared
with trickling lters and activated sludge processes. In a single unit
operation of BAF, carbonaceous BOD removal, solids ltration and
nitrication can be achieved [3]. In addition, BAF can be applied to
the treatment of refractory wastewater such as textile and oil eld
wastewater using attached biomass on media [4,5].
Although BAF system has many advantages, it has difculty of
application to the treatment of raw wastewater which contains
high concentration of suspended solid (SS). For this reason,
BAF was commonly applied to the treatment of primary-treated

* Corresponding author. Tel.: +82 43 261 2469; fax: +82 43 272 2469.
E-mail addresses: nanumenv@hanmail.net (H.-D. Ryu), dkeuny@gmail.com
(D. Kim), hunulnim@nate.com (H.-E. Lim), gatorlee@chungbuk.ac.kr (S.-I. Lee).
1
Tel.: +82 43 261 3625; fax: +82 43 272 2469.
1359-5113/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2008.02.018

wastewater or municipal wastewater for tertiary nitrication

alone [68]. For the treatment of raw wastewater with BAF system,
SS should be removed properly from the wastewater because the
SS of inuent deteriorates the BAF performance by interfering with
mass transfer of oxygen and substrates to biolms. Westerman
et al. [9] initiated BAF process in treating swine wastewater, in
which ushed wastes were primarily collected in the settling basin
and the overow from the settling basin was used as the inuent
for BAF process. However, it should be noted that the pretreatment
of raw wastewater for SS removal may cause loss of organic matter
and then decrease the carbon-to-nitrogen ratio (C/N ratio) of
wastewater. Sufcient carbon must be available in order to
completely denitrify the nitrate formed during nitrication in the
nitrogen removal [10]. In full-scale pre-denitrication study [11],
incomplete denitrication was found in the feed COD/TKN ratio
less than 15.
Therefore, it is urged to modify existing BAF systems in which
denitrication performance can be enhanced in treating low C/N
ratio wastewater.
To the best of our knowledge, a few studies discussed BAF system
with respect to nitrogen removal. Nevertheless, its denitrication
efciency was quite low due to the low C/N ratio of inuent
wastewater [12,13]. In order to solve denitrication problem, some
researchers proposed a post-denitrication BAF process using
methanol as an external carbon source in order to increase the
C/N ratio [14,15]. However, it might cause a cost burden. Chiou and
Ouyang [16] alternatively suggested two-stage pre-denitrication/

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nitrication biolter system, in which the original carbon source in

the inuent was used for denitrication in order to substitute the
additional carbon source. However, its denitrication efciency was
not high enough to be effective even though feed wastewater had
relatively high SCOD to TKN ratio of 6:7.
Hence, further attention needs to be undertaken for the
successful utilization of the BAF system in treating the low C/N
ratio wastewater. Especially, a BAF system suitable for denitrication of low C/N ratio municipal wastewater is needed desperately
in Korea because the raw municipal wastewater has very low COD/
TKN ratio (COD/TKN ratio of 3:4 as annual average value).
For this purpose, we propose a four-stage BAF process, within
which both nitrication and denitrication steps were incorporated. Fig. 1 shows the operational concept of the four-stage BAF
process with two-step cycle employed in this study. Each step was
carried out in sequence by switching the ow directions as shown
in Fig. 1. In the 1st anoxic column, part of substrate in the inuent is
adsorbed on the attached biomass of the media and accumulated
within bacteria cells, which enable the ammonia-rich liquid
streams to go to the next column. Fast nitrication without
interfering with organic matter then occurs in the next column (1st
oxic column) with oxic condition. After that, the 2nd anoxic
column functions as denitrication. Finally, the last oxic column
(2nd oxic column) works for purication. When switching the ow
direction (step 2), carbonaceous substrates previously adsorbed on
the media (1st anoxic column in step 1) will be used as the
substrate for the denitricaion reaction. The use of bio-sorbed
carbonaceous substrates for denitrication in 1st and 2nd anoxic
columns by switching the ow direction is an important
distinction of the proposed four-stage BAF system in this study.
In addition, independent nitrication can be led by the separation
of heterotrophs and autotrophs. This conguration of proposed
four-stage BAF system would maximize its nitrogen removal
efciency although the system seems not economical in comparison with other existing BAF systems such as two-stage submerged
biological lter.
In this method, the experimental plan was then designed to
evaluate the performance of the process proposed in this study.
The evaluations were performed on nitrogen removal in the

Fig. 1. Operational concept of four-stage BAF process. Functions of each column

were indicated in parentheses.

system at two different hydraulic retention times (HRTs). The

effectiveness of the proposed system was also veried by
observing the microbial community structure during an operational period of nitrication in the system.
2. Materials and methods
2.1. System setup and operation
A lab-scale BAF system was prepared for this study (Fig. 2). The main system was
composed of four upow biolter columns connected in series. Each column was
1.90 m in height and 0.09 m in diameter with an effective volume of 12.1 L. The
columns were packed with expanded polystyrene (EPS) media (which is
approximately 23 mm in diameter and over 2000 m2/m3 on an average specic
surface area) to a depth of about 0.95 m. The density of packing media was 15 kg/
m3. In oxic columns, air was provided to maintain the concentration of dissolved
oxygen (DO) of 5  0.5 mg/L through diffusers located at the bottom of the column. In
contrast, DO was not provided in anoxic columns. The system was operated in two-step
cycle, i.e., step 1 and step 2, based on the direction of inuent ow (see Fig. 2). The
duration of each step was set at 45 min, i.e., two-step cycle operation was completed
for 90 min. The cycle operation has been repeated by switching inuent ow. The
activated sludge obtained from an oxic basin of Cheongju municipal wastewater
treatment plant was used as the inoculating sludge in the operation.
In order to remove excess biomass and solid particles accumulated on the media,
each column was backwashed in a counter-current manner at regular intervals of
12 h using nal efuent liquid from the BAF system. Each step of backwash lasted
during 1 min. The backwash sequence included air scour, followed by air scour and
water backwash. The water and air backwash application rates were set at 10 L/min
and 12 L/min, respectively.
The system has been operated at room temperature ranging from 18.3 8C to
25.1 8C, and averaged 21.3  0.1 8C. The ow rates applied were 38.66 m3/d and
19.33 m3/d, corresponding to HRT of 3 h and 6 h, respectively.

Fig. 2. Schematic of BAF system operated in step 1 (upper) and step 2 (bottom)
depending on the direction of inuent ow: (1) 1st anoxic column; (2) 1st oxic
column; (3) 2nd anoxic column; (4) 2nd oxic column; (5) inuent wastewater tank;
(6) inuent pump; (7) efuent storage tank (backwashing water tank); (8)
backwashing pump; (9) air blower; (10) level switch.

H.-D. Ryu et al. / Process Biochemistry 43 (2008) 729735

731

Table 1
Average inuent characteristics
Parameter

Average value

Suspended solid (SS) (mg/L)

Total chemical oxygen demand (TCOD) (mg/L)
Soluble chemical oxygen demand (SCOD) (mg/L)
Total kjeldahl nitrogen (TKN) (mg/L)
NH4-N (mg/L)
PO4-P (mg/L)
pH
TCOD/TKN
SCOD/TKN

65.1  24.1 a
116.9  32.2
77.9  1.9
34.1  3.1
30.3  3.1
2.0  0.6
7.82  0.5
4.3  1.1
2.1  0.6

Standard deviation.

2.2. Characteristics of municipal wastewater

The municipal wastewater was collected from a wastewater treatment plant,
which is located in Cheongju, Korea. The raw inuent was characterized in Table 1.
Note that the TCOD/TKN ratio of inuent was found to be 4:3. Based on the report of
Water Pollution Control Federation [11], this TCOD/TKN ratio was insufcient to
complete the reaction of denitrication within the system.
2.3. Analytical procedures
Chemical analyses of samples were performed according to the procedures
described in Standard methods [17]. Samples were analyzed for SS (standard code:
2540 D), TCOD & SCOD (standard code: 5220 D), TKN (standard code: 4500-N B),
PO4-P (standard code: 4500-P E), NO2-N (standard code: 4500-NO2 B) and NO3-N
(4500-NO3 B). NH4-N was determined using the HACH Nessler method.
Scanning electron microscopy (SEM) analysis was used to observe and analyze
the surface of EPS media of the 1st oxic column before and after the run of BAF
system. The EPS media before and after the run of BAF system were cut into
approximately 0.5-cm sized pieces. Cut media were freeze-dried to maintain the
structure of EPS media and stored in a desiccator before SEM analysis. Dried media
were analyzed with a scanning electron microscope equipped with energy
dispersive X-ray spectrometer (SEM 515, Philips).
2.4. Community structure analysis
The community structure of 1st oxic column was analyzed by terminal
restriction fragment length polymorphism (T-RFLP) method. The media and
efuents from the 1st oxic column for each operational step as shown in Fig. 2 were
collected for the analysis. DNA was extracted using the bead beating procedure
(UltraCleanTM DNA Purication kit, Mo Bio Laboratories Inc., Solana Beach, CA,
USA) according to the study by Miler et al. [18] The polymerase chain reaction (PCR)
targeting 16S ribosomal RNA (rRNA) genes of eubacteria were performed. The 27F
(E. coli numbering 827: 50 -AGAGTTTGATCMTGGCTCAG-30 ) and 785R (E. coli
numbering 785804: 50 -ACTACCRGGGTATCTAATCC-30 ) were used for eubacterial
primer. The PCR reaction mixture contained 1 PCR buffer, 1.5 mM MgCl2, 160 mM
each deoxynucleoside triphosphate (dNTP), 0.3 mM each primer, approximately
10 ng of the DNA template, and 1.5 U of Ex Taq DNA polymerase in a nal volume of
50 ml. The PCR was performed in a thermal cycler. The amplication was done with
on denaturation step 95 8C for 3 min, followed by 30 cycles of denaturation at 95 8C
for 30 s, annealing at 52 8C for 30 s, and extension at 72 8C for 10 min. Amplicons
form duplicate PCR reaction mixtures were pooled using a PCR purication kit
(UltaClean PCR clean-up kit, MoBio Laboratories Inc., Carlsbad, CA, USA). HhaI
(TaKaRa, Japan) was used for restriction enzymes. Runs were performed on
BaseStationTM DNA Fragment Analyzer (MJ Research, USA).

Fig. 3. NH4-N removal characteristics of the BAF system.

The efuent NH4-N concentration of the BAF system was

presented in Fig. 3. The average efuent concentrations of
ammonia nitrogen were 1.1 mg/L and 1.4 mg/L for HRT of 6 h
and 3 h, respectively. Correspondingly, the removal efciencies of
ammonia nitrogen were 96.3% and 95.4% for the HRTs of 6 h and
3 h, respectively, revealing relatively high nitrogen removal in this
system. This observation may be explained by the fact that the
application of attached growth shows higher performance relative
to suspended growth. This was found in other studies that much
higher nitrication rate was observed in the attached growth
process (AGP) rather than the suspended growth process (SGP)
while receiving the same nitrogen loadings [19,20]. In addition, the
unique conguration of the BAF system allowed for independent
nitrication in column 2 and it would be responsible for an increase
in the nitrication capacity of the BAF system at short HRT.
The nal efuent NOx-N concentration at the HRTs of 6 h and 3 h
was 18.7 mg/L and 13.8 mg/L on average, respectively, as shown in
Fig. 4. Note here that most of the efuent NOx-N was nitrate (nitrite
concentration was almost zero) and the average TCOD/TKN ratio of
inuent at the HRTs of 6 h and 3 h was 3:4 and 5:2, respectively. In
the operation of the four-stage BAF system, the low efuent NOx-N
concentration would act as the benet of its application compared
with other existing BAF systems operated in pre-denitrication
conguration. Comparing the average nal efuent NOx-N concentration of the four-stage BAF system with that of two-stage

3. Results and discussion

3.1. Overall performance
The BAF system showed quite a high removal for SS at the HRTs
of 6 h and 3 h, respectively. It was found that the SS in efuent at
the HRT of 6 h was in the range of 0.76.7 mg/L (average of 3.4 mg/
L), and average SS removal efciency was about 95%. As to the HRT
of 3 h, there had an average SS removal efciency of 94% and
efuent SS was in the range of 1.37.1 mg/L (average of 4.2 mg/L).
These high SS removal performances would be due to bioltration
through a submerged medium that serves two purposes: biological
conversion of organic matter by the biomass attached to the large
support medium surface and physical retention of suspended
particles by ltration through the deep lter bed.

Fig. 4. Final efuent NOx-N concentration.

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H.-D. Ryu et al. / Process Biochemistry 43 (2008) 729735

Fig. 6. Dominating group of bacteria identied in the BAF system using T-RFLP: (A)
media in the 1st oxic column for step 1; (B) efuent from the 1st oxic column for
step 1; (C) media in the 1st oxic column for step 2; (D) efuent from the 1st oxic
column for step 2.

Fig. 5. Efuent proles of TCOD, NH4-N, NO2-N and NO3-N in step 1 at the HRT of
3 h: initial TCOD concentration was 202.6 mg/L; initial NH4-N concentration was
34.9 mg/L; initial concentrations of NO2-N and NO3-N were almost zero; initial
TCOD to TKN ratio (TCOD/TKN) was 3:6.

pre-denitrication/nitrication biolter system proposed by Chiou

and Ouyang [16], the nal efuent NOx-N concentration of the fourstage BAF system was much lower than that of that of two-stage predenitrication/nitrication biolter system although the synthetic
wastewater used as inuent had quite a high SCOD to TKN ratio of
6:7. They found that the nal efuent NOx-N concentration was
28 mg/L on average. It is speculated that the excellent denitrication
performance of the four-stage BAF system proposed in this study
resulted from effective use of carbonaceous substrates in raw
municipal wastewater. In the operation of the four-stage BAF
system, the biosorption of available carbon sources of inuent in
column 1 and column 3 by the effective arrangement of aerobic and

Fig. 7. SEM images: (a) EPS media (200T); (b) microbes attached on the EPS media (2000T).

H.-D. Ryu et al. / Process Biochemistry 43 (2008) 729735

anoxic phase and the switch of ow direction would be maximized.

Therefore, the BAF system proposed in this study are particularly
suitable for low C/N ratio wastewater, where the lack of carbon
affects the nutrient removal efciency. The better performance in
the HRT of 3 h for NOx-N removal would be due to the increased
C/N ratio of inuent at the HRT of 3 h. For the purpose of nitrogen
removal, the effectiveness of the BAF system proposed in this system
was allowed by both changing ow direction of inuent and
employing four columns. Each column has a distinguished function
in the system. It is shown in Fig. 5 that each functional column in step
1 had different concentration proles of COD and nitrogen. The
detailed function of each column of BAF system is provided in the
following sections.

733

During the 107-day operational period, the average quantity of

backwashed sludge to be wasted from the BAF system was about
0.30 kg SS/(m3 d).
3.2. Column 1 representing 1st anoxic reactor
In column 1 functioning as biosorption, efuent TCOD
concentration was much lower relative to inuent (see Fig. 5).
On average, 75% of inuent TCOD was bio-sorbed during the
operation period of 45 min and it would be utilized as the substrate
for denitrication reaction in step 2. Most of ammonia nitrogen
was not removed and the concentrations of NO2-N and NO3-N
were almost zero in column 1.

Fig. 8. Effects of applied mass loadings on the removals of TCOD, NH4-N, TKN, and TN, respectively.

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H.-D. Ryu et al. / Process Biochemistry 43 (2008) 729735

3.3. Column 2 representing 1st oxic reactor

In column 2, i.e., 1st oxic reactor, NH4-N in inuent was almost
completely converted into nitrate with 96.1% efciency on average
(see Fig. 5). It is inferred that nitriers in column 2 would dominate
and nitrication was then enhanced. By performing the community
structure analysis (T-RFLP analysis), the dominating group of
bacteria on the media was identied as shown in Fig. 6. The terminal
restriction fragments (T-RF) of 565 bp, 364 bp, and 205 bp were
observed. It illustrates that the dominating group was ammonia
oxidizing bacteria including Nitrosomonas and Nitrosolobus, of which
group was also identied in the efuent from this column. T-RF of
151 bp also illustrates that Cardococcus existed in the community. It
is well known that Cardococcus is Gram-negative bacteria which
prevents sludge bulking in activated sludge system [21].
Enhanced nitrication in column 2 can be related to the fact that
the process is AGP indicated in Section 3.1. The enriched microorganism particles of AGP, as presented in the SEM result of Fig. 7,
could contribute to increase of nitrication efciency. Nevertheless,
it is believed that high nitrication rate and the domination of
nitrifying bacteria in column 2 was mainly due to the removal of
organic substrates by biosorption in column 1. It decreased the C/N
ratio of efuent of column 1 and caused predominance of nitrifying
bacteria in growth competition between autotrophic and heterotrophic micro-organism population in column 2. Some authors
reported the relationships between C/N ratio and nitrication
performance. Carrera et al. [22] showed in the study of a modied
LudzackEttinger pilot-plant conguration for 365 days that an
exponential decrease of nitrication rate was observed when the
inuent COD/N ratio increased. Also, Komorowska-Kaufman et al.
[23] reported that when the COD/N ratio was low the nitrication
process was stable in Bardenpho mode reactors.
The most BAF process previously studied and applied was
single-stage where simultaneous removal of organic matter and
ammonia nitrogen was also available [3,24]. However, this
application was practically limited since nitrication was inhibited
depending on the C/N ratio of inuent. This problem can be solved
by applying the proposed four-stage BAF system.
3.4. Column 3 representing 2nd anoxic reactor
It is also shown in Fig. 5 that nitrate produced in column 2
functioning as nitrication was almost denitried in column 3. The
efuent concentration of nitrate nitrogen in column 3 was about
2.7 mg/L on average. In contrast, high efuent concentrations of
TCOD and NH4-N were observed. This was due to the biosorption of
organic matter and NH4-N on the media occurring in this column
during the operation period of the previous step. TCOD bio-sorbed
on the media would be utilized as the substrate for the
denitrication reaction. Hence, efuent TCOD concentrations
decreased with time since those substrates would be locally
limited within this column. This speculation would explain the
observation that the denitrication performance was improved in
column 3, i.e., 2nd anoxic column. Considering the low carbon-tonitrogen ratio in raw wastewater (C/N = 3:6), the results observed
in Fig. 5 were remarkable as compared with other BAF studies [12
16] and published results of Water Pollution Control Federation
[11] in which it was recommended in full-scale pre-denitrication
study that the inuent COD/N ratio should be higher than 15.
3.5. Column 4 representing 2nd oxic reactor
It is important to note that the nal efuent concentration
of NOx-N was relatively high. In Fig. 5, efuent concentration of
NOx-N from column 4 was 17.5 mg/L on average during an expe-

rimental period employing the HRT of 3 h. Since the nal column,

i.e., column 4 has been maintained in the aerobic condition and
received relatively high concentration of NH4-N from the previous
column, i.e., column 3, nitrication partially occurred in column 4.
However, column 4, originally functioning purication, discharged
relatively the low concentration of COD and total nitrogen.
3.6. Effect of inuent loading on removals of organic matters and
nitrogen
A plot of removed TCOD, NH4-N, TKN and TN versus their
loading rates for the four-stage BAF system is shown in Fig. 8 at the
HRTs of 6 h and 3 h, respectively. The linear regressions of their
data are also shown to indicate the trend. The TCOD applied loads
varied from 0.3 kg TCOD/(m3 d) up to 1.4 kg TCOD/(m3 d). With
these very high carbonaceous loads, the BAF system achieved the
TCOD removal efciencies of 76% and 80% at the HRTs of 6 h and
3 h, respectively. In terms of removing ammonia, it can be seen that
the average ammonia nitrogen removal was consistently at 95
96% at loading up to 0.29 kg NH4-N/(m3 d). It was reported by
previous researches that NH4-N removal efciency increased at the
applied load ranges of 0.050.8 kg NH4-N/(m3 d) with the
regressed lines indicated [7,12]. Most of applied TKN loads were
varied from 0.12 kg TKN/(m3 d) up to 0.33 kg TKN/(m3 d), and the
percentage TKN removed was 89% and 90% at the HRTs of 6 h and
3 h, respectively. In TN removal, linear regression of removed TN
with applied loading rates had an R2 of 0.32 and 0.53 at the HRT of
6 h and 3 h, respectively. TN removal performance increased as
the applied TN loadings increased.
4. Conclusions
The proposed four-stage BAF system for enhancing nitrogen
removal in the treatment of low C/N ratio municipal wastewater
was evaluated. Based on the results of experimental tests, the
following conclusions were drawn:
(1) The BAF system proposed in this study was able to effectively
nitrify ammonia nitrogen with about 95% efciency.
(2) The proposed system was very effective on denitrication at
low inuent TCOD to TKN ratio. It was revealed that the system
can minimize the organic loss and maximize the better use of
bio-sorbed organics for the denitrication reaction.
(3) Overall, nitrogen removal performance was enhanced by both
changing ow direction of inuent and employing four
functional columns with roles of sorption, nitrication,
denitrication, and purication, respectively.

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