COMPARATIVE STUDY ON DIFFERENT
ANAMMOX SYSTEMS
Grzegorz Cema
October 2009
TRITA-LWR PhD Thesis 1053
ISSN 1650-8602
ISRN KTH/LWR/PHD 1053-SE
ISBN 978-91-7415-501-3
Grzegorz Cema TRITA LWR PhD Thesis 1053
ii
Comparative study on different Anammox systems
Dedicated to my parents Małgorzata and Paweł
Harvard Law:
Under the most rigorously controlled conditions of pressure, temperature, humidity, and other
variables, the organism will do as it damn well pleases
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Grzegorz Cema TRITA LWR PhD Thesis 1053
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Comparative study on different Anammox systems
ACKNOWLEDGEMENTS
This thesis would not be possible without my colleagues and the support of my friends and fam-
ily.
I would like to thank to my supervisor Prof. Elżbieta Płaza for giving me the opportunity to join
a special research team and to participate in the deammonification project. I appreciate your
support, constructive advices and suggestions. Thank you for many  brain storm discussions.
I would like to express my gratitude to my second supervisor Prof. Joanna Surmacz-Górska, for
guiding me through the process of becoming a researcher. I would like to thank for the right
amount of freedom, supporting me in my work and for finding her office door always open for
any questions I had. Thank you, for many fruitful and stimulating discussions.
I acknowledge Dr Józef Trela for the leadership of deammonification project and devoting lots
of his time on discussion about the Anammox tests. I would like to sincerely thank to Prof.
Bengt Hultmant for his guidance and support. I respect your huge knowledge and never ending
ideas.
Beata Szatkowska and Luiza Gut were two other Ph.D. students involved in the deammonifica-
tion project and sharing with me experimental and analytical work. Beata, there is not enough
space in this thesis to write down all the support and help I got from you in my time in Stock-
holm. However, most of all thank you for your friendship. Luiza, I am indebted to you for intro-
ducing me and explaining the research conducted in Stockholm. Thank you also for your friend-
ship that developed during our Ph.D. studies. I also respect your professionalism. I would like to
thank Ania Raszka for FISH analyses and the most important for her great friendship. I hope we
can do a real good project together again and I hope for some mountains trips. Special thanks to
Maja Długołęcka for many our helpful and stimulating discussions. Maja, you are also an excel-
lent discussion partner over the everyday  cup of coffee .
Thanks go also to master students Giampaolo Mele, Aleksandra Pietrala, Anna Chomiak and
Arkadiusz Stachurski, for their help in experiments. Giampaolo, you wrote that there was no
need to mention that the  something of me was in your thesis. I could say the same, but there is
 something of you all in this thesis and without your help, it would be impossible to finish it.
You are also more friends than colleagues.
Special thanks have to be done to Jan Bosander, Expert Process Engineer at the Himmerfj�den
WWTP for all his help, continuous availability. His competence and great professionalism have
been one of the best tools in the practical things during my research. He and the staff of the
WWTP created an unforgettable working environment.
Dr Ewa Zabłocka-Godlewska, I appreciate your support in microbiological analyses.
Monica L�w�n, Marek Tarłowski and Katarzyna Radziszewska, thanks for their aid in laboratory
works.
I am very grateful to Aira Saarelainen and Elżbieta Tarłowska for pleasant help with administra-
tive matters.
Jerzy Buczak thank you for the help with computer problems and being so friendly.
Dr Lesław Płonka, thank you for your help with computer issues, but mainly for being a friend
more than a colleague. Many people at the Environmental Biotechnology Department (EBD)
deserve my gratitude. Hereby I would like to thank especially to: Ewa, Jarek, Dorota, Ola and
Sebastian. Thank you for your friendship. Of course, I would like to thank you all the members
of the EBD for friendliness.
Most of all, my family deserves the biggest appreciation. To my Mother, for all her support, en-
couragement and love. My brother Qba and his wife Ula for being always supportive. Qba, you
are the best brother. To all my family for helping me every time I need it. Last but not least, my
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Grzegorz Cema TRITA LWR PhD Thesis 1053
love Oleńka  thank you for your patience, support and understanding and just for being with
me!
This thesis was realized as a joint PhD study at Royal Institute of Technology (KTH), Stockholm,
and Silesian University of Technology, Gliwice.
In Poland: study was funded by Ministry of Science and Higher Education (Project reference
number 1 T09D 030 30 - 0359/H03/2006/30).
In Sweden: Financial support was obtained from VA-FORSK, SYVAB, J. Gust Richert Founda-
tion and Lars Eric Lundbergs Foundation. The pilot plant was build by PURAC AB and has been
operated with co-operation of the Royal Institute of Technology (KTH) and SYVAB.
I wish to acknowledge all people, whom I might have not mentioned here and who have - either
directly or indirectly  affected my professional life.
Thank you
Stockholm, October 2009.
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Comparative study on different Anammox systems
TABLE OF CONTENT
ACKNOWLEDGEMENTS ........................................................................................................................... V
ACRONYMS AND ABBREVIATIONS....................................................................................................... IX
LIST OF PAPERS......................................................................................................................................... XI
ABSTRACT ..................................................................................................................................................... 1
I  INTRODUCTION .................................................................................................................................... 2
II  BACKGROUND ...................................................................................................................................... 2
II-1. The Anammox process .................................................................................................................................. 2
Nitrogen cycle and the discovery of the Anammox process .............................................................................................................. 2
Brief process overview ..................................................................................................................................................................... 4
Application of the Anammox bacteria .......................................................................................................................................... 5
Summary....................................................................................................................................................................................... 9
II-2. Landfill leachate - characteristics and treatment methods for nitrogen elimination........................................ 9
Landfill leachate generation ......................................................................................................................................................... 10
Landfill leachate composition ....................................................................................................................................................... 10
Landfill leachate treatment review  nitrogen removal .................................................................................................................. 11
Summary..................................................................................................................................................................................... 15
II-3. Reject water - characteristics and treatment method for nitrogen removal .................................................. 15
Chemical and physical methods.................................................................................................................................................... 16
Biological treatment ..................................................................................................................................................................... 17
Summary..................................................................................................................................................................................... 18
III  AIM OF THE THESIS ......................................................................................................................... 19
IV  MATERIALS AND METHODS ........................................................................................................... 19
IV-1. Membrane assisted bioreactor (MBR) ......................................................................................................... 19
Reactor operation......................................................................................................................................................................... 19
Analytical procedure .................................................................................................................................................................... 20
Membrane cartridge ..................................................................................................................................................................... 21
Batch tests ................................................................................................................................................................................... 21
IV-2. Moving Bed Biofilm Reactor (MBBR)  Two step process ........................................................................ 21
Reactor operation......................................................................................................................................................................... 21
Biofilm carrier material ............................................................................................................................................................... 22
Analytical procedure .................................................................................................................................................................... 23
Batch tests ................................................................................................................................................................................... 23
IV-3. Moving Bed Biofilm Reactor (MBBR)  one step process .......................................................................... 24
Reactor operation......................................................................................................................................................................... 24
Determination of the dry weight of biomass developed on Kaldnes cerrier....................................................................................... 25
Batch tests ................................................................................................................................................................................... 25
Oxygen uptake rates tests ............................................................................................................................................................ 25
IV-4. Rotating Biological Contactor (RBC) .......................................................................................................... 26
Reactor operation......................................................................................................................................................................... 26
Analytical procedure .................................................................................................................................................................... 28
FISH  Fluorescent in situ Hybridization.................................................................................................................................. 28
Denitrifying bacteria analysis....................................................................................................................................................... 29
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Grzegorz Cema TRITA LWR PhD Thesis 1053
Batch tests ................................................................................................................................................................................... 29
V  RESULTS AND DISCUSSION ............................................................................................................. 29
V-1. Membrane Assisted Bioreactor (MBR) ......................................................................................................... 29
Process performance evaluation..................................................................................................................................................... 29
Nitrogen conversion ..................................................................................................................................................................... 33
V-2. Moving Bed Biofilm Reactor  two step process ......................................................................................... 34
Process performance evaluation..................................................................................................................................................... 34
Assessment of bacterial activity in biofilm and activated sludge..................................................................................................... 37
Estimation of kinetic parameters ................................................................................................................................................. 39
V-3. Moving Bed Biofilm Reactor  from two-step towards one-step process ..................................................... 40
V-4. Moving Bed Biofilm Reactor  one-step process ......................................................................................... 41
Process performance evaluation..................................................................................................................................................... 41
Influence of conditions in the pilot-plant on nitrogen removal dynamics ......................................................................................... 44
Dissolved oxygen influence on the nitrogen removal rate ................................................................................................................ 44
Evaluation of kinetic parameters ................................................................................................................................................. 45
V-5. Rotating Biological Reactor  two step process............................................................................................ 46
Process performance evaluation..................................................................................................................................................... 46
Kinetic evaluation of process ......................................................................................................................................................... 49
Looking for bacteria populations ................................................................................................................................................. 49
V-6. Rotating Biological Reactor  one-step process ........................................................................................... 51
Process performance evaluation..................................................................................................................................................... 51
Nitrogen conversion ..................................................................................................................................................................... 52
Kinetic evaluation of process ......................................................................................................................................................... 52
Looking for bacteria populations ................................................................................................................................................. 53
VI  SYSTEMS COMPARISON ................................................................................................................... 54
VII  CONCLUSIONS ................................................................................................................................. 58
Membrane assisted BioReactor (MBR) ....................................................................................................................................... 58
Moving Bed Biofilm Reactor (MBBR)  two-step process ............................................................................................................ 59
Moving Bed Biofilm Reactor (MBBR)  one-step process ............................................................................................................ 59
Rotating Biological Contactor (RBC)  two-step process .............................................................................................................. 60
Rotating Biological Contactor (RBC)  one-step process .............................................................................................................. 60
General ....................................................................................................................................................................................... 61
VIII  FUTURE RESEARCH ....................................................................................................................... 61
REFERENCES ............................................................................................................................................. 63
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Comparative study on different Anammox systems
ACRONYMS AND ABBREVIATIONS
A  disc surface area, [m2]
AAOB  anaerobic ammonium oxidizing bacteria
Anammox  Anaerobic Ammonium Oxidation
AOB  aerobic Ammonium Oxidizing Bacteria
AOX  adsorbable organic halogen
ATP  Adenosine5 -triphosphate
ATU  allylthiourea
B  dry weight of biomass developed on carriers, [mg d.w.]
BABE  Bio Augmentation Batch Enhanced
BOD  biochemical oxygen demand, [g O2 m-3]
CANON  Completely Autotrophic Nitrogen Removal Over Nitrite
COD  chemical oxygen demand, [g O2 m-3]
d  mass of 50 kaldnes carriers after drying, [mg]
d.w.  dry weight
DEAMOX  DEnitrifying AMmonium OXidation
DEMON  pH-controlled deammonification system, names only refers to the process in a
SBR
denammox  DENitrification-anAMMOX process
DIB  Deammonification in Internal-aerated Biofilm system
DO  Dissolved oxygen, [g O2 m-3]
Dwa  diffusivity coefficient of electron acceptor in water
Dwd  diffusivity coefficient of electron donor in water
e  mass of 50 kaldnes carriers after washing, [mg]
ET  actual evaporative losses from the bare-soil/evapotranspiration losses from a
vegetated surface
FISH  Fluorescent in situ Hybridization
GFP  granular floating polystyrene
H  heterotrophs
HDPE  high density polyethylene
HRT  hydraulic retention time
K  proportionality coefficient
KB  the saturation value constant, [g m-2d-1]
KI  Haldane inhibition coefficient, [g m-3]
KIA  Aiba inhibition coefficient, [g m-3]
KM  Michaelis constant [g m-3]
L  leachate production
M  mol
MAP  magnesium ammonium phosphate
MBBR  Moving Bed Biofilm Reactor
MBR  Membrane assisted BioReactor
MLSS  Mixed Liquors Suspended Solids, [g l-1]
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Grzegorz Cema TRITA LWR PhD Thesis 1053
MSW  municipal solid wastes
mwa  molecular weight of electron acceptor
mwd  molecular weight of electron donor
NO  nitrite oxide
NOB  aerobic Nitrite Oxidizing Bacteria
OLAND  Oxygen-Limited Autotrophic Nitrification Denitrification
OUR  Oxygen Uptake Rate
P  precipitation
PBS  phosphate-buffered saline
PFC  polyurethane foam cubes
Q  inflow rate, [m3 d-1]
r  substrate utilization rate, [g m-3d-1]
R  surface run-off
rA  substrate utilization rate, [g m-2d-1]
RBC  Rotating Biological Contactor
rpm  revolutions per minute
S  substrate concentration, [g m-3]
Sba  bulk liquid electron acceptor substrate concentration
Sbd  bulk liquid electron donor substrate concentration
SBR  sequencing batch reactor
Se  effluent substrate concentration, [g m-3]
Sharon  Single reactor system for High Ammonium Removal Over Nitrite
Si  inflow substrate concentration, [g m-3]
SNAP  Single-stage Nitrogen removal using the Anammox and Partial nitritation
SS  Suspended Solids, [g l-1]
TOC  total organic carbon, [g O2 m-3]
UASB  Upflow Anaerobic Sludge Bed reactor
VFA  volatile fatty acids
Vmax  the maximum utilization rate constant, [g m-2d-1]; [g m-3d-1]
VSS  Volatile Suspended Solids, [g l-1]
WWTP  WasteWater Treatment Plant
XOcs  xenobiotic organic compounds
"Us  change in soil moisture storage
"Uw  change in moisture content of the refuse components
�a  molar stoichiometric reaction coefficient for electron acceptor (moles)
�d  molar stoichiometric reaction coefficient for electron donor (moles
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Comparative study on different Anammox systems
LIST OF PAPERS
This thesis is based on the following papers, which are appended at the end of the thesis and
referred to by their Roman numbers:
I. Cema G., Plaza E., Surmacz-Górska J., Trela J., Miksch K. (2005). Study on evaluation
of kinetic parameters for Anammox process. In: Proceedings of the IWA Specialized Conference
Nutrient Management in Wastewater Treatment Processes and Recycle Streams, Krakow Poland, 19-
21 September 2005, 379-388.
II. Cema G., Szatkowska B., Plaza E., Trela J., Surmacz-Górska J. (2006). Nitrogen removal
rates at a technical-scale pilot plant with the one-stage partial nitritation/Anammox
process. Water Science and Technology, 54(8), 209-217.
III. Szatkowska B, Cema G., Plaza E., Trela J., Hultman B. (2007). One-stage system with
partial nitritation and Anammox processes in moving-bed biofilm reactor. Water Science
and Technology, 55(8-9), 19-26.
IV. Cema G, Płaza E., Trela J., Surmacz-Górska J., (2008). Dissolved oxygen as a factor in-
fluencing nitrogen removal rates in a one-stage system with partial nitritation and Anam-
mox process. In: Proceedings of the IWA Biofilm Technologies Conference, 8  10 January 2008,
Singapore. Submitted for publication in Water Science and Technology.
V. Cema G., Pietrala A., Płaza E., Trela J., Surmacz-Górska J. (2009). Activity assessment
and kinetic parameter estimation in single stage partial nitritation/Anammox. Submitted
for publication in Journal of Hazardous Materials.
VI. Cema G., Wiszniowski J., Żabczyński S., Zabłocka-Godlewska E., Raszka A., Surmacz-
Górska J. (2007). Biological nitrogen removal from landfill leachate by deammonification
assisted by heterotrophic denitrification in a rotating biological contactor (RBC). Water
Science and Technology, 55(8-9), 35-42.
VII. Cema G., Raszka A., Stachurski A., Kunda K., Surmacz-Górska J., Płaza E. (2009). A
one-stage system with partial nitritation and Anammox processes in Rotating Biological
Contactor (RBC) for treating landfill leachate. In: Proceedings of the IWA Conference Processes
in Biofilms: Fundamentals to Applications, Davis, USA, 13-16 September 2009. Submitted for
publication in Journal of Hazardous Materials.
Other publications related to this research not appended in the thesis:
International journals/books
Cema G., Wiszniowski J., Żabczyński S., Zabłocka-Godlewska E., Raszka A., Surmacz-Górska
J., Płaza E., (2008). Simultaneous nitrification, anammox and denitrification in aerobic rotating
biological contactor (RBC) treating landfill leachate. In: Management of pollutants emission from
landfills and sludge. Pawłowska & Pawłowski (eds). Taylor & Francis Group, London, 211-218.
Conference publications
Żabczyński S., Raszka A., Cema G., Surmacz-Górska J. (2009). Nitrifiers populations and kinetic
parameters analysis of membrane  assisted bioreactors. In: Proceedings of the IWA 2nd Specia-
lized Conference Nutrient Management in Wastewater Treatment Processes. Kraków, Poland, 6-
9 September 2009.
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Grzegorz Cema TRITA LWR PhD Thesis 1053
Cema G., Płaza E., Surmacz-Górska J. and Trela J. (2005). Activated sludge and biofilm in the
Anammox reactor  cooperation or competition? In: Integration and optimization of urban sanitation
systems, Joint Polish-Swedish Seminars, Cracow, 2005, TRITA-LWR.REPORT 3018, 129  138.
Cema G., Surmacz-Górska J. and Miksch K. (2004). Implementation of anammox process in the
membrane assisted bioreactor. In: Integration and optimization of urban sanitation systems, Joint Polish-
Swedish Seminars, Stockholm, 2005, TRITA-LWR.REPORT 3017, 81-92.
Surmacz- Górska J., Cema G., and Miksch K. (2004). Deamonification process in membrane
assisted bioreactors. In: Integration and optimization of urban sanitation systems, Joint Polish-Swedish Semi-
nars, Wisła, 2003, TRITA-LWR.REPORT 3007, 81-91.
Reports & compendia
Trela J., Płaza E., Hultman B., Cema G., Bosander J., Levlin E. (2008). Evaluation of one-stage
deammonification. VA-Forskrapport nr 2008-18, (in Swedish)
Trela J., Hultman B., Płaza E., Szatkowska B., Cema G., Gut L., Bossander J. (2006). Develop-
ment of a basis for design, operation and process monitoring of deammonification at municipal
wastewater treatment plants. VA-Forskrapport nr 2006-15, (in Swedish).
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Comparative study on different Anammox systems
ABSTRACT
The legal requirements for wastewater discharge into environment, especially to zones exposed to
eutrophication, lately became stricter. Nowadays wastewater treatment plants have to manage
with the new rules and assure better biogenic elements removal, in comparison with the past.
There are some well-known methods of diminishing concentrations of these compounds, but
they are ineffective in case of nitrogen-rich streams, as landfill leachate or reject waters from
dewatering of digested sludge. This wastewater disturbs conventional processes of nitrification-
denitrification and raise necessity of building bigger tanks. The partial nitritation followed by
Anaerobic Ammonium Oxidation (Anammox) process appear to be an excellent alternative for
traditional nitrification/denitrification. The process was investigated in three different reactors 
Membrane Bioreactor (MBR), Moving Bed Biofilm Reactor (MBBR) and Rotating Biological
Contactor (RBC). The process was evaluated in two options: as a two-stage process performed in
two separate reactors and as a one-stage process. The two-step process, in spite of very low ni-
trogen removal rates, assured very high nitrogen removal efficiency, exceeding even 90% in case
of the MBBR. However, obtained results revealed that the one-step system is a better option than
the two-step system, no matter, what kind of nitrogen-rich stream is taken into consideration.
Moreover, the one-step process was much less complicated in operation. Performed research
confirmed a hypothesis, that the oxygen concentration in the bulk liquid and the nitrite produc-
tion rate are the limiting factors for the Anammox reaction in a single reactor. In order to make a
quick and simple determination of bacteria activity, the Oxygen Uptake Rate (OUR) tests were
shown as an excellent tool for evaluation of the current bacteria activity reliably, and without a
need of using expensive reagents. It was also shown, that partial nitritation/Anammox process,
could be successfully applied at temperatures much lower than the optimum value. Performed
Fluorescent in situ Hybridization (FISH) analyses, proved that the Anammox bacteria were
mainly responsible for the nitrogen removal process.
Key words: Anammox; biofilm system; landfill leachate; nitrogen removal; reject water; removal
rates
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Grzegorz Cema TRITA LWR PhD Thesis 1053
tion/denitrification. These processes proceed
I  INTRODUCTION
well with typical municipal wastewater. Nev-
Nowadays, water is one of the most precious ertheless, there are also nitrogen-rich waste-
components on earth. It is part of all living water streams like landfills leachate or reject
cells and is a key resource in society. Over waters from dewatering of digested sludge
70% of our planet is covered by surface for which, traditional nitrifica-
water. However, around 97% is comprised of tion/denitrification can be generally ineffec-
salty water in the oceans. The rest is freshwa- tive due to free ammonia inhibition and
ter. Most of this, about 69%, is locked up in unfavourable biodegradable carbon content
the glaciers and icecaps. From the remaining for denitrification. Because of high require-
freshwater, most occurs as a ground water ments for oxygen and necessity of addition
and only about 0.3% is found as surface external carbon source, treating such nitro-
water in lakes and rivers. With growing peo- gen-rich streams with traditional nitrifica-
ple population and the same increasing hu- tion/denitrification would become expensive
man consumption of water combined with and not sustainable. Ammonia can be also
increasing pollution of water sources, careful removed by physical/ chemical processes.
use, management, treatment and water reuse However, they have several disadvantages
becomes therefore absolutely essential. like odour production, air pollution or high
cost of chemicals.
One of the problems associated with fresh-
water pollution is nutrients discharge into Partial nitritation followed by Anaerobic
surface water causing acceleration of the Ammonium Oxidation (Anammox) may be
eutrophication process. Although, natural an alternative for such streams. In the partial
eutrophication may take a thousand of years, nitritation, only half of ammonium is con-
due to human activity, this process is rapidly verted to nitrite and then ammonium and
intensified by increasing aquatic plant nutri- nitrite are transformed into nitrogen gas in
ent inputs to water bodies. Thus, the term the Anammox reaction. The ammonium is
 eutrophication has become synonymous oxidized under anoxic conditions with nitrite
with  excessive fertilization or the input of as electron acceptor. Hence, the combination
sufficient amounts of aquatic plant nutrients, of partial nitritation with the Anammox
which causes the growth of excessive process results in reduction of energy con-
amounts of algae and/or aquatic macro- sumption for aeration and additionally no
phytes in a water body (Petts, 2005). During external electron donor has to be added. For
last few decades, a huge numbers of land these reasons, it is a very interesting way of
waters areas all over the world have been wastewater management with comparison to
affected by eutrophication. It is also a serious traditional nitrification/denitrification.
problem concerning the Baltic Sea, which
due to its special geographical and clima- II  BACKGROUND
tological characteristics is highly sensitive to
II-1. The Anammox process
the environmental impacts of human activi-
ties. Hence, wastewater, in its untreated form,
As the Anammox process is object of study
cannot be discharged directly into environ-
in this thesis, the introduction to the process
ment and there is a need for its appropriate
and its characteristics organisms are de-
treatment.
scribed briefly in this chapter.
Nitrogen is essential for living organisms as a
Nitrogen cycle and the discovery of the
part of proteins; however, it is also one of the
Anammox process
nutrients causing eutrophication problems.
In nature inorganic nitrogen atoms can exist
Nowadays, the biological methods are com-
in different oxidation states from -3 (NH4+)
monly used for treatment of municipal
to +5(NO3-). Most of the nitrogen com-
wastewater and some industrial sewage. In
pounds representing these oxidation states
most wastewater treatment plants (WWTP),
can be converted to each other through mi-
nitrogen is removed by biological nitrifica-
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Comparative study on different Anammox systems
Norg.
Fig. 1. Scheme of the
N2 nitrogen cycle.
Nitrificationn
N2H4
Denitrification
NH3
N2O
Anammox
NH2OH Assimilation
NO
Fixation
NO2 Ammonification
Dissimilation
NO3
crobial activity (Kartal, 2008). The turnover nature . Mulder et al. (1995) experimentally
of nitrogen in biosphere is known as the confirmed Broda s prediction two decades
nitrogen cycle (Fig. 1). Nitrogen oxidation later. However, they hypothesized that am-
state is changed by different microorganisms, monium conversion was nitrate-depended.
that carry out catabolic reactions (nitritation, Van de Graaf et al. (1995) proved the biologi-
nitratation, denitrification, dissimilatory ni- cal character of the process. Van de Graaf
trate reduction and Anaerobic Ammonium and co-workers (1996) showed that presence
Oxidation (Anammox), anabolic reactions of nitrite as electron acceptor is essential for
(ammonium uptake, assimilatory nitrate re- Anammox activity and not nitrates as it was
duction and nitrogen fixation), and ammoni- initially supposed. In 1999, Strous et al.
fication (Dapena Mora, 2007). (1999), basing on analysis of the 16S rRNA
gene sequence, identified  missing lith-
In the beginning of the 20th century, most of
otrophs as a new, autotrophic member of
reactions depicted in the N-cycle were al-
the order Planctomycetales. They were found in
ready known for a long time, and the N-cycle
both wastewater treatment plants and natural
was assumed to be complete. In this com-
systems (Table 1) (Zhang et al., 2007). Almost
plete N-cycle, there was no reaction account-
30  50% of gaseous nitrogen production is
ing for the possibility of the anaerobic oxida-
attributed to the Anammox bacteria in nitro-
tion of ammonium (Kartal, 2008). In 1977,
gen cycle (Dalsgaard et al., 2005; Arrigo,
Engelberd Broda used thermodynamic calcu-
2005; Op den Camp et al., 2006). Their dis-
lation  standard free energy values of
tinct phenotypic characteristics involve red
chemical reaction  to make a prediction on
colour, budding production, crateriform
the existence of chemolitoautotrophic bacte-
structure on the cell surface, intracellular
ria capable of oxidizing ammonium using
compartment  anammoxosome , and intra-
nitrite as electron acceptor. These bacteria
cytoplasmic membrane containing ladderane
were known as  Lithotrophs missing from
Table 1. Anammox bacteria discovered up-to-date (after Zhang et al., 2007).
Genus Species Source
Brocadia Candidatus Brocadia anammoxidans Wastewater
Candidatus Brocadia fulgida Wastewater
Candidatus Kuenenia stuttgartiensis
Kuenenia Wastewater
Scalindua Candidatus Scalindua brodae Wastewater
Candidatus Scalindua wagneri Wastewater
Candidatus Scalindua sorokinii Seawater
Others Candidatus Jettenia asiatica Not reported
Candidatus Anammoxoglobus propionicus
Synthetic water
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Grzegorz Cema TRITA LWR PhD Thesis 1053
lipid. As a special organelle in the cell, nium oxidation. Ammonium is oxidized by
anammoxosome was considered to have hydroxylamine (NH2OH) to form hydrazine.
three functions: (1) providing a place for Reducing equivalents derived from N2H4
catabolism; (2) generating energy for ATP then reduce nitrite to form hydroxylamine
synthesis through proton motive force across and N2 (Fig. 2A). Nitrate formation could
the anammoxsome membrane; (3) protecting generate reducing equivalents for biomass
the bacteria from the proton diffusion and growth. Strous et al. (2006) based on genomic
intermediate toxicity due to their imperme- analysis of Kuenenia Stuttgartiensis indicated
able membranes (Zhang et al., 2007). that nitrite oxide (NO) could also be an in-
termediate. According to this new pathway,
Brief process overview
nitrite was first reduced to nitric oxide; am-
In the Anammox process, ammonium is
monium was then combined with NO to
converted with nitrite as electron acceptor in
form hydrazine, which was later oxidized to
a ratio 1:1.32, respectively, to dinitrogen gas
dinitrogen gas (Kartal, 2008)(Fig. 2B). Differ-
(Strous et al., 1998) (eq. 1).
ent metabolic pathway was proposed by
-
NH+ +1,32NO- + 0,066HCO3 + 0.13H+ Kartal (2008). According to his suggestion
4 2
- Anammox catabolism starts with one elec-
1,02N2 + 0,26NO3 + 0,066CH2O0.5N0,15
tron reduction of NO2- to NO, this is poten-
+ 2.03H2O (1)
tially followed by a three electron reduction
of NO to NH2OH. This step could be fol-
The main product of the anaerobic ammonia
lowed by the condensation of hydroxylamine
oxidation is dinitrogen gas, nevertheless
and ammonia to form hydrazine and the to
around 10% of nitrogen in the influent is
converted to nitrate nitrogen. General nitro- dinitrogen gas (Fig. 2C).
gen balance shows ammonium to nitrite to
Generally, nitrite is not directly converted to
nitrate ratio of 1:1.32:0.26. The Anammox
hydrazine but via hydroxylamine and/or
bacteria have very strong affinity for their
nitrite oxide (van der Star, 2008). Figure 2
substrates, ammonium and nitrite. The affin-
shows a schematic representation of three
ity constant values for ammonium and nitrite
possible metabolic pathways.
are below 5�M (Kartal et al., 2007).
Recent studies showed that Anammox bacte-
Substantial uncertainty exists on the interme-
ria were capable of nitrate reduction with
diates in the catabolism (van der Star, 2008).
organic acids as electron donors and the
15
Based on the N-labelling experiments hy-
same out-compete heterotrophic denitrifiers
drazine (N2H4) was identified as an interme-
for these compounds. The end product of
diate of the process. The occurrence of free
nitrate reduction by Anammox bacteria is
hydrazine, a rocket fuel, in microbial nitrogen
dinitrogen gas. It was also showed that
metabolism is rare, if not unique (Kartal e al.,
Anammox bacteria are also able to reduce
2007). Van de Graaf et al. (1997) based on
nitrate to ammonium using organic acids as
15
N-labbeling experiments proposed a possi-
electron donor (Fig. 3). In this way, Anam-
ble metabolic pathway for anaerobic ammo-
mox bacteria are capable of producing their
Fig. 2. Different hypo-
theses on the Anam-
A B
C
mox catabolic path-
NO2� NO2� NO2�
way. Additional
NO
potential intermediate
NH4+ NH2OH NH4+ NO NH4+ NH2OH
are: A) hydroxylamine,
B) nitric oxide, C) or
hydroxylamine and
N2H4 N2H4 N2H4
nitric oxide (van der
Star, 2008).
N2 N2 N2
4
Comparative study on different Anammox systems
2007). It was also demonstrated that com-
denitrification
mon denitrification substrates methanol and
NO N2O N2
ethanol severely inhibited Anammox bacteria
at a concentration below 1 mM. High salinity
dissimilatory nitrate reduction
up to 30 g l-1 salt concentration cause reversi-
NO3 NO2 NH4+
ble inhibition (Kartal et al., 2007).
Application of the Anammox bacteria
Anammox
Operation and investment cost of wastewater
N2
treatment plant can be decreased by using
innovative technologies based on new bio-
Fig. 3. Two possible routes of nitrate reduc-
logical conversion methods. Due to negative
tion by Anammox bacteria (Kartal, 2008).
environmental aspects of nitrogen discharge
to recipients and increasingly stringent efflu-
own ammonium (and nitrite) to perform
ent standards, the effective nitrogen removal
their  standard catabolism (Kartal et al.,
is necessary. Biological removal of high ni-
2007; Kartal, 2008, van der Star, 2008).
trogen concentrations from wastewater is
Egli et al. (2001) demonstrated that the very expensive when there is a lack of biode-
Anammox bacteria were active within the gradable organic carbon. Increasing require-
range of temperatures from 6 to 43�C with ments concerning nitrogen concentration in
the optimum at 37�C. For the optimal tem- treated wastewater and increasing cost of the
perature, the pH range is between 6.5 and 8.5 treatment exert a necessity of development a
(Gut, 2006). Inhibition studies showed, that new method for biological nitrogen removal.
Anammox bacteria are reversibly inhibited by Recently, Anammox process was developed
very low levels (< 1�M) of oxygen concentra- and proposed as a new technology for treat-
tions and irreversibly inhibited by high nitrite ing streams containing high concentration of
concentrations (>10 �M). Egli et al. (2001) ammonia nitrogen and low concentration of
showed that Kuenenia Stuttgartiensis has a organic carbon. However, the Anammox
higher, but still low, tolerance to nitrite. process requires nitrite as electron acceptor
When the nitrite concentration was more for anaerobic oxidation of ammonium, and
than 5 mM for a longer period (12h), the for its application in wastewater treatment,
Anammox activity was completely lost. How- different setups are used to provide nitrite:
ever, the activity could be restored by addi- 1-reactor or 2-reactors systems. The common
tion of trace amounts (ą50 �M) of the purpose in the application of all the systems
Anammox intermediate, hydrazine (Li et al., is providing Anammox bacteria with nitrite
2004; Op den Camp et al., 2007; Kartal et al., (Kartal et al., 2007). Generally, part of am-
Aerated reactor
NO3- +sulfide/COD NO2-
NH4+ + oxygen NO2-
NH4+ + oxygen NO2- denitrifiers
nitrifiers
nitrifiers
NH4+ + NO2- N2
NH4++ NO2- N2
NH4+ + NO2- N2
Anammox Anammox
Anammox
One aerated reactor
One non aerated reactor
Non aerated reactor
A B C
Fig. 4. Simple scheme illustrating different Anammox configurations and different sources of
nitrite: A) Nitritation and Anammox in Two-reactors in series, B) Nitritation and Anammox in
one single reactor, C) Partial denitrification of nitrates to nitrites with the Anammox process in
one non-aerated reactor.
5
Grzegorz Cema TRITA LWR PhD Thesis 1053
monium is converted to nitrite and then the
" The anammox process for the anoxic con-
remaining ammonium and the formed nitrite
version of ammonium and nitrite to dini-
is converted to dinitrogen gas by Anammox
trogen gas.
bacteria. Additionally, recently a new process
" One-reactor nitritation-anammox process as the
was developed which combines the anaerobic
occurrence of the nitrite production and
ammonium oxidation with denitrifying condi-
the anammox process in one reactor.
tions using sulphide as an electron donor for
" Two-reactor nitritation-anammox process for
the production of nitrite from nitrate within
the partial oxidation of ammonium to ni-
anaerobic biofilm (Kalyuzhnyi et al., 2006). In
trite in an aerated reactor, followed by an
Figure 4 there is shown the Anammox proc-
anoxic reactor, where only anammox
ess in different configurations and different
process takes place.
sources of nitrite.
" One-reactor denitrification-anammox process for
The processes of nitritation and Anammox
the anoxic processes of denitrification
were observed and studied in different con-
from nitrate to nitrite, combined with the
figurations, types of reactors and under vari-
anammox process.
ous conditions. Parallel research, performed
by a few research groups in different coun-
" The anammox reactor for the reactor in
tries, has led to several names for processes
which only the anammox process takes
where Anammox organisms play a major role
place.
(Table 2). This situation leads to an unclear
" Anammox organism: the dedicated organ-
terminology in the literature. Van der Star et
isms capable of performing the anammox
al. (2007) proposed to clarify this situation by
process.
using the following descriptive terms:
Two reactor nitritation-Anammox
Partial nitritation/Anammox process is based
on two processes. First assumed, that ammo-
Table 2. Process options and names for nitrogen removal systems involving the Anammox proc-
ess (after van der Star et al., 2007)
Process name Number of Source of nitrite Alternative process names
reactors
Two-reactor 2 NH4+ Nitritation SHARONa  anammox
Nitritation-anammox Two stage OLANDb
Two stage deammonifiation
One-reactor 1 NH4+ Nitritation Aerobic deammonification
Nitritation-anammox OLANDb
CANONc
Aerobic/anoxic deammonification
deammonification
SNAPd
DEMONe
DIBf
Two-reactor 1 NO3Ż DEAMOXg
denitrification - Denitrification denammoxh
anammox anammoxi
a
 acronym of Sustainable High rate Ammonium Removal Over Nitrite,
b
 acronym of Oxygen-Limited Autotrophic Nitrification Denitrification,
c
 acronym of Completely Autotrophic Nitrogen removal Over Nitrite,
d
 acronym of Single-stage Nitrogen removal using the Anammox and Partial nitritation,
e
 names only refers to the process in a SBR under pH-control,
f
- acronym of Deammonification in Internal-aerated Biofilm system,
g
 DEnitrifying AMmonium OXidation
h
 DENitrification-anAMMOX process
i
 System where anammox was found originally. Whole process was originally designated as
 anammox
6
Comparative study on different Anammox systems
sludge liquor generally contains enough alka-
influent
linity (in the form of bicarbonate) to com-
pensate for the acid production if only 50%
of the ammonium is oxidized. In this man-
ner, exact ratio for full nitrogen removal in
Partial nitritation
(Partial SHARON) the Anammox process can be obtained (van
Dongen et al., 2001a).
One reactor Nitritation-Anammox
The ability of bacterial cultures to create
biofilm brings a possibility to enhance bio-
Anammox
logical wastewater treatment efficiency.
Moreover, the ability of Anammox and Ni-
trosomonas species to grow within the same
effluent
biofilm layer enabled to design a one-stage
system for nitrogen removal. Simultaneous
Fig. 5. Scheme of the two-stage partial
performance of nitritation and Anammox
nitritation/Anammox process.
processes can lead to a complete autotrophic
nitrogen removal in one single reactor. In a
nium is partly oxidized to nitrite in the partial
one-stage process ammonium oxidizers in
nitritation stage and then nitrite react with
the outer layer of the biofilm can co-exist
remaining ammonium in the Anammox
with the Anammox organisms present in the
stage. The nitritation of ammonium to nitrite
inner layer. In this way, oxygen that inhibits
is conducted by aerobic ammonium oxidizing
the Anammox process is consumed in the
bacteria (AOB), a total nitrification should be
outer layer of the biofilm and Anammox
avoided and the effluent should contain
bacteria are protected from oxygen. The
around 50% of the ammonium and 50% of
combination of these two processes - partial
nitrite. Different strategies can be used to
nitritation and Anammox- in one reactor is
selective retention of AOB bacteria in the
illustrated in Fig. 6.
system and to prevent further nitrite oxida-
Simultaneous nitritation and Anammox were
tion to nitrate by aerobic nitrite oxidizers,
observed and studied in various types of
including the control of temperature, hydrau-
reactors under different conditions. Aeration
lic retention time, alkalinity, the pH-value,
devices and reactor configuration determine
dissolved oxygen concentration in the reactor
the transfer of air to the bulk phase. A trans-
as well as the amount of free ammonia
fer from the bulk phase over a boundary
(Paredes et al., 2007; Zhang et al., 2007). The
layer to the biofilm limits oxygen transfer to
combination of two processes - partial nitrita-
the bacteria. Also the limitation determined
tion and Anammox - in two reactors in series
by hydrodynamics conditions is very impor-
is illustrated in Figure 5.
tant (van Hulle et al., 2003). In the moving
Van Dongen et al. (2001a) showed that the
bed bioreactor, the oxygen concentration has
Sharon (Single reactor system for High Am-
a great influence on the nitrification rate
monium Removal Over Nitrite) process
when the oxygen is rate-limiting (Hem et al.,
could be successfully combined with the
1994). Also, it was proved that nitrite produc-
Anammox, creating two-stage process for
tion rate is the rate-limiting step for the
treating reject water originating from dewa-
Anammox process in a single-stage system
tering of digested sludge. When the Sharon
(Szatkowska et al., 2007). Intermittent aera-
reactor is used to provide the feed for the
tion can also be used to secure a suitable ratio
Anammox process, only 50% of the ammo-
of oxygen and oxygen free conditions in the
nium needs to be converted to nitrites. Since
biofilm.
7
Grzegorz Cema TRITA LWR PhD Thesis 1053
Fig. 6. Scheme of one-
step partial nitrita-
wastewater
tion/Anammox
process within the
aerobic
Partial
biofilm.
zone
nitritation
anaerobic
zone
carrier
A combination of partial nitrita- conditions for the CANON process can be
tion/Anammox process can also be establish achieved in different kind of systems like
in one single reactor under oxygen limited SBR and gas-lift (V�zquez-Pad�n et al., 2008).
conditions what is principle of the so-called
One-reactor denitrification-Anammox
CANON process. The CANON process was
Recently, a new process was developed. It is
investigated as an alternative to use conven-
a combination of the anaerobic ammonium
tional activated sludge for treatment of
oxidation with denitrifying conditions using
wastewater limited by organic carbon sub-
sulphide as an electron donor for production
strate (Third, 2003). Appropriate ammonium
of nitrite from nitrate within anaerobic
and DO (dissolved oxygen) concentration
biofilm (Kalyuzhnyi et al., 2006). The princi-
enable the consumption of oxygen by AOB
pal flow diagram of this concept for treat-
(aerobic Ammonium Oxidizing Bacteria) to
ment of high strength, strong nitrogenous
an extent in which DO concentration is not
and sulphate bearing wastewater is shown in
over the threshold toxic to the Anammox
Fig. 7.
bacteria. The oxygen-limited conditions be-
In the first stage of this process, anaerobic
low 0.5% air saturation provide an adequate
mineralization of organic nitrogen takes
environment on a stable interaction between
place. Next, the effluent from this reactor
Nitrosomonas-as aerobic microorganisms
(rich in ammonia and sulphide) is partly fed
and Planctomycete-like anaerobic bacteria
to the nitrifying reactor to generate mainly
(Sliekers et al., 2002; Ahn, 2006). The growth
nitrate and the rest directly to the
of NOB (aerobic Nitrite Oxidizing Bacteria)
DEAMMOX reactor. In the final DEAMOX
(and subsequent nitrate production) is pre-
stage, both flows are mixed together for
vented due to their lower affinity for oxygen
consecutive realisation of nitrite production
compared to AOB and for nitrite compared
mainly from nitrate using sulphide as an
to Anammox bacteria. Subsequently, the
electron donor and for the Anammox proc-
produced nitrite, an inhibitor to AOB, is used
ess (Kalyuzhnyi et al., 2006; Szatkowska,
as an electron acceptor by the Anammox
2007).
bacteria (Zhang et al., 2007; V�zquez-Pad�n et
al., 2008). The obtaining of the micro-aerobic
Nitrifying
NO3- + (NO2-)
Anaerobic
Reactor
influent effluent
DEAMOX
Reactor
(NR)
reactor
(AR)
NH4+, (NS-)
Fig. 7. Flow diagram of the DEAMOX concept (Kalyuzhnyi et al., 2006).
8
Comparative study on different Anammox systems
Summary to implementation of the EU Landfill Direc-
tive (1999/31/EC) (Kohler N. & Perry,
Application of the Anammox process in
2005). Nevertheless, municipal waste landfill-
wastewater treatment can lead to significant
ing is still a very important issue in the waste
reduction of operational costs. Compared to
management system in Europe and the rest
conventional nitrification-denitrification
of the world.
dependent nitrogen removal systems, the
Anammox allows over 50% of the oxygen to
Waste disposal to landfills, in general, is an
be saved (only half of the ammonia has to be
easy and low-cost waste management option
oxidized to nitrite instead of full oxidation to
but it raises environmental concerns. During
nitrates). Furthermore, because The Anam- the process of waste degradation, landfills
mox is an autotrophic process, the problem
produce waste products in three phases.
regarding the supply of an electron donor (to
These are solid (i.e., degraded waste); liquid
support conventional denitrification) is cir- (i.e., leachate, which is water polluted with
cumvented and no organic carbon source is
wastes); and gas (usually referred to as landfill
needed. Additionally, Anammox bacteria
gas) (Butt et al., 2007). The major potential
oxidize ammonium under anoxic conditions
environmental impact related to landfill
with nitrite as the electron acceptor, and
leachate generation is pollution of groundwa-
converse energy for CO2 fixation. This is in
ter and surface water (El-Fadel et al., 1997;
great concern because taxes on CO2 may
Kjeldsen et al., 2002).
even incur further significant cost in future if
In Poland due to stricter regulations (Act on
WWTPs are not excluded from this charge.
Wastes of 27 April 2001 with following
Hence, the cost and CO2 emission are re-
changes), which transposed EU legislation
duced by 60% to 90%, respectively
requirements on waste management into the
(Fux, 2003; Op den Camp et al., 2007; Kartal
Polish national legislation, the amount of
et al., 2007).
deposited wastes has to be decreased. Im-
The Anammox process is particularly suited
plementing this legislation is connected with
for high nitrogen loaded industrial wastewa- the necessity of taking up a number of im-
ters that lack a carbon source. Different
portant actions, including limiting the
ammonium-rich streams (piggery manure,
amounts of biodegradable waste sent to the
urine, digested fish canning effluents, tannery
dumping sites. However, because of eco-
wastewater, landfill leachate, sludge liquors)
nomical issues, landfills are the most attrac-
have been studied with regard to the Anam- tive disposal route for municipal solid waste
mox process application for its treatment. As
in Poland (Wiszniowski 2006a). About 90%
the application of the Anammox process for
of municipal solid waste is currently disposed
landfill leachate and sludge liquors originating
of in landfill sites ( Environment 2007 by
from dewatering of digested sludge, treat- Central Statistical Office). Many existing
ment is an objective of this study, the charac- landfills are of an ageing design with no
teristics and different treatment methods for
properly designed foundations, so the
nitrogen elimination from these streams are
leachate can easily penetrate into the sur-
introduced briefly here.
rounding groundwater (Suchecka et al., 2006).
The problem with landfill leachate produc-
II-2. Landfill leachate - characteristics
tion and management is one of the most
and treatment methods for nitrogen eli-
important issues associated with the sanitary
mination
landfills. Environmental regulations require
In most countries, sanitary landfilling is the
controlling the leachate level, which means
most common way to eliminate municipal
that excess leachate must be removed and
solid wastes (MSW) (Renou et al., 2008). Up
disposed of. Because of variable leachate
to 95% of total MSW collected worldwide is
composition from different landfills, leachate
disposed of in landfills (Kurniawan et al.,
treatment methods have not been unified so
2006). In Europe, the number of permitted
far (Kulikowska and Klimiuk, 2007).
or legal landfills appears to have declined due
9
Grzegorz Cema TRITA LWR PhD Thesis 1053
Landfill leachate generation
" Inorganic macrocomponents  Ca2+,
Leachate is produced when water and/or Mg2+, Na+, K+, NH4+, Fe2+, Mn2-, Cl-,
other liquids seep through the wastes depos- SO42-, and HCO3-,
ited in a landfill. Its production is the result
" Heavy metals  Cd, Cr, Cu Pb, Ni and
of precipitation, surface runoff, infiltration,
Zn,
storage capacity, etc. (Heyer and Stegmann,
" Xenobiotic organic compounds (XOCs)
2002). Biochemical conditions, seasonal
 originating from households or indus-
water regime of the landfill and changes in
trial chemicals and present in relatively
the solid waste composition affect both the
low concentrations in the leachate (usu-
quality and the quantity of these wastewaters
ally less than 1.0 g m-3 of individual com-
(Gut, 2006). The water balance on the landfill
pound). These compounds include,
site can be summarized as follows (Blakey,
among others, a variety of aromatic hy-
1992):
drocarbons, phenols, chlorinated aliphatic
L = P  R  "Us - ET  "Uw (2)
and adsorbable organic halogens (AOX).
Where:
Leachate composition may also be character-
L  leachate production, P  precipitation, R
ized by different toxicity, determined using
 surface run-off, "Us  change in soil mois-
toxicological tests (Vibrio fischeri, Daphnia
ture storage, ET  actual evaporative losses
similes, Artemia salina etc.), which proved
from the bare-soil/evapotranspiration losses
indirect information on the content of pol-
from a vegetated surface, "Uw  change in
lutants that may be harmful to a particular
moisture content of the refuse components.
class of organisms (Kjeldsen et al., 2002;
Renou et al., 2008). Toxicity is a consequence
of contaminants mixture, their synergistic or
In addition, the climate has a great influence
antagonistic effects, and different physical-
on leachate generation because the input of
chemical properties, and toxicity tests may
precipitation and loses through evaporation.
thus give more information about potential
Moreover, leachate production depends also
environmental impact than do chemical
on the nature of the wastes themselves (Re-
analyses alone (Marttinen et al., 2002). The
nou et al., 2008).
toxicity tests have confirmed the potential
Landfill leachate composition
dangers of landfill leachate and the necessity
The landfill leachate is very high and complex
of treating it (Kjeldsen et al., 2002; Renou et
polluted wastewater. The mixtures of high
al., 2008).
organic and inorganic contaminants may be
There are many factors affecting the quality
found there as a result of biological, chemical
of the leachate, however among many others,
and physical processes at landfills, which are
the age of the landfill in particular influences
combined with waste composition and land-
the composition of the leachate (Renou et al.,
fill water regime (Heyer and Stegmann, 2002;
2008). Data presented by Kulikowska and
Poznyak et al., 2008). The composition of
Klimiuk (2007) indicate that the landfill age
landfill leachate depends on many various
has a significant effect especially on organic
factors like age of landfill, climate, nature of
compounds and variation of these parame-
deposited wastes and also varies in composi-
ters with time may have important implica-
tion from site to site. Landfill leachate con-
tions in leachate management. Three types of
tains four main groups of compounds (Chris-
leachate can be classified by landfill age:
tensen et al., 2001; Kjeldsen et al., 2002):
young, intermediate and stabilized (Amok-
" Dissolved organic matter  expressed as
rane et al., 1997; Poznyak et al., 2008). Gener-
Chemical Oxygen Demand (COD) or
ally, young landfills contain large amounts of
Total Organic Carbon (TOC), including
readily biodegradable organic matters and as
CH4, volatile fatty acids and more refrac- a result of rapid anaerobic fermentation of
tory compounds,
this matter leachate normally contains high
concentration of volatile fatty acids (VFA).
10
Comparative study on different Anammox systems
With time, when landfill enters the methano- options available. There are many advantages
genic phase, the biodegradable fraction of of operating a landfill as a bioreactor. The
organic pollutants decreases and the VFA are leachate recycling not only improves their
converted to biogas. Consequently, organic quality, but also shortens the time required
matter and BOD (Biochemical Oxygen De- for waste stabilization. Among others, addi-
mand) to COD ratio decreases significantly tional advantages are: in situ leachate treat-
and the organic compounds are dominated ment and improvement of the landfill gas
by refractory compounds (Welander et al., production rate, which may be favourable for
1998; Neczaj et al., 2007). In contrast, the energy recovery. This will tend to produce
concentration of ammonia does not decrease, stabilized leachate containing relatively low
and often constitutes a major long-term concentration of biodegradable organic car-
pollutant in leachate (Kjeldsten et al., 2002). bon but high concentrations of ammonia and
Authors also suggested that neither heavy persistent organic compounds (Knox, 1985;
metals nor xenobiotic organic compounds, Jianguo et al., 2007; Renou et al., 2008). How-
but ammonia would be the most concern in ever, Price et al. (2003) showed that it is pos-
the long run as theory and model simulations sible to remove ammonia from leachate by
show. The main sources of nitrogen are ex-situ nitrification of ammonia followed by
proteins, which accounts for approximately usage of the landfill as an anaerobic bioreac-
0.5% of dry weight of municipal solid wastes. tor for denitrification.
The hydrolysis of the polypeptide chains is
Few years ago, the treatment of landfill
disadvantaged in energetic terms and this is
leachate together with municipal wastewater
apparently the reason for the slow kinetics of
was a common solution. However, this op-
protein hydrolysis, which in turn causes the
tion is not advised due to presence of organic
slow release of ammonia. Nitrogen can trig-
inhibitory compounds and accumulation of
ger off eutrophization in receiving water-
hazardous compounds from the leachate,
courses and therefore its removal from
which consequently leads to reduce treatment
landfill leachate, e.g., by biological treatment,
efficiency and increase the effluent concen-
is required (Jokela et al., 2002).
tration (Waleander et al., 1998; Renou et al.,
2008). Moreover, Aktas and �e�en (2001)
Landfill leachate treatment review  nitrogen
observed nitrification inhibition and nitrite
removal
accumulation to about 85  100% of the total
As mentioned above, landfills leachate char-
NOx-N, when leachate was mixed with do-
acteristics depend on several factors, as type
mestic wastewater.
of wastes collected, seasonal variation of the
Chemical and physical methods
precipitation, the age of landfill and others.
These factors show the complexity of this Because of toxic nature of stabilized leachate,
wastewater and therefore indicate that there these effluents are difficult to deal with and
is no universal solution for its treatment. biological processes are very inefficient.
According to Renou et al. (2008), conven- Therefore, alternative technologies based on
tional landfill leachate treatment can be per- physical-chemical stages are required (Rivas et
formed in three ways: al., 2004). These processes include reduction
of suspended solids, colloidal particles, float-
" leachate transfer  recycling and com-
ing material, colour and toxic compounds by
bined treatment with domestic sewage,
flotation, coagulation/flocculation, adsorp-
" chemical and physical methods,
tion, chemical oxidation and air stripping.
" biological treatment  aerobic and an- Physical-chemical treatments for the landfill
leachate are used in addition at the treatment
aerobic processes.
line (pre-treatment or last purification) or to
Leachate transfer
treat a specific pollutant (e.g. stripping - for
Recycling of the leachate back through the
ammonia removal) (Renou et al., 2008).
top has been largely used in the past decade,
Specifically, ammonia has been identified, as
because it was one of the least expensive
one of the major toxicants to microorganisms
11
Grzegorz Cema TRITA LWR PhD Thesis 1053
in the treatment system, suggesting that pre- effective method for removal of ammonium,
treatment prior to the biological treatment because of its high reaction rate and low
system is required to reduce the concentra- residual ammonium concentration (Li et al.,
tion of NH4-N (Kim et al., 2007). Generally, 1999). On the other hand, in spite of very
it is a well known fact, that volatile fatty acid high ammonia removal exceeding even 98%,
content decrease with landfill age and neither struvite precipitation may be expensive due
biological nitrification nor denitrification is to high cost of chemicals, especially magne-
not appropriate due to low COD to NH4-N sium chloride (Ozturk et al., 2003; Calli et al.,
ratio (the lack of sufficient electron donors in 2005; He et al., 2007). However He et al.
leachate and the high energy requirements for (2007) demonstrated that about 44% of
aeration) (He et al., 2007). chemical cost might be saved by using the
MAP decomposition residues as the sole
The most common physical-chemical
magnesium and phosphate sources. Addi-
method for ammonia removal from leachate
tionally, Li et al. (1999) pointed out that high
is air stripping which allows removing up to
salinity formed in the treated leachate during
93% of ammonia (Li et al., 1999; Marttinen et
precipitation by using MgCl2�6H2O and
al., 2002; Renou et al., 2008). If this method is
NaHPO4�12H2O, which may affect microbial
to be efficient, the medium needs to have
activity in the following biological processes.
high pH value and the contaminated gas
phase must be treated with either H2SO4 or Other solution for ammonium removal from
HCl. A major concern about ammonia air landfill leachate is ion exchange as an alterna-
stripping is releasing NH3 into the atmos- tive treatment option. The ion exchange is
phere, which causes severe air pollution if more competitive to other methods because
ammonia cannot be properly absorbed by of little influence of the low temperature.
neither H2SO4 nor HCl. Other drawbacks are Clinoptilolite, one of natural zeolites, was
the calcium carbonate scaling of the stripping found very effective in removing ammonia
tower, when lime is used for pH adjustment, from water and wastewater (Wang et al.,
and the problem of foaming which imposes 2006). Zeolite is known to possess a higher
to use a large stripping tower (Li et al., 1999). selective ion-exchange capability for ammo-
Additionally, since the leachate from an aged nium ion than Ca2+ and Mg2+, even when the
landfill contains high alkalinity just like a concentration of the latter is higher than the
strong pH buffering system, the pH variation former (Junga et al., 2004). Nevertheless, the
before and after stripping, will consume a presence of competitive ions such as K+,
large amount of alkali and acid (Li et al., Na+, Ca2+ and Mg2+ in landfill leachate can
1999). Moreover, Marttinen et al. (2002) reduce the ammonium adsorption capacity
reported that in some cases stripping and and increase equilibrium-making time. How-
ozonation increased toxicity in spite of COD ever, experimental results indicate that am-
and ammonia removal. This may be a result monia can be removed by 84% from leachate
of oxidation of specific organic compounds using clinoptilolite as an ion exchanger (Kiet-
to more toxic ones. This is on great impor- lińska and Renman, 2005).
tance in case following biological treatment
Biological treatment
or discharges into environment.
In spite of stable treatment effects, and pref-
As an alternative to eliminate high level of
erable adaptability to the changes of wastewa-
NH4-N in leachate, the precipitation of NH4-
ter quality and quantity, physical/chemical
N by forming magnesium ammonium phos-
methods have several shortcomings: odour,
phate (MAP, struvite, MgNH4PO4�6H2O)
air pollution, high chemical costs, high-
can be applied. Kim and co-workers (2007)
energy consumption and excess sludge pro-
demonstrated that struvite precipitation is an
duction (Bae et al., 1997; Liang and Liu,
excellent pre-treatment process. Formation
2007). The main reason to select a biological
of magnesium ammonium phosphate, a
process for nitrogen removal is the lower
crystal with a solubility as low as 0.0023 g per
price compared to the physicochemical
100 ml H2O, has been considered to be an
methods (Dapena Mora, 2007). The biologi-
12
Comparative study on different Anammox systems
cal nitrification/denitrification is probably rial, whereas activated sludge without carriers
the most efficient and the cheapest process was able to remove only 61% of ammonium.
to eliminate nitrogen from leachate. How- Authors suggested that nitrifying microorgan-
ever, specific toxic substances and/or pres- isms were attached to the carrier material and
ence of bio-refractory organics can inhibit somehow stabilised the process at low tem-
biological treatment (Wiszniowski et al., perature. Also Welander et al. (1997) con-
2006b). Moreover, there are other problems ducted nitrification of landfill leachate at
associated with biological nitrogen removal. temperature of 10�C, using plastic carrier
Young landfills contain higher concentration material for biofilm growth. Authors proved
of biodegradable organic matter and ammo- that using suspended-carrier biofilm technol-
nia, what is conductive factor for biological ogy brings no risk for loss of biomass due to
denitrification. On the other hand, the ma- separability problems. Moreover, low tem-
ture landfill leachate contains relatively lower peratures have only weak negative effect on
concentration of degradable organic material nitrification rate. They suggested that this
but higher concentrations of ammonia and phenomenon could be explained by oxygen
the same the external carbon source addition diffusion into the biofilm, so the decreased
is needed. The activity of nitrifying bacteria is specific reaction rate at lower temperatures is
a function of temperature, pH, ammonia masked by deeper penetration of oxygen in
concentration and nitrifying biomass concen- the biofilm, resulting in larger mass of active
tration. The growth rate of nitrifying micro- nitrifiers. Also Jokela et al. (2002) confirmed
organisms is also slow and might be inhibited that nitrification of landfill leachate at low
by metals and hazardous materials. The low temperatures at range of 10 and even 5�C is
amount of bioavailable phosphorus in landfill possible using biofilm systems.
leachate may limit the nitrification process or
The most important issue concerning N
cause bulking sludge problems. Additionally,
removal is to ensure appropriate C to N
landfill sites exist in a wide range of envi-
ratio. The optimum COD/NO3-N ratio for
ronments, including areas with cool climate
denitrification depends on the nature of the
and achieving nitrification at low temperature
carbon source and on operating conditions of
requires large aeration basin volume or high
the system. Generally, the biological process
biomass concentration in the aeration unit
is especially efficient in treatment young
(Hoilijoki et al., 2000; Isaka et al., 2007).
landfill leachate rich in volatile fatty acids
Ilies and Mavini%0ń (2001) investigated the (Wiszniowski et al., 2006b). For leachate
nitrification and denitrification processes at characterized by BOD5/COD ratio above
operating temperatures down to 10�C in the 0.5, Surmacz-Górska (2000) proposed three
activated sludge system. The nitrification systems for biological treatment: membrane
process appeared to be unaffected (to any bioreactor, rotating biological contactor and
great extent) by a decrease in ambient tem- system with pre-denitrification and microor-
peratures to 14�C. However, when tempera- ganisms immobilized on suspended carrier.
ture dropped to 10�C, the nitrification inten- The best results were obtained in the system
sity drop between 10 and 30% was observed. consisted of activated sludge with pre-
Authors primarily attributed nitrification denitrification and microorganisms immobi-
inhibition to low operating temperature, lized on suspended carrier. However, the
although they identified also other possible post-denitrification with the external source
factors like too short aerobic hydraulic reten- of organic carbon is recommended to re-
tion time (HRT) and increasing level of ni- move remaining nitrites and nitrates. Im and
trous acid associated with low pH-value. But, co-workers (2001) proposed anaerobic-
Hoilijoki et al. (2000) proved that nitrification aerobic system including simultaneous
is feasible even at temperature as low as 10, 7 methanogenesis and denitrification to treat
and even 5�C. However, at 5�C complete organic and nitrogen compounds in imma-
nitrification was obtained in the activated ture leachate. The BOD5/COD and C/N
sludge system with addition of carrier mate- ratio were 0.44 and 14, respectively and com-
13
Grzegorz Cema TRITA LWR PhD Thesis 1053
plete N removal was achieved with raw land- able nitrogen loads, such as landfill leachate,
fill leachate as a carbon source. Much more because of possible stability problems when
complicated is situation with mature leachate receiving ammonium loading shocks
containing relatively low concentration of (Ganigu� et al., 2007). Canziani and co-
biodegradable organic material but high con- workers (2006) tested nitrogen removal from
centration of ammonia. For that reason a mature leachate by partial nitritation in mem-
supplementary source of organic carbon is brane bioreactor and subsequent denitrifica-
needed (Wiszniowski et al., 2006b; Zhang et tion in a moving bed biofilm reactor. It was
al., 2007). Kaczorek and Ledakowicz (2006) shown that a stable partial nitritation of am-
achieved the 99% removal of inorganic ni- monium to nitrite might be accomplished
trogen compounds using sodium acetate as with temperature higher than 30�C and free
external carbon source in two-sludge system ammonia concentration higher than 2.5 g m-3.
with secondary denitrification. Ilies and Mav- However, a low dissolved oxygen concentra-
ini%0ń (2001) used methanol as supplementary tion, kept under 0.5 g m-3, remains the key
carbon source for denitrification in 4-stage control parameter for partial nitritation. Un-
Bardenpho system (without anaerobic stage). der these conditions; it was possible to oxi-
Additionally, the authors reported inhibition dize more than 85% of influent nitrogen to
of denitrification when operating temperature nitrite. Peng and co-workers (2008) reported
dropped from 20 to 17�C and finally to 10�C. other possibility for controlling partial nitrita-
Welander and co-workers (1998) accom- tion of ammonium to nitrite. They showed
plished denitrification using initially acetic that the main factor achieving and maintain-
acid and later methanol as external carbon ing nitritation is a proper range of free am-
source. The study was performed under monia concentration obtained by dilution
realistic conditions (variations in temperature recycled final effluent. It inhibits nitrite oxi-
and leachate composition, etc.), and the tem- dizing bacteria but not ammonium oxidizing
perature of the leachate varied between 10 bacteria. Isaka et al. (2007) observed a partial
and 26�C. One of the main advantages of this nitrification performed by nitrifying bacteria
system compared to activated sludge is weak entrapped in a gel carrier. In the study, nitri-
temperature dependence. In addition, Louki- fication reactor was operated at high dis-
dou and Zoubolis (2001) performed denitrifi- solved oxygen concentration levels; low
cation using methanol and found out that the temperature, and infinite sludge retention
attached-growth biomass treatment method time, therefore partial nitrification was possi-
may be an interesting option comparing to ble because of free ammonia inhibition. One
the conventional activated sludge process. more method for controlling the oxidation of
ammonium to nitrite is presented by Ganigu�
Due to shortage of organic carbon source for
and co-workers (2007). In their study, the
denitrification of mature landfill leachate and
ammonium conversion to nitrite was
cost of external carbon source addition it was
achieved and controlled by alkalinity avail-
important to obtain technology that facili-
ability. Nevertheless, in this study occurred
tates enhanced nitrogen removal under the
only partial ammonium oxidation to nitrite
condition of low carbon source. Denitrifica-
occurred, because the researchers wanted the
tion via nitrite instead of nitrate saved the
Anammox process to take place.
oxygen requirement for nitrite oxidation and
40% of the carbon demand. A biodegradable Due to the limitations of biological processes
COD to NO2-N ratio greater than 2.5 may associated with low C/N ratio, high-energy
ensure complete denitrification when short- consumption and unstable running, there was
cut nitrification takes place (Bae et al., 1997; a need to search for new treatment methods.
Canziani et al., 2006; Zhang et al., 2007). The It appeared that the Anammox process could
oxidation of ammonium to nitrite has usually be a good alternative for traditional nitrifica-
been carried out using the Sharon technol- tion denitrification of mature landfill leachate
ogy. However, this technology could not be by reducing oxygen and carbon source re-
suitable for treatment of influents with vari- quirements and assuring high nitrogen re-
14
Comparative study on different Anammox systems
moval efficiency. However, in spite that the tics, technical applicability and constraints,
Anammox process was known in the mid effluent discharge alternatives, cost-
nineties, there are still very few papers con- effectiveness, regulatory requirements and
cerning the process for landfill leachate environmental impact. A combination of
treatment. In the end of that decade, Hele- physicochemical and biological treatments is
mer and Kunst (1998) observed a loss of required to achieve effective removal of
inorganic nitrogen of up to 90% in the nitri- NH4 N and COD with a substantial amount
fication step of rotating biological contactor of biodegradable organic matter. In most
under low DO conditions. Moreover, Siegrist cases, physicochemical treatments are suit-
and co-workers (1998) also observed exten- able for pre-treatment of stabilized leachate
sive loss of nitrogen (up 70% of ammonium to complement the biological degradation
oxidized) in a nitrifying rotating biological process (Kurniawan et al., 2006). In case of
contactor treating ammonium rich landfill the Anammox process, there is a need to
leachate. Authors suggested two hypotheses make more detailed research concerning on
for this autotrophic nitrogen removal: auto- dependence the process on temperature
trophic denitrification by Nitrosomonas or by decrease.
the Anammox process. Performed detailed
II-3. Reject water - characteristics and
analyzes of composition and spatial structure
treatment method for nitrogen removal
of the microbial community in the biofilm on
the RBC (Egli et al., 2001; Egli et al., 2003)
Not only landfill leachate is with reason con-
proved that the Anammox bacteria were
sidered as problematic. As it turns out, recy-
exclusively present in the deeper part of the
cled streams within the wastewater treatment
biofilm. Zhang and Zhou (2006) used a
plant may also contribute to magnitude ni-
bench scale UASB reactor for removal of
trogen load in the inlet. Liquors arising from
nitrogen from leachate by means of the
dewatering of sludge by belt presses, centri-
Anammox process. The results showed that
fuges or alternative dewatering measures are
the average removal efficiencies of ammo-
referred to as sidestreams (Thornton et al.,
nium, nitrite and total nitrogen were 87.5%,
2007). Usually during anaerobic sludge diges-
74.9% and 79.6% respectively, corresponding
tion, organic carbon is partially converted to
to the average ratio of removed nitrite-to-
methane gas, while about 50% of the nitro-
ammonium equal to 1.14 during the steady
gen, bound in the sludge, is released as am-
phase of the Anammox activity. Liang and
monium (Siegrist, 1996; van Dongen et al.,
Liu (2008) used bench scale up-flow fixed
2001a). This sludge liquor contains relatively
bed biofilm reactors for two-step partial
high concentration of ammonium nitrogen
nitritation and the Anammox process to
(typically 200  700 g m-3) and a relatively low
remove nitrogen. About 60% of ammonium
content of biodegradable organic matter.
and 64% of nitrite nitrogen were simultane-
Additionally in the reject water the ratio of
ously removed in the Anammox reactor.
HCO3-:NH4+ is normally equal to 1.1:1 (van
Dongen et al., 2001a; van Dongen et al.,
Summary
2001b; Thorton et al., 2007). In Table 3 the
It is important to note that the selection of
example of average reject water composition
the most suitable treatment methods for
is presented.
landfill leachate depends on their characteris-
15
Grzegorz Cema TRITA LWR PhD Thesis 1053
Component Unit Values Table 3. Example of
average reject water
SS g m-3 675
composition (Dosta et
COD g m-3 1500  2000
al., 2007).
NH4+-N g m-3 800  900
P-total g m-3 19.3
HCO3- g m-3 3000
HCO3-/N ratio mol HCO3- (mol NH4+-N)-1 0.98
Temperature �C 35
pH - 8.2
Despite of the fact that the volumetric su- crystals from impurities of the anaerobic
pernatant flow is only around 2% of the total digester. First, precipitates were dissolved in
influent wastewater, contains up to 25% of acid and the pollutions were removed by
the total nitrogen load into WWTP. Addi- centrifugation. The clarified supernatant was
tionally it is usually returned to the head of re-precipitated by adjusting its pH with caus-
the sewage treatment works (Siegrist, 1996; tic. It was shown that in the two steps proc-
Janus and van der Roest, 1997; Thornton et ess white MAP crystals could be obtained
al., 2007; Dosta et al., 2007). This is especially with over 85% recovery. However, to per-
dubious in case when the latter has limited form precipitation the pH-value must be
aeration/nitrification/denitrification capacity. increased up to 9 usually with NaOH solu-
Conventional biological extension requires tion, what generates additional cost. Gener-
additional volume of aeration tanks and con- ally, struvite precipitation may be expensive
sequently a substantial investment (Janus and because of high chemical cost, especially
van der Roest, 1997; Volcke et al., 2006). In magnesium chloride (Ozturk et al., 2003).
some cases enlarge or modifying the opera- Besides this, sludge disposal costs also in-
tion of the mainstream processes already on crease since use of MAP could increase the
site and this may be the most cost-effective mass of dry solids for disposal by 50%. Addi-
options. However, some works may not have tionally, the lacks of reliable ammonia moni-
this option available and on sites, where tor for such dirty liquors make the process
sidestreams comprise a significant proportion difficult to control (Jeavons et al., 1998).
of the total ammonium loading on the main-
As an alternative to eliminate high level of
stream processes, a possible option is to treat
NH4-N from reject water, there is used am-
directly the sidestream (Thornton et al., 2007).
monia air or steam stripping. Ammonia in
Generally, the chemical, physical and biologi- reject water is mainly present as ammonium
cal processes are feasible to recycle or elimi- ion. By raising the pH-value the ammonium
nate ammonium from reject waters. is converted to ammonia, which is readily
soluble in water. When contacted with a
Chemical and physical methods
gaseous phase, the ammonia will be trans-
One of the methods is precipitation of am-
ferred from water phase to the gaseous
monium in the form of magnesium ammo-
phase. The main difference between air and
nium phosphate MgNH4PO4 (MAP) by addi-
steam stripping is the treatment of the am-
tion of phosphoric acid and magnesium
monia-rich gaseous phase (Janus and van der
oxide. The process is according to following
Roest, 1997). In order to achieve an increase
reaction when the thermodynamic solubility
of the reject water pH-value above 10,
product is exceeded (Siegrist, 1996; �elen and
NaOH or Ca(OH)2 is added. Sludge flocks
T�rker, 2001):
and precipitated as CaCO3 due to pH in-
Mg2+ + NH+ + PO3- + 6H2O crease and have to be removed in a pre-
4 4
(3)
sedimentation step (Siegrist, 1996). Siegrist
MgNH4PO4 �" 6H2O
(1996) demonstrated NH3 removal efficiency
�elen and T�rker (2001) developed a two-
up to 97% at supernatant temperature be-
step purification process to recover MAP
tween 10  22�C. The greatest advantage of
16
Comparative study on different Anammox systems
the ammonia stripping is relatively simple content of the supernatant was low, therefore
operation not affected by wastewater fluctua- a carbon source (methanol) had to be added
tion if pH and temperature remain stable for appropriate denitrification performance.
(Szatkowska, 2007). A major concern about The process temperature was close to the
ammonia air stripping is releasing NH3 as it inlet temperature of supernatant which was
was mentioned before. about 20  30 �C. Moreover, author stated
that addition of excess sludge from super-
Other available method of treatment is ion
natant treatment would enhance performance
exchange by materials selective to the ammo-
of nitrification in wastewater treatment in
nium ion. Most of the research undertaken
winter. Hwang et al. (2000) analysed two
has studied the effectiveness of naturally
types of upflow biofilm reactor packed with
occurring materials, mainly zeolites. Today
granular floating polystyrene (GFP) and
the most suitable zeolite for this process is
polyurethane foam cubes (PFC) to investigate
clinoptilolite, a selective aluminosilicate of
the denitrification performance in reject
volcanic provenance, which shows an am-
waters. The results showed that very high
monium exchange capacity in the range 0.94 -
denitrification rate was achieved in both
21.52 g NH4+ N kg-1 (Thornton et al., 2007).
types of biofilm reactor because the high
Mackinnon et al. (2003) demonstrated that
concentration of volatile fatty acids contained
ion exchange using a new material, known by
in reject water provided the effective donors
the name MesoLite, shows strong potential
for denitrification.
for the removal of ammonia from sludge
liquors and an opportunity to concurrently Operational costs of the biological nitrogen
reduce phosphate levels. MesoLite shows an removal process stem from oxygen and or-
ammonium exchange capacity in the range 45 ganic matter requirements for nitrification
- 55 g NH4+ N kg-1. Using MesoLite materi- and denitrification, respectively. Due to the
als, over 90% reduction of ammonia was fact, that nitrite is intermediary compound in
achieved in the side stream. Thornton and both steps: nitrification and denitrification, it
co-workers (2007) confirmed that MesoLite would be convenient to procure a partial
is highly selective for the ammonium ion. nitrification up to nitrite and then denitrifica-
Authors showed that over 95% of it was tion starting from nitrite. This approach will
removed from belt press liquors with an produce savings in the oxygen needs during
initial ammonium nitrogen concentration nitrification, a reduction in the denitrification
exceeding 600 g m-3. The ion exchange is organic matter requirements, plus a decrease
more competitive comparing to others meth- in surplus sludge production (Ciudad et al.,
ods because of little dependency at the low 2004). Fux et al. (2004a) investigated nitrogen
temperatures. removal by nitritation followed by denitrifica-
tion with carbon addition in the SBR reactor.
Biological treatment
They obtained efficient and robust nitrogen
Biological treatment is generally considered
elimination at a total hydraulic retention time
as the best available treatment of sludge
of 1 day via the nitrite pathway. The removal
liquors. However, high strength ammonia
efficiency was amounted to 85  90% and
liquors are proved to be difficult to treat
ethanol was used as electron donor for deni-
biologically, due to the toxicity of ammonia
trification at a ratio of 2.2 gCOD g-1 N re-
and its oxidation products at particular pH
moved. Another way to eliminate nitrogen
values. The alkalinity in digested sludge liquor
from the sludge liquor is the Sharon process.
is generally not sufficient to allow full nitrifi-
Due to a short retention time of biomass
cation and an additional alkalinity source
(approximately 1 day) and high temperature
needs to be added to maintain the pH
(35�C), the nitrite oxidisers are washed out
(Jeavons et al., 1998).
and only nitrites are formed in the Sharon
Siegrist (1996) investigated separate intermit-
reactor. The process is performed without
tent nitrification/denitrification of digester
sludge retention. For oxidation of ammo-
supernatant. Because degradable organic
nium to nitrite, 25% less oxygen is necessary
17
Grzegorz Cema TRITA LWR PhD Thesis 1053
than for the oxidation of ammonium to ni- trite-to-ammonium ratio through setting an
trate. For denitrification of nitrite, 40% less O2-set-point that is tracked by adjusting the
methanol is needed than for denitrification of air flow rate. Additionally, an economic
nitrate. Denitrification is used to control the analysis of operating cost index shows that
pH-value (Hellinga et al., 1998; van Dongen et implementation of this operation mode war-
al., 2001a). The first full-scale application of rants the associated investment costs (Volcke
the Sharon process has been constructed at et al., 2005; Volcke et al., 2006). Van der Star
the Rotterdam Dokhaven WWTP. It was et al. (2007) described the first full-scale
possible to achieve an N-removal efficiency Anammox reactor. The operation was com-
of 90%, and the process was stable despite pared with parameters previously reported in
load variations and some process distur- studies on laboratory scale. The maximum
bances. The process requires high tempera- attained conversion of 9.5 kg N m-3d-1 was
ture; however, heat production by biological limited by the available influent load and was
conversion appeared to be significant, due to not a maximum volumetric conversion of the
the high inlet concentration, and contributes Anammox reactor.
to the optimal operating temperature of 30 -
Rosenwinkel et al. (2005) investigated the
40�C (Mulder et al., 2001).
simultaneous process in the Moving Bed
The BABE (Bio Augmentation Batch En- Biofilm Reactor (MBBR) treating reject wa-
hanced) process is another technique for ter. It was possible to remove up to 80% of
treatment of sludge liquor. The principle of the ammonia nitrogen load from sludge
this process is to implement nitrification dewatering on Hattingen WWTP. The nitro-
reactor in the sludge return line, the so-called gen removal from reject water led to higher
BABE reactor. It can be fed with an internal stability of the activated sludge tanks of the
N-rich flow. Also, limited amount of acti- WWTP and reduced nitrogen load in the
vated sludge from main process is recycled influent (Rosenwinkel and Cornelius, 2005).
over the BABE reactor. Hence, the nitrifica-
Recently, a full-scale application of partial
tion capacity of an activated sludge process
nitritation/Anammox process was success-
can be augmented by the addition of nitrifiers
fully achieved in SBR reactor at the wastewa-
cultivated in the BABE reactor. Practical
ter treatment plant in Strass, Austria (In-
results of the full-scale application of the
nerebner et al., 2007). This pH-controlled
BABE technology showed that the augmen-
system reached the designed elimination
tation effect of applying the BABE process
capacity of 300 kg N day-1 at the end of 2004.
improved the nitrification rate of the sludge
Both aerobic and anaerobic ammonia-
with almost 60% (Salem et al., 2003; Salem et
oxidizing bacteria were enriched in the bio-
al., 2004).
mass and due to the high NH4-N/COD ratio
Since the Anammox bacteria were discov- of 2.5 to 3 in the influent flow, autotrophic
ered, a new alternative appeared for treat- processes were dominant. The process was
ment of sludge liquor (Gut, 2006). In order operated in a SBR system providing intermit-
to relieve the main wastewater treatment tent aeration governed by three control
plant, reject water treatment with a combined mechanisms: time-, pH-, and dissolved oxy-
Sharon-Anammox process seems to be a gen (DO) control.
promising option. The Sharon process can be
Summary
operated without denitrification, what is
Generally, separate sludge dewatering liquor
especially interesting in view of coupling the
treatment mostly requires new tanks or a
former with the Anammox process (van
comprehensive modification of available
Dongen et al., 2001a). The simulation results
reactors. The overall investment costs are
indicate that significant improvements of the
therefore significantly reduced if the separate
effluent quality of the main wastewater
treatment operates at high volumetric reac-
treatment plant can be realized. The best
tion rates. Furthermore, the dewatering liq-
results were obtained by means of cascade
uor normally needs to be stored prior sepa-
feedback control of the Sharon effluent ni-
18
Comparative study on different Anammox systems
rate treatment, as the sludge dewatering facili-
" To establish potential process bottle-
ties are often operated intermittently (Fux et
necks and influence of operation pa-
al., 2004a). With a wide variety of technolo-
rameters on process performance,
gies for reject water treatment, the question
" Estimation of kinetic constants of nitro-
rises, which process is the best? For each
gen removal process by usage of batch
treatment processes there is case-specific
tests.
limitation, which sets the main goals for the
The more specific objectives of the research
side-stream treatment. Besides pure process
included:
engineering aspects, also other aspects such
as: start-up time, risk of failure, flexibility and
" studying the Anammox process in Mov-
others have to be taken into account (van
ing Bed Biofilm Reactor treating reject
Loosdrecht and Salem, 2005).
water originating from dewatering of di-
gested sludge, and to obtain long-lasting
III  AIM OF THE THESIS
and stable Anammox process,
Biological nitrogen removal used to purify " studying the Anammox process in the
wastewater with high ammonium content can Rotating Biological Contactor treating
become a major cost factor of wastewater real landfill leachate, and to obtain long-
treatment, in particular when the wastewater lasting and stable Anammox process,
contains little amounts of biologically de-
" finding correlations between the factors
gradable carbon compounds. Moreover, the
that influence the functioning of the
operational and investment cost can be saved
Anammox bacteria and to determine
with the use of new biological conversion
suitable conditions in order to obtain
methods and technology. Partial nitrita-
maximum removal rates,
tion/Anammox is an excellent example of
" defining the most important parameters
such new process, where oxygen and aeration
influencing bacterial activity to oxidize
energy are largely reduced and no external
ammonium with nitrite to nitrogen gas,
carbon source is required. The partial nitrita-
tion/Anammox process has the potential to " investigating the optimal oxygen condi-
be more cost-efficient than nitrifica- tion for purpose of maximum nitrogen
tion/denitrification. The process can be removal rate by batch tests application,
applied for treatment of ammonium-rich
" determination of the respiration rate for
wastewaters such as landfill leachate or sludge
the partial nitritation process by applica-
liquor originating from dewatering of di-
tion of OUR (oxygen uptake rate) tests
gested sludge.
with addition of ATU (inhibition of ni-
The aim of these studies was to investigate
tritation step) and NaClO3 (inhibition of
nitrogen removal from ammonium-rich
nitratation step),
streams with application of the Anammox
" developing the Anammox process in the
process.
Membrane assisted BioReactor.
The main goal of the investigations was to
study the process performance and to esti-
IV  MATERIALS AND METHODS
mate nitrogen removal rates in the partial
nitritation/Anammox process applied in
IV-1. Membrane assisted bioreactor
different systems. The process was estab-
(MBR)
lished in three different reactors (Membrane
Reactor operation
assisted BioReactor, Moving Bed Biofilm
The Anammox process for nitrogen removal
Reactor and Rotating Biological Contactor).
from synthetic wastewater was investigated in
The main objectives of this study were:
the laboratory scale Membrane assisted Bio-
" To compare different systems applied
Reactor (MBR). The liquid volume in the
for the Anammox process,
reactor was 0.036 m3 and the reactor was
provided with synthetic wastewater by
19
Grzegorz Cema TRITA LWR PhD Thesis 1053
medium composition were gradually
changed. The scheme of the MBR is shown
in Figure 8, while the changes in operational
conditions of the MBR are listed in Table 4.
Analytical procedure
During the research period, the samples were
taken from influent, effluent and the mixed
liquor three times a week. The efficiency of
the biological treatment was followed in
terms of the general parameters such as:
COD (dichromate method), ammonia
(Kjeltec System 1026 Tecator), nitrite and
nitrate (colorimetric method). Moreover, the
process was monitored by measurements of
the following parameters: flow rate, pH,
temperature, dissolved oxygen and the bio-
mass concentration in the bulk liquid. The
Fig. 8. Scheme of the investigated MBR free ammonia and free nitrous acid concen-
system.
trations were calculated according to An-
thonisen et al. (1976).
peristaltic pump and the same way permeate
Additionally, respiratory activity of the first
was sucked out. The effluent pump was
and the second stage of the nitrification were
connected with flat sheet membrane cartridge
measured as described by Surmacz-Górska et
(Fig. 8).
al. (1996). Mensuration of dissolved oxygen
Synthetic medium was made of tap water and
uptake by the bacterial culture and during the
additionally adjusted by NaHCO3, Na2HPO4,
subsequent addition of the selective inhibi-
NaNO2 and NH4Cl in order to reach nitrite-
tors was conducted (Fig. 9).
to-ammonium ratio of 1.32:1 (Strous et al.
A completely closed reactor vessel (volume =
1998). To check the reactor s management
110 ml) was used for the measurements. The
with virtual medium, at the end of the ex-
reactor was equipped with oxygen electrode
periment the landfill leachate from the mu-
connected with the recorder. Mixed liquors
nicipal landfill in Gliwice (Poland) was added
samples were collected from the MBR reac-
to the synthetic medium up to 10% of its
tor, aerated by shaking the sample, and trans-
volume. The reactor was equipped with a
ferred to the vessel, which was carefully
heater to keep temperature stable above
closed, with no remaining air bubbles.
30�C, and with vertical stirrer to ensure
During the measurement, samples were
proper mixing.
mixed using a magnetic stirrer.
During the research period, the Hydraulic
Retention Time (HRT) and the synthetic
Table 4. Operation conditions of the MBR.
Parameter Unit value
Reactor volume m3 0.036
Membrane cartridge surface m2 0.1
Flow rate m3 d-1 0.009  0.023
Hydraulic retention time (HRT) d 4  1.52
COD0 g O2 m-3 4.9  60
NH4+ - N0 g m-3 17.8  74.5
NO2- - N0 g m-3 21.8  98.1
Biomass concentration g MLSS l-1 3.5  12.4
Temperature �C 33.8 ą 0.6
pH - 7.9 ą 0.1
20
Comparative study on different Anammox systems
to determine nitrogen removal rates. Tests
Nitrite Inhibited
were performed in 2 L reactor. Activated
oxidation by NaClO3
sludge from membrane assisted bioreactor
+
Ammonia Ammonia Inhibited was stirred for 24 hours without aeration and
oxidation oxidation by ATU
feeding in order to remove substrate and
+ +
starving the sludge. The water bath was used
Organic Organic Organic
to keep the same temperature like in the
carbon carbon carbon
oxidation oxidation oxidation
reactor. After 24 hours, sludge was thickened
to 0.2 L and then reactor was filled with
Fig. 9. Schematic representation of the synthetic feeding media up to 2 L. Collected
action of NaClO and ATU on the respirato- samples were immediately analysed for inor-
3
ry activity (Surmacz-Górska et al., 1996).
ganic nitrogen compounds. Additionally,
temperature, dissolved oxygen and pH were
Sodium chlorate (2ml of 12% NaClO3 solu-
measured during the batch tests.
tion) was used as an inhibitor of the nitrite
oxidizers whereas allylthiourea (2ml of
IV-2. Moving Bed Biofilm Reactor
0.028% ATU solution) as an inhibitor of
(MBBR)  Two step process
ammonium oxidizing bacteria. The respira-
Reactor operation
tory activity of heterotrophs was also calcu-
lated as the remaining oxygen uptake after The Moving Bed Biofilm Reactor (MBBR)
additions of inhibitors. was constructed by the PURAC Company
and was located at the Himmerfj�rden Waste
Membrane cartridge
Water Treatment Plant (WWTP), southwest
KUBOTA membrane cartridge type 203 was
of Stockholm by the Himmerfj�rden bay.
used. Membrane sheets are ultrasonic-welded
The pilot plant was running since 2002. Re-
on both surfaces of the membrane panel.
sults from the start-up period are presented
They are made of chlorinated polyethylene
in Trela et al., (2004) and Szatkowska (2004).
with nominal 0.4 �m pore size. Effective
The technical-scale pilot plant was designed
surface area was equal to 0.1 m2. Between the
for studies of the two step partial nitrita-
panel and the membrane sheets, a spacer is
tion/Anammox process in two reactors of
laid to distribute filtered liquid into series of
2.1 m3 each, in series. A partial nitritation
channels that lead to a nozzle on top of the
process was in the first reactor with Hydrau-
cartridge. Moreover, the spacer prevents
lic Retention Time (HRT) of 2 days followed
membrane sheets from sticking onto mem-
by the Anammox process in the second
brane panel (Shino et al., 2004).
reactor with HRT of 3 days. Reactors were
Batch tests filled, to 50% of their volume, with Kaldnes
biofilm carriers. The influent reject water,
Two batch tests were performed during the
from dewatering of the digested sludge, was
research period in order to confirm presence
continuously pumped to the first reactor and
of the Anammox process in the reactor and
Conductivity Conductivity
Conductivity
Settling
Settling
pH, T
DO pH, T
Settling tank
tank
tank (buffer)
Effluent
Deoxidising
R2
R1
Air Dilution with tap water
column
Internal recirculation
External recirculation
Fig. 10. Pilot plant configuration; R1  partial nitritation reactor, R2  Anammox reactor ( inter-
nal and external recirculation introduced in the transition period from 2-step towards 1-step),
(based on Gut, 2006).
21
Grzegorz Cema TRITA LWR PhD Thesis 1053
Fig. 11. Photo of the
technical-scale pilot
plant. Partial nitrita-
tion reactor on the
right side and the
Anammox reactor on
the left side.
next the Anammox reactor was fed with density polyethylene HDPE. Their density
effluent from the first step (Fig. 10). The was equal to 0.96 kg l-1. The standard types,
effluent from the first reactor was diluted used in this research, had dimension of 9.1
with tap water. The nitrogen influent load to mm in diameter and the length of 7.2 mm.
the Anammox reactor was increased during Each Kaldnes carrier is shaped as a small
February  July 2004 period by stepwise cylinder with a cross on the inside of the
decrease of the flow rate of the tap water cylinder and  fins on the outside (Fig. 12).
used. In the start-up period of the Anammox The carriers were suspended in the reactors
reactor operation, the pH was adjusted by and were in continuous movement. Since the
dosage of a Na2CO3 and NaHCO3 solution biomass is growing primary on the protected
to keep pH-value around 8. Both reactors surface on the inside of the carriers (Rusten et
were divided into three zones and each zone al., 2006), only the effective biofilm surface
had a mechanical stirrer. To keep appropriate area of the carriers is given in Table 5. The
temperature conditions, the heaters were total surface area consists of both inner and
situated in the first zone of each reactor. outer surfaces, while the effective surface
Additionally, in the partial nitritation reactor area is that where biofilm seems to be at-
blowers were used, to ensure aeration for tached. Bur the outer surface of the carrier is
ammonium oxidation. The technical details continuously cleaned from the biofilm by the
of pilot plant are presented in Table 5. frequent collisions between carriers' ele-
ments. Application of these carriers allowed
Biofilm carrier material
achieving total specific surface area of the
The Kaldnes carriers were made of a high-
biofilm equal to 690 m2 in 1 m3 of carriers
Parameter Unit value Table 5. Technical
parameters of technic-
No. of reactors - 2
al scale pilot plant 
Volume of each reactor m3 2.1
two-stage process.
No. of zones in each reactor - 3
Reactor 1 filling by Kaldnes carriers % 46
Reactor 2 filling by Kaldnes carriers % 51
Effective surface area in R1 m2 483.5
Effective surface area in R2 m2 530.2
Volume of settling tank (buffer) m3 0.8
Volume of settling tanks m3 0.125
22
Comparative study on different Anammox systems
" pH-meter  WTW pH 330,
" DO probe  YSI 550A (YSI incorpo-
rated),
" thermometer  HI 9063 microcomputer
K-thermo couple thermometer
(HANNA instruments, Labassco),
" conductivity meter  TDS Meter
Fig. 12. Photos of the Kaldnes carrier ma-
(HACH).
terial and biofilm on it.
Additionally, the pilot plant was equipped
with on-line devices. The on-line equipment
and an effective specific surface area of the
consisted of Cerlic BB2 device measuring the
biofilm equal to 500 m2 in 1 m3 of carriers
oxygen concentration. The pilot plant was
(Welander et al., 1998; �degaard et al., 2000;
also provided with two conductivity meters
�degaard et al., 2006).
(Dr Lange Analon Cond 10), located in the
Analytical procedure
settling tanks.
During the study, samples of both influent
Batch tests
and effluent (each reactor) were collected and
In order to estimate nitrogen removal rates in
analysed for inorganic nitrogen forms, alka-
different periods of pilot plant operation,
linity, total nitrogen and Chemical Oxygen
several batch tests for studies of nitrogen
Demand (COD). Chemical analyses were
uptake rates were performed. One series of
conducted using the Dr. Lange test tubes.
these tests were divided into four groups
Samples from batch tests were analysed using
(Table 6) in order to recognize significance of
AQUATEC-TECATOR 5400 ANALYZER
the activated sludge and Kaldnes biofilm. In
(flow-injection system based on VIS spectro-
each test, in one bottle, condensed sludge
photometry). Before analyses, all samples
from the Anammox reactor was used while
were filtrated through 25-źm prefilter fol-
medium used in the second bottle was differ-
lowed by a 0.45-źm filter. Moreover, the
ent in each group. In the first group, Kaldnes
process was monitored on a daily basis by
carriers and sludge from R2 were used
manual measurements of physical parameters
whereas in the second group Kaldnes carriers
(flow rate, pH, temperature, conductivity and
and filtrated supernatant. In the third group,
dissolved oxygen concentration in the bulk
Kaldnes was rinsed out in order to remove
liquid) in the influent, effluent and within the
sludge covering the carriers during pulling
reactor. Moreover, a biofilm thickness was
out from the reactor. The first time the
measured. For measurement, several Kaldnes
Kaldnes were rinsed out in tap water and the
carriers were randomly collected from the
second time by filtered supernatant (to elimi-
pilot plant. To get a proper picture of the
nate negative impact of tap water on biofilm)
biofilm, the carriers were cut into thin slices.
having the same temperature like in the pilot.
Then the carriers were scanned with a resolu-
Additionally, to confirm previous results, in
tion of 2400 pixels per cal. and the biofilm
the fourth group three tests were made: one
thickness was measured using Gimp2 soft-
with activated sludge, second with medium
ware. Twenty different measurements of
from R2 and the third with rinsed Kaldnes
biofilm thickness were made for each carrier.
carriers and filtered supernatant.
The physical parameters were measured using
Additionally for estimation of the Michaelis-
following equipments:
Table 6. Different batch tests combinations (K  Kaldnes; S  concentrated sludge; NR  not
rinsed; R  rinsed).
Group 1 Group 2 Group 3 Group 4
Test 1 and Test 2 Test 3 and Test 4 Test 5 and Test 6 Test 7
S K+S S K (NR) S K (R) S K+S K (R)
23
Grzegorz Cema TRITA LWR PhD Thesis 1053
Menten kinetic parameters for the Anammox
Conductivity
Conductivity
reaction, series of four batch tests were per-
DO Settling
pH, T
formed. The values of Vmax and KM were Settling
tank
tank (buffer)
estimated separately for ammonium and
nitrite using Lineweaver-Burk, Eadie-Hofstee
and Hanes-Woolf. The procedure of test
performance is presented in Paper I.
Air
Fig. 13. Flow diagram of the pilot-plant with
IV-3. Moving Bed Biofilm Reactor
one-stage partial nitritation and Anammox
(MBBR)  one step process
processes.
Reactor operation
The pilot plant experiment has been run for carriers providing an effective surface area of
more than 3 years as a two-stage process 250 m2 m-3 of the reactor volume. The Kald-
where partial nitritation and Anammox took nes carriers in the reactor were in a continu-
place in two separate reactors (see chapter ous movement due to work of vertical mixers
IV-2). The reactors might be operated in and air supply from the bottom of the reac-
series or parallel. In March 2005 the pilot tor. The MBBR was supplied with reject
plant was modified and an experiment with water from sludge dewatering after anaerobic
recirculation of a nitrate-rich Anammox digestion in the preliminary phase, the hy-
effluent to the nitritation reactor was started draulic retention time (HTR) was equal to 1
(Fig. 10). It was intended to denitrify nitrates day. To estimate influence of a nitrogen load
in the first zone of reactor 1; zones 2 and 3 increase in the following period HRT was
of the nitritation reactor were connected and shortened to 16 hours. Then, the HRT was
in zone 1 the aeration was ceased to obtain again increased to one day. The technical
oxygen-free conditions for denitrification. details of pilot plant are presented in Table 7.
After a few weeks, nitrogen loss was ob-
Previous work in this area (Szatkowska and
served in reactor 1. It was due to the fact that
Płaza, 2006), demonstrated that the Anam-
Anammox bacteria found excellent condi-
mox process could be operated at a tempera-
tions to develop its bacteria culture in zone 1.
ture range below 30-35oC, therefore, the
Hence, Anammox was seeded throughout
process proceeded at a natural temperature
the reactor. Then recirculation was stopped
of incoming supernatant. Only in winter time
and aeration was switched on again in zone 1
an additional heater was supplied to keep
to develop a two-step biofilm layer, an outer
temperature above 20�C. The scheme of the
layer with oxygen-rich conditions for nitrifi-
pilot plant is presented in Figure 13.
ers and an inner layer for the Anammox
Analytical procedure and biofilm carriers
growth. At first the reactor was divided into
were described above in chapter IV-2.
two zones (zones 2 and 3 were connected),
later on all partitions were opened and reac-
tor was set as completely mixed reactor. The
reactor was filled up to 50% of its volume
with Kaldnes biofilm carriers, as biofilm
Parameter Unit value
Table 7. Technical
parameters of technical
No. of reactors - 1
scale pilot plant  one-
Volume of reactor m3 2.1
stage process.
No. of zones in reactor - 2, later 1
Reactor filling by Kaldnes carriers % 46
effective surface area m2 483.5
Volume of settling tank (buffer) m3 0.125
Volume of settling tank m3 0.125
24
Comparative study on different Anammox systems
were placed in a water bath to keep the tem-
Determination of the dry weight of biomass
perature constant. Additionally, the magnetic
developed on Kaldnes carrier
stirrers were used to assure appropriate mix-
To evaluate dry weigh of the biomass at-
ing of medium during the tests. The vessels
tached to the carriers, 50 carriers were taken
could be supplied with air and/or additions
from the reactor and dried in 105�C up to
of nitrite. Samples were taken every half hour
stable weight. Each series consisted of three
for measurement of inorganic nitrogen com-
independent repeats in order to avoid poten-
ponents. Parallel to the sampling, measure-
tial measurement errors. Then the biofilm
ments of pH-value, DO, conductivity and
was carefully removed under water stream. In
temperature were performed. The specific
order to completely get rid of remaining
conditions of each series of test were differ-
biomass, the carriers were placed in vessel
ent and specific test performance according
with 2M sodium hydroxide solution. A week
to tests objectives can be found in Paper II,
later, carriers were rinsed again by tap water
III, IV and V.
stream, dried in 105�C and weighted again.
The dry weight of biomass developed on
Oxygen uptake rates tests
carrier can be expressed as follows:
Six series of OUR test were performed. Se-
ries 1-4 were conducted on fresh medium,
d - e
B = (4)
taken directly form the pilot-plant. Series 5th
50
and 6th were conducted on medium after one
Where: and two days starving, respectively. At every
test day, the suspended solids and volatile
d  mass of 50 Kaldnes carriers after drying
suspended solids were analyzed. Moreover,
[mg]; e  mass of 50 Kaldnes carriers after
immobilized biomass concentration was
washing the biomass from carriers and drying
analyzed.
[mg]; B  the dry weight of biomass devel-
oped on carrier [mg d.w.] The tests were performed in a tightly closed
vial equipped with a DO probe and a stirrer,
Batch tests
and lasted about 10 minutes each. The con-
In order to estimate nitrogen removal rates in
centrations of sodium chlorate and allylthio-
different periods of the pilot plant operation,
urea were 17 mM and 43 �M, respectively.
five series of batch tests for studies of nitro-
The concentration of the former compound
gen uptake rates were performed. The spe-
was chosen according to Gut et al. (2005).
cific objective of each series is presented in
The research proved that the dose of sodium
Table 8.
chlorate at level of 20 mM proposed by Sur-
All batch tests were performed in a one-litre
macz-Górska et al. (1996) had an inhibitory
vessel that was filled with 50% Kaldnes carri-
effect on ammonia oxidizing bacteria in this
ers from the pilot plant reactor. An effective
particular system, and a lower one was de-
specific biofilm surface of 0.25 m2 per 1 dm3
termined.
of a reactor bottle was provided. The bottles
Table 8. Objectives of the following batch tests series.
Tests Paper
Series Objective
amount no
" Estimation of the influence of conditions in the pilot-plant on
1 19 II
nitrogen removal rates.
" Test with addition of allylthiourea to examine more closely the
2 8 II
nature of nitrogen removal mechanism.
" Estimation of the nitrogen removal rate at oxygen rich, oxygen
3 12 III
free and oxygen rich with artificial nitrite conditions.
" Study of the influence of different dissolved oxygen
4 20 IV
concentrations on nitrogen removal rates.
" Evaluation of bacteria population activity and estimation of
5 32 V
nitrogen removal kinetic parameters.
25
Grzegorz Cema TRITA LWR PhD Thesis 1053
Fig. 14. Flow scheme
Effluent
Effluent
discs
discs
of the RBC.
Influent
Influent
Drive motor
Drive motor
External carbon source
External carbon source
The aim of the tests performed on fresh Moreover, in few cases it was impossible to
medium was to evaluate changes in bacteria fit Aiba model at all.
activity during the pilot-plant performance.
The Aiba model:
Whereas the tests performed on starved
Vmax �"S �# ś#
S
medium were made in order to evaluate
ś#
rA = expś#- ź# (6)
kinetic parameters of the aerobic ammonium KM + S KIA ź#
�# #
oxidizing bacteria (AOB). The detailed test
Where:
performance can be found in Paper V.
rA  the substrate utilization rate [g m-2d-1];
For calculation of inhibitory effect observed
Vmax  the maximum substrate uptake rate
due to substrate excess, the Haldane model
[g m-2d-1]; S  the substrate concentration [g
was used (eq. 5).
m-3]; KM  half-saturation (Michaelis) coeffi-
Vmax �"S
cient [g m-3]; KIA  the Aiba inhibition coeffi-
rA = (5)
cient [g m-3].
S2
KM + S +
KI
IV-4. Rotating Biological Contactor
Where:
(RBC)
rA  the substrate utilization rate [g m-2d-1];
Reactor operation
Vmax  the maximum substrate uptake rate
A lab-scale Rotating Biological Contactor
[g m-2d-1]; S  the substrate concentration [g
(RBC) with partially immersed discs was
m-3]; KM  half-saturation (Michaelis) coeffi-
used. The RBC consisted of three equally
cient [g m-3]; KI  the Haldane inhibition
sized stages. For each stage there were four
coefficient [g m-3].
discs fixed to the centre horizontal shaft. The
effective disc area in each stage was 0.87 m2
The Aiba model (eq. 6) was also checked, and the disc submergence was 41%. A devel-
however, the correlation coefficient in each oped surface area for biofilm was provided
case was worse then in the Haldane model. with doormat (PE) fixed to discs. Oxygen
Fig. 15. Photos of the
lab-scale rotating
biological contactor.
26
Comparative study on different Anammox systems
Parameter Unit value Table 9. Characteris-
tics of the rotating
No. of stages - 3
biological contactor.
No. of discs per stage - 4
Total number of discs - 12
Disc diameter m 0.225
Total surface area available for growth m2 2.61
Disc submergence % 41
Liquid volume m3 0.014
transfers from the air to the RBC unit in in order to denitrify nitrites and nitrates after
three ways: oxygen absorption at the liquid the nitrification and Anammox process.
film over the biofilm s surface when the
In the later period, the glucose dosage was
biofilm is in the air; direct oxygen transfer
stopped and the characteristics of the influent
happening at the air water interface caused
was changed. For development of the simul-
by the turbulence created by the rotator
taneous nitritation/Anammox process, the
movement; and direct oxygen absorption by
contactor was supplied only with the ammo-
the microorganisms during the air exposure
nium nitrogen and no longer supplied with
(Rodgers and Zhan, 2003). The RBC unit
additional nitrite. In the first period, the
was covered by polystyrene foam to prevent
contactor was provided with leachate from
the growth of algae by light exclusion. The
old landfill site in Gliwice. The leachate was
scheme of the RBC is shown in Figure 14
collected from the pond and later on from
and the design characteristics of the RBC are
the concrete chamber being a retention tank.
listed in Table 9.
Due to the fact, that ammonium concentra-
The contactor was supplied with municipal tion in the leachate was low, not exceeded
landfill leachate and artificial NaNO2 and 100 g N-NH4+ m-3, the NH4Cl solution was
NH4Cl solutions to reach high nitrogen con- added to reach high nitrogen concentration
centration and appropriate excess of nitrite around 700 ą 50 g N-NH4+ m-3. In the later
over ammonium oscillating around the period of research, the leachate form Gli-
NO2:NH4 ratio 1.3:1 for Anammox process. wice s landfill was replaced by leachate from
young landfill in Zabrze. The change of the
Nitrogen loading rates applied to the first
leachate feeding the contactor was gradual.
stage of the RBC were gradually increased
The leachates from both landfill sites were
from 3 to 6 g N m-2d-1. Three research peri-
mixed gradually in the following ratios: 2:1,
ods can be differentiated basing on the in-
1:1, 1:2, 0:1 for leachate from Gliwice and
creasing nitrogen concentration in the influ-
Zabrze, respectively. As a matter of fact that
ent (period I: day 1 to 36; period II: day 36 to
the landfill leachate from Zabrze was charac-
91; period III: day 91 to 176). Additionally,
terized by high concentration of ammonia
an appropriate dose of sodium bicarbonate
nitrogen exceeding 1000 g N-NH4+ m-3, the
NaHCO3 was added to neutralize the alkalin-
addition of artificial NH4Cl solutions was
ity decrease during the nitrification process.
stopped. The characteristics of pure landfill
Glucose, as an external carbon source, was
leachates from landfill sites in Gliwice and
introduced in the third part of the contactor
Zabrze are presented in Table 10.
Parameter Unit Gliwice Zabrze Table 10. The charac-
teristics of the leachate
pond chamber
from landfill sites In
NH4+-N g m-3 < 10 100 1100
Gliwice and Zabrze.
NO2--N g m-3 0,5 0,1 0,1
NO3--N g m-3 76 17 0,7
COD g O2 m-3 250 370 1500
BOD g O2 m-3 100 120 950
27
Grzegorz Cema TRITA LWR PhD Thesis 1053
Analytical procedure probes; the sequences and targeted sites are
listed in Table 11.
During the research, samples were collected
from the influent, effluent and each stage of
The probes EUB338, EUB338 II and
the contactor twice a week. The efficiency of
EUB338 III were mixed together (EUB338
the biological treatment was followed in
mix) in proportion 1:1:1 in order to detect all
terms of the general parameters such as:
bacteria.
COD (dichromate method), ammonia and
The probes, chosen from the probeBase
organic nitrogen (Kjeltec System 1026 Teca-
database (Loy et al., 2003), were 5 labeled
tor), nitrite, and nitrate. Moreover, the proc-
with the dye FLUOS (5(6)-
ess was monitored by measuring other pa-
carboxyfluorescein-N-hydroxysuccinimide
rameters: flow rate, pH, temperature and
ester), Cy3 or Cy5. Both the probes and
dissolved oxygen (DO). No specific heating
unlabeled competitor oligonucleotides were
was applied and the temperature was kept
obtained from Biomers, Ulm, Germany.
constant at 17 ą 2.4 �C.
Prior to microscope observations samples
were embedded in Citifluor (Citifluor Ltd,
FISH  Fluorescent in situ Hybridization
UK) to reduce the fluorochrome fading
Detached biofilm samples were fixed with
(bleaching). A scanning confocal microscope
paraformaldehyde solution (4% paraformal-
(Zeiss LSM 510) equipped with an Ar-ion
dehyde in phosphate-buffered saline (PBS),
laser (488nm) and two HeNeLasers (543nm
pH 7.2) at 4 �C for 3 hours and washed sub-
and 633nm) were used to examine the mi-
sequently in PBS. Fixed samples were stored
crobial community. Image processing was
in PBS: ethanol (1:1) solution at -20 �C. In
performed using the standard software pack-
situ hybridization was performed as described
age delivered with the instrument (Zeiss LSM
previously by Daims et al. (2005). 16S rRNA
version 3.95).
targeted fluorescence labelled oligonucleotide
Table 11. List and description of probes used for the analysis.
FA Ref
probe target organisms probe sequence target site
% .
most halophilic and
NEU
halotolerant Nitrosomonas CCC CTC TGC TGC ACT CTA 653  670 40 1
spp.
CTE-
competitor TTC CAT CCC CCT CTG CCG 653  670 1
NEU
Nitrosomonas oligotropha CTT TCG ATC CCC TAC TTT
Claster 6a192 192  211 35 2
lineage CC
Competitor CTT TCG ATC CCC TGC TTC
192  211 2
claster 6a192 C
Ntspa662
genus Nitrospira GGA ATT CCG CGC TCC TCT 662-679 35 3
Competitor
GGA ATT CCG CTC TCC TCT 662-679 3
Ntspa662
NIT3 CCT GTG CTC CAT GCT CCG 1035 
Nitrobacter spp. 40 4
1052
Competitor 1035 
CCT GTG CTC CAG GCT CCG 4
NIT3 1052
EUB338 most Bacteria GCT GCC TCC CGT AGG AGT 338 - 355 35 5
EUB338 II Planctomycetales GCA GCC ACC CGT AGG TGT 338 - 355 35 6
EUB338 III Verrucomicrobiales GCT GCC ACC CGT AGG TGT 338 - 355 35 6
Pla46 Planctomycetales GAC TTG CAT GCC TAA TCC 46 - 63 30 7
anaerobic ammonium-
oxidizing bacteria,
Candidatus `Brocadia AAA ACC CCT CTA CTT AGT
Amx820 820 - 841 40 8
anammoxidans' and GCC C
Candidatus `Kuenenia
stuttgartiensis'
Ref: 1  Wagner et al., 1995, 2  Adamczyk et al., 2003, 3  Daims et al., 2001, 4  Wagner et al.,
1996, 5  Amann et al., 1990, 6  Daims et al., 1999, 7  Neef et al., 1998, 8  Schmid et al., 2001.
28
Comparative study on different Anammox systems
Denitrifying bacteria analysis Batch tests
Material used for microbiological analysis was During operation of the lab-scale rotating
collected from three stages of the contactor. biological contactor, it became possible to
Two 10 g samples of biofilm were prepared perform some batch tests for studies of ni-
from each stage. One of them was used for trogen uptake rates. The most essential thing
determination of dry mass. The second sam- associated with this RBC reactor was that test
ple was inserted into Erlenmeyer flask con- had to be done directly in the reactor. It was
taining 90 ml of 0.85% NaCl and then was impossible to take part of the rotating disc
vigorously shaken on the rotary shaker for 15 for test without destroying of the whole
min. Afterwards the sample was left for 2 reactor. That was the main disadvantage of
min. to allow sedimentation of large particles these tests as they could negatively influenced
and was then used for preparing various the continuous process occurring in the
dilutions. Suspensions prepared in this way reactor. One day before the test, the feed of
were used for isolation of denitrifying bacte- the leachate to the contactor was stopped in
ria. For titre determination of denitrifying order to starve the bacteria and to remove
bacteria liquid Giltay medium was used. remaining COD to exclude heterotrophic
Inoculated samples were incubated at 26 �C denitrification during the test. Additionally,
for 48 hours. After incubation from positive the first stage of the contactor was tightly
samples 0.1 ml of inoculum was taken and separated from the rest of the reactor. After
transferred onto nutrient agar plates for 24 hours, NaNO2 and NH4Cl solutions mix-
isolation of pure denitrifying bacteria cul- ture were added to the first stage of the con-
tures. Inoculated plates were incubated at 26 tactor to obtain initial ammonium and nitrite
�C for 48 hours. After incubation, single concentrations. The test lasted two hours and
cultures of bacteria were isolated and purified samples were collected every 15 minutes and
on Petri dishes with nutrient agar. Pure analyzed for inorganic nitrogen forms. Addi-
strains were used for inoculation of the liquid tionally, temperature, dissolved oxygen and
Giltay medium to recheck their ability for pH were measured during the batch test.
denitrification and their morphology, Gram
reaction and oxidase reaction were deter- V  RESULTS AND DISCUSSION
mined. For identification of oxidase-positive
V-1. Membrane Assisted Bioreactor
bacteria strains API 20 NE (standardized
(MBR)
system for non-enteric bacteria strains) was
used and for oxidase-negative bacteria strains
Process performance evaluation
API 20 E (standardized system for enteric
Implementation of the Anammox process
bacteria strains).
into the Membrane Assisted BioReactor
(MBR) was the aim of performed
Table 12. Characteristics of the influence medium and operational parameters of the MBR reac-
tor.
Samples
Parameter Unit Average Min. Max. St. dev.
No.
NH4-N in. g m-3 49.5 17.8 74.5 14.5 53
NO2-N in. g m-3 53.9 21.8 98.1 20.3 53
NO2-N/NH4-N - 1.08 0.59 1.44 0.18 53
N removal efficiency % 38.9 0 74.9 16.4 53
COD0 gO2 m-3 43.9 4.9 60 17.2 12
Biomass concentration kg MLSS m-3 7.3 3.5 12..4 2.8 38
Flow rate m3 d-1 0.016 0.009 0.023 0.005 47
HRT d 2.58 1.52 4.1 0.9 37
Temperature �C 33.9 32 35.8 0.7 52
pH-value in. - 8.0 7.6 8.1 0.1 52
pH-value reactor - 7.9 7.5 8.1 0.1 52
29
Grzegorz Cema TRITA LWR PhD Thesis 1053
inorg N in inorg N out N-NO3 out
200
N-NO2 out N-NH4 out
180
HRT
160
140
120
100
80
60
40
20
0
1 23 54 78 96 105 114 126 138 148 161 170 180
Time [days]
Fig. 16. Nitrogen conversion during start-up of the Anammox process in the membrane assisted
bioreactor (Cema et al., 2004).
experiment. Therefore, it was necessary to medium a very low content of biodegradable
provide condition favourable for growth of organic carbon was maintained in order to
appropriate bacteria. The bacteria cultivation prevent over-growth of the heterotrophs.
took place at the temperature above 30�C, at Moreover, the nitrite-to-ammonium ratio in
very low dissolved oxygen concentration the influent to the reactor was around 1:1
(below 0.3 g O2 m-3) and at average pH-value which is close to the stoichiometric value of
in the influent corrected and maintained 1.32:1 (Strous et al., 1998). During the ex-
around 8. The pH in the reactor was usually periment, the nitrogen load to the reactor
the same as in the influent, what agrees with was gradually increased from 0.01 to 0.09 kg
theories (Schalk et al., 1998, Siegrist et al., N m-3d-1. The operational parameters and
1998). Exceptional occurrence of the process characteristics of the influent medium are
brake-downs caused the pH drop, as the first presented in Table 12.
stage of nitrification prevailed over the
At the beginning of the experiment, nitrite
Anammox.
nitrogen was the main product of ammonia
The temperature was kept above 30�C, with oxidation (Fig. 16).
average value of 33.8 ą 0.6�C. In the influent
removal % of removal NO2-N/NH4-N ratio
1.8 140
1.6
120
1.4
100
1.2
80
1
0.8
60
0.6
40
0.4
20
0.2
0 0
1 23 54 78 96 105 114 126 138 148 161 170 180 189
Time [days]
Fig. 17. Nitrogen removal and nitrogen removal efficiency in the membrane assisted bioreactor
(Cema et al., 2004).
30
-3
[g N m
]
Nitrogen concentration
-3
2
4
NO -N/NH -N ratio
N removal [g m
] and
N removal efficiency[%]
Comparative study on different Anammox systems
0.04
0.08
Fig. 18. A) Correlation
y = 0.5202x - 0.0054
0.035
0.07 between nitrogen
R� = 0.9656
loading and nitrogen
0.03
0.06
removal rate, B) Corre-
0.025
0.05
lation between nitro-
0.02
0.04
gen loading and nitro-
0.015 0.03
gen removal rate (for
0.01 0.02 stable level of loading
y = 0.8654x - 0.0245
R� = 0.1599
after 121st day of expe-
0.01
0.005
riment), C) variations
0.00
0
of nitrogen loading
0.07 0.08 0.09 0.10
0 0.02 0.04 0.06 0.08
rate, nitrogen removal
B
A
N loading rate [kg N m-3d-1]
N loading rate [kg N m-3d-1]
rate and biomass con-
centration in the MBR
0.14 14
NItrogen removal rate Nitrogen loading rate MLSS
(Cema et al., 2004).
0.12 12
0.10 10
0.08 8
0.06 6
0.04 4
0.02 2
0.00 0
0 20 40 60 80 100 120 140 160 180 200
C
Time [days]
During first 30 days of the experiment, nitro- thermore, hydraulic retention time (HRT)
gen removal efficiency decreased from 27 to was gradually diminished from 4 to 1.5 days.
10 % (Fig. 17). It seems most probable, that The changes, made the nitrate production
this reduction of nitrogen removal efficiency drop and slight increase of nitrite and ammo-
was mainly caused by high nitrite nitrogen nia concentration was noticed. Total nitrogen
concentration in the reactor equal to 64.8 g removal efficiency again increased within 14
NO2-N m-3 on average. This concentration days from 4.1% to 63.8%. Moreover, very
was even higher than 60 g NO2-N m-3 re- intensive gas production in the reactor was
ported by Fux et al. (2002) as the toxic value observed. Due to the fact, that nitrite nitro-
for the Anammox bacteria activity, even gen was being completely removed in the
causing its loss. Therefore, the total nitrogen reactor, the nitrite-to-ammonium ratio in the
concentration in the influent had to be re- influent was changed from 1:1 to 1:32 ac-
duced from 100 g N m-3 to 40 g N m-3. As a cording to stoichiometric value. After this
result of this change, the nitrite concentration change, nitrogen removal efficiency increased
in the reactor dropped significantly to level to the maximum value equal to 74.9%. Since
below 6 g NO2-N m-3. At the same time then, process break-down and drastically
nitrate nitrogen was the main product of drop of nitrogen removal efficiency to 25.4%
ammonia oxidation. In order to suppress the were observed (Figure 17). Also, gas produc-
nitrate production, the inorganic nitrogen tion in the reactor stopped. Moreover, nitrite
load to the reactor was raised. For this pur- nitrogen concentration rose to value of 74 g
pose, total nitrogen concentration in the NO2Ż-N m-3, which is a toxic value for the
influent to the reactor was increased gradually Anammox activity (Fux et al., 2002, Schmidt
up to the value around 100 g N m-3. Fur- et al., 2003). It seems probable, that such an
31
-3
-1
-3
-1
N removal rate [kg N m d ]
N removal rate [kg N m d ]
-1
-3
-1
MLSS [g l ]
[kg N m d ]
Grzegorz Cema TRITA LWR PhD Thesis 1053
12
OUR Ammonium oxidizing bacteria Fig. 19. Oxygen up-
take rate of ammo-
OUR Nitrite oxidizing bacteria
10
nium oxidizing and
8
nitrite oxidizing bacte-
ria in the MBR (Cema
6
et al., 2004).
4
2
0
0 25 50 75 100 125 150 175 200
Time [days]
unexpected breakdown of the Anammox increase (Fig. 18c). After increasing of the
process was caused by very intensive growth nitrogen-loading rate, the intensive rise of
of algae (Chlorophyta) on the wall of the reac- nitrogen removal rate was observed and also
tor. Due to high nitrite concentration, influ- nitrogen removal efficiency has been im-
ent nitrogen load had to be reduced to nearly proved. It is highly probable, that the
50% (Figure 16). Additionally, reactor was Anammox bacteria work better with higher
covered with aluminium foil to protect from capacity. The correlation between nitrogen
the light and algae growth. These changes loading rate on the stable level 0.075  0.09
caused increase of nitrogen removal effi- kg N m-3d-1 and nitrogen removal rate (Fig.
ciency to 51%. However, the process was 18b) is very low. It is rather unlikely that
very unstable. On the other hand, it seems these nitrogen removal rates are correlated
most probable that the salt precipitation with nitrogen loading rate. Therefore, the
which interfered with microbial activity could good linear correlation was found only within
be other reason of the nitrogen removal the low range of the nitrogen loads. For high
efficiency decrease (Trigo et al., 2006). This range of the nitrogen-loading rate, the
could be an explanation of such sudden mechanism of nitrogen removal is more
breakdown of the process and its further complex, what indicated that there were
instability. additional inhibitors. It could be possible,
that such a high nitrogen-loading rate was
The other possibility of these problems was
too high for bacteria activity and it was the
the fact, that the mineral elements like K, Fe
first signal of the process breakdown after
or Mg was not added to the influent medium
142nd days of the experiment. The other
even as it is obvious that they were present in
possibility is that the Anammox bacteria had
the tap water used for the influent medium
lost in a competition for ammonia with aero-
preparation. Nevertheless, it was decided to
bic ammonium oxidizing bacteria.
introduce these mineral elements to the reac-
tor by addition of real landfill leachate to the Interesting information about microorgan-
synthetic wastewater up to 5-10% of its vol- isms activity gave Oxygen Uptake Rate
ume. This caused slight increase of the nitro- (OUR) measurements (Fig. 19).
gen removal, which reached 44%, on average,
The Anammox process is strictly anaerobic
at the end of research period.
process; however, no one has grown pure
Figure 18a shows that the nitrogen removal cultures of these bacteria in the laboratory.
rate increased parallel with growth of the Other microorganisms are essential to re-
nitrogen-loading rate. On the one side, this move one or more toxic products  nitrite,
may be due to adaptation of the bacteria; on oxygen, organic matter or free radicals  or
the other hand, when the nitrogen-loading they might be required to provide essential
rate was stable between 86th and 110th day of nutrient (Mohan et al., 2004). Membrane
the experiment, also nitrogen removal rate assisted bioreactor used for start-up of the
was stable despite biomass concentration Anammox process was used earlier for nitri-
32
-3
-1
2
OUR [g O m h ]
Comparative study on different Anammox systems
NO2/NH4 NO3/NH4
2.5
1.6
y = -0.0163x + 1.5011
1.4
2.0
R2 = 0.6142
1.2
1.5
1.0
1.0
0.8
0.5
0.6
0.0
0.4
-0.5
0.2
0.0
-1.0
20 30 40 50 60 70 80
0 25 50 75 100 125 150 175 200
A
B
Time [days] N removal efficiency [%]
Fig. 20. A) Conversion ratio of nitrite and ammonium and between nitrate production and am-
monium conversion (negative values  nitrite build up took place), B) Relationship between
nitrogen removal efficiency and conversion ratio of nitrate production and ammonium conver-
sion (data from 78th to 148th day  from start of the nitrogen removal to process breakdown).
fication of high ammonia nitrogen concentra- (Strous et al., 1998). On the other hand, the
tion, therefore it is possible that the nitrifiers conversion ratio of nitrate production to
were still present in the reactor. Measure- ammonium conversion was 0.76 ą 0.31 what
ments of OUR activity of ammonium oxidiz- was much higher than the stoichiometric one.
ers and nitrite oxidizers gave much lower This phenomenon showed that high nitrite
results than in earlier research (Surmacz- oxidizers activity occurred in the reactor,
Górska et al. 2004), but these bacteria were what is in agreement with results obtained in
still present in the MBR. There were some OUR tests. Interesting information could be
relationship between OUR of the ammonium seen from the correlation between nitrogen
and nitrite oxidizers and nitrite and nitrate removal efficiency and nitrate production to
nitrogen concentration in the reactor. It was ammonium conversion ratio in period from
especially clearly seen during the process start of the nitrogen removal to process
breakdown. At the same time nitrite concen- breakdown in 148th day. Along with nitrogen
tration significantly increased and the OUR removal efficiency increase, the nitrate pro-
of the ammonium oxidizers increased con- duction to ammonium conversion ratio was
siderably and along with increase of OUR of decreasing (Fig. 20B) whereas at the same
nitrite oxidizers. Also increase of nitrate time, nitrite to ammonium conversion ratio
concentration was observed (Fig. 19). was stable amounted to 1.31. These facts
indicate that with decreasing activity of nitri-
More information about process stoichiome-
fiers, the Anammox activity and probably
try, especially in relation to results of OUR
amount of the Anammox bacteria was rising.
test, are given by the ratio of nitrate produc-
After process breakdown, the nitrate produc-
tion to ammonium consumption
tion to ammonium conversion ratio rapidly
(NO3-/NH4+), as well as the ratio between
increased up to 1.04 in average, consequently
nitrite conversion and ammonium conversion
leading to loss of the Anammox activity.
(NO2-/NH4+) (Fig. 20) (the ratios were calcu-
lated as the molar ratio of nitrogen).
Nitrogen conversion
The average conversion ratio of nitrite and
The tests were carried out in order to verify
ammonium nitrogen during the whole re-
whether the Anammox process did take in
search was 1.05 ą 0.69. However, from start
the reactor. During first hours of the test, the
of the nitrogen removal to process break-
nitrogen removal was not observed. At the
down in 148th day, the average ratio was equal
same time, the parallel removal of ammo-
to 1.31 ą 0.26, which is similar to value
nium and nitrite nitrogen with increase of
stemming from the Anammox stoichiometry
33
-
+
3
4
-
+
3
4
NO
/NH
-
+
2
4
NO
/NH
and NO
/NH
Grzegorz Cema TRITA LWR PhD Thesis 1053
NO3-N NO2-N NO3-N NO2-N
Fig. 21. Example of
140 140
NH4-N Total inorg. N
NH4-N inorg. N
nitrogen conversion in
inorg. N t=0
inorg. N - t=0
120 120
the batch tests, A)
125th day of the expe-
100 100
riment, B) 132nd day
80 80
of the experiment
60 60
(Cema et al., 2004).
40 40
20
20
0
0
0 4 8 12 16 20 24
0 4 8 12 16 20 24
A B
Time [hours]
Time [hours]
nitrates concentration was observed. Despite
V-2. Moving Bed Biofilm Reactor  two
the fact, that there was no nitrogen removal
step process
on the beginning of the tests, after 24 hours,
decrease of inorganic nitrogen concentration
Process performance evaluation
was noticed on the level of 34.5 and 24%,
The partial nitritation/Anammox system was
respectively. Nitrates were the main product
designed as a two-step process consisting of
of nitrogen oxidation. The nitrite-to-
an initial partial nitritation reactor (R1) fol-
ammonium removal rate ratio during the
lowed by an Anammox reactor (R2), where
whole test was equal to 2.59, and was much
the nitrogen removal took place. The process
higher than the stoichiometric one for the
was operated at a temperature above 30�C to
Anammox process. It confirms the result of
keep favorable conditions for growth of the
OUR tests that the aerobic nitrite oxidizing
Anammox bacteria for which 37�C is the
bacteria are still present and active in the
optimal temperature (Egli et al., 2001). The
reactor; but on the other hand, the Anam-
pH-value in the effluent was within the range
mox process occurs in the MBR. It seems
from 7.6 to 9.2 and was higher than in the
probable that lower than in the MBR nitro-
influent (equal to 7.3ą0.4). This phenome-
gen removal efficiency and none nitrogen
non is in accordance with theory of the
removal during first few hours of the tests
Anammox process, in which a certain
were caused by too high nitrite concentration
pH-value increase is expected due to con-
in the reactor, at the beginning of the tests.
sumption of hydrogen ions during cell syn-
Nitrogen removal started after partial nitrite
thesis. Consequently, there was no need for
consumption by nitrite oxidizers (Fig. 21A
pH-value adjustment by dosage of a Na2CO3
and B).
and NaHCO3 solutions as it was in the start-
Table 13. Characteristics of the influent medium and operational parameters of the two-stage
MBBR reactor.
Samples
Parameter Unit Average Min. Max. St. dev.
No.
NH4-N in g m-3 124.4 32.3 201.0 31.0 41
NO2-N in g m-3 157.3 71.5 220.5 29.5 41
NO2-N/NH4-N - 1.3 0.7 2.2 0.3 41
N removal efficiency % 86.0 49.4 97.4 8.6 53
COD0 gO2 m-3 185.4 100.0 374.0 76.3 20
Suspended solids g SS m-3 670 335 1165 312 13
Flow rate m3 d-1 0.52 0.36 0.85 0.04 184
HRT d 3.1 1.8 5.1 0.3 184
Temperature reactor �C 31.4 21.4 42.7 2.7 199
pH-value in - 7.3 5.7 8.5 0.4 199
pH-value out - 8.3 7.6 9.2 0.3 199
34
-3
-3
N concetration [g N m ]
N concetration[g N m ]
Comparative study on different Anammox systems
Ninorg. reduction NO2-N/NH4-N ratio N load N removal rate
120 2,4
2,2
100 2,0
1,8
80 1,6
1,4
60 1,2
1,0
40 0,8
0,6
20 0,4
0,2
0 0,0
0 25 50 75 100 125 150 175 200 225 250 275 300
Time [days]
Fig. 22. Variations of nitrite-to-ammonium ratio, nitrogen removal efficiency and nitrogen load
and removal rate.
up period of the Anammox reactor operation the reactor. However, the Anammox process
(Gut, 2006). The DO concentration was kept was the main cause of nitrogen removal due
at a low level of 0.14 ą 0.07 g O2 m-3 in spite to average low drop in the COD concentra-
that the reactor was not airtight. Moreover, tion equal to 63.7 g O2 m-3. The stoichiomet-
the nitrite-to-ammonium ratio in the influent ric use of COD in heterotrophic denitrifica-
to the reactor was kept around the stoichi- tion (neglecting cell synthesis) is 1.72 g COD
ometric value equal to 1.32:1. Table 13 shows per gram of reduced nitrite nitrogen, what
the parameters of the Anammox MBBR and indicated that less than 15% of nitrogen
characteristics of the influent medium. could have been removed due to heterotro-
phic denitrification. Also other factors, like
The Anammox reactor worked as a moving-
presence of nitrite and ammonia in an opti-
bed reactor that combined activated sludge
mal ratio and efficient biomass retention
and biofilm cultures. The Anammox reactor
(biofilm system), indicate dominant role of
was operated steadily obtaining high removal
the Anammox process. Moreover presence
of total inorganic nitrogen with an average
of the Anammox bacteria (Brocadia anammoxi-
value of 254 g N m-3, which corresponded to
dans and Kuenenia stuttgartiensis) were proved
average efficiency of 87%. The reactor was
by means of using the FISH technique (FISH
loaded with nitrogen with the average value
analysis for the pilot plant reactor were made
of 0.28 g N m-2d-1 corresponded to an aver-
thank to courtesy of the research group from
age nitrogen removal rate amounting to
the Delft University of Technology, Kluyvert
0.25 g N m-2d-1. The maximum obtained
Laboratory for Biotechnology, the Nether-
nitrogen removal rate was equal to 0.39 g N
lands). Later tests, performed by a research
m-2d-1 (Fig. 22).
group of the Gotheborg University, Depart-
During the process, the ammonium and
ment of Chemistry, Sweden, confirmed that
nitrite nitrogen were removed almost com-
Brocadia anammoxidans was present in the
pletely and the nitrate nitrogen was the main
Anammox reactor (Gut, 2006). Despite of
nitrogen form in the effluent from the reac-
fluctuations in nitrogen load and variable
tor. The nitrate nitrogen formation was
nitrite-to-ammonium ratio in the influent
measured to 6.9% (on average) of the re-
(Fig. 22), there were no problems with keep-
moved inorganic nitrogen with comparison
ing stable values of nitrogen, below 50 g m-3,
to theoretically expected value of 11%. Dif-
in the effluent from the reactor. However,
ferences between experimental data and
too high nitrite-to-ammonium ratio has an
theory can be explained by the fact that the
adverse influence on the Anammox process.
heterotrophic bacteria were also present in
35
-2
-1
r
a
N
r
2
4
N eduction efficiency [%]
NO -N/NH -N atio nd itrogen
load and removal rate [g m
d ]
Grzegorz Cema TRITA LWR PhD Thesis 1053
2.5
NO3/NH4 NO2/NH4
NO2-/NH4+ = 1.32 according
to stoichiometry
2.0
1.5
1.0
NO3-/NH4+ = 0.26 according to
stoichiometry
0.5
0.0
0 25 50 75 100 125 150 175 200 225 250 275 300
Time [days]
Fig. 23. Conversion ratio of nitrite and ammonium and between nitrate production and ammo-
nium conversion.
The nitrite-to-ammonium ratio exceeding 2.0 dominating in the system; however, the lower
caused significant decrease of nitrogen re- production of nitrates and higher conversion
moval efficiency. of nitrite than stemming from stoichiometry
was mainly caused by some heterotrophic
The ratio of nitrate production to ammonium
denitrification process.
consumption (NO3-/NH4+), as well as the
ratio between nitrite conversion and ammo- During the research period, concentration of
nium conversion (NO2-/NH4+) gave some suspended solids in the Anammox reactor
additional information about the process was very changeable (Fig. 24). The average
stoichiometry (Fig. 23). values were 1.57 kg m-3 and 1.14 kg m-3 for
SS and VSS respectively. It was obvious that
The average conversion ratio of nitrite and
such high concentration of suspended bio-
ammonium (NO2-/NH4+) during the whole
mass must have had some impact on process
research period was 1.35 ą 0.27 and was a
performance. Owing to stable results, it
little bit higher than these values found by
seems probable that the activated sludge had
Strous and co-workers (1998). Additionally,
rather positive influence on the process.
the conversion ratio between nitrate produc-
tion and ammonium conversion was 0.15 ą
0.07 what was much lower than the
stoichiometric value of 0.26. These data
suggested that the Anammox process was
6
Fig. 24. Variations of
SS VSS
the suspended solids
5
(SS) and volatile sus-
4
pended solids (VSS) in
3
the Anammox reactor
(Cema et al., 2005).
2
1
0
z1 z3 z1 z3 z1 z3 z1 z3 z1 z3 z1 z3 z1 z3 z1 z3 z1 z3 z1 z3 z1 z3 z1 z3 z1 z3 z1 z3
1 31 50 64 78 92 106 120 132 150 199 251 274 295
Time [days]
36
-
+
3
4
-
+
2
4
Conversion ratios:
NO
:NH
and NO
/NH
-3
Sludge concentration [kg m ]
Comparative study on different Anammox systems
Table 14. Nitrogen
Nitrogen removal rate Sludge concentration
removal rates and
gN m-3d-1 gN/gVSS�d gSS/L gVSS/L
sludge concentrations
Group 1
in batch tests (K=S 
Sludge 82.8 0.069 3.30 1.20
Kaldnes carrier and
Test 1
K + S 140.6 n/a 0.73 0.37
sludge; NR  not
Sludge 283.2 0.102 3.80 2.77
rinsed; n/a  not ana-
Test 2
K + S 197.7 n/a 1.20 0.88
lysed).
Group 2
Sludge 62.6 0.020 4.50 3.21
Test 3
Kaldnes
77.9 n/a 0.39 0.30
(NR)
Sludge 136.1 0.051 3.77 2.67
Test 4
Kaldnes
76.0 n/a 0.33 0.23
(NR)
Group 3
Sludge 58.0 0.030 2.60 1.96
Test 5
Kaldnes (R) 0 n/a 0.05 0.04
Sludge 139.7 0.052 3.90 2.70
Test 6
Kaldnes (R) 0 n/a 0.08 0.06
Group 4
Sludge 91.2 0.031 3.86 2.90
Test 7 K + S 37.4 n/a 0.31 0.26
Kaldnes (R) 0 n/a 0.02 0.02
were obtained in the test with condensated
Assessment of bacterial activity in biofilm and
activated sludge. However, in these tests the
activated sludge
nitrification activity and oxidation of ammo-
To confirm the hypothesis of the positive
nia nitrogen prevailed over nitrite removal.
effect of activated sludge on the process, as
Additionally, high pH drop was noticed, what
well as to check if the Anammox process
confirms the presence and activity of aerobic
occurs mainly in biofilm, several batch tests
ammonium oxidizing bacteria. Nevertheless,
were performed. In Table 14 concentrations
removal of total inorganic nitrogen indicated,
of SS, VSS and nitrogen removal rates are
that the Anammox bacteria could also live in
presented for each batch test.
the suspended biomass. In the test with not
In Figure 25 example profiles of nitrogen
rinsed out Kaldnes carriers and filtrated su-
conversion during batch tests are presented.
pernatant, the VSS concentration was equal
Generally, the highest nitrogen removal rates
to 0.23 kg m-3 as the sludge stuck to the
60 60
N-NH4 N-NO2 N-NO3 65
55 55
55
50 50
45
45 45
40 40 35
35 35
25
N-NH4 N-NO2 N-NO3
N-NH4 N-NO2 N-NO3
30 30
15
25 25
5
20 20
0 60 120 180 240 300 360 420
0 30 60 90 120150180210 240 270 0 30 60 90 120150180210240270
A B C
Time [min]
Time [min] Time [min]
Fig. 25. Example of nitrogen conversion during batch test A) for condensed sludge, B) Test for
Kaldnes carriers and filtrated supernatant, C) Test for Kaldnes carriers (rinsed-out) and filtrated
supernatant.
37
-3
-3
-3
N concentration [g m ]
N concentration [g m ]
N concentration [g m ]
Grzegorz Cema TRITA LWR PhD Thesis 1053
Kaldnes carriers while they were taken out of The amount of nitrifiers in the biofilm on
the liquor. In these tests, the average nitrogen Kaldnes carriers is insufficient to perform
removal rates were lower than in the tests deoxidation. Interesting is also, that in the
with condensed sludge. Also, the ratio of tests with the activated sludge, dissolved
nitrite-to-ammonium removal rate was below oxygen concentration was always a little bit
1 what indicated that there was also some lower than in the tests with the mixture of
nitrifiers contribution in the biofilm popula- Kaldnes carriers and sludge. These results,
tion. Based on calculations of nitrogen re- might confirm the hypothesis that nitrifiers
moval by activated sludge, and assuming that are present mainly in the activated sludge.
sludge, in bottle with Kaldnes carriers, works
Both in the mixture of Kaldnes carriers and
in the same way like activated sludge alone, it
sludge and in the tests with the activated
was proved that mainly biofilm is responsible
sludge alone, it could be observed that along
for nitrogen removal. The results for the tests
with the increase of VSS concentration, also
with rinsed Kaldnes carriers and filtrated
nitrogen removal rate was increasing (Fig.
supernatant (Fig. 25C) were surprising, be-
26A and 26B). However, much smaller con-
cause nitrogen removal was not detected.
centration of VSS in the tests performed with
Instead, minor oxidation of ammonium to
combined biofilm and activated sludge is
nitrite was noticed. High oxygen concentra-
related to much higher nitrogen removal than
tion during the test could be the main reason
that observed in the test with activated sludge
of this situation, which could inhibit the
only. It can indicate two hypotheses: firstly,
Anammox process. Before the test, the bottle
the Anammox bacteria are present in higher
was flushed with nitrogen gas to obtain oxy-
percentage in biocenosis of biofilm than in
gen free condition. However, during the
the biocenosis of activated sludge. Secondly,
experiment oxygen concentration exceeded
it is possible that nitrifiers, present in the
0.5 g O2 m-3. It seems most probable that
sludge, create favourable conditions for
nitrifiers, which are present mostly in acti-
Anammox bacteria on the biofilm by con-
vated sludge, play a role of oxygen removers.
300
300
Fig. 26. A) Correlation
y = 291.18x + 2.6776
between VSS concen-
250
250
R2 = 0.8959
tration and average N
y = 115.65x - 176.69
200
200
removal rate in the
R2 = 0.9027
tests with mixture of
150
150
Kaldnes carriers and
100
100
sludge, B) Correlation
between VSS concen-
50
50
tration and average N
0
0
removal rate in the
0 0.2 0.4 0.6 0.8 1
1.9 2.1 2.3 2.5 2.7 2.9
A B tests with condensed
VSS [g l-1] VSS [g l-1]
sludge, C) Relation-
180 0.09
300 ship between N con-
centration (in t = 0) to
150 0.075
250
VSS ratio and N re-
120 0.06
y = 115.65x - 176.69
200
moval rate (test with
R2 = 0.9027
90 0.045
condensed sludge), D)
150
60 0.03
Relationship between
100
VSS concentration, N
30 0.015
removal rate and N to
0 0
50
VSS ratio in the tests
2.382.772.671.96 2.7 2.9
0
with condensed
VSS [g VSS l-1]
1.9 2.1 2.3 2.5 2.7 2.9
sludge.
C D
N rem. gN/gVSS
VSS [g l-1]
38
-3
-1
-3
-1
N rem. [g N m
d ]
N rem. [g N m
d ]
-3
-1
-3
-1
[gN/gVSS]
[g N m
d ]
N remmoval rate
Nitrogen to VSS ratio
N rem. [g N m
d ]
Comparative study on different Anammox systems
sumption of oxygen that can diffuse into the process efficiency in the pilot plant operated
liquid, which do not happen in activated at Himmerfj�rden WWTP was on average
sludge. 84% also confirms this hypothesis.
On Figure 26C and 26D, relationships be- The moving-bed system is adequate to gain
tween nitrogen to VSS ratio and nitrogen cooperation of many bacterial cultures in
removal rates are presented for the tests removing nitrogen. The results from this
performed only with the concentrated sludge study on the Anammox reactor demonstrated
alone. The decrease of nitrogen removal rate that there is the nitrifying activity present in
along with the increase of nitrogen to VSS the Anammox reactor and it is concentrated
ratio was observed in different tests. Because chiefly in the activated sludge. The coopera-
in none of the tests with the activated sludge tion of activated sludge and biofilm on Kald-
there were no problems with sustaining nes carriers is responsible for total effect of
proper oxygen concentration, it seems most nitrogen removal but Anammox activity
probable that it is mainly due to increase focuses on biofilm on Kaldnes carriers.
nitrogen load in the tests. Moreover, with the
Estimation of kinetic parameters
increase in nitrogen load, the nitrogen re-
One of the main aims of this thesis was to
moval rates were decreasing. It could be a
estimate the kinetic parameters of nitrogen
little surprising that at such low nitrogen load
removal. Due to this reason several batch test
during the test the efficiency in nitrogen
were performed to evaluate bacteria activity
removal deteriorated. Explanation of this
in the reactor and to determine kinetic con-
could be that in the batch tests very high
stants of ammonium and nitrite removal for
concentration of nitrogen forms are reached
the Anammox process were calculated (Pa-
at the beginning of the test, which is signifi-
per I). Three different methods were used
cantly higher than in the system with con-
for determination of the kinetic parameters,
tinuous flow. Such high concentration of the
and the highest correlation was reached for
nitrogen could suppress the bacteria respon-
the Hanes-Woolf method. According to this
sible for nitrogen removal.
method, value of Km and Vmax for ammonium
Cooperation or competition?
removal was 5.74 gNH4-N m-3 and 77.52 g
Performed batch tests proved that in the
NH4-N m-3d-1 (0.31 g NH4-N m-2d-1) and for
Anammox reactor nitrifiers were present. It
nitrite removal 6.53 gNO2- N m-3 and 90.09
seems probable that it is mainly due to the
gNO2- N m-3d-1 (0.36 gNO2- N m-2d-1), re-
spreading of nitrifying bacteria from the
spectively. Since, in the reactor were present
partial nitritation reactor to the second reac-
Anammox bacteria as well as nitrifiers and
tor. Obtained results show that nitrifiers are
heterotophs, the obtained results of the ki-
mostly in the activated sludge and their
netic parameters refer to the complex sys-
amount on biofilm is insignificant. The re-
tems more than to the particular microorgan-
sults of these batch tests were also confirmed
ism. In such system, the interaction between
by OUR tests performed by Gut (2006). By
different microorganisms may have a big
OUR test it was also proved that AOB cul-
influence on the received results. Received
ture was more dynamic than NOB culture.
Michaelis constant both for nitrite and am-
Some substrate competition between nitrifi-
monium nitrogen are quite high. Additionally,
ers and the Anammox bacteria could be
van Dongen and co-workers (2001a) discov-
possible. On the other hand, the tests
ered that a biofilm thickness of even 0.2 mm
showed that nitrifiers are responsible for
is yet active. Up to this depth, the conver-
oxygen consumption. Oxygen diffusion into
sions can be calculated without taking into
mixed liquor can inhibit the nitrogen re-
account diffusion limitation. These facts
moval. It appears that it is rather some coop-
indicate that the mass transfer effect in the
eration of different type of bacteria than
biofilm and the laminar layer above the
competition, what was proven by the per-
biofilm is negligible compared to the Micha-
formed batch tests. The fact that under the
elis constant.
period of tests execution nitrogen removal
39
Grzegorz Cema TRITA LWR PhD Thesis 1053
1000 in NH4-N out NH4-N out NO2-N out NO3-N N inorg out
900
external
800
recirculation
700
600
500
400
300
200
100
0
0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450
Time [days]
Fig. 27. Nitrogen conversion in the partial nitritation reactor.
the whole period, it was possible to achieve
V-3. Moving Bed Biofilm Reactor  from
nitrite-to-ammonium ratio in the effluent
two-step towards one-step process
from the reactor equal to 1.3 ą 0.2, which is
Originally, the technical-scale pilot-plant was
close to stoichiometry of the Anammox
designed for studies of two-stage partial
reaction. It was feasible to reach such favour-
nitritation/Anammox process in two reactors
able ratio by combination of several factors
in series. Stable partial nitritation is an essen-
that favour ammonium oxidizing bacteria
tial precondition for Anaerobic Ammonium
over nitrite oxidizers. The sludge liquor, used
Oxidation in a two-step process (Fux, 2003).
in presented study, had an influent alkalinity-
The HRT fluctuated from 1.6 to 3.3 days,
to-ammonium ratio around 1.3 - 1.4 resulting
with an average value equal to 2 ą 0.3 days.
in 58 ą 7% ammonium oxidation ensuring
The reactor was supplied directly with reject
proper feeding media for the Anammox
water from dewatering of digested sludge that
reactor. Drop in pH-value, presence of free
contained high concentration of ammonium
ammonia that exceeded 20 g NH3 m-3 (in the
nitrogen varying from 260 to 917 g m-3 with
first zone of the reactor) and free nitrous acid
an average value amounted to 609.4 ą 167 g
up to 5 g HNO2 m-2 (in zone 2 and 3 of R1),
m-3 (Fig. 27).
temperature exceeding 30�C allowed to sup-
press nitrite-oxidizing bacteria. As a result,
The average ammonium loading rate was
the nitrate nitrogen concentration in the
1.08 g NH4-N m-2d-1 and the specific nitrite
effluent from the partial nitritation reactor
production rate during stable partial nitrita-
not exceeded 20 g m-3 on average. Fux and
tion period was 0.57 g NO2-N m-2d-1. During
800 Fig. 28. Total inorgan-
influent effluent
ic nitrogen in the
700
influent and effluent
600
from partial nitritation
500
reactor.
400
300
200
100
0
275 300 325 350 375 400 425 450
Time [days]
40
-3
Nitrogen concentration [g m
]
-3
m
Total nitrogen
concentration [g
]
Comparative study on different Anammox systems
co-workers (2004b) reported that after eleven mox reactor. Rosenwinkel and Cornelius
months of operation of partial nitritation (2005) described the same phenomenon that
MBBR reactor, significant nitrate production after decrease of DO concentration in the
occurred. Authors explained this phenome- partial nitritation reactor the nitrogen loss
non as a result of the adaptation of existing occurred which was not caused by heterotro-
nitrite oxidizers or the accumulation of a new phic denitrification (based on the COD bal-
species. The average DO concentration was 3 ance). The authors suggested that it is neces-
g O2 m-3 and decrease of this value to sary to first build up a biofilm structure in the
1.5 g O2 m-3 caused only temporal decrease carrier material, which can be in the next step
of nitrate production and it increased again seeded with the Anammox microorganisms.
within 40 days. Jianlong and Ning (2004)
Due to the high nitrogen removal in the first
demonstrated that partial nitritation was
reactor, it was decided to change process
steadily obtained at DO concentration of 1.5
from two-step into one-step process.
g O2 m-3. In our research, it was possible to
obtain stable partial nitritation with only
V-4. Moving Bed Biofilm Reactor  one-
minor production of nitrates with the average
step process
DO concentration equal to 1 g O2 m-3. Gen-
Process performance evaluation
erally, at low DO, Nitrosomonas is growing
Both the partial nitritation and the Anammox
faster than Nitrobacter, so nitrite will be
processes took place in a single reactor. Table
enriched (Rosenwinkel and Cornelius, 2005).
15 presents statistical evaluation of the pa-
In 339th day of the experiment, the external
rameters measured during operation of the
recirculation from the effluent of the Anam-
pilot plant.
mox reactor to the first zone of the partial
During the operational period, a pH-value in
nitritation reactor was applied. Simultane-
the effluent from the reactor was equal to the
ously the aeration of the first zone of the R1
influent value. While the partial nitritation
was swiched-off. The aim of introduced
reaction takes place, a large decrease in pH
changes was to remove the nitrate nitrogen
value is normally observed, whereas a rise of
generated in the Anammox process by het-
pH value is characteristic for the Anammox
erotrophic denitrification by remaining or-
reaction as cell synthesis occurs. Simultane-
ganic acids from anaerobic digestion. How-
ous performance of both processes resulted
ever, introduced recirculation initiated a
in ions compensation and therefore the pH
significant reduction of nitrogen (Fig. 28) that
drop in the one-stage system was minimal.
could not be explained by heterotrophic
The pH level was rather constant, with the
denitrification. The average nitrogen removal
average values of 7.84 ą 0.11 in the influent
efficiency was equal to 37% during that pe-
to the reactor and 7.84 ą 0.24 in the outlet of
riod.
the system. The sporadic drop of the pH
It seems that created condition turned out to
value in the outlet was mainly as a result of
be favourable to Anammox microorganisms.
stops in the inflow to the pilot plant.
High nitrogen removal in the first reactor
Temperature is a very important factor influ-
was observed just after one-month period of
encing the biological processes. It is espe-
recirculation. Such fast observed activity of
cially key factor for the Anammox process,
the Anammox bacteria could be explained by
which has optimum temperature on the level
two phenomena. At first, the Anammox
of 37 oC. When the reactor was operated as a
microorganisms were already present in the
partial nitritation reactor in two-step process,
nitrifying biofilm. It was proved by the FISH
the average temperature exceeded 30oC.
analysis that in partial nitritation reactor
However, due to the fact that it was proved
except Nitrosomonas sp. also some Anammox
that the Anammox process can be operated
bacteria were present in the biofilm. More-
at a temperature below the range of 30-35oC
over, the external recirculation allowed seed-
(Szatkowska and Płaza, 2006; Szatkowska,
ing the partial nitritation reactor with the
2007), the process did not required additional
Anammox bacteria derived from the Anam-
41
Grzegorz Cema TRITA LWR PhD Thesis 1053
Table 15. Characteristics of the influent medium and operation parameters of the one-stage
MBBR reactor.
Samples
Parameter Unit Average Min. Max. St. dev.
No.
NH4-N in g m-3 577.0 351.0 945.0 87.4 102
N removal efficiency % 58.7 32.8 90.4 12.6 102
COD0 gO2 m-3 209.9 177.0 271.0 27.1 14
Suspended solids g SS m-3 261 87 950 267 10
Flow rate m3 d-1 2.0 1.5 2.9 0.3 102
HRT d 1.1 0.7 2.7 0.3 197
Temperature reactor �C 24.7 17.0 31.9 2.44 397
pH-value in - 7.84 7.38 8.30 0.11 377
pH-value out - 7.84 6.87 8.60 0.24 389
DO reactor gO2 m-3 2.03 0.07 7.13 0.95 391
heating as the digester supernatant tempera- but is mainly consumed by the Anammox
ture was at 25 ą 2.4�C. Due to this fact, the culture in inner layer what means that oxygen
system worked under natural temperature of is therefore the main limiting factor control-
incoming supernatant with the average tem- ling the overall rate of the partial nitrita-
perature in the reactor equal to 24.2 ą 2.6 �C. tion/Anammox process in biofilm reactors.
Additional heater was supplied only during Particularly, it should be taken under consid-
the winter period to keep temperature stable. eration that dissolved oxygen is also respon-
sible for inhibition of the Anammox process.
The dissolved oxygen (DO) concentration
Under high values of oxygen concentration
(Fig. 29), as the most vital one for proper
process efficiency can be significantly re-
course of the simultaneous partial nitrita-
duced. The average value of dissolved oxygen
tion/Anammox process, was monitored over
concentration during operational period until
the period described.
656th day of research was 2.3 ą 0.8 g O2 m-3.
Since ammonium nitrogen concentration in
A big fluctuation, which can be observed in
the bulk liquid is much higher than the oxy-
Fig. 29, was mainly due to centrifuges break-
gen or nitrite nitrogen concentration, ammo-
downs, which resulted in no influent coming
nium diffusion will not limit the process.
to the system, as well as electricity break-
Nitrite is produced in outer layer by nitrifiers
downs that caused diffusers stops. After 656th
7.0
6.0
average DO concentration 2.3ą0.8
Interm ittent
aeration
5.0
4.0
3.0
2.0
1.0
0.0
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
Time [days]
Fig. 29. Variations of dissolved oxygen concentration in the reactor. From 656th day of research,
intermittent aeration was applied (dashed line stands for average values during aeration mode).
42
-3
DO concentration [g
m
]
Comparative study on different Anammox systems
8
in
out
7
6
5
4
3
2
1
0
0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800
Time [days]
Fig. 30. Variations of the conductivity in the inflow and effluent from the reactor.
day of research, the intermittent aeration carbonate resulted also in conductivity deple-
system was tested with cyclic turn on/off the tion. The drop in conductivity value in the
aeration system. The aeration mode was 35 reactor is because of ammonia oxidation to
minutes with 25 minutes mode without aera- nitrite and in the same time alkalinity con-
tion. After 723rd day of research, the cycles sumption in the nitritation process as well as
were changed to 30 minutes of aeration and nitrogen loss during the Anammox process.
30 minutes without. The average oxygen It can be also observed that curves of con-
concentration during aeration time was equal ductivity measurements at the influent and
to 3.1 ą 0.6 g O2 m-3. During that period, the effluent run parallel.
values of DO were more stable in spite of
The system was supplied directly with super-
more often breakdown noticed in the pilot
natant that contained high concentrations of
plant.
ammonium varying from 351-945 g m-3 (Fig.
The process was also monitored by conduc- 31) with an average value amounted to 577 ą
tivity measurements since Szatkowska (2004, 87.4 g m-3 (Table 15). During the process,
2007) proved, that it an excellent indicator part of ammonium was oxidised to nitrite
for process monitoring (Figure 30). that reacted with remaining ammonium to
dinitrogen gas and nitrates. The total inor-
The removal of two main ions coexisting in
ganic nitrogen elimination for the whole
supernatant as ammonium and hydrogen
1000
NH4-N in N inorg. out NO2-N out NH4-N out NO3-N out
900
800
700
600
500
400
300
200
100
0
1 36 71 94 113 127 154 197 241 283 316 350 388 435 479 522 570 612 675 724 765
Time [days]
Fig. 31. The nitrogen variations in the partial nitritation/Anammox reactor.
43
-1
Conductivity [mS cm
]
-3
Nitrogen concentration [g N m ]
Grzegorz Cema TRITA LWR PhD Thesis 1053
analysed period was 58.7 ą 12.6 % on aver- II). However, after short time, the adaptation
age. At the same time the average removal of to the new condition was observed. The
ammonium nitrogen amounted to 66.1 ą average nitrogen removal rate was higher
13.9%, what indicates that, in the outlet from than compared to the period with lower flow
the pilot plant, there was still high concentra- rate. Batch tests confirmed, that in spite of
tion of ammonia. The mean value of ammo- initial negative effect of increased flow rate,
nium nitrogen in the effluent from the reac- the system could easily adapt to the new
tor was equal to 198.6 ą 98.6 g NH4-N m-3. conditions. The investigations performed by
During the operational period, about 38.5 g Jin et al. (2008), showed the similar results
m-3 of nitrates was produced, what was concerning tolerance of the reactor to the
around 10.1% of the removed ammonium flow rate shock. Additionally, authors stated
nitrogen. This value is very close to a that the tolerance of Anammox reactor to
stoichiometric one, which is equal to 11%. substrate concentration shock could be
Figure 31 shows the nitrogen variations in weaker than to flow rate shock. In practice, it
the partial nitritation/Anammox reactor. is suggested that mixing characteristics is a
key factor in selection of reactor configura-
The detailed description of the pilot plant
tions for Anammox and an equalizing tank or
operations is described in Paper II, III, IV
recycling line is necessary to cope with se-
and V.
rious influent substrate concentration shock
Influence of conditions in the pilot-plant on
(Strous et al., 1997; Jin et al., 2008).
nitrogen removal dynamics
In order to more detailed examine of nitro-
One of the crucial things concerning design
gen elimination mechanism, the tests with
of reactor for nitrogen removal is their stabil-
addition of allylthiourea (selective inhibitor of
ity in case of hydraulic and substrate concen-
Nitrosomonas bacteria) were performed.
tration shocks. It is especially relevant in case
These tests proved that mainly the Anammox
of the Anammox process, which is known as
bacteria are responsible for nitrogen removal
very sensitive one. In order to investigate of
(Paper II).
the sudden change in the hydraulic regime in
Dissolved oxygen influence on the nitrogen
the pilot plant, several batch tests were per-
removal rate
formed. It was shown, that the shock change
in the flow rate and the same in the substrate In simultaneous partial nitritation/Anammox
loading rate to the reactor could temporarily process, nitrite availability for the Anammox
decrease the nitrogen removal efficiency and bacteria has a great impact on overall nitro-
the same  the nitrogen removal rates (Paper gen removal rates. Generally, it is dependent
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
119 day 126 day 128 day 162 day 119 day 126 day 128 day 162 day 119 day 126 day 128 day 162 day
no DO DO DO+NO2
Nrem NH4-N NO2-N
Fig. 32. Juxtaposition of the tests with anoxic conditions (noDO), aerobic conditions (DO) and
aerobic conditions with addition of a NaNO solution (DO+NO
2 2-N).
44
-2
-1
N removal rate [g N m
d ]
Comparative study on different Anammox systems
Table 16. Kinetic pa-
Vmax KM KI
series
rameters estimated for
g m-2d-1 g (g d.w.)-1d-1 gN-NH4 m-3 gN-NH4 m-3
batch tests.
5th 1.64 0.13 3.01 1417
6th 2.03 0.10 3.38 1519
on the nitritation rate. In order to determine
Evaluation of kinetic parameters
the significance of the presence of the nitrite
Previous tests confirmed that in the single
on the overall nitrogen removal rate, several
stage partial nitritation/Anammox process,
batch tests were performed. Estimation of
the activity of the aerobic ammonium oxidiz-
the nitrogen removal rate at oxygen rich and
ing bacteria and the nitrite production rated
oxygen free conditions was made. Moreover,
had a great impact on the overall reactor
additional series of batch tests in aerobic
performance (Paper III and IV). For that
condition with addition of a NaNO2 solution
reason, it was necessary to evaluate a simple
were performed. Investigation of the influ-
method, allowing to monitor the activity of
ence of additional nitrite supply to the batch
aerobic ammonium oxidizing bacteria. The
volume was the main aim of these tests (Pa-
oxygen uptake rate (OUR) measurements
per III). Figure 32 shows the results from all
turned out to be an excellent tool for estima-
batch tests.
tion of the activity of different microorgan-
Obtained results proved, that the nitrite
ism (Paper V). Application of OUR tests
concentration seems to be the rate-limiting
allows measuring the bacteria activity quickly
factor for the simultaneous nitrita-
and with good repeatability, and without a
tion/Anammox process. Moreover, the tests
need of using expensive chemicals. Other
showed that the best results were obtained
tests were designed to determine kinetic
for those performed under anoxic conditions.
parameters of reactions performed by aerobic
It could be explained by competition for
organisms in bacterial community. The expe-
substrate by aerobic and anaerobic ammo-
riments showed that due to very high inhibi-
nium oxidizers or by partial penetration of
tion coefficient the aerobic ammonium oxi-
the oxygen into the inner, Anammox layer of
dizing bacteria seems to be very resistant to
the biofilm. These results revealed that more
high substrate concentration. Generally it can
detailed investigation on dissolved oxygen
be stated, that inhibition effect of ammonia
influence is necessary. Due to this reason the
nitrogen can be neglected.
impact of the oxygen concentration on the
Additionally, for evaluation of the different
nitrogen removal rates was examined (Paper
bacteria activity and to determine kinetic
IV). It turned out, that the highest nitrogen
parameters of nitrogen removal several series
removal rates were obtained for the dissolved
of batch test were performed (Paper V).
oxygen concentration around 3 g O2 m-3.
Tests were performed in different periods of
However, it can be stated, that the dissolved
pilot-plant operation in order to evaluate the
oxygen concentration above 2 g O2 m-3 had
influence of long time process operation on
not significant effect on the nitrogen removal
kinetic parameters. The results of calculated
rates. On the other hand, at a DO concentra-
parameters are presented in table 16.
tion of 4 g O2 m-3 increases of nitrite and
Obtained results of Vmax, are significantly
nitrate nitrogen concentration in the batch
higher than the results obtained for two-step
reactor were noticed. It was also observed
process (Paper I). These results showed that
that increase of biofilm thickness during the
one-step simultaneous partial nitrita-
operational period had no influence on nitro-
tion/Anammox process is a better option
gen removal rates in the pilot plant.
that two-step system.
45
Grzegorz Cema TRITA LWR PhD Thesis 1053
Table 17. Characteristics of the influent medium and operational parameters of the two-stage
RBC.
Samples
Parameter Unit Average Min. Max. St. dev.
No.
NH4-N in g m-3 421.0 176.0 658.8 148.8 44
NO2-N in g m-3 524.1 215.4 830.0 181.0 44
NO2-N/NH4-N - 1.3 0.7 2.3 0.3 44
N removal efficiency % 71.7 4.4 98.4 18.6 41
COD0 gO2 m-3 669.2 200.0 1700.0 217.5 42
Flow rate m3 d-1 0.004 0.002 0.006 0.001 44
HRT d 3.3 2.2 6.9 0.8 44
Temperature reactor �C 17.0 12.0 23.6 2.7 43
pH-value in - 8.1 7.0 8.6 2.4 41
pH-value out - 7.9 6.5 9.0 0.7 43
DO I stage gO2 m-3 2.6 1.6 4.4 0.7 31
During the operational period, a slight de-
V-5. Rotating Biological Reactor  two
crease in pH-value between influent and
step process
effluent was noticed. The pH-value in the
influent was equal to 8.1 ą 2.4 whereas in the
Process performance evaluation
effluent it was 7.9 ą 0.7. During the partial
The RBC was run for six months at 17 ą
nitritation reaction, a large decrease in pH
2.4�C which was much lower than the opti-
value is normally observed, while a rise of pH
mum temperature reported for the Anam-
value is characteristic for the Anammox
mox process (Paper VI). However, Siegrist et
reaction due to cell synthesis. The decrease of
al. (1998) observed a significant nitrogen loss
pH value indicates that there is intensive first
(ranging from 27 to 68%) in RBC during the
step of nitrification in the first stage of the
treatment of stabilized leachate at tempera-
reactor.
tures ranging from 15 to 20�C, but they were
not sure whether it resulted from the Anam- Nitrogen loading rates applied to the first
mox process or from autotrophic denitrifica- stage of the RBC were gradually increased
tion. Table 17 presents statistical evaluation from 3 to 6 g N m-2d-1, and based on the
increasing nitrogen concentration in the
of the parameters measured during operation
of the RBC. influent three research period can be differ-
entiated (Fig. 33). The influent ammonium
1600 influent Stage I effluent
1400
Period I Period II Period III
1200
1000
800
600
400
200
0
1 8 18 25 32 38 50 57 64 71 81 88 106 113 121 127 136 143 148 155 165 171
Time [days]
Fig. 33. Inorganic nitrogen removal in RBC.
46
-3
Nitrogen concentration [g N m ]
Comparative study on different Anammox systems
The process of nitrogen removal predomi-
1400 NO3-N NO2-N NH4-N COD
nated in the first stage of the contactor, pro-
1200
viding 88 and 95% ammonium and nitrite
nitrogen removal, respectively, during the
1000
whole period of operation. In Figure 34 an
example of nitrogen profiles in the RBC unit
800
in period I (day 18), period II (day 53) and
600
period III (day 116) are shown. These pro-
files confirm that the process of inorganic
400
nitrogen removal predominated in the first
200
stage of the contactor. Moreover, it was
associated with high nitrate production as a
0
result of aerobic nitrification. Further re-
moval of remaining nitrates took place in the
third stage of the contactor where glucose as
18 53 116
external carbon source was added. Addition-
Fig. 34. Example of nitrogen profile in the
ally, the nitrite-to-ammonium ratio was also
RBC (days: 18, 53 and 116).
changeable in the influent and varied from
0.7 to 2.3 (Paper VI). However, it seems that
and nitrite nitrogen concentration were
this variation had no influence on the inor-
gradually increased and remained within the
ganic nitrogen removal efficiency.
range of 176  658.8 g N m-3 and 215.4 -
Additional information about process
839.0 g N m-3 respectively (Fig. 33). The
stoichiometry are given by the ratio between
inorganic nitrogen removal efficiency in the
nitrate production and ammonium consump-
first stage of the contactor was 58 ą 17% on
tion (NO3-/NH4+), as well as the ratio be-
average during the whole operating time and
tween nitrite conversion and ammonium
it was comparable during subsequent periods
conversion (NO2-/NH4+) (Fig. 35). The aver-
I, II and III (58, 55 and 57% respectively).
age conversion ratio of nitrite-to-ammonium
This indicates that the nitrogen removal
was equal to 1.46, varied from 0.7 to 4.3, and
efficiency was independent of the nitrogen
was higher than value described by Strous
load in the applied range. Most probably, the
and co-workers (1998). However, this ratio
Anammox process was the main cause of
was comparable to 1.43 obtained in the rotat-
nitrogen removal (Paper VI).
ing biological contactor by Wyffels and co-
workers (2003). Substantial fluctuations of
4.5
NO2/NH4 NO3/NH4
4.0
NO2-/NH4+ = 1.32 according
3.5
NO3-/NH4+ = 0.26 according
to stoichiometry
to stoichiometry
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0 20 40 60 80 100 120 140 160 180
Time [days]
Fig. 35. Conversion ratio of nitrite and ammonium and between nitrate production and ammo-
nium conversion.
47
-3
Nitrogen Compounds and COD [g N m ]
In
In
In
Out
Out
Out
St. I
St. I
St. I
St. II
St. II
St. II
St. III
St. III
St. III
-
+
3
4
-
+
2
4
Conversion ratios:
NO
:NH
and NO
/NH
Grzegorz Cema TRITA LWR PhD Thesis 1053
100
90
80
70
60
50
40
30
20
10
0
1 15 25 36 50 60 71 85 106 116 127 141 148 163 171
Time [days]
% N inrg removal % NH4-N removal % NO2-N removal
Fig. 36. Ammonium, nitrite and inorganic nitrogen removal efficiency in the first stage of the
RBC.
the ratio can be explained by the competition earlier consumption of substrates for nitrifi-
between nitrifiers and Anammox bacteria. cation in the first stage and there was no
The occurrence of nitrification is also con- reaction in the second stage of the contactor.
firmed by the drop in pH from 8.1 in the The aerobic conditions, in stage I, were con-
influent to 7.1 in stage I. The conversion ducive to nitrification proceeding in the sys-
ratio between nitrate production and ammo- tem, and resulted in nitrate production
nium conversion was 0.84 ą 0.44 what was amounting to 50% of nitrogen inflow. Ac-
much higher than the stoichiometric value of cording to the stoichiometry of the Anam-
0.26 (Strous et al., 1998). This phenomenon mox reaction (Strous et al. 1998), nitrate
shows that high nitrite oxidizers activity production should be equal to 11% nitrogen
occurred in the first stage of the contactor. inflow, and the difference was caused by
Based on the Anammox process stoichiome- presence of nitrite oxidizers in the biofilm.
try, no more than 30% of produced nitrates Nitrobacter and Nitrospira were the species
were due to anaerobic ammonium oxidation responsible for oxidation of nitrite to nitrate
activity. and they competed for substrate with the
Anammox bacteria.
The ammonium, nitrite and inorganic nitro-
gen removal efficiencies in the first stage of Between the 130th and the 150th day of the
the RBC unit are shown in Figure 36. During experiments, a temporary breakdown of the
the whole research period, the average am- process performance was observed. It is
monium and nitrite nitrogen removal effi- difficult to explain, because no operating
ciency were on the level of 87.8 ą 15.8% and problem was noticed. It is conceivable that
94.8 ą 4.8%. the Anammox bacteria are inhibited (reversi-
bly) if exposed to aerobic conditions
The maximum ammonium and nitrite re-
(Schmidt et al., 2003). For two weeks before
moval rates were 3.0 and 3.9 g N m-2d-1,
this breakdown, the ammonium concentra-
respectively. The maximum inorganic nitro-
tion in stage I dropped much below 20 g
gen removal rates were 5.8 g N m-2d-1 (0.93
NH4-N m-3; this could lead to the process
kg N m-3d-1) with an average value of 2.8 g N
inhibition due to the deeper penetration of
m-2d-1 (Paper VI). The aeration of treated
oxygen into the biofilm. Siegrist et al. (1998)
medium resulted in the DO concentration
also observed this phenomenon. It could be
amounting to 2.6 ą 0.7 g O2 m-3 in the first
also caused by the increase of nitrite nitrogen
stage of the contactor. In the second stage
up to 90 g m-3 on the 123rd day of the ex-
the DO concentration rose to
periment, however, on the contrary high
4.8 ą 0.9 g O2 m-3 probably as a result of
peaks of nitrite concentration on the 67th and
48
Nitrogen removal [%]
Comparative study on different Anammox systems
71st days (around 90 g m-3) did not cause such Kinetic evaluation of process
problems. The Anammox process (for the
It was shown that the Stover-Kincannon
type Brocadia Anammoxidans) is irreversibly
model can be used to describe the ammo-
inhibited by nitrite at concentrations exceed-
nium and nitrite removal rates in the RBC
ing 70 g N m-3 for longer period (Schmidt et
(Paper VI). However, it appeared that the
al., 2003; Dapena Mora 2007). Egli and co-
Stover-Kincannon model was not appropri-
workers (2001) showed that Kuenenia stutt-
ate for the process of the inorganic nitrogen
gartiensis has a tolerance to nitrite even up to
removal (correlation coefficient R = 0.62).
180 g N m-3. However, there is no informa-
This suggested the existence of different
tion about short-term effects of high nitrite
enzyme systems acting in the nitrogen trans-
nitrogen concentration in the Anammox
formations. Probably the lower correlation
reactor. This could mean that the process is
was due to participation of other microorgan-
insensitive to short-term high nitrite concen-
isms in nitrogen conversion. For instance
trations in the reactor (even up to 100 g
Nitrosomonas is able to deammonify and,
NO2-N m-3 for several hours). It resulted in
perhaps to a small extent, heterotrophic
only a temporary decrease of nitrogen re-
denitrifiers able to use small amounts of
moval rates. Therefore, the breakdown of the
biodegradable carbon in the influent. The
process efficiency could be due to the over-
estimated values of the maximum substrate
lap of these two phenomena: deeper oxygen
utilization rates (16.72 and 44.05 g m-2d-1 for
penetration into the biofilm due to low am-
ammonium and nitrite nitrogen, respectively)
monium concentration in the bulk liquid, as
showed the possibility of a still higher nitro-
well as high nitrite concentration. The other
gen load to the RBC.
possibility is that the Candidatus Kuenenia
Looking for bacteria populations
stuttgatiensis was the dominating Anammox
The presence of the Anammox bacteria
bacteria in the reactor and consequently there
belonging to Candidatus Brocadia anammoxidans
were more resistant to high nitrite concentra-
and/or Candidatus Kuenenia stuttgatiensis in the
tion.
first two stages of the contactor was also
The final nitrogen removal was performed in
proved (by FISH analyses), confirming the
the stage III of the RBC unit by using a sup-
main role of the Anammox process in the
plementary biodegradable organic (glucose)
nitrogen removal in the first stage of the
as an external carbon source for denitrifica-
RBC (Paper VI). Additionally, microbial
tion. The average COD/N ratio was equal to
analysis confirmed he presence of nitrifiers in
6.2 ą 0.7. The removal of nitrate entering the
the biofilm (Table 18). These data suggested
third stage of the contactor was 34 % on
that the two simultaneous processes of am-
average, which corresponded to a nitrogen
monium oxidation and Anammox could
removal rate of 0.68 g NO3-N m-2d-1. The
occur in one single RBC unit.
average efficiency of inorganic nitrogen re-
Referring to studies of Egli et al. (2003), it
moval achieved in the whole RBC was 77%.
was proved that within the RBC biofilm
depth, ammonium oxidizing bacteria are on
Table 18. Identified nitrifiers and Anammox bacteria.
Anaerobic ammonium-
Most
oxidizing bacteria,
Nitrosomonas halophilic and
Nitrobacter genus Candidatus  Brocadia
oligotropha halotolerant
spp. Nitrospira anammoxidans and
lineage Nitrosomonas
Candidatus  Kuenenia
spp.
stuttgartiensis
Stage I - +++ ++ +++ +++
Stage II + ++ ++ ++ +
Stage III + + - + -
Scale: (-)  absence; (+)  few; (++)  middle; (+++)  high availability
49
Grzegorz Cema TRITA LWR PhD Thesis 1053
Bacteria identified Stage I Stage II Stage III
Table 19. Identified
Acinetobacter calcoaceticus + - -
denitrifying bacteria
Pseudomonas fluorescence + + -
strains.
Alcaligenes xylodoxidans + - -
Pseudomonas alcaligenes - ++ -
Proteus vulgaris -+-
Aeromonas salmonicida - - ++
Aeromonas hydrophila - - ++
Pseudomonas earuginosa - - +
Shigella spp - - +
Acinetobacter lwoffi - - +
++ - Dominating strain
the outer layer, and the Anammox bacteria phila were the dominating denitrifying strains
were only detected in the lower part of the in the third stage of the contactor. They are
biofilm, defined by penetration depth of the Gram-negative, ubiquitous organisms. They
oxygen into the biofilm. are widespread in the mixed liquor of acti-
vated sludge plant when they are involved in
Using commercial identification kits API
the degradation of organic matter. They are
20NE and API 20E, in the biofilm from all
known as incompletely denitrifying hetero-
three stages of the reactor, the denitrifying
trophic bacteria showing strong reduction of
bacteria strains have been identified (Table
nitrates to nitrites (Drysdale et al., 1999).
19).
Moreover, some authors have observed that
Microbial analysis in the stage I of RBC re-
these bacteria preferred oxic conditions for
vealed the presence of denitrifying bacteria
growth but were still able to produce nitrites
such as: Acinetobacter calcoaceticus, Pseudomonas
from nitrates reduction while simultaneously
fluorescence and Alcaligenes xylodoxidans, fol-
utilising oxygen as a final electron acceptor.
lowed by: Pseudomonas fluorescence, Pseudomonas
The domination of denitrifiers responsible
alcaligenes, Proteus vulgaris in stage II (Table 19).
for partial denitrification of nitrates only to
However, the biodegradable organic carbon
nitrites and the presence of nitrite oxidizers
to nitrogen ratio was unfavourable for het-
in the stage III may be an explanation of the
erotrophic denitrification. Therefore, hetero-
low efficiency of nitrate removal. The average
trophic denitrification could not be responsi-
oxygen concentration in the bulk liquid was
ble for nitrogen removal in the first stage of
3.0 ą 1.5 g O2 m-3; this created favourable
the RBC. The presence of Nitrosomonas spp.
conditions for nitrifiers that oxidized nitrites
in the biofilm suggested that part of the ni-
produced by Aeromonas spp.
trogen loss could be also attributed to denitri-
fying Nitrosomonas cells located in the lower
part of the biofilm. They could use ammo-
nium as electron donors and nitrite as elec-
tron acceptor in case of oxygen lack (Bock et
al., 1995; Helmer and Kunst, 1998).
The results confirmed presence of Pseudomo-
nas considered as a species responsible for
denitrification. It is in agreement with the
recent results of Ju et al. (2005) who found
that Pseudomonas earuginosa is able to denitrify
under aerobic condition when DO concen-
tration exceeded 1 g O2 m-3. Likewise, in an
early observation, Bang et al. (1995) reported
the presence of aerobic denitrification in the
RBC reactor even when the DO concentra-
tion exceeded 3 g O2 m-3 in the treated me- Fig. 37. Photos of the biofilm in the denitri-
dium. Aeromonas salmonicida, Aeromonas hydro- fication part of the RBC.
50
Comparative study on different Anammox systems
1800
influent Stage I effluent
1600
Period II
Period
Period I
PerioIII
1400
1200
1000
800
600
400
200
0
1 16 31 46 61 76 91 106 121 136 151 166 181 196 211 226
Time [days]
Fig. 38. Inorganic nitrogen removal in RBC.
Glucose appears to be the least efficient
V-6. Rotating Biological Reactor  one-
carbon source in comparison with the
step process
methanol and acetic acid used in previous
research. Efficiencies of 83 to 97% in nitro-
Process performance evaluation
gen removal respectively were achieved for
The Rotating Biological Contactor (RBC)
the both organic carbon sources (unpub-
treating real landfill leachate from two Polish
lished data).
landfill sites was running for 8 months with
Additionally, it seems that with glucose as an
average operational temperature equal to 20.6
external carbon source, intensive biofilm ą 1.1�C. Three research periods can be dif-
growth accompanied nitrate reduction and ferentiated based on the increasing nitrogen
aerobic degradation. In the third stage of the
concentration in the influent (period I: day 1
RBC very intensive growth was observed, to 45; period II: day 45 to 159; period III: day
and discs were completely covered with a 159 to 230). (Fig. 38). During the first one,
white, fluffy biofilm (Fig. 37)
the start up of the Anammox process took
place. The second one was the period of the
Moreover, different denitrifying bacteria
stable work of the reactor. The last one was
strains were noticed when methanol and
the period when the process inhibition oc-
acetic acid were applied (unpublished data).
curred (Paper VII).
For biofilm fed with methanol the following
bacteria strains have been identified: Aeromo- The reactor was supplied with landfill
nas hydrofilla, Aeromonas salmonicida (dominat- leachate that contained high concentration of
ing strain), Aeromonas mausoucida, Pseudomonas
ammonium nitrogen varying from 891 to
aeruginosa (dominating strain), Pseudomonas 1562 g m-3 with an average value amounted
fluorescence and for biofilm fed with acetic acid: to 1173 ą 234 g m-3 (Fig. 38).
Pseudomonas shigellides, Pseudomonas alcaligenes
In the third period of reactor operation,
and Aeromonas sorbia.
strong process inhibition was noticed (Paper
During the operational period it was proved VII). The nitrite nitrogen concentration in
that, the process is insensitive to short-term
the reactor exceeded 100 g m-3 and due to
high nitrite concentrations in the reactor
this, the nitrogen concentration in the influ-
(even up to 100 g NO2-N m-3 for several ent, and the same nitrogen load, had to be
hours) (Paper VI). decreased from above 1000 g m-3 to 300 
400 g m-3.
After process breakdown, changes in biofilm
characteristics in the first stage of the contac-
tor were noticed. During stable process op-
51
-3
N concentration [g N m
]
Grzegorz Cema TRITA LWR PhD Thesis 1053
Fig. 39. Photos of the
biofilm in the first
stage of the contactor
A) 69th day, Period II 
stable process perfor-
mance, B) 222th day,
Period III  after
process inhibition.
eration, intensive biofilm growth was ob- decreased. Interesting is also comparison of
served. Process inhibition caused significant the test from second and third period of
biofilm detachment from the disc, and at the experiment. In the test performed in the third
same time biofilm changed colouring from period, a slight increase of nitrite nitrogen
light to very dark brown (Fig. 39). concentration after 60 minutes was observed,
whereas in second period nitrites concentra-
Nitrogen conversion
tion decreased for whole test. It could be
Additional information about process per-
concluded, that mainly the Anammox proc-
formance in the first stage of the RBC gave
ess was inhibited during the breakdown of
batch tests carried out in second and third
the process performance, and it seemed that
period of the experiment. Fig. 40A and B
partial nitritation was not affected.
shows the nitrogen conversion during the
Kinetic evaluation of process
batch tests.
The analysis of data indicated that the Stover-
In both test, the ammonium nitrogen re-
Kincannon model well described the process
moval during the whole test was higher than
of ammonium and inorganic nitrogen re-
nitrite nitrogen while in the Anammox proc-
moval (Paper VII). Due to the fact, that
ess should be the opposite situation accord-
obtained value of KB (the saturation con-
ing to the stoichiometric reaction. However,
stant), the equation become the equation of
two subsequent processes  aerobic oxida-
the first order reaction (Gonz�lez-Mart�nez
tion to nitrite and the Anammox process,
and Duque-Luciano, 1991). The Stover-
used ammonium nitrogen. It was also inter-
Kincannon equation can be rewritten as:
esting that the nitrogen removal was higher
during first 40 minutes of the test. This phe-
Q �"Si
rA = K �" (7)
nomenon could be explained by very high
A
nitrite nitrogen concentration during the
Where:
batch tests, which exceeded 100 g m-3 what
K  is a proportionality coefficient.
caused the Anammox process inhibition and
the same nitrite removal rate after 40 minutes
200 200
NH4-N NO2-N NO3-N
N-NH4 N-NO2 N-NO3 Fig. 40. Example of
180 180
nitrogen conversion in
160 160
the batch tests A)
140
140
Period II, B) Period
120
120
III.
100
100
80
80
60
60
40
40
20
20
0
0
0 20 40 60 80 100 120 140
0 20 40 60 80 100 120 140
A B
Time [min]
Time [min]
52
-3
-3
Nitrogen concentration [g m ]
Nitrogen concentration [g m ]
Comparative study on different Anammox systems
Table 20. Identified nitrifiers and the Anammox bacteria in the first stage of the contactor (AOB
 ammonium oxidizing bacteria; NOB  nitrite oxidizing bacteria; AAOB  anaerobic ammo-
nium oxidizing bacteria).
AOB NOB AAOB; B.
anammoxidans
Day
Nitrosomonas
and/or K.
Nitrosomonas sp. Nitrobacter sp. Nitrospira sp.
oligotropha
stuttgartiensis
16 +++ ++ ++ + -
44 ++++ ++ +++ + -
75 ++++ + - - -
110 ++++ + - - +
135 +++ - - - +++
169 +++ - - - +++
200 +++ - - - +++
236 ++++ - - - +++
Scale: (-)  absence; (+)  few; (++)  middle; (+++)  high availability; (++++) - dominant
Nitrosomonas oligotropha were present only
Vmax
K = (8)
during the first period of the experiment and
KB
in the first stage of the contactor also at the
The relation between load and the removal
beginning of the second period, and it did
rate results is linear for the tested values of
not develop to a larger sized population
load to the reactor, according to the model
under the operating conditions of the reactor.
proposed by Eckenfelder (Gonz�lez-
Egli and co-workers (2003) analysed changes
Mart�nez and Duque-Luciano, 1991).
in bacteria population during the start-up of
the simultaneous partial nitrita-
Looking for bacteria populations
tion/Anammox process in the RBC. They
Microbial analysis (FISH) confirmed the
identified Nitrosomonas eurpaea/eutropha, Nitro-
coexistence of nitrifiers and the Anammox
somonas urea/oligotropha and Nitrosomonas com-
bacteria belonging to Candidatus Brocadia
munis in the reactor, however, N. euro-
anammoxidans and/or Candidatus Kuenenia
pea/eutropha become the dominating stain in
stuttgatiensis in the rotating biological contac-
the reactor. This might indicating, that also in
tor (Paper VII). These data confirm the
presented research this bacteria strain was
main role of the simultaneous partial nitrita-
dominating aerobic ammonium oxidizer in
tion/Anammox process in the nitrogen re-
the system.
moval. In table 20, 21 and 22 result of bacte-
Nitrobacter sp. and Nitrospira sp. were identified
ria population analysis in the first, second and
as bacteria responsible for aerobic nitrite
third stages of the contactor are presented,
oxidation. However, theses bacteria were
respectively.
identified only during the first period of the
During the whole research period the Nitro-
experiment when nitrates were the main
somonas sp. were the main aerobic ammonium
product of ammonium oxidation. In the later
oxidizers all stages of the contactor. The
period when nitrate production was much
Table 21. Identified nitrifiers and the Anammox bacteria in the second stage of the contactor.
AOB NOB AAOB; B.
anammoxidans
Day
Nitrosomonas
and/or K.
Nitrosomonas sp. Nitrobacter sp. Nitrospira sp.
oligotropha
stuttgartiensis
16 +++ + ++ + -
44 +++ ++ +++ + -
75 +++ + - - -
110 +++ + - - +
135 +++ - - - +++
169 +++ - - - +++
200 +++ - - - +++
236 ++++ - - - +++
Scale: (-)  absence; (+)  few; (++)  middle; (+++)  high availability; (++++) - dominant
53
Grzegorz Cema TRITA LWR PhD Thesis 1053
Table 22. Identified nitrifiers and the Anammox bacteria in the third stage of the contactor.
AOB NOB AAOB; B.
anammoxidans
Day
Nitrosomonas
and/or K.
Nitrosomonas sp. Nitrobacter sp. Nitrospira sp.
oligotropha
stuttgartiensis
16 +++ + +++ + -
44 +++ + +++ + -
75 +++ - - - -
110 +++ - - - +
135 +++ - - - +
169 +++ - - - +
200 +++ - - - +
236 +++ - - - +
Scale: (-)  absence; (+)  few; (++)  middle; (+++)  high availability; (++++) - dominant
lower, probably some other bacteria, which inner part of the biofilm (Egli et al., 2003), it
were not identified, were responsible for may explain such late detection of these
second step of nitrification. Generally, nitrite bacteria. The Anammox bacteria predomi-
oxidizing bacteria were present in much nated mainly in the first and second stage of
lower quantity than the aerobic ammonium the contactor, however they were also de-
oxidizers. tected in the last stage. Interesting is, that
amount of the Anammox bacteria became
The presence of Candidatus Brocadia anam-
stable after process breakdown. It was possi-
moxidans and/or Candidatus Kuenenia stuttgatien-
ble that there was rather inhibition of bacteria
sis, especially in the first two stages of the
activity than cell s death.
RBC, was also proved. However, they were
detected in the middle of the second period
VI  SYSTEMS COMPARISON
in 110 day of experiment, whereas, high
nitrogen removal efficiency exceeding 70%
Table 23 compiles results from performed
were form 45th day. It can be as a result of
studies with operation of one- and two-step
the methodology of samples collection for
partial nitritation/Anammox in different
FISH analysis. For bacteria analysis, detached
reactors  MBR, MBBR and RBC.
biofilm samples from bottom of the reactor
During the performed research, it was possi-
were collected. Taking under consideration,
ble to achieve high nitrogen removal effi-
that the Anammox bacteria are present in the
ciency in all systems. In all systems the
Table 23. Results and operational parameters for all described systems.
MBBR*** MBBR RBC RBC****
parameter unit MBR**
two-step one-step two-step one-step
N rem. % 47.5ą12.7 86.0ą8.6 58.7ą12.6 58 ą17* 74.6ą9.5*
N-load kg m-3d-1 0.06ą0.02 0.07ą0.01 0.6ą0.1 0.9ą0.24* 1.12ą0.23*
N-load g m-2d-1 - 0.28ą0.06 2.42ą0.06 4.74ą1.1* 5.99ą1.24*
N rem. rate kg m-3d-1 0.03ą0.018 0.06ą0.01 0.34ą0.08 0.53ą0.20* 0.83ą0.32*
N rem. rate g m-2d-1 - 0.25ą0.05 1.39ą0.31 2.80ą1.20* 2.55ą1.73*
NH4-N in g m-3 49.5ą14.5 124.4ą31.0 577.0ą87.4 421.0ą148.8 755.5ą380.9
NO2-N in g m-3 53.8ą20.3 157.3ą29.5 - 524.1ą181.0 -
NO2-Nconv/NH4-Nconv - 1.05ą0.69 1.35ą0.27 - 1.46ą0.57 -
NO3-Nprod/NH4-Nconv - 0.8ą0.46 0.15ą0.07 0.1ą0.05 0.84ą0.44* 0.32ą0.29*
pH-value in - 8.0ą0.1 7.3ą0.4 7.8ą0.1 8.1ą2.4 8.1ą0.5
pH-value out - 8.0ą0.1 8.3ą0.3 7.8ą0.2 7.9ą0.7 8.3ą0.7
DO g m-3 <0.2 0.14ą0.07 2.03ą0.95 2.6ą0.7* 1.8ą0.7*
Temp. reactor �C 33.9ą0.7 31.4ą2.7 24.7 17.0ą2.7 20.6ą1.5
* - for the first stage of the RBC where the Anammox process took place; ** - For MBR results
for period from 78th to 148th day  from start of the nitrogen removal to process breakdown; ***
- Results only for the Anammox reactor; **** - Results for stable process operation  period II.
54
Comparative study on different Anammox systems
120
N inorg. NH4-N NO2-N
100
80
60
40
20
0
MBR MBBR two-step MBBR one-step RBC two-step RBC one-step
Fig. 41. Ammonium, nitrite and inorganic nitrogen removal efficiency  comparison of different
systems. (For MBR results for period from start of the nitrogen removal to process breakdown;
for RBC one-stage results for stable process operation  period II; for RBC two-stage as well
one-stage results are for the first stage of the contactor where Anammox process took place).
Anammox process took place what was tor. Very high removal efficiency of ammo-
confirmed by FISH analysis (with except for nium and nitrite nitrogen achieved for the
MBR  no FISH assay was performed). Reac- MBBR two-step and RBC two-step and the
tors were supplied with different feeding values were on the similar level. However, in
media. The MBR was supplied with synthetic the RBC reactor the inorganic nitrogen re-
wastewater, MBBR with reject water from moval efficiency was much lower than in the
dewatering of digested sludge and the RBC MBBR and was equal to 58 ą 17% on aver-
was supplied with municipal landfill leachate age during the whole operating time.
from two landfill sites. The highest average
The difference between these two systems
nitrogen removal efficiency equal to 86%
was mainly in the nitrate production to am-
during the whole research was achieved for
monium conversion ratio. In the MBBR two-
two-step partial nitritation/Anammox proc-
step, this ratio was equal to 0.15ą 0.07 and
ess in the Moving Bed Biofilm Reactor, but
was lower than the stoichiometric one,
the N-load was very low. The lowest value
whereas in the two-step RBC the ratio was
was achieved for the MBR and was equal to
equal to 0.84 ą 0.44 indicating high nitrite
47.5 ą 12.7%. However it should be stated
oxidizers activity in the first stage of the
that in the MBBR reactor, the Anammox
contactor. It should be stated that in the two-
process was already working, whereas in the
step RBC the DO concentration was much
MBR, the start-up of the process took place.
higher, on the level of 2.6 g m-3, while in the
In Figure 41 the comparison of ammonium,
two-step MBBR it was 0.14 g m-3 on average.
nitrite and inorganic nitrogen removal effi-
The DO concentration in the two-step RBC
ciency in different system are presented. For
was comparable rather with values obtained
the MBR, the results for period from 78th to
in single stage systems. Comparing single
148th day are presented  from start of the
stage systems, where partial nitritation and
nitrogen removal to process breakdown.
Anammox took place in one single reactor,
Additionally, also for RBC one-step system,
the nitrogen removal efficiency was higher in
the results from stable process operation are
the RBC than MBBR, and was equal to
presented. Generally, the highest removal
75.7% on average (in the second period)
efficiency was achieved for the MBBR two-
compared to 58.7% achieved in the MBBR.
step process, and the lowest for MBR reac-
55
removal efficiency [%]
Grzegorz Cema TRITA LWR PhD Thesis 1053
0,9 5
4,5
0,8
4
0,7
3,5
0,6
3
0,5
2,5
0,4
2
0,3
1,5
0,2
1
0,1
0,5
0 0
MBR MBBR MBBR RBC two- RBC one- MBR MBBR MBBR RBC two- RBC one-
two-step one-step step step two-step one-step step step
A B
Fig. 42. A) Nitrogen removal rates in kg N m-3d-1  comparison of different systems; B) Nitrogen
removal rates in g N m-2d-1 - comparison of different systems. (For MBR results for period from
start of the nitrogen removal to process breakdown; for RBC one-stage results for stable process
operation  period II; for RBC two-stage as well one-stage results are for the first stage of the
contactor where Anammox process took place).
It can also be noticed (from standard devia- 0.08 kg N m-3d-1, whereas in single unit a
tion values), that during whole operational mean nitrogen removal rate of
period in the RBC much higher fluctuation in 0.8 kg N m-3d-1 was observed.
the nitrogen removal efficiency was recorded.
Conducted studies showed that nitrogen
However also there were higher fluctuations
removal rates in the two-step RBC system
in influent ammonium concentration than in
(equal to 2.8 ą g N m-2d-1 and 0.53 ą 0.20 kg
the one-step MBBR. It also seems that char-
N m-3d-1, in the first stage of the contactor)
acteristics of the landfill leachate influenced
were lower than for one-step process (4.57 ą
the process performance in a higher extend
1.10 g N m-2d-1 and 0.83 ą 0.17 kg N m-3d-1,
than the reject water.
in the first stage of the contactor for stable
Obtained values of nitrogen removal rates in process operation - period II). Additionally in
MBBR system, showed that one-step partial the two-step RBC higher nitrate production
nitritation/Anammox process is a better to ammonium conversion ratio was noticed,
option for reject water treatment that two- and it was 0.84 ą 0.44 and 0.32 ą 0.29 (0.17
step process. The average inorganic nitrogen for stable process operation) for two-step
removal rate in two-step MBBR system was and one-step respectively. In the two-step
0.25 ą 0.05 g N m-2d-1 (0.06 ą 0.01 kg N m- RBC nitrite nitrogen from influent was oxi-
3
d-1) and was five time lower than in one-step dized by nitrite oxidizers to nitrates, whereas
MBBR system where achieved 1.39 ą 0.31 g in one-step process nitrite oxidizers loosed
N m-2d-1 (0.34 ą 0.08 kg N m-3d-1). Figure the competition for nitrite with the Anam-
42A and B show the nitrogen removal rates mox bacteria. During the whole research, the
in all investigated systems. highest removal rates were achieved for the
one-step RBC and were 2.5 time higher that
V�zquez-Pad�n and co-workers (2009) ob-
for one-step MBBR system.
served similar difference in nitrogen removal
rates between two-step and one step process. AOB and Anammox bacteria are slow grow-
They used sequencing batch reactors (SBR) ing microorganisms which optimal tempera-
for treating supernatant of an anaerobic ture of operation is around 30�C. For this
digester. The combined system allowed the reason, Anammox and CANON process
removal of nitrogen loading rates around were mostly applied to effluents with tem-
56
-3
-1
-2
-1
N removal rate [kg N m d ]
N removal rate [g N m d ]
Comparative study on different Anammox systems
peratures higher than 30�C (V�zquez-Pad�n et the temperature dependence is strongly af-
al., 2009). Therefore, the MBR and two-step fected by the type of Anammox culture used.
MBBR systems were operated at tempera-
In table 24 there is a comparison of condi-
tures exceeding 30�C. However, Szatkowska
tions (temperature and DO concentration)
and Płaza (2006) and Dosta et al. (2008)
and results in different systems for one-step
demonstrated that the impact of temperature
partial nitritation/Anammox process.
on Anammox process is significant in case of
Due to the fact, that the Anammox bacteria
sudden decrease of temperature. Moreover,
are reversibly inhibited by very low concen-
authors proved that bacteria exposed for low
trations (<1 �M) of oxygen (Kartal et al.,
temperatures for longer period are able to
2007), the Anammox process in the MBR
adapt to new conditions and work more
reactor and two-step MBBR was operated
efficiently. Due to this reason, the one-step
without any aeration with DO concentration
MBBR system worked under natural tem-
in the bulk liquid below 0.2 g O2 m-3. Addi-
perature of incoming reject water with the
tionally, nitrifiers present in both reactors
average temperature in the reactor equal to
managed with the increased oxygen concen-
24.3 ą 2.6�C. An additional heat was sup-
tration. Different situation took place in the
plied only during winter period to keep tem-
two-step RBC reactor, in which frequent
perature stable. Moreover, the average opera-
rotation of the media by the shaft supplies
tional temperature for the one-step as well
oxygen to the microorganisms both in the
two-step RBC system was even lower and
biofilm and in the bulk liquid. As a result, the
was equal to 20.6 and 17�C for two-step and
average DO concentration in the first zone
one-step system, respectively. Also Siegrist et
of the contactor was equal to 2.6 ą 0.7
al. (1998) and Egli et al. (2001) observed a
g O2 m-3. Such a high DO concentration
significant nitrogen removal in the RBC at
resulted in significant nitrate nitrogen pro-
temperature below 20�C. Recently a few
duction. For one-step process, the partial
authors demonstrated that the Anammox
nitritation is a bottleneck of overall reaction.
process can be operated at temperature be-
Generally, nitrification rate in fixed-film
low 20�C. Isaka and co-workers (2008) dem-
systems is often limited by the bulk liquid
onstrated that removal activity decreased
DO concentration (Metcalf and Eddy, 2004).
gradually with decreasing operating tempera-
Additionally, DO concentration is one of the
ture, however, it still occurred at 6�C with
most often discussed parameter when eco-
nitrogen conversion rate equal to 0.36 kg N
nomic factors are considered in treatment
m-3d-1 with comparison to 2.8 and 6.2 at 22
plant operation (Szatkowska, 2007). On the
and 32�C, respectively. Authors stated that
other hand, oxygen affects the Anammox
Table 24. Comparison of conditions and nitrogen removal in a one-stage nitrogen removal
process.
Reactor type Application T DO N rem N rem Ref
kg m-3d-1
�C g m-3 %
(g m-2d-1)
MBBR Sludge liquor 28.0-29.0 0.8  2.0 71 - 75 (1.2  2.2) 1.
MBBR Sludge liquor 20 - 35 0 - 4 80 (2  2.5) 2.
MBBR Sludge liquor 24.3 2.03 58.7 0.34 (1.39) 3.
RBC Landfill leachate 20.6 1.8 75 0.83 (4.57) 4.
RBC Synthetic wastewater 29.1 0.57 89 (7.4) 5.
RBC Landfill leachate 15 - 20 1 - 3 30 - 70 (0.4  1.2) 6.
RBC High salinity s.w. n.d. <1 84 0.48 7.
SBR Synthetic wastewater 30.0 0.1 n.d. 0.08 8.
SNAP biofilm reactor Synthetic wastewater 35.0 2 - 3 60 - 80 n.d. 9.
Gas-lift reactor Synthetic wastewater n.d. 0.5 42 1.5 10.
SBR Sludge liquor 18 - 24 0.5 85 0.45 11.
1. Seyfried et al., 2002; 2. Rosenwinkel et al., 2005b; 3. This study; 4. This study; 5. Pynaert et al.,
2003 ; 6. Siegrist et al., 1998; 7.Windey et al., 2005; 8. Third. et al., 2005 ; 9. Furukawa et al., 2006;
10. Sliekers et al., 2003; 11. V�zquez-Pad�n et al., 2008; n.d. - no data; s.w.  synthetic wastewater.
57
Grzegorz Cema TRITA LWR PhD Thesis 1053
process since it is a process inhibitor. The process inhibitor in higher concentration.
average DO oxygen concentration measured The one-step process seems to be more cost
in the bulk liquid was equal to 2.03 g O2 m-3 effective since there need only one reactor
for one-step MBBR reactor and 1.8 g O2 m-3 and due to higher nitrogen removal capacity,
for one-step RBC system. However, as it was the smaller reactor volume can be used.
mentioned before, the higher nitrogen re- Moreover, process can be operated under
moval rates were obtained for the RBC. natural temperature of incoming sludge liq-
Other researchers who worked with the uor and in case of RBC reactor even at tem-
MBBR technology report DO concentration peratures below 20�C, therefore the expenses
between 0.8 and 2 g m-3 (Hippen, 2000; Sey- could be reduced.
fried et al., 2002). Under such condition, it
VII  CONCLUSIONS
was possible to reach 75 and 70% of nitrogen
removal, respectively. At the DO value equal
The combined partial nitritation/Anammox
to 5.9 g m-3, the nitrogen removal amounted
process was investigated in three different
only to 10%. According to Johansson et al.
reactors. Additionally process was investi-
(1998), the nitrogen efficiency removal of 60-
gated in two different system configurations
70% can be obtained under the DO concen-
as one- and two-step process. The partial
tration below 1 g m-3. The full-scale MBBR in
nitritation/Anammox process could be suc-
Germany where one-stage partial nitrita-
cessfully applied to treat nitrogen rich
tion/Anammox process was applied gave
streams as landfill leachate and reject water
80% of nitrogen removal efficiency at DO in
originating from dewatering of digested
the range of 0-4 g m-3 (Rosenwinkel and
sludge. Performed research allowed to show
Cornelius, 2005). Oxygen condition and
differences among used systems and to point
nitrogen removal rates presented in this the-
out the best system solution. This research
sis are comparable to results reported by
has shown that during the start-up period,
Seyfried et al. (2002). However, the German
the Anammox process is very sensitive and
experiments were run under higher tempera-
unstable. The conclusion are summarised
tures. Pynaert et al (2003) demonstrated ni-
briefly below:
trogen removal in the RBC at the level of
Membrane assisted BioReactor (MBR)
89% at the very high nitrogen surface load
ranging from 4 to 8 g N m-2d-1. In compari- 1. The experiments carried out proved that
son to RBC presented in this study, they retention of biomass in Membrane as-
achieved DO concentration equal to 0.57 g sisted BioReactor and long sludge age give
O2 m-3. However, their reactor was operated possibility for implementation of the
at temperature 29.1�C, what was significantly Anammox process.
higher value than 20.6�C presented in this
2. At the temperature above 30�C, dissolved
study.
oxygen concentration below 0.3 g O2 m-3
Conducted experiments on different systems and at very low contents of biodegradable
with both two- and one-stage partial nitrita- organic compounds within 5 months of
tion/Anammox process demonstrated that process operation over 75% of nitrogen
one-stage system is a better option for nitro- removal was reached and very intensive
gen removal from ammonium rich streams gas production was observed.
irrespective of kind of wastewater. It was
3. The research confirmed that very long
possible to obtain higher nitrogen removal
time, over fourth months, is needed for
rates and moreover, one-step system was less
implementation of the Anammox process.
complicated in operation as there was no
4. Performed OUR tests bear out presence
need for nitrite-to-ammonium control in the
of ammonium and nitrite oxidizing bacte-
influent. Additionally, there was no risk with
ria in the Anammox reactor.
overloading the system with NO2-N, how-
ever, the production of NO2-N in the reactor
should be controlled as nitrite nitrogen is
58
Comparative study on different Anammox systems
Moving Bed Biofilm Reactor (MBBR)  two- plant although the basic conditions like
step process DO concentration, pH and temperature
were similar.
1. Performed research proved, that in spite
of observed changes in activities of differ-
Moving Bed Biofilm Reactor (MBBR)  one-
ent groups of bacteria in Anammox reac-
step process
tor, it is possible to obtain stable results of
1. The biofilm with bacterial culture was able
nitrogen removal and high process effi-
to perform two processes simultaneously.
ciency of nitrogen removal equal to
Adequate hydraulic retention time com-
86.0 ą 8.6%.
bined with proper oxygen conditions and
2. There is a cooperation of both bacterial
nitrogen loads are essential for high nitro-
cultures (Aerobic ammonium oxidizers
gen removal rates.
and Anammox bacteria) in activated
2. The maximum removal rate, obtained for
sludge and biofilm in the nitrogen conver-
pilot plant amounted to 2.6 g N m-2d 1,
sions. Due to the fact, that nitrifiers play
with the average value equal to 1.39 ą
role of oxygen removers, it was possible
0.31 g N m-2d-1, during the whole research
to establish stable Anammox process in
period.
the reactor.
3. The coexistence of aerobic and anaerobic
3. In the batch test with both Kaldnes and
ammonium oxidizers within the biofilm
sludge, the nitrogen removal capacity is
was confirmed by FISH analysis.
much higher in the tests with concen-
4. In conducted batch tests it was shown
trated sludge only. The contribution of
that the best results are achieved for oxy-
biofilm on Kaldnes carriers made up the
gen concentration around 3 g O2 m-3 with
efficient Anammox performance.
the average nitrogen removal rates on the
4. It is interesting to notice that nitrifiers are
level of 1.8 ą 0.31 g N m-2 d-1 The highest
outcompeted by Anammox bacteria in the
nitrogen removal rate in batch tests were
biofilm so that their population is very
at DO concentration equal to
small, but still active.
3.15 g O2 m-3 and amounted to
5. The kinetic constants of ammonium and
2.09 g N m-2d-1. At DO concentration
nitrite removal for the Anammox process
equal to 4 g O2 m-3 increase of nitrite con-
in pilot plant at the Himmerfj�den WWTP
centration in the batch reactor was no-
were calculated. The best correlation coef-
ticed, and in the same time decrease of in-
ficients were found for Hanes-Woolf
organic nitrogen removal rates.
method. The average value of Km and
5. It was proved, that the Oxygen Uptake
Vmax for ammonium removal was 6.18
Rate tests, as well as the batch tests are
gNH4-N m-3 and 78.21 gNH4-N m-3d-1 and
great tools that can be applied to the esti-
for nitrite removal 6.76 gNO2-N m-3 and
mation of kinetic parameters of reaction
90.80 gNO2-N m-3d-1, respectively.
performed by nitrifiers and heterotrophs
6. Obtained results of kinetic parameters
involved in the one-stage partial nitrita-
were not stable during the research period,
tion/Anammox process.
probably because of most probable very
6. The kinetic constants of ammonium re-
dynamic changes in activities and biomass
moval for the partial nitrita-
amounts of different groups of bacteria,
tion/Anammox process in pilot plant at
which are responsible for Anammox and
the Himmerfj�den WWTP were calcu-
nitrification process in the reactor.
lated. The best correlation coefficients
7. High free ammonia and nitrite concentra-
were found for Lineweaver-Burk and
tion during the batch tests (not observed
Hanes-Woolf methods. The average value
in the pilot) affected nitrifiers and Anam-
of KM and Vmax for ammonium removal
mox populations and proved that such
was 17.92 g NH4-N m-3 and 6.45 gNH4-N
type of batch tests are not excellent simu-
m-2d-1. Six months later, measured Vmax
lation of condition in the Anammox pilot
was on the similar level equal to
59
Grzegorz Cema TRITA LWR PhD Thesis 1053
6.92 g NH4-N m-2d-1, whereas KM was sig- 4. The Stover-Kincannon model can be used
nificantly higher and equal to to describe the ammonium and nitrite re-
37.84 g NH4-N m-3. moval rates in the RBC. The estimated
values of the maximum substrate utiliza-
7. It was shown, that both aerobic ammo-
tion rates (16.72 and 44.05 g N m-2d-1 for
nium oxidizers and the ANAMMOX bac-
ammonium and nitrite nitrogen respec-
teria reveal strong affinity to the substrate,
tively) showed the possibility of still
and relatively high resistance for high con-
higher nitrogen load to the RBC.
centrations of it. The nitrite oxidizing bac-
teria were much more sensitive, and thus 5. The rotating biological contactor with the
they will always be partly inhibited, which Anammox process can be successfully op-
is good from the point of view of the best erated even at temperatures below 20 �C.
partial nitritation/Anammox process per-
6. The process was insensitive to short-term
formance.
high nitrite concentrations in the reactor
8. Additionally, the activity of ammonia of up to 100 g NO2-N m-3 for several
oxidizing nitrifiers was very close to the hours.
maximal velocity, which indicates, that the
Rotating Biological Contactor (RBC)  one-
operational conditions serve them good.
step process
9. The highest nitrogen removal rate in the
1. Removal of total inorganic nitrogen has
batch test simulating the simultaneous
been achieved with combination of nitrifi-
partial nitritation/Anammox process
cation, Anammox and denitrification
amounted to 2.96 g N m-2d-1, for simula-
processes.
tion of the Anammox process only it was
2. FISH analysis confirmed coexistence of
equal to 5.18 g N m-2d-1.
nitrifiers and the Anammox bacteria in
10. The batch tests showed that the nitrite
one reactor.
production seems to be the rate-limiting
3. The maximum inorganic nitrogen removal
step for the Anammox reaction in a single
rates amounting to 6.1 g N m-2d-1
reactor.
(1.14 kg N m-3d-1) in the first stage of the
Rotating Biological Contactor (RBC)  two-
contactor. The average inorganic nitrogen
step process
removal rate during the stable process op-
1. Removal of total nitrogen has been eration was equal to 4.45 g N m-2d-1
achieved with a combination of different (0.83 kg N m-3d-1). However, during the
processes: nitrification, Anammox and whole research period the average inor-
heterotrophic denitrification processes in ganic nitrogen removal rate during the
one RBC. Moreover, it is also possible stable process operation was equal to
that some part of nitrogen was removed 2.55 g N m-2d-1 (0.46 kg N m-3d-1).
due to autotrophic denitrification con-
4. The RBC reactor could be highly loaded
ducted by Nitrosomonas spp.
with ammonium nitrogen and is able to
2. FISH analysis confirmed coexistence of treat medium of very high its concentra-
nitrifiers and the Anammox bacteria in tion even exceeding 1500 g N m-3.
one reactor. Additionally used API tests
5. Generally, the process of nitrogen re-
proved presence of aerobic denitrifying
moval predominated in the first stage of
bacteria in the reactor.
the contactor, providing 92 and 61% of
3. The maximum removal rates for ammo- ammonium and inorganic nitrogen re-
nium and nitrite nitrogen amounting to moval, respectively, during the whole pe-
3.0 g N m-2d-1 (0.56 kg N m-3d-1) and riod of operation.
3.9 g N m-2d-1 (0.76 kg N m-3d-1) respec-
6. The Stover-Kincannon model can be used
tively, moreover the maximum inorganic
to describe the inorganic and ammonium
nitrogen removal rates (5.0 g N m-2d-1
nitrogen removal in the RBC. The values
(0.93 kg N m-3d-1)) has been achieved
of Vmax and KB were estimated as 264.2
60
Comparative study on different Anammox systems
and 346.4 g N m-2d-1, respectively for in- mox populations and proved that such
organic nitrogen and 64.94 and type of batch tests are not excellent simu-
64.80 g N m-2d-1, respectively, for ammo- lation of condition in the Anammox pilot
nium nitrogen in the first stage of the con- plant although the basic conditions like
tactor. DO concentration, pH and temperature
were similar.
7. The obtained value of KB for nitrogen
removal was much higher than tested
VIII  FUTURE RESEARCH
loading rates indicating inorganic nitrogen
removal according to the first order reac-
It has been shown that the systems combin-
tion. The relation between load and the
ing partial nitritation/Anammox process can
removal rate results is linear for the tested
be successfully applied for separate treatment
values of load to the reactor.
of different types of nitrogen rich wastewa-
8. It was proved that RBC with the simulta- ter. Due to increasing pollution of water
neous partial nitritation/Anammox proc- sources and stricter regulations, interest in
ess can be successfully operated even at new processes like the Anammox is rising.
temperatures around 20 �C. There is still a lot of problems, which need to
be solved. The following aspects may be
9. The high nitrite concentration can inten-
investigated in future.
sify the inhibition effect of the toxic sub-
stances present in the landfill leachate.
" The slow growth rate of the Anammox
organisms is one of the most important
General
of the process bottlenecks. The cultiva-
1. The partial nitritation/Anammox process
tion of slow-growing microorganisms re-
can be successfully applied for separate
quires efficient retention time of biomass.
treatment of reject water from dewatering
The process start-up can be impeded by
of digested sludge as well as for nitrogen
insufficient biomass build-up and the
removal from landfill leachate.
same, the full-scale implementation re-
2. Obtained values of nitrogen removal rates
quires a longer start-up period compared
showed that one-step simultaneous partial
to other nitrogen removal technologies.
nitritation/Anammox process is a better
Therefore, one of the most important
option than two-step system irrespective
things in future research is to speed-up
of kind of wastewater.
this stage.
3. The obtained nitrogen removal rates were
" The Anammox process is sensitive to
higher in the one-step system which was
high oxygen and nitrite concentrations. In
additionally less complicated in operation
case of lost of the bacteria activity a lot of
and there was no need for nitrite-to-
time is needed for process recovery. That
ammonium control in the influent.
is why, there is a need to draw up a moni-
4. In spite of the low nitrogen removal rates, toring system for process performance as
the two-step MBBR system assured the well as for control of quality of seeding
highest nitrogen removal efficiency and media to prevent process inhibition by
stable long-term process operation. some toxics substances.
" There is strong need to identify a toxic
5. Partial nitritation/Anammox process can
substances present in landfill leachate and
be successfully applied at temperatures
in others wastewater to prevent future
much lower than the optimum value.
process inhibition.
6. The oxygen concentration in the bulk
" In biofilm systems, biokinetic parameters
liquid and the nitrite production rates
such as substrate affinities, maximum
seem to be the rate-limiting step for the
growth rate or maintenance need can not
Anammox reaction in a single reactor.
be well assessed due to diffusion limita-
7. High free ammonia and nitrite concentra-
tion (van der Star, 2008). Therefore, the
tion during the batch tests (not observed
enrichment of the Anammox bacteria in
in the pilot) affected nitrifiers and Anam-
61
Grzegorz Cema TRITA LWR PhD Thesis 1053
suspended culture, give the great oppor-
" Due to an influence of mass transfer in
tunity for the research on Anammox
biofilm, the biofilm kinetics should be ex-
physiology. The Membrane Assisted bio-
amined.
reactor seems to be promising and excel-
" Process optimization by using of the
lent tool for study of the Anammox bac-
mathematical modelling would be an in-
teria.
teresting task, which allows also checking
the influence of different condition with-
out risk of process inhibition.
62
Comparative study on different Anammox systems
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