Sustain. Environ. Res., 20(4), 205-211 (2010)
(Formerly, J. Environ. Eng. Manage.)
205
TREATMENT OF MUNICIPAL WASTEWATER USING AN ANAEROBIC-ANOXICOXIC BIOLOGICAL FILTER REACTOR PACKED WITH CARBON FIBERS AND
AERATED WITH MICROBUBBLES
Takahiro Yamashita1,*, Ryoko Yamamoto-Ikemoto2, Eiji Sakurai3,
Kouhei Aikawa4 and Erika Kaneko5
1
Pollution Control Research Team
National Institute of Livestock and Grassland Science
Ibaraki 305-0901, Japan
2
Institute of Science and Engineering
Kanazawa University
Ishikawa 920-1192, Japan
3
Spring Field Ltd.
Ishikawa 921-8034, Japan
4
Graduate School of Natural Science and Technology
Kanazawa University
Ishikawa 920-1192, Japan
5
Nippon Rensui Co., Ltd.
Tokyo 170-0005, Japan
Key Words: Municipal wastewater, nutrient removal, sulfate-reducing prokaryotes, nested PCRDGGE, carbon fibers, microbubbles
ABSTRACT
An anaerobic-anoxic-oxic biological filter reactor was applied to sewage treatment for nutrient
removal. Average concentrations of total organic carbon (TOC), suspended solids (SS), total N (TN)
and total P (TP) in the influent of the reactor were 46, 46, 37 and 5.8 mg L-1, respectively. Effluent
TOC was consistently about 5 mg L-1 and the average concentration of SS was 11 mg L-1. Although
the average removal efficiency of TN was 52%, it increased to over 70% when most of ammonium
was nitrified to nitrate in the oxic bed under the conditions of hydraulic residence time > 1.5 h and
dissolved oxygen > 2 mg L-1. Average removal efficiency of TP was 50%. In the anaerobic bed, 80%
of sulfate decreased and 59% of dissolved organic carbon (DOC) was removed. The decrease in
DOC tended to correlate with a decrease in sulfate. Although the excess sludge produced in the oxic
bed was put into the anaerobic bed, the effluent water quality did not become poor. These results
suggested that sulfate-reducing prokaryotes played an important role in organic removal and excess
sludge reduction in the anaerobic bed. Subsequently, the microbial community of sulfate-reducing
prokaryotes in the anaerobic biofilm was examined by the dissimilatory (bi)sulfite reductase genetargeting nested PCR-DGGE, and the sequences of ten excised bands from DGGE profiles were
determined.
INTRODUCTION
Excess sludge generated from sewage treatment
plants has increased due to the increase in coverage of
sewer systems. A great amount of excess sludge is
also generated from industrial wastewater treatment
plants. Methods to decrease excess sludge by biological treatment are urgently needed. Recently, we pro*Corresponding author
Email: [email protected]
posed a cylindrical anaerobic-oxic biological filter reactor packed with carbon fibers and aerated with microbubbles. As the result of an examination using dye
works wastewater, it was reported that suspended solids (SS) and the colorization removal of the proposed
reactor was better than that of an activated sludge reactor, and excess sludge production was very low [1].
In the anaerobic bed, sulfate reduction was predomi-
206
Sustain. Environ. Res., 20(4), 205-211 (2010)
nant. It was estimated that sulfate reduction played an
important role in organic removal and sludge decomposition. However, the sulfate-reducing prokaryotes in
the anaerobic bed had not been identified.
Since all the produced sulfide could be reoxidized in an oxic bed placed on top of the anaerobic
bed of the reactor, it was shown that no sulfide gas
had escaped from the reactor. However, oxygen was
consumed for sulfide removal. On the other hand, sulfide can be utilized for denitrification. In this study, a
reactor was developed using an anaerobic-anoxic-oxic
process for nutrient removal, and was applied to municipal wastewater treatment. Furthermore, sulfatereducing prokaryotes carrying the dissimilatory
(bi)sulfite reductase (dsr) genes in the anaerobic
biofilm were evaluated by a nested PCR-DGGE
analysis [2] and the sulfate reduction activity was analyzed.
MATERIALS AND METHODS
Figure 1 shows the anaerobic-anoxic-oxic biological filter reactor used in the experiment. The reactor was made of an acrylic resin column 50 cm in diameter and 200 cm in height. The total reactor volume
was 340 L. The reactor was divided into three beds.
The bottom bed was packed with hanging carbon fibers. The middle and upper beds were packed with
carbon fibers coiled around the mesh cylinders. In addition, 1.8 kg of iron ribbon (including 0.52-0.58% of
carbon and 0.6-0.9% of manganese), which was 0.10.2 mm thick and 0.5-1.0 mm wide, was packed in the
middle bed. The reactor was set up in the Johoku
Wastewater Treatment Plant in Kanazawa City and
seeded with 18 L of return sludge. Settled sewage was
placed into the bottom anaerobic bed. The upper bed
was aerated with microbubbles. Part of the effluent
from the upper bed was returned to the middle anoxic
bed. The carbon fibers had high biological affinity.
Since such carbon fibers were broken under heavy
aerating conditions, microbubbles were used for aeration in the oxic bed. The circulation water with microbubbles produced a unique rotation flow in the reactor,
and SS were trapped by the carbon fibers [1]. The reactor hydraulic retention time (HRT) was regulated to
10 h. After 202 d of operation, HRT was extended to
24 h, and at 370 d, it was shortened to 12 h.
The temperature and dissolved oxygen (DO) of
the oxic bed in the reactor were measured once a week.
The influent and effluent of each bed in the reactor
were collected and taken to the laboratory. The concentrations of SS, pH, biochemical oxygen demand
(BOD), total organic carbon (TOC) and total N (TN)
(TOC and TN analyzer, Shimadzu, Co. Ltd., TOC-V
CPN, TNM-1) were analyzed. Total P (TP) (phosphorus analyzer, Bran + Luebbe GmbH, AACS-III) was
analyzed after degradation of organic phosphorus by
potassium peroxydisulfate. The concentrations of sul
Effluent
20
cm
v
Oxic bed
v
Generator
with
micro-bubble
v
Iron bed
Anoxic bed
64 cm
5 cm
11 cm
50 cm
Pump
Influent
Anaerobic bed
50 cm
50 cm
Fig. 1. Experimental set-up.
fate, nitrate, nitrite, thiosulfate, ammonium (Ion
Chromatograph, Shimadzu Co., Ltd., IC-10AD), organic acids and bicarbonate (High Performance
Liquid Chromatography with post column detection,
Shimadzu, Co., Ltd., LC-10AD), dissolved organic
carbon (DOC) (TOC analyzer) and PO4 (phosphorus
analyzer) in the filtered samples were analyzed.
After 670 d of operation, the biofilm in the anaerobic bed was taken out, and the sulfate reduction
was examined using batch experiments according to
the following procedures. One g of biofilm and the
substrates purged with nitrogen gas were put into a 50
mL plastic syringe and stirred by a magnetic stirrer at
30 or 20 °C. The mixed liquor was taken out of the syringe at 0, 3, 6, 12, 24 and 48 h, and the concentrations of sulfate, organic acids and bicarbonate in the
filtered sample were analyzed. Municipal wastewater
and artificial organic wastewater (polypeptone = 200,
CH3COOK = 100, yeast extract = 20, NaHCO3 = 71,
KH2PO4 = 44, CaCl2•2H2O = 29.44; all in mg L-1)
were used as the substrates. Sulfate (MgSO4•7H2O =
257 mg L-1)-supplemented substrates were also used.
The microbial community of sulfate-reducing
prokaryotes in the anaerobic biofilm was examined by
the dsr gene targeting nested PCR-DGGE. After the
anaerobic biofilm was taken out of the reactor, DNA
was extracted using the UltraClean Soil DNA kit (Mo
Bio Laboratories, Inc., USA). The dsrB fragments of
the extracted DNA were amplified with a two-step
nested PCR protocol using combinations of primers
targeting the dsr genes (Table 1). First, PCR amplification of an about 1900-bp dsrAB fragment was per-
Yamashita et al.: Anaerobic-onoxic-oxic Biological Filter Reactor
207
Table 1. Dissimilatory (bi)sulfite reductase gene (dsrAB)-targeted primers used in this study
Primer
DSRmix 1F
DSRmix4R
DSR1F
DSR1Fa
DSR1Fb
Sequence (5’-3’)a
ACS CAC TGG AAG CAC G
ACC CAY TGG AAA CAC G
GGC CAC TGG AAG CAC G
DSR4R
DSR4Ra
DSR4Rb
DSR4Rc
GTG ATG CAG TTA CCG CA
GTG TAA CAG TTT CCA CA
GTG TAA CAG TTA CCG CA
GTG TAG CAG TTK CCG CA
Reference
[4]
[3]
[3]
[4]
[3]
[3]
[3]
DSRp2060Fb
CAA CAT CGT YCA YAC CCA GGG
[5]
a
Ambiguities: R (G or A); Y (C or T); K (G or T); M (A or C); S (G or C); W (A or T).
b
A 40-bp GC clamp was added to the 5’ end (CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC) when the PCR
product was used for DGGE analysis (DSRp2060F-GC).
formed using the primers DSRlFmix (equimolar mixture of DSRlF, DSRIFa, and DSRlFb) and DSR4Rmix
(equimolar mixture of DSR4R, DSR4Ra, DSR4Rb,
DSR4Rc) described by Loy et al. [3]. A dsrB fragment
of about 350-bp was amplified from dsrAB PCR
products using the primer pair DSRp2060F-GC and
DSR4R described by Miletto et al. [2]. DGGE was
performed using a D-Code system (Bio-Rad Laboratories, Japan) according to the instruction manual. To
identify DGGE bands, a small piece of gel from the
middle of the target band was excised from the gel using a gel chip (Funa gel chip, Funakoshi Co., Ltd.)
and incubated in 20 μL of distilled water for 24 h at
4 °C. The eluted DNA was reamplified by PCR using
primers DSRp2060F and DSR4R without a GC clamp.
PCR products for sequencing were purified using
GenElute PCR Clean-Up Kit (Sigma-aldrich, Inc.,
USA) and sequenced using the DYEnamic ET Terminator Cycle Sequencing Kit and an ABI PRISM 3100
Genetic Analyzer (Applied Biosystems Japan Ltd.).
To determine the closest known relatives of the partial
DNA sequences obtained, searches were performed in
the public data library DDBJ (http://www.ddbj.
nig.ac.jp) using FASTA search tool. Phylogenetic
trees were calculated and drawn using Clustal X and
njplot. All dsrB gene sequences were deposited in the
database under the accession numbers AB507433AB507445.
RESULTS AND DISCUSSION
1. Reactor Performances
The composition of municipal wastewater is
shown in Table 2. Average concentrations of TOC, SS,
TN and TP in the influent of the reactor were 46, 46,
37 and 5.8 mg L-1, respectively. The courses of HRT,
water temperature, TOC, DOC, SS, TN and TP concentrations are shown in Fig. 2. When the HRT was
controlled at 10 h, the reactor did not show good performance. Therefore, HRT was extended to 24 h after
202 d of operation. Since the reactor showed improved wastewater treatment performance, the HRT
Table 2. Composition of the municipal wastewater used
in the experiment
TOC (mg L-1)
SS (mg L-1)
TN (mg L-1)
TP (mg L-1)
SO42- (mg L-1)
NH4+ (mg L-1)
pH
Average
46
46
37
5.8
23
35
-
Range
16.1-72.3
20-90
21.3-61.0
2.9-9.0
14.9-31.2
14.6-50.9
6.7-7.6
was shortened to 12 h after 370 d operation. Effluent
TOC and DOC concentrations were consistently about
5 mg L-1, and the average SS was 11 mg L-1. When
the air diffuser was used for oxygen supply due to the
breaking down of the microbubbles generator from
day 271 to 326 of operation, the effluent SS temporarily increased. This result suggests that the water flow
around the carbon fibers produced by microbubbles
was important in the biofilm filtration. Almost no organic acids such as acetate and propionate were detected during the operational period in the aerobic bed
or the iron bed. The TN in the influent and effluent
were 21-61 and 6-35 mg L-1, respectively, and the average removal efficiency of TN was 52%. TP in the
municipal wastewater decreased in the iron bed. However, the phosphate removal efficiency decreased
gradually due to iron corrosion.
Figure 3 shows the effects of HRT and DO in the
oxic bed on the nitrification and TN removal efficiencies in the reactor. The nitrification efficiency was
high when HRT was over 1.5 h and DO was over 2
mg L-1 in the oxic bed. The TN removal efficiency
was over 70% under the same condition. These results
suggest that the nitrification and TN removal efficiency improved when the HRT was over 1.5 h and
the DO was over 2 mg L-1 in the oxic bed.
2. Effects of Sulfate Reduction in the Anaerobic Bed
The courses of the sulfate and DOC concentrations through the reactor are shown in Fig. 4. In the
HRT (ｈ）
Water temperature（℃）
50
45
40
35
30
25
20
15
10
5
0
Water temperature
0
100
200
HRT
300
400
Period
（days）
Period
(d)
In fluent
Effluent
60
40
20
0
80
70
60
50
40
30
20
10
0
In fluent
Effluent
500
80
70
60
50
40
30
20
10
0
10
600
700
In fluent (TOC)
Effluent (TOC)
In fluent (DOC)
Effluent (DOC)
Influent
Effluent
8
TP （mg L-1）
SS （mg L-1）
80
TN （mg L -1）
TOC and DOC (mg L-1)
100
10
9
8
7
6
5
4
3
2
1
0
DO in the oxic bed
DO in the oxic bed （mg L -1）
Sustain. Environ. Res., 20(4), 205-211 (2010)
208
6
4
2
0
0
100
200
300
400
500
600
0
700
100
200
300
400
500
600
700
Period (days)
Period (days)
Period (d)
Period (d)
Fig. 2. Courses of effluent characteristics.
50
Sulfate （mg L -1）
80
60
40
L -1)
DO (mg
Not over 2
Over 2
20
TN removal efficiency (%)
0
100 0
1
2
HRT (hrs)
3
Influent
4
80
60
Anaerobic bed
Anoxic bed
Effluent
40
30
20
10
0
50 0
DOC （mg L-1）
Nitrification efficiency (%)
100
100
200
300
400
500
600
700
500
600
700
Period (days)
40
30
20
10
0
0
40
DO (mg L -1)
Not over 2
Over 2
20
100
200
300
400
Period
(days)
Period
(d)
Fig. 4. The courses of the sulfate and DOC concentrations in the reactor.
0
0
1
2
HRT
(hrs)
HRT (h)
3
4
Fig. 3. The relationship between HRT in the oxic bed
and the nitrification and TN removal efficiency.
anaerobic bed, sulfate and DOC decreased by sulfate
reduction. In the anoxic bed, sulfate increased by sulfur denitrification, and the remaining sulfur was oxidized in the oxic bed. Figure 5 shows the relationship
between sulfate reduction and the DOC removal effi
ciencies in the anaerobic bed. When sulfate reduction
occurred predominantly, the DOC removal efficiency
tended to increase. Although excess sludge produced
in the oxic bed was returned to the anaerobic bed, the
effluent water quality did not become poor. These results suggest that sulfate-reducing prokaryotes played
an important role in organic removal and excess
sludge reduction in the anaerobic bed.
Yamashita et al.: Anaerobic-onoxic-oxic Biological Filter Reactor
sulfate-reducing prokaryote. On the other hand, when
artificial wastewater was used at 20 °C, acetate and
bicarbonate increased in sulfidogenic conditions.
Propionate was accumulated without sulfate. Some
sulfate-reducing prokaryotes oxidized propionate to
acetate. It was considered that both prokaryotes coexisted in the anaerobic bed.
HRT
10 h
12 h
24 h
60
40
4. Microbial Community of Sulfate-reducing
Prokaryotes in the Anaerobic Bed
60
70
80
90
Sulfate reduction efficiency (%)
100
Fig. 5. Relationship between sulfate reduction efficiency
and the DOC removal efficiency in the anaerobic
bed.
3. Sulfate Reduction in the Batch Experiment
400
(a1)
120
300
90
200
60
100
30
0
0
400
Concentrations (mg L-1)
150
(a2)
120
300
90
200
60
100
30
0
0
0
12
24
Time (h)
36
48
Bicarbonate concentration (mg L-1)
Concentrations (mg L-1)
150
Bicarbonate concentration (mg L-1)
The sulfate-reducing activity of the anaerobic
biofilm in the reactor was examined by batch experiment. Figure 6 shows typical results of the batch experiments using the anaerobic biofilm. When municipal wastewater was used for the substrate at 30 °C, bicarbonate increased by sulfate reduction. After 12 h, a
small amount of acetate was accumulated. Organic
matter in the sewage was decomposed completely by
The microbial community of sulfate-reducing
prokaryotes in the anaerobic biofilm was examined by
dsr gene-targeting nested PCR-DGGE. Figure 7
shows DGGE profiles. The DGGE profiles of the dsr
gene extracted from the biofilm on day 538 to 670 of
operation showed similar patterns. The sequences of
ten bands excised from the DGGE profiles were determined. Table 3 shows the closest species match obtained from FASTA search between DNA of the excised DGGE bands and sequences from the DDBJ database. DGGE bands at the same horizontal positions
(bands 3-1 to 3-3, 7-1 and 7-2, respectively) were considered to represent the same prokaryotes. The dominant sulfate-reducing prokaryotes in the anaerobic
biofilm on days 538-670 of operation was most
closely related to Desulfotomaculum nigrificans (band
3) by FASTA search. Prior to the 1980s, the prokary150
400
(b1)
120
300
90
200
60
100
30
0
150
0
400
(b2)
120
300
90
200
60
100
30
0
0
0
12
24
36
48
Bicarbonate concentration (mg L-1)
50
Bicarbonate concentration (mg L-1)
0
Concentrations (mg L-1)
20
Concentrations (mg L-1)
DOC removal efficiency (%)
100
80
209
Time (h)
Fig. 6. Typical results of the batch experiments using the anaerobic biofilm. (a) Municipal wastewater (30 °C); (b)
Artificial wastewater (20 °C ); (a1 and b1) with SO4; (a2 and b2) without SO4. Symbols: sulfate (●), acetate
(▲), propionate (■), bicarbonate (◆).
Sustain. Environ. Res., 20(4), 205-211 (2010)
210
a
b c d
e f
35%
7-1
8
9
10
3-2
3-3
4
5
6
7-2
Denaturant gradient
1
2
3-1
80%
Fig. 7. Denaturing gradient gel electrophoresis of dsrB
PCR product from the anaerobic biofilm in the
reactor. Lanes: a, the anaerobic biofilm (538
days); b, the anaerobic biofilm (559 days); c, the
anaerobic biofilm (608 days); d, the anaerobic
biofilm (670 days); e, PCR negative control; f,
marker. Labelled DGGE bands represent bands
that were excised and sequenced.
ote had gone by two names, from Desulfovibrio desulfuricans and Clostridium nigrificans [6]. D. nigrificans can utilize sulfate, sulfite and thiosulfate as electron acceptors, and oxidize organic substrates incom-
pletely [7]. In addition, close relations to prokaryotes
Desulfacinum infernum (bands 4 and band 7), Desulfovibrio aminophilus (band 5), Desulfovibrio fructosovorans (band 6), Desulfovibrio longus (band 8)
and Syntrophobacter fumaroxidans (bands 9, 10) were
detected. The detected prokaryotes except for D. infernum oxidize organic substrates incompletely [7].
Since organic acids were not detected in the effluent
of the anaerobic bed, methanogenic prokaryotes might
coexist with sulfate-reducing prokaryotes.
CONCLUSIONS
Treatment of municipal wastewater was examined using an anaerobic-anoxic-oxic biological filter
reactor packed with carbon fibers and aerated with
microbubbles. The results can be summarized as follows.
1. The reactor showed good performance for organic
and SS removal when HRT was over 10 h.
2. The nitrification and TN removal efficiency were
improved when HRT was over 1.5 h and DO was
over 2 mg L-1 in the oxic bed.
3. TP from the municipal wastewater decreased in
the iron bed. However, the phosphate removal
efficiency decreased gradually due to iron
corrosion.
4. In the anaerobic bed, abut 80% of DOC could be
removed. It was suggested that sulfate-reducing
prokaryotes played an important role in organic
removal and excess sludge reduction.
5. The dominant sulfate-reducing prokaryote
Table 3. Closest species matches obtained from FASTA search between DNA of excised DGGE bands and sequences
from the DDBJ database
FASTA
Sequence
Closest relative
Accession No.b
Band
% Identityc
Overlap (bp)
length (bp)
1
334
Desulfotomaculum nigrificans
AF482466
67.4
316
2
334
Desulfotomaculum nigrificans
AF482466
68.0
316
3-1
329
Desulfotomaculum nigrificans
AF482466
68.4
313
3-2
337
Desulfotomaculum nigrificans
AF482466
67.8
320
3-3
337
Desulfotomaculum nigrificans
AF482466
67.2
317
4
289
Desulfacinum infernum
AF418194
77.3
282
5
327
Desulfovibrio aminophilus
AY626029
69.3
257
6
320
Desulfovibrio fructosovorans
AF418187
73.2
250
7-1
345
Desulfacinum infernum
AF418194
70.0
340
7-2
318
Desulfacinum infernum
AF418194
67.1
295
8
341
Desulfovibrio longus
AB061540
71.0
314
9
334
Syntrophobacter fumaroxidans
CP000478
69.6
336
10
326
Syntrophobacter fumaroxidans
CP000478
68.7
332
a
Band numbers correspond to those presented in Fig. 7.
b
Accession number of sequence of the closest relative found by FASTA search.
c
Percentage of identical nucleotides in the sequence obtained from the DGGE band and the sequence of the closest relative found in
the GenBank database.
a
Yamashita et al.: Anaerobic-onoxic-oxic Biological Filter Reactor
detected by dissimilatory (bi)sulfite reductase
gene- targeted nested PCR-DGGE was D.
nigrificans.
4.
ACKNOWLEDGMENTS
The authors wish to express their thanks to
graduate students, Ms. Namiko Tsukihashi and Mr. Jianqing Zhu, Kanazawa University for their technical
assistance.
5.
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Manuscript Received: October 13, 2009
Revision Received: March 10, 2010
and Accepted: March 26, 2010