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Anoxic ammonium oxidation by application of a down-flow hanging sponge


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J. Environ. Eng. Manage., 18(6), 409-417 (2008)

ANOXIC AMMONIUM OXIDATION BY APPLICATION OF A DOWN-FLOW


HANGING SPONGE (DHS) REACTOR

Hui-Ping Chuang,1 Takashi Yamaguchi,2 Hideki Harada3 and Akiyoshi Ohashi1,*


1
Department of Social and Environmental Engineering
Hiroshima University
Hiroshima 739-8527, Japan
2
Department of Environmental and Systematic Systems Engineering
Nagaoka University of Technology
Niigata 940-2188, Japan
3
Department of Civil Engineering
Tohoku University
Sendai 980-8579, Japan

Key Words: Anoxic ammonium oxidation (anammox), down-flow hanging sponge (DHS) reactor,
gaseous nitrogen, sludge development

ABSTRACT

Anoxic ammonium oxidation (anammox) process has been recommended as an energy-saving


technology in the removal of nitrogen during the treatment of wastewater. In this study we
investigated propagation and activity of anammox microbes in a closed down-flow hanging sponge
(DHS) reactor. Ability to retain high biomass and maintain long sludge retention time has made DHS
reactor a suitable system for development of slow-growing anammox bacteria. The closed reactor
was fed with artificial wastewater containing NH4Cl and NaNO2, and run at four differently
controlled operating parameters; temperature, influent flow rate, total nitrogen concentrations and
effluent recirculation. Ten months of continuous operation showed that anammox bacteria could be
effectively enriched in a DHS in which “Candidatus Kuenenia stugartiensis” dominated. Favorable
nitrogen removal rate attained was 1.85 kg N m-3 sponge d-1 with 95% of removal efficiency. It was
concluded that DHS technology in combination with anammox process benefited in developing a
low-cost nitrogen-removal system.

INTRODUCTION era have been reported as the known anammox bacte-


ria [1,3-5]. However, anammox bacteria has slow
Nitrogen removal is an important aspect of ad- growth rate with a doubling time of 11 d, and the
vanced wastewater treatment process. The conven- process has a long history and unstable startup period
tional process of nitrogen removal by nitrification and [6]. Strous et al. [7] indicated the optimal pH and
denitrification requires considerable amount of energy temperature for anammox process are 8 and 40 ± 3 °C,
and cost for aeration and carbon supply. An innova- respectively. Besides, a drawback of the anammox
tive nitrogen removal process, anoxic ammonium oxi- process is its high sensitivity to substrates and oxygen.
dation (anammox), offers an energy efficient way of For example, even 2 μM of oxygen concentration and
removing nitrogen in the wastewater. This process has 5-10 mM of nitrite concentration were reported to in-
been reported to be a powerful piece of technology hibit anammox activity [8].
and its application for nitrogen removal has several A reactor system having good biomass retention
advantages as lowering operation costs by 90%, and efficiency is suitable for anammox process. Therefore,
energy requirement by 66% [1,2]. selection of an appropriate reactor system is one of the
Anammox process is the major contributor to the decisive factors to carry out the process successfully.
application of shortcut nitrogen removal system in A down-flow hanging sponge (DHS) reactor has been
wastewater. This process is completed by a newly successfully applied for removal of organic matter in
identified Planctomycete bacteria, in which five gen- wastewater [9]. This system has a remarkable charac-

*Corresponding author
Email: ecoakiyo@hiroshima-u.ac.jp
410 J. Environ. Eng. Manage., 18(6), 409-417 (2008)

3. Analysis

To monitor the anammox performance of a


closed DHS reactor, NH4+, NO2- and NO3- in influent
and effluent were regularly measured by the colori-
metric method (Hach, USA). The compositions of the
off-gas were determined by gas chromatography
(Shimadzu GC-8AIT for CO2 and N2, Japan). The
biomass inside the sponge was taken out and the con-
centration was measured at the end of the experiment.

4. Construction of Anammox-specific 16S rRNA


Fig. 1. Experimental set-up. Clone Library

teristic of retaining high concentration of biomass Extraction of deoxyribonucleic acid from the
fairly elongating the sludge retention time of the sys- sludge was done and amplification of the 16S ribo-
tem, which is an amiable environment for slow grow- somal ribonucleic acid (rRNA) gene was carried out
ing anammox bacteria. In this study, DHS reactor was by polymerase chain reaction (PCR) according to the
first applied for the enrichment of an anammox cul- previously described method [10]. Amplification of
ture with emphasis on improving efficiency of nitro- anammox-specific 16S rRNA gene was performed
gen removal by the controlling different operational with the primer pair of Amx368f/Univ. 1392r [11,12].
parameters. The objective is to evaluate the applicabil- The PCR products were purified with MinEluteTM
ity of DHS system for reduction of ammonium to PCR purification kit (QIAGEN) and subsequently
gaseous nitrogen under anoxic condition. cloned into Escherichia coli using the TOPO TA clon-
ing® kit (Invitrogen, USA). All 16S rRNA gene clones
MATERIALS AND METHODS were randomly selected. The sequences of the clones
were determined by dye terminator cycle sequencing
1. Experimental Apparatus with a Quick Start Kit (Beckman Coulter Inc., USA)
and an automatic sequencer (CEQ2000XL, Beckman
DHS were placed inside a closed 2.5 L rectangu- Coulter Inc., USA). A 16S rRNA gene-based phy-
lar column and had a working volume of 0.753 L, logenetic tree was constructed by applying the
based on sponge material having a void ratio of 97.1% neighbor-joining method with the ARB program.
(Fig. 1). Triangular sponge (3 × 3 × 4 cm) strips were Bootstrap re-sampling analysis for 1,000 replicates
tiled opposite each other on the inner wall of the col- was performed with the PAUP 4.0 package to estimate
umn. The height of the column was 1 m, but the effec- the confidence of tree topologies [10].
tive height was 2 m as the two sponge columns on op-
posite walls were connected in series by circulating RESULTS AND DISCUSSIONS
the flow through one sponge column to the opposite
one. The reactor column was made anaerobic by 1. Performance of the DHS System
flushing (5 mL min-1) an Ar/CO2 (95/5%) mixture for
10 min before start-up. Gas produced from the reactor Application of a DHS reactor for anammox reac-
was collected using a gas bag. tion was examined under the anoxic condition, with
the results illustrated in Fig. 2. During the startup pe-
2. Substrate Composition and Operation riod (phase-1) the reactor was operated at a low nitro-
gen-loading rate of 0.48 kg N m-3 sponge d-1, corre-
Before the start-up of the system, sponge mate- sponding to the hydraulic retention time (HRT) of 2.0
rial was inoculated by anammox granule and a small h and a temperature of 30 °C. After nitrite concentra-
amount of activated sludge. An artificial wastewater tion in the effluent decreased to 5.5 mg N L-1, total ni-
constituting NH4Cl and NaNO2 was fed to the system trogen concentration in the influent doubled to 80 mg
as main substrate with the addition of minerals. Sub- N L-1. However, a low nitrogen-removal rate was ob-
strate mixture ratio given by Jetten et al. [8] was re- served in this phase and residual levels of nitrogen
ferred for the preparation of influent. The pH in the compounds in the effluent were above 25.8 mg N L-1.
feed was maintained at 8.0 by the addition of KHCO3, With an objective of raising the activity of anammox
and the temperature inside the column was controlled bacteria at the same substrate concentration, the tem-
at 30-35 °C. The nitrogen-loading rate was increased perature of the system was then increased to 35 °C in
by increasing the influent flow rate and total nitrogen phase-3. Here, the nitrogen-removal rate increased
concentrations, dividing the entire experimental peri- from 0.26 to 0.45 kg N m-3 sponge d-1.
ods into 11 different phases. In phase-4, the nitrogen-loading rate was further
Chuang et al: Anoxic Ammonium Oxidation in a DHS 411

Time (d) Time (d)


(kg N m-3 sponge d-1)

pH

Time (d) Time (d)


Fig. 2. The performance of the closed DHS reactor. (a) Influent flow rate and temperature (b) Nitrogen concentrations
(c) Nitrogen loading rate and removal efficiency (d) pH profile during the experiment.

increased to 1.94 kg N m-3 sponge d-1 by increasing have a significant effect on nitrogen removal.
influent flow rate (18 L d-1). According to [13], in- In an attempt to raise nitrogen load, total nitro-
creased flow rate allows deeper penetration of waste- gen concentration in the influent was increased to 160
water into the sponge material. Increases in nitrogen mg N L-1 in phase-7, which corresponded to a nitro-
removal and dinitrogen production were observed in gen-loading rate of 5.96 kg N m-3 sponge d-1. In this
this phase. phase, a two-fold increase in nitrogen removal was
Subsequently, in phase-5, the HRT of the system achieved; however, nitrogen removal efficiency was
was further reduced to 0.7 h, which corresponded to a still low (36%).
nitrogen-loading rate of 2.98 kg N m-3 sponge d-1. The In order to increase the removal efficiency, recir-
removal rate further increased to 1.12 kg N m-3 culation was increased to 300% in phase-8. The
sponge d-1, showing that the system could be operated maximum removal rate attained at this phase was 2.27
with stability even at an HRT of 0.7 h. We suggest kg N m-3 sponge d-1; however, there was still signifi-
that the incremental increase in nitrogen removal is cant residual ammonium and nitrite in the effluent. A
due to the increase in bacterial growth and effective large amount of biomass grew along the path of the
substrate utilization. flow, but some dead space was observed in the sponge.
In order to evaluate the effect of recirculation on This occurrence of dead space was likely the main
nitrogen removal, 100% effluent recirculation was in- cause of nitrogen removal limitation in the system. An
troduced in phase-6 of experiment, without changing obvious decrease in nitrogen removal was observed in
the nitrogen-loading rate. Although the distribution of the beginning of each phase (Fig. 2c), probably due to
biomass in the reactor became more homogenous the change in flow patterns through the sponge mate-
compared to that of previous phase, the removal effi- rial following the adjustment of influent flow rate.
ciency increased only slightly from 38 to 40%. This To further confirm the efficiency of nitrogen re-
indicates that the recirculation of effluent does not moval, in phase-9, the reactor was operated with the
412 J. Environ. Eng. Manage., 18(6), 409-417 (2008)

same parameters as in phase-8, but the attached sludge


was disturbed to reduce the dead space. However, an
unfortunate decrease in nitrogen removal at the initial
stage was observed, which might be due to altered
flow patterns through the sponge-cube. The maximum
nitrogen-removal rate attained at this phase was 1.95
kg N m-3 sponge d-1, less than that of the previous
phase.
In addition to removal rate, removal efficiency is
another key indicator of the anammox process. How-
ever, removal efficiencies of the preceding phases 2-9
were about 40%. In subsequent phases, loading rates
were reduced and the performance of the system was
investigated. Total nitrogen concentration in influent
(g L-1 based on sponge vol.)
was reduced to 80 mg N L-1 in phase-10 (Fig. 2b), cor-
responding to a nitrogen loading rate of 2.98 kg N m-3 Fig. 3. The variation of biomass concentration along the
sponge d-1. The nitrogen removal rate attained in this height of the reactor.
phase was 2.01 kg N m-3 sponge d-1, a 68% efficiency.
In phase-11, the nitrogen load decreased further to
1.94 kg N m-3 sponge d-1 by decreasing the influent uniform biomass retention with considerable dead
flow rate to 18 L d-1. Nitrogen removal efficiency space restricted the removal capacity of applying a
(95%) was satisfactory obtained in this phase. In addi- DHS reactor for anoxic ammonium oxidation; how-
tion, variation of pH in the reactor is shown in Fig. 2d. ever, a relatively high removal efficiency (95%) was
During the operation, pH was observed to be the range attained at a nitrogen-loading rate of 1.92 kg N m-3
of 7.7-8.3. sponge d-1.
Based on the results of the operation, the conver-
sion ratios between NO2--Nutilized/NH4+-Nremoved 2. Sludge Development in the DHS Reactor
(YNO −2 /NH + ) and NO3--Nproduction/NH4+-Nremoved
4
(YNO 3− /NH + ) are evaluated as a function of the total ni- The amount and the quality of retained sludge is
4
trogen conversion. The average value for YNO− /NH + a very important factor in the nitrogen removal system.
2 4
during the operation is 1.25 ± 0.08 and the YNO − /NH + The variation of biomass concentration along the
3 4
is 0.25 ± 0.04, showing that anammox was the domi- height of the reactor is shown in Fig. 3. Since the ef-
nant process in this system [7]. fective height of the reactor was 2 m, the sludge con-
The amount of biomass retained in the DHS re- centration along the total effective height is shown. It
actor was quantified after the completion of phase-11. is apparent that the biomass concentration in the upper
The reactor had an average VS concentration of 5.59 g part of the DHS was higher than that in the other re-
VS L-1 based on sponge volume. As calculated based gions. This could be the result of entrapment of bio-
on this sludge concentration, the anammox activity of mass during inoculation or entrapment of minerals.
the retained biomass was established at 0.32 kg N kg-1 After attaining a stable state in removal rate, the
VS d-1 in phase-11. The ratio of volatile solids (VS) to sludge was more uniform than during the start-up pe-
total solids (TS) was 0.17, which suggests high accu- riods. However, some dry portions were still observed
mulation of inorganic matter, probably due to high in the final phase and the VS/TS ratio was unexpect-
concentrations of minerals in the substrate. An accu- edly very low (0.17). Similar conditions were also ob-
mulation of white precipitant was found in the middle served in another study of partial nitrification [16]. In
layer between the biomass and sponge in the DHS re- comparison with other conventional biological sys-
actor, which could be an obstacle to normal develop- tems, sponges in the DHS hanging in the air, not sub-
ment of microorganisms [14]. The white precipitant mersing in the liquid phase, resulted in formation of
was likely calcium or magnesium because high con- dry portions and tended to accumulate the precipitated
centrations of both were used in the medium [15]. inorganic matter because of weak dissolution.
Our study confirmed that the incremental As previously mentioned, DHS systems have the
changes in operating parameters such as temperature, capacity to retain high TS including biomass and min-
influent flow rate and total nitrogen concentrations, eral in the sponge material, and no excess sludge was
benefited nitrogen removal in the DHS reactor without observed in the effluent (data not shown). Trigo et al.
any significant inhibitory effects. Moreover, the in- [17] suggested that low calcium concentration in the
crease in down-flow rate by introducing effluent recir- medium retards the accumulation of salt precipitation
culation, which resulted in the retained biomass being in the system. Hence appropriate influent flow-rate
more homogenous, raised the removal efficiency from plus the suitable salt concentrations in substrate will
35 (phase-4) to 95% (phase-11). Additionally, non- facilitate the improvement of sludge development in
Chuang et al: Anoxic Ammonium Oxidation in a DHS 413

Table 1. Nitrogen removal characteristic for different system


Reactor Temp. HRT Inf. NH4+-/NO2--N NLRb NRRc N removal
Substrate Inocula pH References
typea (°C) (h) conc. (g L-1) (kg N m-3 d-1) (kg N m-3 d-1) (%)
FBR Synthetic Denitrifying 36 4.2 7.0 0.09-0.13 – 0.70-1.50 – [18]
Synthetic Denitrifying 30 4.2 7.0 0.42/0.55 – 4.80 – [19]
Synthetic Denitrifying 36 22-42 8.0 0.07-0.84/0.07-0.84 – 1.80 83-85 [33]
D Effluentd Denitrifying 36 3.5-264 8.0 1.10-2.10/0.07-0.84 – 1.50 81-99 [33]
FB Synthetic Denitrifying 36 6-23 8.0 0.07-0.84/0.07-0.84 – 1.10 92-99 [33]
Synthetic – 26-27 – 7.3 – – 3.50 80 [26]
Synthetic Nitrifying 35 – – – – 0.35f – [34]
SBR Synthetic Denitrifying 32-33 6.2-48 7.0-8.0 0.30/0.40 – 1.00 – [20]
Synthetic Anammox – – – 0.45/0.46 – 0.75 – [6]
Synthetic Anammox 35 6.0 7.8-8.0 0.38/0.38 0.75 0.59 78 [23]
Synthetic Anammox – 18 7.5-8.0 0.06-0.21/0.06-0.24 0.60 – 67-99e [24]
DSg Sludge from CFh 31 – 7.5-8.0 – – 2.40 – [21]
Synthetic Anammox (80%) 30 24 7.8 0.26/0.20 – 0.31 – [22]
Synthetic Activated sludge 30 24 7.8 – – 0.60 – [25]
CSTR Synthetic Anammox 30 120 7.0-8.0 0.007-0.75/0.008-1.15 0.004-0.662 0.003-0.58 90-94 [35]
Gas-lift Synthetic Anammox (80%) – 6.7-43 7.5 1.36/1.37 – 8.90 83 [27]
Synthetic Anammox 30 – 8.0 0.90/1.10 2.00 1.76 88 [23]
UASB Synthetic Anammox – – – 0.71-1.03/0.027-1.41 – 2.3-6.4 86-92 [36]
PWi UASB granule 35 120 8.4-8.6 2.16/2.50 – 0.6-0.7 65-71 [37]
Synthetic Activated sludge 30 12 – 0.27-0.52/0.35-0.57 2.18 – 33-86 [38]
Batch Urine Anammox 30 – 8.0 4.10/3.40 – 1.0-1.3 – [39]
PWi UASB granule 35 120 – 1280/2070 – 1.15 70 [40]
MSBR Synthetic Anammox 35 24 8.0 0.01-0.39/0.01-0.39 – 0.71 – [17]
UFBCR Synthetic Denitrifying 37 8-1.4 7.0-7.5 – 0.10-9.40 6.00 64 [28]
Synthetic Denitrifying 37 8-0.2 7.0-7.5 – 0.10-58.0 26.0 45 [28]
ABF Synthetic Anammox 37 3.0-0.67 7.2 – 19.1 11.5 60 [41]
Synthetic Anammox 20-22 3.0-0.67 7.2 – 12.5 8.10 65 [41]
GSR Synthetic Nitrifying+Anammox 32-35 19.4 – – – 10.0 – [29]
USFF Synthetic Activated sludge 30 12 7.5-8.0 0.28-0.52/0.31-0.63 1.28-2.57 – 28-90 [38]
ASBR Synthetic Activated sludge 30 16 7.5-8.0 0.28-0.53/0.31-0.59 0.86-1.60 – 54-97 [38]
j
DHS Synthetic AN(80%)+AS (20%) 30-35 0.7-2.0 7.6-8.2 0.02-0.08/0.02-0.08 0.48-5.96 0.26-2.27 38-95 This study
a
FBR: Fluidized bed reactor; FB: Fixed bed reactor; SBR: Sequencing batch reactor; RBC: Rotating biological contactor; UASB: Up-flow anaerobic
sludge bed reactor; MSBR: Membrane sequencing batch reactor; UFBCR: Up-flow stationary fixed-bed column reactor; ABF: Anaerobic biological
filtrated reactor; GSR: Granule sludge reactor; MB: Moving bed reactor; USFF: Up-flow stationary fixed film reactor; ASBR: Anaerobic sequencing
batch reactor; DHS: Down-flow hanging sponge reactor
b
NLR: Nitrogen loading rate
c
NRR: Nitrogen removal rate
d
D Effluent: Anaerobic digester effluent
e
Nitrite (limiting substrate) removal percentage
f
Urea nitrogen removal (kg (NH2)2CO-N L-1 d-1)
g
DS: Digester supernatant
h
Sludge from CF: Sludge collected from effluent of Cloth filter system
i
PW: Piggery waste
j
AN (80%) + AS (20%): Anammox (80%) + activated sludge (20%)

the sponge system. process has garnered an increased attention in research


and application. Different reactor configurations have
3. Nitrogen Removal Characteristics for Different been tried for anammox processes. Nitrogen removal
Systems efficiencies by various reactor systems are summa-
rized in Table 1. Anammox activity was first observed
Various nitrogen removal processes have been in a denitrifying fluidized bed reactor [18] and suc-
researched and developed. However, the anammox cessfully enriched by van de Graaf et al. [19]. Later,
414 J. Environ. Eng. Manage., 18(6), 409-417 (2008)

the sequencing batch reactors were widely applied further manipulation in reactor operation or design.
[6,21-25] because of reliable biomass retention and Although there were some problems experienced in
complete bulk mixing. However, wash-out of bio- this system, a relatively high nitrogen removal was
mass is a problem affecting the total nitrogen removal accomplished (95%).
due to the slow-growth of anammox bacteria.
Efficient sludge retention is the crucial factor in 4. Microbial Community of the DHS Reactor
carrying out the process successfully, and biofilm sys-
tems were recommended as a good enriched system To investigate the microbial diversities in the
for anammox. Fux et al. [26] demonstrated a nitrogen- DHS reactor that had performed anammox reaction,
removal rate of 3.5 kg N m-3 d-1 in a fixed-bed reactor. we conducted anammox-specific 16S rRNA-gene
An elevated nitrogen-removal rate of 8.9 kg N m-3 d-1 based cloning analysis. We could obtain PCR ampli-
was obtained in a gas-lift reactor by Sliekers et al. [27]. cons and the anammox-specific 16S rRNA gene clone
Among all systems, the greatest nitrogen-removal rate library was constructed. These clones were randomly
of 26.0 kg N m-3 d-1 has been reported in an up-flow selected and categorized into 10 phylotypes based on
fixed-bed column reactor by Tsushima et al. [28]. In partial sequence of approximately 500 bp in length.
addition, the first full-scale anammox reactor was es- The sequences of the phylotypes were fully deter-
tablished in Rotterdam [29]. mined and the representative sequences were affiliated
The DHS systems with high biomass retention with 5 operational taxonomic units. The phylogenetic
were successfully developed and applied to sewage affiliation of all analyzed phylotypes is shown in Fig. 4.
treatment in combination with an upflow anaerobic The results of microbial analysis showed the
sludge bed (UASB) reactor. In this study, application most abundant microbes (31 clones in a total of 65
of a DHS reactor for the anammox process indicated clones) in this system were closely related to Kue-
that the maximum nitrogen-removal rate attained was nenia stugartiensis (99% 16S rRNA gene sequence
2.27 kg N m-3 sponge d-1, which is not satisfactory in similarity). Kuenenia stugartiensis has been found to
comparison with other systems (see Table 1). A likely be the dominant microbes responsible for removal of
reason for this poor performance is the material and nitrogen compounds in the wastewater systems
pore-size of the sponge used, which may not be suit- [30,31]. In addition to Kuenenia, Anammoxoglobus
able for retainment of anammox biomass. In addition, propionicus (sequence similarity 99%) were also ob-
limitation of sludge development, caused by non- served in a DHS during the anammox process.
uniform flow patterns, formation of dry portions and Anammoxoglobus propionicus is an anammox bacte-
accumulation of considerable inorganic material in the rium capable of propionate oxidation along with
sponge restricted nitrogen removal. This necessitates anammox reaction [5]. Besides, large amount of un-

Fig. 4. Anammox-specific 16S rRNA-based populations identified in the closed DHS reactor was constructed by the
neighbor-joining method based on 16S rRNA sequences. Bootstrap value (> 70%) for analysis of 1000
replicates was shown at each node of the tree. The tree was rooted with 16S rRNA sequence of Mathanosaecina
frisius.
Chuang et al: Anoxic Ammonium Oxidation in a DHS 415

cultured Chlorobi microbes were also observed in this 5. Kartal, B., J. Rattray, L.A. van Niftrik, van de
system, which probably utilized organic matter de- Vossenberg, J.M.C. Schmid, R.I. Webb, S.
rived from the microbial catabolism of anammox bac- Schouten, J.A. Fuerst, J. Sinninghe Damsté,
teria [32]. It was obtained that DHS system supplied a M.S.M. Jetten and M. Strous, Candidatus
good growth environment for anammox microbes, "Anammoxoglobus propionicus" a new propionate
wherein K. stugartiensis dominated over other species oxidizing species of anaerobic ammonium
for anammox reaction.
oxidizering bacteria. Syst. Appl. Microbiol., 30(1),
CONCLUSIONS 39-49 (2007).
6. Van Dongen, U., M.S.M. Jetten and M.C.M. van
Application of the DHS reactor for the anammox Loosdrecht, The SHARON®-Anammox® process
process is a new attempt to develop a low-priced ni- for the treatment of ammonium rich wastewater.
trogen-removal process. As a result, a significant in- Water Sci. Technol., 44(1), 153-160 (2001).
cremental increase in nitrogen removal was achieved 7. Strous, M., J.G. Kuenen and M.S.M. Jetten, Key
by raising the influent flow rate, which increased bac- physiology of anaerobic ammonium oxidation.
terial growth and effective substrate utilization. Nitro- Appl. Environ. Microb., 65(7), 3248-3250 (1999).
gen removal rate attained 1.85 kg N m-3 sponge d-1 8. Jetten, M.S.M., M. Strous, K.T. van de Pas-
with 95% efficiency where “Candidatus Kuenenia Schoonen, J. Schalk, U.G.J.M. van Dongen, A.A.
stugartiensis” was mainly responsible for anammox van de Graaf, S. Logemann, G. Muyzer, M.C.M.
reaction. However, some problems such as non-
van Loosdrecht and J.G. Kuenen, The anaerobic
uniform flow patterns, dry space formation and accu-
mulation of inorganic matter restricted the nitrogen- oxidation of ammonium. FEMS Microbiol. Rev.,
removal capacity of DHS system. Further investiga- 22(5), 421-437 (1999).
tion for improving sludge development is needed to 9. Tandukar, M., S. Uemura, I. Machdar, A. Ohashi
enhance the nitrogen efficiency. The results of this and H. Harada, A low-cost municipal sewage
study will supply an experience for development of a treatment system with a combination of UASB
low-cost nitrogen removal system in wastewater. and the "fourth-generation" down-flow hanging
sponge reactors. Water Sci. Technol., 52(1-2),
ACKNOWLEDGEMENTS 323-329 (2005).
10. Sekiguchi, T., Y. Kamagata, K. Nakamura, A.
The authors thank Kenichi Abe at Nagaoka Uni- Ohashi and H. Harada, Fluorescence in situ
versity of Technology for assistance in this study.
hybridization using 16S rRNA-targeted
oligonucleotides reveals localization of
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39. Udert, K.M., C. Fux, M. Münster, T.A. Larsen, H. sion section of a future issue. All discussions should
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autotrophic denitrification of source-separated of publication.
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40. Ahn, Y.H. and H.C. Kim, Nutrient removal and Revision Received: September 27, 2008
microbial granulation in anaerobic process and Accepted: September 30, 2008

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