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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-

Nitrogen removal is an important aspect of ad-
vanced wastewater treatment process. The conven-
tional process of nitrogen removal by nitrification and
denitrification requires considerable amount of energy
and cost for aeration and carbon supply. An innova-
tive nitrogen removal process, anoxic ammonium oxi-
dation (anammox), offers an energy efficient way of
removing nitrogen in the wastewater. This process has
been reported to be a powerful piece of technology
and its application for nitrogen removal has several
advantages as lowering operation costs by 90%, and
energy requirement by 66% [1,2].
Anammox process is the major contributor to the
application of shortcut nitrogen removal system in
wastewater. This process is completed by a newly
identified Planctomycete bacteria, in which five gen-
ria [1,3-5]. However, anammox bacteria has slow
growth rate with a doubling time of 11 d, and the
process has a long history and unstable startup period
[6]. Strous et al. [7] indicated the optimal pH and
temperature for anammox process are 8 and 40 ± 3 °C,
respectively. Besides, a drawback of the anammox
process is its high sensitivity to substrates and oxygen.
For example, even 2 μM of oxygen concentration and
5-10 mM of nitrite concentration were reported to in-
hibit anammox activity [8].
A reactor system having good biomass retention
efficiency is suitable for anammox process. Therefore,
selection of an appropriate reactor system is one of the
decisive factors to carry out the process successfully.
A down-flow hanging sponge (DHS) reactor has been
successfully applied for removal of organic matter in
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)
Fig. 1. Experimental set-up.
teristic of retaining high concentration of biomass
fairly elongating the sludge retention time of the sys-
tem, which is an amiable environment for slow grow-
ing anammox bacteria. In this study, DHS reactor was
first applied for the enrichment of an anammox cul-
ture with emphasis on improving efficiency of nitro-
gen removal by the controlling different operational
parameters. The objective is to evaluate the applicabil-
ity of DHS system for reduction of ammonium to
gaseous nitrogen under anoxic condition.
MATERIALS AND METHODS
1. Experimental Apparatus
DHS were placed inside a closed 2.5 L rectangu-
lar column and had a working volume of 0.753 L,
based on sponge material having a void ratio of 97.1%
(Fig. 1). Triangular sponge (3 × 3 × 4 cm) strips were
tiled opposite each other on the inner wall of the col-
umn. The height of the column was 1 m, but the effec-
tive height was 2 m as the two sponge columns on op-
posite walls were connected in series by circulating
the flow through one sponge column to the opposite
one. The reactor column was made anaerobic by
flushing (5 mL min-1) an Ar/CO2 (95/5%) mixture for
10 min before start-up. Gas produced from the reactor
was collected using a gas bag.
2. Substrate Composition and Operation
Before the start-up of the system, sponge mate-
rial was inoculated by anammox granule and a small
amount of activated sludge. An artificial wastewater
constituting NH4Cl and NaNO2 was fed to the system
as main substrate with the addition of minerals. Sub-
strate mixture ratio given by Jetten et al. [8] was re-
ferred for the preparation of influent. The pH in the
feed was maintained at 8.0 by the addition of KHCO3,
and the temperature inside the column was controlled
at 30-35 °C. The nitrogen-loading rate was increased
by increasing the influent flow rate and total nitrogen
concentrations, dividing the entire experimental peri-
ods into 11 different phases.
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
Clone Library
Extraction of deoxyribonucleic acid from the
sludge was done and amplification of the 16S ribo-
somal ribonucleic acid (rRNA) gene was carried out
by polymerase chain reaction (PCR) according to the
previously described method [10]. Amplification of
anammox-specific 16S rRNA gene was performed
with the primer pair of Amx368f/Univ. 1392r [11,12].
The PCR products were purified with MinEluteTM
PCR purification kit (QIAGEN) and subsequently
cloned into Escherichia coli using the TOPO TA clon-
ing® kit (Invitrogen, USA). All 16S rRNA gene clones
were randomly selected. The sequences of the clones
were determined by dye terminator cycle sequencing
with a Quick Start Kit (Beckman Coulter Inc., USA)
and an automatic sequencer (CEQ2000XL, Beckman
Coulter Inc., USA). A 16S rRNA gene-based phy-
logenetic tree was constructed by applying the
neighbor-joining method with the ARB program.
Bootstrap re-sampling analysis for 1,000 replicates
was performed with the PAUP 4.0 package to estimate
the confidence of tree topologies [10].
RESULTS AND DISCUSSIONS
1. Performance of the DHS System
Application of a DHS reactor for anammox reac-
tion was examined under the anoxic condition, with
the results illustrated in Fig. 2. During the startup pe-
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-
sponding to the hydraulic retention time (HRT) of 2.0
h and a temperature of 30 °C. After nitrite concentra-
tion in the effluent decreased to 5.5 mg N L-1, total ni-
trogen concentration in the influent doubled to 80 mg
N L-1. However, a low nitrogen-removal rate was ob-
served in this phase and residual levels of nitrogen
compounds in the effluent were above 25.8 mg N L-1.
With an objective of raising the activity of anammox
bacteria at the same substrate concentration, the tem-
perature of the system was then increased to 35 °C in
phase-3. Here, the nitrogen-removal rate increased
from 0.26 to 0.45 kg N m-3 sponge d-1.
In phase-4, the nitrogen-loading rate was further
 Chuang et al: Anoxic Ammonium Oxidation in a DHS
Time (d)
(kg N m-3 sponge d-1)
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
influent flow rate (18 L d-1). According to [13], in-
creased flow rate allows deeper penetration of waste-
water into the sponge material. Increases in nitrogen
removal and dinitrogen production were observed in
this phase.
Subsequently, in phase-5, the HRT of the system
was further reduced to 0.7 h, which corresponded to a
nitrogen-loading rate of 2.98 kg N m-3 sponge d-1. The
removal rate further increased to 1.12 kg N m-3
sponge d-1, showing that the system could be operated
with stability even at an HRT of 0.7 h. We suggest
that the incremental increase in nitrogen removal is
due to the increase in bacterial growth and effective
substrate utilization.
In order to evaluate the effect of recirculation on
nitrogen removal, 100% effluent recirculation was in-
troduced in phase-6 of experiment, without changing
the nitrogen-loading rate. Although the distribution of
biomass in the reactor became more homogenous
compared to that of previous phase, the removal effi-
ciency increased only slightly from 38 to 40%. This
indicates that the recirculation of effluent does not
411
Time (d)
pH
Time (d)
have a significant effect on nitrogen removal.
In an attempt to raise nitrogen load, total nitro-
gen concentration in the influent was increased to 160
mg N L-1 in phase-7, which corresponded to a nitro-
gen-loading rate of 5.96 kg N m-3 sponge d-1. In this
phase, a two-fold increase in nitrogen removal was
achieved; however, nitrogen removal efficiency was
still low (36%).
In order to increase the removal efficiency, recir-
culation was increased to 300% in phase-8. The
maximum removal rate attained at this phase was 2.27
kg N m-3 sponge d-1; however, there was still signifi-
cant residual ammonium and nitrite in the effluent. A
large amount of biomass grew along the path of the
flow, but some dead space was observed in the sponge.
This occurrence of dead space was likely the main
cause of nitrogen removal limitation in the system. An
obvious decrease in nitrogen removal was observed in
the beginning of each phase (Fig. 2c), probably due to
the change in flow patterns through the sponge mate-
rial following the adjustment of influent flow rate.
To further confirm the efficiency of nitrogen re-
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
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
sponge d-1. The nitrogen removal rate attained in this
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
flow rate to 18 L d-1. Nitrogen removal efficiency
(95%) was satisfactory obtained in this phase. In addi-
tion, variation of pH in the reactor is shown in Fig. 2d.
During the operation, pH was observed to be the range
of 7.7-8.3.
Based on the results of the operation, the conver-
sion ratios between NO2--Nutilized/NH4+-Nremoved
(YNO −2 /NH + ) and NO3--Nproduction/NH4+-Nremoved
4
(YNO 3− /NH + ) are evaluated as a function of the total ni-
4
trogen conversion. The average value for YNO− /NH +
2 4
during the operation is 1.25 ± 0.08 and the YNO − /NH +
3 4
is 0.25 ± 0.04, showing that anammox was the domi-
nant process in this system [7].
The amount of biomass retained in the DHS re-
actor was quantified after the completion of phase-11.
The reactor had an average VS concentration of 5.59 g
VS L-1 based on sponge volume. As calculated based
on this sludge concentration, the anammox activity of
the retained biomass was established at 0.32 kg N kg-1
VS d-1 in phase-11. The ratio of volatile solids (VS) to
total solids (TS) was 0.17, which suggests high accu-
mulation of inorganic matter, probably due to high
concentrations of minerals in the substrate. An accu-
mulation of white precipitant was found in the middle
layer between the biomass and sponge in the DHS re-
actor, which could be an obstacle to normal develop-
ment of microorganisms [14]. The white precipitant
was likely calcium or magnesium because high con-
centrations of both were used in the medium [15].
Our study confirmed that the incremental
changes in operating parameters such as temperature,
influent flow rate and total nitrogen concentrations,
benefited nitrogen removal in the DHS reactor without
any significant inhibitory effects. Moreover, the in-
crease in down-flow rate by introducing effluent recir-
culation, which resulted in the retained biomass being
more homogenous, raised the removal efficiency from
35 (phase-4) to 95% (phase-11). Additionally, non-
(g L-1 based on sponge vol.)
Fig. 3. The variation of biomass concentration along the
height of the reactor.
uniform biomass retention with considerable dead
space restricted the removal capacity of applying a
DHS reactor for anoxic ammonium oxidation; how-
ever, a relatively high removal efficiency (95%) was
attained at a nitrogen-loading rate of 1.92 kg N m-3
sponge d-1.
2. Sludge Development in the DHS Reactor
The amount and the quality of retained sludge is
a very important factor in the nitrogen removal system.
The variation of biomass concentration along the
height of the reactor is shown in Fig. 3. Since the ef-
fective height of the reactor was 2 m, the sludge con-
centration along the total effective height is shown. It
is apparent that the biomass concentration in the upper
part of the DHS was higher than that in the other re-
gions. This could be the result of entrapment of bio-
mass during inoculation or entrapment of minerals.
After attaining a stable state in removal rate, the
sludge was more uniform than during the start-up pe-
riods. However, some dry portions were still observed
in the final phase and the VS/TS ratio was unexpect-
edly very low (0.17). Similar conditions were also ob-
served in another study of partial nitrification [16]. In
comparison with other conventional biological sys-
tems, sponges in the DHS hanging in the air, not sub-
mersing in the liquid phase, resulted in formation of
dry portions and tended to accumulate the precipitated
inorganic matter because of weak dissolution.
As previously mentioned, DHS systems have the
capacity to retain high TS including biomass and min-
eral in the sponge material, and no excess sludge was
observed in the effluent (data not shown). Trigo et al.
[17] suggested that low calcium concentration in the
medium retards the accumulation of salt precipitation
in the system. Hence appropriate influent flow-rate
plus the suitable salt concentrations in substrate will
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

3. Nitrogen Removal Characteristics for Different
Systems
Various nitrogen removal processes have been
researched and developed. However, the anammox
and application. Different reactor configurations have
been tried for anammox processes. Nitrogen removal
efficiencies by various reactor systems are summa-
rized in Table 1. Anammox activity was first observed
in a denitrifying fluidized bed reactor [18] and suc-
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
[6,21-25] because of reliable biomass retention and
complete bulk mixing. However, wash-out of bio-
mass is a problem affecting the total nitrogen removal
due to the slow-growth of anammox bacteria.
Efficient sludge retention is the crucial factor in
carrying out the process successfully, and biofilm sys-
tems were recommended as a good enriched system
for anammox. Fux et al. [26] demonstrated a nitrogen-
removal rate of 3.5 kg N m-3 d-1 in a fixed-bed reactor.
An elevated nitrogen-removal rate of 8.9 kg N m-3 d-1
was obtained in a gas-lift reactor by Sliekers et al. [27].
Among all systems, the greatest nitrogen-removal rate
of 26.0 kg N m-3 d-1 has been reported in an up-flow
fixed-bed column reactor by Tsushima et al. [28]. In
addition, the first full-scale anammox reactor was es-
tablished in Rotterdam [29].
The DHS systems with high biomass retention
were successfully developed and applied to sewage
treatment in combination with an upflow anaerobic
sludge bed (UASB) reactor. In this study, application
of a DHS reactor for the anammox process indicated
that the maximum nitrogen-removal rate attained was
2.27 kg N m-3 sponge d-1, which is not satisfactory in
comparison with other systems (see Table 1). A likely
reason for this poor performance is the material and
pore-size of the sponge used, which may not be suit-
able for retainment of anammox biomass. In addition,
limitation of sludge development, caused by non-
uniform flow patterns, formation of dry portions and
accumulation of considerable inorganic material in the
sponge restricted nitrogen removal. This necessitates
further manipulation in reactor operation or design.
Although there were some problems experienced in
this system, a relatively high nitrogen removal was
accomplished (95%).
4. Microbial Community of the DHS Reactor
To investigate the microbial diversities in the
DHS reactor that had performed anammox reaction,
we conducted anammox-specific 16S rRNA-gene
based cloning analysis. We could obtain PCR ampli-
cons and the anammox-specific 16S rRNA gene clone
library was constructed. These clones were randomly
selected and categorized into 10 phylotypes based on
partial sequence of approximately 500 bp in length.
The sequences of the phylotypes were fully deter-
mined and the representative sequences were affiliated
with 5 operational taxonomic units. The phylogenetic
affiliation of all analyzed phylotypes is shown in Fig. 4.
The results of microbial analysis showed the
most abundant microbes (31 clones in a total of 65
clones) in this system were closely related to Kue-
nenia stugartiensis (99% 16S rRNA gene sequence
similarity). Kuenenia stugartiensis has been found to
be the dominant microbes responsible for removal of
nitrogen compounds in the wastewater systems
[30,31]. In addition to Kuenenia, Anammoxoglobus
propionicus (sequence similarity 99%) were also ob-
served in a DHS during the anammox process.
Anammoxoglobus propionicus is an anammox bacte-
rium capable of propionate oxidation along with
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
cultured Chlorobi microbes were also observed in this
system, which probably utilized organic matter de-
rived from the microbial catabolism of anammox bac-
teria [32]. It was obtained that DHS system supplied a
good growth environment for anammox microbes,
wherein K. stugartiensis dominated over other species
for anammox reaction.
CONCLUSIONS
Application of the DHS reactor for the anammox
process is a new attempt to develop a low-priced ni-
trogen-removal process. As a result, a significant in-
cremental increase in nitrogen removal was achieved
by raising the influent flow rate, which increased bac-
terial growth and effective substrate utilization. Nitro-
gen removal rate attained 1.85 kg N m-3 sponge d-1
with 95% efficiency where “Candidatus Kuenenia
stugartiensis” was mainly responsible for anammox
reaction. However, some problems such as non-
uniform flow patterns, dry space formation and accu-
mulation of inorganic matter restricted the nitrogen-
removal capacity of DHS system. Further investiga-
tion for improving sludge development is needed to
enhance the nitrogen efficiency. The results of this
study will supply an experience for development of a
low-cost nitrogen removal system in wastewater.
ACKNOWLEDGEMENTS
The authors thank Kenichi Abe at Nagaoka Uni-
versity of Technology for assistance in this study.
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Discussions of this paper may appear in the discus-
sion section of a future issue. All discussions should
be submitted to the Editor-in-Chief within six months
of publication.
Manuscript Received: July 15, 2008
Revision Received: September 27, 2008
and Accepted: September 30, 2008