N2O production in a single stage nitritation/anammox MBBR process

Water and Environmental Engineering
Department of Chemical Engineering
N 2 O production in a single stage
nitritation/anammox MBBR process
Master’s Thesis by
Sara Ekström
January 2010

Vattenförsörjnings- och Avloppsteknik
Institutionen för Kemiteknik
Lunds Universitet
Water and Environmental Engineering
Department of Chemical Engineering
Lund University, Sweden
N 2 O production in a single stage
nitritation/anammox MBBR process
Master Thesis number: 2010-01 by
Sara Ekström
Water and Environmental Engineering
Department of Chemical Engineering
March 2007
Supervisors:
Professor Jes la Cour Jansen
Doctor Magnus Christensson, AnoxKaldnes
Examiner:
Associate professor Karin Jönsson
Picture on front page:
1
1. K1 carriers with anammox biofilm from the laboratory nitritation/anammox
MBBR.
Postal address: Visiting address: Telephone:
P.O Box 124 Getingevägen 60 +46 46-222 82 85
SE-221 00 Lund. +46 46-222 00 00
Sweden,
Telefax:
+46 46-222 45 26
Web address:
www.vateknik.lth.se

Summary
Wastewaters contain abundant nitrogen that causes eutrophication in the receiving
recipient if not removed before the water is released into nature. Common nitrogen
removal is performed through nitrification and denitrification which are biologic
processes. Microorganisms are utilised to convert inorganic nitrogen compounds into
dinitrogen gas through different chemical reactions in there metabolism. Nitrous oxide,
(N2O), can be an intermediate or end product in the metabolism of both nitrification and
denitrification. N2O is a greenhouse gas, 320 times stronger than carbon dioxide (CO2).
The gas is contributing to global warming and is also taking part in depletion of the
protecting ozone layer in the stratosphere. If large amounts of N2O are emitted from
wastewater treatment facilities the problem with abundant nitrogen in aquatic
environments is only transferred into the atmosphere and N2O emissions should
therefore be avoided.
The energy demand for biologic nitrogen removal is high since aeration is needed for the
aerobe nitrification process. Denitrification requires an organic carbon source that often
has to be added to the process, generally in the form of methanol. Burning of fossil fuels
for energy coverage of the wastewater treatment plant and during transportation of
carbon source is leading to CO2 emissions with negative effects on the climate. If an
additional organic carbon source is used in the denitrification process this will also
contribute to increased CO2 emissions from the wastewater treatment plant.
Biological nitrogen removal through anaerobic ammonium oxidation (anammox) is a
relatively new process solution in wastewater treatment. Anammox has the potential to
replace common nitrogen removal of recycled internal wastewater streams with high
strength of ammonium and low COD content. The bacteria responsible for the anammox
process are converting ammonium to dinitrogen gas with nitrite as electron acceptor
making a short cut in the nitrogen cycle. Only 50% of the influent nitrogen load in the
form of ammonium has to be converted to nitrite by the nitrifiers and no additional
carbon source is needed. This means that the process offers great saving possibilities,
economical as well as environmental.
Different system configurations are available for the anammox process, it can either be
operated as a two stage process where nitritation and anammox are performed in
separate reactors, or in a single stage process where both processes are taking part in
one reactor. A MBBR is suitable as a single stage nitritation/anammox process since a
biofilm with an outer aerobe layer for the nitrifiers and one inner anoxic layer for the
anammox bacteria can develop. To allow the build up of a biofilm structure with
different oxic layers the process has to be operated at low dissolved oxygen
concentrations. Insufficient oxygenation in the nitrification process is known to enhance
nitrous oxide emissions from ammonium oxidising bacteria. Since the single stage
anammox process involves nitritation at low dissolved oxygen concentrations the
process might lead to significant N2O emissions.
i

The main objective with this master thesis work was to study the production of nitrous
oxide from a laboratory single stage nitritation/ anammox MBBR. The N2O
measurements were performed online in the water phase with a Clark–type
microsensor developed by Unisense, Århus, Denmark. The reactor was operated at both
intermittent and continuous aeration. The results from the experiments are summarised
below:
At intermittent aeration % reduction and removal rate in gN/m 2 d were in the range of
47-59% and 0.9-1.1 gN/m 2 d respectively. As the reactor mode was shifted into
continuous aeration at a lower DO concentration both % reduction and removal rate in
gN/m 2 d was more stable and higher than during intermittent aeration. % nitrogen
reduction was between64-65% and the removal rate in the interval of 1.3-1.6 gN/m 2 d.
The MBBR system produced N2O regardless of operation mode. The nN2O production
was determined through measurements of initial accumulation of nitrous oxide in the
water phase when aeration was turned off Intermittent aeration at high dissolved
oxygen concentrations 3 mg/l was resulting in significant nitrous oxide production
ranging from 6-11% of removed inorganic nitrogen. Operation at continuous aeration
yielded nitrous oxide emissions corresponding to about 2-3% of removed inorganic
nitrogen. Higher process performance may be an explanation to smaller amounts of
emitted N2O.
Conclusions that can be made from the experiments are summarised below:
• The single stage nitritation/anammox system produced significant amounts of
N2O with a minimum production of 2% of removed inorganic nitrogen.
• Operating the MBBR at intermittent aeration with a DO of ~3 mg/l gave the
highest N2O production with initial and maximum productions of 6-11% and 10-
30% respectively.
• Smaller amounts of N2O were produced by the partial/nitritation anammox
system during continuous operation at DO in the interval 1-1.5 mg/l. The initial
N2O production was found to be 2-3% and the maximum N2O production
corresponded to 2-6%.
• When the MBBR was exposed to a longer period of anoxic conditions both
ammonium oxidation and N2O production ceased.
• From results of mixing with N2 gas during the anoxic period it cannot be said
with certainty that the N2O production is the same during aeration and anoxic
phase. The absolute number on overall N2O production for an operation mode
(based on the measurements of N2O accumulating during the anoxic phase) could
be both overestimated or underestimated and should therefore be used as a
comparative tool.
ii

Acknowledgements
I would like to express my gratitude to everyone who have inspired and supported me
during the work with my master thesis.
My genuine appreciation goes to:
My supervisor Magnus Christenson at AnoxKaldnes for all guidance, support, sharing off
valuable knowledge and experiences, also for giving me the opportunity to get to know
the fascinating anammox process.
My supervisor Professor Jes la Cour Jansen at Water and Environmental Enigneering
Department of Chemical Engineering, Lund University for scientific guidance and
encouragement, for all your valuable aspects on my work and always reminding me of
looking into things from a wider perspective.
To Lars H. Larsen at Unisense for all help and support with the microsensors.
To everyone at AnoxKaldnes for all kindness, support and for creating an inspiring
environment to work in. Special thanks to Maria Ekenberg for always answering my
questions about the laboratory MBBR process and for all help in the laboratory. To
Carolina Shew Cammernäs for all patients and time while helping me with the FIA
analyses. To Stig Stork for all technical support.
To David Gustavsson at VA SYD for exchanging your ideas and knowledge about N2O
emissions in wastewater treatment processes.
Last but not least I would like to thank my mother and my sister, Gustav and Helena for
always encouraging and supporting me.
Thank you!
iii

Glossary
Aerobic – in the presence of oxygen in the form of O2
Anaerobic – an oxygen free environment
Anoxic – environment where oxygen is present as nitrite or nitrate
Autotrophic – organism that can produce organic compounds from carbon dioxide with
light or inorganic chemical compound as energy source
Carbon dioxide equivalent – is a measurement standard where the weight of a
greenhouse gas released in to the atmosphere is converted into the weight of carbon
dioxide that would cause the same temperature rise in Earths ecosystem as the gas in
question
Global warming potential – a measure of how much a given amount of a greenhouse
gas would contribute to global warming in comparison with the same amount of carbon
dioxide. The global warming potential of a greenhouse gas depends on (i) the absorption
of infrared radiation of the gas, (ii) atmospheric life time, (iii) spectral location of
absorbing wavelengths, where the global warming potential of carbon dioxide is 1
Heterotrophic – organism requiring organic compounds as energy source
Lithotrophic – organism using inorganic nutrients to obtain energy
Oxic – environment where oxygen is present
Abbreviations
Anammox – anaerobic ammonium oxidation
AOB – ammonium oxidising bacteria
ATP – adenosine triphosphate
Canon – completely autotrophic nitrogen removal over nitrite
Deamox – denitrifying ammonium oxidation
DO – dissolved oxygen
FIA – flow injection analysis
MBBR – moving biofilm bed reactor
NH4 + – ammonium
NO2 − – nitrite
NO3 − – nitrate
N2O – nitrous oxide
ppm – part per million
Sharon – Single reactor system for High rate Ammonium Removal Over Nitrite)
v

Table of content
Chapter 1 .................................................................................................................................... 1
1. Introduction ............................................................................................................................ 1
1.3 Objectives ............................................................................................................................................... 3
1.4 Accomplishment and scope ............................................................................................................. 3
Chapter 2 .................................................................................................................................... 5
2. Background ............................................................................................................................. 5
2.1 Biological nitrogen removal in wastewater treatment ......................................................... 5
2.1.1 Nitrification ................................................................................................................................... 5
2.1.2 Denitrification .............................................................................................................................. 6
2.1.3 Anaerobic ammonium oxidation........................................................................................... 6
2.2. Environmental factors ...................................................................................................................... 8
2.2.1 Dissolved oxygen ......................................................................................................................... 8
2.2.2 Temperature ................................................................................................................................. 9
2.2.3 pH and alkalinity ......................................................................................................................... 9
2.2.4 Substrate...................................................................................................................................... 10
2.2.5 Mixing ........................................................................................................................................... 10
2.3 Biofilm reactors ................................................................................................................................. 11
2.3.1 Trickling filter ............................................................................................................................ 11
2.3.2 Biofilters ...................................................................................................................................... 12
2.3.3 Fluidised bed .............................................................................................................................. 12
2.3.4 Moving bed reactor ................................................................................................................. 13
2.3.5 Rotating disc .............................................................................................................................. 13
2.4 Biofilm kinetics .................................................................................................................................. 13
2.5 System configurations for nitrogen removal by anammox .............................................. 15
2.5.1 Sharon/Anammox ................................................................................................................... 15
2.5.2 Canon ............................................................................................................................................ 16
2.5. 3 Deammonification .................................................................................................................. 17
2.5.4 Deamox ........................................................................................................................................ 18
2.5 N2O emissions from wastewater treatment ........................................................................... 18
2.5.1 Nitrification as a source of N2O emissions ..................................................................... 19
2.5.2 Denitrification as a source of N2O emissions ................................................................ 19
2.5.3 Chemical production of N2O ................................................................................................ 20
2.6 Microsensors ...................................................................................................................................... 22
2.6.1 Nitrous oxide sensor ............................................................................................................... 22
2.6.2 Nitrite biosensor....................................................................................................................... 23
Chapter 3 .................................................................................................................................. 25
3. Material and Methods .......................................................................................................... 25
3.1 Partial nitritation/anammox laboratory MBBR . ................................................................. 25
3.2 Reactor medium ................................................................................................................................ 26
3.3 Analytical methods .......................................................................................................................... 27
3.3 Cycle studies ....................................................................................................................................... 27
3.3.1 Intermittent aeration .............................................................................................................. 27
vii

3.3.2 Prolonged study, intermittent aeration........................................................................... 28
3.3.3 Continuous aeration ................................................................................................................ 28
3.4 Calibration of microsensors ......................................................................................................... 28
3.5 Diffusivity tests of N2O ................................................................................................................... 30
Chapter 4 .................................................................................................................................. 31
4. Results .................................................................................................................................. 31
4.1 Process performance ...................................................................................................................... 31
4.3 N2O emissions from partial nitritation/anammox MBBR ................................................ 33
4.3.2 Intermittent aeration. ............................................................................................................. 34
4.3.2 Prolonged unaerated period. ............................................................................................... 35
4.3.3 Continuous operation at DO ~1.5 mg/l ........................................................................... 36
4.3.4 Continuous operation at DO ~1.0 mg/l ........................................................................... 37
4.3.5 Effect of mixing with N2 gas during unaerated phase, continuous operation at
DO ~1.0 mg/l and ~1.5 mg/l .......................................................................................................... 38
4.4 NO2-N biosensor ............................................................................................................................... 40
4.5 Diffusivity and stripping test of N2O ......................................................................................... 41
Chapter 5 .................................................................................................................................. 44
5. Discussion ............................................................................................................................. 44
5.1 Process performance ...................................................................................................................... 44
5.2 N2O production .................................................................................................................................. 44
5.3 Measurements with NO2-N biosensor ...................................................................................... 47
5.4 Diffusivity and stripping test of N2O ......................................................................................... 48
6. Conclusions ........................................................................................................................... 51
7. Future research .................................................................................................................... 53
8. References ............................................................................................................................ 55
Appendix A ............................................................................................................................... 63
Calculation of concentrations in calibration solutions for N2O and NO2-N microsensors
......................................................................................................................................................................... 63
Appendix B ............................................................................................................................... 67
Calculations of N2O emissions ............................................................................................................ 67
Appendix C................................................................................................................................ 71
Microsensor measurements ................................................................................................................ 71
Appendix D ............................................................................................................................... 77
Nitrogen grab samples ........................................................................................................................... 77
Appendix E Scientific Article ..................................................................................................... 87
viii

Chapter 1
1. Introduction
Nitrogen is one of the main building blocks in proteins and is therefore a vital element
for all living organisms. The elemental form of nitrogen is made available to the
biosphere through microbial fixation of dinitrogen gas which constitutes 79% of the
atmosphere. Combustion of fossil fuels, the use of nitrogen in industry and fertilizers,
waste and wastewater streams results in large amounts of anthropogenic nitrogen lost
to nature. The human contribution to nitrogen cycling impacts the environment
negatively through eutrophication of aquatic environments and emissions of
nitrogenous compounds to the atmosphere. Release of binary nitrogenous gases
contributes to the greenhouse effect and depletion of ozone layer with consequences on
a global scale lasting for centuries.
Since the start of the industrialisation human activity has increased the emissions of
greenhouse gases (carbon dioxide, chlorofluorocarbons, methane, ozone and nitrous
oxide), to the atmosphere with about 30%, with global warming as a result (Liljenström
& Kvarnbäck, 2007). In 2004 the global amount of anthropogenic emitted greenhouse
gases corresponded to 49 billion tons carbon dioxide equivalents, (a measurement
standard where the weight of a greenhouse gas released in to the atmosphere is
converted into the weight of carbon dioxide that would cause the same temperature rise
in Earths ecosystem). Carbon dioxide stands for the greatest proportion of the emissions
with 79% followed by methane and nitrous oxide contributing with 14% and 8%
respectively, (Naturvårdsverket, 2009).
Wastewater treatment plants produces greenhouse gases through; (i) burning of fossil
fuels for coverage of the energy demand, (ii) transportation of chemicals for on-site
usage and final disposal of solids, (iii) biologic treatment processes where nutrients,
(organic matter, nitrogen and phosphorus) are removed through microbial processes.
Biologic wastewater treatment processes are known to produce three of the major
greenhouse gases carbon dioxide(CO2), methane (CH4) and nitrous oxide (N2O) (Bani
Shahabadi et al., 2009). Nitrous oxide which is the strongest of these greenhouse gases is
known to be produced during nitrification and denitrification, processes used to remove
nitrogen from the wastewater. The global warming potential of N2O is 320 times
stronger than that of CO2. Release in to the atmosphere not only amplifies the warming
of Earth’s surface temperature it also contributes to depletion of the ozone layer (Jacob,
1999). During a thirty year period from 1990 to 2020 the N2O emissions associated with
microbial nitrogen degradation of both treated and untreated wastewaters are
estimated to increase with 25% from 80 to 100 megaton carbon dioxide equivalents.
Emissions from the post-consumer waste sector are approximately 1300 megaton
carbon dioxide equivalents which corresponds to

stricter it might lead to elevated emissions of nitrous oxide from the biological removal
processes. It is therefore of great importance to design and operate these processes to
minimise the emissions of nitrous oxide to the atmosphere.
Wastewater treatment plants using biologic treatment processes for nutrient removal
are producing excessive sludge giving rise to ammonium rich effluent from the
anaerobic sludge digestion. This internal wastewater stream is recombined with the
influent of the treatment plant and corresponds to 15-20% of the total nitrogen load of
the wastewater treatment plant (Fux et al., 2003). In the early 1990s a new biological
treatment process for nitrogen removal through anaerobic ammonium oxidation
(anammox) was discovered by research teams in Holland, Germany and Switzerland
(Mulder et al., 1995, Hippen et al., 1997, Siegrist et al., 1998). The technology has turned
out to be suitable for treatment of reject waters and other problematic wastewaters
with a low COD/N ratio and high ammonium concentrations. The bacteria performing
the microbial conversion of nitrite into dinitrogen gas are strict anaerobe autotrophs
and the process has the potential to replace conventional nitrification/denitrification of
recirculated high strength ammonium streams within the wastewater treatment plant
(Strous et al., 1997). No additional carbon source is needed, the oxygen demand is
reduced by 50% in the nitrifying step and the aeration can thereby be strongly reduced
(Jetten et al., 2001, Fux et al., 2002). This means that the process offers an opportunity to
decrease the carbon footprint of the wastewater treatment plant in terms of saving
possibilities of both additional carbon source and power consumption (Jetten et al.,
2004). Further advantages with the anammox process is that the production of surplus
sludge is minimized and that high volumetric loading rates can be obtained resulting in
reduced operational and investment costs (Abma et al., 2007). However there are
doubts that the process could produce significant amounts of N2O gas with negative
environmental impacts detracting the process advantages.
2

1.3 Objectives
The aim with this master thesis was to estimate the N2O emissions from a partial
nitritation/anammox laboratory moving bed biofilm reactor (MBBR) treating
ammonium-rich synthetic wastewater. Measurements were carried out with a
microsensor recording the N2O concentration online in the water phase, main objectives
were:
• To determine the N2O emission from the system under initial operation
conditions which were intermittent aeration at a dissolved oxygen, (DO),
concentration of ~3 mg/l.
• To evaluate N2O production when changing the operation mode into continuous
aeration to a lower DO concentration. Continuous operation at two different DO
concentrations were tested ~1.5 mg/l and ~1.0 mg/l.
• Determine how the N2O production was influenced during a longer period of
anoxic conditions.
• Investigate whether the N2O accumulation observed in the water phase as
aeration is turned off was due to termination of N2O stripping or if the N2O
production actually increases during the anoxic period.
• To examine if a biosensor for online measurements of NO2-N concentrations can
replace traditional analyse methods for determination of NO2-N during this
master thesis work.
1.4 Accomplishment and scope
The master thesis was based on both experimental work and a literature study.
Laboratory studies were performed on an existing partial laboratory
nitritation/anammox MBBR at AnoxKaldnes in Lund. The study took it’s start with a
definition of the existing system and setting up the equipment for the microsensors used
during online measurements. The laboratory work proceeded with measurement
sessions where the N2O concentration was registered in the water phase at different
operational conditions of the MBBR. Since no equipment for measurement of N2O in the
off-gas were available experiments to estimate how much N2O that was stripped off
from the water phase by diffusion and aeration were made.
The collected data was analysed and the N2O production from the MBBR could be
estimated with mass balance calculations of the system. The evaluation of the estimated
N2O emission from the nitritation/anammox MBBR laboratory system was based on a
literature study of N2O emissions in wastewater treatment. No calculations were made
to estimate whether the anammox process is reducing or increasing the carbon footprint
in comparison to common nitrogen removal processes.
3

For simplicity a synthetic wastewater was used, this water may both be easier to
degrade and not as complex as a normal wastewater which may impact the microbial
performance.
4

Chapter 2
2. Background
2.1 Biological nitrogen removal in wastewater treatment
Nitrogen removal is one of the major tasks in wastewater treatment. Biologic nitrogen
removal is the most efficient way to eliminate nitrogen from the wastewater and a
variety of system configurations like activated sludge plants with suspended growth,
biofilters designed for attached growth and combinations of the two have been
developed. These systems are built on the knowledge of microbial nitrogen cycling
where nitrogen compounds like NH4+, NO2 − and NO3 − are removed by conversion into
elemental N2 gas released to the atmosphere, see Figure 1. The well known nitrification
and denitrification processes are commonly used to achieve satisfactory nitrogen
removal. Today anaerobic ammonium oxidation, a relatively new technology for
nitrogen removal is also in use at several places.
Figure 1. Major biological transformations of nitrogen in wastewater treatment. (Kampschreur et
al., 2009).
2.1.1 Nitrification
Nitrification is oxidation of ammonium into nitrate under aerobic conditions, the
process occurs in two separated reaction steps, each involving different species of
bacteria. The nitrifiers are chemo-lithoautotrophs which means that they use carbon
dioxide or carbonate as carbon source and inorganic nitrogen is used for both energy
supply and cellular growth (Gray, 2004). In the first nitritation step ammonium
oxidising species like Nitrosomonas and Nitrosospira oxidises ammonium into nitrite:
5

NH 1.5O NO 2H 2H O (2.1.1)
The intermediate of the nitrification process (NO2 − ) is then further oxidised into nitrate
by nitrite oxidisers:
NO 0.5O NO
(2.1.2)
The nitratation step is performed by species like Nitrobacter and Nitrococcus (Prescott
et al., 2005). The overall nitrification reaction can be described by:
NH 2O NO 2H 2H O (2.1.3)
Energy gained by the bacteria during nitrification is used in the electron transport chain
to make adenosine triphosphate, (ATP is the energy currency of the cell making
chemical transport possible), (Prescott et al., 2005). Nitrifying bacteria are slow
growers since nitrification processes gives a low energy yield, (see Table 1) and the
nitrifiers have to oxidise large amount of inorganic material for their growth and
reproduction, (Prescott et al., 2005).
2.1.2 Denitrification
Denitrification is nitrate respiration under anoxic conditions carried out by a large
number of different heterotrophic bacteria. Nitrate is used to oxidate organic carbon
into elemental nitrogen and carbon dioxide:
NO organic carbon N CO (2.1.4)
Denitrifying bacteria need an easily biodegradable carbon source and their demand for
removal of one gram of nitrogen corresponds to 3-6 grams of chemical oxygen demand,
(COD). If the COD/N ratio of the wastewater becomes too low an additional carbon
source like methanol must be added in order to achieve nitrogen removal of nitrate
through denitrification (Gillberg et al., 2003)
Pseudomonas, Paraccocus, and Bacillus are examples of bacteria denitrifiying under
anoxic conditions. Most denitrifiers are facultative anaerobes which means that they
generally respire with oxygen as final electron acceptor, this since the oxygen route
yields more energy than nitrate respiration (Prescott et al., 2005).
2.1.3 Anaerobic ammonium oxidation
Anammox bacteria are obligate anaerobe autotrophs using inorganic nitrogen and
carbon for energy supply and growth, the process offers a short cut in the nitrogen cycle
as illustrated in Figure 1(Jetten et al., 1999). Ammonium is converted into dinitrogen gas
with nitrite as electron acceptor (2.1.6), hydrazine (N2H4) and hydroxylamine (NH2OH)
6

are intermediates in the chemical reaction. This reaction is the catabolic and energy
supplying part in anammox metabolism, it has to be carried out 15 times to fix one
molecule of carbon dioxide with nitrite as electron donor in the cellular synthesis or
anabolism (2.1.7) which produces nitrate (van Niftrik et al., 2004).
NH NO N 2H O (2.1.6)
CO 2NO
H O CH O 2NO (2.1.7)
Broda, (1977), predicted this microbial process through thermodynamic calculations for
over thirty years ago. The anammox process was discovered in the early nineties in a
rotating-disk plant treating landfill leachate at Mechernich, Germany (Rosenwinkel &
Cornelius, 2005). Mulder et al. also identified the anammox process in a denitrifying
fluidised bed reactor in Deltft, the Netherlands, at about the same time (Mulder et al.,
1995). Total stoichiometry of the anammox process has been estimated by Strous et al.,
(1998):
1NH
1.32NO 0.066HCO
0.13H
1.02 N 0.26NO
0.066CH2O . N . 2.03 H O. (2.1.7)
The bacterium performing the anammox reaction has been identified as a new member
of the order Planctomycete (Strous et al., 1999). Until now totally five anammox genera
have been identified, four from enriched wastewater sludge: Kuenenia, Brocadia,
Anammoxoglobus and Jettenia, the fifth genera of anammox bacteria Scalindua is often
found in marine environments (Jetten et al., 2009).
Planctomycetes are gram-negative bacteria with phenotypic properties such as absence
of peptidoglycan in the cell wall, budding reproduction and internal cell
compartmentalisation due to two membranes on the inside of the cell wall (Prescott et
al., 2005). Anammox bacteria are characterized by their deep red colour and ability to
form bio-films (Abma et al., 2006). Anammox cell structure is divided into three
compartments. The outer region closest to the cell wall called the paryphoplasm
encloses the second compartment which is the riboplasm. The riboplasm contains the
nucleoid and the anammoxosome, the third compartment where anammox catabolism
takes place, see Figure 2. All compartments are separated by bilayer membranes
constituted of impermeable and high density ladderane lipids (van Niftrik et al., 2004).
The higher membrane density is of importance to the anammox bacteria of two reasons,
one that it creates an electro potential force driving the ATP synthesis, and two it keeps
the toxic intermediates from the anammox process hydroxylamine and hydrazine inside
the anammoxosome (van Niftrik et al., 2004).
7

Figure 2. Illustration of anammox bacteria. (Adapted from van Niftrik et al., 2004).
Anammox bacteria are extremely slow growers, their doubling time has been found to
11 days in activated sludge (Strous et al., 1999). However it might be possible to
increase this doubling rate with optimal operation conditions since other researchers
have found a much shorter doubling rate of 3.6-5.4 days for anammox bacteria in a upflow
fixed-bed biofilm column reactor (Tsushima et al., 2007).
The microbial processes and chemical reactions of nitrification, denitrification and
anammox are summarized in Table 1.
Table 1. Microbial processes and chemical reactions taking part in the nitrogen cycle showed in
Figure 1.
Energy yield
ΔG ̊’ eq.
Process
Chemical reaction
+
kJ/mol NH 4
Nitritation: NH 1.5O NO 2H H O -271 (2.1.1)
Nitratation: NO 0.5O
NO -72.8 (2.1.2)
Nitrification: NH 2O NO 2H H O - (2.1.3)
Denitrification: NO org. carbon N 2CO
-
(2.1.4)
Anammox: NH
NO N 2H O -358.8 (2.1.6)
2.2. Environmental factors
Dissolved oxygen, temperature, pH, substrate concentrations and turbulence are abiotic
conditions that are of great importance for the growth and survival of the microbiology
in a wastewater treatment system.
2.2.1 Dissolved oxygen
Depending on the electron donor in the respiratory chain of the microorganism can be
limited or inhibited by either to low or to high DO concentrations. It is the oxygen
concentration within the biofilm experienced by the bacteria that is of importance for
the wellness of the organism (Henze et al., 1997).
Nitrifying bacteria utilising oxygen as electron donor are sensitive for too low oxygen
concentrations and are limited by DO concentrations

minimum concentration of 2 mg/l should be maintained (Gray, 2004). The nitrifying rate
increases up to DO levels of 3-4 mg/l, (Metcalf & Eddy). All figures given here yields for
DO concentrations in the water bulk phase of activated sludge processes, higher DO
concentrations are needed to satisfy the microbial oxygen demand in biofilm processes.
This since the oxygen concentration in the biofilm depends on diffusion of oxygen from
the water phase into the biofilm which is further explained in chapter 2.4.
Both denitrification and anammox processes are inhibited by oxygen. Denitrification has
been observed to be inhibited at DO concentrations above 0.2 mg/l (Metcalf & Eddy,
2003) and anammox organisms are reversibly inhibited by DO concentrations as low as
2 µmole/l or 0.032 mg/l, (Jetten et al., 1998).
2.2.2 Temperature
The temperature impacts the structure of the microbial community and is crucial for
growth and reaction rates in the system. Microbial reactions are often dependent on
enzyme-catalysed reactions that increase in velocity at higher temperatures. When the
time for a reaction to be catalysed is shortened the metabolism is more active and the
microorganism is allowed to grow faster (Prescott et al., 2005). Temperature does also
impact non viable factors like settling characteristics, gas solubility and transfer rates
(Gray, 2004).
Nitrification can be operated in a temperature interval of 0-40 °C with a temperature
optimum between 30-35 °C (Gray, 2004). Denitrifying bacteria are less sensitive to
temperature than nitrifiers and denitrification can take place in a temperature interval
from 2-75 °C with an optimum around 30 °C (Pierzynski et al., 2005)
Anammox bacteria are active in temperature range from 6-43 °C with an optimum at 30
̊C (Anammox online).
2.2.3 pH and alkalinity
pH, which is the measurement of a solutions acidity or alkalinity, is another important
environmental factor that impacts the growth rate of the microbial community. Since pH
is defined as the inverse logarithm of H + ions in solution a change of one pH unit
corresponds to a tenfold increase in the activity of H + ions. Each bacteria species have a
pH growth range and optimum.
Nitrification consumes alkalinity since two moles of OH − are used per mole ammonium
oxidised. Nitrification is favoured by mild alkaline conditions with pH optimum in the
range of pH 8.0-8.4 (Gray, 2004). The nitrification rate is significantly declined by low
pH values

Denitrification produces alkalinity and pH is generally raised by the process. pH
optimum is ranging from 7-9 depending on local conditions (Henze et al., 1997).
Cell synthesis in the anammox reaction is increasing pH and the process is active in a pH
range from 6.5-9 with an optimum around 8 (Egli et al., 2001).
2.2.4 Substrate
Nitrifying, denitrifying and anammox bacteria are all dependent on different substrates
for energy and cellular growth as discussed above. The ability to utilise their substrate
varies between bacterial species. This implies that a species with high affinity for its
substrate will be better at utilising the substrate at low concentration and therefore
outcompete species with lower affinity for the substrate. The half saturation constant,
which is the substrate concentration when the growth rate is half of maximum, is often
used to compare how well adapted different microorganisms are to their substrates.
The half saturation constant or Ks value for ammonium oxidisers in a nitrifying biofilm
airlift reactor was found to correspond to a NH4-N concentration of 11 mg/l. And the
microorganisms were inhibited by NH4-N concentrations of 3300 ±1400 mg/l. (Carvallo
et al., 2002)
Denitrification rate and capacity is very dependent on available organic carbon source.
External carbon sources like methanol, ethanol and acetic acid are readily biodegradable
and give much higher denitrification rates than denitrification with organic compounds
found in the waste water (Ødegaard, 1993).
Anammox bacteria have high affinity for their substrates ammonia and nitrite, the Ks
values are below chemical detection level (

2.3 Biofilm reactors
Biofilm reactors can be used for nutrient removal in wastewater treatment and are
commonly used in biological nitrogen removal. Bacteria with the ability to adhere to
solid surfaces are colonising and growing in high concentrations in a biofilm attached to
a fixed surface. The carrier material can be solid or free moving made out of stone, wood
or plastic. Biofilm thickness varies with the hydrodynamics and growth conditions of the
system, (Metcalf & Eddy, 2003). The fixed polymer film formed by the bacteria protects
them from toxics and being washed out of the system (Henze et al., 1997).
Figure 3. Illustration of different types of bioflim reactors. (Adapted from Ødegaard, 1993).
Biofilters are designed to achieve high and efficient nutrient removal rates in compact
and energy efficient systems. Operating conditions should be such that transfer rates of
substrates from the water bulk phase to the microbial community assures efficient
removal rates and development of a biofilm thickness satisfying the microbial demands
in a certain biologic process. This makes different available biofilm technologies suitable
for varied microbial processes. Figure 3 illustrates some of the available biofilm
technologies shortly described in the following text.
2.3.1 Trickling filter
Trickling filters are biological reactors where the wastewater is sprinkled over a filter
bed at the top. The water is then allowed to percolate through a fixed bed material made
out of stone or plastic. Volumetric flow rates are controlling the biofilm thickness and
11

aeration takes place through self drag from bottom to top of the filter (Henze et al.,
1997). Since the wastewater is sprinkled over and percolated through the filter media
there is little hydraulic control of the biofilm thickness resulting in uneven growth
throughout the filter. This causes local clogging that hinders free flow of water and air
through the filter resulting in decreased nutrient removal rate. Modern filling materials
in plastic have a specific filling area of about 100-250 m 2 /m 3 while the carrying material
in older treatment plants often is crushed stone or pumice that only has a specific area of
40-60 m 2 /m 3 , (Gillberg et al., 2003).
2.3.2 Biofilters
The carrier material in these filters can be either a granulated media or corrugated
sheets. In the granulated reactor wastewater passes through a stationary filter bed made
out of sand or plastic beads, the filter media is aerated from the bottom in the aerobic
version. The specific area of the filter media is high and ranges from 1000-1200 m 2 /m 3 ,
the effective area is however not this high since only 50% of the reactor volume is filled
with the carrier material (Ødegaard, 1993). This reactor clogs easily and has to be
backwashed. Clogging and substrate decrease from top to bottom in the reactor rules
out efficient nutrient removal throughout the whole reactor volume. The second type of
media is a stationary carrier material constituted of plastic sheets that are welded
together in cubes, the specific area of these filters ranges from 150-200 m 2 /m 3 . The
system is aerated from the bottom with blower systems and has to be backwashed at
intervals to prevent clogging (Ødegaard, 1993).
2.3.3 Fluidised bed
In a fluidised bed the biofilm grows on sand grains with a size of 0.4-0.5 mm. To keep the
sand grains in suspension at all times wastewater is pumped through the bottom of the
reactor at a high constant flow rate. The turbulence created from high volumetric flow
rates passing through the reactor implies great shear forces and very thin biofilms. With
bed depths ranging from 3 to 4 m a specific surface area of 1000 m 2 /m 3 can be achieved
(Metcalf & Eddy, 2004). High turbulence in combination with very high contact area
between microorganisms and wastewater assures efficient substrate transmission and
high conversion rates. The draw back with this process design in nitrification is that
oxygen transfer rates to the water phase are too slow to maintain sufficient oxygen
concentrations for microbial activity (Gillberg et al., 2003) Sine the biofilm is very thin in
this reactor configuration it does not allow development of a biofilm with layers of
different oxygen concentration. This reactor type can there for not combine microbial
communities with different dissolved oxygen demands. Recirculation is necessary to
maintain the high fluid velocity.
12

2.3.4 Moving bed reactor
Another way to design a compact biofilm process is to use a suspended inert carrier that
moves freely within the reactor. The first carrier materials were small polyethylene
(density 0.95 g/cm 3 ) cylinders with a cross in side providing the microorganisms with a
protected surface to grow on. The carriers are kept in motion by aeration or mechanical
stirring, a sieve in the outlet keeps the moving carriers in the reactor. The reactor does
not clog and there is no need for backwashing or biomass recycling. (Ødegaard et al.,
1994). Different shapes and sizes of the carrier material provides an effective specific
area ranging from 220 -1200 m 2 /m 3 (AnoxKaldnes, 2009). Biofilm thickness depends on
carrier design and hydraulic conditions in the reactor. If a stagnant laminar layer is
formed around the carrier material this will increase the diffusional resistance that is
limiting in biofilm processes. The moving bed technology can also be combined with the
activated sludge process resulting in higher removal rates and more compact systems.
2.3.5 Rotating disc
Rotating filters are constituted of flat discs often made out of plastic, 2-3 meters in
diameter mounted in rows on a horizontal shaft. The filter medium that is semisubmerged
is alternately rotated through the water phase at right angles to the flow.
The filters are 10-20 mm thick, spaced about 20 mm apart and have an active surface
area of about 150-200 m 2 /m 3 (Gray N, 2004). Rotation of the filter discs keeps the
biofilm oxygenated and the motion creates an efficient contact between the water phase
and biofilm. Revolution speed of the filter is controlling the biofilm thickness (Henze et
al., 1997).
2.4 Biofilm kinetics
The kinetics of substrate conversion in a biofilm reactor is dependent of the reactor
configuration that decides the biofilm structure and of available nutrients in the
wastewater. Substrates in the water bulk phase are converted to biomass, energy and
end products through cellular metabolism in the bacteria. The mass balance for an
infinitely small section of the biofilm is described by:
(2.4.1)
(2.4.2)
where: Q is the volumetric flow, (dimension L -3 ∙T -1 ), C is the concentration, (dimension
M∙L -3 ), r describes the biological growth rate, (dimension M∙L -3 ∙ T -1 ) and V is the reactor
volume, (dimension L -3 ), (Warfvinge, 2008).
The substrate conversion rate in a biofilm reactor is dependent on three main
mechanisms, the diffusion resistance of substrates from the well mixed water bulk phase
13

to the liquid film, diffusion of substrates through the biofilm and substrate turnover in
the cellular mass (Ødegaard, 1993), see Figure 4. The diffusion dependent transport of
substrate from the liquid bulk and the diffusion within the biofilm is driven by a
concentration gradient. The substrate concentration profile decreases with biofilm
depth and has a downward curvature due to the substrate utilisation rate illustrated in
Figure 4, (Henze et al., 1997). Conversion rate on cellular level is dependent on
enzymatic processes. Available amount of enzymes handling the specific substrate will
decide how fast the conversion takes place. When substrate concentrations are low, the
accessible substrate S is the rate limiting factor and the rate equation is said to be of first
order with respect to S, see (2.4.3). At high substrate concentrations the substrate
turnover is limited by the amount of available enzymes. The reaction rate is now of zero
order since it is independent of the substrate concentration, see (2.4.4), (Warfvinge,
2008).
First and zero order approximations are given by the following equations:
,
·
(2.4.3)
,
(2.4.4)
where: rv,s describes the biological growth rate in a certain volume of biofilm at a certain
substrate concentration, (dimension M∙ L -3 ∙ T -1 ), µmax is the maximum specific growth
rate, (dimension T -1 ), XB is the concentration of biomass, (dimension M∙L -3 ), Ymax gives
the maximum yield constant, (dimension MXB∙Ms -1 ) and Ks is the saturation constant for
the substrate, (dimension M∙L -3 ), (Henze et al., 1997).
Figure 4. Schematic overview of substrate transport from liquid bulk phase to microorganisms
growing on carrier material. The concentration gradient profile in the biofilm depends on
transport into the biofilm and substrate utilisation rate in the film. (Adapted from Metcalf & Eddy,
2003, and Warfvinge, 2008).
14

To enable efficient nutrient removal the hydraulic conditions should prevent the buildup
of a laminar layer and the contact surface between water phase and biofilm should be
as large as possible (Metcalf & Eddy, I. 2003).
2.5 System configurations for nitrogen removal by anammox
Nitrogen removal by anammox can be implemented either as a two stage or one stage
system. 50% of the influent ammonium is oxidised to nitrite by nitrifying bacteria in
both cases. In a two stage system this conversion takes place in a nitritation reactor
followed by an anammox reactor where the oxidation of ammonium into dinitrogen gas
with nitrite takes place. In single stage technology both processes takes place in the
same reactor (Abma et al., 2007). Since the anammox process was discovered by
different research teams and in slightly diverse environments the system configurations
evolved have been given different names but are in practice quite similar. Main
differences are reactor configuration and operational mode. Some of the available
anammox processes are shortly described in this chapter.
2.5.1 Sharon/Anammox
A Sharon (single reactor system for high rate ammonium removal over nitrite)
reactor followed by an anammox reactor is a possible system configuration to achieve
nitrogen removal with anammox bacteria. The Sharon process is used to nitrify 50% of
the influent ammonium to nitrite. The effluent from the Sharon reactor is then used as
feed for the anammox reactor where nitrite and ammonium is converted to elemental
nitrogen, see Figure 5, (Stowa, 2009).
Figure 5. Sharon/Anammox process scheme.
To achieve partial nitrification to only 50% the Sharon reactor is operated in a manner
that benefits the ammonium oxidizers, washing out the nitrite oxidizers from the system
(van Dongen et al., 2001). This is obtained by operation above 25 ̊C, keeping the sludge
15

age equal to the hydraulic retention time, and by controlling the pH to get the desired
ammonium/nitrite ratio:
NH HCO 0.75O 0.5NH 0.5NO CO 1.5H O (2.5.1)
The effluent from the Sharon reactor is then feeding the anammox process that converts
nitrite and ammonium to elemental nitrogen according to eq. (2.1.7).
Some nitrate is formed in the anammox process as biomass is formed from inorganic
carbon with nitrite as electron donor (van Dongen et al., 2001).
2.5.2 Canon
The Canon process (completely autotrophic nitrogen removal over nitrite) is a single
stage process for nitrogen removal with ammonium oxidisers and anammox bacteria,
(Third et al., 2001), see Figure 6 for system description.
Figure 6. Canon process scheme
The Canon reactor has to be oxygen limited to allow the co-existence of both ammonium
oxidisers and anammox bacteria in the same environment. Ammonium oxidation into
nitrite is performed under oxygen limitation by aerobic ammonium oxidisers eq. (2.5.3).
Anammox bacteria are oxidising ammonium with nitrite into dinitrogen gas eq. (2.5.4).
The resulting over all chemical reaction for the Canon process is described by eq. (2.5.5),
(Third et al., 2005).
Partial nitrification:NH 1.5O NO 2H H O (2.5.3)
Anammox: NH 1.3NO N 0.26NO 2H O (2.5.4)
Canon process: NH 0.85O 0.4N 0.13NO 1.3H O 1.4H (2.5.5)
16

The Canon process is often implemented with granular sludge and it is important that
large biomass flocs with decreasing oxygen gradient within the floc are allowed to form.
This in order to achieve an environment suitable for both aerobic ammonium oxidisers
and anammox bacteria in the same reactor set up (Third et al., 2005).
2.5. 3 Deammonification
The deammonification process is also a one stage process for nitrogen removal through
partial nitrification and anammox. The process scheme resembles that of the Canon
process illustrated in Figure 6. The greatest difference between deammonification and
the Canon process is that deammonification is a biofilm process utilising a carrier
material to support biofilm growth.
Conversion of ammonium into dinitrogen gas takes place at different biofilm depths.
Nitrification of ammonium into nitrite is carried out by nitrifiers in the outer aerobic
layers of the biofilm. This process together with diffusion provides the anammox
bacteria in the deeper anaerobic layers with their substrates (Egli et al., 2003), see
Figure 7. The diffusion depth of oxygen is dependent on the DO concentration in the
water bulk phase and it influences to which extent the conventional nitritation process
takes part in the biofilm (Helmer et al., 2001).
Figure 7. Conversion of ammonium to dinitrogen gas takes place through two separate reactions at
different biofilm depths. (Adapted from Rosenwinkel and Cornelius, 2005)
To ensure anoxic conditions for the anammox bacteria the system must be operated at
low oxygen concentrations or with alternating aeration. Low oxygen concentrations in a
biofilm system also inhibits aerobic nitrite oxidisers which is needed to make sure that
only the first step of nitrification is performed. The growth rate of aerobic ammonium
oxidisers (AOB) is higher than for nitrite oxidisers at low oxygenation, AOB are also
faster at recovering in activity after the anoxic phase.Temperatures over 20°C are also
favouring the growth rate of AOB over nitrite oxidisers (Rosenwinkel and Cornelius,
2005). Biofilm systems are suitable for the anammox process since the bacteria are slow
growers that require high biomass retention (Abma et al., 2006).
17

2.5.4 Deamox
Deamox (denitrifying ammonium oxidation) combines the anammox process with
autotrophic denitrification utilising sulphide as an electron donor for production of
nitrite from nitrate. The Deamox reactor can therefore be used in the treatment process
of wastewaters containing organic bound nitrogen and SO4 2− (Kalyuzhnyi et al., 2006).
The organic nitrogen content in these wastewaters firstly has to be anaerobic
mineralised before nitrification can proceed. If the Deamox process is utilised not all
effluent water from the anaerobic mineralisation reactor has to be nitrified, it can
instead be partially fed to the Deamox reactor. Anammox activity is stimulated by the
denitrifying conditions in the Deamox reactor and since nitrite concentrations are kept
low the process is not thought to produce unwanted emissions of NOx-gases (Kalyuzhnyi
et al., 2006). Since sulphide rich waters are not common in municipal wastewater
treatment the Deamox process has been further developed utilising volatile fatty acids
as a more widespread electron donor for the partial denitrification (Kalyuzhnyi et al.,
2008).
2.5 N2O emissions from wastewater treatment
It has been known for decades that N2O is produced both as an intermediate and end
product in the metabolism of microorganisms performing nitrification and
denitrification processes (Hooper, 1968, Poth and Focht, 1986, Firestone et al., 1979).
Until recently, anammox activity has not been believed to produce any N2O, however
Kartal et al., (2007) have shown that anammox bacteria produces small amounts of N2O
as a result of detoxification of NO which is an intermediate in the anammox process.
Variable temperature and loading rates of inorganic nitrogen compounds, low pH,
alternating aerobic and anaerobic conditions together with growth rate and microbial
composition are parameters that have great influence on N2O emissions from a
wastewater treatment plant (Kampschreur et al., 2008 b). N2O production as a
consequence of these environmental conditions during nitrification and denitrification
will be described in the chapter 2.5.1-2.5.2.The possibility of chemical N2O production in
wastewater treatment is shortly described in 2.4.3. Table 2 gives molecular weight of
N2O and the water solubility both in mol/l and g/l.
Table 2. Physical properties of N 2O
Property:
Unit:
Molecular weight 44.0 g/mol
Water solubility (0 salinity at 20 ̊C)
27.05∙10 − 3 mol/l
1.19 g/l
18

2.5.1 Nitrification as a source of N2O emissions
Ammonium oxidising bacteria (AOB) are the organisms believed to be responsible for
N2O production during nitrification. N2O can be produced both through aerobic
oxidation of ammonium and through nitrifier denitrification of nitrite with ammonium
as an electron donor (Schmidt and Bock, 1997, Kampschreur et al., 2006).
In the presence of oxygen N2O is produced during oxidation of ammonium with oxygen.
2NH O 2HCO N O H O CO (2.5.1)
(Trela et al., 2005)
Hooper, (1968) detected hydroxylamine-nitrite reductase, an enzyme in Nitrosomonas
europaea that reduces nitrite in the presence of hydroxylamine with NO and N2O as
products. Nitrite is reduced anaerobically to N2O with hydroxylamine:
HN OH HNO N O 2H O (2.5.2)
The denitrifying activity of Nitrosomonas is only related to life supporting energy yield
and is probably a survival mechanism in anaerobic habitats (de Bruijn et al., 1995). Low
DO concentrations in the nitrification process has been shown to give higher N2O
emissions than a process operated under well aerated conditions (Magnaye et al., 2008).
High nitrite and ammonium concentrations, high organic loading, low temperature
together with short sludge age are other factors known to give rise to increased N2O
emissions in the nitrification process (Kampschreur et al., 2009).
2.5.2 Denitrification as a source of N2O emissions
Denitrifying organisms are producing N2O as an intermediate when nitrate or nitrite is
reduced to N2 (Kampschreur et al., 2007). Production of N2O takes place as the nitrate
reductase system for electron transport is induced to produce ATP under anoxic
conditions (Gray, 2004), the process occurs in the following sequence:
NO
(2.5.3)
A low pH (

found that when the added quantities of carbon source only allowed between 66-88%
denitrification, N2O emissions from the system were increased from an average of 0.2%
up to 1.3% of reduced nitrate. Long residence times for N2O in denitrifying sludge have
been shown to result in smaller amounts of emitted N2O. Since N2O is an intermediate in
the denitrification process dissolved N2O in the water phase can be turned over by the
denitrifiers and a long residence time for N2O increases the possibility that the gas is
converted into dinitrogen gas. The experiments showing these results were performed
in 100 ml bottles with different sludge volumes which also indicates that N2O emission
is greater from wastewater treatment basins with large surface to volume ratio
(Gejlsbjerg et al., 1997).
2.5.3 Chemical production of N2O
N2O can be produced by chemical denitrification in a wastewater treatment plant, the
reduction follows the same pathway as during biologic denitrification shown in eq.
(2.5.3). The difference is that chemical reductants are reducing the nitrogen compounds
instead of microbial enzymes (Debruyn et al., 1994).
Figures of N2O emissions from microbial nitrogen conversion found during the literature
study are summarised in Table 3.
20

Table 3. N 2O emission from different wastewater treatment facilities given in % of influent N-concentrations.
Reactor type
Influent N-concentrations mg/l
DO N 2O emission % of influent
NH 4-N NO 2-N NO 3-N COD mg/l N-concentration
Reference
nitritation – anammox SBR 650 ±50

2.6 Microsensors
Microsensors measure changes in the chemical composition of complex and
heterogeneous environments in a micrometer scale with a very short response time.
The sensors can therefore be used in a broad range of scientific research, for example in
cell and tissue analysis, microrespiration, marine ecology, biofilm analysis and
wastewater treatment. Laboratory experiments in this master thesis are based on online
measurements with one microsensor for nitrous oxide and one for nitrite. Both sensors
that are developed by Unisense, Århus, Denmark rely on electrochemical detection of
N2O.
2.6.1 Nitrous oxide sensor
The N2O microsensor is a Clark-type microsensor constituted of a cathode shaft (tapered
glass casing) equipped with silicone tip membrane. A N2O reducing cathode is
positioned in an electrolyte behind the silicone membrane. The reference for the N2O
reduction is a silver anode and the sensor is equipped with an oxygen front guard (with
an silicone membrane in the tip. The front guard prevents oxygen from interfering with
the nitrous oxide measurements. The guard is filled with an alkaline ascobate solution,
an effective reducing agent, to prevent oxygen interference with nitrous oxide
measurements (Andersen et al., 2001). The silicone membranes in the sensor tip only
allows passage of gases and small uncharged molecules, shielding the electrolyte from
the outer environment (Unisense b, 2007).
Figure 8. Photo of the N 2O microsensor.
22

The microsensor is connected to a piccoameter that polarises the cathode surface where
the nitrous oxide that diffuses through the silicone membrane is reduced to N2 gas. As
nitrous oxide is reduced at the cathode surface two electrons from the silver anode is
used for each reduced N2O molecule. The electron transport gives rise to a current
proportional to the amount of reduced nitrous oxide. The current is registered and
converted to an out signal by the piccoameter. The guard cathode is also polarised to
deplete oxygen in the electrolyte which minimizes zero current (Unisense b, 2007).
The nitrous oxide sensor has a measuring range of about 0-1 atmosphere pN2O with a
response time less than 10 seconds. The stirring sensitivity is smaller than 2% and the
out signal is temperature dependent with a temperature coefficient of about 2-3% per
°C. Interference in the out signal might occur from electrical noise in the surrounding
environment (Unisense b, 2007).
2.6.2 Nitrite biosensor
The nitrite biosensor is a nitrous oxide sensor equipped with a replaceable biochamber
(Figure 9a), (Unisense e, 2009). A plastic tube containing a carbon source and a bacterial
culture constitutes the biochamber that is mounted in the front of the sensor tip (Figure
9b), (Unisense e, 2009). The biomass in the reaction chamber is positioned between the
carbon source required for their growth and an ion-permeable membrane separating
the microorganisms from the external environment (Unisense c, 2007).
The denitrifying bacterial culture used in the biochamber is deficient in NO3 − and N2O
reductase which means that it is only able to reduce NO2 − into N2O. As NO2 − diffuses into
the biochamber it is reduced to N2O by the biomass (Nielsen et al., 2004). Since
denitrifying bacteria are facultative aerobic they can use both oxygen and nitrite as
oxidation agent for their respiration (Larsen et al., 1997). Oxygen is used preferential to
nitrite as it results in a higher energy yield. This will create a NO2 − reducing gradient in
the biochamber with the bacteria closest to the membrane respiring with oxygen. The
NO2 − reducing capacity of the biosensor will depend on the length of the aerobic zone
resulting in higher maximum detectable concentrations of NO2 − in anaerobic
environments (Larsen et al., 1997).
Produced N2O diffuses through the silicone membrane and is reduced at the cathode in
the transducer part of the biosensor. A piccoameter measures the current arising from
the electron transport just as in the case with the nitrous oxide sensor. The output signal
is proportional to the amount of NO2 − that has been reduced after diffusion into the
biochamber (Unisense c, 2007).
23

Figure 9 A) Nitrite biosensor with removable biochamber(Unisense e, 2009). B) Enlargement of
biochamber (Unisense e, 2009).
At 20 °C the biosensor has a measuring range in the interval 0-1000 µM NO2-N, (0-14
mg/l), and gives about 1.25-4 nA in output signal per 100 µM NO2-N (1.4 mg/l) added.
The sensor signal depends on both ionic composition and temperature of the sample. It
might vary up to 30% due to salinity and it has a temperature coefficient of about 2-4%
per °C. Sensitivity to stirring of the sample depends both on temperature and salinity.
The 90% response time in a stirred sample is less than 90 seconds (Unisense c, 2007).
Nitrous oxide diffusing into the biochamber from the external environment is detected
by the N2O transducer and is therefore interfering with the NO2 − signal. The theoretical
sensitivity to N2O should be a signal 2.5 times higher than for equal concentrations of
NO2 − . This since it takes two NO2 − molecules to form one N2O molecule and the diffusion
coefficient for NO2 − is 0.8 times that of N2O (Nielsen et al., 2004).
24

Chapter 3
3. Material and Methods
3.1 Partial nitritation/anammox laboratory MBBR .
A 7.5 litre laboratory MBBR fed with a synthetic medium was used to estimate the N2O
emissions from a single stage nitritation/anammox system. The reactor was initially
started in October 2008 with a carrier material with already established biofilm derived
from Himmerfjärdsverkets full scale DeAmmon ® reactor which is a single stage reactor
for ammonium reduction to dinitrogen gas. The used carrier was AnoxKaldnes K1
biocarrier with a protected surface area of 500 m 2 /m 3 . The total volume of carriers in
the reactor was 3.5 litres which corresponds to about 3400 carriers, a total protected
area of 1.7 m 2 and a filling degree of 46.7%. Figure 10 shows the laboratory set up of the
MBBR system.
Figure 10. The left part of the figure shows a photograph of the MBBR system, the schematic
drawing to the right shows the main features of the MBBR system.
The temperature of the MBBR was kept at around 30 °C. A thermostat bath recirculating
warm water through the jacketed double walls of the reactor was used to maintain the
temperature. pH of the reactor was controlled with a pH electrode connected to a
regulator unit. The regulator controlled a peristaltic pump supplying the reactor with
2M H2SO4 when needed. The synthetic medium was fed to the reactor with a Watson
Marlow peristaltic pump. Aeration and mixing of the system was obtained with two
aquarium pumps that supplied the reactor with air through a punched bottom plate, (2
mm Ø). A top mounted stirrer was used to keep the system mixed during anoxic periods,
see Figure 10 for system description. A timer was used to control the duration of aerated
and mechanical mixed periods.
25

3.2 Reactor medium
The reactor was fed with a synthetic wastewater with an inorganic nitrogen
concentration corresponding to 314 mg/l. The synthetic wastewater contained all vital
nutrients the microorganisms needed, including trace elements, see Table 4 and Table 5
for composition of reactor medium and trace element stock solution.
Table 4. Composition of synthetic medium fed to the MBBR.
Component
NaHCO3 2.6
NH4Cl 1.2
Concentration g/l
KH2PO4 5.67∙10 -3
Peptone 3.0∙10 -3
trace element solution 1 0.40 ml/l
trace element solution 2 0.40 ml/l
Table 5. Composition of trace element stock solution.
Component
Stock solution 1
MgSO4∙7H2O 4.80
MnCl2∙2H2O 1.60
CoCl2∙6H2O 0.48
NiCl2∙6H2O 0.24
ZnCl2 0.26
CuSO4∙5H2O 0.10
FeCl2∙4H2O 1.44
BH3O3 0.104
Na2MoO4∙2H2O 0.440
Na2SeO3∙5H2O 0.288
Na3WO3∙2H2O 0.280
Stock solution 2
CaCl2∙2H2O 5.80
Concentration g/l
Initially the medium was mixed in a 600 litres container situated in the workshop and
pumped to the second floor where the laboratory is situated. Microbial growth in the
tank and pump tubing were causing large differences in composition of the influent
medium to the reactor. Due to these problems the medium was mixed in a 100 litres
tank kept in the laboratory in close connection to the reactor set up.
26

3.3 Analytical methods
Concentrations of NH4-N, NO2-N and NO3-N were determined with Dr Lange’s
spectrophotometry kit after filtration through Munktel 1.6 µm glass fibre filters. During
cycle studies NO2-N and N-tot were analyzed directly with Dr Lange’s method. Samples
were frozen and flow-injection analysis was used to determine NH4-N and NOx, the sum
of NO2-N and NO3-N. The NO3-N content was calculated by subtraction of NO2-N from the
sum of the two NOx species. Dissolved oxygen and pH was sampled with a portable
meter, HQ40d with mounted oxygen and pH probe. Parameters analysed and method
used are summarised in Table 6.
Table 6. Analysed parameters and methods.
Analysed parameter Method
NH4-N
LCK 303/FIA
NO2-N
LCK 342/341/Bio sensor
NO3-N LCK 339
NOx
FIA
N-tot LCK 238
DO
HQ40d
pH
HQ40d
3.3 Cycle studies
To examine which operation conditions which seemed to produce the largest amounts
of N2O gas, the reactor was operated at different DO concentrations during intermittent
and constant aeration. A study where the anoxic phase was prolonged to two hours was
performed to observe how the N2O production was influenced. Mixing with N2 gas at the
same aeration flow as in the aerated period was tested to see how stripping influenced
the amount of N2O in the water phase.
Parameters monitored online in the reactor, every minute were; DO, pH, N2O and NO2-N.
To examine the concentration changes of NH4-N, NO2-N and NO3-N grab samples were
taken in both influent and effluent water.
3.3.1 Intermittent aeration
The reactor was operated at a DO concentration of ~3 mg/l during the aeration phase.
One reactor cycle lasted for one hour with 40 minutes of aeration and 20 minutes of
mechanical mixing. The study started at the same moment as aeration went on after the
anoxic period and lasted for 66 minutes in order to overlap the initial conditions.
Grab samples in the effluent were taken with 6 minute intervals to get two measure
points in the anoxic phase. Only three measurements of the influent medium was taken
27

in one cycle ( 0, 36 and 66 minutes) considered that the variation of the influent medium
during one hour should not be significant.
3.3.2 Prolonged study, intermittent aeration
A study of the effect of prolonged, intermittent aeration was made in order to observe
how the N2O production was influenced by a longer anoxic period. During the first hour
the reactor was operated in the same manner as above and the same sampling
procedure was applied. The anoxic period of 20 minutes during a normal cycle was
prolonged with 2 hours.
Grab samples were taken in the effluent with 6 minute intervals and every 36 minutes in
the feeding medium.
3.3.3 Continuous aeration
Measurements during continuous aeration were performed at DO concentrations of
~1.5 mg/l and ~1 mg/l. To determine the production of N2O gas during these operation
conditions the aeration was turned off and the unaerated period was determined to 20
minutes to be comparable to the measurements done during intermittent aeration.
Measurements proceeded 20 minutes after the aeration was switched on again.
To examine if the accumulation of N2O gas in the water phase during the anoxic period
depended on increased production or was an effect of stripping during aeration, mixing
with pure N2 gas was used during the anoxic period. The N2 gas flow was equal to the
airflow during the aerated period.
Grab samples were taken in the effluent every 6 th minute and every 36 th minute in the
influent medium.
3.4 Calibration of microsensors
The N2O production and NO2 − concentration profile were measured online with Clarktype
microelectrodes described in chapter 2.6. Before usage the microsensors were
calibrated separately in a jacketed, temperature controlled beaker with 300 ml of pH
regulated synthetic medium to assure the same salinity, temperature and pH of
calibration solution and reactor, see Figure 11.
The sensor to be calibrated was mounted in the calibration beaker, a stable sensor signal
was awaited and the zero value was read and registered with Unisense’s software
SensorTrace BASIC. A top mounted stirrer was used for fast mixing and uniform
concentration of the calibration solution.
28

Figure 11. Calibration setup for microsensors.
Both sensors were calibrated by stepwise addition and signal reading at known
concentrations of N2O and NO2-N respectively. The resulting calibration curves are
illustrated in Figure 112. The linear regression shown in the figure is only based on one
single point registered by the computer software at each concentration. Both
concentration profile and linear regression obtained during calibration are drawn to
illustrate the procedure.
Figure 12. Examples of calibration points and concentration profiles for N 2O and NO 2-N
microsensors during calibration procedure. The first calibration point represents the signal
obtained in mV without any addition of N 2O or NO 2-N. As stepwise concentrations of N 2O and NO 2-N
were added during the calibration the sensor signal increased. A stable signal was awaited before
a voltage corresponding to the added concentration was registered.
Addition to known N2O concentrations was achieved by adding a defined volume of a
saturated N2O solution. The saturated N2O solution was prepared by bubbling N2O gas
through distilled water with a flow rate of 1 l/min for at least 30 minutes. For calibration
of the biosensor additions were made from a bulk solution with NaNO2 with a NO2-N
concentration of 5 mg/l. See Appendix A for calculated volume additions of the saturated
N2O and NO2-N solutions. After calibration both sensors were mounted directly into the
MMBR reactor, the N2O sensor was placed in a small metal-mesh basket for protection
from the moving carriers.
29

3.5 Diffusivity tests of N2O
The diffusivity of N2O was examined experimentally since no off-gas equipment was
available during N2O measurements and the calculations of produced N2O are based on
the assumption that the diffusivity of N2O can be neglected.
To get as close as possible to the real conditions in the MBBR process but without any
N2O producing bacteria a 7.5 l reactor of the same type as used for the MBBR process
was utilised during the diffusivity experiments. The influent synthetic wastewater was
selected to get a medium similar in salinity to that in the real process. The reactor was
heated to 30 ̊C with a thermostat bath and K1 heavy carriers (same type of carrier as K1
used in the MBBR, but with slightly higher density) without biomass was used. K1 heavy
was used to keep the carrier material without biofilm in the water phase and not floating
on top of the water surface.
To examine how fast N2O diffuses from the water phase during mechanical mixing N2O
saturated water was added to a concentration of ~11 µM. The decrease of N2O in the
water phase was registered with the N2O microsensor during a period of eight hours.
The stripping effect from aeration was also observed by registering the decrease of N2O
in the water phase during aeration at three different air flow rates. N2O was added to a
concentration of 12 µM.
30

Chapter 4
4. Results
4.1 Process performance
Variations in nitrogen concentration of the influent medium, flow rate, temperature and
pH are shown in Table 7 and Figure 13. Variations in nitrogen concentration shown in
Figure 13 are the sum of influent nitrogen compounds accounted for in Table 7. The
figure also shows variations in oxygen concentrations during aeration.
Table 7. Characteristics of influent feed, flow rates, temperature and pH during the operation
period 09/07/09-15/12/09.
Nitrogen mg/l
NH 4-N NO 2-N NO 3-N Q l/h T °C pH
264.5 ± 24.5 19.7 ±12.4 3.6 ±2.1 0.48 ±0.06 30.0 ±0.8 7.3-7.8
Changes of nitrogen concentrations in the effluent water are shown together with %
nitrogen reduction and the total nitrogen removal in Table 8 and Figure 13. The Effluent
nitrogen illustrated in Figure 13 is the sum of effluent nitrogen compounds seen in Table
8.
Table 8. Average concentrations of inorganic nitrogen in effluent water, reduction rates and
removal rates during the operation period 09/07/09-15/12/09.
Nitrogen mg/l
NH 4-N NO 2-N NO 3-N
Reduction % Removal gN/m 2 d
82.4 ±44.0 6.3 ±1.7 32.3 ±9.5 58 ±13 1.1 ±0.2
On the 28 th of September the operation mode was changed to continuous aeration,
aiming at a DO level of ~1.5 mg/l. It took around one week to get a stable performance
at the desired oxygen level. For measurements at even lower oxygenation the oxygen
concentration was further decreased to ~1.0 mg/l.
31

10
8
DO concentration, pH & temperature
35
28
DO (mg/l), pH
6
4
2
21
14
7
Temp °C
DO (mg/l)
pH
Temperature (°C)
0
0
06/07/09
26/07/09
15/08/09
04/09/09
24/09/09
Date
14/10/09
03/11/09
23/11/09
13/12/09
350
Concentrations of influent and effluent total inorganic nitrogen
Total inorganic nitrogen (mg/l)
300
250
200
150
100
50
0
Σ TIN in (mg/l)
Σ TIN out (mg/l)
06/07/09
26/07/09
15/08/09
04/09/09
24/09/09
Date
14/10/09
03/11/09
23/11/09
13/12/09
Removal rate (gN/m²d)
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Removal rate (gN/m²d) & % reduction of incomming nitrogen
90
80
70
60
50
40
30
20
10
0
% reduction
Removal rate
(gN/m2d)
% reduction
06/07/09
26/07/09
15/08/09
04/09/09
24/09/09
Date
14/10/09
03/11/09
23/11/09
13/12/09
Figure 13. Reactor operation and process performance during the period 09/07/09-15/12/09.
Top graph illustrates concentration of DO during aeration, pH and temperature. The second graph
shows total concentration of inorganic nitrogen in influent feed and effluent water. The third
shows the removal rate gN/m 2 d and the reduced nitrogen in %.
Figure 14 shows the process performance during different operation modes. Green
series are representing measurements done at intermittent aeration during the period
090918-090927. Blue series are representing measurements at continuous operation,
DO ~1.5 mg/l during the period 091006-091010. Orange series are representing
measurement at continuous operation, DO ~1mg/l during the period 091013-091016.
32

10
8
DO concentration, pH & temperature
35
28
DO (mg/l), pH
6
4
21
14
Temp °C
DO (mg/l)
pH
Temperature (°C)
2
7
0
0
14/09/09
19/09/09
24/09/09
29/09/09
Date
04/10/09
09/10/09
14/10/09
19/10/09
350
Concentrations of influent and effluent total inorganic nitrogen
Total inorganic nitrogen (mg/l)
300
250
200
150
100
50
0
Σ TIN in (mg/l)
Σ TIN out (mg/l)
14/09/09
19/09/09
24/09/09
29/09/09
Date
04/10/09
09/10/09
14/10/09
19/10/09
Removal rate (gN/m²d)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Removal rate (gN/m²d) & % reduction of incomming nitrogen
90
80
70
60
50
40
30
20
10
0
% reduction
Removal rate
(gN/m2d)
% reduction
14/09/09
19/09/09
24/09/09
29/09/09
Date
04/10/09
09/10/09
14/10/09
19/10/09
Figure 14. Illustration of how the process performance changes with changed operation mode, The
top graph illustrates DO during the aerobe phase, pH and temperature. The second graph shows
total concentration of inorganic nitrogen in influent feed and effluent water. The third graph
shows the removal rate gN/m 2 d and the reduced nitrogen in %.
4.3 N2O emissions from partial nitritation/anammox MBBR
The microsensor measured the N2O in the water phase during different operation modes
and aeration rates of the MBBR. The actual N2O production was not measured since N2O
left the water phase continuously through stripping and or by diffusion. However an
estimation of the N2O production was obtained by measuring the accumulation of the
N2O directly after the airflow is turned off assuming the N2O diffusion from water to air
is negligible. If corrections for the N2O leaving the reactor with the effluent water is
33

made the increase in N2O accumulation will then be equal to the N2O production rate,
(see appendix B for calculation example). The N2O production is calculated as % of
removed inorganic nitrogen. Two N2O production rates are estimated and referred to as
initial and maximum production rates. The initial production rate is calculated from the
increase of N2O that can be seen in the water phase immediately after switching of
aeration. Maximum N2O production is estimated between the two measuring points
where the increase in N2O has its maximum during the unaerated period. Mean N2O
concentration in the water phase when the MBBR is aerated, calculated initial and
maximum N2O production rates, mean O2 concentrations during the aerated period,
mean nitrogen concentrations, reduction and removal rates for all measurements are
summarised in Table 9 -Table 14. One figure of typical N2O and O2 profiles for each
operation mode is shown in Figure 15 -Figure 20, N2O and O2 profiles for all
measurements made are found in appendix C.
4.3.2 Intermittent aeration.
Measurements of produced N2O at intermittent aeration, (DO ~3.0 mg/l) were
performed at four different occasions. Typical profiles of how N2O and DO changed
during the cycle are shown in Figure 15. The N2O concentration measured in the water
phase varied with the aeration of the MBBR. When aeration started in the beginning of
the cycle the airflow striped N2O out of the water phase and the concentration decreased
to a constant minimum level. As soon as the aeration was shut off the N2O started to
accumulate in the water phase until aeration was switched on again and the procedure
started over as shown in Figure 15.
N₂O (µmol/l)
12
10
8
6
4
2
0
4.5
3.75
2.25
1.5
0.75
0
0 20 40 60 80
Time (min)
3
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
Figure 15. Concentration profiles of N 2O and O 2 during a cycle of intermittent aeration, DO ~3 mg/l
in the aerated phase. The cycle study started in the beginning of the aerated period. N 2O gas was
stripped from the water phase at the same time as the oxygen concentration rose.
Initial N2O production varied between 5.6-11% of influent nitrogen concentration that
was converted into dinitrogen gas (here after referred to as removed inorganic N-
34

concentration), while the maximum production ranged from 11-16% of removed
inorganic nitrogen, see Table 9.
Table 9. Average N 2O concentration in the water phase during aeration. Calculated initial and
maximum N 2O production rates, mean O * 2 concentrations during the aerated period, mean
nitrogen concentration, reduction and removal rates for studies of intermittent aeration at a DO
concentration of ~3.0 mg/l.
Date
Average
N 2O
µmol/l
Produced N 2O in % of
removed inorganic N-
concentration O 2
mean N-concentration
mg/l
initial max mg/l NH 4-N NO 3-N NO 2-N
N-red.
%
Removal
gN/m 2 d
090918 3.2 11.0 16.3 3.22 300 - - 54 1.1
090921 2.0 5.6 11.0 3.49 293 - - 56 1.1
090922 2.6 9.9 13.9 3.10 287 - - 56 1.1
*Mean O2 concentration from the moment when the DO level reaches its maximum
concentration until aeration is shut off and oxygen starts to decrease again.
4.3.2 Prolonged unaerated period.
Three studies of a prolonged unaerated period were made to examine for how long the
accumulation of N2O proceeded. The measurement started in the beginning of a normal
cycle when the airflow was switched on. Aeration lasted for forty minutes followed by
an unaerated period of two hours and twenty minutes, typical profiles of how N2O and
O2 changes during the cycle are illustrated in Figure 16.
12
10
4.5
3.75
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 50 100 150 200
Time (min)
Figure 16. Concentration profiles of N 2O and O 2 during prolonged unaerated period, DO ~3 mg/l
during aerated phase. (Only manually registered O 2 concentrations every sixth minute are
available during the first fifty minutes, due to problems with overwriting of data in the DO meter).
N2O decreased in the water phase as aeration was switched on and the oxygen
concentration started to increase, the concentration profiles resembles the cycle profile
shown in Figure 15 until the prolonged unaerated period started. At first N2O
accumulation was rather linear, when DO decreases under 1 mg/l the accumulation rate
of N2O was reduced until a maximum concentration was reached at DO concentrations
35

close to 0 mg/l. The N2O concentration is constant under a period of 20-50 minutes and
then slowly started to decrease as seen in Figure 16. Initial and maximum production
rates of N2O calculated during the prolonged cycles as are shown in Table 10. Initial N2O
production rates varied between 6.2-11% while maximum production varied between
10-30% of removed inorganic nitrogen.
Table 10. Prolonged measurement: Average N 2O concentration in the water phase during aeration.
Calculated initial and maximum N 2O production rates, mean O 2* concentrations during aerated
period, mean nitrogen concentration, reduction and removal rates.
Date
Average
N 2O
µmol/l
Produced N 2O in % of
removed inorganic N-
concentration O 2
mean N-concentration
mg/l
initial max mg/l NH 4-N NO 3-N NO 2-N
N-red.
%
Removal
gN/m 2 d
090925 0.9 6.2 30.3 3.0 234 - - 59 1.0
090926 2.5 10.7 10.7 2.8 237 - - 47 0.9
090927 2.2 9.5 9.5 3.1 228 - - 57 1.0
*Mean O2 concentration from the moment when DO concentration reached its maximum
level until aeration is shut off.
4.3.3 Continuous operation at DO ~1.5 mg/l
The MBBR was operated at continuous aeration which was switched off for twenty
minutes in order to estimate the N2O accumulation. Figure 17shows the concentration
profiles of N2O and O2. As seen in the figure they resemble the profiles obtained during
cycle studies of intermittent aeration. The N2O accumulation increased as O2 decreased
but not as fast as before.
12
10
4.5
3.75
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 20 40 60 80
Time (min)
Figure 17. Concentration profiles of N 2O and O 2 obtained from measurement during the period of
continuous reactor operation at a DO concentration of ~1.5 mg/l.
Twenty minutes of the anoxic period was enough to reach the maximum N2O
concentration and the period where N2O production seems to be in equilibrium with the
amount of N2O leaving the system. Figure 17 also illustrates that the mean concentration
36

of N2O during aeration was slightly higher and that the maximum concentration reached
was lower than during intermittent aeration.
Initial and maximum % N2O production was calculated to be in the range of 2-3.2% and
5.6.-6.2% of removed inorganic nitrogen respectively, the result is presented in Table
11.
Table 11. Average N 2O concentration in the water phase during aeration. Calculated initial and
maximum N 2O production rates, mean O 2 concentrations during aeration, mean nitrogen
concentration, reduction and removal rates for studies at continuous operation mode DO ~1.5
mg/l.
Produced N 2O in % of
Average
N 2O
removed inorganic N-
concentration O 2
mean N-concentration
mg/l
N-red. Removal
Date µmol/l initial max mg/l NH 4-N NO 3-N NO 2-N % gN/m 2 d
091006 4.3 2.8 5.6 1.3 249 25.2 0.3 66 1.3
091007 2.9 2.1 5.6 1.8 246 28.9 0.0 64 1.3
091010 3.3 3.2 6.2 1.8 220 36.3 0.0 72 1.3
4.3.4 Continuous operation at DO ~1.0 mg/l
Two measurements were performed at a constant aeration with a DO concentration of 1
mg/l. Registered N2O accumulation was the lowest so far and the initial N2O production
was below 2% of reduced inorganic nitrogen. Maximum production varied between 2-
4.3%. Table 12 presents the results from continuous operation at DO concentration of~1
mg/l.
12
10
4.5
3.75
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 20 40 60 80
Time (min)
Figure 18. Concentration profiles of N 2O and O 2 obtained from measurement during the period of
continuous reactor operation at a DO concentration of ~1.0 mg/l.
As seen in Figure 18 N2O was only accumulating for the first 5-6 minutes of the
unaerated period then there was a short time span when produced N2O and N2O flows
that left the reactor were in equilibrium. The N2O concentration measured in the water
phase decreased before aeration started again.
37

Table 12. Average N 2O concentration in the water phase during aeration. Calculated initial and
maximum N 2O production rates, mean O 2 concentrations during the aerated period, mean
nitrogen concentration, reduction and removal rates for studies at continuous operation mode DO
~1.0 mg/l.
Date
Average
N 2O
µmol/l
Produced N 2O in % of
removed inorganic N-
concentration O 2
mean N-concentration
mg/l
initial max mg/l NH 4-N NO 3-N NO 2-N
N-red.
%
Removal
gN/m 2 d
091013 2.5 1.7 4.3 1.0 285 0.0 0.0 85 1.6
091014 2.3 2.0 2.0 1.0 284 0.0 0.3 73 1.3
4.3.5 Effect of mixing with N2 gas during unaerated phase, continuous
operation at DO ~1.0 mg/l and ~1.5 mg/l
Pure N2 gas was used during the anoxic period instead of mechanical mixing, in order to
evaluate the stripping effect from the gas (the same gas flow rate was used as during
aeration with air). A small accumulation of N2O was observed right after the switch from
aeration with air to N2 gas, see Figure 19 The increase was followed by a sharp decrease
in the N2O concentration profile. When aeration was switched on again, the N2O
concentration increased faster than during previous measurements.
12
10
4.5
3.75
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 20 40 60 80
Time (min)
Figure 19. Concentration profiles of N 2O and O 2 when N 2 gas was used for mixing during anoxic
period, reactor was operated with continuous aeration at a DO level of ~1.0 mg/l.
The accumulation of N2O that can be seen was converted to a corresponding % N2O
production calculated to be

Table 13. Average N 2O concentration in the water phase during aeration. Calculated initial and
maximum N 2O production rates, mean O 2 concentrations during the aerated period, mean nitrogen
concentration, reduction and removal rates during measurements with N 2 gas in anoxic phase,
operation at DO ~1.0 mg/l.
Date
Average
N 2O
µmol/l
Produced N 2O in % of
removed inorganic N-
concentration O 2
mean N-concentration
mg/l
initial max mg/l NH 4-N NO 3-N NO 2-N
N-red.
%
Removal
gN/m 2 d
091015 1.9 0.4 - 1.0 279 0.4 0.0 79 1.2
091016 1.1 0.8 - 1.3 278 0.3 0.0 77 1.2
When the MBBR was operated at a DO concentration of 1.5 mg/l instead of 1 mg/l, a
slightly higher N2O accumulation was observed right after the shift from aeration with
air to mixing with N2 gas. Accumulation proceeded for about 5 minutes and there after
the N2O concentration started to decrease. The rate with which N2O left the water phase
increased as aeration with air was switched on, see Figure 20
12
10
4.5
3.75
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 20 40 60 80
Time (min)
Figure 20. Concentration profiles of N 2O and O2 when N 2 gas is used for mixing during anoxic
period, reactor operated with continuous aeration at a DO level of ~1.5 mg/l.
Table 14 presents the results from the twomeasurements done when N2 gas was used
for mixing during the anoxic period.
Table 14. Average N 2O concentration in the water phase during aeration. Mean calculated initial
and maximum N 2O production rates, mean O 2 concentrations during aerated period, mean
nitrogen concentration, reduction and removal rates during measurements with N 2 gas in anoxic
phase, operation at DO ~1.5 mg/l.
Produced N 2O in % of
Average
N 2O
removed inorganic N-
concentration O 2
mean N-concentration
mg/l
N-red. Removal
Date µmol/l initial max mg/l NH 4-N NO 3-N NO 2-N % gN/m 2 d
091017 1.4 1.4 - 1.5 289 0.3 0.0 71 1.2
091018 1.1 2.1 - 1.8 289 0.3 0.0 73 1.3
39

4.4 NO2-N biosensor
The biosensor was used to register changes in NO2-N online during measurements of
N2O production. The purpose was both to examine NO2-N concentration changes in the
anammox process and to see if it was possible to replace the traditional NO2-N analysis
with Dr Lange kit (LCK 342/341). Two typical measurement occasions where the
biosensor has been used are shown here in . All concentrations profiles achieved
through measurements made with the biosensor can be found in appendix C, the sensor
was not in use during measurements of N2O production 14-15 of September. The reason
for not using the sensor was that there were problems in achieving a stable sensor signal
during calibration and the biochamber had to be exchanged.
Figure 21 shows the obtained NO2-N concentrations from online measurements with the
biosensor and from grab samples analysed with Dr Lange’s method, LCK 342, during a
prolonged unaerated measurement period.
DO, NO₂-N (mg/l)
10
9
8
7
6
5
4
3
2
1
0
0.00 50.00 100.00 150.00 200.00
Time (min)
NO₂-N
biosensor
(mg/l)
NO₂-N LCK
342
(mg/l)
Figure 21. NO 2-N concentration profiles obtained with biosensor and with Dr Lange’s method, LCK
342, during prolonged unaerated measurement.
As can be seen in Figure 21 there was a difference in concentrations obtained from the
two different measurement methods. The concentration of NO2-N registered by the
biosensor is 2-3 mg/l higher than NO2-N concentrations obtained from grab samples
analysed with LCK 342. Even if NO2-N concentrations registered with the biosensor
were higher than concentrations obtained from analysis with LCK 342 both methods
showed the same trends in NO2-N concentration profiles during the measurement.
Figure 22 shows the result from online measurements with the biosensor compared to
grab samples analysed with LCK 342 from a measurement occasion when the MBBR was
operated at continuous aeration at a DO level of ~1.5 mg/l. The NO2-N concentrations
obtained from both methods correlated much better during this measurement. The
deviation in NO2-N concentrations was below 1 mg/l during this measurement and the
concentration profile given from the two methods correlates well.
40

12
NO₂-N (mg/l)
10
8
6
4
2
NO₂-N
biosensor
(mg/l)
NO₂-N LCK 342
(mg/l)
0
0 20 40 60 80
Time (min)
Figure 22. NO 2-N concentration profiles obtained with biosensor and with Dr Lange’s method, LCK
342, from measuring session when the reactor was operated at continuous aeration.
Changes in measured NO2-N concentration with LCK 342 varied between 4-6 mg/l,
while the NO2-N concentrations registered with the biosensor stayed in a slightly
narrower range of 4-5 mg/l. Figure 22 also shows that the biosensor seems to have a lag
phase, a phenomenon which can be seen in Figure 21 as well.
4.5 Diffusivity and stripping test of N2O
The diffusivity test of N2O performed in a reactor with carriers without biofilm during
mechanical mixing showed that N2O dissolved in the water phase left the system slowly.
Figure 23 shows how the initial N2O concentration decreased from ~11- 4 µmol N2O/l
during a period of about 8 hours, (500 minutes). Linear regression of the diffusion rate
showed that ~0.0132 µmol N2O left the reactor per minute. This molar concentration
corresponds to < 1% of N2O present in the water phase at all times during the
measurement. In order to validate the assumption that N2O diffusion can be neglected
during calculations of produced N2O the diffusivity rate/min has to be compared to the
production rate/min. The result of this comparison gives that diffusion corresponds to
about 10% of produced N2O during one minute.
41

N₂O µmol/l
12
10
8
6
4
2
0
0 100 200 300 400 500
N₂O (µmol/l)
y = -0.0132x + 10.59
Time (min)
Figure 23. N 2O decrease in the water phase due to diffusion during mechanical mixing.
When the system was aerated dissolved N2O was stripped out of the water phase at a
much higher speed compared to the diffusion rate, see Figure 24-Figure 26. However the
stripping rate did not change much with the different aeration rates. When the reactor
was aerated with an airflow corresponding to 1.2 l/min, dissolved N2O left the water
phase at a rate of ~0.55 µM/min, if linearly approximated, see Figure 24. The linear that
is drawn in Figure 24 shows that a linearization is not a good approximations of how
N2O was stripped out of the water phase. As the stripping rate decreased with
decreasing N2O concentration a potential curve fitting might be a better option which is
also shown in Figure 24.
14
Aeration 1.6 (l/min) with carriers
N₂O (µmo/l)
12
10
8
6
4
2
0
0 5 10 15 20 25
Time (min)
N2O (µmol/l)
y = -0.6482x + 10.193
y = 12.95e -0.143x
Figure 24. Stripping of N 2O from the water phase during aeration with an airflow of 1.2 l/min.
As the aeration rate was increased to 1.6 l/min the linear stripping rate increased to
~0.65 µM/min, see Figure 25.
42

Aeration 1.6 (l/min) with carriers
N₂O (µmo/l)
14
12
10
8
6
4
2
0
0 5 10 15 20 25
Time (min)
N2O (µmol/l)
y = -0.6482x + 10.193
y = 12.95e -0.143x
Figure 25. Stripping of N 2O from the water phase during aeration with an aeration rate of 1.6
l/min.
When the aeration rate was further increased to 2.0 l/min the linear stripping rate
increased to ~0.67 µM/min, see Figure 26.
14
Aeration 2.0 (l/min) with carriers
N₂O (µmo/l)
12
10
8
6
4
2
0
0 5 10 15 20 25
Time (min)
N2O (µmol/l)
y = -0.6722x + 10.714
y = 13.571e -0.141x
Figure 26.Stripping of N 2O from the water phase during aeration with an aeration rate of 2.0 l/min.
With a starting concentration of ~12 µmol/l, it took between 15 and 20 minutes to strip
N2O out of the water phase to a concentration ~1µmol/l.
43

Chapter 5
5. Discussion
5.1 Process performance
Theoretical maximum nitrogen removal by the anammox process is 88%, (Strous et al.,
1998), highest achieved performance in the MBBR during the period of this master
thesis work was about 80% with fluctuations down to a reduction corresponding to only
20%, the mean nitrogen conversion was 58%. Non stable process performance is
probably due to operation disturbances like; stop in the influent flow, power failure,
fluctuations in influent nitrogen compounds, (caused by microbial conversion of
nitrogen compounds in the synthetic wastewater).
As the reactor operation mode was shifted into continuous aeration at a lower DO
concentration both % reduction and removal rate in gN/m 2 d was more stable and
higher than the average during intermittent aeration shown in Figure 13 and Figure 14.
This is consistent with results obtained by Szatkowska et al., (2003) who showed that
higher DO concentrations impact a MBBR anammox process negatively with decreased
nitrogen conversion rates as a result. Since the oxygen penetration depth within the
biofilm increases with increasing DO (Henze et al., 1997) the anaerobic layer where
anammox activity is taking part will be thinner which is causing a lower inorganic
nitrogen conversion rate.
One drawback with the anammox process is that NO3-N is produced during cell
synthesis of anammox bacteria. However this problem is not significant since the
effluent from the anammox process can be re-circulated with the influent water to the
wastewater treatment plant.
5.2 N2O production
Compared to N2O emissions from nitrogen removal processes found in literature (Table
3) the emissions from the single stage nitritation/anammox system examined in this
work can be regarded as relatively high.
During this study N2O production have been higher at intermittent aeration where the
dissolved oxygen concentration averaged around 3 mg/l in the aerated period. Lowest
N2O production recorded was during continuous aeration at DO concentrations
corresponding to 1.0-1.5 mg/l. The emissions during continuous aeration are in the
same range as emissions from a nitrifying SBR reactor (2.8%) and a Sharon reactor
(1.7%) reported by Kampschreur et al., (2008 b and a). R 2 value obtained when
comparing % N2O production of removed inorganic nitrogen with DO concentrations
shows that there is a correlation between the higher N2O emissions and DO in the, see
Figure 27.
44

% produced N₂O
12
10
8
6
4
2
Produced N₂O in
relation to DO
concetration
R² = 0.6859
0
0 1 2 3 4
DO (mg/l)
Figure 27. Correlation between the % N 2O production and DO concentration.
It has been observed that changing environmental conditions can give rise to higher N2O
emissions (Kampschreur et al., 2008, b) and the shifting oxygen conditions during
intermittent aeration can be an explanation to higher N2O emissions during this
operation mode.
If the concentration profiles registered with the biosensor during intermittent aeration
are considered it is shown that nitrite concentrations are actually increasing during the
anoxic phase which is the opposite situation to what could be expected. Since nitritation
is inhibited by low oxygen concentrations the conversion of ammonium into nitrite
should decrease and anammox activity should consume nitrite leading to a total
decrease in nitrite concentrations. High influent nitrite concentrations and the possible
presence of nitrite oxidisers are two likely explanations to increasing nitrite
concentrations during the anoxic period. Since increasing NO2-N concentrations have
been observed to give higher N2O emissions (Tallec et al., 2006,a) rising nitrite
concentrations during the anoxic period observed in this study can also be a reason for
higher emissions during intermittent operation of the MBBR.
Process performance seems to influence the extent of N2O emitted from the MBBR since
less N2O was produced when higher nutrient removal was achieved during periods of
continuous aeration. Figure 28 which shows the correlation between % N2O production
and % N-reduction indicates that process performance might influence the N2O
emissions from the system (R 2 =0.70).
45

% produced N₂O
12
10
8
6
4
2
Produced N₂O in
relation to % N-
reduction
R² = 0.7029
0
0 20 40 60 80 100
% N-reduction
Figure 28. Correlation between % N 2O production and % N-removal.
NO2-N concentrations during continuous aeration decreased as the aeration was
switched off, at the same time N2O production was not as high as during intermittent
aeration. This result can be partly explained with better control of influent nitrogen
fractions during these measurements. A different microbial composition in the MBBR
during continuous aeration or that conditions are not favouring N2O production to the
same extent as during intermittent aeration are other possible explanations to lower N2
O emissions during continuous operation of the reactor. In this study it is not possible to
determine whether better process performance was the reason or if lower N2O
production might be a result of other reasons such as different composition of the
microbial community during continuous aeration.
Increased NH4-N concentrations and decreasing NO2-N concentrations recorded during
prolonged unaerated studies showed that the nitrifying activity decreased as the MBBR
was left without oxygen supply for a longer period. N2O production within the system
ceased at the same time indicating that nitrifier denitrification of ammonium with nitrite
performed by AOB was the reason to N2O emissions. Why N2O production was not
taking part as long as there was NO2-N available for nitrifier denitrification in the water
phase is unknown. One explanation might be that the NO2-N concentration in the biofilm
was below concentrations that the bacteria can utilise.
Stripping tests of N2O and mixing with pure N2 gas during the anoxic phase indicates
that the N2O accumulation registered by the microsensor is due to the microbial activity
producing N2O and to termination in stripping N2O out of the water. It is not possible to
say if the production rate is the same during aeration and the anoxic phase.
Uncertainties and sources of errors can be many during laboratory work some are
shortly discussed here. During these experiments a synthetic wastewater was used, this
might influence the N2O production from the system, a real waste water is more complex
and might give other emission results, both higher and lower. The fact that diffusion
corresponded to 10% of produced N2O in the MBBR indicates that emissions from the
46

laboratory system might be underestimated. If calibrations have been performed with
an unsaturated N2O solution this will give rise to overestimated N2O productions from
the partial nitritation/anammox MBBR. As pointed out by Kampschreur et al., (2009)
changing environmental conditions might lead to higher N2O emissions and short term
laboratory scale measurements might therefore give over estimated N2O emissions.
Since the anammox process needs less resources and produces less CO2 than common
nitrogen removal, anammox has been pointed out as a more environmental friendly
alternative (Fux and Siegrist, 2004). Kuenen and Robertson, (1994) are calling attention
to that wastewaters in the Netherlands generally have a nitrogen content between 40-60
mg/l, each person produces about 150 l/d which gives a nitrogen production of 2.2 kg
nitrogen per person and year and that even a small N2O production corresponding to
0.1% of the nitrogen concentration will result in significant N2O emissions. Considering
that the MBBR process has been found to produce N2O corresponding to a minimum of
2% of removed inorganic nitrogen it has to be further examined whether this single
stage anammox process is more environmental friendly than common nitrogen removal
processes. Even if rather high N2O production was obtained in this study, experiences in
pilot scale trials with similar operation modes has given N2O production as low as 0.2 %
of removed inorganic nitrogen (Christensson, 2010). Additional research is needed to
determine if the N2O production from a full scale process would be as high as the
production found from the laboratory MBBR system. It also has to be determined which
bacteria that are responsible for producing N2O, whether the relatively high N2O
emissions found from the laboratory MBBR are due to biofilm structure with oxygen
pore conditions. Amounts of N2O emissions have to be further evaluated in correlation
to process operation and performance. A single stage biofilm system might not be the
best solution for the partial nitritation anammox process if this process design always
gives rise to high N2O emissions.
5.3 Measurements with NO2-N biosensor
The biosensor gave results that correlated very well with concentrations obtained with
LCK 342 at some occasions and the fluctuations in NO2-N concentration measured with
the biosensor always showed the same trends as achieved with LCK 342. However the
NO2-N biosensor did not give reliable results at all times in use and could never replace
LCK 342 for determination of NO2-N concentrations during this master thesis work.
Some measurements performed with the biosensor recorded much higher NO2-N
concentrations than obtained with the Dr. Lange kit. This was probably due to electric
disturbances that caused electric migration which is the transport of a charged body in
an electric field. Kjær et al., (1999), have shown that this phenomenon can be used to
force negatively charged NO3 − ions over the semi permeable membrane of the
biochamber increasing the ion sensitivity by a factor of more than 10.000. The electric
disturbances can have been caused by other laboratory equipment or since the ground
channel on the backside of the piccoameter was used. This ground port has another
47

electrical potential than the sensor port which can create an electric potential and
increased nitrate flux over the biochamber membrane. (There are two different
possibilities to ground the environment in the close range of the microsensors. The first
option is to use the ground channel connected to sensor port on the piccoameter, the
electric potential of the sensor and ground channel is the same. The other option is use
the ground port on the backside of the piccoameter, this port has another electric
potential than the sensor port).
Since the biosensor relies on denitrifying bacteria converting NO2-N to N2O, the sensor
was hard to work with. The bacteria in the biochamber are changing and adapting their
metabolism as their physical environment with available substrates changes (Larsen et
al., 1997). This means that their metabolism might be influenced by moving from the
environment in which they are kept in between measurements, via the calibration setup
into the MBBR where measurements are performed. At some occasions the biosensor
had to be recalibrated one to three times before giving a stable signal, which is very time
consuming. The sensor also has to be well nursed in between measurements in order to
keep the microorganisms viable.
To obtain the same salinity during calibration and measurement the biosensor was
calibrated in the synthetic wastewater feeding the MBBR. The microbial nitrogen
conversion in the MBBR is changing the ionic composition of the influent wastewater
with a difference in ionic strength of influent medium and effluent as result. Since the
biosensor is sensitive to ionic strength as well as salinity (Nielsen et al., 2004) better
results might have been obtained by calibration of the biosensor in the effluent water.
(Since the effluent water contains NO2-N this calibration method gives a background
signal of NO2-N which has to be corrected for).
The biosensor might be a good option if changes of NO2-N are going to be studied during
cyclic changes of a microbial process. However the sensitivity of the sensor and the fact
that it has to be well looked after in between measurements has to be taken into account
when considering the biosensor as an option to conventional methods of determining
the NO2-N concentrations. The biosensor and required equipment is also a significant
investment cost.
5.4 Diffusivity and stripping test of N2O
Testing the diffusivity of N2O through mechanical mixing with K1–heavy carriers
without biofilm showed that

6. Conclusions
The following conclusions concerning, process performance of the laboratory MBBR,
produced N2O and evaluation of the NO2-N biosensor can be made:
• The single stage nitritation/anammox system produced significant amounts of
N2O with a minimum production of 2% of removed inorganic nitrogen.
• Operating the MBBR at intermittent aeration with a DO of ~3 mg/l gave the
highest N2O production with initial and maximum productions of 6-11% and 10-
30% respectively.
• Smaller amounts of N2O were produced by the partial/nitritation anammox
system during continuous operation at DO in the interval 1-1.5 mg/l. The initial
N2O production was found to be 2-3% and the maximum N2O production
corresponded to 2-6%.
• When the MBBR was exposed to a longer period of anoxic conditions both
ammonium oxidation and N2O production ceased.
• From results of mixing with N2 gas during the anoxic period it cannot be said
with certainty that the N2O production is the same during aeration and anoxic
phase. The absolute number on overall N2O production for an operation mode
(based on the measurements of N2O accumulating during the anoxic phase) could
be both overestimated or underestimated and should therefore be used as a
comparative tool.
• It was not possible to replace conventional methods for determination of NO2-N
concentrations with the NO2-N biosensor since stable operation of the sensor
could not be obtained at all times.
51

7. Future research
Better understanding off which mechanisms and organisms that are responsible for N2O
production in the nitrifying/anammox MBBR system is needed. There is also a need for
better accuracy in the measurements of emitted N2O from the process.
• Measurement should be done where N2O is measured both in the water phase
and in the off-gas simultaneously, this would both help to better understand
when the N2O is produced in the system and it would give a much better accuracy
of how much N2O that is produced and emitted by the MBBR system.
• Since the biofilm creates a microenvironment with anoxic conditions which are
believed to enhance the N2O production by AOB the importance of biofilm
structure and thickness should be investigated.
• Disturbances are believed to cause higher N2O production from the
microorganisms. It should be examined whether intermittent aeration could be
considered a disturbance to the bacteria performing the nitrogen removal
causing higher N2O emissions from the process.
• To be able to operate the MBBR in a manner that gives as small amounts of
emitted N2O as possible it is of great importance to understand which
microorganisms within the system that are responsible for the N2O emissions.
Measurements of the N2O production during batch tests with inhibitors should be
performed to gain this kind of knowledge.
• Since substrate concentrations (NH4 + , NO2 − and NO3 − ) are known to influence the
amount of produced N2O, it would be interesting to evaluate their impact on
emitted N2O in both batch tests and with operation at different influent inorganic
nitrogen concentrations. Performing the tests in this manner could give answers
to whether it is the increased nitrogen concentrations /disturbance that causes
the increase in N2O production or the actual higher substrate concentration.
• As volume to surface ratios are of importance to emitted N2O from a wastewater
treatment process and since there are further differences between full scale and
laboratory systems the emitted N2O from full scale systems should be
determined.
• Measurement from a process operated with real wastewater is needed for
determination of N2O emissions during real conditions.
• Examine the influence of aeration rate on N2O emission by continuous aeration
with pure oxygen, (a much lower aeration rate can maintain a sufficient oxygen
concentration in the reactor if pure oxygen is used.)
53

8. References
Abma W.R., Schultz C.E., Woutres J.W., Mulder J.W., van Loosdrecht M.C.M., van der Star
W., Strous M., Tokutomi T. (2006) Full Scale Granular Sludge ANAMMOX® process. 4th
CIWEM Annual Conference, New Castle, 12-14 September 2006.
Abma W.R., Schultz C.E., Mulder J.W., van Loosdrecht M.C.M., van der Star W., Strous M.,
Tokutomi T., (2007). The advance of Anammox. Water 21, February 2007.
Andersen K., Thomas K., Revsbech N.P., (2001). An oxygen insensitive microsensor for
nitrous oxide. Sensors and Actuators B-Chemical, 81 (2001), 42-48.
Bani Shahabadi M., Yerushalmi Y., Haghighat F., (2009). Impact of process design on
greenhouse gas (GHG) generation by waste water treatment plants. Water research, 43,
2679-2687.
Bock E., Scmidt I., Stüven R., Zart D., (1994). Nitrogen loss caused by denitrifying
Nitrosomonas cells using ammonium or hydrogen gas as electron donors and nitrite as
electron acceptor. Archives of Microbiology (1995), 163, 16-20.
Bogner, J., Abdelrafie Ahmed M., Diaz C., Faaij A., Gao Q.,. Hashimoto S., Mareckova K.,
Pipatti R., Zhang T., (2007). Waste Management, In Climate Change 2007: Mitigation.
Contribution of Working Group III to the Fourth Assessment Report of the
Intergovernmental Panel on Climate Change [B. Metz, O.R. Davidson, P.R. Bosch, R. Dave,
L.A. Meyer (eds)], Cambridge University Press, Cambridge, UnitedKingdom and New
York, NY, USA.
Broda E., (1977). Two kinds of lithotrophs missing in nature. Zeitschrift für allgemeine
mikrobiologi, 17, (6), 491-493.
de Bruijn P., vad der Graaf A., Jetten M., Robertson L., Kuenen G., (1995). Growth of
Nitrosomonas europaea on hydroxylamine. FEMS Microbiology Letters 125, 179-184.
Carvallo L., Carrera J., Chamy R., (2002). Nitrifying activity monitoring and kinetic
parameters determination in a biofilm airlift reactor by respirometry. Biotechnology
Letters, 24, 2063-2066.
Chirstensson, Magnus, personal communications, 2010.
Debruyn W., Lissens G., van Rensbergen J., Wevers M., (1994). Nitrous oxide emissions
from waste water. Environmental Monitoring and Assessment, 31, 159-165.
55

Egli K., Fanger U., Alvarez P. J., Siegrist H., van der Meer J. R., Zehnder A. J., (2001).
Enrichment and characterization of an anammox bacterium from a rotating biological
contactor treating ammonium-rich leachate. Archives of microbiology 175, (3), 198-207.
Egli K., Bosshard F., Werlen C., Lais P., Siegrist H., Zhender A.J.B., vad der Meer J.R.,
(2003). Microbial composition and structure of a rotating biological contactor biofilm
treating ammonium-rich wastewater without organic carbon. Microbial Ecology, 45, 419-
432.
Firestone M., Smith M., Firestone R., Tiedje J., (1979). The influence of nitrate, nitrite and
oxygen on the composition of the gaseous products of denitrification in soil. Soil Science
Society of America Journal, 43, 1140-1144.
Fux C., Boehler M., Huber P., Brunner I., Siegrist H., (2002). Biological treatment of
ammonium-rich wastewater by partial nitritation and subsequent anaerobic ammonium
oxidation (anammox) in a pilot plant. Journal of Biotechnology, 99, 295-306.
Fux C., Egli K., van der Meer J.R., Siegrist H., (2003). The anammox process for nitrogen
removal from waste water. EAWAG news 56 (November 2003), 20-21.
Fux C., Siegrist H., (2004). Nitrogen removal from sludge digester liquids by
nitrification/denitrification or partial nitritation/anammox: environmental and
economical considerations. Water Science and Technology, 50, (10), 19-26.
Gejlsbjerg B., Frette L., Westermann P., (1997). Dynamics of N2O production from
activated sludge. Water research, 32, (7), 2113-2121.
Gillberg L., Hansen B., Karlsson I., Nordstöm Enkel A., Pålsson A., (2003). About water
treatment. ISBN 91-631-4344-5, Helsingborg, Kemira Kemwater.
Gray N., (2004). Biology of wastewater treatment (2nd Edition). Imperial College Press,
London. Series editor. Bell J. Centre of environmental technology, imperial college.
Gut L. (2006) Assessment of a partial nitriation/ Anammox system for nitrogen removal.
Licentiate thesis, Department of Land and Water Resources Engineering, Royale
Institute Of Technology, Stockholm, Sweden
Helmer C., Tromm C., Hippen A., Rosenwinkel K-H., Seyfried C.F., Kunst S., (2001). Single
stage biological nitrogen removal by nitritation and anaerobic ammonium oxidation in
biofilm systems. Water Science and Technology, 43, (1), 311-320.
Henze, M., HarremoësP., la Cour Jansen J., Arvin E., (1997), Wastewater Treatment 2nd
edition. ISBN 3-540-62702-2. Berlin Heidelberg, Springer-Verlag.
56

Hippen A., Rosenwinkel K-H., Baumgarten G., Seyfried C.F., (1997). Aerobic
deammonification: A new experience in the treatment of wastewaters. Water Science and
Technology, 35, (10), 111-120.
Hooper A., (1968). A nitrite reducing enzyme from Nitrsomonas europaea preliminary
charachterization with hydroxylamine as electron donor. Biochimica et Biophysica Acta,
162, 49-65.
Itokawa H., Hanaki K., Matsuo T., (2001). Nitrous oxide production in high loading
biological nitrogen removal process under low COD/N ratio condition. Water Research, 35,
(3), 657-664.
Jacob D., (1999). Introduction to atmospheric chemistry. ISBN 0-691-00185-5, Princeton:
Princeton University Press.
Jetten M.S.M, Strous M, van de Pas-Schoonen K.T., Schalk J., van Dongen U.G.J.M., van de
Graaf A.A., Logemann S., Muyzer G., van Loosdrecht M.C.M., Kuenen J.G., (1999). The
anaerobic oxidation of ammonium. FEMS Microbiology Reviews, 22, (5), 421-437
Jetten M., Wagner M., Fuerst J., van Loosdrecht M., Kuenen J., Strous M., (2001).
Microbiology and application of the anaerobic ammonium oxidation ('anammox') process.
Current Opinion in Biotechnology 12, (3), 283-288.
Jetten M.S.M., Cirpus I., Kartal B., van Niftrik L., van de Pas-Schoonen K.T., Sliekers O.,
Haajier S., van der Star W., Schmid M., van de Vossenberg J., Schmidt I., Harhangi H., van
Loosdrecht M., Kuenen J., Op den Camp H., Strous M., (2004). 1994-2004: 10 years of
research on the anaerobic oxidation of ammonium. Biochemical Society Transactions 33,
(1), 119-123.
Jetten M.S.M., van Niftrik L., Strous M., Kartal B., Keltjens J.T., Op den Kamp H.J.M.,
(2009). Biochemistry and molecular biology of anammox bacteria. Critical Reviews in
Biochemistry and Molecular Biology, 44, (2), 65-84.
Joss A., Salzberger D., Eugster J., König R., Rottermann K., Burger S., Mohn J., Siegrist H.,
(2009). Full-Scale Nitrogen Removal from Digester Liquid with Partial Nitritation and
Anammox in one SBR. Environmental Science & Technology, 43,(14), 5301-5306.
Jung J.Y., Kang S.H., Chung Y.C., Ahn D.H., (2007). Factors affecting the activity of
anammox bacteria during start up in the continuous culture reactor. Water Science and
Technology, 55, (1), 459-468.
57

Kalyuzhnyi S., Gladchenko M., Mulder A., Versprille B., (2006). New anaerobic process for
nitrogen removal. Water Science and Technology, 54, (8), 163-170.
Kalyuzhnyi S., Gladchenko M., Kang H., Mulder A., Versprille B., (2008). Development and
optimisation of VFA driven DEMAOX process for treatment of strong nitrogenous
anaerobic effluents. Water Science and Technology, 57, (3), 323-328
Kampschreur M.J., Tan N.C.G, Picioreanu C., Jetten M.S.M., Schmidt I., van Loosdrecht
M.C.M ., (2006). Role of nitrogen oxides in the metabolism of ammonia-oxidising bacteria.
Biochemical Society Transactions 34, (1), 179-181.
Kampschreur M.J., van der Star W.R.L., Wielders H.A., Mulder J.W., Jetten M.S.M., van
Loosdrecht M.C.M., (2008, (a)). Dynamics of nitric oxide and nitrous oxide emission during
full-scale reject water treatment. Water Research 42, (3), 812-826.
Kampschreur M.J., Tan N.C.G, Kleerebezem R., Picioreanu C., Jetten M.S.M., and van
Loosdrecht M.C.M ., (2008, (b)). Effect of dynamic processes on nitrogen oxides emission
from an nitrifying culture. Environmental Science Technology, 42, 429-435.
Kampschreur M.J., Temmink H., Kleerebezem R., Jetten M.S.M., van Loosdrecht M.C.M.,
(2009). Nitrous oxide emission during waste water treatment. Water Research 43, (17),
4093-4103.
Kartal B., Kuypers M.M.M., Lavik G., Schalk J., Op den Camp H.J.M., Jetten M.S.M., Strous
M., (2007). Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas
via nitrite and ammonium. Environmental Microbiology, 9, (3), 635-642.
Kuenen G., Robertson L., (1994). Combined nitrification-denitrification processes. FEMS
Microbiology Reviews, 15, 109-117.
Kjær T., Huer Larsen L., Revsbech N-P., (1999). Sensitivity of ion-selective biosensors by
electrophoretically mediated analyte transport. Analytica Chimica Acta, 391, 57-63.
Larsen L.H., Kjaer T & Revsbech N.P., (1997). A microscale NO3− biosensor for
environmental applications. Analytical Chemistry, 69, (17), 3527-3531.
Liljenström S., Kvarnbäck M., (red), (2007). Miljömålen i ett internationellt perspektiv, de
facto 2007. Naturvårdsverket, Bromma.
Magnaye F.A., Gaspillo Pag-asa D., Auresenia J.L., Kampschreur M.J., Kleerebezem R., van
Loosdrecht M., (2008). Nitrous and nitric oxides and the effect of oxygen level and nitrite
concentration on its emission from nitriation and nitrification-denitrification reactors.
Water Research 42, (3), 812-826.
58

Metcalf & Eddy I. (2003). Wastewater engineering: treatment and reuse. New York, NY,
McGraw-Hill.
Mulder A.V.A, Robertson L.A., Kuenen J.G.,(1995). Anaerobic ammonium oxidation
discovered in a denitrifying fluidized-bed reactor. FEMS Microbiology Ecology 16(3), p.
177-184.
Nielsen M., Larsen L.H., Jetten M.S.M., Revsbech N.P., (2004). Bacterium-Based NO2−
Biosensor for environmental Applications. Applied and Environmental Micorbiology, 70
(11), p. 6551-6558.
Pierzynski A.M., Sims J.T., Vance G.F., (2005). Soils and environmental quality. 3. ed. Boca
Raton: Taylor & Francis Group, LCC.
Poth M., Focht D., (1985). 15N Kinteci analysis of N2O production by Nitrosomonas
europaea: an examination of nitrifier denitrification. Applied and Environmental
Microbiology, 49, (5), 1134-1141.
Prescott L.M, Harley J. P., Klein D.A., (2005). Microbiology. 6. ed. New York: McGraw Hill.
Rosenwinkel K., Cornelius A., (2005). Deammonification in the moving-bed process for the
treatment of wastewater with high ammonia content. Chemical Engineering Technology,
28, (1), 49-52.
Siegrist H., Reithaar S., Koch G., Lais P., (1998). Nitrogen loss in a nitrifying rotating
contactor treating ammonium rich wastewater without organic carbon. Water Science
and Technology, 38, (8-9), 241-248.
Schmidt I., Bock E., (1997). Anaerobic ammonia oxidation with nitrogen dioxide by
nitrosomonas eutropha. Archives of Microbiology 167, 106-111.
Strous M., van Gerven E., Zheng P., Kuenen J.G., Jetten M.S.M., (1997). Ammonium removal
from concentrated waste streams with the anaerobic ammonium oxidation (anammox)
process in different reactor configurations. Water Research, 31,(8), 1955-1962.
Strous M., Heijnen J., Kuenen J., Jetten M., (1998). The sequencing batch reactor as a
powerful tool for the study of slowly growing anaerobic ammonium-oxidizing
microorganisms. Applied Microbiology Biotechnology, 50, 589-596.
Strous M, F. J., Kramer E.H.M., Logemann S., Muyzer G., van de Pas-Schoonen K.T., Webb
R., Kuenen J.G., Jetten M.S.M., (1999). "Missing lithotroph identified as new planctomycete"
Nature, 400, (6743), 446-449.
59

Szatkowska B., Plaza E., Trela J., Bakowska A., (2003). Influence of dissolved oxygen
concentration on deammonification process performance. Integration and optimisation of
urban sanitation systems, Joint Polish-Swedish Reports, NO 11. Royal Institute of
Technology, Stockholm.
Tallec G., Garnier J., Billen G., Gousailles M., (2006 (a)). Nitrous oxide emission from
secondary activated sludge in nitrifying conditions of urban waste water treatment plants:
Effect of oxygenation level. Water Research, 40, 2972-2980.
Tallec G., Garnier J., Gousailles M.,(2006 (b)). Nitrogen removal in a waste water
treatment plant through biofilters: nitrous oxide emission during nitrification and
denitrification. Bioprocess Biosystem Engineering, 29, 323-333.
Tallec G., Garnier J., Billen G., Gousailles M., (2008). Nitrous oxide emissions from
denitrifying activated sludge of urban waste water treatment plants under anoxia and low
oxygenation. Bioresource Technology, 99, 2200-2209.
Third K.A., Sliekers A.O, Kuenen J.G., Jetten M.S.M., (2001). The CANON System
(Completely Autotrophic Nitrogen-removal Over Nitrite) under Ammonium Limitation:
Interaction and Competition between Three Groups of Bacteria. Systematic and Applied
Microbiology, 24, 588-596.
Third K.A., Paxman J., Schmid M., Strous M., Jetten M.S.M., Cord-Ruwisch R., (2005).
Treatment of nitrogen-rich wastewater using partial nitrification and Anammox in the
CANON process. Water Science and Technology, 52, (4), 47-54.
Trela J., Plaza E., Gut L., Szatkowska B., Hultman B., Bosander J., (2005).
Deammonifikation, en ny process för behandling av avloppsströmmar med hög kvävehalt
– fortsatta pilot-plant experiment. Rapport NR 2005-14, Svensk Vatten Utveckling.
Tsushima I., Ogasawara Y., Kindaichi T., Satoh H., Okabe S., (2007). Development of highrate
anaerobic ammonium-oxidizing (anammox) biofilm reactors. Water Research 41,
1623-1634.
Unisense, b (2007). Nitrous oxide sensor manual, version 20070912. Unisense A/S.
Unisense, c (2007). NOx- biosensor manual, version 20071001. Unisense A/S.
van Dongen U., Jetten M.S.M., van Loosdrecht M.C.M., (2001). The SHARON® -Anammox®
process for treatment of ammonium rich wastewater. Water Science and Technology,
44,(1), 153-160.
60

van Niftrik L.A., Fuerst J.A., Sinninghe Damsté J.S., Kuenen J.G., Jetten M.S.M., Strous M.,
(2004). The anammoxosome an intracytoplasmic compartement in anammox bacteria.
FEMS Microbiology Letters, 233 (1), p. 7-13.
Warfvinge P., (2008). Mass balances and reactor design- Study book for environmental
engineering. Department of Chemical Engineering, Box 124, Lund.
Ødegaard H., (ed.), (1993). Nitrifying and denitrifying biofilms for wastewater treatment.
Nordic Collaborative Project on Environmental Biotechnology. Nordic Council of
Ministers.
Ødegaard H., Rusten B., Westrum T., (1994). A new moving biofilm reactor – applications
and results. Water Science and Technology, 29( 10-11), 157-165.
Internet källor:
AnoxKaldnes /Produkter och tjänster/Bioprocesser/MBBR TM (online). Available
at:

Appendix A
Calculation of concentrations in calibration solutions for N2O and NO2-
N microsensors
During calibration of the microsensors solutions with known concentration of N2O and
NO2-N are used from the start. The volume in the calibration chamber is known and the
final concentration for each calibration step is also known. The initial volume that has to
be added to get the correct concentration in the calibration solution is obtained with:
(A.1)
where:
Mi = the initial molar concentration mol/l, Vi = the initial volume (l), Mf = the final molar
concentration mol/l and Vf = the final volume (l).
The initial volume is then added in each calibration step until the final concentration is
reached.
Equilibrium nitrous oxide concentrations at different temperatures and salinities are
obtained from the nitrous oxide sensor users manual. The saturated water solution is
prepared from distilled water at 20 °C corresponding to an equilibrium N2O
concentration of 27.05mmol/l or ~1.2 g/l. 2 µM (88 µg/l )N2O was added in each step to
perform a five point calibration up to 10 µM, (440 µg/l ). See Table A1 for calculated
initial volume, (Vi), of saturated N2O solution added to the calibration chamber, Mi, Mf,
and Vf are also given in the table.
A stock solution with a NO2-N concentration of 5 g/l NO2-N was used for calibration of
the biosensor. 2 mg/l NO2-N was added in each step to perform a five point calibration
up to 10 mg/l NO2-N. See Table A1 for calculated initial volume of NO2-N stock solution
added to the calibration chamber, Mi, Mf, and Vf are also given in the table.
Table A1. Parameters used to calculate the volume of concentrated N 2O and NO 2-N solutions that
has to be added during the calibration procedure of the sensors, calculated values for V i is also
shown.
N2O calibration solution NO2-N calibration solution
Mi 27.05∙10 -3 mol/l Mi 5∙10 -3 g/l
Mf 2∙10 -6 mol/l Mf 2∙10 -3 g/l
Vf 0.300 L Vf 0.300 l
Vi 22∙10 -6 L Vi 120∙10 -6 l
22 µl 120 µl
63

Appendix B
Calculations of N2O emissions
The purpose is to calculate the produced amount of nitrous oxide as percentage of
removed inorganic nitrogen.
It is assumed that the MBBR is behaving like an ideal completely stirred tank reactor,
(CSTR), and that the general mass balance equation for a given component can be
implied:
eq.(3.1)
The in and output terms are molar fluxes over the reactor boundary, acquired as the
product of the volumetric flow rates, Q (m/s) and the concentrations, c (mole/l).
Production within the system is described by the kinetic rate equation, r (mole/m 3 s)
times the reactor volume, V (m 3 ), (negative sign indicating consumption instead of
production). Accumulation is quantified by the molar change of a substance per unit
time, described by a time dependent differential including the concentration, c (mol/l)
and the reactor volume, V (m 3 ). The mass balance equation for a component j can be
rewritten as:
( )
mol/s
eq.(3.2)
For a reacting system like the MBBR where some substances are consumed and others
are produced various kinds of substances will be passing the system borders in the
influent, effluent and through the gas phase, see Figure B1.
Figure B1. Mass transfer over the MBBR system boundaries.
67

With the considerations; (i) that there is no N2O gas in the influent medium, (ii) the
reactor volume is constant and (iii) Qin and Qout are equal the mass balance for the
system can be described by:
eq.(3.3)
: ( )
eq.(3.4)
: 0 ( )
To get the consumption and production rates equation 3 and 4 are rewritten:
eq.(3.5)
:
( )
eq.(3.6)
:
( )
rN in the first equation is describing the consumption rate of the influent nitrogen, if
there were no N2O or any other gaseous production in the system this term would
entirely correspond to the production of N2 gas leaving the system. Here it is assumed
that all removed inorganic nitrogen is leaving the system in gaseous form as N2 or N2O.
The accumulation term that would correspond to assimilation in equation 3 is neglected.
Calculation example (091007)
Used parameters to calculate the consumption (rN) and production (rN2O ) rates are
shown in Table B1.
Table B1. Calculation parameters used to caluclate r N and r N2O.
Parameter value unit
Q 0.540 l /h
V 7.5 l
cinN 273.904∙10 -3 g/ l
coutN 97.593∙10 -3 g/l
c N2O t1 2.82414∙10 -6 mol/l
c N2O t2 2.97601∙10 -6 mol/l
0.1519∙10 -6 mol/lmin
0.540 · (273.904 · 10 97.593 · 10 )
0.2116 · 10
7.5 · 60
0.540 · (2.97601 · 10 )
0.1519 · 10 0.1555 · 10
7.5 · 60
g/lmin
mol/lmin
68

The rN value is calculated with the mean in and effluent concentrations during one cycle/
measurement session. For the approximation of the total N2O production in the MBBR it
is assumed that the production corresponds to the initial value of rN2O seen when
aeration is turned off (Figure B2) and that this assumption is valid at all times.
12
N₂O (µmol/l),DO (mg/l)
10
8
6
4
2
N₂O
(µmol/l)
DO
(mg/l)
N₂O (µmol/l), DO (mg/l)
0
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0 20 40 60 80
Time (min)
Initial N₂O production
2.82414
2.97601
20 22 24 26 28 30
N₂O
(µmol/l)
DO
(mg/l)
y = 0.1519x - 0.8207
Time (min)
Figure B2. The accumulation term in the production rate equation is obtained as the initial k-value
of the N 2O curve when N 2O starts to accumulate in the reactor. The lower part of the figure shows
an enlargement of the area.
The percentage of removed inorganic nitrogen emitted as N2O is finally obtained by:
· 2
· 100
where rN2O given in mole N2O/l min is converted to g N/l min by multiplying with 2MN,
the molar weight of N in g/mole. MN is multiplied by 2 since the molar ratio for produced
N2O-N to removed inorganic N is 2:1.
69

Table B2. Calculation parameters used to calculate the percentage N 2O-N produced by removed
inorganic N.
Parameter value
unit
rN2O 0.1555∙10 -6 mole/l min
MN 14.01 g/mol
0.2116 · 10 mg/l
rN
0.1555 · 10 · 2 · 14.01
0.2116 · 10 · 100 2.1%
Assumptions made for this calculation are; (i) that the microorganisms in the system are
unaffected of the changed operation conditions during the time span of one minute
when the production rate is approximated, (ii)that the mass transfer of N2O through the
phase boundary between liquid and air is negligible during the time interval of one
minute, (iii) that there is no net change in production due to operating conditions during
one cycle.
70

Appendix C
Microsensor measurements
12
090918 Intermittent aeration, DO 3.0 (mg/l)
4.5
12
090921 Intermittent aeration, DO 3.0 (mg/l)
4.5
10
3.75
10
3.75
N₂O (µmol/l)
8
6
4
3
2.25
1.5
DO (mg/l)
N₂O sensor
(µmol/l)
DO (mg/l)
NO₂-N (mg/l)
8
6
4
3
2.25
1.5
DO (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
2
0.75
2
0.75
0
0
0 20 40 60 80
Time (min)
0
0
0 20 40 60 80
Time (min)
Figure C1.
Figure C4.
30
090918 Intermittent aeration, DO 3.0 (mg/l)
4.5
12
090922 Intermittent aeration, DO 3.0 (mg/l)
4.5
25
3.75
10
3.75
NO₂-N (mg/l)
20
15
10
5
3
2.25
1.5
0.75
DO (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 20 40 60 80
Time (min)
0
0
0 20 40 60 80
Time (min)
Figure C2.
Figure C5.
12
10
090921 Intermittent aeration, DO 3.0 (mg/l)
4.5
3.75
40
090922 Intermittent aeration, DO 3.0 (mg/l)
4
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O sensor
(µmol/l)
DO (mg/l)
NO₂-N (mg/l)
30
20
10
3
2
1
DO (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
0
0
0 20 40 60 80
Time (min)
0
0
0 20 40 60 80
Time (min)
Figure C3.
Figure C6.
71

12
090925 Intermittent aeration, DO 3.0 (mg/l)
4.5
12
090925 Prolonged unaerated period, DO 3.0 (mg/l)
4.5
10
3.75
10
3.75
N₂O (µmol/l)
8
6
4
3
2.25
1.5
DO (mg/l)
N₂O sensor
(µmol/l)
DO (mg/l)
NO₂-N (mg/l)
8
6
4
3
2.25
1.5
DO (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
2
0.75
2
0.75
0
0
0 20 40 60 80
Time (min)
0
0
0 50 100 150 200
Time (min)
Figure C7.
Figure C10.
12
090925 Intermittent aeration, DO 3.0 (mg/l)
4.5
12
090926 Prolonged unaerated period, DO 3.0 (mg/l)
4.5
10
3.75
10
3.75
NO₂-N (mg/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 20 40 60 80
Time (min)
0
0
0 50 100 150 200
Time (min)
Figure C8.
Figure C11.
12
090925 Prolonged unaerated period, DO 3.0 (mg/l)
4.5
12
090926 Prolonged unaerated period, DO 3.0 (mg/l)
10
3.75
10
3.75
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O sensor
(µmol/l)
DO (mg/l)
NO₂-N (mg/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
NO₂-N (mg/l)
DO (mg/l)
0
0
0 50 100 150 200
Time (min)
0
0
0 50 100 150 200
Time (min)
Figure C9.
Figure C12.
72

12
090927 Prolonged unaerated period, DO 3.0 (mg/l)
4.5
12
091006 Continously operation, DO 1.5 (mg/l)
10
3.75
10
3.75
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO (mg/l)
NO₂-N (mg/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
NO₂-N (mg/l)
DO (mg/l)
0
0
0 50 100 150 200
Time (min)
0
0
0 20 40 60 80
Time (min)
Figure C13.
Figure C16.
12
090927 Prolonged unaerated period, DO 3.0 (mg/l)
4.5
12
091007 Continously operation, DO 1.5 (mg/l)
4.5
10
3.75
10
3.75
NO₂-N (mg/l)
8
6
4
3
2.25
1.5
DO (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
N₂O (µmol/l)
8
6
4
3
2.25
1.5
DO (mg/l)
N₂O
(µmol/l)
DO (mg/l)
2
0.75
2
0.75
0
0
0 50 100 150 200
Time (min)
0
0
0 20 40 60 80
Time (min)
Figure C14.
Figure C17.
12
091006 Continously operation, DO 1.5 (mg/l)
4.5
12
091007 Continously operation, DO 1.5 (mg/l)
10
3.75
10
3.75
N₂O (µmol/l)
8
6
4
3
2.25
1.5
DO (mg/l)
N₂O
(µmol/l)
DO (mg/l)
NO₂-N (mg/l)
8
6
4
3
2.25
1.5
DO (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
2
0.75
2
0.75
0
0
0 20 40 60 80
Time (min)
0
0
0 20 40 60 80
Time (min)
Figure C15.
Figure C18.
73

12
091010 Continously operation, DO 1.5 (mg/l)
4.5
12
091014 Continously operation, DO 1.0 (mg/l)
4.5
10
3.75
10
3.75
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO (mg/l)
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 20 40 60 80
Time (min)
0
0
0 20 40 60 80
Time (min)
Figure C19.
Figure C22.
12
091010 Continously operation, DO 1.5 (mg/l)
12
091015 Continuously operation, DO 1.0 (mg/l), aeration with
N2 gas.
4.5
10
3.75
10
3.75
NO₂-N (mg/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
NO₂-N (mg/l)
DO (mg/l)
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 20 40 60 80
Time (min)
0
0 20 40 60 80
Time (min)
0
Figure C20.
Figure C23.
12
091013 Continously operation, DO 1.0 (mg/l)
4.5
12
091016 Continuously operation, DO 1.0 (mg/l), aeration with
N2 gas.
4.5
10
3.75
10
3.75
N₂O (µmol/l)
8
6
4
3
2.25
1.5
DO (mg/l)
N₂O
(µmol/l)
DO (mg/l)
N₂O (µmol/l)
8
6
4
3
2.25
1.5
DO (mg/l)
N₂O
(µmol/l)
DO (mg/l)
2
0.75
2
0.75
0
0
0 20 40 60 80
Time (min)
0
0 20 40 60 80
Time (min)
0
Figure C 21
Figure C24.
74

12
091016 Continuously operation, DO 1.0 (mg/l), aeration with
N2 gas.
4.5
12
091018 Continuously operation, DO 1.5 (mg/l), aeration with
N2 gas.
10
3.75
10
3.75
NO₂-N (mg/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0 20 40 60 80
Time (min)
0
0
0 20 40 60 80
Time (min)
0
Figure C25.
Figure C 28
12
091017 Continuously operation, DO 1.5 (mg/l), aeration with
N2 gas.
4.5
12
091018 Continuously operation, DO 1.5 (mg/l), aeration with
N2 gas.
10
3.75
10
3.75
N₂O (µmol/l)
8
6
4
3
2.25
1.5
DO (mg/l)
N₂O
(µmol/l)
DO (mg/l)
N₂O (µmol/l)
8
6
4
3
2.25
1.5
DO (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
2
0.75
2
0.75
0
0 20 40 60 80
Time (min)
0
0
0 20 40 60 80
Time (min)
0
Figure C26.
Figure C29.
12
091017 Continuously operation, DO 1.5 (mg/l), aeration with
N2 gas.
N₂O (µmol/l)
10
8
6
4
2
3.75
3
2.25
1.5
0.75
DO (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
0
0 20 40 60 80
Time (min)
0
Figure C27.
75

Appendix D
Nitrogen grab samples
090913 NH₄-N
090913 DO, NO₂-N biosensor, NO₂-N LCK 342
NH₄-N (mg/l)
300
250
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
DO, NO₂-N (mg/l)
12
10
8
6
4
2
NO₂-N out
(mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
0
0
0 20 40 60
0 20 40 60
Time (min)
Time (min)
Figure D1.
Figure D4.
090913 NO₃-N
090918 NH₄-N
40
350
NO₃-N (mg/l)
35
30
25
20
15
10
5
0
NO₃-N in
(mg/l)
NO₃-N out
(mg/l)
NH₄-N (mg/l)
300
250
200
150
100
50
0
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
0 20 40 60
0 20 40 60
Time (min)
Time (min)
Figure D2.
Figure D5.
090913 NO₂-N
090918 NOx-N
NO₂-N (mg/l)
30
25
20
15
10
5
0
NO₂-N in
(mg/l)
NO₂-N out
(mg/l)
NOx-N (mg/l)
50
45
40
35
30
25
20
15
10
5
0
NOx-N in
NOx-N ut
0 20 40 60
0 20 40 60
Time (min)
Time (min)
Figure D3.
Figure D6.
77

350
090921 NH₄-N
350
090922 NH₄-N
300
300
NH₄-N (mg/l)
250
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
NH₄-N (mg/l)
250
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
0
0 20 40 60
0
0.000 20.000 40.000 60.000
Time (min)
Time (min)
Figure D7.
Figure D10.
70
090921 NOx-N
70
090922 NOx-N
60
60
NOx-N (mg/l)
50
40
30
20
10
NOx-N in
(mg/l)
NOx-N out
(mg/l)
NOx-N (mg/l)
50
40
30
20
10
NOx-N in
(mg/l)
NOx-N in
(mg/l)
0
0 20 40 60
0
0 20 40 60
Time (min)
Time (min)
Figure D8.
Figure D11.
DO, NO₂-N (mg/l)
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
090921 DO, NO₂-N biosensor, NO₂-N LCK 342
0 20 40 60
Time (min)
Figure D9.
NO₂-N
biosensor
(mg/l)
DO (mg/l)
DO, NO₂-N (mg/l)
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
Figure 29
090922 DO, NO₂-N biosensor, NO₂-N LCK 342
0 20 40 60
Time (min)
Figure D12.
NO₂-N
biosensor
(mg/l)
DO (mg/l)
78

090925 NH₄-N
090925 Prolonged unaerated period NH₄-N
300
250
250
200
NH₄-N (mg/l)
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
NH₄-N (mg/l)
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
0
0
0 20 40 60
0 50 100 150 200
Time (min)
Time (min)
Figure D13.
Figure D16.
090925 NOx-N
090925 Prolonged unaerated period NOx-N
70
70
60
60
NOx-N (mg/l)
50
40
30
20
10
NOx-N in
(mg/l)
NOx-N out
(mg/l)
NOx-N (mg/l)
50
40
30
20
10
NOx-N in
(mg/l)
NOx-N out
(mg/l)
0
0
0 20 40 60
0 50 100 150 200
Time (min)
Time (min)
Figure D14.
Figure D17.
DO, NO₂-N (mg/l)
10
9
8
7
6
5
4
3
2
1
0
090925 DO, NO₂-N biosensor (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
DO, NO₂-N (mg/l)
8
7
6
5
4
3
2
1
0
090925 Prolonged unaerated period DO, NO₂-N
biosensor (mg/l)
NO₂-N
biosensor
(mg/l)
DO (mg/l)
0 20 40 60
0 50 100 150 200
Time (min)
Time (min)
Figure D15.
Figure D18.
79

300
090926 Prolonged unaerated period NH₄-N
60
090927 Prolonged unaerated period NOx-N
250
50
NH₄-N (mg/l)
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
NOx-N (mg/l)
40
30
20
10
NOx-N in
(mg/l)
NOx-N out
(mg/l)
0
0 50 100 150 200
0
0.00 50.00 100.00 150.00 200.00
Time (min)
Time (min)
Figure D19.
Figure D22.
NOx-N (mg/l)
50
45
40
35
30
25
20
15
10
5
0
090926 Prolonged unaerated period NOx-N
0 50 100 150 200
Time (min)
Figure D20.
NOx-N in
(mg/l)
NOx-N out
(mg/l)
NOx-N (mg/l)
60
50
40
30
20
10
0
090927 Prolonged unaerated period NOx-N
0.00 50.00 100.00 150.00 200.00
Time (min)
Figure D23.
NOx-N in
(mg/l)
NOx-N out
(mg/l)
DO, NO₂-N (mg/l)
9
8
7
6
5
4
3
2
1
0
090926 Prolonged unaerated period DO, NO₂-N
biosensor (mg/l)
0 50 100 150 200
Time (min)
Figure D21.
NO₂-N
biosensor
(mg/l)
DO (mg/l)
DO, NO₂-N (mg/l)
090927 Prolonged unaerated period DO, NO₂-N
biosensor (mg/l), NO₂-N LCK 342
10
8
6
4
2
0
0.00 50.00 100.00 150.00 200.00
Time (min)
Figure D24.
NO₂-N
biosensor
(mg/l)
80

NH₄-N (mg/l)
300
250
200
150
100
50
0
091006 NH₄-N
0 20 40 60
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
NOx-N (mg/l)
45
40
35
30
25
20
15
10
5
0
091007 NOx-N
0 20 40 60
NOx-N in
(mg/l)
NOx-N out
(mg/l)
Time (min)
Time (min)
Figure D25.
Figure D28.
DO, NO₂-N (mg/l)
6
5
4
3
2
1
0
091006 DO, NO₂-N biosensor (mg/l)
0 20 40 60
NO₂-N
biosensor
(mg/l)
DO (mg/l)
DO, NO₂-N (mg/l)
7
6
5
4
3
2
1
0
091007 DO, NO₂-N biosensor (mg/l)
0 20 40 60
NO₂
biosensor
(mg/l)
DO (mg/l)
NO₂-N LCK
342 out
(mg/l)
Time (min)
Time (min)
Figure D26.
Figure D29.
300
091007 NH₄-N
300
091010 NH₄-N
250
250
NH₄-N (mg/l)
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
NH₄-N (mg/l)
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
0
0 20 40 60
0
0 20 40 60
Time (min)
Time (min)
Figure D27.
Figure D30.
81

091010 NOx-N
091013 NOx-N
NOx-N (mg/l)
50
45
40
35
30
25
20
15
10
5
0
NOx-N in
(mg/l)
NOx-N out
(mg/l)
NOx-N (mg/l)
40
35
30
25
20
15
10
5
0
NOx-N in
(mg/l)
NOx-N out
(mg/l)
0 20 40 60
0 20 40 60
Time (min)
Time (min)
Figure D31.
Figure D34.
091010 DO, NO₂-N biosensor (mg/l)
091013 NO₂-N
DO, NO₂-N (mg/l)
7
6
5
4
3
2
1
0
NO₂-N
biosensor
(mg/l)
DO (mg/l)
NO₂-N LCK
342 out
(mg/l)
NOx-N (mg/l)
5
4
4
3
3
2
2
1
1
0
NO₂-N in
(mg/l)
NO₂-N out
(mg/l)
0 20 40 60
0 20 40 60
Time (min)
Time (min)
Figure D32.
Figure D35.
091013 NH₄-N
091013 NO₃-N
300
35
250
30
NH₄-N (mg/l)
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
NOx-N (mg/l)
25
20
15
10
5
NO₃-N in
(mg/l)
NO₃-N out
(mg/l)
0
0
0 20 40 60
0 20 40 60
Time (min)
Time (min)
Figure D33.
Figure D36.
82

300
091014 NH₄-N
30
091014 NO₃-N
250
25
NH₄-N (mg/l)
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
NOx-N (mg/l)
20
15
10
5
NO₃-N in
(mg/l)
NO₃-N out
(mg/l)
0
0 20 40 60
0
0 20 40 60
Time (min)
Time (min)
Figure D37.
Figure D40.
NOx-N (mg/l)
35
30
25
20
15
10
5
0
091014 NOx-N
0 20 40 60
NOx-N in
(mg/l)
NOx-N out
(mg/l)
NH₄-N (mg/l)
350
300
250
200
150
100
50
0
091015 NH₄-N
0 20 40 60
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
Time (min)
Time (min)
Figure D38.
Figure D41.
NOx-N (mg/l)
5
4
4
3
3
2
2
1
1
0
091014 NO₂-N
0 20 40 60
NO₂-N in
(mg/l)
NO₂-N out
(mg/l)
NOx-N (mg/l)
70
60
50
40
30
20
10
0
091015 NOx-N
0 20 40 60
NOx-N in
(mg/l)
NOx-N out
(mg/l)
Time (min)
Time (min)
Figure D39.
Figure D42.
83

35
091015 NO₃-N
70
091016 NOx-N
30
60
NOx-N (mg/l)
25
20
15
10
5
NO₃-N in
(mg/l)
NO₃-N out
(mg/l)
NOx-N (mg/l)
50
40
30
20
10
NOx-N in
(mg/l)
NOx-N out
(mg/l)
0
0 20 40 60
0
0 20 40 60
Time (min)
Time (min)
Figure D43.
Figure D46.
NOx-N (mg/l)
12
10
8
6
4
2
0
091015 NO₂-N
0 20 40 60
NO₂-N in
(mg/l)
NO₂-N out
(mg/l)
DO, NO₂-N (mg/l)
12
10
8
6
4
2
0
091016 DO, NO₂-N biosensor (mg/l),
0 20 40 60
NO₂-N
biosensor
(mg/l)
DO (mg/l)
NO₂-N out
LCK 342
(mg/l)
Time (min)
Time (min)
Figure D44.
Figure D47.
350
091016 NH₄-N
35
091016 NO₃-N
300
30
NH₄-N (mg/l)
250
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
NOx-N (mg/l)
25
20
15
10
5
NO₃-N in
(mg/l)
NO₃-N out
(mg/l)
0
0 20 40 60
0
0 20 40 60
Time (min)
Time (min)
Figure D45.
Figure D48.
84

NOx-N (mg/l)
12
10
8
6
4
2
0
091016 NO₂-N
0 20 40 60
NO₂-N in
(mg/l)
NO₂-N out
(mg/l)
DO, NO₂-N (mg/l)
12
10
8
6
4
2
0
091017 DO, NO₂-N biosensor (mg/l),
0 20 40 60
NO₂-N
biosensor
(mg/l)
DO (mg/l)
NO₂-N out
(mg/l)
Time (min)
Time (min)
Figure D49.
Figure D52.
350
091017 NH₄-N
35
091017 NO₃-N
300
30
NH₄-N (mg/l)
250
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
NOx-N (mg/l)
25
20
15
10
5
NO₃-N in
(mg/l)
NO₃-N out
(mg/l)
0
0 20 40 60
0
0 20 40 60
Time (min)
Time (min)
Figure D50.
Figure D53.
70
091017 NOx-N
12
091017 NO₂-N
NOx-N (mg/l)
60
50
40
30
20
10
NOx-N in
(mg/l)
NOx-N out
(mg/l)
NOx-N (mg/l)
10
8
6
4
2
NO₂-N in
(mg/l)
NO₂-N out
(mg/l)
0
0 20 40 60
0
0 20 40 60
Time (min)
Time (min)
Figure D51.
Figure D 54
85

350
091018 NH₄-N
35
091018NO₃-N
300
30
NH₄-N (mg/l)
250
200
150
100
50
NH₄-N in
(mg/l)
NH₄-N out
(mg/l)
NOx-N (mg/l)
25
20
15
10
5
NO₃-N in
(mg/l)
NO₃-N out
(mg/l)
0
0 20 40 60
0
0 20 40 60
Time (min)
Time (min)
Figure D55.
Figure D58.
70
091018 NOx-N
12
091018 NO₂-N
NOx-N (mg/l)
60
50
40
30
20
10
NOx-N in
(mg/l)
NOx-N out
(mg/l)
NOx-N (mg/l)
10
8
6
4
2
NO₂-N in
(mg/l)
NO₂-N out
(mg/l)
0
0 20 40 60
0
0 20 40 60
Time (min)
Time (min)
Figure D56.
Figure D59.
091018 DO, NO₂-N biosensor (mg/l),
DO, NO₂-N (mg/l)
12
10
8
6
4
2
0
0 20 40 60
NO₂-N
biosensor
(mg/l)
DO (mg/l)
NO₂-N out
(mg/l)
Time (min)
Figure D57.
86

Appendix E Scientific Article
N2O production in a single stage nitritation/anammox MBBR process.
Sara Ekström
Water and Environmental Engineering Department of Chemical Engineering, Lund
University, Sweden.
Abstract. The nitrous oxide (N 2O) production from a laboratory nitritation/anammox MBBR reactor was
determined from N 2O measurements in the water phase with a Clark-type microsensor. The reactor was
operated at intermittent and continuous aeration to evaluate which operation mode that gives the highest
N 2O production. Different aeration rates were used during continuous operation to examine the influence
of dissolve oxygen (DO) on N 2O emissions. Measurements of N 2O production during prolonged unaerated
periods were performed to examine possible mechanisms of the N 2O production. The MBBR produces 6-
11% of removed inorganic nitrogen as N 2O during intermittent operation, whereas only 2-3% was
produced during continuous operation at low oxygen concentrations. Higher inorganic nitrogen removal
was achieved during continuous operation and better process performance is thought to be one
explanation of lower N 2O emissions during continuous operations of the laboratory MBBR.
Introduction
Nitrous oxide, a greenhouse gas with a global warming potential 320 times stronger
than that of CO2, is known to be produced during nitrification and denitrification
processes used to remove nitrogen from wastewaters (Jacob, 1999). Variable
temperature and loading rates of inorganic nitrogen compounds, low pH, alternating
aerobic and anaerobic conditions together with growth rate and microbial composition
are parameters that have great influence on N2O emissions from a wastewater treatment
plant (Kampschreur et al., 2008).
Wastewater treatment plants using biologic treatment processes for nutrient removal
are producing excessive sludge giving rise to ammonium rich effluent from the
anaerobic sludge digestion. This internal wastewater stream is recombined with the
influent of the treatment plant and corresponds to 15-20% of the total nitrogen load of
the wastewater treatment plant (Fux et al., 2003). In the early 1990s a new biological
treatment process for nitrogen removal through anaerobic ammonium oxidation
(anammox) with nitrite as electron acceptor was discovered by research teams in
Holland, Germany and Switzerland (Mulder et al., 1995, Hippen et al., 1997, Siegrist et
al., 1998). Total stoichiometry of the anammox process has been estimated by Strous et
al., (1998):
1NH
1.32NO 0.066HCO
0.13H
1.02 N 0.26NO
0.066CH2O . N . 2.03 H O.
Anammox has turned out to be suitable for treatment of reject waters and other
problematic wastewaters with a low COD/N ratio and high ammonium concentrations.
87

The bacteria performing the microbial conversion of nitrite into dinitrogen gas are strict
anaerobe autotrophs and the process has the potential to replace conventional
nitrification/denitrification of recirculated high strength ammonium streams within the
wastewater treatment plant (Strous et al., 1997). No additional carbon source is needed,
the oxygen demand is reduced by 50-60% in the nitrifying step and the aeration can
thereby be strongly reduced (Jetten et al., 2001, Fux et al., 2002). This means that the
process offers an opportunity to decrease the carbon footprint of the wastewater
treatment plant in terms of saving possibilities of both additional carbon source and
power consumption (Jetten et al., 2004). Further advantages with the anammox process
is that the production of surplus sludge is minimized and that high volumetric loading
rates can be obtained resulting in reduced operational and investment costs (Abma et
al., 2007). Indications that the process may produce significant amounts of N2O gas with
negative environmental impacts detracting the process advantages. The aim of this
study was to determine the amount of N2O produced in a nitritation/ anammox MBBR
process during different operation modes.
Materials and methods
MBBR system
A 7.5 litre laboratory MBBR (see Figure 1) fed with a synthetic medium was used to
determine the N2O emissions from a single stage nitritation/anammox system. The
reactor was originally started up in October 2008 with a carrier material with already
established biofilm taken from Himmerfjärdsverkets full scale DeAmmon ® reactor. The
used carrier was AnoxKaldnes carrier media type K1 with a protected surface area of
500 m 2 /m 3 . The total volume of carriers in the reactor was 3.5 litres which corresponds
to a total protected area of 1.7 m 2 and a filling degree of 46.7%.
Figure 1. The left part of the figure shows a photograph of the MBBR system, the schematic
drawing to the right shows the main features of the MBBR system.
88

Cycle studies
To examine what operation conditions that seem to produce the largest amounts of N2O
gas, the reactor was operated at different DO concentrations during intermittent and
constant aeration. A study where the anoxic phase was prolonged to two hours was
made to observe how the N2O production was influenced. Parameters monitored every
minute on-line in the reactor were; DO, pH, N2O and NO2-N. To examine the
concentration changes of NH4-N, NO2-N and NO3-N grab samples were taken in both
influent and effluent water.
Analytical methods
N2O concentrations were measured in the water phase with a Clark-type microelectrode
sensor developed by Unisense, Århus, Denmark.
Concentrations of NH4-N, NO2-N and NO3-N were determined with Dr Lange
spectrophotometry kit after filtration through Munktel 1.6 µm glass fibre filters. During
cycle studies NO2-N and N-tot were analyzed directly with Dr Lange’s method. Samples
were frozen and flow-injection analysis was used to determine NH4-N and NOx, the sum
of NO2-N and NO3-N. The NO3-N content was calculated by subtraction of NO2-N from the
sum of the two NOx species.
Dissolved oxygen and pH was measured with a portable meter HQ40d with mounted
oxygen and pH probe. Parameters analysed and method used are summarised in Table
15.
Table 15 Analysed parameters and methods.
Analysed parameter Method
N2O
Unisense N2O microsensor
NH4-N
LCK 303/FIA
NO2-N
LCK 342/341/biosensor
NO3-N LCK 339
NOx
FIA
N-tot LCK 238
DO
HQ40d
pH
HQ40d
Results and discussion
Intermittent aeration
The reactor was operated at a DO concentration of ~3 mg/l during the aeration phase.
One reactor cycle lasted for one hour with 40 minutes of aeration and 20 minutes of
mechanical mixing. Grab samples in the effluent were taken every 6 th minute. Only three
measurements of the influent medium was taken in one cycle ( 0, 36 and 66 minutes)
since it was considered that the variation of the influent medium during one hour should
not be significant.
89

Typical profiles of how N2O and DO changes during the cycle are shown in Figure . The
N2O concentration measured in the water phase varies with the aeration of the MBBR.
When aeration starts at the beginning of the cycle the airflow strips N2O out of the water
phase and the concentration decreases to a constant minimum level. As soon as the
aeration is shut of the N2O starts to accumulate in the water phase until aeration is
switched on again and the procedure starts over as shown in Figure 2.
N₂O (µmol/l)
12
10
8
6
4
2
0
4.5
3.75
2.25
1.5
0.75
0
0 20 40 60 80
Time (min)
3
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
Figure 2. Concentration profiles of N 2O and O 2, reactor is operated with intermittent aeration at a
DO concentration of~3mg/l in the aerated phase. The cycle study starts at the beginning of the
aerated period. N 2O gas is stripped from the water phase at the same time as the oxygen
concentration rises.
Initial N2O production varies between 6-11% of influent nitrogen concentration that is
converted into dinitrogen gas (here after referred to as removed inorganic N-
concentration), while the maximum production ranges from 11-30% of removed
inorganic nitrogen.
Prolonged study, intermittent aeration
A study of the effect of prolonged, intermittent aeration was made in order to observe
how the N2O production was influenced by a longer anoxic period, results are shown in
Figure 3. The reactor was operated in the same manners as above and the same
sampling procedure was applied. After the anoxic period of 20 minutes when the
aeration usually went on during a normal cycle the mechanical mixing proceeded for
another two hours.
90

12
10
4.5
3.75
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 50 100 150 200
Time (min)
Figure 3. Concentration profiles of N 2O and O 2 during prolonged unaerated period.
At first the N2O accumulation is rather linear, when DO decreases under 1 mg/l the
accumulation rate of N2O is reduced until a maximum concentration is reached at DO
concentrations close to 0 mg/l. The N2O concentration is constant under a period of 20-
50 minutes and then slowly starts to decrease as seen in Figure 3.
Continuous aeration
Measurements with continuous aeration were performed at DO concentrations of ~1.5
mg/l and ~1 mg/l. To be able to determine the production of N2O gas during these
operation conditions the aeration was turned off. The unaerated period was chosen to
20 minutes to be comparable with the measurements done during intermittent aeration.
Measurement proceeded 20 minutes after the aeration was switched on again. Two
measurements were performed at a constant aeration with a DO concentration of ~1.5
mg/l and ~1 mg/l respectively a typical profile of O2 and N2O concentrations are shown
in Figure 4.
91

12
10
091014 Continously operation, DO 1.0 (mg/l)
4.5
3.75
N₂O (µmol/l)
8
6
4
2
3
2.25
1.5
0.75
DO (mg/l)
N₂O
(µmol/l)
DO
(mg/l)
0
0
0 20 40 60 80
Time (min)
Figure 4. Concentration profiles of N 2O and O 2 during measurement at continuous aeration with
DO ~1.0 mg/l.
As seen in Figure 4 N2O is only accumulating for the first 5-6 minutes of the unaerated
period then there is a short time span when production and N2O flows leaving the
reactor are in equilibrium. The N2O concentration measured in the water phase is
decreasing before aerations starts again which have not been noticed in any of the
former cases. Initial N2O production is below 2% of reduced inorganic nitrogen and
maximum production is also very low.
Conclusions
Conclusions that can be made from the experiments are summarised below:
• The single stage nitritation/anammox system produced significant amounts of
N2O with a minimum production of 2% of removed inorganic nitrogen.
• Operating the MBBR at intermittent aeration with a DO of ~3 mg/l gave the
highest N2O production with initial and maximum productions of 6-11% and 10-
30% respectively.
• Smaller amounts of N2O were produced by the partial/nitritation anammox
system during continuous operation at DO in the interval 1-1.5 mg/l. The initial
N2O production was found to be 2-3% and the maximum N2O production
corresponded to 2-6%.
• When the MBBR was exposed to a longer period of anoxic conditions both
ammonium oxidation and N2O production ceased.
• The absolute number on overall N2O production for an operation mode (based on
the measurements of N2O accumulating during the anoxic phase) could be both
overestimated or underestimated and should therefore be used as a comparative
tool.
92

Acknowledgements
The experiments were carried out at AnoxKaldnes in Lund during the work with my
master thesis and I would like to thank my supervisor my supervisor Magnus
Christenson for all guidance, support, sharing off valuable knowledge and experiences,
also for giving me the opportunity to get to know the fascinating anammox process.
I would also like to thank my supervisor Professor Jes la Cour Jansen at Water and
Environmental Engineering Department of Chemical Engineering, Lund University for
scientific guidance and encouragement, for all your valuable aspects on my work and
always reminding me of looking into things from a wider perspective.
References
Abma W.R., Schultz C.E., Mulder J.W., van Loosdrecht M.C.M., van der Star W., Strous M.,
Tokutomi T., (2007). The advance of Anammox. Water 21, February 2007.
Fux C., Boehler M., Huber P., Brunner I., and Siegrist H., (2002). Biological treatment of
ammonium-rich wastewater by partial nitritation and subsequent anaerobic ammonium
oxidation (anammox) in a pilot plant. Journal of Biotechnology, 99, 295-306.
Fux C., Egli K., van der Meer J.R., Siegrist H., (2003). The anammox process for nitrogen
removal from waste water. EAWAG news 56 (November 2003), 20-21.
Hippen A., Rosenwinkel K-H., Baumgarten G., and Seyfried C.F., (1997). Aerobic
deammonification: A new experience in the treatment of wastewaters. Water Science and
Technology, 35, (10), 111-120.
Jacob D., (1999). Introduction to atmospheric chemistry. ISBN 0-691-00185-5,Princeton:
Princeton University Press.
Jetten M., Wagner M., Fuerst J., van Loosdrecht M., Kuenen J., Strous M., (2001).
Microbiology and application of the anaerobic ammonium oxidation ('anammox') process.
Current Opinion in Biotechnology 12, (3), 283-288.
Jetten M.S.M., Cirpus I., Kartal B., van Niftrik L., van de Pas-Schoonen K.T., Sliekers O.,
Haajier S., van der Star W., Schmid M., van de Vossenberg J., Schmidt I., Harhangi H., van
Loosdrecht M., Kuenen J., Op den Camp H. and Strous M., (2004). 1994-2004: 10 years of
research on the anaerobic oxidation of ammonium. Biochemical Society Transactions 33,
(1), 119-123.
Kampschreur M.J., Tan N.C.G, Kleerebezem R., Picioreanu C., Jetten M.S.M., and van
Loosdrecht M.C.M ., (2008, (b)). Effect of dynamic processes on nitrogen oxides emission
from an nitrifying culture. Environmental Science Technology, 42, 429-435.
93

Mulder A.V.A, Robertson L.A., Kuenen J.G.,(1995). Anaerobic ammonium oxidation
discovered in a denitrifying fluidized-bed reactor. FEMS Microbiology Ecology 16, (3),
177-184.
Siegrist H., Reithaar S., Koch G., and Lais P., (1998). Nitrogen loss in a nitrifying rotating
contactor treating ammonium rich wastewater without organic carbon. Water Science
and Technology, 38, (8-9), 241-248.
Strous M., van Gerven E., Zheng P., Kuenen J.G., and Jetten M.S.M., (1997). Ammonium
removal from concentrated waste streams with the anaerobic ammonium oxidation
(anammox) process in different reactor configurations. Water Research, 31, (8), 1955-
1962.
Strous M., Heijnen J., Kuenen J., Jetten M., (1998). The sequencing batch reactor as a
powerful tool for the study of slowly growing anaerobic ammonium-oxidizing
microorganisms. Applied Microbiology Biotechnology, 50, 589-596.
94