N O production in a single stage nitritation/anammox … and Environmental Engineering Department of...

Water and Environmental Engineering

Department of Chemical Engineering

N2O 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


Lund University, Sweden

N2O production in a single stage

nitritation/anammox MBBR process

Master Thesis number: 2010-01 by

Sara Ekström Water and Environmental Engineering


March 2007

Supervisors: Professor Jes la Cour Jansen

Doctor Magnus Christensson, AnoxKaldnes

Examiner: Associate professor Karin Jönsson

Picture on front page:

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

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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.

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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/m2d were in the range of 47-59% and 0.9-1.1 gN/m2d respectively. As the reactor mode was shifted into continuous aeration at a lower DO concentration both % reduction and removal rate in gN/m2d 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/m2d. 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.

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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!

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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)

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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

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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

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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 <5% of total emissions, (Bogner et al., 2007). As legislation on nitrogen removal in wastewater treatment plants becomes

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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.

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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.

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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.

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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:

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NH�� 1.5O � NO

� � 2H� � 2HO (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� � 2HO (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)

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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 � 2HO (2.1.6)

CO � 2NO� �HO � CHO � 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 HO. (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).

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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 up-flow 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.

Process Chemical reaction

Energy yield

ΔG ̊’

kJ/mol NH4⁺

eq.

Nitritation: NH�� 1.5O � NO

� � 2H� � HO -271 (2.1.1)

Nitratation: NO� � 0.5O � NO�

� -72.8 (2.1.2)

Nitrification: NH�� 2O � NO�

� � 2H� � HO - (2.1.3)

Denitrification: NO�� org. carbon � N � 2CO

- (2.1.4)

Anammox: NH�� NO

� � N � 2HO -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 <1 mg/l in the water bulk phase. To reassure that the DO concentration doesn’t affect the nitrification negatively a

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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 <6.8. A neutral pH of around 7 is normally maintained to achieve satisfactory nitrification rates (Metcalf & Eddy, 2004).

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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 (<5 µM or < 70 µg/l) and the bacterial biomass yield is 0.07 C-mole fixed/ mole ammonia oxidized (Jetten et al., 2004). Nitrite is known to inhibit anammox activity irreversible at NO2-N concentrations ranging from 70-100 mg/l, (Gut., 2006, Jung et al., 2007, Strous et al., 1999).

2.2.5 Mixing

Turbulence is a factor important for keeping both substrate and DO concentrations at constant concentrations throughout the whole reactor. The turbulence also impacts the biofilm thickness and substrate transfer from the water bulk to the biofilm surface. Efficient mixing can be achieved by aeration from the bottom of the reactor. During anoxic periods or in an anoxic reactor mixing can be maintained by mechanical stirring or through a gas flow of N2 gas.

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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

12

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 m2/m3 while the carrying material in older treatment plants often is crushed stone or pumice that only has a specific area of 40-60 m2/m3, (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 m2/m3, 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 m2/m3. 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 m2/m3 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.

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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/cm3) 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 m2/m3 (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 semi-submerged 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 m2/m3 (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: <= > ?@ABC@D � EFG (2.4.1) HIJKIJ > LM � HNOPKNOP (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

14

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:

L�Q,S >TUVW

XUVW·

S

Z[\] (2.4.3)

L�Q,S >TUVW

XUVW (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).

15

To enable efficient nutrient removal the hydraulic conditions should prevent the build-up 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 C̊, keeping the sludge

16

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 � 1.5HO (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� � HO (2.5.3)

Anammox: NH�

� � 1.3NO� � N � 0.26NO�

� � 2HO (2.5.4) Canon process: NH�

� � 0.85O � 0.4N � 0.13NO�� 1.3HO � 1.4H� (2.5.5)

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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).

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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 SO42⁻ (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 N2O

Property: Unit:

Molecular weight 44.0 g/mol Water solubility (0 salinity at 20 C̊) 27.05∙10⁻3 mol/l

1.19 g/l

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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�� NO � HO � 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: HNOH � HNO � NO � 2HO (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��

ÎP_`Pa _abOcP`daeffffffffffffg hE

�ÎP_IP _abOcP`daefffffffffffg hE

ÎP_Ic NiIba _abOcP`daeffffffffffffffffghE

ÎP_NOd NiIba _abOcP`daefffffffffffffffffgh (2.5.3)

A low pH (<7) in the denitrification process impacts the end products towards a higher N2O/N2 ratio (Firestone et al., 1979, Henze et al., 1997). Since dissolved oxygen impacts denitrification negatively increased levels of oxygen are also expected to give a higher emission ratio of N2O/N2 in the denitrification process. The O2 effect could result from different mechanisms; (i) competition as electron acceptor; (ii) favoured inhibition of N2O reductase; (iii) e general slowdown of denitrification with greater escape of N2O as a result, (iv) a combination of these processes (Firestone et al., 1979). Low COD/N ratio is also affecting the amount of N2O produced by denitrification. Tallec et al., (2006 b)

20

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.

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Table 3. N2O emission from different wastewater treatment facilities given in % of influent N-concentrations.

Reactor type Influent N-concentrations mg/l

COD

DO

mg/l

N2O emission % of influent

N-concentration Reference NH4-N NO2-N NO3-N

nitritation – anammox SBR 650 ±50 <0.2 <0.2 - <1.0 0.4-0.6 Joss et al., 2009

denitrifying fluidized bed reactor (23 l) 90-130 - (5.6-6.8)10^3 - <65ppma Mulder et al., 1995

anammox SBR (15 l) 63-420 63-420 - - - 0.03-0.06 Strous et al., 1998

Sharon SBR 584 <1 <1 - 4 Jetten et al., 1999

anammox – fluidized bed reactor 267 227 64 - - <1 Jetten et al., 1999

nitrifying activated sludge (2.3 l) 15-49 0-5 0-6 - 0.1-6 0.1-0.4b Tallec et al., 2006 (a)

nitrifying biofilter reactor (7 l) 30 0 0 - 0.5-9.2 0.4c Tallec et al., 2006 (b)

denitrifying biofilter reactor (7 l) 1 0.1 21 - 0.2d Tallec et al., 2006 (b)

denitrifying activated sludge (3 l) - 5 15 - - 0.4d Tallec et al., 2007

nitrifying SBR (2 l) 1145 - - - 0-5.5 2.8 Kampschreur et al., 2008 b

nitrifying-denitrifying bioreactor (1.6 l) 1700-1800 - - 2.4-3.5 20-30 Itokawa et al., 1999

Sharon reactor (1500 m3) 980e - - - 2.5 1.7 Kampschreur et al., 2008 a

anammox reactor (70 m3) 372 414 17 - - 0.6 Kampschreur et al., 2008 a

a) below detection limit of GC. b) given in % of oxidized NH4-N. c) emitted average given in % of oxidized NH4-N. d) emitted average in % of reduced NO3-N.

e) N-load given as NKj.

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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 N2O microsensor.

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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).

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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).

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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 m2/m3. 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 m2 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.

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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 Concentration g/l

NaHCO3 2.6 NH4Cl 1.2 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 Concentration g/l

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

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.

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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

28

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 6th minute and every 36th minute in the influent medium.

3.4 Calibration of microsensors

The N2O production and NO2⁻ concentration profile were measured online with Clark-

type 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.

29

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 N2O and NO2-N

microsensors during calibration procedure. The first calibration point represents the signal

obtained in mV without any addition of N2O or NO2-N. As stepwise concentrations of N2O and NO2-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.

30

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.

31

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

Q l/h T °C pH NH4-N NO2-N NO3-N

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

Reduction % Removal gN/m2d NH4-N NO2-N NO3-N

82.4 ±44.0 6.3 ±1.7 32.3 ±9.5 58 ±13 1.1 ±0.2

On the 28th 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.

32

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/m2d 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.

0

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35

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/12

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Te

mp

°C

DO

(m

g/

l),

pH

Date

DO concentration, pH & temperature

DO (mg/l)

pH

Temperature (°C)

0

50

100

150

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300

350

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To

tal

ino

rga

nic

n

itro

ge

n (

mg

/l)

Date

Concentrations of influent and effluent total inorganic nitrogen

Σ TIN in (mg/l)

Σ TIN out (mg/l)

0

10

20

30

40

50

60

70

80

90

0.0

0.2

0.4

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% r

ed

uct

ion

Re

mo

va

l ra

te (

gN

/m

²d)

Date

Removal rate (gN/m²d) & % reduction of incomming nitrogen

Removal rate

(gN/m2d)

% reduction

33

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/m2d 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

0

7

14

21

28

35

0

2

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10

14

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09

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/09

14

/10

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19

/10

/09

Te

mp

°C

DO

(m

g/

l),

pH

Date

DO concentration, pH & temperature

DO (mg/l)

pH

Temperature (°C)

0

50

100

150

200

250

300

350

14

/09

/09

19

/09

/09

24

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/09

29

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09

/10

/09

14

/10

/09

19

/10

/09

To

tal

ino

rga

nic

n

itro

ge

n (

mg

/l)

Date

Concentrations of influent and effluent total inorganic nitrogen

Σ TIN in (mg/l)

Σ TIN out (mg/l)

0

10

20

30

40

50

60

70

80

90

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

14

/09

/09

19

/09

/09

24

/09

/09

29

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/09

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14

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/09

19

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/09

% r

ed

uct

ion

Re

mo

va

l ra

te (

gN

/m

²d)

Date

Removal rate (gN/m²d) & % reduction of incomming nitrogen

Removal rate

(gN/m2d)

% reduction

34

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.

Figure 15. Concentration profiles of N2O and O2 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. N2O 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-

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(m

g/l)

N₂O

(µ

mo

l/l)

Time (min)

N₂O

(µmol/l)

DO

(mg/l)

35

concentration), while the maximum production ranged from 11-16% of removed inorganic nitrogen, see Table 9. Table 9. Average N2O concentration in the water phase during aeration. Calculated initial and

maximum N2O production rates, mean O2* 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

N2O

µmol/l

Produced N2O in % of

removed inorganic N-

concentration O2

mg/l

mean N-concentration

mg/l N-red.

%

Removal

gN/m2d initial max NH4-N NO3-N NO2-N

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.

Figure 16. Concentration profiles of N2O and O2 during prolonged unaerated period, DO ~3 mg/l

during aerated phase. (Only manually registered O2 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

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 50 100 150 200

DO

(m

g/l)

N₂O

(µ

mo

l/l)

Time (min)

N₂O

(µmol/l)

DO

(mg/l)

36

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 N2O concentration in the water phase during aeration.

Calculated initial and maximum N2O production rates, mean O2* concentrations during aerated

period, mean nitrogen concentration, reduction and removal rates.

Date

Average

N2O

µmol/l



concentration O2

mg/l


mg/l N-red.

%

Removal


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.

Figure 17. Concentration profiles of N2O and O2 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

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(m

g/l)

N₂O

(µ

mo

l/l)

Time (min)

N₂O

(µmol/l)

DO

(mg/l)

37

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 N2O concentration in the water phase during aeration. Calculated initial and

maximum N2O production rates, mean O2 concentrations during aeration, mean nitrogen

concentration, reduction and removal rates for studies at continuous operation mode DO ~1.5

mg/l.

Date

Average

N2O

µmol/l



concentration O2

mg/l


mg/l N-red.

%

Removal


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.

Figure 18. Concentration profiles of N2O and O2 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.

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(m

g/l)

N₂O

(µ

mo

l/l)

Time (min)

N₂O

(µmol/l)

DO

(mg/l)

38

Table 12. Average N2O concentration in the water phase during aeration. Calculated initial and

maximum N2O production rates, mean O2 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

N2O

µmol/l



concentration O2

mg/l


mg/l N-red.

%

Removal


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.

Figure 19. Concentration profiles of N2O and O2 when N2 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 <1% of removed inorganic nitrogen, see Table 13. However this value is lower than the real production since N2 gas stripped N2O from the water phase at all times.

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(m

g/l)

N₂O

(µ

mo

l/l)

Time (min)

N₂O

(µmol/l)

DO

(mg/l)

39

Table 13. Average N2O concentration in the water phase during aeration. Calculated initial and

maximum N2O production rates, mean O2 concentrations during the aerated period, mean nitrogen

concentration, reduction and removal rates during measurements with N2 gas in anoxic phase,

operation at DO ~1.0 mg/l.

Date

Average

N2O

µmol/l



concentration O2

mg/l


mg/l N-red.

%

Removal


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

Figure 20. Concentration profiles of N2O and O2 when N2 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 N2O concentration in the water phase during aeration. Mean calculated initial

and maximum N2O production rates, mean O2 concentrations during aerated period, mean

nitrogen concentration, reduction and removal rates during measurements with N2 gas in anoxic

phase, operation at DO ~1.5 mg/l.

Date

Average

N2O

µmol/l



concentration O2

mg/l


mg/l N-red.

%

Removal


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

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(m

g/l)

N₂O

(µ

mo

l/l)

Time (min)

N₂O

(µmol/l)

DO

(mg/l)

40

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.

Figure 21. NO2-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.

0

1

2

3

4

5

6

7

8

9

10

0.00 50.00 100.00 150.00 200.00

DO

, NO

₂-N

(m

g/l

)

Time (min)

NO₂-N

biosensor

(mg/l)

NO₂-N LCK

342

(mg/l)

41

Figure 22. NO2-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.

0

2

4

6

8

10

12

0 20 40 60 80

NO

₂-N

(m

g/l

)

Time (min)

NO₂-N

biosensor

(mg/l)

NO₂-N LCK 342

(mg/l)

42

Figure 23. N2O 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.

Figure 24. Stripping of N2O 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.

y = -0.0132x + 10.59

0

2

4

6

8

10

12

0 100 200 300 400 500

N₂O

µm

ol/

l

Time (min)

N₂O (µmol/l)

y = -0.6482x + 10.193

y = 12.95e-0.143x

0

2

4

6

8

10

12

14

0 5 10 15 20 25

N₂O

(µ

mo

/l)

Time (min)

Aeration 1.6 (l/min) with carriers

N2O (µmol/l)

43

Figure 25. Stripping of N2O 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.

Figure 26.Stripping of N2O 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.

y = -0.6482x + 10.193

y = 12.95e-0.143x

0

2

4

6

8

10

12

14

0 5 10 15 20 25

N₂O

(µ

mo

/l)

Time (min)


N2O (µmol/l)

y = -0.6722x + 10.714

y = 13.571e-0.141x

0

2

4

6

8

10

12

14

0 5 10 15 20 25

N₂O

(µ

mo

/l)

Time (min)


N2O (µmol/l)

44

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/m2d 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). R2 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.

45

Figure 27. Correlation between the % N2O 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 (R2=0.70).

R² = 0.6859

0

2

4

6

8

10

12

0 1 2 3 4

% p

rod

uce

d N

₂O

DO (mg/l)

Produced N₂O in

relation to DO

concetration

46

Figure 28. Correlation between % N2O 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

R² = 0.7029

0

2

4

6

8

10

12

0 20 40 60 80 100

% p

rod

uce

d N

₂O

% N-reduction

Produced N₂O in

relation to % N-

reduction

47

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

48

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 <1% of dissolved N2O present in the water phase left through diffusion during the interval of one minute. The test was performed since calculations of produced N2O are based on the assumption that the amount of N2O leaving the water phase through diffusion can be neglected. When diffusion and production rates were compared it was shown that the diffusion rate corresponded to about 10% of the production rate. This means that the N2O production from the MBBR process might have been under estimated.

51

6. Conclusions

The following conclusions concerning, process performance of the laboratory MBBR, produced N2O and evaluation of the NO2-N biosensor can be made:





• 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.

53

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.)

55

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.

56

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.

57

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.

58

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.

59

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.

60

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 high-

rate 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.

61

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/MBBRTM (online). Available at:<http://www.anoxkaldnes.com/Sve/c1prodc1/mbbr.htm (2009-10-28). Naturvårdsverket. /klimat/utsläppstatistik och klimatdata/utsläpp av växthusgaser/ globala utsläpp (online). Available at:<http://www.naturvardsverket.se/sv/Klimat-i-forandring/Utslappsstatistik-och-klimatdata/Utslapp-av-vaxthusgaser/Globala-utslapp (2009-11-20). Stowa/links/ Overzicht buitenlandse zuiveringstechnieken/ www.stowa-selectedtechnologies.nl/fact sheets (online). Available at:<http: http://www.stowa-selectedtechnologies.nl/Sheets/index.html (2009-12-15). The anammox online resource/research/anammox physiology (online). Available at: http://www.anammox.com/research.html#biodiversity (2009-10-10). Unisense, a/sience (online). Available at: <http://www.unisense.com/Default.aspx?ID=49> (2009-09-10). Unisense, d/sience/products/sensors and electrodes/ Nitrous oxide sensor (online). Available at http://www.unisense.com/Default.aspx?ID=437> (2009-09-10) Unisense, e/sience/products/sensors and electrodes/ NOx- biosensor (online). Available at:< http://www.unisense.com/Default.aspx?ID=436> (2009-09-10).

63

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:

MI >��Q�

�� (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 N2O and NO2-N solutions that

has to be added during the calibration procedure of the sensors, calculated values for Vi 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

67

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: <= � �LBD > EFG � �� 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/m3s) times the reactor volume, V (m3), (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 (m3). The mass balance equation for a component j can be rewritten as:

HIJ�IJ� � L�M > HNOP�NOP� � b(c�Q)

bP 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.

68

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)

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 rN and rN2O.

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 D�^�

DG 0.1519∙10-6 mol/lmin

L� >0.540 · (273.904 · 10�� 97.593 · 10��)

7.5 · 60� 0.2116 · 10�� g/lmin

L̂��

>0.540 · (2.97601 · 10��)

7.5 · 60� 0.1519 · 10�� 0.1555 · 10�� mol/lmin

h: H�IJ^ � L̂ M > H�NOP^ � MD(�^)

DG

hE: 0 � L̂��M > H�NOP^��

� MD(�^��

)

DG

h: L̂ > H

M(�IJ^ � �NOP^)

hE: L̂��

> H

M�NOP^��

� D(�^��

)

DG

69

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.

Figure B2. The accumulation term in the production rate equation is obtained as the initial k-value

of the N2O curve when N2O 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:

�@L�@=G hE � h �LBDF�@D B� �B=C@LG@D h >L̂

��· 2�^

L�· 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.

0

2

4

6

8

10

12

0 20 40 60 80

N₂O

(µm

ol/

l),D

O (

mg

/l)

Time (min)

N₂O

(µmol/l)

DO

(mg/l)

2.82414

2.97601

y = 0.1519x - 0.8207

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

20 22 24 26 28 30

N₂O

(µ

mo

l/l)

, D

O (

mg

/l)

Time (min)

Initial N₂O production

N₂O

(µmol/l)

DO

(mg/l)

70

Table B2. Calculation parameters used to calculate the percentage N2O-N produced by removed

inorganic N.

Parameter value unit

rN2O 0.1555∙10-6 mole/l min MN 14.01 g/mol

rN 0.2116 · 10�� mg/l

�@L�@=G hE � h �LBDF�@D B� �B=C@LG@D h >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.

71

Appendix C

Microsensor measurements

Figure C1.

Figure C2.

Figure C3.

Figure C4.

Figure C5.

Figure C6.

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg/

l)

N₂O

(µ

mo

l/l)

Time (min)

090918 Intermittent aeration, DO 3.0 (mg/l)

N₂O sensor

(µmol/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

5

10

15

20

25

30

0 20 40 60 80

DO

(m

g/l

)

NO

₂-N

(mg/

l)

Time (min)


NO₂-N

biosensor

(mg/l)DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg

/l)

N₂O

(µm

ol/

l)

Time (min)


N₂O sensor

(µmol/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(m

g/l)

NO

₂-N

(mg/

l)

Time (min)


NO₂-N

biosensor

(mg/l)DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg

/l)

N₂O

(µm

ol/

l)

Time (min)


N₂O

(µmol/l)

DO

(mg/l)

0

1

2

3

4

0

10

20

30

40

0 20 40 60 80

DO

(m

g/l)

NO

₂-N

(mg/

l)

Time (min)


NO₂-N

biosensor

(mg/l)DO (mg/l)

72

Figure C7.

Figure C8.

Figure C9.

Figure C10.

Figure C11.

Figure C12.

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg/

l)

N₂O

(µ

mo

l/l)

Time (min)


N₂O sensor

(µmol/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg/

l)

NO

₂-N

(mg/

l)

Time (min)


NO₂-N

biosensor

(mg/l)DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 50 100 150 200

DO

(mg

/l)

N₂O

(µm

ol/

l)

Time (min)

090925 Prolonged unaerated period, DO 3.0 (mg/l)

N₂O sensor

(µmol/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 50 100 150 200

DO

(m

g/l)

NO

₂-N

(mg/

l)

Time (min)


NO₂-N

biosensor

(mg/l)DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 50 100 150 200

DO

(mg

/l)

N₂O

(µm

ol/

l)

Time (min)


N₂O

(µmol/l)

DO

(mg/l)

0

0.75

1.5

2.25

3

3.75

0

2

4

6

8

10

12

0 50 100 150 200D

O (m

g/l)

NO

₂-N

(mg/

l)

Time (min)


NO₂-N (mg/l)

DO (mg/l)

73

Figure C13.

Figure C14.

Figure C15.

Figure C16.

Figure C17.

Figure C18.

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 50 100 150 200

DO

(mg/

l)

N₂O

(µ

mo

l/l)

Time (min)


N₂O

(µmol/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 50 100 150 200

DO

(mg/

l)

NO

₂-N

(mg/

l)

Time (min)


NO₂-N

biosensor

(mg/l)DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg/

l)

N₂O

(µm

ol/

l)

Time (min)

091006 Continously operation, DO 1.5 (mg/l)

N₂O

(µmol/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

0

2

4

6

8

10

12

0 20 40 60 80

DO

(m

g/l

)

NO

₂-N

(mg/

l)

Time (min)


NO₂-N (mg/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg/

l)

N₂O

(µm

ol/

l)

Time (min)


N₂O

(µmol/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

0

2

4

6

8

10

12

0 20 40 60 80D

O (m

g/l)

NO

₂-N

(mg/

l)

Time (min)


NO₂-N

biosensor

(mg/l)DO (mg/l)

74

Figure C19.

Figure C20.

Figure C 21

Figure C22.

Figure C23.

Figure C24.

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg/

l)

N₂O

(µm

ol/

l)

Time (min)


N₂O

(µmol/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg/

l)

NO

₂-N

(mg/

l)

Time (min)


NO₂-N (mg/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg

/l)

N₂O

(µm

ol/

l)

Time (min)


N₂O

(µmol/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg/

l)

N₂O

(µ

mo

l/l)

Time (min)


N₂O

(µmol/l)

DO

(mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg

/l)

N₂O

(µm

ol/

l)

Time (min)

091015 Continuously operation, DO 1.0 (mg/l), aeration with

N2 gas.

N₂O

(µmol/l)

DO

(mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80D

O (m

g/l

)

N₂O

(µm

ol/

l)

Time (min)


N2 gas.

N₂O

(µmol/l)

DO (mg/l)

75

Figure C25.

Figure C26.

Figure C27.

Figure C 28

Figure C29.

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(m

g/l

)

NO

₂-N

(mg/

l)

Time (min)


N2 gas.

NO₂-N

biosensor

(mg/l)DO (mg/l)

0

0.75

1.5

2.25

3

3.75

4.5

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg

/l)

N₂O

(µm

ol/

l)

Time (min)


N2 gas.

N₂O

(µmol/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg

/l)

N₂O

(µm

ol/

l)

Time (min)


N2 gas.

NO₂-N

biosensor

(mg/l)

DO (mg/l)

0

0.75

1.5

2.25

3

3.75

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg/

l)

N₂O

(µ

mo

l/l)

Time (min)


N2 gas.

N₂O

(µmol/l)

DO

(mg/l)

0

0.75

1.5

2.25

3

3.75

0

2

4

6

8

10

12

0 20 40 60 80

DO

(mg

/l)

N₂O

(µm

ol/

l)

Time (min)


N2 gas.

NO₂-N

biosensor

(mg/l)

DO (mg/l)

77

Appendix D

Nitrogen grab samples

Figure D1.

Figure D2.

Figure D3.

Figure D4.

Figure D5.

Figure D6.

0

50

100

150

200

250

300

0 20 40 60

NH

₄-N

(m

g/l

)

Time (min)

090913 NH₄-N

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

0

5

10

15

20

25

30

35

40

0 20 40 60

NO

₃-N

(m

g/l

)

Time (min)

090913 NO₃-N

NO₃-N in

(mg/l)

NO₃-N out

(mg/l)

0

5

10

15

20

25

30

0 20 40 60

NO

₂-N

(m

g/l

)

Time (min)

090913 NO₂-N

NO₂-N in

(mg/l)

NO₂-N out

(mg/l)

0

2

4

6

8

10

12

0 20 40 60

DO

, NO

₂-N

(m

g/l

)

Time (min)

090913 DO, NO₂-N biosensor, NO₂-N LCK 342

NO₂-N out

(mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

0

50

100

150

200

250

300

350

0 20 40 60

NH

₄-N

(m

g/l

)

Time (min)

090918 NH₄-N

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

0

5

10

15

20

25

30

35

40

45

50

0 20 40 60

NO

x-N

(m

g/l

)

Time (min)

090918 NOx-N

NOx-N in

NOx-N ut

78

Figure D7.

Figure D8.

Figure D9.

Figure D10.

Figure D11.

Figure 29

Figure D12.

0

50

100

150

200

250

300

350

0 20 40 60

NH

₄-N

(m

g/l

)

Time (min)

090921 NH₄-N

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

0

10

20

30

40

50

60

70

0 20 40 60

NO

x-N

(m

g/l

)

Time (min)

090921 NOx-N

NOx-N in

(mg/l)

NOx-N out

(mg/l)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

0 20 40 60

DO

, NO

₂-N

(m

g/l

)

Time (min)


NO₂-N

biosensor

(mg/l)

DO (mg/l)

0

50

100

150

200

250

300

350

0.000 20.000 40.000 60.000

NH

₄-N

(m

g/l

)

Time (min)

090922 NH₄-N

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

0

10

20

30

40

50

60

70

0 20 40 60

NO

x-N

(m

g/l

)

Time (min)

090922 NOx-N

NOx-N in

(mg/l)

NOx-N in

(mg/l)

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

0 20 40 60

DO

, NO

₂-N

(m

g/l

)

Time (min)


NO₂-N

biosensor

(mg/l)

DO (mg/l)

79

Figure D13.

Figure D14.

Figure D15.

Figure D16.

Figure D17.

Figure D18.

0

50

100

150

200

250

300

0 20 40 60

NH

₄-N

(m

g/l

)

Time (min)

090925 NH₄-N

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

0

10

20

30

40

50

60

70

0 20 40 60

NO

x-N

(m

g/l

)

Time (min)

090925 NOx-N

NOx-N in

(mg/l)

NOx-N out

(mg/l)

0

1

2

3

4

5

6

7

8

9

10

0 20 40 60

DO

, NO

₂-N

(m

g/l

)

Time (min)

090925 DO, NO₂-N biosensor (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

0

50

100

150

200

250

0 50 100 150 200

NH

₄-N

(m

g/l

)

Time (min)

090925 Prolonged unaerated period NH₄-N

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

0

10

20

30

40

50

60

70

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)

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biosensor (mg/l)

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biosensor

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Figure D19.

Figure D20.

Figure D21.

Figure D22.

Figure D23.

Figure D24.

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Figure D25.

Figure D26.

Figure D27.

Figure D28.

Figure D29.

Figure D30.

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342 out

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(mg/l)

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82

Figure D31.

Figure D32.

Figure D33.

Figure D34.

Figure D35.

Figure D36.

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342 out

(mg/l)

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₄-N

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(m

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NO₃-N in

(mg/l)

NO₃-N out

(mg/l)

83

Figure D37.

Figure D38.

Figure D39.

Figure D40.

Figure D41.

Figure D42.

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NH

₄-N

(m

g/l

)

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(mg/l)

NH₄-N out

(mg/l)

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x-N

(m

g/l

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(mg/l)

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(mg/l)

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x-N

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(mg/l)

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(mg/l)

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₄-N

(m

g/l

)

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NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

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x-N

(m

g/l

)

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091015 NOx-N

NOx-N in

(mg/l)

NOx-N out

(mg/l)

84

Figure D43.

Figure D44.

Figure D45.

Figure D46.

Figure D47.

Figure D48.

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x-N

(m

g/l

)

Time (min)

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NO₃-N in

(mg/l)

NO₃-N out

(mg/l)

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x-N

(m

g/l

)

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(mg/l)

NO₂-N out

(mg/l)

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₄-N

(m

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(mg/l)

NH₄-N out

(mg/l)

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x-N

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)

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(mg/l)

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₂-N

(m

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091016 DO, NO₂-N biosensor (mg/l),

NO₂-N

biosensor

(mg/l)

DO (mg/l)

NO₂-N out

LCK 342

(mg/l)

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x-N

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)

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(mg/l)

NO₃-N out

(mg/l)

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Figure D49.

Figure D50.

Figure D51.

Figure D52.

Figure D53.

Figure D 54

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x-N

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(mg/l)

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(mg/l)

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Figure D55.

Figure D56.

Figure D57.

Figure D58.

Figure D59.

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₂-N

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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 (N2O) production from a laboratory nitritation/anammox MBBR reactor was determined from N2O 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 N2O production. Different aeration rates were used during continuous operation to examine the influence of dissolve oxygen (DO) on N2O emissions. Measurements of N2O production during prolonged unaerated periods were performed to examine possible mechanisms of the N2O production. The MBBR produces 6-11% of removed inorganic nitrogen as N2O 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 N2O 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 HO.

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.

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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 m2/m3. The total volume of carriers in the reactor was 3.5 litres which corresponds to a total protected area of 1.7 m2 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.

89

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 6th 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.

90

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.

Figure 2. Concentration profiles of N2O and O2, 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. N2O 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.

0

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(m

g/l)

N₂O

(µ

mo

l/l)

Time (min)

N₂O

(µmol/l)

DO

(mg/l)

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Figure 3. Concentration profiles of N2O and O2 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.

0

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(µ

mo

l/l)

Time (min)

N₂O

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DO

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Figure 4. Concentration profiles of N2O and O2 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 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.

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l)

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DO

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93

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.

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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.

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