Sustain. Environ. Res., 21(3), 155-160 (2011)
155
USE OF TWO-STAGE BIOLOGICAL PROCESS IN TREATING THIN FILM
TRANSISTOR LIQUID CRYSTAL DISPLAY WASTEWATER OF
TETRAMETHYLAMMONIUM HYDROXIDE
Han-Lin Lin, Bo-Kuang Chen, Hui-Ping Hsia, Gen-Hao Yang, Ya-Fei Yang, Yu-Chieh Chao and
Sheng-Shung Cheng*
Department of Environmental Engineering
National Cheng Kung University
Tainan 701, Taiwan
Key Words: Anaerobic hydrolysis, anaerobic ammonium oxidation (ANAMMOX), anaerobic-anoxic
process, tetramethylammonium hydroxide (TMAH), total organic carbon (TOC)/NH4+-N,
TFT-LCD wastewater
ABSTRACT
Anaerobic ammonium oxidation (ANAMMOX) is a newly discovered and cost-effective
process to remove nitrogen compounds in wastewater treatment. The process is the anoxic oxidation
of ammonium converting to nitrogen with nitrite as electron acceptor. However, many organic
inhibitors and unknown niche of ANAMMOX are the challenges to apply ANAMMOX process to
industrial wastewater treatment. In this study, two-stage bioprocess of anaerobic hydrolysis and
anoxic ANAMMOX were performed for tetramethylammonium hydroxide (TMAH) wastewater
treatment. In the first anaerobic hydrolysis process, the organic nitrogen from TMAH wastewater
containing 110-250 mg L-1 (TMAH 600-2000 mg L-1) could be hydrolyzed into ammonium. A 95%
hydrolysis efficiency of TMAH could be achieved in this process. In the second ANAMMOX
process, the effluent from the first process with a total organic carbon (TOC)/NH4+-N ratio of 0.10.17 mg C mg-1 N is mixed with NO2- (50-250 mg N L-1) as electron acceptor. The total inorganic
nitrogen conversion was up to 90% under volumetric loading rate of 100-500 mg N L-1 d-1.
Consortia in the second bioreactor were detected with molecular biomonitoring of 16S rDNA during
401 d continuous culture. ANAMMOX clusters, Brocadia anammoxidans and Kuenenia
stuttgartiensis, are identified in this study. Process evaluation and ecological study were conducted
on the use of two-stage biological process in treating thin film transistor liquid crystal display
wastewater.
INTRODUCTION
The main organic compounds of thin film transistor liquid crystal display (TFT-LCD) wastewater
are tetramethylammonium hydroxide (TMAH), monoethanolamine (MEA) and dimethylthylsuphoxide.
Among these compounds, TMAH and MEA are the
main nitrogen source, which provides a large amount
of organic nitrogen. Furthermore, the total Kjeldahl
nitrogen/chemical oxygen demand (TKN/COD) ratio
of TFT-LCD wastewater (0.17-0.2) is much higher
than the nutrient ratio of conventional activated sludge
(0.05). Due to the low C/N ratio, TFT-LCD wastewater is a typical nitrogen-rich wastewater [1]. TMAH is
*Corresponding author
Email: [email protected]
chemically classified among the quaternary ammonium compounds, which are considered to be nearly
immune to attack by microorganisms. Therefore,
TMAH is difficult to be bio-degraded in activated
sludge process [2]. However, previous studies also
showed that TMAH could be degraded under aerobic
conditions by some specific microorganism and under
anaerobic condition by methanogen [3-5].
Based on the wastewater characteristics, a specific ecological consortia community provides biochemical electron transfers and biosynthetic growth. A
special bioreactor combination is necessary to be conducted for the development of microbial ecology and
substrate balance. In this study, an anaerobic hydroly-
Sustain. Environ. Res., 21(3), 155-160 (2011)
156
sis reactor could sustain relatively high organic loading rate and toxic compounds, which is designed in
the first stage of two-stage bioprocess. Nevertheless,
either the low C/N ratio of the first stage effluent, or
the raw TFT-LCD wastewater, is ineffective for heterotrophic denitrification. Without any organic carbon
source, autotrophic anaerobic ammonium oxidation
(ANAMMOX) process could be used under anoxic
conditions to oxidize ammonium to N2 with nitrite as
electron acceptor [6]. Therefore, an anoxic ANAMMOX reactor was designed for nitrogen removal in
the second stage. The overall stoichiometry of
ANAMMOX process is shown below [7]:
NH4+ + 1.32 NO2- + 0.066 HCO3- + 1.3 H+
→ 1.02 N2 + 0.26 NO3- + 0.066 CH2O0.5N0.15
+ 2.03 H2O
Previously, it was found that some organic inhibitory compounds caused many risks to apply
ANAMMOX to industrial wastewater treatment [6,8].
Interestingly, a new species of ANAMMOX could
grow mixtrophically and co-oxidize propionate and
ammonium [9]. Two possible approaches could be
applied to industrial wastewater treatment. One is that
more mixtrophic species of ANAMMOX consortia
would be found and adopted. The other is a bioprocess
combination for ecological development and substrate
balance. In this study, an anaerobic-anoxic bioprocess
was established in treating TFT-LCD wastewater of
TMAH.
METHODS AND MATERIALS
1. Parameters
Bioreactors
Design
of
Anaerobic-anoxic
In industrial wastewater treatment, fluidizedbed-like reactors could sustain a relatively higher organic loading rate with small footprint. In the first
stage, a reverse flow jet loop reactor (RFJLR) was designed as a continuous stirred tank reactor (CSTR)
with high internal recirculation and dilution rate (Fig.
1). The hydrodynamic parameter of NCSTR and D/μL
of RFJLR were 1.26 and 1.36, respectively, which
represented the CSTR mode. The well-mixed reactor
is beneficial for treating highly toxic compounds in
wastewater with high mass transfer, high mix-up and
high bioconversion efficiency. The operating parameters of RFJLR are shown in Table 1 (Fig. 1, insert).
In the second stage, a powdered activated carbon
attached biofilm expanded bed (PABEB) was initially
inoculated with nitrifying sludge from a petrochemical
wastewater treatment plant in southern Taiwan (Fig.
1). Due to the very low growth rate of ANAMMOX
bacteria [7], a three-phase separator was located on
the top of reactor for biomass retention. The feed was
introduced from the bottom of the reactor and mixed
with an additional recirculation flow from the effluent
to maintain adequate superficial velocity and shear
stress to favor granule formation. The operating parameters of PABEB are shown in Table 2 (Fig. 1, insert).
2. Start-up and Continuous Operation of Two-stage
Bioprocess
The operation involved two phases. During the
first 158 d, the ratio of NO2--N:NH4+-N of 1:1 with
NO2--N 50-100 mg L-1 at a non-inhibition level was
used for the start-up strategy of ANAMMOX reactor
(PABEB) using a synthetic wastewater [10,11]. During day 108-138, the anaerobic reactor (RFJLR) was
start-up in batch mode with full-scale TFT-LCD
wastewater of TMAH. After day 139, the first anaerobic reactor was running as a CSTR with TMAH
wastewater. The TMAH wastewater provided about
99% org-N of total N, which was diluted with water
for controlling org-N volumetric loading rate (VLR)
(29-94 mg N L-1 d-1). The organic nitrogen concentration of the feed was 110-270 mg L-1 (TMAH 6002000 mg L-1). In the second phase (159-401 d), the
first anaerobic reactor was combined with the second
anoxic reactor. The feed of the second reactor was the
effluent from first reactor with a TOC/NH4+-N ratio of
0.1-0.17 mg C mg-1 N and NO2--N (50-200 mg L-1)
was added as electron acceptor. In order to control
enough electron donor and VLR of the second reactor,
NH4+-N (25-150 mg L-1) was added for adjusting the
fluctuated flow rate and N content of the full-scale
wastewater. The maximum total inorganic nitrogen
(TIN) VLR of second ANAMMOX reactor was then
500 mg N L-1 d-1.
3. Genomic DNA Extraction, PCR Amplification
and 16s DNA Cloning Library
Extraction of DNA from the sludge was done
and amplification of the 16S rDNA gene was carried
out by polymerase chain reaction (PCR) according to
the previously described method [12]. Amplification
of ANAMMOX-specific 16S rDNA gene was performed with the primer pair of Amx368f/1392r
[13,14]. The PCR products were purified with purification kit and subsequently cloned into Escherichia
coli using the TA cloning kit (TOPO TA cloning kit,
Invitrogen, Carlsbad, CA). All 16S rDNA gene clones
were randomly selected. The sequences of the clones
were determined by dye terminator cycle sequencing
with a Quick Start Kit (GenomeLab DTCS Quick
Start Kit, Beckman Coulter, Fullerton, CA) and an
automatic sequencer (CEQ2000XL, Beckman Coulter,
Fullerton, CA). A 16S rDNA gene-based phylogenetic
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
Lin et al.: Biological Treatment of TMAH
157
Table 2. Dimension and operating parameters of
PABEB
Table 1. Dimension and operating parameters of RFJLR
Fig. 1. Schematic diagram of two-stage bioprocess in treating TMAH wastewater.
PAUP 4.0 package to estimate the confidence of tree
topologies [12].
4. Other Analytical Methods
TKN, NH4+, NO2- and NO3- were detected according to Standard Method [15].
RESULTS AND DISCUSSION
1. Performances of Org-N Hydrolysis in First Stage
During the second phase, the first anaerobic reactor was combined with the second anoxic reactor.
From 159-220 d, the org-N VLR was 16-29 mg L-1 d-1
and the hydrolysis efficiency of org-N could achieve
80-95% (Fig. 2a). It indicated that TMAH was hydrolyzed efficiently with a relatively low VLR and/or a
long hydraulic retention time, HRT (4 d). Subsequently, the org-N VLR (29-60 mg L-1 d-1) was raised
by decreasing the proportion of water during day 221383. Unfortunately, the org-N VLR could not be controlled well because of the fluctuation in water quality
of the full-scale wastewater. However, org-N was almost converted to ammonium in this period (Fig. 2b).
After day 384, the org-N hydrolysis efficiency was
still above 85% even when HRT was shortened from 4
to 2 d. Hence, it showed TMAH could be degraded effectively under the operation condition and function of
the RFJLR was demonstrated.
2. Performances of Nitrogen Removal in Second
Stage
During the first 158 d, start-up and enrichment of
the second reactor with synthetic wastewater: the reactor was initially inoculated with nitrifying sludge from
a full-scale petrochemical wastewater treatment plant
and fed with synthetic wastewater contained a NO2-N:NH4+-N ratio of 1:1. The NO2- and NH4+ conversion
was low during the start-up period, probably due to
the slow growth rate of ANAMMOX microbes (doubling time 11 d) and low ANAMMOX biomass from
the seed resulting in a long start-up period [7]. The
TIN conversion rate was attained a value higher than
50 mg L-1 d-1 (50% of TIN VLR) only from day 97
(Fig. 3a). The NO2- conversion was coupled with
NH4+ oxidation with NO3- production during this period (Fig. 3b). Subsequently, the TIN conversion was
gradually increased as doubling VLR for intensive enrichment.
During 159-401 d, periods of two-stage biochemical process, the effluent of first reactor was introduced into the second reactor in this phase. The
feed of second reactor with a low TOC/NH4+-N ratio
of 0.1-0.17 mg mg-1 indicated that electron was donated from ammonium. In previous studies, organic
carbon caused ANAMMOX unable to compete with
heterotrophic denitrifiers or enzyme activity was in-
Concentration (mg L-1) Org-N VLR (mg L-1 d-1)
100
100
Org-N VLR
Hydrolysis efficiency
80
80
60
60
40
40
20
0
300
250
200
150
100
50
0
20
(A)
(A)
0
Org-N Inf.
NH4-N Inf.
Hydrolysis efficiency (%)
Sustain. Environ. Res., 21(3), 155-160 (2011)
158
Org-N Eff.
NH4-N Eff.
(B)
(B)
1
51
101
151
201
251
301
351
401
Operation time (d)
600
500
400
300
200
100
0
300
250
100
TIV VLR
TIN Conversion
TIN Conversion Efficiency
(A)
40
20
0
NH4- N Inf.
NO2- N Inf.
NO3- N Inf.
200
150
100
50
0
80
60
Conversion efficiency (%)
Concentration (mg L-1) LVR, Conversion (mg L-1 d-1)
Fig. 2. The variation of (a) Organic nitrogen volumetric loading rate and hydrolysis efficiency; (b) Organic nitrogen
and ammonium concentration versus operation time.
NH4- N Eff.
NH2- N Eff.
NH3- N Eff.
(B)
1
51
101
151
201
251
301
351
401
Operation time (d)
Fig. 3. The variation of (a) Total inorganic nitrogen volumetric loading rate and conversion efficiecny; (b) Nitrogen
concentration versus operation time.
hibited by intermediates of the degradation pathway
[6,8]. Actually, NO2- could be converted with NH4+ as
electron donor after anaerobic-anoxic system established, due to an insufficient organic carbon source.
The TIN conversion was 70-90%, which showed
ANAMMOX could compete electron acceptor (NO2-)
with other denitrifiers. The NO3- production was still
significant, even low TOC was present in the anoxic
system. In addition, the residual TOC was not inhibitory to ANAMMOX process. The granule formation
was also observed from day 203. Figure 3a shows an
increase of VLR and conversion, which indicates that
ANAMMOX could be applied in treating wastewater
with a low TOC/NH4+-N ratio (0.1-0.17 mg mg-1).
The ratio of NO2- conversion and NO3production rates of NH4+ conversion was calculated to
compare with TIN conversion rate. Both ratios were
fluctuated considerably when TIN conversion rate was
below 250 mg L-1 d-1, but gradually approached
constant when TIN conversion rate was above 250 mg
L-1 d-1. The average ratio of NO2--N conversion to
NH4+-N conversion (YNO2 /NH4 ) at TIN conversion rate
above 250 mg L-1 d-1 was 1.2 ± 0.2 and the ratio of
NO3--N production to NH4+-N conversion (YNO3-/NH4 )
was 0.08 ± 0.04 (Fig. 4). Comparing the average value
with the theoretical ANAMMOX stoichiometry, the
YNO2-/NH4 of 1.2 was closed to theoretical value 1.3,
but the YNO3-/NH4 of 0.08 is much lower than
theoretical value 0.26. The lower NO3- production
maybe caused by heterotrophic denitrification under
low TOC residuals from the effluent of the first
reactor. However, it still showed ANAMMOX could
compete electron acceptor with heterotrophic
denitrifers when a low TOC/NH4+-N ratio wastewater
was adopted.
−
+
+
+
+
3. Application
Technology
of
Molecular
Biomonitoring
Molecular BioMonitoring technique was applied
Conversion ratio
(NO2--N/NH4+-N and NO3--N/NH4--N)
Lin et al.: Biological Treatment of TMAH
159
3
64
100
100
2
LU24
LU16
LU44
98
Candidatus Brocadia anammoxidans (AF375994)
Candidatus Brocadia fulgida (DQ459989)
37
1.32
Candidatus Jettenia asiatica (DQ301513)
68
1
Candidatus Anammoxoglobus propionicus (DQ317601)
87
Kuenenia stuttgartiensis (CT573071)
100
0
-0.26
-1
Candidatus Kuenenia stuttgartiensis (AF375995)
72
LU37
63
LU30
Candidatus Scalindua wagneri (AY254882)
Candidatus Scalindua marina clone (EF602039)
100
0
50
100
150
250
300
200
TIN conversion rate (mg L-1 day-1)
350
400
450
Candidatus Scalindua brodae (AY254883)
97
99
Candidatus Scalindua sorokinii (AY257181)
Nitrospira sp. strain RC25.(Y14639)
Fig. 4. Conversion ratio between nitrite conversion rate
of ammonium conversion rate (YNO2-/NH4+); ratio
between nitrate production rate of ammonium
conversion rate (YNO3-/NH4+). ◇ △NO2-N/△NH4-N;
□ △ NO -N/△NH -N.
3
4
to the second ANAMMOX reactor during phase 2 to
investigate the ANAMMOX community. Amplifica
tion of ANAMMOX-specific 16S rRNA gene was
performed with the primer pair of Amx368f/1392r
[13,14]. The result showed that ANAMMOX bacteria
could be detected in this system. The sequence results
were computed and matched with database of National Center for Biotechnology Information (NCBI).
The similarity of Brocadia anammoxidans and Kuenenia stuttgartiensis were 97 and 99% respectively
(Table 3). Clearly, ANAMMOX bacteria truly existed
in this anoxic system. The relationship of ANAMMOX genus was also depicted in a phylogenetic tree
(Fig. 5). Further studies still need to be conducted to
evaluate the dominant species in this system (e.g.,
fluorescence in situ hybridization and real-time PCR
[14,16]).
CONCLUSIONS
With the first anaerobic reactor of high bioconversion efficiency, the conversion from organic nitrogen (TMAH) to ammonium could be achieved at least
80-100%. It showed that TMAH wastewater could be
degraded effectively by acclimated anaerobes in
RFJLR. In addition, the HRT of RFJLR could be decreased from 4 to 2 d without significant impact on
the hydrolysis efficiency. Degrading TMAH is a very
uncommon case with a relatively short HRT in the anaerobic hydrolysis process. Consequently, a relatively
high dilution rate and more flexible parameters (e.g.,
0.0 2
Fig. 5. Anammox-specific 16S rDNA-based population
identified in the second reactor was constructed
by the neighbor-joining method based on 16S
rDNA sequences.
With two-stage bioprocess operation, the maxi VLR)
could be used in the full-scale industrial wastewater
treatment. mum nitrogen compound conversion
reached 90% within TIN VLR of 200-500 mg L-1 d-1
in the second ANAMMOX process. It indicated that
ANAMMOX bacteria could be applied to degrade
TFT-LCD wastewater with a low TOC/NH4+−N ratio
of 0.1-0.17 mg C mg-1 N. Both undegraded TMAH
and/or intermediates of the degrading pathway contributed to the TOC compounds in effluent of the first
reactor. Although, it appears that the residual TOC is
not inhibitory to ANAMMOX, the inhibition level and
TOC/NH4+ threshold should be determined in the future.
With molecular biomonitoring technology of 16s
rDNA cloning library, ANAMMOX microorganisms
of the second anoxic reactor were detected (clusters, B.
anammoxidans and K. Stuttgartiensis). The similarity
of each species was 97 and 99%, matched with database on NCBI. With such low TOC/NH4+ ratio, it is
not possible for heterotrophic denitrifers converting
nitrite. Furthermore, a low anaerobic oxidation rate of
nitrifers could not remove the major part of nitrogen
under the anoxic condition [11]. There the role of
ANAMMOX is apparent. However, the dominant
species still need to be evaluated in the future. In short,
two-stage anaerobic-anoxic bioprocess could be used
in treating TFT−LCD wastewater.
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Table 3. Phylogenetic distribution of 16s rDNA clone sequence retrieved from the second reactor clone library
Clone number
Sequence length
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LU16
LU44
LU37
LU30
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993/1019
994/1019
1016/1020
1016/1017
Phylogenetic relationship
Closest species in Genebank
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Candidatus Brocadia anammoxidans
Candidatus Kuenenia stuttgartiensis
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Discussions of this paper may appear in the discussion section of a future issue. All discussions should
be submitted to the Editor-in-Chief within six months
of publication.
Manuscript Received: June 15, 2009
Revision Received: December 29, 2009
and Accepted: April 26, 2010