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熊本大学学術リポジトリ
Kumamoto University Repository System
Title Studies on fixed bed anammox process treating low
strength ammonium containing wastewater with high
…
Author(s) 劉, 成良
Citation
Issue date 2009-03-25
Type Thesis or Dissertation
URL http://hdl.handle.net/2298/14296
Right
Studies on fixed bed anammox process treating low strength
ammonium containing wastewater with high salinity
(高塩分含有低濃度アンモニア含有排水の固定床アナッモックスプロセスに関する研究)
Doctoral Dissertation
March ,2009
By
CHENGLIANG LIU
SupervIsor
Pro f. KENJI FURUKAWA
Department ofNew Frontier Science
Graduatc School of Science and Tcchnology
KUMAMOTO UNIVERSITY , JAPAN
Acknowledgement
I would like to acknowledge with deep gratitude to all those who helped me finish this
proJ ect.
I want to thank my advisor, Kenji Furukawa, professor of the Graduate School of
Science and Technology in Kumamoto University, for his assistance, guidance and support
during my four years of study (guest researcher, one year; Doctor Study, three years).
Without his constant consultant in my writing this thesis, it would not have been possible.
Prof. Furukawa is a very warmhearted man, his kindness and emotion investment to his
Chinese students is famous in Department of Science and Technology. I can never forget that
it was professor Furukawa who supports me with his own research fund. I will not forget this
matter during all my life.
I am very gratefu1 to Prof. Shinichi Abe, Prof. Susmu Takio, Prof. Yoshito Kifazono of
Kumamoto University and Prof. Haobo Hou of Wuhan University, China, deeply appreciate
for their encouragements and supports in my study and care on my life in Japan. Thanks ProÅí
Takao Fujii and Dr. Takashi Nishiyama for their kind helps to my experiment.
' I have a real debt of gratitude to Miss Seiko Seitoh and Miss Aya Fujimoto for their
enthusiastic assistance ofmy applying arbiter bill and experimental help, respectively.
Thanks for help of Mr. Xiaochen Xu, who always gave me encouragement. He is the
man who has the willing to reason and comprehend someone not by appearance phenomenon
but by the essence of spirit at early time he contact. I am particularly indebted to Chunfang
Zhang, Mr. Chunlai Li, Mrs. Jiali Yang, Mrs. Yingiun Cheng, Mr. Yuanhua Xie, Mrs. Yunling
Feng, Mr. Cheng Chen, Mr. Zhigang Li, Mr. Jiachun Yang, Mr. Xiangsheng Cao, Mr. Wenjie
Zhang, Mrs. Li Zhang, Mr. Yongguang Ma, Dr. Hu jing, Mrs. Yanning Gao, Mr. Chuangming
Zhou and Vice prof. Changcun Chao for their kindly supports and encouragements in usual
life and study in Japan.
My heartfelt thanks are due to Japanese students of Furukawa's laboratory for their
kindly help and warm friendship. Thanks M). Taichi Yamamoto for his kind advice in my
research work. I am particularly indebted to Mr. Lai Minh Quan, Mr. Hashimoto Yoshinori,
Mr. Do Phoung Khanh, Mr. shinohara Takehhiko, Mr. wakamatsu Shingo, Mr. Tran Thanh
Liem, Mr. Okuda Yutaro, Mr. kaneshiro Kosuke, Mr. Nakashima Ryosuke and Mrs.
matsumoto Noriko for their kindly supports and encouragements in usual life and study in
Japan.
Finally, I would like to give my special thanks to my wife and lovely son for supporting
my scientific career. Because of my busy study, all the housework was on my wife's
shoulders. But she had never complained. Her encourage in hard times and warmhearted
love enabled me to complete this work.
Abstract
The anammox process (!2Ltaerobic ammonium g2tgiidation), a new process for ammonium
removal from wastewater, can remove ammonium without oxygen and with nitrite as the
electron acceptor. The costs of conventional ammonium removal via
nitrification-denitrification from sludge reject waters are mainly associated with the
reduction in aeration-cost and supplementation cost for extemal carbon source such as
methanol in denitrification step. The anammox process provides an alternative for the
nitrification, with no requirement for an external carbon source. Combined with partial
nitritation (PN) process such as SHARON-process, the total aerati•on costs are reduced with
25 O/o. Compared to conventional nitrificationldenitrification, PN-anammox process can save
100 O/o of the required carbon source (i.e. methanol) and 50 O/o of the required oxygen. This
leads to a reduction of operational costs of 90 O/o, a decrease in C02 emissions of more than
100 O/o (the process actually consumes C02), and a decrease in energy demand. Wastewaters
that are suitable for the treatment with anammox is sludge reject waters ("sludge liquor") and
industrial wastewater (such as sour water) and brine wastewater from natural gas production.
The two processes can usually be engineered in two separate reactors, or in a single vessel
(the CANON-concept, "gompletely Autotrophic N-removal 9ver N"i'trite").
In this dissertation I have developed a novel anammox reactor using small carriers and
succeeded in its operation under high salts concentration.
The first research work was focused on "Anammox treatment of low-strength
ammonium-containing wastewater under room temperature". The anammox is commonly
used for the treatment of high-strength ammonia-containing wastewater at rather high
temperature of 30-40 Åé. Novel up-flow anammox fixed-bed reactor using a thin sheet of
polyester nonwoven biomass carrier could be established within 3 months of operation. This
fixed-bed anammox reactor was operated at room temperature for the treatment of
i
low-strength ammonium wastewater for 900 days. Influent containing ammonium and nitrite
in the forms of (NH4)2S04 and NaN02 with other nutrients and buffer was supplied to the
reactor in up-flow mode. The ratio ofNH4-N and N02-N in the influent was maintained at
'1.0 to ensure N02-N limiting conditions during treatment. At t-he first step, 8 strips of
-3original nonwoven were used and the maximum TN removal rate (TNRR) of 1.2 kg-TN m
d-,i was obtained. At the second step, 16 strips nonwoven was used as biomass carriers and
the maximum TN loading rate (TNLR) of2.4 kg-N m-3 d"i was obtained. At the third step,
small size biomass caniers composing nonwoven cutting average length of 1-2 mm were
used as biomass caniers and fi11ed in the gap of strips. High TNRR of 4.9 kg-TN m-3 d-i
was obtained finally. At the last step, eleven layers of small biomass beds were used, and the
maximum TNRR of 9.93 kg-TN m'3 d-i was obtained after 720 days of operation with TNLR
of 12.5 kg-TN m-3 d-i . When the system had been fu11y developed, a stable anammox
treatment lasted for three months. The anammox reactor was operated under N02-N limiting
condition for whole experimental period during which anammox treatment of a relatively
low-strength ammonium-containing wastewater at 20-25 eC was successfu11y demonstrated.
tt The second research was focused on "Effect of salt concentration in anammox treatment
' ' 'using non-woven biomass carrier". Effects of salt concentration on fixed-bed anammox
' 'reactor using non-woven biomass canier was investigated. A freshwater anammox sludge
'was used for the inoculum, and the salt concentration was gradually incrgased from 2.5 g 1-i
' tt tt 'to 33 g 1'i. The anammox treatment was stable at the salt concentration of 30 g 1-i and
'TNRR of 1.7 kg-N m-3 d-i was stably kept for 65 days. However, the NRR was sharply
/. t tt tt t. ' t .t.deteriorated above the salt concentration more than 30 g 1-i. The bacterial cgnsortia were also
examined by 16S rRNA gene analysis before and after acclimation of the anammox sludge to
ttt t t tthigh galt concentrations. Although all results of major clgne were rel.ateq, to .uncultured
'bacterium clone the two entries were affiliated with candidate division OPIO. And the
' t tt/ tfreshwater anammox bacteria KU2 and KSU-1 were detected at the salt concentration of30 pe tt tt . . .tt ' ttt t ttg 1- 1. Ip addition, Lysobacter, sp. belonging to 7 Proteobact,eria were revealed to coexist with
anarhmox bqcteria.
t/ .t The third research was focused on "Nitrogen removal capabilities of two kinds of
' tt t ttt t
ii
fixed-bed anammox reactors for treating partial nitrified brine wastewater". Fixed-bed
anammox reactors using non-woven biomass carrier and small size biomass caniers were
operated under salinity condition' comparable to sea water level. Fixed-bed anammox reactor 'using nonwoven biomass carrier could be successfu11y adapted to high salinity conditions by
elevating influent NaCl concentrations from 2.5 to 33 g 1-i stepwisely. This anammox
reactor could keep stable operation even under salt concentration comparable to sea water
( 30 g l-i ofNaCl), and the highest TNRR of 1.7 kg-N m-3 d-i was obtained After about one
year of continuous operation under salt concentration comparable to sea water, the synthetic
inorganic salt wastewater was replaced with the effluent from partial nitritation reactor
treating brine wastewater containing NH4-N from natural gas producing company. The
reactor was operated for more than 80 days by feeding the partial nitrified brine wastewater
and the maximum TNRR of 1.9 kg-N m-3 d]i was obtained. •It was difficult to increase TNRR
'more than this value in this fixed-bed anammox reactor. Then, the nonwoven biomass canier
was replaced with small size biomass carriers for getting higher TNRR. TNLRs of the
fixed-bed anammox reactor using small size biomass carrier could be successfu11y increased
stepwise from O.89 to 2.41 kg-N m-3 d-i within 40 days of continuous operation. TNRR of
'2.52 kg-N m-3 d-i was achieved under TNLR of 3.18 kg-N m-3 d-i without clogging and
' ' 'channeling phenomena, which were observed in the operation of fixed-bed reactor using
' tt tnonwoven biomass carrier. The color of anammox sludge in this fixed bed reactor using
small size biomass carriers was changed to weak red from black on day 85. TN removal
tt ' ' 'capability of fixed-bed anammox reactor using small size biomass carriers was much higher
'than that of fixed-bed anammox reactor using nonwoven biomass carrier, and the usefulness
of small size carriers as biomass attachment was verified.
' t/t There are two types of anammox sludge, freshwater type and salinity water type. The
tt t tt tt tabove mentioned experimental data indicated clearlY the way of acclimation for freshwater
t/ t ' t
anammox sludge to the ammonium•-containing wastewater with high salinity comparable to
.t/ /t 'sea water level. As to the freshwater anammox sludge, the difficult point in its establishment
'of stable anammox consortia is to increase TNRR under low ammonium concentrations
without temperature control. As to the anammox sludge operated uhder high salt
tt ttt t t tt tconcentrtations, the difficult point in its establishment of stable anammox consortia is to
iii
obtain high anammox reaction rates. These difficulties could be overcome in this present
studies. Our obtained results will give a new open and perspective road for the further
application of anammox reaction to different kinds of ammonium containing wastewaters.
iv
List of contents
Acknowledgements
Abstract
List of contents
List of abbreviations
List of Tables
List of Figures
Chapterl Introduction
1.1 Biologicalnitrogencycle
1.2 Anamniox
1.3 Anammoxcellstructureandbiochemistry
1.3.1 Planctomycetes
1.3.2 Anammoxcellstmcture
1.3.3 Anammoxlipids
1.3.4 Anammoxbiodiversity
1.3.5 Biochemicalmechanism
1.4 Traditionalbiologicalnitrificationldenirificationprocess
1.5 Anammoxprocess
1.6 ,Partialnitrifications/anammoxprocess
1.7 SHARONandanammoxprocess
1 .8 OLAND and anammox process
1.9 CANONprocess
1.10 SNAPprocess
1.11 Researchwork
1.11.1 Problemstatement
1.11.2 Obj ectives of this study
1.11.3 Researchplan
1.12 ReferencesChapter 2 Anammox Treatment of Low-strength Ammonium-containing Wastewater at Room Temperature
2.1 Introduction
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2.2 Materialsandmethods ./ 2.2.1 Experimentalsetup
2.2.2 Seedsludge
2.2.3 Biomasscanier
2.2.4 Attachimmobilizationofanammoxsludgeonbiomasscarrier
2.2.5 Syntheticinfiuent
2.2.6 Protocol for increasing in TN loading rate
2.2.7 Operationalconditioninwholeperiod
2.2.8 Analyticalitemsandmethods
2.3 Resultsanddiscussion
2.3.1 Observationoftheattachedbiomass
2.3.2 Changes in total nitrogen loading rates and removal rates
2.3.3 PerformancesofTotalnitrogenremovalefficiencies
2.3.4 NH4-Nremovalperfomiances
2.3.5 N02-Nremovalperformances
2.3 .6 Removal ratio of NH4-N, N02-N and N03-N
2.3.7 Comparisonofanammoxtreatmentcapabilities
2.3.8 The reasons forhigh removal rate
2.4 Conclusions
2.5 ReferepcesChapter 3 Effect of Salt Concentration in Anammox Treatment Non-Woven Biomass Carrier
3.1 Introduction
3.2 Materialsandmethods
3.2.1 Set up and operational conditions ofanammox reactor
3.2.2 Analyticalmethods
3.2.3 DNAextractionandPCRamplification
3.2.4 Cloning and sequencing of 16S rRNA
3.2.5 Denaturinggradientgel-electrophoresis(DGGE)
3.2.6 Nucleotidesequenceaccessionnumbers '
3.3 Resultsanddiscussion
3 .3 . 1 Reactor performances under high salt conditions
3.3.2 Bacterialcommunityundersaltcondition
Using
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3.4 ReferencesChapter 4 Nitrogen Removal Capabilities of Two Kinds of Fixed-bed Anammox Reactors for Treating Partial Nitrified Brine Wastewater
4.1 Introduction
4.2 Materialsandmethods
4.2.1 Anammoxreactors
4.2.2 Biomasscaniers
4.2.3 Brine water used in nature gas company
4.2A Partialnitritationprocess
4.2.5 Influentofanammoxreactors
4.2.6 Start-up and operational strategy ofanammox reactors
4.2.7 Analyticalmethods
4.3 Results and discussion
4.3.1 Performanceofthefixed-bedreactors 4.3.2 Changes in biomass color in fixed-bed anarmnox reactors
4.3.3 Small size biomass caniers for enhancing anammox performance
4.3.4 Comparison of anammox treatment capabilities under high salinity
condition
4.4 Conclusions
4.5 ReferencesChapter 5 Study on Long-term Stable Operation of High-rate Fixed-bed Anammox Reactor Using Different Biomass Carriers at Moderately Low Temperature
5.1 Introduction
5.2 Materialsandmethods
5.2.1 Anammoxreactor
5.2.2 Nowoven biomass carriers and small size biomass carriers packed in small carrier bed
5.2.3 Inoculumsandsyntheticinfluent
5.2.4 Start-up and operational conditions offixed-bed anammox reactors
5.2.5 Analyticalmethods
5.3 Resultsanddiscussion
5.3.1. Perfotmanceofanammoxreactor
5.3.2 Observationoftheattachedbiomass
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5.3.3 Small size biomass caniers for enhancing anammox performance
5.3.4 Mathematic simulating model for increasing TNLR when using small size biomass canier
5.3.5 Comparisonofanammoxtreatmentcapabilities
5.4 Conclusions
5.5 References
Chapter6 ConclusionsandRecommendations
6.1 Conclusions
6.2 Recommendations
Appendix: Publications Related to This Dissertation
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ANAMMOX
AOB
CANON
DGGE
DO
FBR
HRT
KSU-1
KU1, KU2
PN
NOB
OLAND
PCR
PE
TNLR
MRRSEM
SHARON
SNAP
TN
MLSS
MLVSS
List of abbreviations
' 'Anaerobic ammonium oxidation
' ' ' t tttAmmonium-oxidizing bacteria
Completely autotrophic nitrogen removal over nitrite
' 'Denaturing gradient gel electrophoresis
Dissolved oxygen
Fluidized'bed reactor
Hydraulic retention time
t.registered names of anammox strains in NCBI database
registered names of anammox strains in NCBI database
Partial nitritation
Nitrite-oxidizing bacteria
Oxygen-limited autotrophic nitrification-denitrification
Polymerase chain reaction
Polyethylene
Total nitrogen loading rate
Total nitrogen removal rate
Scanning electron micrograph
Single reactor system for high ammonium removal over nitrite
Single-stage nitrogen removal using anammox and partial nitritation
Total nitrogen
Mixed liquor suspended solids
Mixed liquor volatile suspended solids
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Table
Table 2-1
Table 2-2
Table 2-3
Table 3-1
Table 3-2
Table 4-1
Table 4-2
Table 4-3
Table 5-1
Table 5-2
Table 5-3
List of Tables
Title
Composition of synthetic wastewater
Minimum HRT for each Run
Comparison of anamniox removal capabilities
Composition of synthetic wastewater
Homology search results for 16S rRNA gene sequences ofbacterial
members in the community existing under salt condition (day 320).
Composition ofbrine water and sea water
Operational conditions during the whole experimental period
Comparison of different anammox reactors under high salinity
condtion
' 'Corpposition of synthetic wastewater
'Operational conditions during the whole experimental period
Comparison of different anammox reactors under different
operational conditiOns
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Etigy!ere
Fig. 1-1
Fig. 1-2
Fig. 1-3
Fig.
Fig.
Fig.
1-4
1-5
1-6
Fig. 1-7
Fig. 1-8
Fig. 1-9
Fig. 1-10
Fig. 1-11
Fig. 1-12
Fig. 2-1
Fig. 2-2
Fig. 2-3
Fig. 2--4
Fig. 2-5
Fig. 2-6
Fig. 2-7
Fig. 2-8
List of Figures
Title
Biological nitrogen cycle
Anammox reaction pathway
The first full scale anammox reactor (2002) at the Dokhaven
wastewater treatment plant, Rotterdam, the Netherlands.( Photo:
Paques BV.)
Anammox granules from Dokhaven anammox reactor
Cell structure of anammox bacterium
TEM image of Candidatus "Brocadia anammoxidans" (photo John
FuerstlRick Webb)
Example of a chemical structure of a natural ladderane (ladderane Y)
from anammox cells. (above, left) , place of ladderanes X and Y in
anammox lipids electron microscopic image of two isolated(above,
middle) and anammoxosomes next to a panially lysed anammox cell
(above,right)
Biochemical mechanisms of the anammox process.
HH: hydrazine hydrolase, the hydrazine-forming enzyme;
HZO: hydrazine-oxidizing enzyme; NR: nitrite-reducing enzyme.
Nitrificationfdenitrification process
Partial nitrificationlAnammox
The SHARON process in a well mixed continuous flow reactor
Reactor configuration for PN (left) and anammox oxidatjon (rjght)
Schematic diagram ofexperimental system
Thin strips of nonwovens
Small-carrier of nonwoven
Color changes in anammox biofilm
Changes in TN loading/removal rate and N03-N production
Changes in influent and effluent TN concentrations and TN removal
efflciencies
NH4-N removal perfomances
N02-N removal performances
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Fig. 2-9
Fig
Fig
Fig
Fig
Fig
2-1O
2-11
2-12
3-1
3-2
Fig. 3-3
Fig. 3-4
Fig. 4-1
Fig. 4--2
Fig. 4-3A
Fig. 4-3B
Fig. 4-4A
Fig. 4-4B
Fig. 4-5
Fig. 4-6
Fig. 4-7
TN removal, N02•-N removal and N03-N production rates with
respect to NH4-N removal rate
Thin strips ofnonwoven carrier
Eight strips ofnonwoven carrier
Undoing nonwoven fiber mixed with anammox sludge
Schematic diagram of fixed bed anammox reactor
Time courses of nitrogen concentrations under various salt
concentratlons
Relationship between the nitrogen removal rate (NRR) and salt
concentration. Bars indicate standard deviation
Denaturing gradient gel electrophoresis (DGGE) profiles for 16S
rRNA gene sequences of bacterial members in the community
existing under high salt condition (day 320).
Fixed-bed anammox reactors
Original and split nonwoven biomass caniers (top view)
Nitrogen removal performance ofthe reactors. (Panel A)
Nitrogen removal performance of the reactors. (PanelB)
Ratios of TNRR, N02-N consumption and N03-N production rates to
NH4•-N consumption rate
Ratios of TNRR, N02-N consumption and N03-N production rates to
NH4-N consumption rate
Changes in color of anammox biomass during the whole experimental
period
Microscopic observations of anammox biomass attached on small size
carriers on day 148: (a) biomass attached on hydrated lime, (b)
biomass attached on nonwoven filaments, (c, d) biomass attached on
the mixture of small size caniers.
SEM images of small size biomass carriers and attached biomass. (A)
Hydrated small carriers without surface modification by hardcore-1,
without biomass attaclment; (B) hydrated small caniers after surface
modification by hardcore-1 without biomass attachment; (C, D)
biomass attaching on small size biomass carriers at different
magnifications on day 150
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Fig. 5-1
Fig. 5-2
Fig. 5-3
Fig. 5-4
Fig. 5-5
Fig. 5-6
Fig. 5-7
Fig. 5•-8
Fig. 5-9
Fig. 5-10
Fixed-bed'reactor
Original and split nonwoven biomass carriers (top view)
TN removal performance of the reactors
NH4-N and N02-N removal performance ofthe reactors
Ratios of TNRR, N02-N consumption and N03-N production rate to
NH4-N consumption rate. .External appearance of the anammox reactor during the whole
experlment
Appearance of the anammox biofilm token from the reactor
Size of small carrier bed
SEM images ofsmall size biomass caniers
Mathematic simulating model obtained in the end of the experiment.
X axis: time (days)
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Chapter 1 Introduct .Ion
Concentrated wastewaters produced in many agriculture and food industries are now
commonly treated by anaerobic digestion process. High nitrogenous pollutants are also
produced from livestock farms, sites of aquaculture and landfi11 sites, etc. However, high
concentration of ammonium can not be removed in the anaerobic digestion process, thereby
yielding an effluent containing a high concentration of ammonia and low biodegradable
COD. The discharge of ammonium-rich wastewater caused nitrogen pollution for receiving
water bodies such as lake, inner sea and underground water. Nitrogen pollution is now the
critical environmental problem for the protection of water environment. These
ammonium-rich wastewaters are usually treated by biological nitrogen removal process, but
it is costly and troublesome. The original methods of nitrogen removal are physical and
ttchemical processes. Several phYsical and chemical processes, such as breakpoint
chlorination, air stripping and selective ion exchange have been used for nitrogen removal.
However, these processes are not applied widely due to their high treatment cost, operational
and maintenance problems and environmental concerns about the use of chemicals (Sedlak R.
et al., 1991). Therefore, traditional biological nitrificationldenitrification process is the next
option because of high potential removal efficiency, high process stability and reliability
(Sedlak R. et al., 1991, Watson S.W. et al., 1991). In the 1980s and 1990s, there were several
indications that nitrificationldenitrification are not the only processes that can remove
ammonium, but that there is an organism that can oxidize ammonium to nitrogen gas using
nitrite instead of oxygen as its terminal electron acceptor. This process is, therefore, called
"aLtaerobic ammonium gLtidation" (anammox). A lot of research works in this anammox
field are still going and its application to ammonium-rich wastewaters will contribute to the
prevention ofnitrogen pollution to receiving water bodies.
1.1 Biologicalnitrogencycle
The biological nitrogen cycle plays an
1
lmportant role in the mamtenanceof global
biosphere. It has been a focus of microbiological investigations since the late nineteenth
century. In the past 100 years, applied interests in the nitrogen cycle have shifted from
improving agricultural crop yields to concerning about surface water pollution, destruction of
the ozone layer and global wamiing. The biological nitrogen cycle, with the processes of
nitrification, denitrification, N-fixation and anammox, is shown in Fig. 1-1.
pt.ESX# Siee
xA '"
'
tisY"Xeeptfgee?dvSaj tpsIx"SS
Nesi
Fig. 1-1 Biological nitrogen cycle
1.2 Anammox
Anaerobic ammonlumOXIdation
es .R.
wt..
(anammox), i.e. the microbiological
"sSg,•pt&tt•ren
rdtwffTEImm. iEiam, iaj.
'
ee#si4
' t ttt ttt
Nge2ow
/
.t ttt
•• •• ••1•
•-
--•- -- eeltaj"•,
'
conversion of
uaftsr•
Fig. 1-2 Anammox reaction pathway
2
ammonium and nitrite to dinitrogen gas, is a very recent addition to our understanding of the
biological nitrogen cycle (Kuenen et al., 2001; Strous et al., 1999a). Discovered as late as
1986, it is so far the most unexplored part of the cycle. Given its basic features, the anammox
process is a viable option for biological wastewater treatment (Jetten et al., 1998;Jetten et al.,
2001;Strous et al., 1997b). Very recently, it was discovered that anammox made a significant
(up to 70 O/o) contribution to nitrogen cycling in the World's oceans (Thamdrup & Dalsgaard
2002). The anammox reaction pathway is shown in Fig. 1-2. The first fu11 scale anammox
plant has been installed in Dokhaven (Rotterdam, the Netherlands) for the ammonium
removal of digester liquor of domestic wastewater plant (Fig. 1-3). In this actual plant,
anammox granules were applied. Anammox granules from the Dokhaven anammox reactor
are shown in Fig. 1-4.
v
;.'"'•,E/.//./).,tt
""••'
ifl f•v"
Fig. 1-3 The first fu11 scale anammox Fig. 1-4 Anammox granules reactor (2002) at the Dokhaven Dokhavenanammoxreactor wastewater treatment plant, Rotterdam,
the Netherlands. ( Photo: Paques BV. )
1.3 Anammox cell structure and biochemistry
1.3.1 Planctomycetes
The planctomycetes are an interesting group of bacteria with many rare
3
from
or unlque
properties. The order Planctomycetes were described in detail (Schlesner H. et al., 1996) and
included only four genera (Planctomyces, Pirellula, Gemmata, and Isosphaera) with seven
validly described species (Fuerst J.A., 1995). They lack the otherwise universal bacterial cell
wall (Fuerst J.A. 1995, Konig, E. et al., 1984, Liesack W. et al., 1986) polymer
peptidoglycan, they divide by budding, and they have a differentiated cytoplasm, with
different membrane-bounded compartments apparently allocated to different cellular
functions. They are separated from other bacteria and amongst themselves by large
evolutionary distances. Recently, it appeared that planctomycetes might be the most ancient
group of bacteria, at the very root of the bacterial tree of life. Other species of
planctomycetes are aerobic chemoorganoheterotrophs, very different from the anammox
bacteria (which are anaerobic chemolithoautotrophs) (Fuerst J.A., 1995, Liesack W. et al.,
1986, Stackebrandt, E. et al., 1986)
1.3.2 Anammox cell structure
The bacterium has two main parts, inner and outer space separated by cell inner
membrane. There are nucleoid, cytoplasm, ribosome and anammoxosome enclosed by
bilayer in inner space, versus paryphoplasm, cell wall in outer space in anammox bacterium.
Anammox bacterium is a coccoid bacterium with diameter of less than 1 ptm. They are
physiologically different from the other known Planctomycetes and are anaerobic
chemolithoautotrophs (Strous M. et al., 1998). In anammox bacterium (Fig. 1-5), catabolism
),ljg'?. .
ssxf'l,k,.tX 'i' .
\$tKk•x-
...kl}.S..'gva'bet.v"rv"k•hrttl'llliibu••'es.".',•$ti•:.tfidi,,,
.•.x-'Svsff W•• ,'S;t'"ef ;' x ..I}}t? .x ...,,iiig,e•Z,,.,f.//,r,lllllli•ll/l//ii.,1///111ilii•lilillilili/ILI•,lilll!lillliilfil•illli.iiii.•i,l
',•,"','/.ut.tF-k".."svai,,'),;y.' ""'tt"':' in"i"• •f'g•n,E. {'+': ••.•.
.t tlt"trf
.tsts- '-'X'.
.I".;tl...:S'.ti.{.I
paryphoplasm z e=,pt,tt,fea,V-es
. .,pt-"•#s' cellnucleoid
{} hmaT,, ,' anammoxosome "k.."'4""//li.l,ii.l.ii/f/..-E....••••tse'e{$ts" intracytopiasm1
•N. lt, ',,, ',Sl. i .
J;'t•E-:,i'•,/z'ir'
i/i/I'•l',l,lll.l,ll:11,,/r:11'111i/11i'f////:///.Xikna•"iXeilii"i•
"•\i"'.:•Pil{•S..l':':,t.:.i,1/,,"..W•pt
"•{•-;t•rs•//1/,/1'k"'tww'g'.tr[/111SIii/..
t "t t.t "kufw w i."'/
ecr: yew.,.:twt•wapssc1,.:••;pt.A-.:f.r.rS.+
f.4I..-•-f-
ltt
lIx:
Ex'
' 't' ; tttk\t iv"n'w.z,vm / ''.s' k'v' u'•" ftsik•tii'w' '-" bilayer
ftf /k"sili'IL...g•.."l.k.si,rv. .gts"`S" rf
' ""+"t
.sS
t.
)e"tftt,tttstt.wnttxmss,mmtwe-
Fig. 1--5 Cell structure ofanammox bacterium
4
mner space
cell membrane
outer space
cell wall
•l
es
Fig. 1-6 TEM image of Candidatus
"Brocadia anammoxidans" (photo
John FuerstlRick Webb)
takes place in the compartments which are separated by a single bilayer membrane and is
called the anammoxosome. The cytoplasm in anammox bacteria is divided into three
compartments by single bilayer membranes: (1) the outer region is the paryphoplasm which
is bounded on the outer side by the cytoplasmic membrane and cell wall and on the inner
side by the intracytoplasmic membrane, (2) the riboplasm, containing the nucleoid and (3)
the inner ribosome-free compartment is the anammoxosome bounding by the
anammoxosome membrane (Van Niftrik L.A. et al., 2004). Scheme of anammox cell
structure is shown in Fig. 1-5. Fig. 1-6 indicates the TEM image of Candidatus "Brocadia
anammoxidans".
1.3.3 Anammox lipids
The anammoxosome is surrounded by a bilayer membrane that consists of unique and
bizarre lipids. The anammoxosome lipids contain 'ladderane' moieties, rigid and dense
ladders of concatenated cyclobutane rings. Both experimental evidence and molecular
modeling have shown that a membrane with ladderanes in its core is extremely impermeable
towards passive diffusion ofchemicals. From an evolutionary perspective, it is interesting to
note that part ofthe ladderane tails are ether-linked to the glycerol backbone; up to now, only
a minority of bacteria (either thermophiles or sulfate reducers) have ether linked to lipids.
Ladderane lipids will be used as biomarkers for past and present anammox activity in natural
ecosystems. Ladderane molecules with unique feature are now considered to be used as
opto-electronics. The anammox process is the only known natural source so far, and
,,i,..,,(i'
llg'
w
-]
t
t v y
1
k)
1
mo t,tS's..,,v,"tJVX,t,vvt--Lx-,,..y"za- .s.it.X C)r Y'
#gi"
"N.tf"OV-..""th'X..."S"-uwS-'X....t"""h.x
LL.o.f'N.......v"Kx".N'fNx."x"'x...x'X CY Y
!i
,'
" lli!!.-11N
Fig. 1-7 Example ofa chemical structure ofa natural ladderane (ladderane Y) from anammox cells. (above,
left) , place of ladderanes X and Y in anammox lipids electron microscopic image oftwo isolated(above,
middle) and anammoxosomes next to a partially lysed anammox cell (above,right)
5
ladderanes are very difficult to produce synthetically. Anammox bacteria contain a variety of
unconventional membrane lipids (Schmid M. et al., 2003, Kuypers M.M.M. et al., 2003,
Damste J.S.S. et al., 2003). Like all other Planctomycetes, anammox bacteria lack
peptidoglycan and have a proteinaceous cell wall instead ( Damste J.S.S. et al., 2002, Liesack
W. et al., 1986, Stackebrandt, E. et al., 1986). The lipids contain one, two or both of two
different ring-systems (X and Y) as shown in Fig. 1-7(Van Niftrik L.A. et al., 2004, Jetten
M.S.M. et al., 2003). Lipids from anammox bacteria are characterized by substantial}y lower
i3 C content than their carbon source(Schmid M. et al., 2003, Schouten S. et al., 2004). The
structure of the ladderane membrane lipids is unique in nature (Van Niftrik L.A. et al., 2004,
Damste J.S.S. et al., 2002) that can use as biomarkers for the presence of anammox cells in
environment (Schmid M. et al., 2003, Kuypers M.M.M. et al., 2003).
1.3.4 Anammoxbiodiversity
Currently, three genera of anammox bacteria have been discovered: Brocadia, Kuenenia
and Scalindua. The first two anammox bacteria ha' ve been found in wastewater treatment
systems. The latter, Scalindua, has also been detected in many marine ecosystems, such as
the Black Sea. The three genera share a common ancestor, but are evolutionally quite far
apart (less than 85 O/o sequence similarity on the 16S level). Still, all anammox bacteria seem
to be very similar phenotypically: they all grow at very slow rate (doubling time of about
1ldays), they all have an anammoxosome and ladderane lipids. The differences that must
exist among the three genera are the topic of ongoing research.
1.3.5 Biochemicalmechanism
The anammox pathway was determined by using i5N-labelling experiments, which
showed that hydrazine was an important intermediate (Jetten M.S.M. et al., 2003, Van de
GraafA.A. et al., 1997). The occurrence of free hydrazine in microbial nitrogen metabolism
was quite unique. The hydrazine oxidoreductase was purified from B. anammoxidans (Schalk
6
J., De Vries S. et al., 2000). Anammox catabolism takes place inside the anammoxosome.
The proposed biochemical mechanism of anammox reaction is shown in Fig. 1-8. (Schalk J.,
De Vries S. et al., 2000). In this mechanism, ammonium combined with hydroxylamine to
form hydrazine by hydrazine hydrolase (HH), a hydrazine-forming enzyme. Subsequently,
hydrazine is oxidized by a hydrazine--oxidizing enzyme (HZO), a key enzyme. HZO was
similar to hydroxylamine oxidoreductase (HAO) of Nitrosomonas europaea (Watson S.W. et
al., 1991). The oxidation of hydrazine produces dinitrogen gas, four protons and four
electrons. Consequently, these four electrons are combined with five protons from the
cytoplasm to reduce nitrite to hydroxylamine by a nitrite-reducing enzyme (NIR) (Kuenen J.
G. et al., 2001) as shown in Fig. 1-8.
cyteplasm - NH3
1N2}k"
NH20H
"Ng 4me
xxNe2
5H"
HN02 + H20 +N.K)" ptHNe3 +N.XDH,
Fig. 1-8 Biochemical mechanisms ofthe anammox process.
HH: hydrazine hydrolase, the hydrazine-forming enzyme;
HZO: hydrazine-oxidizing enzyme; NR: nitrite-reducing enzyme.
Anammox culture cell-free extracts showed a strong absorption at 468 nm in reduced
cytochrome spectra. The enzyme appeared to have some similarity to HAO ofNitrosomonas
europaea. HAO of Nitrosomonas europaea has a similar peak at 460 nm. Both enzymes
could oxidize both hydroxylamine and hydrazine (Schalk J. et al., 2000).
1.4 Traditional biological nitrification/denitrification process
Nitrogen elimination is commonly regarded as the conversion of biologically available
7
nitrogen compounds such as ammonium
(NH1), nitrite (NOi), or nitrate (NOi),
to dinitrogen gas (N2), which is released
into the atmosphere as a harmless
product (Fig.1-9). Today, wastewater
treatment plants commonly adopt a
biological nitrificationldenitrification
process for the purpose of nitrogen
removal from wastewater. Nitrification
means aerobic oxidation of ammonium
by nitrifying bacteria. Ammonium (NH;
nitrite (NO5) to nitrate (NOi).
. 20, (1 000/,) -
,,.,,,:••, , elgl{.paio
ti"i,in.x,..k,,:,.1///klj/i.i.l,,(e.gig•/
.{.l{-"f•*kg;•'
tt.:}}'.{tttti',t/•1' ..,+•,,,v,/•
ttt tt lttttuttttttt tt tt t
NMIicitiiim l,'
.•;-S.i •i;iti'i• NH;
2 e, (roo%}
k/,Eill/11Ii,lll,I/g•e•s,S••i••,i:•ii',r/'i•l,•,g•,l••g'scK•\••e•'T"r'5i"tw""•va•g•tt"`"ci;iii'ilva,i,,tll.i/s,,x•i.i'i:/l/11/i"i'I'iillllllii.,i}••,/it;.,,,.,.•.gN•,II,lll,i,,.,/I/,,.,,,,,•,,,,..•,,•t,eg.ili'1111•lg,;1/L/g.iigli:/:;
. . ."..... .ut +... t tt t ttt z';•••,': '•1/l•//,:?get•iv'//:ndgtiwh}tt.il"l'lil',i"//g'k•-"'i"':'''
getTe•i'X/',tE•(,:1'i'•"-•i)•s••"il•,".,/./)•••+•••-./l.,/3S'i/i.••. •it'sc-"
ill/lliPenitrifi,,,,,'I,,..l.,,,,g'I'g'S'iE•k"e,,y,.:"'/e'•,g,t//tts•,/k/,11/s,11ii•f•i,/,gi,/L/.ge..pt..i,?i'TS,g;i}l//;.,I-//,,,,./J//,r/:,/l•li•/E,//,,L,/,e.i•i-•/k,/•,//,••ee'•,,L:;•t,`iJif•y,Eii•i.
Fig. 1-9 Nitrificationldenitrification process
) is oxidized by oxygen (02) via the intermediate
Nitrification and denitrification reactions are as follows:
Organic - Carbon(e.g., methanol3.4kg l kgN)NH, ->NO, (1-1) -> O.5N2 Nitnfication Denitripcation
NH; +1.5 02 - NOi +H20+2H' (1-2)
N05 +02 - NOi (1-3)
Denitrification means nitrate respiration by denitrifying bacteria. Nitrate is reduced
under anaerobic conditions to dinitrogen gas (N2) with the addition of external organic
carbon source such as methanol.
NOi+OrganicCarbon . N2+2C02 (1-4)
1.5 Anammox process
Ammonium is oxidized to dinitrogen gas by nitrite under anaerobic condition through
anammox reactlon.
8
3•4kg.f}cag}.•il,,,.....,.,,{/f//E.i,,l,,//,g-/)ttew••xxii'III,,
. ... -t .. tt
s. '1ge.i//':
e.5 M2
+
s•f,di,.ee,i•"t2,•,gj,/L/1•i•l•l/
'"1'2P,i•,.{•.},••,.
,/t;•iil,S•kI.•••i
•..,tYl'l.Å}l':11•
NHI +NOi-N2+H20 (1-5) Anammox process (Fig.1-2) is a biological autotrophic process to removal ammonium
from wastewater. Ammonium is converted to dinitrogen gas with nitrite as electron accepter
under anaerobic conditions and without adding external organic carbon. The anammox
bacteria form a monophyletic cluster branching off deep in the order Planctomhycetales
(Fuerst J.A. 1995, Stackebrandt, E. et al., 1986), and were previously discovered in
wastewater treatment plants systems. Based on mass balances over anammox enrichnent
cultures, the anammox stoichiometry was estimated as following (Strous M., Heijnen J.J. et
al., 1998):
NH2 +1.32NOE +O.066 HCOi +O.13 H' .
1.02 N2+O.26 NOi +O.066 CH20o.sNo.s+2.03 H20 (1-6)
1.6 Partial nitrifications/anammox process
Two kinds of reactor are 'Pardal nibitadonrequired for ammoniumig?c?;oZipaXfa'7.g,,,,,.1",X.Rl,ll&i /NKZ x/?4g5%S
(about 50 O/o of influent
ammonium is converted to
nitrite) treatment is the
anammox process. For the
removal of nitrogen by nitrification-denitrification process, influent total organic carbon to
nitrogen ratio (CfN) is an important parameter to take into account for getting high nitrogen
removal efficiencies. When the available amount of carbon source is not enough to complete
the denitrification reaction, the addition of external organic carbon source is required. PN
process can reduce the oxygen requirements by 60 O/o compared with nitirification (Fig.
9
1-1O).
Nitrification is a two step biological processes mediated by two kinds of nitrifying
bacteria. First step is the conversion of ammonium to nitrite by ammonium oxidizing
bacteria (AOB) and the second step is the conversion of nitrite to nitrate by nitrite oxidizing
bacteria (NOB). Owing to the higher reaction rate of NOB compared with that of AOB,
accumulation of nitrite during nitrification reaction is quite rare. Nitrite is a substrate for
anammox reaction, so that inhibition ofNOB is essential for partial nitritation process. There
are different approaches for restricting NOB activity as follows.
1. Addition of specific inhibitors for NOB (Schmid M. et al., 2003). This option is not
easy to implement due to the difficulty to find a compound able to inhibit only the second
step of the nitrification. Besides the microorganisms can adapt to the inhibitor after long
periods of application, decrease its inhibitory efficiency must be considered. (Kuypers
M.MM, 2003).
2. Control of the dissolved oxygen concentration at low values to avoid the oxidation of
nitrite to nitrate due to the higher affinity for oxygen ofAOB than NOB ( Damste J.S.S. et al.,
2004).
3. The use of pure cultures of ammonia oxidizing bacteria is proposed. This method is
not usefu1 in practice, because the wastewater itself contains different kinds of
microorganisms, which quickly grow and contaminate the pure culture in the reactor.
4. To control the pH value and the ammonia concentration in the reactor for inhibition
of NOB according to the findings of Anthonisen et al., (Van Niftrik L.A., 2004). In this
approach, the adaptation of NOB to these controlled operational conditions will become a
problem. ' 5. Selection of nitrifying population based on the different growth rates of AOB and
NOB. This is the concept applied in the SHARON process. This process is carried out in a
chemostat operated ataHRT (equal to the SRT) of1day and 30 Åé which is favorable for
the growth of AOB. NOB are finally washed out from the reactor under these operational
condition. (Liesack W. et al., 1986).
PN lanammox process (Fig. 1-3) is shown as follows.
10
NH2 p..Oti'7.5192iit,OiOtX.')ti.. -'O'5NO5+O'5NH4' O'i5.0.in(ÅíO.'.YO) N'O'5N2 (1-6)
'
Compared to two step process ((1-1) & (1-6)), the PNIanammox process has several
'advantages over traditional nitrificationldenitrification process. Oxygen supply can be
reduced by 60 O/o. This means the reduction in the energy requirement for aeration. Also,
autotrophic anammox bacteria do not require the addition of external carbon source. The cell
yield of autotrophic anammox bacteria is quite low, so that the treatment cost for excess
sludge can be minimized. The new PN!anammox process uses not only less energy and fewer
resources, but it is also less expensive than conventional nitrificationldenitrification.
1.7 SHARON and anammox process
SHARON is an abbreviation of
Single reactor system for Uigh
Ammonium Removal Over Nitrite.
SHARONIanammox system iscomposed of two reactors. In the original
SHARON reactor, ammonium iscompletely transformed to nitrite (Fig.
1-11). Nitrite oxidation by NOB can be
prevented and heterotrophic denitrification
of nitrite using methanol as external
' 'carbon source occur under high 'temperature (35 O
of the carbon demand can be saved
.pt El 3
Hc•eS
S\es-k-asu'eZ'
c,e1,
ll
LXT•e2
S•L,WMGE
e es.
-p 't
.e2" g
-
"t
e
- tsns
" s- ev --
" 1te ,-
,-ms l
C) without sludge retention. Twenty five percent of the oxygen
compared
process. However, an external organic carbon for denitrificaton, i.e, methanol
'effective aeration system are still required (Stackeb
'from SHARON process and original ammonium containing wastewater mixed together by
ratio of 1 : 1 and is fed to the following anammox reactor.
11
Fig. 1-11 The SHARON process in a well
mixed continuous flow reactor
and 40 O/o
with complete nitrificationldenitrification
as well as an ' randt et al.,1986). Full nitrified effluent
In 2002, pilot scale experiment using PNIanammox process, which is shown in Fig.1-12,
for ammonium removal of sludge digester supernatant was carried out. Fifty eight percent of
the ammonium in the supernatant was converted to nitrite and nitrite production was O.35
kg-N m-3 d-i at temperature of 300C during nitritation process. The anammox process was
canied out in a SBR with a nitrogen removal rate of2.4 kg-N m'3 d'i. Over 90 O/o ofthe inlet
nitrogen load to the anammox reactor was removed and the sludge production was negligible
(Van Nifuik L.A. et al., 2004).
Elemonta}"itregen <Nal
dgedigestereMuentMixtureof({ammoniurn-rich)ammontumartdnitriteNH;
Hcoi+methanoiITIg'O.ooQ;oNeq'NH;••
e" fiN;su1{;egieNte`y"io
'1'/'t''/i"''''1'1/'''
tt tttt
Aeration {02>
"We<b.kgts}l.{igg6pte
, ' A
Treated vvater to the prs-settIement tank(low Åëoncant'ratiens of
NHS, N05, NOi}
d'i'),
ww"NC}i , eq ' t tttt tt blgeÅíxxypaggE ,,..,
Fig. 1-12 Reactor configuration for SHARON(left) and anammox oxidation (right).
1.8 OLAND and anammox process
OLAND is an abbreviation of 9xygen Limited Ammonium removal via
Nitrification-Denitrification.
OLAND and anammox reactions were canied out in one reactor. The present lab-scale
research revealed the potential of implementation of OLAND system with normal nitrifying
sludge as the biocatalyst for the nitrogen removal from nitrogen-rich wastewater in one step.
The current treatment capacity of the OLAND system is still low. However, the fact that the
inoculum can readily be grown in large quantities is an important factor which favors the
applicability of the OLAND system for practical purposes. Indeed, the seed nitrifying sludge
can easily be prepared from an activated sludge, and it can be applied directly to OLAND
system without adaptation. Moreover, operation of the OLAND system has no requirement
12
for an NO5 supply. An NH2 rich wastewater can be fed directly at a suitable Ioading rate.
Although the process requires limited dissolved oxygen concentrations, it does not require
strictly anaerobic conditions. Anammox reaction occurrs inside ofbiofilm, which ammonium
and nitrite exist under anaerobic condition. Therefore, inhibition by trace 02 exposure is not
a serious problem of concern in practice. The process operated by a pH controller is simple
and reliable for practical operation (Jetten M.S.M. et al., 2003).
tt '
1.9 CANON process
CANON is an abbreviation ofCompletely Autotrophic Nitrogen removal-Qver Nitrite.
In the CANON process, aerobic and anaefobic ammonia oxidizing bacteria cooperate to
remove ammonia in one oxygen-limited reactor ( Schmid M. et al., 2003). Recent studies
(Schouten S. et al., 2004) showed that the CANON biomass is very resilient against
disturbances in wastewater composition. CANON process becomes a good alternative to
existing nitrificationldenitrification systems for treatment of liquid waste rich in ammonia by
proper control of cultivation condition.
In CANON process, both partial nitrification and anammox reactions occur in one
reactor systems, where AOB and anammox bacteria coexist under oxygen-limited condition.
In this option, limited amounts of oxygen were introduced carefully into CANON reactor.
AOB consume all the dissolved oxygen, thus maintaining a very low dissolved oxygen
concentration in the aggregates of anammox sludge and produce nitrite as the electron
acceptor for anammox bacteria (Kuypers M.M.M. et al., 2003, Van Niftrik L.A. et al., 2004,
Damste J.S.S. et al., 2002). The CANON biomass was analyzed by FISH method and
showed that about 40 O/o ofbacterial community ofCANON process consisted ofAOB, and
about 40 O/e of bacterial community consisted of anammox bacteria. Nitrite oxidizing
bacteria (NOB) such as Nitrospira or Nitrobacter species were not detected in the bacterial
communlty.
Compared to SHARON-anammox reactor, CANON process uses one reactor, whereas
SHARON-anammox needs two reactors. In addition, the TN conversion of
13
SHARON-anammox is limited by the maximal strength of the wastewater being treated.
Compared to nitrificationldenitrification, the N-removal rate of CANON is lower (Damste
J.S.S. et aL, 2002).
1.10 SNAP process
SNAP (Single-stage pitrogen removal using anammox and partial nitritation) process is
a newly developed ammonium removal process using PN and anammox reactions. SNAP
process was successfuliy applied to the ammonium removal of synthetic landfi11 leachate.
During operation of partial nitritation, anammox bacteria grew inside of attached sludge on
the net-type acry1-fiber biomass carrier and enabled the short-cut conversion of ammonium
to dinitrogen gas in only one SNAP reactor. Under volumetric ammonium loading rate ofO.6
kg-N m-3 d"i, the SNAP process achieved nearly 90 O/o of ammonium conversion and about
80 O/o of TN nitrogen removal efficiency. Under higher volumetric loading rate of 1.0 kg-N
m-3 dLi,about 80 O/o of TN removal efficiency was obtained. The SNAP biomass was
demonstrated to be composed of AOB (close relatives of Nitrosomonas europaea), NOB
(close relatives ofNitrospira sp.) and anammox bacteria (close relatives of KU2 and KSU-1
'strains), in which AOB and anammox bacteria were dominant. These results showed the high
applicability of SNAP process to the ammonium removal oflandfi11 lechate.
1.11 Researeh work
A lot of research works were carried out for the development and applying for
anammox technology more than 15 years. Most of the researchers focused on basic studies
and got lots of valuable new information. I had started my research work on anammox about
three years ago after my enrolling Ph.D. study. My main work focusing on anammox
research is how to get a stable high-rate anammox treatment system under poor operational
conditions. With the help of Prof. Furukawa, I was able to obtain various excellent
achievements on anammox treatment process.
14
1.11.1 Problem statement
There are several problems on anammox application as follows:
1) In case of starting and operating an anammox system, the total nitrogen removal rate
goes through a course of increasing firstly and decreasing afterwards.
2) The anammox sludge did not fu1fi11 in reactor, but the sludge volume began to
reduce and anamniox activity was devitalization with sufficient nutrients influent.
3) The total nitrogen loading rate can not increase easily for low-strength
ammomum-contamlng at room temperature. . . 4) How to develop a high-rate anammox treatment system under high salinity
concentration comparable to sea water?
5) How to adapt the freshwater anammox sludge to salt concentration of 30 g 1-i for
short acclimation time?
6) How to solve the problem of system starting, defeat and restarting all the times.
All above mentioned problems puzzled me for about one year. I was able to overcome
those difficult problems with the help of Prof. Furukawa.
1.11.2 Objectives of this study
The objectives ofthis study are mentioned as follows:
1) To evaluate the nitrogen removal capabilities of anammox sludge in a fixed-bed
reactor using various biomass carriers, nonwoven, split nonwoven, splitted nonwoven and
nonwoven powders as caniers.
2) To develop an anammox process using hydrates material ofcement and harecore-1 as
biomass carrier and to compare the nitrogen removal capabilities with anammox process
usmg nonwoven. ' 3) To investigate the nitrogen removal capability of the anammox process using small
carriers (selfmade products) as biomass carrier.
4) To develop an anammox process under salinity concentration of30 g 1-i. First step is
15
to change freshwater type anammox sludge to salt resistance type anammox sludge.
4) To compare the nitrogen removal capabilities of nonwoven biomass caniers and self
made small carriers.
1.11.3 Research plan
This study focuses on the application of anammox process for low strength
ammonium-rich wastewater under low temperature and high salinity condition.
Part 1. Anammox treatment of low-strength ammonium-containing wastewater at room
temperature
In this study, fixed-bed anammox reactor was operated at room temperature for the
treatment of low-strength ammonium containing wastewater. The anammox reactor was
operated under N02-N limiting condition for the treatment of a relatively low-strength
ammonium-containing wastewater at 20-25 Åé.
Part 2. Effect of salt concentration in anammox treatment using non-woven biomass
carrler
This study focuses on the evaluation of anammox process using nonwoven biomass
carriers under high salt concentration. Effect of high salt concentration on the anammox
treatment was investigated for the establishent of acclimation strategy under high salt
concentration conditions. The bacterial community was examined by 16S rRNA gene
analysis and DGGE after the acclimation of the anammox sludge to high salt conditions.
Part 3. Nitrogen removal capabilities of two kinds of fixed-bed anammox reactors for
treating partial nitrified brine wastewater
This experiment was carried out to determine the nitrogen removal capacities of two
types ofbiomass caniers (nonwoven and selfmade small carriers). Two fixed-bed anammox
reactors using nonwoven and small size biomass carriers were operated respectively under
high salinity condition comparable to sea water level, and compared nitrogen removal
capacities ofthis two type biomass carriers.
Part 4. Study on long-term stable operation ofhigh-rate anammox biofilm reactor using
nonwoven carrier under moderately low temperature
' ' '
16
In this study, main task was to improve the activity of anammox sludge. Small biomass
technology for keeping plenty of anammox sludge in the reactor and chemical or physical
methods to enhance anammox activity by killing some part bacteria of anammox sludge was
applied. At last, mathematical methods to simulate and forecast the TNLR 5 or 7 days later
were developed.
1.12 References
Anthonisen AC, Loehr BC, Prakasam TBS, Srinath EG.: Inhibition of nitrification by
ammonia and nitrous acid, J. MPCF., 48(5), 835-852(1976). '
Broda E.: Two kinds of lithotrophs missing in nature, Z. Allg. Mikrobiol., 17 (6), 491493
(1977).
Damste J.S.S., Strous M., Rijpstra W,I.C., Hopmans E.C., Geenevasen JAJ., Van Duin
A.C.T., Van Niftrik L.A. and Jetten M.S.M.: Linearly concatenated cyclobutane lipids
form a dense bacterial membrane, Nature, 419, 708-7 12 (2002).
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' ' biology, Microbiology, 141, 1493-1506 (1995).
Fux C., Boehler M., Huber P,, Brutnner I., Siegrist H.R.: Biological treatment of
' ammonium-rich wastewater by partial nitritation and subsequent anaerobic ammonium
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Ganido JM, Van Benthum WAJ, Van Loosdrecht MCM, Heijnen J. J.: Infiuence of dissolved
oxygen concentration on nitrite accumulation in a biofilm airlift suspension reactor,
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Hellinga C., Schellen A.A.J.C., Mulder J.W., van Loosdrecht M.C.M., Heijnen J. J.: The
' Sharon process: an innovative method for nitrogen removal from ammonium rich
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Jetten M.S.M., Sliekers A.O., Kuypers M.M.M., Dalsgaard T., Van Niftrik L., Cirpus I.,
Van de Pas-Schoonen KT., Lavik G., Thamdrup B. et al.: Anaerobic ammonium
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Jetten M.S.M., Sliekers A.O., Kuypers M.M.M., Dalsgaard T., Van Niftrik L., Cirpus I.,
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Jetten MSM et al.: The anaerobic oxidation of ammonium, FEMS Microhiol. Rev , 22,
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Jetten MSM et al., Microbiology and application of the anaerobic ammonium oxidation
('anammox') process. Curr. Opin.Biotechnol. 12, 283-288, (2001)
Konig, E., Schlesner, H. and Hirsch, P.: Cell wall studies on budding bacteria of the
Planctomyces/Pasteuria group and on a Prosthecomicrobium sp, Arch. Microbiol., 138,
200-205 (1984).
Kuenen J. G., Jetten M. S. M. : Extraordinary anaerobic ammonium-oxidizing bacteria, ASM
News, 67,456-463 (2001)
Kuypers M.M.M., Sliekers A.O., Lavik G., Schmid M., Jorgensen B.B., Kuenen J.G.,
Damster J.S.S., Strous M., Jetten M.S.M.: Anaerobic ammonium oxidation by
Anamniox bacteria in the Black Sea, Nature, 422, 608-61 1 (2003).
Liesack W., Konig H., Schlesner H. and Hirsch P.: Chemical composition of the
peptidoglycan-free cell envelopes of budding bacteria of the Pirella/Planctomyces
group, Arch Microbial., 145, 361-366 (1986).
Lieu P.K., Hatozaki R., Homan H. and Furukawa K.: Single-stage nitrogen removal using
anammox and partial nitritation (SNAP) for treatment of synthetic landfi11 leachate,
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Lieu P.K.: Nitrogen removal from landfi11 leachate using a single-stage process combining
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(2006).
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18
Michael Nielsen , Annette Bollmarm , Olav Sliekers , Mike Jetten , Markus Schmid , Marc
Strous , Ingo Schmidt , Lars Hauer Larsen d, Lars Peter Nielsen , Niels Peter Revsbech:
Kinetics, diffusional limitation and microscale distribution of chemistry and organisms
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Schalk J., De Vries S., Kuenen J.G. and Jetten M.S.M.: Involvement of a novel
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5405-5412 (2000).
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19
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20
van Dongen U, Jetten MSM, Van Loosdrecht M.C.M.: The SHARON ANAMMOX process
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21
22
Chapter 2
Anammox Treatment ofLow-strength Ammonium-Containing Wastewater at Room Temperature
2.1 Introduction
Anaerobic ammonium oxidation (Anammox) is an anoxic microbiological process in
which ammonia, together with nitrite, is oxidized to gaseous nitrogen anaerobically (A. Olav
Sliekers et al., 2002). Anammox is commonly used for the treatment of high-strength
ammonia-containing wastewater at rather high temperature of30-40 Åé. Anammox process
is a new and promising alternative to the conventional nitrogen removal processes. The
application of anammox to nitrogen removal process in wastewater treatment would lead to a
significant reduction of costs for aeration and addition of exogenous electron donor as
compared to the conventional nitrification-denitrification process(van Dongen et al., 2001).
'
NH4' + 1.32 N02-+ O.13 H'+O.066 HC03- -i>
1.e2 N2+ O.26 N03- + 2.03H2Q + Q.66 BiQmass (Eq. 1)
' effluent
outlet
However, anammox process
requires a long start-up time due to
extremely slow growth rate of anammox
bacteria (van Dongen et al., 2001) (the
doubling time was reported to be about
11 days) (Strous et al., 1998). For the
prompt establishrnent of anammox
reactor, the selection of proper seed
sludge and carefu1 increasing protocol in
nitrogen loading rates become important.
temperaturecontroller nonwovens
carrier
entwin6d
heater
S,l,
g/l/l
l-
tpt/ ..
gl {--
influent tank peristaltic pump
Fig.2-1 Schematic diagram of experimental system
ts!t/.111v/'[vvt"':/x/--y'-'T'e .
1::,l•::•i•i,,liliEllEi!'iiii{l
e
[
fixed- edreactor
23
Also, the complete retention of slowly growing anammox bacteria in the reactor is important.
It was reported that it could successfu11y develop high-rate anammox biofilm reactors using
the up-flow fixed-bed biofilm column reactor by inoculating seeding sludge with high
'abundance of anammox bacteria for short periods, operate steadily and achieve a high
nitrogen removal rate of26.0 kg-N m"3 d-i within 250 days(Tsushima et al., 2007). But it was
operated at high temperature and high concentration of ammonium, and the experiment was
not under the control of stability. Up to now, anammox reaction was applied to the high
strength ammonium containing wastewaters such as digestion liquor and landfi11 leachate.
In order to apply this anammox reaction to the conventional nitrogen removal process,
the applicability of anammox process to low strength ammonium containing wastewaters at
room temperature must be evaluated. The objectives of this study were to examine the
start-up anammox reactor using low strength ammonium containing wastewater under room
temperature and make clear the anammox treatment potential of our newly invented up-flow
column reactor.
For these purposes, up-fiow fixed-bed biofilm column reactor with nonwoven fabric
sheets as biomass carrier was used for the cultivation of anammox bacteria and the reactor 'was operated for one and a halfyear.
22 MaterialsandMethods
2.2.1 Experimental setup
The reactor used in this experiment was shown in Fig.2-1. This reactor was made of
glass and the size is Q 95mm x 423mm, with the available volume of 2.8L. Influent was
supplied to the reactor by up-flow mode. Reactor was heated to about 20 OC in winter day
and kept at room temperature in other seasons. The reactor set was shaded from a light with
black vinyl sheet enclosures.
24
2.2.2 Seed sludge
Seed anammox sludge of 1.4g-VSS was obtained from another anammox reactor
cultivated in our laboratory. In order to improve the attachnent of anammox sludge on the
nonwoven biomass canier, granular of anammox sludge was crushed into small particles
using O.5 mm sieve.
2.2.3 Biomass carrier
The 8 strips of polyester nonwovens were outspread evenly by 16 strips appearing a
column liking inside of reactor. The purpose of using thin-nonwovens is to get more
superficial surface area where more biofilm can be attached on it. The nonwoven shape was
shown in Fig. 2-2. After anammox sludge developed fu11y in the reactor, undoing nonwoven
?ww•i/ttt#,/g.,///.i•
•ge
'
t••;l isÅ}:
Fig. 2--2 Thin strips ofnonwovens Fig. 2-3. Carrier ofnonwoven
fibers shown in Fig. 2-3 were added for bridging the gap between thin strips of nonwoven.
The small caniers are like sticks with length no more than 1 mm. The undoing nonwoven
fibers were prepared by smashing thin strips ofnonwoven with cutting method.
2.2.4 Attach immobilization of anammox sludge on biomass carrier
Thin strip of nonwoven carrier was set in the reactor. Three Liter of pretreated crushed
25
anammox sludge suspension was added from the bottom of reactor with flow rate of
1,OOO-2,OOO lh-i. Then, the reactor was purged with nitrogen gas for 20 min. At last, internal
circulation 10 1 h-i was carried out for 12 h. Through this procedure, seed anammox sludge
was evenly attach-immobilized on thin strip polyester nonwoven biomass carrier.
2.2.5 Synthetic influent
Influent wastewater was prepared by adding ammonium and nitrite in the forms of
(NH4)2S04 and NaN02, with other nutrients and buffer according to the composition given in
Table 2-1.
Table2-1 Compositionofsyntheticwastewater
Composition Concentration
(NH4)2S04 70Å}5(mg-N1-')
NaN02 70Å}5(mg-N1-i)f
EDTA 5.0(mg1-i)
FeS04.7H20 9.0(mg1-i)
KHC03 125.1(mgri)
KH2P04 54.4(mg1-')
2.2.6 Protocol for increasing in TN loading rate
Total nitrogen loading rate (TNLR) was increased stepwise by O.05 kg-N m-3 d-i - O.1
'kg-N m"3 d-i every step from O.l kg-N m"3 d-i to 1.2 kg-N m-3 dffi . After loading rate
reached to 1.2 kg-N m-3 d-i , TN loading rate was increased by O.2Å}O.05 kg-N m-3 d-i for
each step. Effluent nitrite concentrations were always kept below 20 mg-N 1-i.
2.2.7 Operational condition in whole period
Temperature was kept at 20Å}2 OC in winter season and at room temperature in other
26
seasons. Concentration of (NH4)2S04 and NaN02 in synthetic wastewater was 75Å}5 mg-N 1-i
and the total nitrogen concentration was 150Å}10 mg-N l-i. TNLR was increased only by
increasing flow rate.
2.2.8 Analytical items and methods
The concentrations ofN02-N and N03-N were measured by the colorimetric method in
accordance with the Standard Methods (APHA, AWWA, WPCF 1995). NH4-N concentration
was measured by the modified phenate method using ortho-phenyl phenol (OPP) (Kanda, J.,
1995). Absorbance and pH values were measured using a spectrophotometer (U-2010;
Hitachi, Tokyo, Japan) and a pH meter (B-21 1; Horiba, Kyoto, Japan), respectively.
2.3 Results and Discussion
2.3.1 Observation of the attached biomass
The changes in anammox biofilm shape and color were shown in Fig. 2-4. The biofilm
developed in thicker biomass and the color of anammox biofilm changed to deep red color
gradually. But after 13 months of operation, the color was not as red as before because of
addition ofundoing nonwoven fibers for bridging the gap between thin strips ofnonwoven in
the anommox reactor.
•• l•Sl,ii1.,,LGg
"'''i'I'}'//'
•k
"txxl'
beeeww.,l
ge
-wwiigeigi
Fig.2-4 Colorchangesinanammoxbiofilm
27
2.3.2 Changes in total nitrogen loading rates and removal rates
Fig. 2-5 showed daily changes in total nitrogen loading rate (TNLR), total nitrogen
removal rate (TNRR) and N03-N production rate. From this graph, whole experiments can
be divided into 5 Runs. The term of Run 1 was days O to 205. The maximum TNLR was 1.63
kg-N m-3 dri and the maximum total nitrogen removal rate (TNRR) of 1.3 kg-N m'3 d"i was
obtained after 205 days of operation. The establishment of anammox reactor using low
strength of influent nitrogen (influent NH4-N N02-N was about 70Å}5 mg 1-i) was proved
possible without temperature control from these results.
Ig .6
,""KTv" ezdn
MK...i
eN
'i6
gE2
Å~tu
sRRz-
.::...3
.3.,i,
l.S
igl -e
I II III IV v
o-A
TN loading ratet( kg-N m-3 d-i)
TN removal rate( kg-N m'3 di) @No3-N production( kg-N m-3 d-i) )L
@- l i ;,,- ,' 'lg'.,./:, ..
x'-" ,,k •-,/ .,./ ':l-ll-ll-!- i" L• i
.i ,-x k 1 ,r, ., r,r,n ' ,',, i' I,l" -lats
s•s•/ 'g: if-}
Fig. 2-5
t, eV
-
@Smallga,rr,ie.r,
•
i-i
"F
Y
'
e,{Fii ,i,i,,,,ii"i'
f
""
6
,E"
L'
'
5
i'.S ,
e l{ie :•gbex :4•rs 2gf,• :,;,tk seci• ajee 4s(: wwe spt.y'
Time(days) -Changes in TN loadinglremoval rate and N03-N production rate
g.,4
'3 .fs•
O.4
.'-'N
Tv9gzda
Mvpoe:
,9689nz
:"oZ
The term of Run 3 was days 235 to 370. We were able to increase TNLR to 2.6 kg-N
m-3 d-i on day 320, but TNRR fluctuated and could not get the stable anammox activities.
After day 365, the reactor system deteriorated quickly due to clogging and channeling inside
of the anammox reactor. This phenomenon was supposed to be caused by no intentional
sludge withdrawal. Thus, 6.5g-VSS of anammox sludge was withdrawn from reactor on day
370.
28
The term of Run 3 was days 235 to 370. 0n day 400, reactor temperature was increased
accidentally to 50 OC owing to failure in temperature control at 20 Åé in winter season.
The color of anammox sludge turned to white color and lost most of its anammox activity.
This dead anammox sludge was taken out from the reactor and restarted experiment under
low loading rates. Just at this time, we added small-caniers into the gaps of thin strips of
nonwoven.
The term of Run 4 was days 41O to 520. TNLR and TNRR were able to increase quickly.
TNLR increased from O.78 kg-N m-3 d-i to 6.5 kg-N m-3 d-i in about 3 months of operation.
Then, the reactor was fu11y occupied with anammox sludge as shown in Fig. 2-4. Maximum
TNLR and TNRR were 6.5 kg-N m-3 d-i and TNRR is 4.68 kg-N mH3 d-i , respectively in this
Run. On day 530, the reactor was stopped for the measurement of anammox sludge grown in
the reactor. Nonwoven biomass carrier was taken out from the reactor and detached the
attached sludge on nonwoven and suspended in 31 ofefiluent, and measured its concentration.
The weight of anammox biomass ofnonwoven small-carrier in the reactor was 40.7 g-MLSS
and 3 1 .2 g-MLVSS. The weight of anammox biomass attached on thin strips of nonwoven in
the reactor was 31.0 g-MLSS and 21.7 g-MLVSS. So, the total weight of anammox
biomass in the reactor was 71.7 g-MLSS and 52.9 g-MLVSS. VSS content of our
cultivated anammox sludge was 73.8 O/o. This VSS value was unexpectedly high compared
with another autotrophic sludge like nitrifying sludge.
Finally, the nonwoven canier was installed into the reactor again. Then, 3 1 of anammox
sludge was fi11ed in the anammox reactor and culture liquid was recycled at 10 1 h'i for
attachment of anammox sludge on nonwoven biomass carrier. After 32 hours of recirculation,
the effluent was almost clear. Then, continuous operation was restarted. Initial anammox
sludge concentration was 19.8 g-MLSS 1-i. Run 5 was started from day 520. Stable and quick
anammox start-up was also realized in Run 5 like Run 4.
Table 2-2 shows the maximum influent flow rate and 'minimum HRT for each Run.
Owing to the low influent nitrogen concentration, high influent flow rate had to be applied
for getting high TNLR. In Run 4, extremely short HRT (O.56 h) was applied. This is the first
report to succeed the stable operation of anammox reactor under such an extremely low HRT
under room temperature.
29
Table 2-2 Minimum HRT for each Run
Run 1 2 3 4 5
Maximuminfluentflowrate(1d
MinimumHRT(h)
-i ) 29.0
2.32
66.2
1.01
14.4
4.67
120
O.56
98
O.69
2.3.3 Performances ofTotal nitrogen removal efficiencies
TN removal performances during whole experiments were shown in Fig. 2-6. At the
starting periods up to 50 days, influent TN concentration was kept at the low level of 50 to
70 mg-N 1-i. From day 51 to day 530, influent TN concentration was kept at 150 mg-N 1-i but
the effluent total nitrogen concentrations were changed depending on the operational
conditions. Before day 340, the effluent total nitrogen concentrations were stable and ranged
from 20 mg l-i to 30 mg-N 1-i But the effluent total nitrogen concentrations were ranged from
30 mg INi to 40 mg-N 1-i after day 340. The reason of these high effluent TN concentrations
was supposed to be the formation of water channel under short HRT. The total nitrogen
removal efficiencies mainly ranged from 70 O/o to 80 O/o before day 340, while ranged mainly
from 60 O/o to 70 O/o after day 340.
g?,$.
'Oi
-aneu.9retsg8gzeu8EEII
a8
ÅëE
a2, 6
g'19
se2.
g,i
•5S
.32
34
'g'7,
it
"-
,gkllk s-.
}il es
g
s
ge'w
Influent TN concentration(mg 1-i)
Effluent TN concentration(mg 1-i)
TN removal efficiency(O/o)
TN removal rate kg-N mt3 d'i
1-i
c'
iitseci
t'
1
:`'' :
I.
"tli s.. :
Å}
s
vatt.
i-
t
pm
g,g)
'7, a)
tw
.)-,a)
:";'t
LTg
gij
/gft
AeXO
Å~8
•g
ur..o
-,ge
oE9zE---,
vg-A
ivveegRm
•# 4iC
Fig. 2- 6
at$ giipt,1 :7L.l o- :.s-e•.g llltg,e 32,tr s6,gj ec.ee `#E#if 4gg) u•,s -sec,
Time (days)Changes in influent and effluent TN concentrations and TN removal efficiencies
30
2.3.4 NH 4-N removal performances
Fig. 2-7 showed daily changes in influent and effluent NH4-N concentrations, NH4-N
removal efficiencies and NH4-N removal rates during whole experiments. NH4-N removal
efficiencies ranged from 75 O/o to 85 O/o and effiuent NH4-N concentrations were ranged from
10 mg 1-i to 25 mg 1-i before day 340. While NH4-N removal efficiency ranged from 60 O/e to
55 O/o and effluent NH4-N concentrations ranged from 20 mg 1-i to 30 mg 1-i after day 340 as
shown in Fig.2-7.
j-.-S
ATv"ez
tSi)
MveE
re>ofi
9zk
=z
3.2
:.-s
2.,S
i.s
g.4
g,x
g.'7,
,g..4
gl .e
,iil)
vaiiiiiidi
tw
ADee
gl}S
Infiuent NH4-N(mg 1-i)
NH4-N removal efficiency(O/o)Effiuent NH4-N(mg 1-i)
NH4-N removal rate( kg-N m-3 d'i)
"
e 4g] gij lg2,ga sgi) :.tw
Fig. 2-7.
k"gck 2-ffo s2g) r,6cf #tt Time(days)NH4-N removal perfomances
4gct k..g soo
pa
gij -A,
,, g-so
ljgfi" fi' ag
wtr-". ev'i#i .St:r•# g•E"
.. gg US',,'i
1ge
2.3.5 NO2-N removal performances
Fig. 2-8 showed daily changes in influent and effluent N02-N concentrations, N02-N
removal efficiencies and N02-N removal rates. N02-N removal efficiencies ranged from 80
to 95 O/o and effluent N02-N concentration were kept below 20 mg-N 1-i before day 340.
After day 340, N02-N removal efficiencies were averaged at 77 O/o and effluent N02-N
concentration were kept below 25 mg-N IMi. Compared with NH4-N removal, N02-N removal
performances were better than NH4-N removal performances in this study. We have operated
our anammox reactor under N02-N limiting condition for feeding equal concentration of
31
NH4-N and N02-N.
T"Ab
v" Ez
e{!)
MUts:ge
oE•9
z &oz
3.j
3.2
2.S
L,.,S
e.g,
E.4
g.a
fi•ij -
pts,ft,,C ,e-
=,I
.e." •-- ,c{
,r
f r'
A Influent N02-N(mg IJi)
O N02-N removal efficiency(O/o)A Effluent N02-N(mg 1-i)
wa N02-N removal rate( kg-N m-3 d-i
'
lc
"
,ll , wt
e-
r
`
r"li f=-
y,
'
L
"
kl,t1
'
"
. F:L
-i
-
-
,g
-
eff.-
va
it s$
(liix
ge .g2.e a•$i) 2•.ee 4-sie )-lx, )-ffe
lle
•ge"
sg
S5
;,s'
3Ll
'l S
Fig. 2-8
24e 2sg ,3Lr,e.4 ,3iec, 4oo tssig{,
Time(days)N02-N removal performances
,$
A-v'
vegbE
Z",5"
gu•tds
ggo
Ae.
oh
g'6.a-Et
ge
o8e
zk
ozvg
2.3.6 Removal ratio of NH4-N, N02-N and N03-N
K2o,
kÅé,
s•E
g•.pa
fi •g,
482thO"n
z
rl.-.-•,
i'("
g.--',
-c,
'i
.cfi
.5
.s
.5
.I}
'j
e.o
Amp
D
TN removal rate( kg-N m'3 d-i)
N02-N removal rate( kg-Nm'3 d "i)
N03-N production( kg-N m-3 d-i )
v= l,.'ge9x,x
,TRf = iO.99i2 "- "
v •== l.;•t g x
R2 = g.gT• r}
-
'
eedwee
rf e
, ,IE' " 'e• ""' '-' il' lf'/ f'
tat. ..t
pt
g.t ="/.Lr23x
,R' = 'i-kt' m'9'$"2'
fi..{p
Fig. 2-9
e-s ge g..i ?...5
Ammonium (NH4-N) removal rate ( kg-N m'j d'`)TN removal, N02-N removal and N03-N production rates with ' respect to NH4-N removal rate
and
Fig. 2-9 showed the relationship between NH4-N removal rates, N02-N removal rates
N03-N production rates. The ratios of TN removal, N02-N removal and N03-N
32
production rates to NH4-N removal rates in this study were shown in Fig. 2-9. The ratio was
1.98 : 1.20 : O.22, which was slightly lower value compared to the theoretical anammox
reaction ratio (Eq. 1)
2.3.7 Comparison of anammox treatment capabilities
We have operated our anammox reactor under poor operational conditions (low nutrient
concentrations, low operational temperature and short HRT), but good treatment
performances were obtained even under poor operational conditions. Our obtained results
were comparable with another results for same type of anammox reactors, which were all
operated under ideal operational conditions with high nutrient level, high operational
temperature(35 OC) and long HRT. Table 2-3 showed that comparison of the total nitrogen
removal capabilities for various kinds of anammox reactors operated in our laboratory. Even
under poor operational conditions, high TN removal rate was successfu11y obtained in this
study.
Table 2-3 Comparison of anammox removal capabilitiesTypeofbiomass Inf.TN Operational TNremovalrate
.carrler cocentration(mg-Nl`i) Temperature( oC)( g-Nm-3d-i)
Nonwoven 250-300 35 3.1
Acrylicresinfiber 90-120 35 2.2(Fujiietal.,2002)
Nonwoven 150 20 4.68thisstud
2.3.8 The reasons for high removal rate
In this study, high nitrogen removal rate was obtained even under poor operational
conditions. There are several reasons responsible for this high total nitrogen removal rate
obtained in this study.
Firstly, 16 thin strips nonwoven biomass carrier provided a high surface area for the
attachment of anammox sludge. Second was the particle size of seed anammox sludge. Our
used small size of seed anammox sludge (<O.5 mm) enabled the efficient entrapment of
33
anammox sludge inside nonwoven biomass carrier. Anammox sludge grew inside of strips
firstly and then grew outside ofthe surface of strip as shown in Fig.2-1O. We have used the 8
strips nonwoven shown in Fig.2-1 1 for long time as the biomass canier for anammox sludge.
Big size of anammox granules were formed on the surface of eight strips of nonwoven and
could not grow inside of strips. After the fu11 development of anammox biomass on
nonwoven biomass carrier, about 10 g-MLSS anammox sludge was taken out from the
reactor and mixed with 30 g of undoing nonwoven fiber shown in Fig.2-12 and added to the
reactor for bridging the gap between thin strips of nonwoven. The gap was fi11ed with
anammox sludge after 145 days ofoperation and extremely high anammox activities of4.68
kg-N m-3 d'i were obtained under HRT ofO.56 hours.
#
Fig. 2-10. Thin strips ofnonwoven carrier
fit t yt
-'tig/ ''f
Fig. 2- 1 1 . Eight strips of nonwoven carrier
Fig. 2-12. Undoing nonwoven fiber in anammox sludge
2.4 Conclusions
We have succeeded in the establishment of anammox reactor using low strength influent
substrate of 150 mg-N 1-i under low temperature of20-250C. TNLR and TNRR reached to 6.5
kg-TN m-3 d-i and 4.68 kg-TN m-3 d-i, respectively. The TNRR achieved in this study was
34
about two times higher than that of others anammox reactor operated in our laboratory. The
results of this study on the establistment of anammox process using low strength TN
concentration under low temperature would be significant in practical industrial application.
2.5 References
A. Olav Sliekers, K.A. Third, Abma,J.G. Kuenen, M.S.M. Jetten.: CANON and Anammox in
a gas-lift reactor, FEMS Microbiol. Letter, 218 ,339-334(2003).
Ikuo Tsushima Yuji Ogasawara, Tomonori Kindaichi, Hisashi Satoh, Satoshi Okabe.:
' Development ofhigh-rate anaerobic ammonium-oxidizing(anammox) biofilm reactors,
Mat. Res., 41,1623 - 1634 (2007).
Strous, M., Heijnen, J.J., Kuenen, J.G,, Jetten, M.S.M.: The sequencing batch reactor as a
powerfu1 tool to study very slowly growing micro-organisms, Appl. Microbiol.
Biotechnol. 50, 589-596( 1998).
Takao Fujii, Kenji Furukawa.: Characterization ofthe microbial community in an anaerobic
ammonium-oxidizing biofilm cultured on a nonwoven biomass carrier, J. Biosci. and
Bioeng., 94(5), 412-418(2002). •
van Dongen, U., Jetten, M.S.M., van Loosdrecht, M.C.M.: The SHARON-anammox process
for treatment of ammonium rich wastewater, Mater Sci. Technol. 44 (1) , 153-160
( 2001).
35
36
Chapter 3
Effect of Salt Concentration in Anammox Treatment Using
Non-woven Biomass Carrier
3.1 Introduction
Nitrification-denitrification process has been widely used for nitrogen removal from
wastewater. However, not only a large amount of oxygen for nitrification and addition of
external carbon sources (i.e., methanol) for denitrification are required but considerable
amount of excess sludge is also produced. In order to overcome these factors, attempts have
been made to develop the alternate biological nitrogen processes. One of the processes
recently developing was the partial nitritation-anammox process, which has many potential
advantages over conventional nitrogen removal processes (van Dongen, U. et al., 2001). In
this nitrogen process, 60 e/o of influent ammonium is converted to nitrite in the aerobic panial
nitritation treatment and then anammox bacteria oxidize remaining ammonium to nitrogen
gas using nitrite as an electron accepter under anoxic conditions, with their growth occurring
by carbon dioxide fixation. For these reasons, the amounts of oxygen supply, external carbon
source, and excess sludge production can be reduced in this partial nitritation-anammox
process (van Dongen, U. et al., 2001).
Since partial nitritation-anammox process was successfu11y applied to the treatment of
'sewage sludge digester liquor in Netherlands (van der Star, W.R.L. et al., 2007), it opened
doors for application to many kinds of wastewater treatments such as industrial wastewater,
livestock wastewater, and landfi11 leachate. However, these wastewater contain high
concentrations of salts which have been considered as an inhibiting factor in biological
nitrogen removal process. Thus, effects of high salt concentration on nitrification and
denitrification have been previously investigated (GIass, C. et al., 1999; Campos, J. L. et al.,
2002; Moussa, M.S. et al., 2006). It was repored in these studies that nitrification and
'denitrification activities were sustained by gradual acclimation of freshwater sludge to high
37
salt conditions until certain salts levels. Halophilic denitrifying bacteria were isolated from
the long-term acclimated sludge, and higher denitrification performances were demonstrated
when the long-term acclimated sludge was used as inoculum (Yoshie, S. et al., 2006).
Furthermore, Furukawa et al., (Furukawa, K. et al., 1993) reported that nitrifying sludge
taken from night soil treatment plant employing a sea-water dilution in summer season could
adapt more smoothly to high salt condition than the case of freshwater sludge. In other
studies, marine anammox bacteria belonging to "Scalindua" genus have been detected in
natural surroundings (Thamdrup, B. et al., 2002; Kuypers, M.M.M. et al., 2003; Schmid,
M.C. et al.,2007) and very recently Nakajima et al., (2008) enriched them from an enclosed
coastal sea in Japan using a continuous culture system. These results suggested that the
anammox bacteria inherently prefening to the culture containing high concentration of salts
and living in the high salt habitats would be accumulated in the cultivations and available for
industrial application. In other hand, there is an inconsistent experiment. Kartal et al., (2006)
adapted the anammox sludge which consisted of 50 O/o of Candidatus "Kuenia
stuttgartiensis" and 50 O/o of Candidatus "Scalindua wagneri" to the salt concentration of 30
g 1-i. Although it would be the culture condition suitable to the growth of Candidatus
'"Scalindua, he reported that the major anammox bacteria after the acclimation were
Candidatus "Kuenenia stuttgartiensis" enriched from freshwater condition. Because Kartal et
'al., (2006) used the seed sludge containing marine anammox bacteria besides freshwater
anammox bacterium, the result that major anammox bacteria at high salt condition were
freshwater anammox specie is open to question. In addition, Kartal et al., (2006) focused on
only the population of anammox bacterium species without the evaluation of coexistent
bacteria community.
In this study, the effect ofhigh concentration of sodium chloride on anammox treatment
was investigated. We used the anammox fixed-bed reactor using non-woven biomass carrier
with a seed containing only freshwater anammox bacteria, strain KU2 and KSU-1, differing
from those used by Kartal et al (2006). The salt concentration was increased stepwise from
2.5 g 1-i to 33 g 1-i. In addition, the bacterial community was also examined by 16S rRNA
gene analysis after the acclimation ofthe anammox sludge to high salt condition.
38
3.2 Materials and methods
3.2.1 Set up and operational conditions of anammox reactor
An up--flow fixed-bed column reactor with an inner diameter of 95 mm, and a height of
400 mm (an effective voiume of2.8 1) was used in this study as shown in Fig. 3-1. Aporous
polyester non-woven fabric canier (1.0 L, Japan
Vilene, Tokyo, Japan) was used as support material
at a packing ratio of 35.7 O/o. The reactor was
Non-woveninoculated with 1.0 g-VSS of freshwater anammox B osludge, which was taken from another anammox : waterjacket
reactor treating synthetic wastewater without salt 'addition (Qiao, S., ph.D. thesis, Kumamoto Settiingarea
University, Kumamoto, 2007).
The reactor temperature was maintained at P lnfluent2soc during the entire operational period• The PH Of Fig. 3-1 schematic diagram of fixed
bed anammox reactor.the reactor was not adjusted, and the pH of influent
and effluent were around 7.6 and 7.7, respectively. The composition of the synthetic
wastewater used in this study is shown in Table 3-1.
Table 3-1. Composition of synthetic wastewater
Efflu
ee
sr.
Composition Concentration
(NH4)2S04 75mg-N1-i
NaN02 75mg-N.1-i
KHC03 125mgl'l
KH2P04 54mg1-1
T.element o.smlrl
NaCl O-33.og1-i
T. element: FeS04- 7H20, 18g 1-i
'
39
•EDTA•,
2Na, 10 mg 1-i
The synthetic wastewater was flushed with nitrogen gas to decrease the dissolved
oxygen (DO) concentration below 1.0 mg 1'i. The hydraulic retention time (HRT) was varied
from 36 h to 1.7 h depending on the nitrogen loading rate (NLR). The reactor was fed with
synthetic wastewater without salt addition until the nitrogen removal rate (NRR) increased to
1.6 kg-N m-3 dHi (day 102). After that, the NLR was maintained at 2.2 kg-N m-3 d-i and the
infiuent salt concentration was increased stepwise from 2.5 g 1-i to 33 g lmi.
3.2.2 Analytical methods
The concentrations ofN02-N and N03-N were measured by the colorimetric method in
accordance with the Standard Methods (APHA, AWWA, WPCF 1995). NH4-N concentration
was measured by the modified phenate method using ortho-phenyl phenol (OPP) (Kanda, J.
1995). Absorbance and pH values were measured using a spectrophotometer (U-2010;
Hitachi, Tokyo, Japan) and a pH meter (B-211; Horiba, Kyoto, Japan), respectively.
3.2.3 DNA extraction and PCR amplification
A sludge sample was taken from the reactor after its acclimation to high salt
concentration (day 320). The sample was suspended in 1 ml of TE buffer with 1 pt1 of
Ready-Lyse Lysozyme Solution (EPICENTRE, U.S.A.) and incubated at 37 OC for 1 h. The
solution was added with 1.5 mg of achromopeptidase (Wako, Osaka, Japan) and incubated at
37 OC for 30 min. Then, bacteriolysis was performed by addition of 100 pl of 10 O/o sodium
dodecyl sulfate solution. Proteins in the supernatant prepared by centrifugation were
decomposed with Proteinase K (Wako) treatment. The supernatant was prepared by
centrifugation and meta-genomic DNA in it was purified by phenoYchloroform extraction.
The DNA was recovered by ethanol. The meta-genomic DNA was dissolved in TE buffer,
treated with RNase A, precipitated by ethanol with 13 O/o PEG8000-1.6 M NaCl, and
dissolved again in TE buffer. The amplification of 16S rRNA gene was performed with KOD
-plus- DNA polymerase (TOYOBO, Osaka, Japan) using eubacterial primers 6F (forward
40
primer: 5'-GGAGAGTTAGATCTTGGCTCAG-3') and 1492R (reverse primer:
5'-GGTTACCTTGTTACGACT-3') (Lane, D.J. 1991; Tchelet, R. et al., 1999). PCR
(polymerase chain reaction) was carried out according to the following thermocycling
parameters: 2 min initial denaturation at 94 OC, 25 cycles of 15 sec at 94 OC, 30 sec at 60 OC,
and 30 sec at 68 OC. The amplified products were electrophoresed on a1 O/o agarose gel and
the DNAs in excided agarose gel were purified using Wizard SV Gel and PCR CIean-Up
System (Promega, U.S.A.).
3.2.4 Cloning and sequencing of 16S rRNA
The purified fragments were ligated into the Hincll site of pBluescript II KS+
(Stratagene, U.S.A.). E. coli DHIOB was transformed using the constructed plasmids. The
plasmids were extracted by the alkaline method from 33 clones carrying them. The
nucleotide sequences ofinserted DNA in them were determined with 3130xl genetic analyzer
and BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems, U.S.A.). The
sequences determined in this study were compared with those in nr database by the basic
local alignment search tool (BLAST) program on the NCBI web site.
3.2.5 Denaturing gradient gel electrophoresis (DGGE)
Partial 16S rRNA gene was amplified by PCR using a eubacterial primer set, 357F
(forward primer: 5'-CCTACGGGAGGCAGCAG-3') and 534R (reverse primer:
5'-ATTACCGCGGCTGCTGG-3') (Muyzer, G et al.,1993), and the extracted meta-genomic
DNA as template. The amplified fragments were purified and added with the GC-clamp
(5'-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG-3') at the 5' termini
by second PCR using a primer set, 357F with GC-clamp and 534R. The products were
resolved by DGGE for 16 h at 100V at 60 OC using DCode system (Bio-Rad, U.S.A.). An 8
O/e acrylamide gel with a 30-65 O/o denaturing gradient was used, where 100 O/o denaturant was
defined as 7 M urea and 40 O/o form amide. The gel was stained with SYBR-Gold solution
(Invitrogen, U.S.A.), and visualized using FLA-2000 system (Fuji Photo Film, Tokyo, Japan).
41
Five bands were excised from the gel to determine the sequences. The gel piece was crushed
with disposable polypropylene pestles and soaked in the DNA extraction buffer (500 mM
sodium acetate (pH 5.2), 1 mM EDTA). After ovemight incubation at 4 OC, the mixture was
centrifuged and the supernatant containing the extracted DNA was transferred into a new
tube. The DNA was ethanol precipitated, dissolved in TE buffer and amplified by PCR with
a primer set, 357F and 534R. The amplified fragment was directly sequenced.
3.2.6 Nucleotide sequence accession numbers
The partial 16S rRNA gene sequences of Operational taxonomic unit (OTU) 1, OTU 2,
OTU 3, OTU4, OTU 5, OTU 6, and OTU 7 were submitted to the DDBJ database under
accession numbers AB434253-AB434256, AB434257, AB434258, AB434259, AB434260,
AB434261, and AB434262, respectively.
3.3 Results and discussion
3.3.1 Reactor performances under high salt conditions
After achieving a NRR of 1.6 kg-N m-3 d-i (day 102) without salt addition, the TNLR
was maintained at 2.2 kg-N m"3 d-i and salt addition was initiated. Fig.3-2 shows the time
courses of nitrogen concentrations under various salt concentrations. When the salt
concentration was set at 5 g 1'i, the effluent NH4-N and N02-N concentrations were increased.
However, the effluent NH4-N and N02-N concentrations decreased by decreasing the salt
concentration from 5 g 1-i to 2.5 g 1'i. Thereafter, although the salt concentration was
increased to 5 g 1-i, the effluent NH4-N and N02-N concentrations did not increase. The salt
concentration was then increased stepwise to 30 g 1-i. During this period, the effluent NH4-N
and N02-N concentrations remained constant demonstrating successfu1 performance of
anammox treatment under high salt conditions. The anammox treatment was successfu11y
maintained at the salt concentration of 30 g 1"i between day 195 to 203. However, when the
salt concentration was further increased above 30 g 1-i, the effluent NH4-N nd N02-N
42
concentrations increased. Consequently, the salt concentration was decreased to 28 g IJi until
the efficiency of anammox treatment was recovered. After recovery, the salt concentration
was again increased to 30 g 1-i. As shown in Fig. 3-2 the anammox treatment then became
stable at the salt concentration of 30 g 1-i and the T]NFRR of 1.7 kg-N m-3 d-i was achieved for
the ensuing 65 days (day 272-337).
-r--l
z/da
S.E'
gg:&.g
scz
foo
ee
ge
'7g
6e
•5if
4ff
3"
21g
Ie
g
Sak c"nc.: 3e g l-a
" gls i?ec sf Lag ,ti/ini• iE x x• -
tUbli va
st
t "tltt
ue':=ajtt,ts"t}tsl'lsl""',t.,"th,k=""ca'fi/fta'wtt .-'xx,ei-.,s"',t've
asx. iiliasrfinv"geaslj.,ge.va-,rm• ee nc ut ee N. S•, *. ili ee lfi
'
p ' "] ewi tsbptP, . S di0 6>
'pm'
iilll81II}}SS/ilii,li,l.le.,.l,CS'"ll.ll•ll,ik:2,inv•IIi•lii:•ts,tl'illtstkmu:,.••.t/,.L,at.ts-ti:h.f.t,:st,:,,tag,i•l;!,.c,kmamul••"kl'th"s•tg"s•"'••ft,'"C'O'•'b•
35
3•e
2-5
2ts
f•5
fie
5
-s
-rpmseti-su
•=ets
stusc
vaiiiEtlR,twngtw"'4-•L'th•,i.ep/ts.,i{[/t/s,tik,,t,t.
fee fsg 2eic 2-sg 3cg ' 3sc Tl lme {d/}
Fig. 3-2 Time courses of nitrogen concentrations under various salt concentrations. Symbols: D influent NH4'; Xinfluent N02-; Q effiuent
NH4'; O effluentNOi; A effluentN03';solidline,salt.
Dapena-Mora et al., (Dapena-Mora, A. et al., 2006) applied anammox treatment to the
digester liquor from fish canning industry using a SBR, and reported that the NRR was O.3
kg-N m-3 d-i at a salt concentration of 10 g 1-i. Kartal et al., (2006) operated a SBR for
anammox treatment with salt addition, and reported that the NRR was 1.0 kg-N m-3 d"i at a
salt concentration of 30 g 1-i. In contrast, the TNRR at the salt concentration of 30 g 1'i was
1.7 kg-N m-3 d-i in this study. Relatively lower NRRs reported by Dapena-Mora et al., (2006)
and Kartal et al., (2006) may be attributed to the fact that because water density was
increased by increasing salt concentration, the sludge with lesser density might have been
washed out of the SBR. On the other hand, high amount of anammox sludge was
consistently maintained in the fixed-bed reactor using non-woven biomass canier in this
study resulting in higher TNRR of 1.7 kg-N m-3 d-i. An Anammox reaction ratio (NH4-N
' 43
consumption, N02-N consumption, N03-N production) of 1 : 1.32 : O.26 is generally
accepted by researchers (Strous, M. et al., 1998; Dapena-Mora, A. et al., 2004). In this study,
the anammox reaction ratio at the salt concentration of 30 g 1-i (day 103-337) was 1 : 1.29 :
O.24, which was close to the accepted ratio (Strous, M. et al., 1998).
The relationship between the TINIRR and
the salt concentration is illustrated in Fig.
3-3. The TNRR was maintained atapproximately 1.65 kg-N m-3 d-i under salt
concentrations ranging between 10 g 1-i and
30 g 1-i. However, the TNRR sharply
declined at a salt concentration of more than
30 g 1-i. Kartal et al., (2006) also observed
that the anammox activity of the SBR was
completely lost when the salt concentration
was increased from 30 g 1-i to 45 g 1-i. Based
on these results, it is suggested that
a
that saltwater acclimated sludge might
concentrations in excess of 30 g 1-i. For
denitrifying sludge which was acclimated to
resulted in high denitrification
Therefore, it is lik
ofmore than 30 g 1-i
"T,
vny:
Ezile,
egif
-N'
>eEfl
EzaEntz
2.e
fe5
1.e
ij.s
e,.e
ll • t l -l ""ti k l
+
freshwater
pplied to anammox treatment at salt concentrations higher th
be
example,
performance
ely that the anammox sludge could p
by long-term acclimation at elevated salt concentrations.
3.3.2 Bacterial community under salt condition
Thirty three 16S rRINA gene sequences ofbacterial members in the community existing
under high salt condition (day 320) were determined. Table 3-2 shows homology search
results for 16S rRNA gene sequences. The major clone in OTU 1 had 100 O/o sequence
identity with uncultured bacterium clone AnDHS-2 (AB430333) which isolated from an
44
e 5 fg {5 2•e 25 •3ts 35 SaEt cenen. (9 }-")
Fig.3-3. Relationship between the nitrogen removal rate (TNRR) and salt concentration. Bars indicate standard deviation.
acclimated anammox sludge can not be
an 30 g 1-i. However, it appears
capable of treating wastewater with salt
Yoshie et al., (2006) reported that a
the salt concentration of 20 g 1-i for 3 years
even at a salt concentration of 100 g 1"i.
rovide treatment at salt concentration
anammox reactor, and this clone might be affiliated with candidate division OPIO (Crocetti,
GR. et al., 2002). Five clones in OTU 2 and 1 clone in OTU 3 had 100 O/o sequence identity
with KU2 and 99 O/o with KSU-1, respectively. These bacteria have been recognized as
anammox bacteria and enriched in the anammox reactor treating synthetic wastewater
without salt addition (Fujii, T. et al., 2002). Two clones in OTU 4 had 99 O/o sequence identity
with Lysobacter sp.
Fig. 3-4 shows DGGE profiles for 16S rRNA gene
sequences of bacterial members in the community
existing under high salt condition (day 320). Four
explicit bands and some minor bands were detected
after the resolution of amplified DNA fragments by
DGGE. The nucleotide sequences of the explicit bands
were directly sequenced. Except for the primer regions,
the determined sequences ofBands 1, 2, and3 were in
agreement with the 16S rRNA sequences of OTUs 4
(AB434259, nts 331-490), 1 (AB434253-AB434256,
nts 319-454), and 2 (AB434257, nts 365-525),
respectively. The nucleotide sequence of Band 4 was
not involved in any sequences of 16S rRNA genes
cloned in this study, and showed low identities (<90 O/o)
to the 16S rRNA gene sequences ofbacteria belonging
to phylum Chloroflexi (data not shown). In addition,
because the intensity of Band 2 was the highest, OTUI
might be dominant in the reactor.
From these results, it was confirmed that
the freshwater anammox bacteria (KU2) and
concentration of 30 g 1'i.
method,
3.
the primer sets in the cloning and DGGE.
45
i'iilii,li
.,I,!-/thi,illlllll
.-.
I' ix•.1
unidentified bacteria which perhaps belong to candidate division OPIO were dominant
Lysobacter
Although Band 4 was not detected and not identified by the cloning
the intensity of the band seemed to be almost the same as those of Band 1 and Band
This result was likely caused by the affmities differences for the 16S rRNA gene between
Fig. 3 - 4. Denaturing gradient gel
electrophoresis (DGGE) profiles for
16S rRNA gene sequences of bacterial
members in the community existing
under salt condition (day 320).
, and
sp. existed under a high salt
ChloroLflexi bacteria and unidentified bacteria K4-33 (AY793665) coexisted with
anammox bacteria (KU2) in the inoculums (Qiao, S., Ph.D. thesis, Kumamoto University,
Kumamoto, 2007). On the other hand, unidentified bacteria which perhaps belong to
candidate division OPIO and Lysobacter sp. coexisted with anammox bacteria under a salt
concentration of 30 g 1'i. Thus, it is assumed that the microbial community shift occurred by
increasing salt concentrations. Since the bacterial members in the community was examined
only at the salt concentration of 30 g 1"i in this study, fUrther research is required to know the
process of microbial community shift. In addition, although anammox bacteria were
normally dominant in the freshwater condition, unidentified bacteria which perhaps belong
to candidate division OP1O were dominant under salt concentration of30 g 1-i (Fujii, T. et al.,
2002; Kindaichi, T. et al., 2007). Thus, the unidentified bacteria seem to be important role in
the reactor. However, since the metabolism of the unidentified bacteria is unknown, further
investigation must be required.
Table 3-2. Homology search results for 16S rRNA gene sequences ofbacterial members in the
communi exjstin undersaltcondition(da 320).
OTU Taxon (Accession No.) Identity
( o/o)
Numberof clones
1
Uncultured bacterium clone AnDHS-2 (AB430333)
Uncultured bacterium clone YCB35 (EF205445)
Uncultured bacterium clone FCPT516 (EF515998)
Uncultured bacterium clone vf23 (DQ975217)
Uncultured candidate division OP1O bacterium clone
HAVOmat40 (EF032775)
99
91
90
90
90
21
2Anoxic sludge bacterium KU2 (AB054007)
Kuenenia stuttgartiensis (CT573071)99 5
3Planctomycete KSU-1 (AB057453)
KIST-JJYOOI (EF515083)99 1
4 Lysobacter sp. (AJ298291) 99 2
5 Mesorhizobium sp. Ala-3 (AM491621) 98 1
6
7
Unclassified
Unclassified
2
1
Total 33
OTU: Operational taxonomic unit. Unclassified: The sequence does not have homology more than85 O/o identity to any one in nr database.
46
There appear to be two plausible explanations of our obtained results that the anammox
treatment was maintained under high salt conditions. One is that anammox bacteria in the
freshwater sludge were replaced by other salt-tolerant anammox strains such as marine
species under high salt conditions. The other is that freshwater anammox bacteria existing in
the inoculums were originally salt-tolerant and could survive even under high salt
concentration of 30 g 1-i. Kartal et al., (2006) adapted the anammox sludge which consisted
of 50 O/o of Candidatus "Kuenia stuttgartiensis" and 50 O/o of Candidatus "Scalindua
wagneri" to the salt concentration of 30 g IHi, and reported that the major anammox bacteria
after the acclimation were Candidatus "Kuenenia stuttgartiensis" enriched from freshwater
condition. Although our cultivation method was different from that of Kartal et al., (2006),
we also confirmed that the major anammox bacteria after the acclimation to high salt
condition were KU2 enriched from freshwater condition. Therefore, it can be deduced that
there is a possibility that freshwater anammox bacteria had the salt-tolerant characteristics
and cloud survive even under high salt conditions.
3.4 References
APHA, AwwA, WPCF: Standard method for the examination ofwater and wastewater, 19th
ed. American Public Health Association, Washington, D.C. (1995).
Campos, J. L., Mosquera-Corral, A., Sanchez, M., Mendez, R., and Lema, J. M.:
Nitrification in saline wastewater with high ammonia concentration in an activated
sludge unit, UlaterRes., 36, 2555-2560 (2002).
Crocetti, G.R., Banfield, J.E, Keller, J., Bond, P.L., and Blackall, L.L.:
Glycogen-accumulating organisms in laboratory-scale and fu11-scale wastewater
treatment processes, Microbiology, 148, 3353-3364 (2002).
Dapena-Mora, A., Campos, J.L., Mosquera-Corral, A., and Mendez, R.: Anammox process
for nitrogen removal from anaerobically digested fish canning efftuents, Mater Sci.
Technol., 53, 265-274 (2006),
Dapena-Mora, A., Campos, J.L., Mosquera-Corral, A., Jetten, M.S.M., and Mendez, R.:
Stability ofthe ANAMMOX process in a gas-lift reactor and a SBR, J. Biotechnol., 110,
47
159-170 (2O04).
Fujii, T., Sugino, H., Rouse, J. D., and Furukawa, K.: Characterization of the microbial
community in an anaerobic ammonium-oxidizing biofilm cultured on a nonwoven
biomass canier, J. Biosci. Bioeng., 94, 412--418 (2002).
Furukawa, K., Ike, A., and Fujita, M.: Preparation of marine nitrifying sludge, J. Ferment.
Bioeng., 76, 134-139 (1993).
Glass, C. and Silverstein, J.: Denitrification ofhigh-nitrate, high-salinity wastewater, Mater
Res., 33, 223-229 (1999).
Kanda, J.: Determination of ammonium in seawater based on the indophenol reaction with
o-phenylphenol (OPP), varateTRes., 29, 2746-2750 (1995).
Kartal, B., Koleva, M., Arsov, R., van der Star, W., Jetten, M.S.M., and Strous, M.: Adaption
of a freshwater anammox population to high salinity wastewater, J. BiotechnoL, 126,
546-553 (2006).
Kindaichi, T., Tsushima, I., Ogasawara, Y., Shimokawa, M., Ozaki, N., Satoh, H., and Okabe,
S.: In situ activity and spatial organization of anaerobic ammonium-oxidizing
(Anammox) bacteria in biofilms, Appl. Environ. MicTobiol., 73, 4931-4939 (2007).
Kuypers, M.M.M., Sliekers, A.O., Lavik, G, Schmid, M., Jorgensen, B.B., Kuenen, J.G.,
Sinninghe Damste, J.S., Strous, M., and Jetten, M.S.M.: Anaerobic ammonium
oxidation by anammox bacteria in the Black Sea, Nature, 422, 608-6l1 (2003).
Lane, D.J.: 16S123S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics
ed, Goodfellow, M. Chichester, UK, Wiley, 115-148 (1991).
Moussa, M.S., Sumanasekera, D.U., Ibrahim, S.H., Lubberding, H.J., Hooijmans, C.M.,
Gijzen, H.J., and van Loosdrecht, M.C.M.: Long term effects of salt on activity,
population structure and floc characteristics in enriched bacterial cultures of nitrifiers,
MaterRes., 40, 1377-1388 (2006).
Muyzer, G., de Waal, E. C., and Uitterlinden, A. G: Profiling of complex microbial
populations by denaturing gradient gel electrophoresis analysis of polymerase chain
reaction-amplified genes coding for 16S rRNA, AppL Environ. Microbiol., 59, 695-700
(1993).
Nakajima, J., Sakka, M., Kimura, T., Furukawa, K., and Sakka, K.: Enrichment of anammox
bacteria from marine environment for the construction ofa bioremediation reactor, Appl.
48
Microbiol. Biotechnol., 77, 1159-1166 (2008).
Schmid, M.C., Risgaard-Petersen, N., van de Vossenberg, J., Kuypers, M.M.M., Lavik, G.,
Petersen, J., Hulth, S., Thamdrup, B., Canfield, D., Dalsgaard, T., Rysgaard, S., Sejr,
M.K., Strous, M., den Camp, H.J.M.O., and Jetten, M.S.M.: naerobic
ammonium-oxidizing bacteria in marine environments: widespread occurrence but low
diversity, Environ. Microbiol., 9, 1476-1484 (2007).
Strous, M., Heijnen, J. J., Kuenen, J. G, and Jetten, M. S. M.: The sequencing batch reactor
as a powerfu1 tool for the study of slowly growing anaerobic ammonium-oxidizing
microorganisms, Appl. Microbiol. Biotechnol., 50, 589-596 (1998).
Tchelet, R., Meckenstock, R., Steinle, P., and van der Meer, J.R.: Population dynamics of an
introduced bacterium degrading chlorinated benzenes in a soil column and in sewage
sludge, Biodegradation, 10, 113-125 (1999).
Thamdrup, B. and Dalsgaard, T.: Production of N2 through anaerobic ammonium oxidation
coupled to nitrate reduction in marine sediments, Appl. Environ. Microbiol., 68,
1312-1318 (2002).
van der Star, W.R.L., Abma, W.R., Blommers, D., Mulder, J.W., Tokutomi, T., Strous, M.,
Picioreanu, C., and van Loosdrecht, M.C.M.: Startup reactors for anoxic ammonium
oxidation: Experiences from the first fu11-scale anammox reactor in Rotterdam, Mater
Res., 41, 4149-4163 (2007).
van Dongen, U., Jetten, M. S. M., and van Loosdrecht, M. C. M.: The SHARON-Anammox
process for treatment of ammonium rich wastewater, Mater Sci. Technol., 44, 153-160
(2001).
Yoshie, S., Ogawa, T., Makino, H., Hirosawa, H., Tsuneda, S., and Hirata, A.: Characteristics
of bacteria showing high denitrification activity in saline wastewater, Lett. in Appl.
Microbiol. , 42, 277-283 (2006).
49
50
Chapter 4
Nitrogen Removal Capabilities of Two Kinds of Fixed-bed Anammox
Reactors for Treating Partial Nitrification Brine Wastewater
4.1. Introduction
Natural gas and iodine dissolved in brine water are now recovered commercially in
Chiba Prefecture, Japan. In the process of natural gas and iodine production, the brine
wastewater containing NH4-N is produced as by-product. The salinity of this brine
wastewater is almost the same level of sea water. For the removal ofNH4-N from this brine
wastewater, the development of an economical nitrogen removal processris urgently
required.
Compared with the traditional biological nitrogen removal process such as
nitrification-denitrification process, anammox process was now regarded to be a novel,
promising, and cost effective alternative. Anammox process has many advantages, e.g., no
requirement of external carbon sources, low oxygen demand, minimized excess sludge
production and reduction in C02 emissions (Op Den Camp et al., 2006; Liu et al., 2008).
Single-reactor high-activity ammonium removal over nitrite (Sharon) process was
successfu11y applied for partial nitritation process, which is the requisite pretreatment before
applying anammox process for NH4-N removal. The Sharon-Anammox system was proved
to be an economical nitrogen removal process for the treatment of wastewaters with low
carbon to nitrogen ratio (C/N), and the running costs and C02 emissions during this new
NH4-N removal process could be decreased up to 60 O/o and 90 O/o, respectively (van Dongen
et al., 2001). In the partial nitritation process, about 50 O/o of influent NH4-N must be nitrified
to nitrite nitrogen (N02-N), and this effluent is ideally suited as the influent for subsequent
anammox process. In the autotrophic anammox process, NH4-N is oxidized by N02-N under
anaerobic conditions and produce dinitrogen gas and a small amount of nitrate nitrogen
51
(N03-N). The generally accepted stoichiometry of anammox process was depicted as follows
(Strous et al, 1998).
NH4' + 1.32N02- + O.13H' + O.066HC03- -)
1.02N2 + O.26N03- + 2.03H20 + O.66Biomass (Eq.1)
Salinity is generally an important factor for wastewater treatment, because many
industrial wastewaters, such as landfi11 leachate, leather industry wastewater, fish-canning
wastewater, tannery wastewater and the above mentioned brine wastewater from natural gas
producing plant, contain both high concentrations of NH4-N and salt. The high salinity of
these kinds of wastewaters had been considered to exert negative effects on biological
nitrogen removal (Chen et al., 1971; Panswad and Anan, 1999; Dincer and Kargi, 1999). The
presence ofhigh salinity in wastewater induces salinity stress to the microbial species, results
in the inhibition of many enzymes, decreases cell activity, and eventually leads to
plasmolysis (Uygur, 2006). Especially for the treatment of brine wastewater from the natural
gas plant, the salinity level is almost the same level as of sea water. However, recent studies
on the effects of high salinity on partial nitritation and anammox processes have illustrated
that both processes could be operated stably after the suitable acclimation period of
freshwater bacterial community to high salt condition. For partial nitritation process, the
Sharon reactor could work stably under high salt concentrations up to 427 mM (about 25 g 1'i)
of NaCl after the adaptation of microorganisms to saline environment (Mosquera-Corral et
al., 2005). On the other hand, for anammox process, the mass cultivation of marine
anammox sludge was not established yet, though Nakajima et al.,(2008) succeeded in the
enrichment of marine anammox bacteria belonging to "Scalindud' genus from an enclosed
coastal sea in Japan (Nakajima et al., 2008). However, there were few reports on the
acclimation of freshwater anammox bacteria to high salt condition. Very recently, some
anammox processes using fixed-bed reactor, sequencing batch reactor (SBR), and rotating
biological contactor (RBC) in an oxygen-limited sutotrophic nitrification-denitrification
(OLAND) system had been successfu11y operated under high salinity condition (Liu et al.,
2009; Kartal et al., 2006; Windey et al., 2005). In these reports, all anammox reactors were
operated with synthetic inorganic saline wastewater and adaptation experiments of
freshwater anammox sludge to cultural condition with high salts concentration were carried
52
out by stepwise increase in influent salt concentrations. Only Dapena-Mora et al.,(2006)
reported a SBR anammox reactor treating real partial nitrification fish-canning wastewater
under relatively low salinity of 10 g NaCl 1-i (Dapena-Mora et al., 2006). Among all these
reactors, the highest TNNR of 1.7 kg-N m-3 d-i was reported under salinity level of 30 g
NaCl 1-i (Liu et al., 2009).
The objectives of this research are to make clear the applicability of two kinds of
fixed-bed anammox reactors to the treatment of partial nitrification brine wastewater and
compare the treatment capabilities of anammox reactors using different kinds of biomass
canier for this wastewater.
4.2. Materials and Methods
4.2.1. Anammox reactors
The reactors used in the r-----;E:;ffiuent rtEffiuent
fixed-bed anammox processes
are shown in Fig. 4-1. The
reactor was made of Perspex
with available volume of 2.81 (rp
95 mm Å~ 423 mm). The influent
was supplied to the reactor by
up-flow mode. The reactor
temperature was maintained at
about25Å}2 OC
and kept at room temperature in
vinyl sheet enclosure.
anammox reactor (a)) for 3 months
biomass carriers
l2u
'x
x, tt•I
i.,{•ecst•i
.ttlg.
•Å}?tt•
}ll•ili,x
"x
{a}
Nen-woven Small carriers
ti
lnfluent lnfluent {b)
Fig. 4-I Fixed-bed anammox reactors ,
oC
sheltered from light by black
by heating through water jacket when the temperature was lower than 25
other days. The reactor was
After a continuous operation of the reactor using nonwoven biomass canier (fixed-bed
, this reactor was changed to a reactor using small size
(fixed-bed anammox reactor (b)). A point of difference for two reactors is
only the difference in applied biomass canier.
53
4.2.2. Biomass carriers
Nonwoven porous polyester coated with a pyridinium-type polymer (Japan Vilene, US
patent 5,185,415; 1993) was used as biomass canier for fixed-bed anammox reactor (a). The
original nonwoven has 8 strips. For the purpose of increasing specific surface area and
nutrients diffusion rate of nonwoven
biomass carrier, 8 strips of nonwoven
was split into 16 strips using razor.
The split column-like nonwoven
canier (100 mm diameter, 2.5 mm
thickness for one trip, 395 mm length,
shown in Fig. 4-2) was mounted into
fixed-bed anammox reactor (a).
In fixed-bed anammox reactor (b),
biomass caniers. The small size
cement (Portland blast-furnace slag),
hardcore-1
,.ffss
Fig. 4-2 Original and split nonwoven biomass carriers (top view)
small size caniers were used instead of nonwoven
biomass caniers consisted of nonwoven small filaments,
calcined lime, steel slag (smelt steel slag) and
(War industry of military department product without opened). Nonwoven small
filaments were prepared with a mean size of lmm length and 50 pm width by untying and
cutting nonwoven strips. The cement was neutralized by soaking it in running tap water for 2
weeks. The steel slag, whose original diameters were from 1 to 5mm, was sieved to obtain a
mean diameter of1 mm. The hardcore-1 was a special high active agent, which was a
powerfu1 nanophase material.
360g of these upper mentioned small materials were mixed together (the dry weight
ratio ofcement:calcined lime & slag:nonwoven filaments:hardcore-1 was 200:200:50:
5) and put into 1.0 1 tap water. This prepared mixed slurry was named as small size biomass
caniers, which was put into fixed-bed anammox reactor (b) as biomass canier after mixing
well with anammox inoculums.
54
42.3. Brine water from Nature Gas Company
Table 4-1 shows the compositon ofwater qualities of the brine water and sea water. The
concentrations, especially of I- and Br- , are obvious different from that of sea water. After
the removal ofusefu1 materials (natural gas and iodine) from this brine water, the remained
brine wastewater contains high concentration of NH4-N. The mean salinity, NH4-N
concentration and pH value of this brine wastewater are 30 g 1-i, 200 mg-N 1-i and 6.9,
respectively. The salinity of the brine wastewater used in this experiemnt is almost the same
as that of sea water.
Table 4-1 Composition ofbrine water and sea water
Component Brine Water(mg 1-i) Sea Water(mg ri)
rCl-
Br-
Na'
K'Ca2'
Mg2'
So42-
HC03- 2-C03
C02HBo32-
Fe (Total)
11O-130
18,OOO-19,500
120
1O,OOO
3OO
190
500
1,OOO
10-30
10
2-5
O.1
18,OOO
60
9,OOO
350
370
1,200
2,500
1OO
5.9
22
02
4.2.4. Partial nitritation process
Nitritation treatment of the brine wastewater was carried out for providing suitable
influent to sebsequent anammox treatment. The partial nitritation process was applied by a
'swim bed reactor using thread type acrylic fiber biomass carrier.
55
4.2.5. Influent of anammox reactors
The partially nitrification brine wasterwater was supplied to the fixed-bed anammox
reactors as influent. The water qualities of the partial nitritation effluent were not stable in
some cases. Therefore, a 300 1 storage tank for collecing the effluent from the partial
nitritation reactor was utilized and the storage nitirified brine wastewater was used as the
influent of the fixed-bed anammox reactors. The storage partial nitrified brine wastewater
was supplied to the anammox reactors directly after measuring its water quality during stable
panial nitritation treatments. In case of unstable partial nitritation periods, the N02-N and
NH4-N concentrations of the partial nitirified brine wastewater was adjusted to a proper
concentration ratio for the subsequent anammox reactor by adding sodium nitrite and
ammonium sulfate. The composition ofboth unadjusted and adjusted influents was 75Å}15
mg-NH4-N l-i, 90Å}20 mg-N02-N 1"i, 160Å}25 mg-TN 1"i (as shown in Table 4-2). The average
ratio of NH4-N to N02-N of the influent fed to anammox reactor during the whole
experiment period was about 1:1.2.
The influent N03-N concentration of the fixed-bed anammox reactors was also fluctuant
sometimes. The influent N03-N is not engaged in the anammox reaction, so that this N03-N
concentration was ignored and excluded in this study. Consequently, the TN concentration
was calculated by the sum ofNH4-N and N02-N concentrations for the influent and plus the
producing N03-N concentration for the effluent. The TNLRs applied to the anammox
reactors were calculated based on the influent TN (NH4-N + N02-N) concentration. The
TNRRs of the anammox reactors were determined by taking into account the producting
N03-N concentration from the anammox reaction.
4.2.6. Start-up and operational strategy of anammox reactors
The fixed-bed anammox reactor using nonwoven biomass carrier was operated
sequentially after the previous study of this reactor treating synthetic high saline wastewater.
This reactor was started-up with the initial TNLR of O.8 kg-N m-3 d-i and a hydraulic
56
retention time (HRT) of 5.6 hr. After the operation of fixed-bed anammox reactor (a) was
stopped, both the anammox biofilm detached from the nonwoven carrier and small amount
of settled sludge in the bottom part of the up-flow reactor were collected. The detached and
collected anammox sludge was mixed completely with the prepared small size biomass
carriers manually, and then the carriers and sludge were put into the reactor. The initial
TNLR of the fixed-bed anammox reactor (b) was O.89 kg-N m-3 d-i with a HRT of 5.17 hr.
The initial TNLRs for both fixed-bed anammox reactors were set at relatively high values
owing to the ' high initial acclimated anammox sludge concentrations. The TNLR was
increased only by increasing flow rate (or reducing HRT). The protocol for increasing in
TNLR was increasing TNLR stepwise by O.18--O.2 kg-N m-3 d-i each time in case of the
effluent N02-N concentration below 20 mg 1-i most oftime.
The experiments were divided into four Runs, the former two Runs were the experiments
for fixed-bed anammox reactor (a) and the later two Runs were the experiments for fixed-bed
anammox reactor (b). In Run I (from day O to 58), reactor (a) was operated with a quick
increase in TNLR. In Run M (from day 85 to 133), fixed-bed anammox reactor (b) was
operated with a quick increase in TNLRs. In Run ]V (from day 134 to 174), the increasing
rate of TNLR was lower than that of Run M due to the shortage of the partial nitrified brine
wastewater and low substrate concentrations. The operational conditions during the whole
experiment were shown in Table 4-2.
The influent dissolved oxygen (DO) concentrations during the whole experiment were
ranged from 5 to 7 mg 1"i (Tabel 2). DO is toxic to anammox consortium, because anammox
activity can be expected only under strict anoxic conditions (Jetten, 2001). However, some
coexisted oxygen consuming microbial consortia, such as aerobic nitrifiers and floc forming
heterotrophs, could protect the anammox bacteria from the effect of oxygen (Fujii et. al.,
2002; Qiao et al., 2008; Liu et al., 2008 b). In order to save the high cost required for keeping
absolutely anaerobic condition, the influent with DO concentration of 5-7 mg 1-i was
supplied directly to the fixed-bed anammox reactors without nitrogen gas flushing.
57
Table 4-2 Operational conditions during the whole experimental period
Parameters
Time range (day)Fixed-bed anammox reactor
Biomass carrier
HRT (hr)InfluentNH4-N (mg-N1-i)
InfluentN02-N (mg-N1-i)
Influent TN (mg-N1-i)
TNLR (kg-N m-3d-i)
Temperature
( .C)
Salinity (g r')DO influent (mg l")
Run I Run ll Run M Run IV
O-58
(a)
Nonwoven5.6-2.17
73'v84
94-103
167-186
O.8-2,12
25Å}2
30
5r"7
59-84
(a)
Nonwoven 2.12-1.64
72-84 97rvl07
184-187 2.12-2.78
27Å}2
30
5-7
Small carriers
85-133
(b)
5.17-1.76
72"v89
88--103
168--188
O.69-2.57
27Å}2
30
5-7
134-174
(b)
Small carriers
1.73-1.12
64ew78
70'v82
137-169 2.51-3.07
25Å}2
30
5-7
There is no pH adjustment to the influent though the pH ofpartially nitrified wastewater
was fluctuant. The temperature was heated to about 25Å}2 @ in cool days and kept at room
temperature in other days. The influent salinity was unchanged at 30 g 1-i in the entire
experimental period.
4.2.7. Analytical methods
The concentrations of N02-N and N03-N were measured by the colorimetric method in
accordance with the Standard Method (APHA, 1995). NH4-N concentration was measured
colorimetrically by the phonate method using ortho-phenylphenol as a substitute for liquid
phenol (Kanda, 1995). DO concentration of the influent was measured by using a digital,
portable DO meter (D-55, Horiba). The concentration of mixed liquor volatile suspended
solids (MLVSS) of sludge samples was deterrnined in accordance with Standard Methods
(APHA, 1995). pH value was determined by using a pH meter (IM-22P, TOA EIectronics).
58
Biomass attached on small biomass caniers was observed by an electron microscope (Nikon
Eclipse E600, Japan) and a digital camera (Nikon 4500, Japan).
Scanning electron microscopy (SEM) was used for the observation of small carriers
with attached biomass, the procedure is as follows. Samples were first washed in a O.1 M
phosphate buffer solution (pH 7.4) for 5 min, and then hardened for 90 min in a 2.5 O/o
glutaraldehyde solution prepared with the buffer solution prepared with the buffer solution.
Next, samples were washed in the buffer solution three times for 1O min once and then fixed
for 90 min in a 1.0 O/o Os04 solution prepared with the buffer solution. After washing
samples three times for 10 min each in the buffer solution, they were dewatered for 10 min
each in serially graded solutions of ethanol at concentrations of 10, 30, 50, 70, 90, and 95 O/o.
SEM observations were conducted using a scanning electron microscope (JEOL, JSM-5310
LV, Japan).
4.3. Results and Discussion
4.3.1. Performances of the fixed-bed anammox reactors
A continuous operation of the anammox reactor applying nonwoven biomass carrier
and small size biomass carriers was carried out for a period of 174 days. During the entire
experimental period, effluent pH values were always O.5--O.7 higher than influent values.
Increase in pH indicated the occurrence of anammox reaction (Yamamoto et al., 2008). The
N03-N production rates were from O.1 to O.3 kg-N m-3 d-i in most ofperiods (Fig. 4-3, Panel
A) which expressed the growth of anammox bacteria (van de Graaf et al., 1996).
The reactor performances in terms ofnitrogen removal during the whole experimental
period is depicted in Fig. 4-3 (Panels A and B). The data in Runs I and ll show the results
for fixed-bed anammox reactor (a), and the data in Runs M and TV present the results for
fixed-bed anammox reactor (b).
In Run I , fixed-bed anammox reactor (a) was operated with a quick increase in TNLR,
from O.8 to 2.12 kg-N m-3 d-i, only in 58 days. The TNRR increased synchronously from O.7
to 1.81 kg-N m-3 d'i due to the high TN removal efficiency ranged from 83 O/o to 88 O/o.
59
tte?
fi
4.tsua
e
S6i
Eg
.so
89za
4oth
z.
Ezi.
MIii
F. .fi
4.5
4,e
:'i
].e
:..s
[•.g
l.S'
'1.C
o.s•
t}.g
1 f)o
l8e
l {i{)/
Eilf)
E:,g
1•ge
ge
tw
40
2, iO
o
.siko-pt.iI ll m N
fi
A
'
evuavaeqwhI op. I l
i ] ?• "f ;t q tizXiipE' -- i,
' ' '
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es
Ir'
l ' rf il l i F, ,J , t, .Yv'' 'ss# l,
iMdeqrvta.
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C''"''
"''
i,tL,,,P,,,,l'rred$'bi,,:,,tisc,[SSi"
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11e 1[o lso 1-to !so loo 1]o leo
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-Ai-Ztsi)
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vg'ktsgg8
z
o lo :c 3a 4f) i.g sf" ?o s.o gf) 1•oo Time (days)
I I ll I. M ,,.,,,.,"-'lit"- - ':i Cb•"V l" -"r "-" ' l. tlS{61S EiS},liSSi{(iii,t,i.?
B t l., l'i,, tJ?/J,.illl.iliiltwIiiilli.liriiilll.lg.•illtliiiillliliiii-••.,th,.ieei,,.L71i.iiiiiliiiillliii..wt,,NvlliiltlSi,lilililiiil,...,.ili,//L.,.l"l,li
: lst itl
' ''' i. T?., ,nt tt't 's. '/' 'lt. i'"'/ {1'' 'i'S pt ,. r StS 'k : '11'- ''t' '.,' /'./l./,. '''/l'Å}'i" '}" lt {'i ""X 'X","'? ag
I " .,• •' "' w, ', ' .. ' .t
l N
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sc
d,if
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( t••fi• •• )
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bg
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oEopt
O ID l.O 3't) 40 50 6" 7e ge 9e 10e 110 120 1.aa- l40 15e L60 17e 1.Re Time (days) Fig.4-3. Nitrogen removal performance of the reactors. Panel A: (tw ) TN removal efficiency;
TNLR; Cll'liiil"'•) TNRR; (i'',) N03-N production rate. Panel B: (O) infiuent TN; (-) N02-N removal
efficiency; (*) NH4-N removal efficiency; (de,) influent N02-N; (O) infiuent NH4-N; effluent TN; (ig•) effluent NH4-N; (A) effluent N02-N.
The effluent TN concentrations were stable and ranged from 20 to 30 mg-N 1'i. Both
effluents ofNH4-N and N02-N concentration were lower than 10 mg-N 1-i with the average
of 5 mg-N 1-i. This perfect TNRR yielded in only two month confirmed that the
saline-resistant anammox culture, which was acclimated to synthetic influent supplemented
with only NaCl (30 g 1-i) (Liu et al., 2008), could adapted quickly to the practical brine
wastewater with same level of salinity, though which contained many other ions (Br-, K',
Mg2' and Ca2', as shown in Table 4-1).
In Run ll , the TNLR was increased to 2.78 kg-N m-3 d-i gradually but the TNRR did
not change much and maintained at a nearly definite value. The effiuent TN concentrations
60
increased from 30 to 60 mg-N 1-i with the TN removal efficiency decreasing to 63 O/o at the
end ofthis Run, when the effluent NH4-N and N02-N concentrations increased to 15 and 40
mg-N 1-i, respectively. The reason ofthis decrease in TN removal efficiency was supposed to
be the limitation of anammox biomass and the occurrence of clogging inside ofthe attached
biomass on nonwoven biomass carrier and some bigger granules, which was indicated by the
color change of anammox biomass from brownish red to black (Fig. 4-5, day 84) in
fixed-bed anammox reactor (a). In Runs I and ll , the highest TNRR reached 1.9 kg-N m-3
d"i was obtained.
Afterwards, a fixed-bed anammox reactor (b) using small size biomass canier was
applied for obtaining higher TNRRs from day 85. The whole anammox sludge in fixed-bed
anammox reactor (a) was transferred to fixed-bed anammox reactor (b), so that the quantity
of anammox sludge was not changed too much between two anammox reactors.
In Run M, the TNLR was exponentially increased from O.89 to 2.41 kg-N m'3 d-i within
40 days, and the TNRR successfu11y increased from O.69 to 1.96 kg-N m'3 d-i during this
period. The TN removal efficiency recovered quickly to 75e-85 O/o with both effluent
NH4-N and N02-N concentrations keeping around 10 mg 1-i. Comparing to the experimental
results for fixed-bed anammox reactor (a), the TNRR for fixed-bed anammox reactor (b) was
increased more quickly. This result indicates that the anammox treatment performance was
improved by using fixed-bed anammox reactor (b). The color of anammox biomass changed
from black to brown gradually, as shown in Fig. 4-5 (dayl33).
Increasing trend in the TNRRs of Run IV was not constant owing to the following two
reasons. One is the improper balance ofpartial nitrified effluent ofthe brine wastewater, and
the other is the insufficient amount of the partial nitrified effluent for the operation of the
anammox reactor under high TNLRs. In Run IV, the maximum TNLR was increased to 3.17
kg-N m'3 d-i and the maximum TNRR reached to 2.52 kg-N m-3 d-i which was higher than
that in Run M. The TN removal efficiency was similar to that obtained in Run M. The
average N02-N removal efficiency was about 88 O/o and the effluent N02-N concentrations
were kept below 1 1 mg 1-i. The color of anammox biomass changed from brown to weak red
finally (Fig. 4-5, day 170). Owing to the lower influent TN concentration around 140 mg-N
61
1-i (Fig. 4-3, Panel B), the maximum influent fiow rate was 60 1 d-i in Run N with the
'shortest HRT of 1.12 hour.
The reaction ratio of TNRR, N02-N consumption and N03-N production rates to
NH4-N consumption rate was 1.81:1.01:O.20 for fixed-bed anammox reactor (a) (Fig. 4-4,
Panel A), and 1.95:1.15:O.20 for fixed-bed anammox reactor (b) (Fig. 4-4, Panel B). The
reaction ratios in two reactors were both slightly lower than the stoichiometric ratios of
anammox reaction as shown in Eq. 1, but the closer reaction ratio with more reliable
correlation were obtained for fixed-bed anarrmiox reactor (b). This indicates that the better
cultural conditions for anammox bacteria were provided and the more stable anammox
treatment performance was obtained in fixed-bed anammox reactor (b) by using small size
biomass caniers.
-A"'E 2' -4 ik.2 ., o.g3 ")E :-4 R"=oys.ua .?-• .{S Mua 2.fig',.i, ge Iiklueagin g',., ;i=-iS,5S,111.Z•li• geS""`5eS"'"/i,,llli,,11,i,,1-f 11i.iklil'• ,..<cc"co" "K,{-:,k,go',,;
g ,,, .. , i' Il'I - 8. i.i. ii,... ., ii, ,.i,.--•ii• iii. ••• •• •'i-i-• g.{I o.e O.4 g.f {k.6 e.7, O.8 O.9 1.0 1.1 1.2 O.r, if.-st C}.5 •O.6 O.? {S.S B.9 1.{e 1.I 1.2 l.3 1.4
NH4-N consumption rate ( kg-N mj day-i) NH4-N consumption rate ( kg-N mj day-i)
Fig.4-4. Ratios of TNRR, N02-N consumption and N03-N production rates to NH4-N consumption rate. Panel A: fixed-bed anammox reactor (a). Panel B: fixed-bed anammox reactor
(b). (op) TNRR; (O) N02-N consumption rate; (l,l,i) N03-N production rate.
Dapena-Mora et al., reported the value for N02-NfNH4-N consumption ratio and
production ratio of N03-N production and NH4-N consumption were 1.67 and O.28,
respectively, in an anammox SBR treating the partial nitrified fish canning effluents under
the salinity of 8•-JIO g NaCl 1-i at 35 OC (Dapena-Mora, 2006). These different experimental
data for anammox reaction ratios indicates that anammox reaction ratios will change
depending on the cultural conditions (substrate composition, salt concentration and
temperature etc.) and the responsible anammox bacteria.
62
4.3.2. Changes in biomass color for fixed-bed anammox reactors
Anammox biomass changed its colour depending on cultural conditions because of the
unique cytochrome content in anammox bacteria (van De Graaf et al., 1996; Ahn et al.,
2004).
day O day 84 day 95 day l03 day l15 day 124 day 133 day 148 day 170 Fig.4-5. Changes in color of anammox biomass during the whole experimental period
The initial inoculums derived from this fixed-bed anammox reactor using nonwoven
carrier by feeding synthetic wastewater with high salinity as influent presented brownish red
color (Fig. 4-5, day O). The color of anammox biomass in fixed-bed anammox reactor (a)
changed to black after using the partial nitrified brine wastewater as influent (Fig. 4-5, day
84). Yamamoto et al., also reported the color of anammox biomass changed from red to
grayish black after switching the influent from synthetic wastewater to the partial nitritation
piggery wastewater (Yamamoto et al., 2008). There are two possible reasons for the color
changes in anammox biomass in this reactor. One reason is the color component such as
fumid acid and many ions contained in the partial nitrified the brine wastewater, and the
color change accounted for the adaptation of anammox bacteria to the brine wastewater. The
other possible reason is the clogging and channeling of biofilm originated from the deeper
part of nonwoven carrier and the inner of some bigger granules, which caused the poor
diffusion of substrate to these part of biomass and consequently decreased the anammox
activity. Ifthese phenomena were kept for long time, the color ofbiofilm tumed to black.
By changing biomass carrier from nonwoven to small size biomass carriers, the color of
anammox biomass changed from black to brown in Run III (Fig. 4-5, day 133), then to weak
red finally (Fig. 4-5, day 170). One reason for this color change is the dissolving the
limitation factor in increasing anammox activity for fixed-bed anammox reactor (a), by the
better living microenvironment of anammox bacteria provided by small size biomass carriers.
63
Another possible reason is the special function of the small size biomass carriers containing
some nanophase materials, which surrounding and enwinding the anammox bacteria could
prevent or relieve the permeation of the color components and ions in the brine infiuent to
anarnmox cells. This assumption should be verified by further study.
In this study, the color of anammox biomass changed from brownish red to black in
fixed-bed anammox reactor (a), and successfu11y rebounded to weak red with high anammox
activity in fixed-bed anammox reactor (b) using small size biomass carriers. But the detailed
mechanism ofcolor change in anammox biomass could not be verified.
4.3.3. Small size biomass carriers for enhancing anammox performance
For the fixed-bed anammox reactor (a), the TNRRs more than 1.9 kg-N m-3 d-i could
not be achieved owing to the limitation of elevating anammox biomass and the decrease in
anammox activity caused by clogging and channeling phenomena. Even though the biomass
carrying capacity of nonwoven carrier was very high (Furukawa, 2003), it was difficult to
increase the amount of anammox biomass in the reactor after reaching maximum value of
sludge retaining capacity of nonwoven carrier. In order to increase the total amount of
anammox biomass in the reactor and reactivate anammox activity, nonwoven biomass canier
was replaced with small size biomass carriers.
The different spatial distributions ofbiomass between in fixed-bed anammox reactor (a)
and in reactor (b) can be distinguished from Fig. 4-6. Anammox biomass mainly attached on
the chrysanthemum shape nonwoven canier in fixed-bed anammox reactor (a), which only
occupied small proportion of the available volume of the reactor. On the contrary, anammox
biomass distributed evenly over the whole space ofthe fixed-bed anammox reactor (b).
Fig. 4-6 shows the microscopic observation ofbiomass attached on small size carriers
on day 148. The brown, brownish red, red anammox biomass attached on hydrated white
lime (Fig. 4-6, a), nonwoven filaments (Fig. 4-6, b) and mixture of small size carriers (Fig.
4-6, c and d). These attachments confirmed the even distribution of anammox biomass in the
whole reactor, deprived from the initial well mixture ofthe inoculums and small size carriers
64
and the continuously homogeneous disturbance of the whole caniers imposed by the
produced micro bubbles from anammox reaction, which were observed at the top ofreactor
all the time.
Fig.4-6. Microscopic observations of anammox biomass attached on small size caniers on day 148: (a)
biomass attached on hydrated lime, (b) biomass attached on nonwoven filaments, (c, d) biomass
attached on the mixture of small size caniers such as cement, lime and nonwoven powders.
The hardcore-1 was a significant component in the small size caniers. After the surface
modification of the small size crystal mixture (hydrated cement, slag and lime) by the
enwrapping ofthis nanophase material, the smooth surfaces and edge angles ofit changed to
coarse surface with passivity angles (Fig. 4-7, a and b). The spatially wrinkled structure with
many needle fibers and micro channels produced after surface modification was in favorable
of the bacterial immobilization and substrate transfers, which consequently resulted in the
high anammox activity. By the surface modification, the whole of small size biomass caniers
presented a very evenly dispersed status rather than irregular aggregations. No clogging and
channeling phenomena were observed during the whole period of fixed-bed anammox
reactor (b). Moreover, the surrounding of nanophase material on the small mixture could
prevent the exudation ofsome heavy metal ions which was supposed to be toxic to biomass.
SEM images of C and D (Fig.4-7) visualize the biomass adhered the modified small
size carriers. Comparing with the microscopic photos in Fig. 4--6 (b), anammox biomass
attached to inorganic small carriers prior to nonwoven filaments. The nonwoven filaments
performed the main function of connecting these inorganic carriers and transfening the
substrates due to its hydrophilicity. Because of the high mean density of the small size
biomass caniers
Attached by anammox biomass tightly, there was almost no any washout of anammox
sludge in Runs III and IV. The MLVSS concentration of anammox biomass (excluding
nonwoven filaments) in fixed-bed anammox (b) was 6,265 mg 1-i on day 148. Dapena-Mora
et al., reported a biomass concentration between 1,OOO and 2,700 mg-VSS 1'i in a similar
65
study (Dapena-Mora et al., 2006).
concentration was much higher. This
small size biomass caniers.
In contrast to this result, our obtained biomass
result verified the excellent sludge retaining ability of
•ts
gtw"
pu
Fig.4-7. SEM images of small size biomass carriers and attached biomass. (A) Hydrated
small carriers without surface modification by hardcore-1, without biomass attachnent; (B)
hydrated small carriers after surface modification by hardcore-1 without biomass
attachment; (C, D) biomass attachment on small size biomass carriers at different
magnifications on day 150.
4.3.4. Comparison of anammox treatment capabilities under high salinity condition
Table 4-3 shows the comparison of our obtained results with other results obtained from
anammox treatment under high salinity concentration.
Our anammox reactors were operated under unfavorable operational conditions, i.e.,
salinity concentration of 30 g NaCl 1-i, practical industrial influent, moderately low nutrient
concentrations, room temperature, no removal of influent DO and short HRT. Two
fixed-bed anammox reactors in this study yielded higher TNRRs than that in others reports.
66
Table 4-3 Comparison of TNRR for different condition anammox reactors under high salini condtion
Process ReactorSalinity(g 1-i)
TNRR( kg-Nm-3d-i)
Substrate Reference
Anammox
OLAND
Anammox
Anammox
Anammox
Anammox
SBR
RBC
SBR
Fixed-bed
Fixed-bed (a)b
Fixed-bed (b)C
10
30
30
30
30
30
O.34
O.61
1.oa
1.7
1.9
2.52
Fish-canning
wastewaterSynthetic
Synthetic
Synthetic
Brine
wastewaterBrine
wastewater
Dapena Moraet al.,(2006)
Windey et al.,(2005)Kartal et al.,(2006)
Liu et al.,(2009)
This study
This study
aThis is the TNLR. In all other cases, the TNRR is presented.
bFixed-bed anammox reactor using nonwoven biomass canier.
CFixed-bed anammox reactor using small size biomass carriers.
Especially, the highest TNRR of 2.52 kg-N m-3 d'i obtained in fixed-bed anammox
reactor (b) using small size biomass carriers was several times higher than the reported
TNRRs by other researchers. Moreover, the limitation of the TNRR in fixed-bed anammox
reactor (b) was caused by the shortage of the influent. Without this limitation, much high
TNRR might be obtained. The biomass concentration in fixed-bed anammox (b) was reached
at 6,265 mg 1-i MLVSS on day 148, and the specific anammox activity was calculated to be
320 mg-N g-MLVSS'i d-i on this day. This high biomass concentrations and high anammox
activity contributed to the excellent treatment result and indicated the advantages of the
application of small size biomass carriers to fixed-bed anammox reactors. By using this
small size of biomass carriers, we were able to obtain stable anammox treatment
performances without clogging and channeling phenomena which were the disadvantages for
fixed-bed anammox reactor using nonwoven biomass carriers.
4.4. Conclusions
During a period of 174 days, we have succeeded in the establishment of anammox
67
reactors treating the partial nitrification brine wastewater, whose salinity was 30 g 1-i, at
about 25 OC. The maximum of TNRR for the fixed-bed reactor using nonwoven biomass
'carrier reached to 1.9 kg-N m-3 d]i, but could not increase more than this value owing to the
clogging, channeling and limitation of retaining biomass in the reactor. The TNRR for the
fixed-bed anammox reactor using small size biomass carrier reached to 2.52 kg-N m-3 d-i and
stable anammox operation was achieved in this reactor. The color ofbiomass in the fixed-bed
anammox reactor using small size caniers turned to weak red from black color finally. For
completely understanding the excellent performance in this fixed-bed anammox reactor
using small size biomass caniers, microbiological community analyses and the batch activity
evaluations of anammox bacteria should be perfomied in future study.
4.5 References
Ahn, Y.H., Hwang, I.S., Min, K.S.: ANAMMOX and panial denitritation in anaerobic
nitrogen removal from piggery waste, Mater Sci. Technol. 49, 145-153(2004).
APHA Standard Methods for the Examination of Water and Wastewater.: American Public ' Health Association, Baltimore, MD. (1995).
Chen, M., Canelli, E., Fush, G.W.: Effect of salinity on nitrification in East River, J. Mater
Pollut. ControlFed. 42, 2474-2481(1971).
Dapena-Mora, A., Campos, J.L., Mosquera-Corral, A. and Mendez, R.: Anammox process
for nitrogen removal from anaerobically digested fish canning effluents, nlater Sci.
Technol. 53 (12), 265-274(2006).
Dincer, A.R., Kargi, F.: Salt inhibition of nitrification and denitrification in saline wastewater,
Environ. Technol., 20 (11), 1147-1153(1999).
Fujii, T., Sugino, H., Rouse, J.D., Furukawa, K.: Characterization of the Microbial
Community in an Anaerobic Ammonium-Oxidizing Biofilm Cultured on a Nonwoven
Biomass Carrier, 1. Biosci. Bioeng., 94 (5), 412-418(2002).
Furukawa, K., Rouse, J.D., Yoshida, N., Hatanaka, H.: Mass cultivation of anaerobic
ammonium-oxidizing sludge using a novel nonwoven biomass canier, J. Chem. Eng.
Jpn., 36, 1163-1169(2003).
68
Jetten, M.S.M., New pathways for ammonia conversion in soil and aquatic systems. Plant
Soil, 230, 9-l9(2001).
Kanda, J.: Determination of ammonium in seawater based on the indophenol reaction with
o-phenylphenol (OPP), MaterRes., 29, 2746-2750(1995).
Kartal, B., Koleva, M., Arsov, R., van der Star, W., Jetten, M.S.M., and Strous, M.: Adaption
of a freshwater anammox population to high salinity wastewater, J. Biotechnol., 126,
546-553(2006).
Liu, C., Yamamoto, T., Nishiyama, T., Fujii, T., and Furukawa, K.: Effect of Salt
Concentration in Anammox Treatment Using Non-woven Biomass Carrier. J. Biosci.
Bioeng., 107 (5) (2009) (in press)
Liu, S., Yang, F., Xue,Y., Gong Z., Chen H., Wang, T., Su, Z.: Evaluation of oxygen
adaptation and identification of functional bacteria composition for anammox
consonium in non-woven biological rotating contactor, Bioresoun Technol. 99,
8273-8279(2008).
Mosquera-Corral, A., Gonzalez, F., Campos, J.L., M6ndez, R.: Partial nitrification in a
SHARON reactor in the presence of salts and organic carbon compounds, Process
Biochem., 40, 3109-3 118(2005).
Nakajima, J., Sakl<a, M., Kimura, T., Furukawa, K., and Sakka, K.: Enrichment ofanammox
bacteria from marine enviroument for the construction of a bioremediation reactor,
Appl. Microbiol. Biotechnol., 77, 1159-1166(2008).
Op Den Camp, H.J.M., Kartal, B., Guven, D., van Niftrik, L.A.M.P., Haaijer, S.C.M., Van
Der Star, W.R.L., Van De PasSchoonen, K.T., Cabezas, A., Ying, Z., Schmid, M.C.,
Kuypers, M.M.M., Van De Vossenberg, J., Harhangi, H.R., Picioreanu, C., Van
Loosdrecht, M.C.M., Kuenen, J.G., Strous, M., Jetten, M.S.M.: Global impact and
application of the anaerobic ammonium-oxidizing (anammox) bacteria, Biochem. Soc.
Trans., 34 (1), l74-178(2006).
Panswad, T., Anan, C.: Specific oxygen, ammonia, and nitrate uptake rates of a biological
nutrient removal process treating elevated salinity wastewater, Bioresoun Technol., 70
(3), 237-243(1999).
Qiao, S., Kawakubo, Y., Cheng, Y., Njshiyama, T., Fujii, T., Furukawa, K.: Identification of
69
bacteria coexisting with anammox bacteria in an upflow column type reactor,
Biodegradation (in press) (2008).
Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M.: The sequencing batch reactor as a
powerfu1 tool for the study of slowly growing anaerobic ammonium-oxidizing
microorganisms, AppL Microbiol. Biotechnol., 50, 589-596(1998).
Uygur A.: Specific nutrient removal rates in saline wastewater treatment using sequencing
batch reactor, Process Biochem., 41 (1), 61-66(2006).
van de Graaf, A.A., de Bruijn, P., Robertson, L.A., Jetten, M.S.M., Kuenen, J.G.: Autotrophic
growth of anaerobic ammonium-oxidizing microorganisms in a fluidized bed reactor,
Microbiology, 142 (8), 2i87-2196(1996).
van Dongen, U., Jetten, M.S.M. and Loosdrecht, M.C.M.: The SHARON@-Anammox@
process for treatment of ammonium rich wastewater, Mater Sci. Technol., 44(1),
153-160(2001).
Windey, K., De Bo, I., Verstraete, W.: Oxygen•-limited autotrophic
nitrification-denitrification (OLAND) in a rotating biological contactor treating
high-salinitywastewater, MaterRes., 39, 4512-4520(2005).
Yamamoto, T., Takaki, K., Koyama, T., Furukawa, K.: Long-term stability of partial
nitritation of swine wastewater digester liquor and its subsequent treatment by
Anammox, Bioresouz Technol., 99, 6419-6425(2008).
70
Chapter 5
Study on Long-term Stable Operation of High-rate Fixed-bed
Anammox Reactor Using Different Biomass Carriers at Moderately Low Temperature
5.1 Intreduction
Anaerobic ammonium oxidation (anammox) process was first experimentally
demonstrated and documented, in a denitrifying pilot plant at Gist-Brocades, Delft, the
Netherlands in 1995 (Mulder et al., 1995). The anammox reaction converts ammonium to
dinitrogen gas with nitrite as electron acceptor, catalyzed by the planctomycete-like bacteria
involving hydrazine as an intermediate. These bacteria have a unique prokaryotic organelle
(anammoxosome) surrounded by ladderane lipids, which exclusively contains the hydrazine
oxidoreductase as the major protein to combine nitrite and ammonia in a one-to-one fashion
(Jetten et al., 2005). Compared with the traditional biological nitrogen removal process such
as nitrification-denitrification process, anammox process is now regarded to be a novel,
promising, and cost effective alternative, which has many advantages, e.g., no requirement of
external carbon sources, low oxygen demand, minimized excess sludge production and
reduction in C02 emissions (Op Den Camp et al., 2006; Liu et al., 2008). In recent years,
there has been an enormous increase in research efforts dedicated to the anammox process,
and a few fu11-scale treatment plants using anammox process have been established (Abma et
al., 2006; van der Star et al., 2007). However, the stringent operation conditions and
extremely slow growth rate of the anammox bacteria has restricted the application of
anammox process (Strous et al., 1999).
One of the stringent conditions is the high optimal temperature for the maximum
anammox activity, which was between 30-35 OC, 37 OC and 35-40 OC in different reports
(Yang et al., 2006; Isaka et al., 2008; Dosta et al., 2008). So most of the anammox studies
were normally carried out at temperature high than 30 OC (Strous et al, 1997; Furukawa et al.,
71
2003; Tsushima et al., 2007). However, very recently, several researches focused on the
effects of moderately low temperatures on the stability of this process (Dosta et al., 2008;
Isaka et al., 2008). Their results indicated that the application of anammox process could not
be restricted to effluents with temperatures around 30 OC. Nitrogen removal activity was
observed even at 6 OC, but gradually decreased with decrease in temperature. The apparent
activation energy was calculated as 93 kJ mol-i and 33 kJ mol"i at temperature range from 22
to 28 OC, and from 28 to 37 OC respectively (Isaka et al., 2008). Moreover, at 20-22 OC, Isaka
et al., achieved stable nitrogen conversion rate of 2.3 kg-N m-3 d-i in 320 days, and high
nitrogen conversion rate of 8.1 kg-N m-3 d-i was reported but this activity was maintained on
this value only for 15 days.
Another drawback of anammox process is the requirement of a long start-up period due
to mainly extremely slow growth rates ofanammox bacteria. The doubling time of anammox
bacteria was reported to be approximately 11 days (Strous et al., 1998), and 9 days under
optimal conditions (Strous et al., 1999). Therefore, the immobilization of anammox bacteria
would be attractive for faster start-up and obtaining high nitrogen removal performances
(Isaka et al., 2007). Up to date, the highest nitrogen removal rate reported in the literature
was 26.0 kg-N m-3 d-i, obtained in a high-rate anammox biofilm reactors using the up-flow
fixed-bed biofilm column reactor within 250 days (Tsushima et al., 2007). But this reactor
was operated at high temperature (37 OC), and such so high removal rate was only challenged
once and decreased quickly. Recently, fixed-bed reactors using nonwoven fabric carriers
became attractive for stably attached immobilization of anammox sludge (Funkawa et al.,
2003; Isaka et al., 2007; Tsushima et al., 2007).
There are many industrial wastewaters containing high concentration of NH4-N with
less organic rnaterial, such as wastewaters coming from fertilizer industry, explosive industry
or some pharmaceutical processes (Wiesmann, 1994) and developers used in
photolithography contain tetramethyl ammonium hydroxide (TMAH). Anammox process
has the great potential for the treatment of these industrial wastewaters. In case of TMAH
containing wastewater, TMAH could be completely degraded to NH4-N through
ammonification (Chang et al., 2008). After proper partial nitritation treatment, such as
Single-reactor high-activity ammonium removal over nitrite (Sharon) process, of these
72
wastewaters, partially nitrified effluent is fed to anammox process. This combined PN and
anammox process has an advantage of cost-saving and environnient friendliness over
physico-chemical method, such as catalytic oxidation process for treating this wastewater
containing TMAH (Hirana et al., 2001). The total nitrogen (TN) concentration of wastewater
containing TNAH from some semi-conductor plants is about 150 mg-N 1-i. Therefore, in the
present experiment, influent TN concentration was set to this value, which is lower than that
used in most studies, especially in high rate anammox reactors, such as approximately 330
and 900 mg-N 1-i in the literatures (Isaka et al., 2007; Tsushima et al., 2007).
In this study, a fixed-bed anammox reactor using different configurations of nonwoven
biomass canier and small size biomass carriers was developed and evaluated for pursuiting
the long-term stability of high nitrogen removal performance using low strength wastewater
under moderately low temperature (19-25 OC).
5.2 Materials and Methods
52.1. Anammoxreactorf
The schematic diagram of fixed-bed anammox reactor used
in this experiment was shown in Fig. 5-1. This reactor was made
up of glass and the size is rp 95 mm x 423 mm, with the available
volume of 2.8 1. Influent was supplied to the reactor by up-flow
mode. The reactor temperature was maintained to about 20 OC by
heating through heater coil outside when the temperature was
lower than 20 OC and kept at room temperature in other days. The
reactor was sheltered from light by black vinyl sheet enclosure.
5.2.2. Nowoven biomass carriers and small size biomass carriers
Nvwy
ar4
'
Fig. 5-1. Fixed-bed reactor
Nonwoven polyester coated with a pyridinium-type polymer (Japan Vilene, US patent
5,185,415; 1993) was used as biomass carrier for fixed-bed anammox reactor. The original
73
strips usmg originalsplitted chrysanthemum shape nonwoven
caniers (100 mm diameter, 5 and 2.5 mm
thickness for one trip, 395 mm length, as
shown in Fig. 5-2) were mounted into
fixed-bed anammox reactor sequentially.
Afterwards, in order to further
immobilize more biomass in the gap
between thin strips or nonwoven, 30
size of 1mm length and 50 pm
In the last period of this experiment,
nonwoven biomass caniers and packed in
nonwoven (shown in Fig.5-7). There are
The small size biomass caniers consisted
blast-furnace slag), and hardcore-1 (War
opened). Nonwoven small filaments were
was neutralized by soaking it in running
were from 1
self-made special high active agent,
cement : nonwoven small filaments :
water. This prepared mixed slurry was
chrysanthemum shape nonwoven carrier has 8 strips, which was used in the first period of
experiment. Then, for the purpose of increasing specific surface area for biomass attachment
and nutrients diffusion rate ofnonwoven biomass canier, 8 strips ofnonwoven was split into
16 ' ' razor. The '' and
Fig. 5-•2 Original and split nonwoven biomass
carriers (top view)
g of undoing nonwoven small filaments with a mean
diameter were added in to the reactor.
small size biomass carriers were used instead of
a kind of small carrier bed made of plastic and
11 layers of beds with 3.6cm height in reactor.
Every bed size was 7.0cmÅ~6.3cm and net made of nonwoven srips was 2cmÅ~2cmÅ~3cm.
of nonwoven small filaments, cement (Portland
industry of military department product without
the same as that above mentioned. The cement
tap water for 2 weeks, whose original diameters
to 5mm, was sieved to obtain a mean diameter of 1 mm. The hardcore-1 was a
which was a powerfu1 nanophase material. These above
mentioned small materials (270 g dry weight) were mixed together (the dry weight ratio of
hardcore-1 was 200:60:10) and put into 1.0 L of tap
named as small size biomass carriers, which was
packed homogeneously into a self-made small canier bed made ofplastic after mixing well
with anammox inoculums collected from the total biomass in the previous period. The small
carrier bed consisted of 1 1 units with the same size (tp 94.5 mm x 36 mm). The cylindrical
unit was hollow and of no cover, with a square open (30 mm x 5 mm) near the circle and
74
many small open holes (tp 2 mm) on the floor. These units packed with mixed small size
biomass carriers and seed sludge were mounted into the reactor sequentially with a stagger
arrangement of the square opens on adjacent ones. The unique structure of the small carrier
bed could produce a main horizontal baffling and subsidiary vertical steam of the current in
the reactor, consequentially provide a perfect niche for anammox consortium with sufficient
contact with subtract.
5.2.3. Inoculumandsyntheticinfluent
The seed anammox sludge was taken from a laboratory scale anammox up-flow column
reactor (Funkawa et al., 2003) and seeded with of 500 mg 1-i. In order to improve the
attachment of anammox sludge on the nonwoven biomass carrier, granular anammox sludge
was crushed into small particles using O.5 mm sieve.
After the pretreated crushed anammox sludge WaS Table s-1
filmed evenly onto nonwoven strips, the reactor was Compositionofsyntheticwastewater
purged with nitrogen gas for 20 min. Then, internal
circulation was carried out for 12 h. Through this
procedure, seed anammox sludge was evenly
attached-immobilized on the nonwoven biomass
carrler.
Synthetic influent was prepared by adding NH4-N and N02-N in forms of (NH4)2S04
and NaN02, with other nutrients and buffer according to the composition given in Table 1.
The pH of the synthetic influent was 7Å}O.2 all the time. The reactor was operated under
N02-N limiting condition by feeding equal concentration ofNH4-N and N02-N, both about
75 mg-N 1-i during the whole experimental period, except the lower concentrations with
same ratio in the first 50 days.
5.2.4. Start-up and operational conditions of fixed-bed anammox reactor
This reactor was started-up with the initial TNLR of O.05 kg-N m-3 d-i with a hydraulic
75
Components Concentration
(NH4)2S04 75Å}5(mg-N1-i)
NaN02 75Å}5(mg-N1-i)
EDTA 5.0(mg1"')
FeS04.7H20 9.0(mg1-')
KHC03 125.1(mg1-')
KH2P04 54.4(mg1-')
retention time (HRT) of 26.7 hr. The experiment was divided into five Runs according to the
usage of different biomass carriers.
In Run I (from day O to 206), the original column-like nonwoven biomass carrier was
used. From day 207 to 234, efforts for recovering the activity of anammox bacteria were
devoted with fluctuant results (data not shown). Then in Run ll (day 235 to 365), the reactor
was operated using splitted chrysanthemum shape biomass carrier. Subsequently, some black
color anammox sludge was removed from the reactor and the TNLR was decreased initially
in Run M (day 366 to 404). Afterwards, nonwoven small filaments were added in to the
reactor in Run N (day 405-518). At last, in Run V (day 519-898), small size biomass
carriers packed in the small carrier bed were used instead of nonwoven biomass caniers.
Except Run M, the anammox biomass in the previous Run was all collected and packed into
the reactor in the next Run. The operational conditions during the whole experiment were
shown in Table 5-2.
In the first 50 days of operation, the TNLR was increased stepwise by O.05 to O.1 kg-N
m-3 d-i every step. After day 50, the TNLR was increased only by increasing flow rate (or
reducing HRT), and the protocol for increasing in TNLR was increasing TNLR stepwise by
O.18-O.2 kg-N m-3 d-i each time in case ofthe effluent NOi -N concentration below 20 mg-N
ri most oftime.
Dissolved oxygen (DO) is toxic to anammox consortium, because anammox activity
can exert only under strict anoxic conditions (Jetten, 2001). In Run I, influent DO
concentrations were reduced to O.5-1 mg IMi by flushing nitrogen gas prior to supplying
influent to the reactor. However, some coexisted oxygen consuming microbial community,
such as aerobic nitrifiers and floc forming heterotrophs, could protect the anammox bacteria
from the effect of oxygen (Fujii et. al. 2002; Qiao et al., 2008; Liu et al., 2008 b). Therefore,
after Run I , influent wastewaters with DO concentration of 5-7 mg 1-i were supplied
directly to the anammox reactor for evaluation of effect of DO on anammox treatment
performances.
76
5.2.5 . Analyticalmethods
Table 5-2 Operational conditions during th ewhole experimental period
Parameters
Time range (day)
Biomass carriers in
reactor
HRT (hr)
Inf. NH4-N( mg-N 1"')
Inf. N02-N( mg-N 1-i)
Influent TN( mg-N
1-i)
TNLR ( kg-N m-3 d"i)
Temperature (OC)
infiuent DO (mg IHi)
Run I Run ll Run M Run IV Run V
O-206
CNBca
26.67-2.32
27.7-79.2
26.8-79.0
54.5-158.9
O.O5--1.63
19-25
O.5-1
235-365
Split CNBC
2.22-1.01
71.0-79.1
71.0-81.5
i412-160.6
158-3.72
19-25
5-7
366-404
Split CNBC
1.77-1A2
702-75.0
71.0-76.4
141.6-151.4
1.9S-2.56
19-25
5-7
405-518
Split CNBC& NSFb
4.67-O.56
72.5-76.5
76.3-81.0
150.1-153.1
O.78-6.56
19-25
5-7
519-898
SSBcc din SCB
1.22-O.32
74.9--83.5
73.3-84.2
149.9-162.1
2.98-12.48
19-25
5-7
CNBC: column-like nonwoven biomass carrier b NSF: nonwoven small filaments
C SSBC: small size biomass canier
dscB: small canier bed
The concentrations ofN02-N and N03-N were measured by the colorimetric method in
accordance with the Standard Method (APHA, l995). NH4-N concentration was measured
colorimetrically by the phonate method using ortho-phenylphenol as a substitute for liquid
phenol (Kanda, 1995). DO concentration of the influent was measured by using a digital,
portable DO meter (D-55, Horiba). The concentration of mixed liquor volatile suspended
solids (MLVSS) of sludge samples was determined in accordance with Standard Methods
(APHA, 1995). PH value was determined by using a pH meter (IM-22P, TOA EIectronics).
Biomass attached on small biomass carriers was observed by an electron microscope (Nikon
Eclipse E600, Japan) and a digital camera (Nikon 4500, Japan).
Scanning electron microscopy (SEM) was used for the observation of small carriers
with attached biomass. The procedure was as follows. Samples were first washed in a O.1 M
phosphate buffer solution (pH 7.4) for 5 min, and then hardened for 90 min in a 2.5 O/o
77
glutaraldehyde solution prepared with the buffer solution prepared with the buffer solution.
Next, samples were washed in the buffer solution three times for 1O min once and then fixed
for 90 min in a 1.0 O/o Os04 solution prepared with the buffer solution. After washing
samples three times for 10 min each in the buffer solution, they were dewatered for 10 min
each in serially graded solutions of ethanol at concentrations of 10, 30, 50, 70, 90, and 95 O/o.
SEM observations were conducted using a scanning electron microscope (JEOL, JSM-5310
LV, Japan).
5.3. ResultsandDiscussion
5.3.1 Anammox reactor performances
An operation of the fixed-bed anammox reactor using different configurations of
nonwoven biomass carrier and small size biomass carriers was sequentially canied out for
900 days. During the entire experiment period, the N03-N production rate was from O.1 to
1.2 kg-N m"3 d-i in most of periods (Fig. 5-3, Panel A) which expressed the growth of
anammox bacteria (van de Graaf et al., 1996).
The TN removal performances in terms of nitrogen removal during the whole
experimental period is depicted in Fig. 5-3 (Panels A and B). Fig. 5-4 shows the NH4-N and
N02-N removal performances in Panel A and Panel B, respectively.
In Run I , the TNLR of the fixed-bed anammox reactor increased from O.05 to 1.63
kg-N m-3 d'i gradually. The TNRR increased synchronously from O.04 to 1.3 kg-N m-3 d-i
with the TN removal efficiency ranged from 63 O/o to 85 O/o. The effluent TN concentrations
ranged from 20 to 50 mg-N 1-i with fluctuations resulting from the intermittent increase of
TNLR. Effluent NH4-N and N02-N concentrations were below 25 and 19 mg-N 1"i
respectively, also with fluctuations. The establishment of fixed-bed anammox reactor using
low strength influent nitrogen was proved possible under moderately low temperature (19-25
Oc) from this result.
However, clogging and channeling phenomena of the anammox biofilm occurred after
200 days of long term operation.The black color of anammox biofilm was observed in deep
78
part of nonwoven biomass canier, but the outside anammox biofilm was still in red color, as
shown in Fig. 5-6 (day 145) and Fig.5-7 (day 200). The black anammox biofilm was
supposed to be cased by the insufficient substrate supply under high sludge concentration.
Therefore, from day 207 to 234, some efforts were devoted to recover the activity of
anammox sludge, such as removing the black sludge from the reactor, increasing to 350C
IS
"Ai
cV.ie
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vee
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o
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g-pt
=
I,'-sy g•"'''i"SIBIIkl,l 11
l
","l R v i
:, za i j i i i i i
\ aj
t 'atl
iilii
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' i
}ll}
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i
eti
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{
lsg
lao
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l{X]
3ij
6t"j
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2• .O
o
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urrSt
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-;
zer
u'-o.
e-
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1. s•o lo•as !iio :•.e{} 2fi,so 3ivg 3io gen 4ie iee ssas 6ijo 6sij 7,pt] 7,-sio ggo gsff g{)o
Time (days)
Fig.5-3. TN removal performance of the reactors. Panel A: (-) TNLR; (ua) TNRR; (A) N03-N production rate. Panel B: (Å~) infiuent TN; ( .th ) TN removal efficiency; (D) effluent TN; (-) HRT.
suddenly and returning to 20 OC after 12 hr, inducing internal recycle for one day, changing
influent concentrations, etc. The combination of these methods retumed the black part of the
biofilm to red color for certain period.
i9
In Run ll , by switching the original chrysanthemum shape nonwoven biomass to split
one, the TNRR reached 2.51 kg-N m'3 d-i with a short HRT of 1.01 hr. The effluent TN
concentrations ranged from 22 to 40 mg 1-i before day 340, but even increased to 80 mg-N 1-i
on day 350 with a low TN removal efficiency of only 50 O/o. A few fioating anammox
granules, as shown in Fig. 5-7 (day 350), were washed out from the reactor and the biofilm
inside the nonwoven biomass carrier turned to black as same as in Run 1 (Fig. 5-7, day 365).
Ieo 1ias
p gn,
vrp ?,8zela 'pt
MUsEg aj'E
a3:g L,,o 1
o
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s / g if
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l -.Aili,, .•. il.f' Etl{;$illljet '• l,.'l•
l!ll •iiii [i /II llIl/ .,II[ Iii
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9
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81:g :,O 1 o
e ie 1fio sg :,ttse :•so 3'tza sso 4/uo 4se s.go ss" "off 6su ?tva 7,s.o gew gss gf?oTime (days)
9{}
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Time (days)Fig.5-4. NH4-N and N02-N removal performance of the reactors. Panel A: (A) NH4-N removalefficiency; (me) influent NH4-N; (D) effluent NH4-N; ( >< ) NH4-N consumption rate. Panel B: ( de..pt"• ) N02-N
removal efficiency; (A) influent N02-N; (O) effluent N02-N; (me/) N02-N consumption rate.
The reason for this deteriorative reactor performances were supposed to be the clogging
and channeling of the biofilm under short HRT. From day 250, the influent was supplied
directly to the reactor without nitrogen gas flushing. However, the TN removal performance
80
was stable before day 340, which elucidated that the biomass in this reactor could adapt the
abrupt increase in infiuent DO in a short time. Compared with the report by Liu et al., (Liu et
al., 2008) with requirement of step-wise increase in influent DO concentration the quick
adaptation of anammox biomass to DO observed in this experiment was possibly attributed
to the biodiversity of anammox bacteria in our system.
For recovery ofthe anammox activity, in Run M, 6.5 g (dry weight) ofblack anammox
biomass was removed from the reactor on day 370. The TNLR was reduced first then
increased gradually to 2.56 kg-N m-3 d-i. The TN removal efficiency increased to 80 O/o with
the NH4-N and N02-N concentrations in the effluent lower than 15 and 10 mg-N 1-i
respectively on day 404. Just on this day, reactor temperature was increased accidentally to
500C owing to failure in temperature control at 200C in winter season. The color of outside
anammox sludge turned to white color and lost most of its anammox activity at one night.
This died anammox sludge was removed from the reactor and restarted experiment under
low TNLR in Run IV.
In Run IV, the nonwoven small filaments (30 g) were added in to the reactor for
increasing attached-immobilized anammox sludge. The quick recovery of anammox activity
was indicated by the quick growth of anammox biofilm on nonwoven small filaments which
was almost fu11 of the reactor and the change in color from white to distinct red color (Fig.
5-6, day 430). The TNLR was
increased from O.78 to 6.56 kg-N
m-3 dmi during 113 days of
operation with a synchronously
increase of TNRR from O.46 to
4.48 kg-N m-3 d-i. The shortest
HRT in this period was O.56 hr.
No clogging and channeling of
biofilm occurred in this Run.
Compared with the previous
Runs, the high TNLR (or the low
rA-V'E
4yx5'
Ng
-o
g9-g
.tt-
E:go
21
IO
9
.g
7
6
5.
4
3
2
1
g
i{lx'
}; =2.05 •x
tsR-= g.99g
k g ..- --{' xz cb
.' ` . ..'
t
A geEi
v= 1.2 9x '1 RL = O.9S •4 b. ..Sts .+, .f:itt-g•'.•"•pt.
t' re "' '
y = oi .L) rt sx- .:
RJ- = O.963
a
"
mv
g.ij O.5 t.ij l.5 2,g L7.5. Ll.fi 3.5 4.0 4.5 tr,.e 5..i NH4-N consumption rate ( kg-N m3 d-i)
Fig. 5-5. Ratios of TNRR, N02-N consumption and N03-N
production rate to NH4-N consumption rate. (A) TNRR; (])
N02-N consumption rate; (-) N03-N production rate.
81
HRT) prevented substrate transport limitation in the biofilms throughout the reactor
(Nicolella et al., 2005). However, all effluent nitrogen concentrations were fluctuanting (Fig
5-3 and Fig. 5-4), which suggested a better operation strategy should be considered in the
next period. The measured MLSS concentration was reached to 18,890 mg 1"i on day 518. '
' '
In Run V, small size biomass carriers packed in the small carrier bed were applied in
the reactor. All ofbiomass at the end of Run IV was used as seed of this Run. The reactor
was operated by a unique operational strategy of increasing the TNLR according to a
mathematic simulating model. The TNLR was exponentially increased from 2.98 to 11.5
kg-N m-3 d'i within 180 days ofoperation, and the TNRR successfu11y increased from O.46 to
8.3 kg-N m-3 d-i during this period. The HRT was shortened to O.32 hr on day 700 and
maintained this short HRT in the following operational period. TN removal efficiencies were
kept about 75 O/o with effluent TN concentrations ranging from 36 to 50 mg-N 1"i. Effluent
NH4-N and N02-N concentrations were below 25 and 10 mg 1"i, respectively. Compared
with former experimental results, effluent nitrogen concentrations and TN removal
efficiencies were surprisingly stable, which demonstrated the favorable application of
mathematic simulating model for increasing strategy in TNLR and the well anammox
performance deriving from the application of small size biomass carriers. From day 700 to
898, the operational condition was not changed, and stable TN removal performances were
achieved for 198 days. In this stable operational period, the TNRRs were kept stable at 8.2 to
9.93 kg-N m"3 d-i under the TNLR from 11.3 to 12.48 kg-N m-3 dHi. Effluent TN
concentrations were around 36 mg 1"i and effluent N02-N concentrations were always
below 1O mg 1-i with an average of3 mg 1-i.
The reaction ratio of TNRR, N02-N consumption and N03-N production rates to
NH4-N consumption rate during the whole experiment was 2.05:1.29:O.23 (Fig. 5-5), which
was slightly lower value compared to the well accepted anammox reaction ratio of
2.06:1.32:O.26 (Strous et al., 1998). However, the ratio of N02-N to NH4-N consumption
rates was 1.29 at TN concentration of 150 mg 1-i in this study, which was very similar to 1.3
at the TN concentration of 140 mg 1-i (Strous et al., 1999). The close reaction ratio with
82
reliable correlation elucidated the good anammox activity under moderately low
temperature.
5.3.2. 0bservationoftheattachedbiomass
Fig. 5-6. External appearance ofthe anammox reactor during the whole experiment
Generally, anammox biomass is apt to change its color (brownish red, red, or dark red
colors) depending on the cultural conditions because of the unique cytochrome content in
Ifi ,.ge.x . t. .. +.ft't}.etlv " .+ [ .. .-I"ts ;-- "/' :"' th ' ' ge "•k•:ilt- "T/
day 350 day 365
;
and 5-7 showed the changes in
color and shape during the whole
reactor from day 404, the
that for chrysanthemum shape
nonwoven filaments in increasing
of anammox biomass using small
83
day 200 day 498 Fig.5-7. Appearance ofthe anammox biofilm token from the reactor
anammox bacteria (van De Graafet al., 1996 Ahn et al., 2004).
Figures 5-6
anammox biomass
experiment. After adding small nonwoven filaments to
the fixed bed anammox samount of attached immobilized anammox biomass was :' emuch larger than
nonwoven strips. This verified the high sludge retammg
ability of small
biomass. The color
-
x
,A
ge:-l-T
B
day 898
7.ecm "K/ sc
s- 6.3cni
2cmx2cmx3cm
c'
Fig. 5-8. Size of small canier bed
size biomass carriers was changed fromgrey black (cement color) to red (Fig. 5-6, day 523,
575, 898), indicating the novel high sludge retaining characteristics of our invented small
size biomass carriers. Comparing the colors and surface structure of anammox biomass using
small size nonwoven filaments and small size caniers (Fig. 5-7, day 498 and 898), the
former was brownish red and loose structure, and the latter was much redder, denser and
homogeneous.
ewtm..
s
,ec
Fig.5-9. SEM images of small size biomass caniers. (A) Hydrated small cement carriers without
surface modification by hardcore-1; (B) hydrated small cement caniers after surface modification
by hardcore-1.
The clogging and channeling of biofilm occurred in the deeper part of nonwoven
biomass carrier and the inner part of big size anammox granules after fu11y developed
anammox reactor. These phenomena caused the poor substrate diffusion and finally resulted
in the decrease of anammox activities. If these poor cultivating conditions were kept for long
time, the color of anammox biomass changed to black (Fig. 5-7, day 200 and 350). It was
confirmed that black color of anammox biomass could return to red color by applying proper
cure for recovering deteriorated anammox activities.
5.3.3. Small size biomass carriers for enhancing anammox performances
The homogeneously spatial distributions of biomass in the anammox reactor using
small size biomass caniers was regarded to originate from the initial well mixing of seed
sludge with small size caniers and the continuous disturbance of reactor content by micro
84
bubbles of nitrogen gas from anammox reaction.
The hardcore-1 was a significant component in the small size carriers. After the surface
modification of the small size hydrated cement by the enwrapping of this nanophase material,
the smooth surfaces and passivity edge angles. (Fig. 5-9, A and B). The spatially wrinkled
structure with many needle fibers and micro channels produced after surface modification by
hardcore-1 was beneficial to the bacterial attached immobilization and substrate diffusion,
which consequently resulted in the high anammox activity. By this surface modification, the
whole of small size biomass caniers presented a homogenous dispersed status rather than
irregular aggregations without surface modification. Moreover, the surrounding ofnanophase
material on the small mixture may prevent the exudation of some heavy metal ions which
were supposed to be toxic to anammox bacteria. No clogging and channeling phenomena
were observed during the whole operational period for fixed-bed anammox reactor using
small size carriers. The nonwoven filaments performed the main function of connecting
these inorganic carriers and transfening the substrates due to its hydrophilicity. Because of
the high density of small size biomass carriers, anammox biomass was attached tightly to
small size biomass caniers, washout of anammox sludge from the reactor could reduce to a
great extent.
5.3.4. Simulation model for increasing TNLR using small size biomass carrier
In Run V, simulation model was used for increasing the TNLR. This model was
derived from two-dimensional nonlinear regression analysis using initial experimental results.
In this simulation model, time and TNLR are independent variables and TNRR was
independent variable. The predicted TNLR could be obtained by this simulation model, and
then this model was corrected by the actual experimental results. The corrected model was
used for further prediction. Based on the actual results at the end of this Run, the final
regression equation was defined as shown in Fig. 5-10. Fig. 5-10 demonstrated that the
TNLR to TNIUI presents a linear relationship, while TNLR and TNRR present exponential
relationship with time. By increasing TNLR based on this simulation model, the reactor
could reach a stable operational condition under which high anammox activities could be
85
realized. The further evaluation of this simulation model and the elucidation
relationship between this model and the growth rate of anammox bacteria are still
mvestlgatlon.
of the
under
Fig.5-ny10. Mathematic simulating model obtained in the end of the experiment. X axis: time
(days); Y axis: TNLR ( kg-N m'3 d-]); Z axis: TNRR ( kg-N m-3 d-').
The formula ofTNRR is Z==Z(X, Y) produced by software of 1stOpt 1.0. Whole formula
is shown as following.
z=exp((1+pl"log(x+3)"O.5+p2"log(y+3)"O.5+p3*log(x+3)+p4"log(y+3)+p5"log(x+3)"1.5+
p6*log(y+3)"1.5+p7*log(x+3)A2+p8'log(y+3)"2+p9"log(x+3)"2.5+plO*log(y+3)"2.5+pll
"log(x+3)"3+p12*log(y+3)"3+p13*log(x+3)AO.5"log(y+3)"O.5+p14"log(x+3)"O.5"log(y+3)
+p15"log(x+3)"O.5"log(y+3)"1.5+p16"log(x+3)*log(y+3)"O.5+p17*log(x+3)"log(y+3)+pl
8"log(x+3)"log(y+3)Al.5+p19"log(x+3)"1.5"log(y+3)"O.5+p20*log(x+3)"1.5"log(y+3)+p2
1"log(x+3)"1.5"log(y+3)"1.5)!(p22"log(x+3)AO.5+p23"log(y+3)"O.5+p24"log(x+3)+p25"lo
g(y+3)+p26"log(x+3)Al.5+p27"log(y+3)"1.5+p28"log(x+3)"2+p29"log(y+3)"2+p30"log(x
+3)"2.5+p31"log(y+3)"2.5+p32"log(x+3)"3+p33*log(y+3)A3+p34"log(x+3)"O.5"log(y+3)"
O.5+p35*log(x+3)"O.5*log(y+3)+p36"log(x+3)AO.5*log(y+3)"1.5+p37"log(x+3)"log(y+3)"
86
O.5+p38*log(x+3)"log(y+3)+p39"log(x+3)"log(y+3)"1.5+p40"log(x+3)"1.5*log(y+3)"O.5+
p41*log(x+3)"1.5"log(y+3)+p42"log(x+3)Al.5*log(y+3)"1.5+p43))-3;
There are 43 parameters. p(43,1)= [-541.17 -308.15 -12.85 639.3 44.00 546.23 22.79
141.88 4.88 -100.10 -1.1615.07 -16.74 147.5281.30-6.90-5.01--3123-20.35
-37.07 -41.06 35.95 86.26 -18.17 12.68 -13.84 44.22 -5.11 73.55 -O.93 51.17 O.42 -30.64
7.59 -3.30 -O.91 -O.50 -3.30 -9.40 1.04 -1.23 -7.91 390.19].
5.3.5. Comparisonofanammoxtreatmentcapabilities
In this study, four kinds of biomass carrier were applied sequentially, i.e., original
chrysanthemum shape with aid of simulating model for increasing TNLR. This result
demonstrate that the small size biomass caniers highest TNRR and the most stable
performance was obtained in Run V applying small size biomass carriers nonwoven
biomass carrier, split chrysanthemum shape nonwoven biomass carrier, splitted
chrysanthemum shape nonwoven biomass canier with undoing nonwoven small filaments ,
' Table 5-3 Comparison of different Anammox reactors under different operational conditions
Process Reactor Temperature TNRR.,, Stabletime (TNRRa) HRT.i. InfluentDO Reference(Oc) (kg-N m"3 d'i) (days) (kg-N m'3 d") (hr) (mg l-')
Anammox Fixed-bedreactor 37(2007)
Anammox ABF 20-22
AnammoxC RBCd 17Anammox Fixed-bedreactor" 18-26
26.0
10.1
O.5
9.93
1
103
15
320
200
(26)
(5•8m14)
(8.1)
(2.3)
(8.2-9.93)
O.24
O.32 hr
<O.5 Tsushima et al.
Se.5 Isaka et al. (2007)b
Cema et aL, (2006)
5-7(most) Thisstudy
aThis value referred to the average TNRR or TNRR scale at the stable operational time. bThe results showed in this study was nitrogen conversion rate calculated from the sum of
the NH1 -N and NOI -N removal rates, which should be lower than the TNRR due to discarding of the NOi -N production rate. CThis anammox process was accompanied with autotrophic nitrification and heterotrophic denitrification. d Rotating biological contactor. eFixed-bed anammox reactor using small size biomass carriers packed in small carrier bed.
and small size biomass carriers. The low-strength wastewater under moderately low
temperature have the advantage over nonwoven biomass carrier in the fixed-bed anammox
reactor treating the relatively Table 3 shows the comparison of our obtained experimental
87
results with other results obtained from various kinds of anammox reactors operated under
different conditions. In spite of our reactor's operation under unfavorable operational
conditions, i.e., moderately low substrate concentrations and operational temperature; report
on successfu1 operation of fixed bed anammox reactor with high TNRR under moderately
low temperature. carriers showes higher TNRRs as shown in Table 3. Especially, after
increasing TNLR based on simulation model, high-rate anammox treatment of TNRRs
ranged from 8.2 to 9.93 kg-N m'3 d'i was lasted stably for 200 days of operation. This result
will provide basic understanding for development of stable high-rate anammox treatment
using fixed-bed anammox reactor under moderately low temperature. To our knowledge, this
paper is the first high influent DO concentration and short HRT, novel fixed-bed anammox
reactor using small size biomass
5.4. Conclusions
During an experimental period for 900 days, we have succeeded in the establishment of
fixed-bed anammox reactor treating low strength influent wastewater of 150 mg-N 1-i under
moderately Iow temperature of 19-25 OC. Using nonwoven biomass canier, the TNRR
reached to 4.48 kg-N mr3 dHi with fluctuant TN removal efficienceies due to intermittent
increase of TNLR. By the application of small size biomass carriers, the TNRR could
increase to 8.3 kg-N m-3 d-i in 180 days of operation with a HRT of O.32 hr, and maintained
from 8.2 to 9.93 kg-N m-3 d-i with stable TN removal efficiency in the following 198 days of
operation. The results of this study on the stable high-rate anammox process treating
relatively low strength wastewater under moderately low temperature would be significant in
practical industrial application of anammox process.
5.5 References
Abma, W.R., Schultz, C.E., Mulder, J.W., Loosdrecht, M.C.M., Star, W.R.L., Strous, M.,
Tokutomi, T.: Full-scale granular sludge anammox process, In: Proceedings of the
International Conference on Biofilm Systems VI, Amsterdam, The Netherlands,
88
171-178(2006).
Ahn, Y.H., Hwang, I.S., Min, KS.: Anammox and partial denitritation in anaerobic nitrogen
removal from piggery waste, ifater Sci. Technol., 49, 145-153(2004)
APHA, Standard Methods for the Examination of Water and Wastewater, American Public
Health Association, Baltimore, MD. (1995).
Cema, G, Wiszniowski, J., Zabczyfiski, S., Zablocka-Godlewska, E., Raszka, A., and
Surmacz-G6rska, J.: Biological nitrogen removal from landfi11 leachate by
deammonification assisted heterotrophic denitrification in a rotating biological
contactor, Mater Sci. Technol. , 55, 35-42(2007).
Chang, K.-F., Yang, S.-Y., You, H.-S., Pan, J.R.: Anaerobic Treatment of Tetra-Methyl
Ammonium Hydroxide (TMAH) Containing Wastewater. Semiconductor
Manufacturing, IEEE Trans, Semicond. Manuf, 21 (3), 486-491(2008).
Dosta, J., Fernandez, I., Vazquez-Padin, J.R., Mosquera-Corral, A., Campos, J.L.,
Mata-Alvarez, J., Mendez, R.: Short- and long-term effects of temperature on the
Anammox process. , J. Hazard. Mater., 154 (1-3), 688-693(2008).
Fujii, T., Sugino, H., Rouse, J.D., Furukawa, K.: Characterization of the Microbial
Community in an Anaerobic Ammonium-Oxidizing Biofilm Cultured on a Nonwoven
Biomass Carrier, J. Biosci. Bioeng., 94 (5), 412-418(2002).
Furukawa, K., Rouse, J.D., Yoshida, N., Hatanaka, H.: Mass cultivation of anaerobic
ammonium-oxidizing sludge using a novel nonwoven biomass canier, J. Chem. Eng.
Jpn., 36, 1163-1169(2003).
Hirano, K., Okamura, J., Taira, T., Sano, K., Toyoda, A., Ikeda, M.: An efficient treatment
technique for TMAH wastewater by catalytic oxidation. Semiconductor
Manufacturing, IEEE Trans, Semicond. Manuf, 14 (3), 202-206(2001).
Isaka, K., Date, Y., Kimura, Y., Sumino, T. & Tsuneda, S.: Nitrogen removal performance
using anaerobic ammonium oxidation at low temperatures, FEMS Microbiol. Lett., 282
(1), 32-38(2008).
Isaka, K., Y., Sumino, and Tsuneda, S.: High nitrogen removal performance at moderately
low temperature utilizing anaerobic ammonium oxidation reactions, J. Biosci.
Bioeng. ,103 (5), 486-490(2007).
89
Jetten, M.S.M.: New pathways for ammonia conversion in soil and aquatic systems, Plant
Soil, 230, 9-19(2001).
Jetten, M.S.M., Cirpus, I, Kartal, B., van Niftrik, L., van de Pas-Schoonen, KT., Sliekers, O.,
Haaijer, S., van der Star, W., Schmid, M., van de Vossenberg, J,, Schmidt, I., Harhangi,
H., van Loosdrecht, M., Kuenen, J.G., Op den,Camp, H., Strous, M.: 1994-2004: 10
years ofresearch on the anaerobic oxidation ofammonium, Biochem.. Soc. Trans., 33,
119-123(2005).
Kanda, J.: Determination of ammonium in seawater based on the indophenol reaction with
o-phenylphenol (OPP), MaterRes. , 29, 2746-2750(1995).
Liu, S., Yang, F., Xue,Y., Gong Z., Chen H., Wang, T., Su, Z.: Evaluation of oxygen
adaptation and identification of functional bacteria composition for anammox
consortium in non-woven biological rotating contactor, Bioresour. Technol., 99,
8273-8279(2008).
Mulder, A., van de Graaf, A.A., Robertson, L.A., and Kuenen, J.G.: Anaerobic ammonium
oxidation discovered in a denitrifying fluidized bed reactor, FEMS Microbiol. Ecol.,
16, 177-183(1995).
Nicolella, C., van Loosdrecht, M.C.M., Heijnen, S.J.: Paricle-based biofilm reactor
technology, Trends BiotechnoL, 18, 3 12-320(2000).
Op Den Camp, H.J.M., Kartal, B., Guven, D., van Niftrik, L.A.M.P., Haaijer, S.C.M., Van
Der Star, W.R.L., Van De PasSchoonen, K.T., Cabezas, A., Ying, Z., Schmid, M.C.,
Kuypers, M.M.M., Van De Vossenberg, J., Harhangi, H.R., Picioreanu, C., Van
Loosdrecht, M.C.M., Kuenen, J.G., Strous, M., Jetten, M.S.M.: Global impact and
application of the anaerobic ammonium-oxidizing (anammox) bacteria, Biochem. Soc.
Trans., 34 (1), 174-178(2006).
Qiao, S., Kawakubo, Y., Cheng, Y., Nishiyama, T., Fujii, T., Furukawa, K.: Identification of
bacteria coexisting with anammox bacteria in an upflow column type reactor,
Biodegradation (in press) (2008).
Strous, M., Heijnen, J.J., Kuenen, J.G., Jetten, M.S.M.: The sequencing batch reactor as a
powerfu1 tool for the study of slowly growing anaerobic ammonium-oxidizing
microorganisms, Appl. Microbiol. Biotechnol.. 50 (5), 589-596 (1998).
90
Strous, M., Kuenen, J.G., Jetten, M.S.M.: Key physiology of anaerobic ammonium oxidation,
Appl. Environ. Microbiol., 65 (7), 3248-3250(1999).
Strous, M., van Gerven, E., Zheng, P,, Kuenen, J.G., Jetten, M.S.M.: Ammonium removal
from concentrated waste streams with the anaerobic ammonium oxidation (Anammox)
process in different reactor configurations, MaterRes., 31 (8), 1955-1962(1997).
Tsushima, I., Ogasawara, Y., Kindaichi, T., Satoh, H., Okabe, S.: Development of high-rate
anaerobic ammonium-oxidizing (anammox) biofilm reactors, Mater Res., 41,
1623-1634(2007).
van de Graaf, A.A., de Bruijn, P., Robertson, L.A., Jetten, M.S.M., Kuenen, J.G.:
Autotrophic growth of anaerobic amnionium-oxidizing microorganisms in a fluidized
bed reactor, Microbiology, 142 (8), 2187-2196(1996).
van der Star, W.R.L., Abma, W.R., Blommers, D., Mulder, J.-W., Tokutomi, T., Strous, M.,
Picioreanu, C., van Loosdrecht, M.C.M.: Startup of reactors for anoxic ammonium
oxidation: Experiences from the first fu11-scale anammox reactor in Rotterdam, Mater
res., 41 (18), 4149-4163(2007).
Wiesmann, U.: Biological nitrogen removal from wastewater, Adv. Biochem. Eng. Biotech.,
51, 113-154(1994).
Yang, Y., Zuo, J.-E, Shen, P., Gu, X.-S.: Influence of temperature, pH value and organic
substance on activity of ANAMMOX sludge, Huan Jing Ke Xue/Environmental Science , 27 (4), 691-695 (in Chinese). (2006).
91
92
Chapter 6
Conclusions and recommendations
6.1 Conclusions
There are several key points for favorable cultural conditions of anammox process.
They are the characteristics of infiuent wastewater (organic and inorganic carbon, salinity,
ammonium, DO concentrations), operational conditions (temperature, pH), suitable biomass
caniers and reactor types. The researchers who are engaged in anammox process have
already obtained extremely high nitrogen removal rates, but the long term and stable
maintenance of these high nitrogen removal rates were not reported. On the other hand, few
research works are canied out for anammox treatment under unfavorable operational
conditions.
Our experimental studies focused on the nitrogen removal by anammox process under
unfavorable operational conditions and evaluated their anammox treatment capabilities
experimentally.
1) Through the long-term stable operation of high-rate anammox biofilm reactor using
nonwoven biomass canier under moderately low temperatures and moderately low strength
ammonium-containing wastewater, we obtained the following results.
(a). The reactor system could operate for about three years without much disturbing the
treatment system.
(b) We have developed the novel small size biomass carriers composed of nonwoven fiber,
cement and hardcore-1. By using this small size biomass carrier technology, high stable
anammox activities were kept in the fixed bed anammox reactor.
(c) The maximum total nitrogen loading rate (TNLR) of 12.5 kg-N m-3 d'i and the maximum
total nitrogen removal rate (TNRR) of9.93 kg-N m-3 d-i with TNR efficiency of 80 O/o were
obtained for anammox reactor using small size biomass caniers. This high anammox activity
was kept for long time showing the stable sludge retaining capability of small size biomass
'carrlers.
93
(d) The hardcore-1 was a very important material in this small size biomass technology.
When the hydrates particles of cement were remodeled by hardcore-1, anammox sludge
could attach tightly on the layers of anaphases surface wrinkled coarse structure with many
needle fibers and micro channels. The high-rate nitrogen removal rates were realized using
application of hardcore- 1 .
(e) The TNRR could be controlled by the empirical mathematic model for increasing TNLR
according to partial derivative.
2) The effect of salt concentration on anammox treatment was experimentally evaluated
using fixed-bed anammox reactor with non-woven biomass canier. Anammox sludge
dominating freshwater anammox bacteria, strain KU2 and KSU-1, was used as seed sludge
of anammox reactor. Influent salt concentrations were increased stepwise from 2.5 g 1-i to
33 g IHi. Bacterial community was also examined by 16S rRNA gene analysis after the
acclimation of the anammox sludge to high salt condition. Based on these results, it was
revealed that freshwater anammox sludge could not be applied to the anammox treatment at
salt concentrations higher than 30 g 1-i. However, it was demonstrated that freshwater
anammox sludge could acclimate to salts concentration comparable to sea water level of30 g
1-1.
3) Nitrogen removal capabilities of two kinds of fixed-bed anammox reactors (one was using
nonwoven biomass carrier and another was small size biomass carriers) for treating partially
nitrified brine wastewater were compared.
(a) At the first stage of experiment, the fixed-bed reactor using nonwoven was fed with
partially nitrified brine wastewater and operated with a quick increase in TNLR, from O.8 to
2.12 kg-N m-3 d-i, only in 58 days. The TNRR increased synchronously from O.7 to 1.81
kg-N m-3 d-i due to the high TN removal efficiencies ranged from 83 O/o to 88 O/o. These
'results showed that fresh anammox sludge acclimated to NaCl concentration comparable to
'sea water level (30 g 1-i) could adapt quickly to the panial nitrified brine wastewater whose
salts concentration was comparable to sea water level. The color of freshwater anammox
sludge in fixed-bed anammox reactor using nonwoven changed to black color after anammox
treatment ofpanially nitrified brine wastewater.
(b) At the second stage experiment, fixed-bed anammox reactor using nonwoven biomass
94
carrier was changed to the fixed-bed anammox reactor using small size biomass carriers,
Maximum TNLR was increased to 3.17 kg-N m-3 d-i and the maximum TNRR reached to
'2.52 kg-N m-3 d-i, which was higher than that obtained in first stage experiments. The
average N02-N removal efficiency was about 88 O/o and the effluent N02-N concentrations
were kept below 11 mg 1ffi. The color of anammox biomass changed from black to weak red
color.
6.2 Recommendations
1) Anammox reactor using small size biomass canier technology has been proved to be an
efficient anammox reactor without clogging and channeling phenomena, which are the
disadvantages of fixed bed anammox reactor using nonwoven biomass carrier. Development
of suitable operational procedure for this novel anammox reactor is required for proper
application of this process to fu11 scale plant.
2) The preparation of nanophase materials of hardcore-1 must be improved in the future
study. It is very important to make hardcore-1 reacting with cement and others minerals
effectively. The favorable chemical bonds and physical connection between hardcore-1 and
cement must be investigated.
4) The development of novel economical nitrogen removal process such as our invented
fixed bed anammox reactor using small size biomass carriers is becoming increasingly
important as more stringent effluent discharge requirements are imposed. In order to answer
these requests, application test of newly developed nitrogen removal process must be canied
out in the fu11 scale plant.
95
Appendix: Publications related to this dissertation
t"inS(
kt Z Dissertation name: rStudies on fixedbed anammox process treating low strength ammonium
containing wastewater with high salinityj
(iE'fiifi'LpNE JSl[k7eeE.,\. 7' )/ ie= 7kJfltik7tc (1) pm J'k-Erttk Ji' }' )>t gi ))2 t7 - 70u -l >< ecFee7v3-6iilFts'Å}i]U)
1 mu
7AYiikl O JijS21 • H{}Jce
ag 2k
1 . Anammox treatment of low-strength ammonium-containing wastewater at room temperature
(5YM v(fk Anammox Er 7iE fB L vc 7' >i e =7JtkgeiftaJtses7j< rb) 6 a)gltS SA3< i21 k PeeH -9' 6 itst)
INTERNATIONAL SYMPOSIUM ON ENVIRONMENTAL SCIENCE AND TECHNOLOGY.
SECTION SIX: Water Pollution and Water Quality Control (653). Beijing, China, November
13-16, 2007. (International conference proceedings). pp.653-659 lff.
SIZ JSZ 19 qi 1 1 fi 1 3 -- 1 6 H rp MdkSl, M
;Ji"-l lg2i: gfJJi"t Elt. thJi(,ta-coVE?
2 . Anammox application to low strength ammonium containing wastewater (Anammox ErVaM Lv(
7 )/ =e J : 7- Jbtilg$kJfi JAt 7S< rb > C> Og$ ve ik eC Fes -g- 6 tfi Etf)
SIZJ5L 1s cEliRÅ}7k"tri L"kilijizzisZgKfiJ}5'I" IEEIIk/Akfi,ii;E,t S:;R CO-ROM. pp.875-876
iPJN 19 4G}li (:Ndi
ZJ2iZ: giJJISZR. diJliXd.,VA
eg 3 if
Effect of salt cncentration in aammox treatment using non-woven bomass crrier (tfi7ee Jxil? rb }" Z< $&
Jfii igreTlsc 2 Ltc Anammox aJLLmpCIJftX"g4Sl""-.,ts'xOimEt)
Journal ofBioscience and Bioengineering, Vol. 107, No. 5 (2009)
(Printed)
Zg21: vas,idJillZR. illlsJJA<-. iiiEiillwa. ecX:Z;Eft. 'Ei-JllR..tep
96
eg 4e
1 . Nitrogen removal capabilities of two kinds of fixed-bed anammox reactors for treating
partial nitrified brine wastewater. ( 2 Tptlfi. (D Anammox maErttK V 7 P l9 Er 7SM Lv( . gK'/z) t-lffk'
mauTL 8 nkza rb>ink vxog$ ua i21E2Obt ptk, )
Japanese journal of water treatment biology( H JzN 7LI< iSQ{lt ]Egl l lttig ':iil` t21 E','us.)
(Submitted)
gg Z : pa,, ti JilSt Ell . Jli] fi ij HJ] . tk HftR. -iliJIl pt.. , i?fi
eg s il3lr
1. Study on long-term stable operation of high-rate anammox biofilm reactor using nonwoven
carrier at moderately low temperature. (i[k?MExl\v(fs7i<filtli Erti;Eo fe Anammox gltbuEes V 7P 6i
Er l]2 .lgl e9 fo >- ) i-(t re'-t=' L k 6([Lt mp e c Pce7 -94 6 JIfi st)
Japanese journal ofwater treatment biology( EI JzN7Ll<pmÅ}llEEIt ltkif,ii!:`kE',tulrN.)
(Submitted)
llli :Z;L Z , M,, il JSL I:l . mu fi il HJ] . tk HjiiER. 'iSJII fiu VA
97
98