N2O production in a single stage nitritation/anammox MBBR process

svensktvatten.se

N2O production in a single stage nitritation/anammox MBBR process

Water and Environmental Engineering

Department of Chemical Engineering

N 2 O production in a single stage

nitritation/anammox MBBR process

Master’s Thesis by

Sara Ekström

January 2010


Vattenförsörjnings- och Avloppsteknik

Institutionen för Kemiteknik

Lunds Universitet

Water and Environmental Engineering

Department of Chemical Engineering

Lund University, Sweden

N 2 O production in a single stage

nitritation/anammox MBBR process

Master Thesis number: 2010-01 by

Sara Ekström

Water and Environmental Engineering

Department of Chemical Engineering

March 2007

Supervisors:

Professor Jes la Cour Jansen

Doctor Magnus Christensson, AnoxKaldnes

Examiner:

Associate professor Karin Jönsson

Picture on front page:

1

1. K1 carriers with anammox biofilm from the laboratory nitritation/anammox

MBBR.

Postal address: Visiting address: Telephone:

P.O Box 124 Getingevägen 60 +46 46-222 82 85

SE-221 00 Lund. +46 46-222 00 00

Sweden,

Telefax:

+46 46-222 45 26

Web address:

www.vateknik.lth.se


Summary

Wastewaters contain abundant nitrogen that causes eutrophication in the receiving

recipient if not removed before the water is released into nature. Common nitrogen

removal is performed through nitrification and denitrification which are biologic

processes. Microorganisms are utilised to convert inorganic nitrogen compounds into

dinitrogen gas through different chemical reactions in there metabolism. Nitrous oxide,

(N2O), can be an intermediate or end product in the metabolism of both nitrification and

denitrification. N2O is a greenhouse gas, 320 times stronger than carbon dioxide (CO2).

The gas is contributing to global warming and is also taking part in depletion of the

protecting ozone layer in the stratosphere. If large amounts of N2O are emitted from

wastewater treatment facilities the problem with abundant nitrogen in aquatic

environments is only transferred into the atmosphere and N2O emissions should

therefore be avoided.

The energy demand for biologic nitrogen removal is high since aeration is needed for the

aerobe nitrification process. Denitrification requires an organic carbon source that often

has to be added to the process, generally in the form of methanol. Burning of fossil fuels

for energy coverage of the wastewater treatment plant and during transportation of

carbon source is leading to CO2 emissions with negative effects on the climate. If an

additional organic carbon source is used in the denitrification process this will also

contribute to increased CO2 emissions from the wastewater treatment plant.

Biological nitrogen removal through anaerobic ammonium oxidation (anammox) is a

relatively new process solution in wastewater treatment. Anammox has the potential to

replace common nitrogen removal of recycled internal wastewater streams with high

strength of ammonium and low COD content. The bacteria responsible for the anammox

process are converting ammonium to dinitrogen gas with nitrite as electron acceptor

making a short cut in the nitrogen cycle. Only 50% of the influent nitrogen load in the

form of ammonium has to be converted to nitrite by the nitrifiers and no additional

carbon source is needed. This means that the process offers great saving possibilities,

economical as well as environmental.

Different system configurations are available for the anammox process, it can either be

operated as a two stage process where nitritation and anammox are performed in

separate reactors, or in a single stage process where both processes are taking part in

one reactor. A MBBR is suitable as a single stage nitritation/anammox process since a

biofilm with an outer aerobe layer for the nitrifiers and one inner anoxic layer for the

anammox bacteria can develop. To allow the build up of a biofilm structure with

different oxic layers the process has to be operated at low dissolved oxygen

concentrations. Insufficient oxygenation in the nitrification process is known to enhance

nitrous oxide emissions from ammonium oxidising bacteria. Since the single stage

anammox process involves nitritation at low dissolved oxygen concentrations the

process might lead to significant N2O emissions.

i


The main objective with this master thesis work was to study the production of nitrous

oxide from a laboratory single stage nitritation/ anammox MBBR. The N2O

measurements were performed online in the water phase with a Clark–type

microsensor developed by Unisense, Århus, Denmark. The reactor was operated at both

intermittent and continuous aeration. The results from the experiments are summarised

below:

At intermittent aeration % reduction and removal rate in gN/m 2 d were in the range of

47-59% and 0.9-1.1 gN/m 2 d respectively. As the reactor mode was shifted into

continuous aeration at a lower DO concentration both % reduction and removal rate in

gN/m 2 d was more stable and higher than during intermittent aeration. % nitrogen

reduction was between64-65% and the removal rate in the interval of 1.3-1.6 gN/m 2 d.

The MBBR system produced N2O regardless of operation mode. The nN2O production

was determined through measurements of initial accumulation of nitrous oxide in the

water phase when aeration was turned off Intermittent aeration at high dissolved

oxygen concentrations 3 mg/l was resulting in significant nitrous oxide production

ranging from 6-11% of removed inorganic nitrogen. Operation at continuous aeration

yielded nitrous oxide emissions corresponding to about 2-3% of removed inorganic

nitrogen. Higher process performance may be an explanation to smaller amounts of

emitted N2O.

Conclusions that can be made from the experiments are summarised below:

• The single stage nitritation/anammox system produced significant amounts of

N2O with a minimum production of 2% of removed inorganic nitrogen.

• Operating the MBBR at intermittent aeration with a DO of ~3 mg/l gave the

highest N2O production with initial and maximum productions of 6-11% and 10-

30% respectively.

• Smaller amounts of N2O were produced by the partial/nitritation anammox

system during continuous operation at DO in the interval 1-1.5 mg/l. The initial

N2O production was found to be 2-3% and the maximum N2O production

corresponded to 2-6%.

• When the MBBR was exposed to a longer period of anoxic conditions both

ammonium oxidation and N2O production ceased.

• From results of mixing with N2 gas during the anoxic period it cannot be said

with certainty that the N2O production is the same during aeration and anoxic

phase. The absolute number on overall N2O production for an operation mode

(based on the measurements of N2O accumulating during the anoxic phase) could

be both overestimated or underestimated and should therefore be used as a

comparative tool.

ii


Acknowledgements

I would like to express my gratitude to everyone who have inspired and supported me

during the work with my master thesis.

My genuine appreciation goes to:

My supervisor Magnus Christenson at AnoxKaldnes for all guidance, support, sharing off

valuable knowledge and experiences, also for giving me the opportunity to get to know

the fascinating anammox process.

My supervisor Professor Jes la Cour Jansen at Water and Environmental Enigneering

Department of Chemical Engineering, Lund University for scientific guidance and

encouragement, for all your valuable aspects on my work and always reminding me of

looking into things from a wider perspective.

To Lars H. Larsen at Unisense for all help and support with the microsensors.

To everyone at AnoxKaldnes for all kindness, support and for creating an inspiring

environment to work in. Special thanks to Maria Ekenberg for always answering my

questions about the laboratory MBBR process and for all help in the laboratory. To

Carolina Shew Cammernäs for all patients and time while helping me with the FIA

analyses. To Stig Stork for all technical support.

To David Gustavsson at VA SYD for exchanging your ideas and knowledge about N2O

emissions in wastewater treatment processes.

Last but not least I would like to thank my mother and my sister, Gustav and Helena for

always encouraging and supporting me.

Thank you!

iii


Glossary

Aerobic – in the presence of oxygen in the form of O2

Anaerobic – an oxygen free environment

Anoxic – environment where oxygen is present as nitrite or nitrate

Autotrophic – organism that can produce organic compounds from carbon dioxide with

light or inorganic chemical compound as energy source

Carbon dioxide equivalent – is a measurement standard where the weight of a

greenhouse gas released in to the atmosphere is converted into the weight of carbon

dioxide that would cause the same temperature rise in Earths ecosystem as the gas in

question

Global warming potential – a measure of how much a given amount of a greenhouse

gas would contribute to global warming in comparison with the same amount of carbon

dioxide. The global warming potential of a greenhouse gas depends on (i) the absorption

of infrared radiation of the gas, (ii) atmospheric life time, (iii) spectral location of

absorbing wavelengths, where the global warming potential of carbon dioxide is 1

Heterotrophic – organism requiring organic compounds as energy source

Lithotrophic – organism using inorganic nutrients to obtain energy

Oxic – environment where oxygen is present

Abbreviations

Anammox – anaerobic ammonium oxidation

AOB – ammonium oxidising bacteria

ATP – adenosine triphosphate

Canon – completely autotrophic nitrogen removal over nitrite

Deamox – denitrifying ammonium oxidation

DO – dissolved oxygen

FIA – flow injection analysis

MBBR – moving biofilm bed reactor

NH4 + – ammonium

NO2 − – nitrite

NO3 − – nitrate

N2O – nitrous oxide

ppm – part per million

Sharon – Single reactor system for High rate Ammonium Removal Over Nitrite)

v


Table of content

Chapter 1 .................................................................................................................................... 1

1. Introduction ............................................................................................................................ 1

1.3 Objectives ............................................................................................................................................... 3

1.4 Accomplishment and scope ............................................................................................................. 3

Chapter 2 .................................................................................................................................... 5

2. Background ............................................................................................................................. 5

2.1 Biological nitrogen removal in wastewater treatment ......................................................... 5

2.1.1 Nitrification ................................................................................................................................... 5

2.1.2 Denitrification .............................................................................................................................. 6

2.1.3 Anaerobic ammonium oxidation........................................................................................... 6

2.2. Environmental factors ...................................................................................................................... 8

2.2.1 Dissolved oxygen ......................................................................................................................... 8

2.2.2 Temperature ................................................................................................................................. 9

2.2.3 pH and alkalinity ......................................................................................................................... 9

2.2.4 Substrate...................................................................................................................................... 10

2.2.5 Mixing ........................................................................................................................................... 10

2.3 Biofilm reactors ................................................................................................................................. 11

2.3.1 Trickling filter ............................................................................................................................ 11

2.3.2 Biofilters ...................................................................................................................................... 12

2.3.3 Fluidised bed .............................................................................................................................. 12

2.3.4 Moving bed reactor ................................................................................................................. 13

2.3.5 Rotating disc .............................................................................................................................. 13

2.4 Biofilm kinetics .................................................................................................................................. 13

2.5 System configurations for nitrogen removal by anammox .............................................. 15

2.5.1 Sharon/Anammox ................................................................................................................... 15

2.5.2 Canon ............................................................................................................................................ 16

2.5. 3 Deammonification .................................................................................................................. 17

2.5.4 Deamox ........................................................................................................................................ 18

2.5 N2O emissions from wastewater treatment ........................................................................... 18

2.5.1 Nitrification as a source of N2O emissions ..................................................................... 19

2.5.2 Denitrification as a source of N2O emissions ................................................................ 19

2.5.3 Chemical production of N2O ................................................................................................ 20

2.6 Microsensors ...................................................................................................................................... 22

2.6.1 Nitrous oxide sensor ............................................................................................................... 22

2.6.2 Nitrite biosensor....................................................................................................................... 23

Chapter 3 .................................................................................................................................. 25

3. Material and Methods .......................................................................................................... 25

3.1 Partial nitritation/anammox laboratory MBBR . ................................................................. 25

3.2 Reactor medium ................................................................................................................................ 26

3.3 Analytical methods .......................................................................................................................... 27

3.3 Cycle studies ....................................................................................................................................... 27

3.3.1 Intermittent aeration .............................................................................................................. 27

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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

viii


Chapter 1

1. Introduction

Nitrogen is one of the main building blocks in proteins and is therefore a vital element

for all living organisms. The elemental form of nitrogen is made available to the

biosphere through microbial fixation of dinitrogen gas which constitutes 79% of the

atmosphere. Combustion of fossil fuels, the use of nitrogen in industry and fertilizers,

waste and wastewater streams results in large amounts of anthropogenic nitrogen lost

to nature. The human contribution to nitrogen cycling impacts the environment

negatively through eutrophication of aquatic environments and emissions of

nitrogenous compounds to the atmosphere. Release of binary nitrogenous gases

contributes to the greenhouse effect and depletion of ozone layer with consequences on

a global scale lasting for centuries.

Since the start of the industrialisation human activity has increased the emissions of

greenhouse gases (carbon dioxide, chlorofluorocarbons, methane, ozone and nitrous

oxide), to the atmosphere with about 30%, with global warming as a result (Liljenström

& Kvarnbäck, 2007). In 2004 the global amount of anthropogenic emitted greenhouse

gases corresponded to 49 billion tons carbon dioxide equivalents, (a measurement

standard where the weight of a greenhouse gas released in to the atmosphere is

converted into the weight of carbon dioxide that would cause the same temperature rise

in Earths ecosystem). Carbon dioxide stands for the greatest proportion of the emissions

with 79% followed by methane and nitrous oxide contributing with 14% and 8%

respectively, (Naturvårdsverket, 2009).

Wastewater treatment plants produces greenhouse gases through; (i) burning of fossil

fuels for coverage of the energy demand, (ii) transportation of chemicals for on-site

usage and final disposal of solids, (iii) biologic treatment processes where nutrients,

(organic matter, nitrogen and phosphorus) are removed through microbial processes.

Biologic wastewater treatment processes are known to produce three of the major

greenhouse gases carbon dioxide(CO2), methane (CH4) and nitrous oxide (N2O) (Bani

Shahabadi et al., 2009). Nitrous oxide which is the strongest of these greenhouse gases is

known to be produced during nitrification and denitrification, processes used to remove

nitrogen from the wastewater. The global warming potential of N2O is 320 times

stronger than that of CO2. Release in to the atmosphere not only amplifies the warming

of Earth’s surface temperature it also contributes to depletion of the ozone layer (Jacob,

1999). During a thirty year period from 1990 to 2020 the N2O emissions associated with

microbial nitrogen degradation of both treated and untreated wastewaters are

estimated to increase with 25% from 80 to 100 megaton carbon dioxide equivalents.

Emissions from the post-consumer waste sector are approximately 1300 megaton

carbon dioxide equivalents which corresponds to


stricter it might lead to elevated emissions of nitrous oxide from the biological removal

processes. It is therefore of great importance to design and operate these processes to

minimise the emissions of nitrous oxide to the atmosphere.

Wastewater treatment plants using biologic treatment processes for nutrient removal

are producing excessive sludge giving rise to ammonium rich effluent from the

anaerobic sludge digestion. This internal wastewater stream is recombined with the

influent of the treatment plant and corresponds to 15-20% of the total nitrogen load of

the wastewater treatment plant (Fux et al., 2003). In the early 1990s a new biological

treatment process for nitrogen removal through anaerobic ammonium oxidation

(anammox) was discovered by research teams in Holland, Germany and Switzerland

(Mulder et al., 1995, Hippen et al., 1997, Siegrist et al., 1998). The technology has turned

out to be suitable for treatment of reject waters and other problematic wastewaters

with a low COD/N ratio and high ammonium concentrations. The bacteria performing

the microbial conversion of nitrite into dinitrogen gas are strict anaerobe autotrophs

and the process has the potential to replace conventional nitrification/denitrification of

recirculated high strength ammonium streams within the wastewater treatment plant

(Strous et al., 1997). No additional carbon source is needed, the oxygen demand is

reduced by 50% in the nitrifying step and the aeration can thereby be strongly reduced

(Jetten et al., 2001, Fux et al., 2002). This means that the process offers an opportunity to

decrease the carbon footprint of the wastewater treatment plant in terms of saving

possibilities of both additional carbon source and power consumption (Jetten et al.,

2004). Further advantages with the anammox process is that the production of surplus

sludge is minimized and that high volumetric loading rates can be obtained resulting in

reduced operational and investment costs (Abma et al., 2007). However there are

doubts that the process could produce significant amounts of N2O gas with negative

environmental impacts detracting the process advantages.

2


1.3 Objectives

The aim with this master thesis was to estimate the N2O emissions from a partial

nitritation/anammox laboratory moving bed biofilm reactor (MBBR) treating

ammonium-rich synthetic wastewater. Measurements were carried out with a

microsensor recording the N2O concentration online in the water phase, main objectives

were:

• To determine the N2O emission from the system under initial operation

conditions which were intermittent aeration at a dissolved oxygen, (DO),

concentration of ~3 mg/l.

• To evaluate N2O production when changing the operation mode into continuous

aeration to a lower DO concentration. Continuous operation at two different DO

concentrations were tested ~1.5 mg/l and ~1.0 mg/l.

• Determine how the N2O production was influenced during a longer period of

anoxic conditions.

• Investigate whether the N2O accumulation observed in the water phase as

aeration is turned off was due to termination of N2O stripping or if the N2O

production actually increases during the anoxic period.

• To examine if a biosensor for online measurements of NO2-N concentrations can

replace traditional analyse methods for determination of NO2-N during this

master thesis work.

1.4 Accomplishment and scope

The master thesis was based on both experimental work and a literature study.

Laboratory studies were performed on an existing partial laboratory

nitritation/anammox MBBR at AnoxKaldnes in Lund. The study took it’s start with a

definition of the existing system and setting up the equipment for the microsensors used

during online measurements. The laboratory work proceeded with measurement

sessions where the N2O concentration was registered in the water phase at different

operational conditions of the MBBR. Since no equipment for measurement of N2O in the

off-gas were available experiments to estimate how much N2O that was stripped off

from the water phase by diffusion and aeration were made.

The collected data was analysed and the N2O production from the MBBR could be

estimated with mass balance calculations of the system. The evaluation of the estimated

N2O emission from the nitritation/anammox MBBR laboratory system was based on a

literature study of N2O emissions in wastewater treatment. No calculations were made

to estimate whether the anammox process is reducing or increasing the carbon footprint

in comparison to common nitrogen removal processes.

3


For simplicity a synthetic wastewater was used, this water may both be easier to

degrade and not as complex as a normal wastewater which may impact the microbial

performance.

4


Chapter 2

2. Background

2.1 Biological nitrogen removal in wastewater treatment

Nitrogen removal is one of the major tasks in wastewater treatment. Biologic nitrogen

removal is the most efficient way to eliminate nitrogen from the wastewater and a

variety of system configurations like activated sludge plants with suspended growth,

biofilters designed for attached growth and combinations of the two have been

developed. These systems are built on the knowledge of microbial nitrogen cycling

where nitrogen compounds like NH4+, NO2 − and NO3 − are removed by conversion into

elemental N2 gas released to the atmosphere, see Figure 1. The well known nitrification

and denitrification processes are commonly used to achieve satisfactory nitrogen

removal. Today anaerobic ammonium oxidation, a relatively new technology for

nitrogen removal is also in use at several places.

Figure 1. Major biological transformations of nitrogen in wastewater treatment. (Kampschreur et

al., 2009).

2.1.1 Nitrification

Nitrification is oxidation of ammonium into nitrate under aerobic conditions, the

process occurs in two separated reaction steps, each involving different species of

bacteria. The nitrifiers are chemo-lithoautotrophs which means that they use carbon

dioxide or carbonate as carbon source and inorganic nitrogen is used for both energy

supply and cellular growth (Gray, 2004). In the first nitritation step ammonium

oxidising species like Nitrosomonas and Nitrosospira oxidises ammonium into nitrite:

5


NH 1.5O NO 2H 2H O (2.1.1)

The intermediate of the nitrification process (NO2 − ) is then further oxidised into nitrate

by nitrite oxidisers:

NO 0.5O NO


(2.1.2)

The nitratation step is performed by species like Nitrobacter and Nitrococcus (Prescott

et al., 2005). The overall nitrification reaction can be described by:

NH 2O NO 2H 2H O (2.1.3)

Energy gained by the bacteria during nitrification is used in the electron transport chain

to make adenosine triphosphate, (ATP is the energy currency of the cell making

chemical transport possible), (Prescott et al., 2005). Nitrifying bacteria are slow

growers since nitrification processes gives a low energy yield, (see Table 1) and the

nitrifiers have to oxidise large amount of inorganic material for their growth and

reproduction, (Prescott et al., 2005).

2.1.2 Denitrification

Denitrification is nitrate respiration under anoxic conditions carried out by a large

number of different heterotrophic bacteria. Nitrate is used to oxidate organic carbon

into elemental nitrogen and carbon dioxide:

NO organic carbon N CO (2.1.4)

Denitrifying bacteria need an easily biodegradable carbon source and their demand for

removal of one gram of nitrogen corresponds to 3-6 grams of chemical oxygen demand,

(COD). If the COD/N ratio of the wastewater becomes too low an additional carbon

source like methanol must be added in order to achieve nitrogen removal of nitrate

through denitrification (Gillberg et al., 2003)

Pseudomonas, Paraccocus, and Bacillus are examples of bacteria denitrifiying under

anoxic conditions. Most denitrifiers are facultative anaerobes which means that they

generally respire with oxygen as final electron acceptor, this since the oxygen route

yields more energy than nitrate respiration (Prescott et al., 2005).

2.1.3 Anaerobic ammonium oxidation

Anammox bacteria are obligate anaerobe autotrophs using inorganic nitrogen and

carbon for energy supply and growth, the process offers a short cut in the nitrogen cycle

as illustrated in Figure 1(Jetten et al., 1999). Ammonium is converted into dinitrogen gas

with nitrite as electron acceptor (2.1.6), hydrazine (N2H4) and hydroxylamine (NH2OH)

6


are intermediates in the chemical reaction. This reaction is the catabolic and energy

supplying part in anammox metabolism, it has to be carried out 15 times to fix one

molecule of carbon dioxide with nitrite as electron donor in the cellular synthesis or

anabolism (2.1.7) which produces nitrate (van Niftrik et al., 2004).


NH NO N 2H O (2.1.6)

CO 2NO

H O CH O 2NO (2.1.7)

Broda, (1977), predicted this microbial process through thermodynamic calculations for

over thirty years ago. The anammox process was discovered in the early nineties in a

rotating-disk plant treating landfill leachate at Mechernich, Germany (Rosenwinkel &

Cornelius, 2005). Mulder et al. also identified the anammox process in a denitrifying

fluidised bed reactor in Deltft, the Netherlands, at about the same time (Mulder et al.,

1995). Total stoichiometry of the anammox process has been estimated by Strous et al.,

(1998):

1NH


1.32NO 0.066HCO


0.13H

1.02 N 0.26NO


0.066CH2O . N . 2.03 H O. (2.1.7)

The bacterium performing the anammox reaction has been identified as a new member

of the order Planctomycete (Strous et al., 1999). Until now totally five anammox genera

have been identified, four from enriched wastewater sludge: Kuenenia, Brocadia,

Anammoxoglobus and Jettenia, the fifth genera of anammox bacteria Scalindua is often

found in marine environments (Jetten et al., 2009).

Planctomycetes are gram-negative bacteria with phenotypic properties such as absence

of peptidoglycan in the cell wall, budding reproduction and internal cell

compartmentalisation due to two membranes on the inside of the cell wall (Prescott et

al., 2005). Anammox bacteria are characterized by their deep red colour and ability to

form bio-films (Abma et al., 2006). Anammox cell structure is divided into three

compartments. The outer region closest to the cell wall called the paryphoplasm

encloses the second compartment which is the riboplasm. The riboplasm contains the

nucleoid and the anammoxosome, the third compartment where anammox catabolism

takes place, see Figure 2. All compartments are separated by bilayer membranes

constituted of impermeable and high density ladderane lipids (van Niftrik et al., 2004).

The higher membrane density is of importance to the anammox bacteria of two reasons,

one that it creates an electro potential force driving the ATP synthesis, and two it keeps

the toxic intermediates from the anammox process hydroxylamine and hydrazine inside

the anammoxosome (van Niftrik et al., 2004).

7


Figure 2. Illustration of anammox bacteria. (Adapted from van Niftrik et al., 2004).

Anammox bacteria are extremely slow growers, their doubling time has been found to

11 days in activated sludge (Strous et al., 1999). However it might be possible to

increase this doubling rate with optimal operation conditions since other researchers

have found a much shorter doubling rate of 3.6-5.4 days for anammox bacteria in a upflow

fixed-bed biofilm column reactor (Tsushima et al., 2007).

The microbial processes and chemical reactions of nitrification, denitrification and

anammox are summarized in Table 1.

Table 1. Microbial processes and chemical reactions taking part in the nitrogen cycle showed in

Figure 1.

Energy yield

ΔG ̊’ eq.

Process

Chemical reaction

+

kJ/mol NH 4

Nitritation: NH 1.5O NO 2H H O -271 (2.1.1)

Nitratation: NO 0.5O


NO -72.8 (2.1.2)

Nitrification: NH 2O NO 2H H O - (2.1.3)

Denitrification: NO org. carbon N 2CO

-

(2.1.4)

Anammox: NH

NO N 2H O -358.8 (2.1.6)

2.2. Environmental factors

Dissolved oxygen, temperature, pH, substrate concentrations and turbulence are abiotic

conditions that are of great importance for the growth and survival of the microbiology

in a wastewater treatment system.

2.2.1 Dissolved oxygen

Depending on the electron donor in the respiratory chain of the microorganism can be

limited or inhibited by either to low or to high DO concentrations. It is the oxygen

concentration within the biofilm experienced by the bacteria that is of importance for

the wellness of the organism (Henze et al., 1997).

Nitrifying bacteria utilising oxygen as electron donor are sensitive for too low oxygen

concentrations and are limited by DO concentrations


minimum concentration of 2 mg/l should be maintained (Gray, 2004). The nitrifying rate

increases up to DO levels of 3-4 mg/l, (Metcalf & Eddy). All figures given here yields for

DO concentrations in the water bulk phase of activated sludge processes, higher DO

concentrations are needed to satisfy the microbial oxygen demand in biofilm processes.

This since the oxygen concentration in the biofilm depends on diffusion of oxygen from

the water phase into the biofilm which is further explained in chapter 2.4.

Both denitrification and anammox processes are inhibited by oxygen. Denitrification has

been observed to be inhibited at DO concentrations above 0.2 mg/l (Metcalf & Eddy,

2003) and anammox organisms are reversibly inhibited by DO concentrations as low as

2 µmole/l or 0.032 mg/l, (Jetten et al., 1998).

2.2.2 Temperature

The temperature impacts the structure of the microbial community and is crucial for

growth and reaction rates in the system. Microbial reactions are often dependent on

enzyme-catalysed reactions that increase in velocity at higher temperatures. When the

time for a reaction to be catalysed is shortened the metabolism is more active and the

microorganism is allowed to grow faster (Prescott et al., 2005). Temperature does also

impact non viable factors like settling characteristics, gas solubility and transfer rates

(Gray, 2004).

Nitrification can be operated in a temperature interval of 0-40 °C with a temperature

optimum between 30-35 °C (Gray, 2004). Denitrifying bacteria are less sensitive to

temperature than nitrifiers and denitrification can take place in a temperature interval

from 2-75 °C with an optimum around 30 °C (Pierzynski et al., 2005)

Anammox bacteria are active in temperature range from 6-43 °C with an optimum at 30

̊C (Anammox online).

2.2.3 pH and alkalinity

pH, which is the measurement of a solutions acidity or alkalinity, is another important

environmental factor that impacts the growth rate of the microbial community. Since pH

is defined as the inverse logarithm of H + ions in solution a change of one pH unit

corresponds to a tenfold increase in the activity of H + ions. Each bacteria species have a

pH growth range and optimum.

Nitrification consumes alkalinity since two moles of OH − are used per mole ammonium

oxidised. Nitrification is favoured by mild alkaline conditions with pH optimum in the

range of pH 8.0-8.4 (Gray, 2004). The nitrification rate is significantly declined by low

pH values


Denitrification produces alkalinity and pH is generally raised by the process. pH

optimum is ranging from 7-9 depending on local conditions (Henze et al., 1997).

Cell synthesis in the anammox reaction is increasing pH and the process is active in a pH

range from 6.5-9 with an optimum around 8 (Egli et al., 2001).

2.2.4 Substrate

Nitrifying, denitrifying and anammox bacteria are all dependent on different substrates

for energy and cellular growth as discussed above. The ability to utilise their substrate

varies between bacterial species. This implies that a species with high affinity for its

substrate will be better at utilising the substrate at low concentration and therefore

outcompete species with lower affinity for the substrate. The half saturation constant,

which is the substrate concentration when the growth rate is half of maximum, is often

used to compare how well adapted different microorganisms are to their substrates.

The half saturation constant or Ks value for ammonium oxidisers in a nitrifying biofilm

airlift reactor was found to correspond to a NH4-N concentration of 11 mg/l. And the

microorganisms were inhibited by NH4-N concentrations of 3300 ±1400 mg/l. (Carvallo

et al., 2002)

Denitrification rate and capacity is very dependent on available organic carbon source.

External carbon sources like methanol, ethanol and acetic acid are readily biodegradable

and give much higher denitrification rates than denitrification with organic compounds

found in the waste water (Ødegaard, 1993).

Anammox bacteria have high affinity for their substrates ammonia and nitrite, the Ks

values are below chemical detection level (


2.3 Biofilm reactors

Biofilm reactors can be used for nutrient removal in wastewater treatment and are

commonly used in biological nitrogen removal. Bacteria with the ability to adhere to

solid surfaces are colonising and growing in high concentrations in a biofilm attached to

a fixed surface. The carrier material can be solid or free moving made out of stone, wood

or plastic. Biofilm thickness varies with the hydrodynamics and growth conditions of the

system, (Metcalf & Eddy, 2003). The fixed polymer film formed by the bacteria protects

them from toxics and being washed out of the system (Henze et al., 1997).

Figure 3. Illustration of different types of bioflim reactors. (Adapted from Ødegaard, 1993).

Biofilters are designed to achieve high and efficient nutrient removal rates in compact

and energy efficient systems. Operating conditions should be such that transfer rates of

substrates from the water bulk phase to the microbial community assures efficient

removal rates and development of a biofilm thickness satisfying the microbial demands

in a certain biologic process. This makes different available biofilm technologies suitable

for varied microbial processes. Figure 3 illustrates some of the available biofilm

technologies shortly described in the following text.

2.3.1 Trickling filter

Trickling filters are biological reactors where the wastewater is sprinkled over a filter

bed at the top. The water is then allowed to percolate through a fixed bed material made

out of stone or plastic. Volumetric flow rates are controlling the biofilm thickness and

11


aeration takes place through self drag from bottom to top of the filter (Henze et al.,

1997). Since the wastewater is sprinkled over and percolated through the filter media

there is little hydraulic control of the biofilm thickness resulting in uneven growth

throughout the filter. This causes local clogging that hinders free flow of water and air

through the filter resulting in decreased nutrient removal rate. Modern filling materials

in plastic have a specific filling area of about 100-250 m 2 /m 3 while the carrying material

in older treatment plants often is crushed stone or pumice that only has a specific area of

40-60 m 2 /m 3 , (Gillberg et al., 2003).

2.3.2 Biofilters

The carrier material in these filters can be either a granulated media or corrugated

sheets. In the granulated reactor wastewater passes through a stationary filter bed made

out of sand or plastic beads, the filter media is aerated from the bottom in the aerobic

version. The specific area of the filter media is high and ranges from 1000-1200 m 2 /m 3 ,

the effective area is however not this high since only 50% of the reactor volume is filled

with the carrier material (Ødegaard, 1993). This reactor clogs easily and has to be

backwashed. Clogging and substrate decrease from top to bottom in the reactor rules

out efficient nutrient removal throughout the whole reactor volume. The second type of

media is a stationary carrier material constituted of plastic sheets that are welded

together in cubes, the specific area of these filters ranges from 150-200 m 2 /m 3 . The

system is aerated from the bottom with blower systems and has to be backwashed at

intervals to prevent clogging (Ødegaard, 1993).

2.3.3 Fluidised bed

In a fluidised bed the biofilm grows on sand grains with a size of 0.4-0.5 mm. To keep the

sand grains in suspension at all times wastewater is pumped through the bottom of the

reactor at a high constant flow rate. The turbulence created from high volumetric flow

rates passing through the reactor implies great shear forces and very thin biofilms. With

bed depths ranging from 3 to 4 m a specific surface area of 1000 m 2 /m 3 can be achieved

(Metcalf & Eddy, 2004). High turbulence in combination with very high contact area

between microorganisms and wastewater assures efficient substrate transmission and

high conversion rates. The draw back with this process design in nitrification is that

oxygen transfer rates to the water phase are too slow to maintain sufficient oxygen

concentrations for microbial activity (Gillberg et al., 2003) Sine the biofilm is very thin in

this reactor configuration it does not allow development of a biofilm with layers of

different oxygen concentration. This reactor type can there for not combine microbial

communities with different dissolved oxygen demands. Recirculation is necessary to

maintain the high fluid velocity.

12


2.3.4 Moving bed reactor

Another way to design a compact biofilm process is to use a suspended inert carrier that

moves freely within the reactor. The first carrier materials were small polyethylene

(density 0.95 g/cm 3 ) cylinders with a cross in side providing the microorganisms with a

protected surface to grow on. The carriers are kept in motion by aeration or mechanical

stirring, a sieve in the outlet keeps the moving carriers in the reactor. The reactor does

not clog and there is no need for backwashing or biomass recycling. (Ødegaard et al.,

1994). Different shapes and sizes of the carrier material provides an effective specific

area ranging from 220 -1200 m 2 /m 3 (AnoxKaldnes, 2009). Biofilm thickness depends on

carrier design and hydraulic conditions in the reactor. If a stagnant laminar layer is

formed around the carrier material this will increase the diffusional resistance that is

limiting in biofilm processes. The moving bed technology can also be combined with the

activated sludge process resulting in higher removal rates and more compact systems.

2.3.5 Rotating disc

Rotating filters are constituted of flat discs often made out of plastic, 2-3 meters in

diameter mounted in rows on a horizontal shaft. The filter medium that is semisubmerged

is alternately rotated through the water phase at right angles to the flow.

The filters are 10-20 mm thick, spaced about 20 mm apart and have an active surface

area of about 150-200 m 2 /m 3 (Gray N, 2004). Rotation of the filter discs keeps the

biofilm oxygenated and the motion creates an efficient contact between the water phase

and biofilm. Revolution speed of the filter is controlling the biofilm thickness (Henze et

al., 1997).

2.4 Biofilm kinetics

The kinetics of substrate conversion in a biofilm reactor is dependent of the reactor

configuration that decides the biofilm structure and of available nutrients in the

wastewater. Substrates in the water bulk phase are converted to biomass, energy and

end products through cellular metabolism in the bacteria. The mass balance for an

infinitely small section of the biofilm is described by:

(2.4.1)

(2.4.2)

where: Q is the volumetric flow, (dimension L -3 ∙T -1 ), C is the concentration, (dimension

M∙L -3 ), r describes the biological growth rate, (dimension M∙L -3 ∙ T -1 ) and V is the reactor

volume, (dimension L -3 ), (Warfvinge, 2008).

The substrate conversion rate in a biofilm reactor is dependent on three main

mechanisms, the diffusion resistance of substrates from the well mixed water bulk phase

13


to the liquid film, diffusion of substrates through the biofilm and substrate turnover in

the cellular mass (Ødegaard, 1993), see Figure 4. The diffusion dependent transport of

substrate from the liquid bulk and the diffusion within the biofilm is driven by a

concentration gradient. The substrate concentration profile decreases with biofilm

depth and has a downward curvature due to the substrate utilisation rate illustrated in

Figure 4, (Henze et al., 1997). Conversion rate on cellular level is dependent on

enzymatic processes. Available amount of enzymes handling the specific substrate will

decide how fast the conversion takes place. When substrate concentrations are low, the

accessible substrate S is the rate limiting factor and the rate equation is said to be of first

order with respect to S, see (2.4.3). At high substrate concentrations the substrate

turnover is limited by the amount of available enzymes. The reaction rate is now of zero

order since it is independent of the substrate concentration, see (2.4.4), (Warfvinge,

2008).

First and zero order approximations are given by the following equations:

,


·


(2.4.3)

,


(2.4.4)

where: rv,s describes the biological growth rate in a certain volume of biofilm at a certain

substrate concentration, (dimension M∙ L -3 ∙ T -1 ), µmax is the maximum specific growth

rate, (dimension T -1 ), XB is the concentration of biomass, (dimension M∙L -3 ), Ymax gives

the maximum yield constant, (dimension MXB∙Ms -1 ) and Ks is the saturation constant for

the substrate, (dimension M∙L -3 ), (Henze et al., 1997).

Figure 4. Schematic overview of substrate transport from liquid bulk phase to microorganisms

growing on carrier material. The concentration gradient profile in the biofilm depends on

transport into the biofilm and substrate utilisation rate in the film. (Adapted from Metcalf & Eddy,

2003, and Warfvinge, 2008).

14


To enable efficient nutrient removal the hydraulic conditions should prevent the buildup

of a laminar layer and the contact surface between water phase and biofilm should be

as large as possible (Metcalf & Eddy, I. 2003).

2.5 System configurations for nitrogen removal by anammox

Nitrogen removal by anammox can be implemented either as a two stage or one stage

system. 50% of the influent ammonium is oxidised to nitrite by nitrifying bacteria in

both cases. In a two stage system this conversion takes place in a nitritation reactor

followed by an anammox reactor where the oxidation of ammonium into dinitrogen gas

with nitrite takes place. In single stage technology both processes takes place in the

same reactor (Abma et al., 2007). Since the anammox process was discovered by

different research teams and in slightly diverse environments the system configurations

evolved have been given different names but are in practice quite similar. Main

differences are reactor configuration and operational mode. Some of the available

anammox processes are shortly described in this chapter.

2.5.1 Sharon/Anammox

A Sharon (single reactor system for high rate ammonium removal over nitrite)

reactor followed by an anammox reactor is a possible system configuration to achieve

nitrogen removal with anammox bacteria. The Sharon process is used to nitrify 50% of

the influent ammonium to nitrite. The effluent from the Sharon reactor is then used as

feed for the anammox reactor where nitrite and ammonium is converted to elemental

nitrogen, see Figure 5, (Stowa, 2009).

Figure 5. Sharon/Anammox process scheme.

To achieve partial nitrification to only 50% the Sharon reactor is operated in a manner

that benefits the ammonium oxidizers, washing out the nitrite oxidizers from the system

(van Dongen et al., 2001). This is obtained by operation above 25 ̊C, keeping the sludge

15


age equal to the hydraulic retention time, and by controlling the pH to get the desired

ammonium/nitrite ratio:

NH HCO 0.75O 0.5NH 0.5NO CO 1.5H O (2.5.1)

The effluent from the Sharon reactor is then feeding the anammox process that converts

nitrite and ammonium to elemental nitrogen according to eq. (2.1.7).

Some nitrate is formed in the anammox process as biomass is formed from inorganic

carbon with nitrite as electron donor (van Dongen et al., 2001).

2.5.2 Canon

The Canon process (completely autotrophic nitrogen removal over nitrite) is a single

stage process for nitrogen removal with ammonium oxidisers and anammox bacteria,

(Third et al., 2001), see Figure 6 for system description.

Figure 6. Canon process scheme

The Canon reactor has to be oxygen limited to allow the co-existence of both ammonium

oxidisers and anammox bacteria in the same environment. Ammonium oxidation into

nitrite is performed under oxygen limitation by aerobic ammonium oxidisers eq. (2.5.3).

Anammox bacteria are oxidising ammonium with nitrite into dinitrogen gas eq. (2.5.4).

The resulting over all chemical reaction for the Canon process is described by eq. (2.5.5),

(Third et al., 2005).

Partial nitrification:NH 1.5O NO 2H H O (2.5.3)

Anammox: NH 1.3NO N 0.26NO 2H O (2.5.4)

Canon process: NH 0.85O 0.4N 0.13NO 1.3H O 1.4H (2.5.5)

16


The Canon process is often implemented with granular sludge and it is important that

large biomass flocs with decreasing oxygen gradient within the floc are allowed to form.

This in order to achieve an environment suitable for both aerobic ammonium oxidisers

and anammox bacteria in the same reactor set up (Third et al., 2005).

2.5. 3 Deammonification

The deammonification process is also a one stage process for nitrogen removal through

partial nitrification and anammox. The process scheme resembles that of the Canon

process illustrated in Figure 6. The greatest difference between deammonification and

the Canon process is that deammonification is a biofilm process utilising a carrier

material to support biofilm growth.

Conversion of ammonium into dinitrogen gas takes place at different biofilm depths.

Nitrification of ammonium into nitrite is carried out by nitrifiers in the outer aerobic

layers of the biofilm. This process together with diffusion provides the anammox

bacteria in the deeper anaerobic layers with their substrates (Egli et al., 2003), see

Figure 7. The diffusion depth of oxygen is dependent on the DO concentration in the

water bulk phase and it influences to which extent the conventional nitritation process

takes part in the biofilm (Helmer et al., 2001).

Figure 7. Conversion of ammonium to dinitrogen gas takes place through two separate reactions at

different biofilm depths. (Adapted from Rosenwinkel and Cornelius, 2005)

To ensure anoxic conditions for the anammox bacteria the system must be operated at

low oxygen concentrations or with alternating aeration. Low oxygen concentrations in a

biofilm system also inhibits aerobic nitrite oxidisers which is needed to make sure that

only the first step of nitrification is performed. The growth rate of aerobic ammonium

oxidisers (AOB) is higher than for nitrite oxidisers at low oxygenation, AOB are also

faster at recovering in activity after the anoxic phase.Temperatures over 20°C are also

favouring the growth rate of AOB over nitrite oxidisers (Rosenwinkel and Cornelius,

2005). Biofilm systems are suitable for the anammox process since the bacteria are slow

growers that require high biomass retention (Abma et al., 2006).

17


2.5.4 Deamox

Deamox (denitrifying ammonium oxidation) combines the anammox process with

autotrophic denitrification utilising sulphide as an electron donor for production of

nitrite from nitrate. The Deamox reactor can therefore be used in the treatment process

of wastewaters containing organic bound nitrogen and SO4 2− (Kalyuzhnyi et al., 2006).

The organic nitrogen content in these wastewaters firstly has to be anaerobic

mineralised before nitrification can proceed. If the Deamox process is utilised not all

effluent water from the anaerobic mineralisation reactor has to be nitrified, it can

instead be partially fed to the Deamox reactor. Anammox activity is stimulated by the

denitrifying conditions in the Deamox reactor and since nitrite concentrations are kept

low the process is not thought to produce unwanted emissions of NOx-gases (Kalyuzhnyi

et al., 2006). Since sulphide rich waters are not common in municipal wastewater

treatment the Deamox process has been further developed utilising volatile fatty acids

as a more widespread electron donor for the partial denitrification (Kalyuzhnyi et al.,

2008).

2.5 N2O emissions from wastewater treatment

It has been known for decades that N2O is produced both as an intermediate and end

product in the metabolism of microorganisms performing nitrification and

denitrification processes (Hooper, 1968, Poth and Focht, 1986, Firestone et al., 1979).

Until recently, anammox activity has not been believed to produce any N2O, however

Kartal et al., (2007) have shown that anammox bacteria produces small amounts of N2O

as a result of detoxification of NO which is an intermediate in the anammox process.

Variable temperature and loading rates of inorganic nitrogen compounds, low pH,

alternating aerobic and anaerobic conditions together with growth rate and microbial

composition are parameters that have great influence on N2O emissions from a

wastewater treatment plant (Kampschreur et al., 2008 b). N2O production as a

consequence of these environmental conditions during nitrification and denitrification

will be described in the chapter 2.5.1-2.5.2.The possibility of chemical N2O production in

wastewater treatment is shortly described in 2.4.3. Table 2 gives molecular weight of

N2O and the water solubility both in mol/l and g/l.

Table 2. Physical properties of N 2O

Property:

Unit:

Molecular weight 44.0 g/mol

Water solubility (0 salinity at 20 ̊C)

27.05∙10 − 3 mol/l

1.19 g/l

18


2.5.1 Nitrification as a source of N2O emissions

Ammonium oxidising bacteria (AOB) are the organisms believed to be responsible for

N2O production during nitrification. N2O can be produced both through aerobic

oxidation of ammonium and through nitrifier denitrification of nitrite with ammonium

as an electron donor (Schmidt and Bock, 1997, Kampschreur et al., 2006).

In the presence of oxygen N2O is produced during oxidation of ammonium with oxygen.

2NH O 2HCO N O H O CO (2.5.1)

(Trela et al., 2005)

Hooper, (1968) detected hydroxylamine-nitrite reductase, an enzyme in Nitrosomonas

europaea that reduces nitrite in the presence of hydroxylamine with NO and N2O as

products. Nitrite is reduced anaerobically to N2O with hydroxylamine:

HN OH HNO N O 2H O (2.5.2)

The denitrifying activity of Nitrosomonas is only related to life supporting energy yield

and is probably a survival mechanism in anaerobic habitats (de Bruijn et al., 1995). Low

DO concentrations in the nitrification process has been shown to give higher N2O

emissions than a process operated under well aerated conditions (Magnaye et al., 2008).

High nitrite and ammonium concentrations, high organic loading, low temperature

together with short sludge age are other factors known to give rise to increased N2O

emissions in the nitrification process (Kampschreur et al., 2009).

2.5.2 Denitrification as a source of N2O emissions

Denitrifying organisms are producing N2O as an intermediate when nitrate or nitrite is

reduced to N2 (Kampschreur et al., 2007). Production of N2O takes place as the nitrate

reductase system for electron transport is induced to produce ATP under anoxic

conditions (Gray, 2004), the process occurs in the following sequence:

NO








(2.5.3)

A low pH (


found that when the added quantities of carbon source only allowed between 66-88%

denitrification, N2O emissions from the system were increased from an average of 0.2%

up to 1.3% of reduced nitrate. Long residence times for N2O in denitrifying sludge have

been shown to result in smaller amounts of emitted N2O. Since N2O is an intermediate in

the denitrification process dissolved N2O in the water phase can be turned over by the

denitrifiers and a long residence time for N2O increases the possibility that the gas is

converted into dinitrogen gas. The experiments showing these results were performed

in 100 ml bottles with different sludge volumes which also indicates that N2O emission

is greater from wastewater treatment basins with large surface to volume ratio

(Gejlsbjerg et al., 1997).

2.5.3 Chemical production of N2O

N2O can be produced by chemical denitrification in a wastewater treatment plant, the

reduction follows the same pathway as during biologic denitrification shown in eq.

(2.5.3). The difference is that chemical reductants are reducing the nitrogen compounds

instead of microbial enzymes (Debruyn et al., 1994).

Figures of N2O emissions from microbial nitrogen conversion found during the literature

study are summarised in Table 3.

20


Table 3. N 2O emission from different wastewater treatment facilities given in % of influent N-concentrations.

Reactor type

Influent N-concentrations mg/l

DO N 2O emission % of influent

NH 4-N NO 2-N NO 3-N COD mg/l N-concentration

Reference

nitritationanammox SBR 650 ±50


2.6 Microsensors

Microsensors measure changes in the chemical composition of complex and

heterogeneous environments in a micrometer scale with a very short response time.

The sensors can therefore be used in a broad range of scientific research, for example in

cell and tissue analysis, microrespiration, marine ecology, biofilm analysis and

wastewater treatment. Laboratory experiments in this master thesis are based on online

measurements with one microsensor for nitrous oxide and one for nitrite. Both sensors

that are developed by Unisense, Århus, Denmark rely on electrochemical detection of

N2O.

2.6.1 Nitrous oxide sensor

The N2O microsensor is a Clark-type microsensor constituted of a cathode shaft (tapered

glass casing) equipped with silicone tip membrane. A N2O reducing cathode is

positioned in an electrolyte behind the silicone membrane. The reference for the N2O

reduction is a silver anode and the sensor is equipped with an oxygen front guard (with

an silicone membrane in the tip. The front guard prevents oxygen from interfering with

the nitrous oxide measurements. The guard is filled with an alkaline ascobate solution,

an effective reducing agent, to prevent oxygen interference with nitrous oxide

measurements (Andersen et al., 2001). The silicone membranes in the sensor tip only

allows passage of gases and small uncharged molecules, shielding the electrolyte from

the outer environment (Unisense b, 2007).

Figure 8. Photo of the N 2O microsensor.

22


The microsensor is connected to a piccoameter that polarises the cathode surface where

the nitrous oxide that diffuses through the silicone membrane is reduced to N2 gas. As

nitrous oxide is reduced at the cathode surface two electrons from the silver anode is

used for each reduced N2O molecule. The electron transport gives rise to a current

proportional to the amount of reduced nitrous oxide. The current is registered and

converted to an out signal by the piccoameter. The guard cathode is also polarised to

deplete oxygen in the electrolyte which minimizes zero current (Unisense b, 2007).

The nitrous oxide sensor has a measuring range of about 0-1 atmosphere pN2O with a

response time less than 10 seconds. The stirring sensitivity is smaller than 2% and the

out signal is temperature dependent with a temperature coefficient of about 2-3% per

°C. Interference in the out signal might occur from electrical noise in the surrounding

environment (Unisense b, 2007).

2.6.2 Nitrite biosensor

The nitrite biosensor is a nitrous oxide sensor equipped with a replaceable biochamber

(Figure 9a), (Unisense e, 2009). A plastic tube containing a carbon source and a bacterial

culture constitutes the biochamber that is mounted in the front of the sensor tip (Figure

9b), (Unisense e, 2009). The biomass in the reaction chamber is positioned between the

carbon source required for their growth and an ion-permeable membrane separating

the microorganisms from the external environment (Unisense c, 2007).

The denitrifying bacterial culture used in the biochamber is deficient in NO3 − and N2O

reductase which means that it is only able to reduce NO2 − into N2O. As NO2 − diffuses into

the biochamber it is reduced to N2O by the biomass (Nielsen et al., 2004). Since

denitrifying bacteria are facultative aerobic they can use both oxygen and nitrite as

oxidation agent for their respiration (Larsen et al., 1997). Oxygen is used preferential to

nitrite as it results in a higher energy yield. This will create a NO2 − reducing gradient in

the biochamber with the bacteria closest to the membrane respiring with oxygen. The

NO2 − reducing capacity of the biosensor will depend on the length of the aerobic zone

resulting in higher maximum detectable concentrations of NO2 − in anaerobic

environments (Larsen et al., 1997).

Produced N2O diffuses through the silicone membrane and is reduced at the cathode in

the transducer part of the biosensor. A piccoameter measures the current arising from

the electron transport just as in the case with the nitrous oxide sensor. The output signal

is proportional to the amount of NO2 − that has been reduced after diffusion into the

biochamber (Unisense c, 2007).

23


Figure 9 A) Nitrite biosensor with removable biochamber(Unisense e, 2009). B) Enlargement of

biochamber (Unisense e, 2009).

At 20 °C the biosensor has a measuring range in the interval 0-1000 µM NO2-N, (0-14

mg/l), and gives about 1.25-4 nA in output signal per 100 µM NO2-N (1.4 mg/l) added.

The sensor signal depends on both ionic composition and temperature of the sample. It

might vary up to 30% due to salinity and it has a temperature coefficient of about 2-4%

per °C. Sensitivity to stirring of the sample depends both on temperature and salinity.

The 90% response time in a stirred sample is less than 90 seconds (Unisense c, 2007).

Nitrous oxide diffusing into the biochamber from the external environment is detected

by the N2O transducer and is therefore interfering with the NO2 − signal. The theoretical

sensitivity to N2O should be a signal 2.5 times higher than for equal concentrations of

NO2 − . This since it takes two NO2 − molecules to form one N2O molecule and the diffusion

coefficient for NO2 − is 0.8 times that of N2O (Nielsen et al., 2004).

24


Chapter 3

3. Material and Methods

3.1 Partial nitritation/anammox laboratory MBBR .

A 7.5 litre laboratory MBBR fed with a synthetic medium was used to estimate the N2O

emissions from a single stage nitritation/anammox system. The reactor was initially

started in October 2008 with a carrier material with already established biofilm derived

from Himmerfjärdsverkets full scale DeAmmon ® reactor which is a single stage reactor

for ammonium reduction to dinitrogen gas. The used carrier was AnoxKaldnes K1

biocarrier with a protected surface area of 500 m 2 /m 3 . The total volume of carriers in

the reactor was 3.5 litres which corresponds to about 3400 carriers, a total protected

area of 1.7 m 2 and a filling degree of 46.7%. Figure 10 shows the laboratory set up of the

MBBR system.

Figure 10. The left part of the figure shows a photograph of the MBBR system, the schematic

drawing to the right shows the main features of the MBBR system.

The temperature of the MBBR was kept at around 30 °C. A thermostat bath recirculating

warm water through the jacketed double walls of the reactor was used to maintain the

temperature. pH of the reactor was controlled with a pH electrode connected to a

regulator unit. The regulator controlled a peristaltic pump supplying the reactor with

2M H2SO4 when needed. The synthetic medium was fed to the reactor with a Watson

Marlow peristaltic pump. Aeration and mixing of the system was obtained with two

aquarium pumps that supplied the reactor with air through a punched bottom plate, (2

mm Ø). A top mounted stirrer was used to keep the system mixed during anoxic periods,

see Figure 10 for system description. A timer was used to control the duration of aerated

and mechanical mixed periods.

25


3.2 Reactor medium

The reactor was fed with a synthetic wastewater with an inorganic nitrogen

concentration corresponding to 314 mg/l. The synthetic wastewater contained all vital

nutrients the microorganisms needed, including trace elements, see Table 4 and Table 5

for composition of reactor medium and trace element stock solution.

Table 4. Composition of synthetic medium fed to the MBBR.

Component

NaHCO3 2.6

NH4Cl 1.2

Concentration g/l

KH2PO4 5.67∙10 -3

Peptone 3.0∙10 -3

trace element solution 1 0.40 ml/l

trace element solution 2 0.40 ml/l

Table 5. Composition of trace element stock solution.

Component

Stock solution 1

MgSO4∙7H2O 4.80

MnCl2∙2H2O 1.60

CoCl2∙6H2O 0.48

NiCl2∙6H2O 0.24

ZnCl2 0.26

CuSO4∙5H2O 0.10

FeCl2∙4H2O 1.44

BH3O3 0.104

Na2MoO4∙2H2O 0.440

Na2SeO3∙5H2O 0.288

Na3WO3∙2H2O 0.280

Stock solution 2

CaCl2∙2H2O 5.80

Concentration g/l

Initially the medium was mixed in a 600 litres container situated in the workshop and

pumped to the second floor where the laboratory is situated. Microbial growth in the

tank and pump tubing were causing large differences in composition of the influent

medium to the reactor. Due to these problems the medium was mixed in a 100 litres

tank kept in the laboratory in close connection to the reactor set up.

26


3.3 Analytical methods

Concentrations of NH4-N, NO2-N and NO3-N were determined with Dr Lange’s

spectrophotometry kit after filtration through Munktel 1.6 µm glass fibre filters. During

cycle studies NO2-N and N-tot were analyzed directly with Dr Lange’s method. Samples

were frozen and flow-injection analysis was used to determine NH4-N and NOx, the sum

of NO2-N and NO3-N. The NO3-N content was calculated by subtraction of NO2-N from the

sum of the two NOx species. Dissolved oxygen and pH was sampled with a portable

meter, HQ40d with mounted oxygen and pH probe. Parameters analysed and method

used are summarised in Table 6.

Table 6. Analysed parameters and methods.

Analysed parameter Method

NH4-N

LCK 303/FIA

NO2-N

LCK 342/341/Bio sensor

NO3-N LCK 339

NOx

FIA

N-tot LCK 238

DO

HQ40d

pH

HQ40d

3.3 Cycle studies

To examine which operation conditions which seemed to produce the largest amounts

of N2O gas, the reactor was operated at different DO concentrations during intermittent

and constant aeration. A study where the anoxic phase was prolonged to two hours was

performed to observe how the N2O production was influenced. Mixing with N2 gas at the

same aeration flow as in the aerated period was tested to see how stripping influenced

the amount of N2O in the water phase.

Parameters monitored online in the reactor, every minute were; DO, pH, N2O and NO2-N.

To examine the concentration changes of NH4-N, NO2-N and NO3-N grab samples were

taken in both influent and effluent water.

3.3.1 Intermittent aeration

The reactor was operated at a DO concentration of ~3 mg/l during the aeration phase.

One reactor cycle lasted for one hour with 40 minutes of aeration and 20 minutes of

mechanical mixing. The study started at the same moment as aeration went on after the

anoxic period and lasted for 66 minutes in order to overlap the initial conditions.

Grab samples in the effluent were taken with 6 minute intervals to get two measure

points in the anoxic phase. Only three measurements of the influent medium was taken

27


in one cycle ( 0, 36 and 66 minutes) considered that the variation of the influent medium

during one hour should not be significant.

3.3.2 Prolonged study, intermittent aeration

A study of the effect of prolonged, intermittent aeration was made in order to observe

how the N2O production was influenced by a longer anoxic period. During the first hour

the reactor was operated in the same manner as above and the same sampling

procedure was applied. The anoxic period of 20 minutes during a normal cycle was

prolonged with 2 hours.

Grab samples were taken in the effluent with 6 minute intervals and every 36 minutes in

the feeding medium.

3.3.3 Continuous aeration

Measurements during continuous aeration were performed at DO concentrations of

~1.5 mg/l and ~1 mg/l. To determine the production of N2O gas during these operation

conditions the aeration was turned off and the unaerated period was determined to 20

minutes to be comparable to the measurements done during intermittent aeration.

Measurements proceeded 20 minutes after the aeration was switched on again.

To examine if the accumulation of N2O gas in the water phase during the anoxic period

depended on increased production or was an effect of stripping during aeration, mixing

with pure N2 gas was used during the anoxic period. The N2 gas flow was equal to the

airflow during the aerated period.

Grab samples were taken in the effluent every 6 th minute and every 36 th minute in the

influent medium.

3.4 Calibration of microsensors

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

microelectrodes described in chapter 2.6. Before usage the microsensors were

calibrated separately in a jacketed, temperature controlled beaker with 300 ml of pH

regulated synthetic medium to assure the same salinity, temperature and pH of

calibration solution and reactor, see Figure 11.

The sensor to be calibrated was mounted in the calibration beaker, a stable sensor signal

was awaited and the zero value was read and registered with Unisense’s software

SensorTrace BASIC. A top mounted stirrer was used for fast mixing and uniform

concentration of the calibration solution.

28


Figure 11. Calibration setup for microsensors.

Both sensors were calibrated by stepwise addition and signal reading at known

concentrations of N2O and NO2-N respectively. The resulting calibration curves are

illustrated in Figure 112. The linear regression shown in the figure is only based on one

single point registered by the computer software at each concentration. Both

concentration profile and linear regression obtained during calibration are drawn to

illustrate the procedure.

Figure 12. Examples of calibration points and concentration profiles for N 2O and NO 2-N

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

obtained in mV without any addition of N 2O or NO 2-N. As stepwise concentrations of N 2O and NO 2-N

were added during the calibration the sensor signal increased. A stable signal was awaited before

a voltage corresponding to the added concentration was registered.

Addition to known N2O concentrations was achieved by adding a defined volume of a

saturated N2O solution. The saturated N2O solution was prepared by bubbling N2O gas

through distilled water with a flow rate of 1 l/min for at least 30 minutes. For calibration

of the biosensor additions were made from a bulk solution with NaNO2 with a NO2-N

concentration of 5 mg/l. See Appendix A for calculated volume additions of the saturated

N2O and NO2-N solutions. After calibration both sensors were mounted directly into the

MMBR reactor, the N2O sensor was placed in a small metal-mesh basket for protection

from the moving carriers.

29


3.5 Diffusivity tests of N2O

The diffusivity of N2O was examined experimentally since no off-gas equipment was

available during N2O measurements and the calculations of produced N2O are based on

the assumption that the diffusivity of N2O can be neglected.

To get as close as possible to the real conditions in the MBBR process but without any

N2O producing bacteria a 7.5 l reactor of the same type as used for the MBBR process

was utilised during the diffusivity experiments. The influent synthetic wastewater was

selected to get a medium similar in salinity to that in the real process. The reactor was

heated to 30 ̊C with a thermostat bath and K1 heavy carriers (same type of carrier as K1

used in the MBBR, but with slightly higher density) without biomass was used. K1 heavy

was used to keep the carrier material without biofilm in the water phase and not floating

on top of the water surface.

To examine how fast N2O diffuses from the water phase during mechanical mixing N2O

saturated water was added to a concentration of ~11 µM. The decrease of N2O in the

water phase was registered with the N2O microsensor during a period of eight hours.

The stripping effect from aeration was also observed by registering the decrease of N2O

in the water phase during aeration at three different air flow rates. N2O was added to a

concentration of 12 µM.

30


Chapter 4

4. Results

4.1 Process performance

Variations in nitrogen concentration of the influent medium, flow rate, temperature and

pH are shown in Table 7 and Figure 13. Variations in nitrogen concentration shown in

Figure 13 are the sum of influent nitrogen compounds accounted for in Table 7. The

figure also shows variations in oxygen concentrations during aeration.

Table 7. Characteristics of influent feed, flow rates, temperature and pH during the operation

period 09/07/09-15/12/09.

Nitrogen mg/l

NH 4-N NO 2-N NO 3-N Q l/h T °C pH

264.5 ± 24.5 19.7 ±12.4 3.6 ±2.1 0.48 ±0.06 30.0 ±0.8 7.3-7.8

Changes of nitrogen concentrations in the effluent water are shown together with %

nitrogen reduction and the total nitrogen removal in Table 8 and Figure 13. The Effluent

nitrogen illustrated in Figure 13 is the sum of effluent nitrogen compounds seen in Table

8.

Table 8. Average concentrations of inorganic nitrogen in effluent water, reduction rates and

removal rates during the operation period 09/07/09-15/12/09.

Nitrogen mg/l

NH 4-N NO 2-N NO 3-N

Reduction % Removal gN/m 2 d

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

On the 28 th of September the operation mode was changed to continuous aeration,

aiming at a DO level of ~1.5 mg/l. It took around one week to get a stable performance

at the desired oxygen level. For measurements at even lower oxygenation the oxygen

concentration was further decreased to ~1.0 mg/l.

31


10

8

DO concentration, pH & temperature

35

28

DO (mg/l), pH

6

4

2

21

14

7

Temp °C

DO (mg/l)

pH

Temperature (°C)

0

0

06/07/09

26/07/09

15/08/09

04/09/09

24/09/09

Date

14/10/09

03/11/09

23/11/09

13/12/09

350

Concentrations of influent and effluent total inorganic nitrogen

Total inorganic nitrogen (mg/l)

300

250

200

150

100

50

0

Σ TIN in (mg/l)

Σ TIN out (mg/l)

06/07/09

26/07/09

15/08/09

04/09/09

24/09/09

Date

14/10/09

03/11/09

23/11/09

13/12/09

Removal rate (gN/m²d)

1.8

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

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

90

80

70

60

50

40

30

20

10

0

% reduction

Removal rate

(gN/m2d)

% reduction

06/07/09

26/07/09

15/08/09

04/09/09

24/09/09

Date

14/10/09

03/11/09

23/11/09

13/12/09

Figure 13. Reactor operation and process performance during the period 09/07/09-15/12/09.

Top graph illustrates concentration of DO during aeration, pH and temperature. The second graph

shows total concentration of inorganic nitrogen in influent feed and effluent water. The third

shows the removal rate gN/m 2 d and the reduced nitrogen in %.

Figure 14 shows the process performance during different operation modes. Green

series are representing measurements done at intermittent aeration during the period

090918-090927. Blue series are representing measurements at continuous operation,

DO ~1.5 mg/l during the period 091006-091010. Orange series are representing

measurement at continuous operation, DO ~1mg/l during the period 091013-091016.

32


10

8

DO concentration, pH & temperature

35

28

DO (mg/l), pH

6

4

21

14

Temp °C

DO (mg/l)

pH

Temperature (°C)

2

7

0

0

14/09/09

19/09/09

24/09/09

29/09/09

Date

04/10/09

09/10/09

14/10/09

19/10/09

350

Concentrations of influent and effluent total inorganic nitrogen

Total inorganic nitrogen (mg/l)

300

250

200

150

100

50

0

Σ TIN in (mg/l)

Σ TIN out (mg/l)

14/09/09

19/09/09

24/09/09

29/09/09

Date

04/10/09

09/10/09

14/10/09

19/10/09

Removal rate (gN/m²d)

1.6

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

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

90

80

70

60

50

40

30

20

10

0

% reduction

Removal rate

(gN/m2d)

% reduction

14/09/09

19/09/09

24/09/09

29/09/09

Date

04/10/09

09/10/09

14/10/09

19/10/09

Figure 14. Illustration of how the process performance changes with changed operation mode, The

top graph illustrates DO during the aerobe phase, pH and temperature. The second graph shows

total concentration of inorganic nitrogen in influent feed and effluent water. The third graph

shows the removal rate gN/m 2 d and the reduced nitrogen in %.

4.3 N2O emissions from partial nitritation/anammox MBBR

The microsensor measured the N2O in the water phase during different operation modes

and aeration rates of the MBBR. The actual N2O production was not measured since N2O

left the water phase continuously through stripping and or by diffusion. However an

estimation of the N2O production was obtained by measuring the accumulation of the

N2O directly after the airflow is turned off assuming the N2O diffusion from water to air

is negligible. If corrections for the N2O leaving the reactor with the effluent water is

33


made the increase in N2O accumulation will then be equal to the N2O production rate,

(see appendix B for calculation example). The N2O production is calculated as % of

removed inorganic nitrogen. Two N2O production rates are estimated and referred to as

initial and maximum production rates. The initial production rate is calculated from the

increase of N2O that can be seen in the water phase immediately after switching of

aeration. Maximum N2O production is estimated between the two measuring points

where the increase in N2O has its maximum during the unaerated period. Mean N2O

concentration in the water phase when the MBBR is aerated, calculated initial and

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

mean nitrogen concentrations, reduction and removal rates for all measurements are

summarised in Table 9 -Table 14. One figure of typical N2O and O2 profiles for each

operation mode is shown in Figure 15 -Figure 20, N2O and O2 profiles for all

measurements made are found in appendix C.

4.3.2 Intermittent aeration.

Measurements of produced N2O at intermittent aeration, (DO ~3.0 mg/l) were

performed at four different occasions. Typical profiles of how N2O and DO changed

during the cycle are shown in Figure 15. The N2O concentration measured in the water

phase varied with the aeration of the MBBR. When aeration started in the beginning of

the cycle the airflow striped N2O out of the water phase and the concentration decreased

to a constant minimum level. As soon as the aeration was shut off the N2O started to

accumulate in the water phase until aeration was switched on again and the procedure

started over as shown in Figure 15.

N₂O (µmol/l)

12

10

8

6

4

2

0

4.5

3.75

2.25

1.5

0.75

0

0 20 40 60 80

Time (min)

3

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

Figure 15. Concentration profiles of N 2O and O 2 during a cycle of intermittent aeration, DO ~3 mg/l

in the aerated phase. The cycle study started in the beginning of the aerated period. N 2O gas was

stripped from the water phase at the same time as the oxygen concentration rose.

Initial N2O production varied between 5.6-11% of influent nitrogen concentration that

was converted into dinitrogen gas (here after referred to as removed inorganic N-

34


concentration), while the maximum production ranged from 11-16% of removed

inorganic nitrogen, see Table 9.

Table 9. Average N 2O concentration in the water phase during aeration. Calculated initial and

maximum N 2O production rates, mean O * 2 concentrations during the aerated period, mean

nitrogen concentration, reduction and removal rates for studies of intermittent aeration at a DO

concentration of ~3.0 mg/l.

Date

Average

N 2O

µmol/l

Produced N 2O in % of

removed inorganic N-

concentration O 2

mean N-concentration

mg/l

initial max mg/l NH 4-N NO 3-N NO 2-N

N-red.

%

Removal

gN/m 2 d

090918 3.2 11.0 16.3 3.22 300 - - 54 1.1

090921 2.0 5.6 11.0 3.49 293 - - 56 1.1

090922 2.6 9.9 13.9 3.10 287 - - 56 1.1

*Mean O2 concentration from the moment when the DO level reaches its maximum

concentration until aeration is shut off and oxygen starts to decrease again.

4.3.2 Prolonged unaerated period.

Three studies of a prolonged unaerated period were made to examine for how long the

accumulation of N2O proceeded. The measurement started in the beginning of a normal

cycle when the airflow was switched on. Aeration lasted for forty minutes followed by

an unaerated period of two hours and twenty minutes, typical profiles of how N2O and

O2 changes during the cycle are illustrated in Figure 16.

12

10

4.5

3.75

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 50 100 150 200

Time (min)

Figure 16. Concentration profiles of N 2O and O 2 during prolonged unaerated period, DO ~3 mg/l

during aerated phase. (Only manually registered O 2 concentrations every sixth minute are

available during the first fifty minutes, due to problems with overwriting of data in the DO meter).

N2O decreased in the water phase as aeration was switched on and the oxygen

concentration started to increase, the concentration profiles resembles the cycle profile

shown in Figure 15 until the prolonged unaerated period started. At first N2O

accumulation was rather linear, when DO decreases under 1 mg/l the accumulation rate

of N2O was reduced until a maximum concentration was reached at DO concentrations

35


close to 0 mg/l. The N2O concentration is constant under a period of 20-50 minutes and

then slowly started to decrease as seen in Figure 16. Initial and maximum production

rates of N2O calculated during the prolonged cycles as are shown in Table 10. Initial N2O

production rates varied between 6.2-11% while maximum production varied between

10-30% of removed inorganic nitrogen.

Table 10. Prolonged measurement: Average N 2O concentration in the water phase during aeration.

Calculated initial and maximum N 2O production rates, mean O 2* concentrations during aerated

period, mean nitrogen concentration, reduction and removal rates.

Date

Average

N 2O

µmol/l

Produced N 2O in % of

removed inorganic N-

concentration O 2

mean N-concentration

mg/l

initial max mg/l NH 4-N NO 3-N NO 2-N

N-red.

%

Removal

gN/m 2 d

090925 0.9 6.2 30.3 3.0 234 - - 59 1.0

090926 2.5 10.7 10.7 2.8 237 - - 47 0.9

090927 2.2 9.5 9.5 3.1 228 - - 57 1.0

*Mean O2 concentration from the moment when DO concentration reached its maximum

level until aeration is shut off.

4.3.3 Continuous operation at DO ~1.5 mg/l

The MBBR was operated at continuous aeration which was switched off for twenty

minutes in order to estimate the N2O accumulation. Figure 17shows the concentration

profiles of N2O and O2. As seen in the figure they resemble the profiles obtained during

cycle studies of intermittent aeration. The N2O accumulation increased as O2 decreased

but not as fast as before.

12

10

4.5

3.75

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 20 40 60 80

Time (min)

Figure 17. Concentration profiles of N 2O and O 2 obtained from measurement during the period of

continuous reactor operation at a DO concentration of ~1.5 mg/l.

Twenty minutes of the anoxic period was enough to reach the maximum N2O

concentration and the period where N2O production seems to be in equilibrium with the

amount of N2O leaving the system. Figure 17 also illustrates that the mean concentration

36


of N2O during aeration was slightly higher and that the maximum concentration reached

was lower than during intermittent aeration.

Initial and maximum % N2O production was calculated to be in the range of 2-3.2% and

5.6.-6.2% of removed inorganic nitrogen respectively, the result is presented in Table

11.

Table 11. Average N 2O concentration in the water phase during aeration. Calculated initial and

maximum N 2O production rates, mean O 2 concentrations during aeration, mean nitrogen

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

mg/l.

Produced N 2O in % of

Average

N 2O

removed inorganic N-

concentration O 2

mean N-concentration

mg/l

N-red. Removal

Date µmol/l initial max mg/l NH 4-N NO 3-N NO 2-N % gN/m 2 d

091006 4.3 2.8 5.6 1.3 249 25.2 0.3 66 1.3

091007 2.9 2.1 5.6 1.8 246 28.9 0.0 64 1.3

091010 3.3 3.2 6.2 1.8 220 36.3 0.0 72 1.3

4.3.4 Continuous operation at DO ~1.0 mg/l

Two measurements were performed at a constant aeration with a DO concentration of 1

mg/l. Registered N2O accumulation was the lowest so far and the initial N2O production

was below 2% of reduced inorganic nitrogen. Maximum production varied between 2-

4.3%. Table 12 presents the results from continuous operation at DO concentration of~1

mg/l.

12

10

4.5

3.75

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 20 40 60 80

Time (min)

Figure 18. Concentration profiles of N 2O and O 2 obtained from measurement during the period of

continuous reactor operation at a DO concentration of ~1.0 mg/l.

As seen in Figure 18 N2O was only accumulating for the first 5-6 minutes of the

unaerated period then there was a short time span when produced N2O and N2O flows

that left the reactor were in equilibrium. The N2O concentration measured in the water

phase decreased before aeration started again.

37


Table 12. Average N 2O concentration in the water phase during aeration. Calculated initial and

maximum N 2O production rates, mean O 2 concentrations during the aerated period, mean

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

~1.0 mg/l.

Date

Average

N 2O

µmol/l

Produced N 2O in % of

removed inorganic N-

concentration O 2

mean N-concentration

mg/l

initial max mg/l NH 4-N NO 3-N NO 2-N

N-red.

%

Removal

gN/m 2 d

091013 2.5 1.7 4.3 1.0 285 0.0 0.0 85 1.6

091014 2.3 2.0 2.0 1.0 284 0.0 0.3 73 1.3

4.3.5 Effect of mixing with N2 gas during unaerated phase, continuous

operation at DO ~1.0 mg/l and ~1.5 mg/l

Pure N2 gas was used during the anoxic period instead of mechanical mixing, in order to

evaluate the stripping effect from the gas (the same gas flow rate was used as during

aeration with air). A small accumulation of N2O was observed right after the switch from

aeration with air to N2 gas, see Figure 19 The increase was followed by a sharp decrease

in the N2O concentration profile. When aeration was switched on again, the N2O

concentration increased faster than during previous measurements.

12

10

4.5

3.75

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 20 40 60 80

Time (min)

Figure 19. Concentration profiles of N 2O and O 2 when N 2 gas was used for mixing during anoxic

period, reactor was operated with continuous aeration at a DO level of ~1.0 mg/l.

The accumulation of N2O that can be seen was converted to a corresponding % N2O

production calculated to be


Table 13. Average N 2O concentration in the water phase during aeration. Calculated initial and

maximum N 2O production rates, mean O 2 concentrations during the aerated period, mean nitrogen

concentration, reduction and removal rates during measurements with N 2 gas in anoxic phase,

operation at DO ~1.0 mg/l.

Date

Average

N 2O

µmol/l

Produced N 2O in % of

removed inorganic N-

concentration O 2

mean N-concentration

mg/l

initial max mg/l NH 4-N NO 3-N NO 2-N

N-red.

%

Removal

gN/m 2 d

091015 1.9 0.4 - 1.0 279 0.4 0.0 79 1.2

091016 1.1 0.8 - 1.3 278 0.3 0.0 77 1.2

When the MBBR was operated at a DO concentration of 1.5 mg/l instead of 1 mg/l, a

slightly higher N2O accumulation was observed right after the shift from aeration with

air to mixing with N2 gas. Accumulation proceeded for about 5 minutes and there after

the N2O concentration started to decrease. The rate with which N2O left the water phase

increased as aeration with air was switched on, see Figure 20

12

10

4.5

3.75

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 20 40 60 80

Time (min)

Figure 20. Concentration profiles of N 2O and O2 when N 2 gas is used for mixing during anoxic

period, reactor operated with continuous aeration at a DO level of ~1.5 mg/l.

Table 14 presents the results from the twomeasurements done when N2 gas was used

for mixing during the anoxic period.

Table 14. Average N 2O concentration in the water phase during aeration. Mean calculated initial

and maximum N 2O production rates, mean O 2 concentrations during aerated period, mean

nitrogen concentration, reduction and removal rates during measurements with N 2 gas in anoxic

phase, operation at DO ~1.5 mg/l.

Produced N 2O in % of

Average

N 2O

removed inorganic N-

concentration O 2

mean N-concentration

mg/l

N-red. Removal

Date µmol/l initial max mg/l NH 4-N NO 3-N NO 2-N % gN/m 2 d

091017 1.4 1.4 - 1.5 289 0.3 0.0 71 1.2

091018 1.1 2.1 - 1.8 289 0.3 0.0 73 1.3

39


4.4 NO2-N biosensor

The biosensor was used to register changes in NO2-N online during measurements of

N2O production. The purpose was both to examine NO2-N concentration changes in the

anammox process and to see if it was possible to replace the traditional NO2-N analysis

with Dr Lange kit (LCK 342/341). Two typical measurement occasions where the

biosensor has been used are shown here in . All concentrations profiles achieved

through measurements made with the biosensor can be found in appendix C, the sensor

was not in use during measurements of N2O production 14-15 of September. The reason

for not using the sensor was that there were problems in achieving a stable sensor signal

during calibration and the biochamber had to be exchanged.

Figure 21 shows the obtained NO2-N concentrations from online measurements with the

biosensor and from grab samples analysed with Dr Lange’s method, LCK 342, during a

prolonged unaerated measurement period.

DO, NO₂-N (mg/l)

10

9

8

7

6

5

4

3

2

1

0

0.00 50.00 100.00 150.00 200.00

Time (min)

NO₂-N

biosensor

(mg/l)

NO₂-N LCK

342

(mg/l)

Figure 21. NO 2-N concentration profiles obtained with biosensor and with Dr Lange’s method, LCK

342, during prolonged unaerated measurement.

As can be seen in Figure 21 there was a difference in concentrations obtained from the

two different measurement methods. The concentration of NO2-N registered by the

biosensor is 2-3 mg/l higher than NO2-N concentrations obtained from grab samples

analysed with LCK 342. Even if NO2-N concentrations registered with the biosensor

were higher than concentrations obtained from analysis with LCK 342 both methods

showed the same trends in NO2-N concentration profiles during the measurement.

Figure 22 shows the result from online measurements with the biosensor compared to

grab samples analysed with LCK 342 from a measurement occasion when the MBBR was

operated at continuous aeration at a DO level of ~1.5 mg/l. The NO2-N concentrations

obtained from both methods correlated much better during this measurement. The

deviation in NO2-N concentrations was below 1 mg/l during this measurement and the

concentration profile given from the two methods correlates well.

40


12

NO₂-N (mg/l)

10

8

6

4

2

NO₂-N

biosensor

(mg/l)

NO₂-N LCK 342

(mg/l)

0

0 20 40 60 80

Time (min)

Figure 22. NO 2-N concentration profiles obtained with biosensor and with Dr Lange’s method, LCK

342, from measuring session when the reactor was operated at continuous aeration.

Changes in measured NO2-N concentration with LCK 342 varied between 4-6 mg/l,

while the NO2-N concentrations registered with the biosensor stayed in a slightly

narrower range of 4-5 mg/l. Figure 22 also shows that the biosensor seems to have a lag

phase, a phenomenon which can be seen in Figure 21 as well.

4.5 Diffusivity and stripping test of N2O

The diffusivity test of N2O performed in a reactor with carriers without biofilm during

mechanical mixing showed that N2O dissolved in the water phase left the system slowly.

Figure 23 shows how the initial N2O concentration decreased from ~11- 4 µmol N2O/l

during a period of about 8 hours, (500 minutes). Linear regression of the diffusion rate

showed that ~0.0132 µmol N2O left the reactor per minute. This molar concentration

corresponds to < 1% of N2O present in the water phase at all times during the

measurement. In order to validate the assumption that N2O diffusion can be neglected

during calculations of produced N2O the diffusivity rate/min has to be compared to the

production rate/min. The result of this comparison gives that diffusion corresponds to

about 10% of produced N2O during one minute.

41


N₂O µmol/l

12

10

8

6

4

2

0

0 100 200 300 400 500

N₂O (µmol/l)

y = -0.0132x + 10.59

Time (min)

Figure 23. N 2O decrease in the water phase due to diffusion during mechanical mixing.

When the system was aerated dissolved N2O was stripped out of the water phase at a

much higher speed compared to the diffusion rate, see Figure 24-Figure 26. However the

stripping rate did not change much with the different aeration rates. When the reactor

was aerated with an airflow corresponding to 1.2 l/min, dissolved N2O left the water

phase at a rate of ~0.55 µM/min, if linearly approximated, see Figure 24. The linear that

is drawn in Figure 24 shows that a linearization is not a good approximations of how

N2O was stripped out of the water phase. As the stripping rate decreased with

decreasing N2O concentration a potential curve fitting might be a better option which is

also shown in Figure 24.

14

Aeration 1.6 (l/min) with carriers

N₂O (µmo/l)

12

10

8

6

4

2

0

0 5 10 15 20 25

Time (min)

N2O (µmol/l)

y = -0.6482x + 10.193

y = 12.95e -0.143x

Figure 24. Stripping of N 2O from the water phase during aeration with an airflow of 1.2 l/min.

As the aeration rate was increased to 1.6 l/min the linear stripping rate increased to

~0.65 µM/min, see Figure 25.

42


Aeration 1.6 (l/min) with carriers

N₂O (µmo/l)

14

12

10

8

6

4

2

0

0 5 10 15 20 25

Time (min)

N2O (µmol/l)

y = -0.6482x + 10.193

y = 12.95e -0.143x

Figure 25. Stripping of N 2O from the water phase during aeration with an aeration rate of 1.6

l/min.

When the aeration rate was further increased to 2.0 l/min the linear stripping rate

increased to ~0.67 µM/min, see Figure 26.

14

Aeration 2.0 (l/min) with carriers

N₂O (µmo/l)

12

10

8

6

4

2

0

0 5 10 15 20 25

Time (min)

N2O (µmol/l)

y = -0.6722x + 10.714

y = 13.571e -0.141x

Figure 26.Stripping of N 2O from the water phase during aeration with an aeration rate of 2.0 l/min.

With a starting concentration of ~12 µmol/l, it took between 15 and 20 minutes to strip

N2O out of the water phase to a concentration ~1µmol/l.

43


Chapter 5

5. Discussion

5.1 Process performance

Theoretical maximum nitrogen removal by the anammox process is 88%, (Strous et al.,

1998), highest achieved performance in the MBBR during the period of this master

thesis work was about 80% with fluctuations down to a reduction corresponding to only

20%, the mean nitrogen conversion was 58%. Non stable process performance is

probably due to operation disturbances like; stop in the influent flow, power failure,

fluctuations in influent nitrogen compounds, (caused by microbial conversion of

nitrogen compounds in the synthetic wastewater).

As the reactor operation mode was shifted into continuous aeration at a lower DO

concentration both % reduction and removal rate in gN/m 2 d was more stable and

higher than the average during intermittent aeration shown in Figure 13 and Figure 14.

This is consistent with results obtained by Szatkowska et al., (2003) who showed that

higher DO concentrations impact a MBBR anammox process negatively with decreased

nitrogen conversion rates as a result. Since the oxygen penetration depth within the

biofilm increases with increasing DO (Henze et al., 1997) the anaerobic layer where

anammox activity is taking part will be thinner which is causing a lower inorganic

nitrogen conversion rate.

One drawback with the anammox process is that NO3-N is produced during cell

synthesis of anammox bacteria. However this problem is not significant since the

effluent from the anammox process can be re-circulated with the influent water to the

wastewater treatment plant.

5.2 N2O production

Compared to N2O emissions from nitrogen removal processes found in literature (Table

3) the emissions from the single stage nitritation/anammox system examined in this

work can be regarded as relatively high.

During this study N2O production have been higher at intermittent aeration where the

dissolved oxygen concentration averaged around 3 mg/l in the aerated period. Lowest

N2O production recorded was during continuous aeration at DO concentrations

corresponding to 1.0-1.5 mg/l. The emissions during continuous aeration are in the

same range as emissions from a nitrifying SBR reactor (2.8%) and a Sharon reactor

(1.7%) reported by Kampschreur et al., (2008 b and a). R 2 value obtained when

comparing % N2O production of removed inorganic nitrogen with DO concentrations

shows that there is a correlation between the higher N2O emissions and DO in the, see

Figure 27.

44


% produced N₂O

12

10

8

6

4

2

Produced N₂O in

relation to DO

concetration

R² = 0.6859

0

0 1 2 3 4

DO (mg/l)

Figure 27. Correlation between the % N 2O production and DO concentration.

It has been observed that changing environmental conditions can give rise to higher N2O

emissions (Kampschreur et al., 2008, b) and the shifting oxygen conditions during

intermittent aeration can be an explanation to higher N2O emissions during this

operation mode.

If the concentration profiles registered with the biosensor during intermittent aeration

are considered it is shown that nitrite concentrations are actually increasing during the

anoxic phase which is the opposite situation to what could be expected. Since nitritation

is inhibited by low oxygen concentrations the conversion of ammonium into nitrite

should decrease and anammox activity should consume nitrite leading to a total

decrease in nitrite concentrations. High influent nitrite concentrations and the possible

presence of nitrite oxidisers are two likely explanations to increasing nitrite

concentrations during the anoxic period. Since increasing NO2-N concentrations have

been observed to give higher N2O emissions (Tallec et al., 2006,a) rising nitrite

concentrations during the anoxic period observed in this study can also be a reason for

higher emissions during intermittent operation of the MBBR.

Process performance seems to influence the extent of N2O emitted from the MBBR since

less N2O was produced when higher nutrient removal was achieved during periods of

continuous aeration. Figure 28 which shows the correlation between % N2O production

and % N-reduction indicates that process performance might influence the N2O

emissions from the system (R 2 =0.70).

45


% produced N₂O

12

10

8

6

4

2

Produced N₂O in

relation to % N-

reduction

R² = 0.7029

0

0 20 40 60 80 100

% N-reduction

Figure 28. Correlation between % N 2O production and % N-removal.

NO2-N concentrations during continuous aeration decreased as the aeration was

switched off, at the same time N2O production was not as high as during intermittent

aeration. This result can be partly explained with better control of influent nitrogen

fractions during these measurements. A different microbial composition in the MBBR

during continuous aeration or that conditions are not favouring N2O production to the

same extent as during intermittent aeration are other possible explanations to lower N2

O emissions during continuous operation of the reactor. In this study it is not possible to

determine whether better process performance was the reason or if lower N2O

production might be a result of other reasons such as different composition of the

microbial community during continuous aeration.

Increased NH4-N concentrations and decreasing NO2-N concentrations recorded during

prolonged unaerated studies showed that the nitrifying activity decreased as the MBBR

was left without oxygen supply for a longer period. N2O production within the system

ceased at the same time indicating that nitrifier denitrification of ammonium with nitrite

performed by AOB was the reason to N2O emissions. Why N2O production was not

taking part as long as there was NO2-N available for nitrifier denitrification in the water

phase is unknown. One explanation might be that the NO2-N concentration in the biofilm

was below concentrations that the bacteria can utilise.

Stripping tests of N2O and mixing with pure N2 gas during the anoxic phase indicates

that the N2O accumulation registered by the microsensor is due to the microbial activity

producing N2O and to termination in stripping N2O out of the water. It is not possible to

say if the production rate is the same during aeration and the anoxic phase.

Uncertainties and sources of errors can be many during laboratory work some are

shortly discussed here. During these experiments a synthetic wastewater was used, this

might influence the N2O production from the system, a real waste water is more complex

and might give other emission results, both higher and lower. The fact that diffusion

corresponded to 10% of produced N2O in the MBBR indicates that emissions from the

46


laboratory system might be underestimated. If calibrations have been performed with

an unsaturated N2O solution this will give rise to overestimated N2O productions from

the partial nitritation/anammox MBBR. As pointed out by Kampschreur et al., (2009)

changing environmental conditions might lead to higher N2O emissions and short term

laboratory scale measurements might therefore give over estimated N2O emissions.

Since the anammox process needs less resources and produces less CO2 than common

nitrogen removal, anammox has been pointed out as a more environmental friendly

alternative (Fux and Siegrist, 2004). Kuenen and Robertson, (1994) are calling attention

to that wastewaters in the Netherlands generally have a nitrogen content between 40-60

mg/l, each person produces about 150 l/d which gives a nitrogen production of 2.2 kg

nitrogen per person and year and that even a small N2O production corresponding to

0.1% of the nitrogen concentration will result in significant N2O emissions. Considering

that the MBBR process has been found to produce N2O corresponding to a minimum of

2% of removed inorganic nitrogen it has to be further examined whether this single

stage anammox process is more environmental friendly than common nitrogen removal

processes. Even if rather high N2O production was obtained in this study, experiences in

pilot scale trials with similar operation modes has given N2O production as low as 0.2 %

of removed inorganic nitrogen (Christensson, 2010). Additional research is needed to

determine if the N2O production from a full scale process would be as high as the

production found from the laboratory MBBR system. It also has to be determined which

bacteria that are responsible for producing N2O, whether the relatively high N2O

emissions found from the laboratory MBBR are due to biofilm structure with oxygen

pore conditions. Amounts of N2O emissions have to be further evaluated in correlation

to process operation and performance. A single stage biofilm system might not be the

best solution for the partial nitritation anammox process if this process design always

gives rise to high N2O emissions.

5.3 Measurements with NO2-N biosensor

The biosensor gave results that correlated very well with concentrations obtained with

LCK 342 at some occasions and the fluctuations in NO2-N concentration measured with

the biosensor always showed the same trends as achieved with LCK 342. However the

NO2-N biosensor did not give reliable results at all times in use and could never replace

LCK 342 for determination of NO2-N concentrations during this master thesis work.

Some measurements performed with the biosensor recorded much higher NO2-N

concentrations than obtained with the Dr. Lange kit. This was probably due to electric

disturbances that caused electric migration which is the transport of a charged body in

an electric field. Kjær et al., (1999), have shown that this phenomenon can be used to

force negatively charged NO3 − ions over the semi permeable membrane of the

biochamber increasing the ion sensitivity by a factor of more than 10.000. The electric

disturbances can have been caused by other laboratory equipment or since the ground

channel on the backside of the piccoameter was used. This ground port has another

47


electrical potential than the sensor port which can create an electric potential and

increased nitrate flux over the biochamber membrane. (There are two different

possibilities to ground the environment in the close range of the microsensors. The first

option is to use the ground channel connected to sensor port on the piccoameter, the

electric potential of the sensor and ground channel is the same. The other option is use

the ground port on the backside of the piccoameter, this port has another electric

potential than the sensor port).

Since the biosensor relies on denitrifying bacteria converting NO2-N to N2O, the sensor

was hard to work with. The bacteria in the biochamber are changing and adapting their

metabolism as their physical environment with available substrates changes (Larsen et

al., 1997). This means that their metabolism might be influenced by moving from the

environment in which they are kept in between measurements, via the calibration setup

into the MBBR where measurements are performed. At some occasions the biosensor

had to be recalibrated one to three times before giving a stable signal, which is very time

consuming. The sensor also has to be well nursed in between measurements in order to

keep the microorganisms viable.

To obtain the same salinity during calibration and measurement the biosensor was

calibrated in the synthetic wastewater feeding the MBBR. The microbial nitrogen

conversion in the MBBR is changing the ionic composition of the influent wastewater

with a difference in ionic strength of influent medium and effluent as result. Since the

biosensor is sensitive to ionic strength as well as salinity (Nielsen et al., 2004) better

results might have been obtained by calibration of the biosensor in the effluent water.

(Since the effluent water contains NO2-N this calibration method gives a background

signal of NO2-N which has to be corrected for).

The biosensor might be a good option if changes of NO2-N are going to be studied during

cyclic changes of a microbial process. However the sensitivity of the sensor and the fact

that it has to be well looked after in between measurements has to be taken into account

when considering the biosensor as an option to conventional methods of determining

the NO2-N concentrations. The biosensor and required equipment is also a significant

investment cost.

5.4 Diffusivity and stripping test of N2O

Testing the diffusivity of N2O through mechanical mixing with K1–heavy carriers

without biofilm showed that


6. Conclusions

The following conclusions concerning, process performance of the laboratory MBBR,

produced N2O and evaluation of the NO2-N biosensor can be made:

• The single stage nitritation/anammox system produced significant amounts of

N2O with a minimum production of 2% of removed inorganic nitrogen.

• Operating the MBBR at intermittent aeration with a DO of ~3 mg/l gave the

highest N2O production with initial and maximum productions of 6-11% and 10-

30% respectively.

• Smaller amounts of N2O were produced by the partial/nitritation anammox

system during continuous operation at DO in the interval 1-1.5 mg/l. The initial

N2O production was found to be 2-3% and the maximum N2O production

corresponded to 2-6%.

• When the MBBR was exposed to a longer period of anoxic conditions both

ammonium oxidation and N2O production ceased.

• From results of mixing with N2 gas during the anoxic period it cannot be said

with certainty that the N2O production is the same during aeration and anoxic

phase. The absolute number on overall N2O production for an operation mode

(based on the measurements of N2O accumulating during the anoxic phase) could

be both overestimated or underestimated and should therefore be used as a

comparative tool.

• It was not possible to replace conventional methods for determination of NO2-N

concentrations with the NO2-N biosensor since stable operation of the sensor

could not be obtained at all times.

51


7. Future research

Better understanding off which mechanisms and organisms that are responsible for N2O

production in the nitrifying/anammox MBBR system is needed. There is also a need for

better accuracy in the measurements of emitted N2O from the process.

• Measurement should be done where N2O is measured both in the water phase

and in the off-gas simultaneously, this would both help to better understand

when the N2O is produced in the system and it would give a much better accuracy

of how much N2O that is produced and emitted by the MBBR system.

• Since the biofilm creates a microenvironment with anoxic conditions which are

believed to enhance the N2O production by AOB the importance of biofilm

structure and thickness should be investigated.

• Disturbances are believed to cause higher N2O production from the

microorganisms. It should be examined whether intermittent aeration could be

considered a disturbance to the bacteria performing the nitrogen removal

causing higher N2O emissions from the process.

• To be able to operate the MBBR in a manner that gives as small amounts of

emitted N2O as possible it is of great importance to understand which

microorganisms within the system that are responsible for the N2O emissions.

Measurements of the N2O production during batch tests with inhibitors should be

performed to gain this kind of knowledge.

• Since substrate concentrations (NH4 + , NO2 − and NO3 − ) are known to influence the

amount of produced N2O, it would be interesting to evaluate their impact on

emitted N2O in both batch tests and with operation at different influent inorganic

nitrogen concentrations. Performing the tests in this manner could give answers

to whether it is the increased nitrogen concentrations /disturbance that causes

the increase in N2O production or the actual higher substrate concentration.

• As volume to surface ratios are of importance to emitted N2O from a wastewater

treatment process and since there are further differences between full scale and

laboratory systems the emitted N2O from full scale systems should be

determined.

• Measurement from a process operated with real wastewater is needed for

determination of N2O emissions during real conditions.

• Examine the influence of aeration rate on N2O emission by continuous aeration

with pure oxygen, (a much lower aeration rate can maintain a sufficient oxygen

concentration in the reactor if pure oxygen is used.)

53


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Strous M., Heijnen J., Kuenen J., Jetten M., (1998). The sequencing batch reactor as a

powerful tool for the study of slowly growing anaerobic ammonium-oxidizing

microorganisms. Applied Microbiology Biotechnology, 50, 589-596.

Strous M, F. J., Kramer E.H.M., Logemann S., Muyzer G., van de Pas-Schoonen K.T., Webb

R., Kuenen J.G., Jetten M.S.M., (1999). "Missing lithotroph identified as new planctomycete"

Nature, 400, (6743), 446-449.

59


Szatkowska B., Plaza E., Trela J., Bakowska A., (2003). Influence of dissolved oxygen

concentration on deammonification process performance. Integration and optimisation of

urban sanitation systems, Joint Polish-Swedish Reports, NO 11. Royal Institute of

Technology, Stockholm.

Tallec G., Garnier J., Billen G., Gousailles M., (2006 (a)). Nitrous oxide emission from

secondary activated sludge in nitrifying conditions of urban waste water treatment plants:

Effect of oxygenation level. Water Research, 40, 2972-2980.

Tallec G., Garnier J., Gousailles M.,(2006 (b)). Nitrogen removal in a waste water

treatment plant through biofilters: nitrous oxide emission during nitrification and

denitrification. Bioprocess Biosystem Engineering, 29, 323-333.

Tallec G., Garnier J., Billen G., Gousailles M., (2008). Nitrous oxide emissions from

denitrifying activated sludge of urban waste water treatment plants under anoxia and low

oxygenation. Bioresource Technology, 99, 2200-2209.

Third K.A., Sliekers A.O, Kuenen J.G., Jetten M.S.M., (2001). The CANON System

(Completely Autotrophic Nitrogen-removal Over Nitrite) under Ammonium Limitation:

Interaction and Competition between Three Groups of Bacteria. Systematic and Applied

Microbiology, 24, 588-596.

Third K.A., Paxman J., Schmid M., Strous M., Jetten M.S.M., Cord-Ruwisch R., (2005).

Treatment of nitrogen-rich wastewater using partial nitrification and Anammox in the

CANON process. Water Science and Technology, 52, (4), 47-54.

Trela J., Plaza E., Gut L., Szatkowska B., Hultman B., Bosander J., (2005).

Deammonifikation, en ny process för behandling av avloppsströmmar med hög kvävehalt

– fortsatta pilot-plant experiment. Rapport NR 2005-14, Svensk Vatten Utveckling.

Tsushima I., Ogasawara Y., Kindaichi T., Satoh H., Okabe S., (2007). Development of highrate

anaerobic ammonium-oxidizing (anammox) biofilm reactors. Water Research 41,

1623-1634.

Unisense, b (2007). Nitrous oxide sensor manual, version 20070912. Unisense A/S.

Unisense, c (2007). NOx- biosensor manual, version 20071001. Unisense A/S.

van Dongen U., Jetten M.S.M., van Loosdrecht M.C.M., (2001). The SHARON® -Anammox®

process for treatment of ammonium rich wastewater. Water Science and Technology,

44,(1), 153-160.

60


van Niftrik L.A., Fuerst J.A., Sinninghe Damsté J.S., Kuenen J.G., Jetten M.S.M., Strous M.,

(2004). The anammoxosome an intracytoplasmic compartement in anammox bacteria.

FEMS Microbiology Letters, 233 (1), p. 7-13.

Warfvinge P., (2008). Mass balances and reactor design- Study book for environmental

engineering. Department of Chemical Engineering, Box 124, Lund.

Ødegaard H., (ed.), (1993). Nitrifying and denitrifying biofilms for wastewater treatment.

Nordic Collaborative Project on Environmental Biotechnology. Nordic Council of

Ministers.

Ødegaard H., Rusten B., Westrum T., (1994). A new moving biofilm reactor – applications

and results. Water Science and Technology, 29( 10-11), 157-165.

Internet källor:

AnoxKaldnes /Produkter och tjänster/Bioprocesser/MBBR TM (online). Available

at:


Appendix A

Calculation of concentrations in calibration solutions for N2O and NO2-

N microsensors

During calibration of the microsensors solutions with known concentration of N2O and

NO2-N are used from the start. The volume in the calibration chamber is known and the

final concentration for each calibration step is also known. The initial volume that has to

be added to get the correct concentration in the calibration solution is obtained with:



(A.1)

where:

Mi = the initial molar concentration mol/l, Vi = the initial volume (l), Mf = the final molar

concentration mol/l and Vf = the final volume (l).

The initial volume is then added in each calibration step until the final concentration is

reached.

Equilibrium nitrous oxide concentrations at different temperatures and salinities are

obtained from the nitrous oxide sensor users manual. The saturated water solution is

prepared from distilled water at 20 °C corresponding to an equilibrium N2O

concentration of 27.05mmol/l or ~1.2 g/l. 2 µM (88 µg/l )N2O was added in each step to

perform a five point calibration up to 10 µM, (440 µg/l ). See Table A1 for calculated

initial volume, (Vi), of saturated N2O solution added to the calibration chamber, Mi, Mf,

and Vf are also given in the table.

A stock solution with a NO2-N concentration of 5 g/l NO2-N was used for calibration of

the biosensor. 2 mg/l NO2-N was added in each step to perform a five point calibration

up to 10 mg/l NO2-N. See Table A1 for calculated initial volume of NO2-N stock solution

added to the calibration chamber, Mi, Mf, and Vf are also given in the table.

Table A1. Parameters used to calculate the volume of concentrated N 2O and NO 2-N solutions that

has to be added during the calibration procedure of the sensors, calculated values for V i is also

shown.

N2O calibration solution NO2-N calibration solution

Mi 27.05∙10 -3 mol/l Mi 5∙10 -3 g/l

Mf 2∙10 -6 mol/l Mf 2∙10 -3 g/l

Vf 0.300 L Vf 0.300 l

Vi 22∙10 -6 L Vi 120∙10 -6 l

22 µl 120 µl

63


Appendix B

Calculations of N2O emissions

The purpose is to calculate the produced amount of nitrous oxide as percentage of

removed inorganic nitrogen.

It is assumed that the MBBR is behaving like an ideal completely stirred tank reactor,

(CSTR), and that the general mass balance equation for a given component can be

implied:


eq.(3.1)

The in and output terms are molar fluxes over the reactor boundary, acquired as the

product of the volumetric flow rates, Q (m/s) and the concentrations, c (mole/l).

Production within the system is described by the kinetic rate equation, r (mole/m 3 s)

times the reactor volume, V (m 3 ), (negative sign indicating consumption instead of

production). Accumulation is quantified by the molar change of a substance per unit

time, described by a time dependent differential including the concentration, c (mol/l)

and the reactor volume, V (m 3 ). The mass balance equation for a component j can be

rewritten as:

( )

mol/s

eq.(3.2)


For a reacting system like the MBBR where some substances are consumed and others

are produced various kinds of substances will be passing the system borders in the

influent, effluent and through the gas phase, see Figure B1.

Figure B1. Mass transfer over the MBBR system boundaries.

67


With the considerations; (i) that there is no N2O gas in the influent medium, (ii) the

reactor volume is constant and (iii) Qin and Qout are equal the mass balance for the

system can be described by:

eq.(3.3)

: ( )


eq.(3.4)

: 0 ( )


To get the consumption and production rates equation 3 and 4 are rewritten:


eq.(3.5)

:

( )


eq.(3.6)

:

( )


rN in the first equation is describing the consumption rate of the influent nitrogen, if

there were no N2O or any other gaseous production in the system this term would

entirely correspond to the production of N2 gas leaving the system. Here it is assumed

that all removed inorganic nitrogen is leaving the system in gaseous form as N2 or N2O.

The accumulation term that would correspond to assimilation in equation 3 is neglected.

Calculation example (091007)

Used parameters to calculate the consumption (rN) and production (rN2O ) rates are

shown in Table B1.

Table B1. Calculation parameters used to caluclate r N and r N2O.

Parameter value unit

Q 0.540 l /h

V 7.5 l

cinN 273.904∙10 -3 g/ l

coutN 97.593∙10 -3 g/l

c N2O t1 2.82414∙10 -6 mol/l

c N2O t2 2.97601∙10 -6 mol/l



0.1519∙10 -6 mol/lmin

0.540 · (273.904 · 10 97.593 · 10 )

0.2116 · 10

7.5 · 60

0.540 · (2.97601 · 10 )

0.1519 · 10 0.1555 · 10

7.5 · 60

g/lmin

mol/lmin

68


The rN value is calculated with the mean in and effluent concentrations during one cycle/

measurement session. For the approximation of the total N2O production in the MBBR it

is assumed that the production corresponds to the initial value of rN2O seen when

aeration is turned off (Figure B2) and that this assumption is valid at all times.

12

N₂O (µmol/l),DO (mg/l)

10

8

6

4

2

N₂O

(µmol/l)

DO

(mg/l)

N₂O (µmol/l), DO (mg/l)

0

4.5

4

3.5

3

2.5

2

1.5

1

0.5

0

0 20 40 60 80

Time (min)

Initial N₂O production

2.82414

2.97601

20 22 24 26 28 30

N₂O

(µmol/l)

DO

(mg/l)

y = 0.1519x - 0.8207

Time (min)

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

of the N 2O curve when N 2O starts to accumulate in the reactor. The lower part of the figure shows

an enlargement of the area.

The percentage of removed inorganic nitrogen emitted as N2O is finally obtained by:

· 2


· 100

where rN2O given in mole N2O/l min is converted to g N/l min by multiplying with 2MN,

the molar weight of N in g/mole. MN is multiplied by 2 since the molar ratio for produced

N2O-N to removed inorganic N is 2:1.

69


Table B2. Calculation parameters used to calculate the percentage N 2O-N produced by removed

inorganic N.

Parameter value

unit

rN2O 0.1555∙10 -6 mole/l min

MN 14.01 g/mol

0.2116 · 10 mg/l

rN

0.1555 · 10 · 2 · 14.01

0.2116 · 10 · 100 2.1%

Assumptions made for this calculation are; (i) that the microorganisms in the system are

unaffected of the changed operation conditions during the time span of one minute

when the production rate is approximated, (ii)that the mass transfer of N2O through the

phase boundary between liquid and air is negligible during the time interval of one

minute, (iii) that there is no net change in production due to operating conditions during

one cycle.

70


Appendix C

Microsensor measurements

12

090918 Intermittent aeration, DO 3.0 (mg/l)

4.5

12

090921 Intermittent aeration, DO 3.0 (mg/l)

4.5

10

3.75

10

3.75

N₂O (µmol/l)

8

6

4

3

2.25

1.5

DO (mg/l)

N₂O sensor

(µmol/l)

DO (mg/l)

NO₂-N (mg/l)

8

6

4

3

2.25

1.5

DO (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

2

0.75

2

0.75

0

0

0 20 40 60 80

Time (min)

0

0

0 20 40 60 80

Time (min)

Figure C1.

Figure C4.

30

090918 Intermittent aeration, DO 3.0 (mg/l)

4.5

12

090922 Intermittent aeration, DO 3.0 (mg/l)

4.5

25

3.75

10

3.75

NO₂-N (mg/l)

20

15

10

5

3

2.25

1.5

0.75

DO (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 20 40 60 80

Time (min)

0

0

0 20 40 60 80

Time (min)

Figure C2.

Figure C5.

12

10

090921 Intermittent aeration, DO 3.0 (mg/l)

4.5

3.75

40

090922 Intermittent aeration, DO 3.0 (mg/l)

4

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O sensor

(µmol/l)

DO (mg/l)

NO₂-N (mg/l)

30

20

10

3

2

1

DO (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

0

0

0 20 40 60 80

Time (min)

0

0

0 20 40 60 80

Time (min)

Figure C3.

Figure C6.

71


12

090925 Intermittent aeration, DO 3.0 (mg/l)

4.5

12

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

4.5

10

3.75

10

3.75

N₂O (µmol/l)

8

6

4

3

2.25

1.5

DO (mg/l)

N₂O sensor

(µmol/l)

DO (mg/l)

NO₂-N (mg/l)

8

6

4

3

2.25

1.5

DO (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

2

0.75

2

0.75

0

0

0 20 40 60 80

Time (min)

0

0

0 50 100 150 200

Time (min)

Figure C7.

Figure C10.

12

090925 Intermittent aeration, DO 3.0 (mg/l)

4.5

12

090926 Prolonged unaerated period, DO 3.0 (mg/l)

4.5

10

3.75

10

3.75

NO₂-N (mg/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 20 40 60 80

Time (min)

0

0

0 50 100 150 200

Time (min)

Figure C8.

Figure C11.

12

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

4.5

12

090926 Prolonged unaerated period, DO 3.0 (mg/l)

10

3.75

10

3.75

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O sensor

(µmol/l)

DO (mg/l)

NO₂-N (mg/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

NO₂-N (mg/l)

DO (mg/l)

0

0

0 50 100 150 200

Time (min)

0

0

0 50 100 150 200

Time (min)

Figure C9.

Figure C12.

72


12

090927 Prolonged unaerated period, DO 3.0 (mg/l)

4.5

12

091006 Continously operation, DO 1.5 (mg/l)

10

3.75

10

3.75

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO (mg/l)

NO₂-N (mg/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

NO₂-N (mg/l)

DO (mg/l)

0

0

0 50 100 150 200

Time (min)

0

0

0 20 40 60 80

Time (min)

Figure C13.

Figure C16.

12

090927 Prolonged unaerated period, DO 3.0 (mg/l)

4.5

12

091007 Continously operation, DO 1.5 (mg/l)

4.5

10

3.75

10

3.75

NO₂-N (mg/l)

8

6

4

3

2.25

1.5

DO (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

N₂O (µmol/l)

8

6

4

3

2.25

1.5

DO (mg/l)

N₂O

(µmol/l)

DO (mg/l)

2

0.75

2

0.75

0

0

0 50 100 150 200

Time (min)

0

0

0 20 40 60 80

Time (min)

Figure C14.

Figure C17.

12

091006 Continously operation, DO 1.5 (mg/l)

4.5

12

091007 Continously operation, DO 1.5 (mg/l)

10

3.75

10

3.75

N₂O (µmol/l)

8

6

4

3

2.25

1.5

DO (mg/l)

N₂O

(µmol/l)

DO (mg/l)

NO₂-N (mg/l)

8

6

4

3

2.25

1.5

DO (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

2

0.75

2

0.75

0

0

0 20 40 60 80

Time (min)

0

0

0 20 40 60 80

Time (min)

Figure C15.

Figure C18.

73


12

091010 Continously operation, DO 1.5 (mg/l)

4.5

12

091014 Continously operation, DO 1.0 (mg/l)

4.5

10

3.75

10

3.75

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO (mg/l)

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 20 40 60 80

Time (min)

0

0

0 20 40 60 80

Time (min)

Figure C19.

Figure C22.

12

091010 Continously operation, DO 1.5 (mg/l)

12

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

N2 gas.

4.5

10

3.75

10

3.75

NO₂-N (mg/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

NO₂-N (mg/l)

DO (mg/l)

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 20 40 60 80

Time (min)

0

0 20 40 60 80

Time (min)

0

Figure C20.

Figure C23.

12

091013 Continously operation, DO 1.0 (mg/l)

4.5

12

091016 Continuously operation, DO 1.0 (mg/l), aeration with

N2 gas.

4.5

10

3.75

10

3.75

N₂O (µmol/l)

8

6

4

3

2.25

1.5

DO (mg/l)

N₂O

(µmol/l)

DO (mg/l)

N₂O (µmol/l)

8

6

4

3

2.25

1.5

DO (mg/l)

N₂O

(µmol/l)

DO (mg/l)

2

0.75

2

0.75

0

0

0 20 40 60 80

Time (min)

0

0 20 40 60 80

Time (min)

0

Figure C 21

Figure C24.

74


12

091016 Continuously operation, DO 1.0 (mg/l), aeration with

N2 gas.

4.5

12

091018 Continuously operation, DO 1.5 (mg/l), aeration with

N2 gas.

10

3.75

10

3.75

NO₂-N (mg/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0 20 40 60 80

Time (min)

0

0

0 20 40 60 80

Time (min)

0

Figure C25.

Figure C 28

12

091017 Continuously operation, DO 1.5 (mg/l), aeration with

N2 gas.

4.5

12

091018 Continuously operation, DO 1.5 (mg/l), aeration with

N2 gas.

10

3.75

10

3.75

N₂O (µmol/l)

8

6

4

3

2.25

1.5

DO (mg/l)

N₂O

(µmol/l)

DO (mg/l)

N₂O (µmol/l)

8

6

4

3

2.25

1.5

DO (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

2

0.75

2

0.75

0

0 20 40 60 80

Time (min)

0

0

0 20 40 60 80

Time (min)

0

Figure C26.

Figure C29.

12

091017 Continuously operation, DO 1.5 (mg/l), aeration with

N2 gas.

N₂O (µmol/l)

10

8

6

4

2

3.75

3

2.25

1.5

0.75

DO (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

0

0 20 40 60 80

Time (min)

0

Figure C27.

75


Appendix D

Nitrogen grab samples

090913 NH₄-N

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

NH₄-N (mg/l)

300

250

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

DO, NO₂-N (mg/l)

12

10

8

6

4

2

NO₂-N out

(mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

0

0

0 20 40 60

0 20 40 60

Time (min)

Time (min)

Figure D1.

Figure D4.

090913 NO₃-N

090918 NH₄-N

40

350

NO₃-N (mg/l)

35

30

25

20

15

10

5

0

NO₃-N in

(mg/l)

NO₃-N out

(mg/l)

NH₄-N (mg/l)

300

250

200

150

100

50

0

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

0 20 40 60

0 20 40 60

Time (min)

Time (min)

Figure D2.

Figure D5.

090913 NO₂-N

090918 NOx-N

NO₂-N (mg/l)

30

25

20

15

10

5

0

NO₂-N in

(mg/l)

NO₂-N out

(mg/l)

NOx-N (mg/l)

50

45

40

35

30

25

20

15

10

5

0

NOx-N in

NOx-N ut

0 20 40 60

0 20 40 60

Time (min)

Time (min)

Figure D3.

Figure D6.

77


350

090921 NH₄-N

350

090922 NH₄-N

300

300

NH₄-N (mg/l)

250

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

NH₄-N (mg/l)

250

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

0

0 20 40 60

0

0.000 20.000 40.000 60.000

Time (min)

Time (min)

Figure D7.

Figure D10.

70

090921 NOx-N

70

090922 NOx-N

60

60

NOx-N (mg/l)

50

40

30

20

10

NOx-N in

(mg/l)

NOx-N out

(mg/l)

NOx-N (mg/l)

50

40

30

20

10

NOx-N in

(mg/l)

NOx-N in

(mg/l)

0

0 20 40 60

0

0 20 40 60

Time (min)

Time (min)

Figure D8.

Figure D11.

DO, NO₂-N (mg/l)

9.0

8.0

7.0

6.0

5.0

4.0

3.0

2.0

1.0

0.0

090921 DO, NO₂-N biosensor, NO₂-N LCK 342

0 20 40 60

Time (min)

Figure D9.

NO₂-N

biosensor

(mg/l)

DO (mg/l)

DO, NO₂-N (mg/l)

45.0

40.0

35.0

30.0

25.0

20.0

15.0

10.0

5.0

0.0

Figure 29

090922 DO, NO₂-N biosensor, NO₂-N LCK 342

0 20 40 60

Time (min)

Figure D12.

NO₂-N

biosensor

(mg/l)

DO (mg/l)

78


090925 NH₄-N

090925 Prolonged unaerated period NH₄-N

300

250

250

200

NH₄-N (mg/l)

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

NH₄-N (mg/l)

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

0

0

0 20 40 60

0 50 100 150 200

Time (min)

Time (min)

Figure D13.

Figure D16.

090925 NOx-N

090925 Prolonged unaerated period NOx-N

70

70

60

60

NOx-N (mg/l)

50

40

30

20

10

NOx-N in

(mg/l)

NOx-N out

(mg/l)

NOx-N (mg/l)

50

40

30

20

10

NOx-N in

(mg/l)

NOx-N out

(mg/l)

0

0

0 20 40 60

0 50 100 150 200

Time (min)

Time (min)

Figure D14.

Figure D17.

DO, NO₂-N (mg/l)

10

9

8

7

6

5

4

3

2

1

0

090925 DO, NO₂-N biosensor (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

DO, NO₂-N (mg/l)

8

7

6

5

4

3

2

1

0

090925 Prolonged unaerated period DO, NO₂-N

biosensor (mg/l)

NO₂-N

biosensor

(mg/l)

DO (mg/l)

0 20 40 60

0 50 100 150 200

Time (min)

Time (min)

Figure D15.

Figure D18.

79


300

090926 Prolonged unaerated period NH₄-N

60

090927 Prolonged unaerated period NOx-N

250

50

NH₄-N (mg/l)

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

NOx-N (mg/l)

40

30

20

10

NOx-N in

(mg/l)

NOx-N out

(mg/l)

0

0 50 100 150 200

0

0.00 50.00 100.00 150.00 200.00

Time (min)

Time (min)

Figure D19.

Figure D22.

NOx-N (mg/l)

50

45

40

35

30

25

20

15

10

5

0

090926 Prolonged unaerated period NOx-N

0 50 100 150 200

Time (min)

Figure D20.

NOx-N in

(mg/l)

NOx-N out

(mg/l)

NOx-N (mg/l)

60

50

40

30

20

10

0

090927 Prolonged unaerated period NOx-N

0.00 50.00 100.00 150.00 200.00

Time (min)

Figure D23.

NOx-N in

(mg/l)

NOx-N out

(mg/l)

DO, NO₂-N (mg/l)

9

8

7

6

5

4

3

2

1

0

090926 Prolonged unaerated period DO, NO₂-N

biosensor (mg/l)

0 50 100 150 200

Time (min)

Figure D21.

NO₂-N

biosensor

(mg/l)

DO (mg/l)

DO, NO₂-N (mg/l)

090927 Prolonged unaerated period DO, NO₂-N

biosensor (mg/l), NO₂-N LCK 342

10

8

6

4

2

0

0.00 50.00 100.00 150.00 200.00

Time (min)

Figure D24.

NO₂-N

biosensor

(mg/l)

80


NH₄-N (mg/l)

300

250

200

150

100

50

0

091006 NH₄-N

0 20 40 60

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

NOx-N (mg/l)

45

40

35

30

25

20

15

10

5

0

091007 NOx-N

0 20 40 60

NOx-N in

(mg/l)

NOx-N out

(mg/l)

Time (min)

Time (min)

Figure D25.

Figure D28.

DO, NO₂-N (mg/l)

6

5

4

3

2

1

0

091006 DO, NO₂-N biosensor (mg/l)

0 20 40 60

NO₂-N

biosensor

(mg/l)

DO (mg/l)

DO, NO₂-N (mg/l)

7

6

5

4

3

2

1

0

091007 DO, NO₂-N biosensor (mg/l)

0 20 40 60

NO₂

biosensor

(mg/l)

DO (mg/l)

NO₂-N LCK

342 out

(mg/l)

Time (min)

Time (min)

Figure D26.

Figure D29.

300

091007 NH₄-N

300

091010 NH₄-N

250

250

NH₄-N (mg/l)

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

NH₄-N (mg/l)

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

0

0 20 40 60

0

0 20 40 60

Time (min)

Time (min)

Figure D27.

Figure D30.

81


091010 NOx-N

091013 NOx-N

NOx-N (mg/l)

50

45

40

35

30

25

20

15

10

5

0

NOx-N in

(mg/l)

NOx-N out

(mg/l)

NOx-N (mg/l)

40

35

30

25

20

15

10

5

0

NOx-N in

(mg/l)

NOx-N out

(mg/l)

0 20 40 60

0 20 40 60

Time (min)

Time (min)

Figure D31.

Figure D34.

091010 DO, NO₂-N biosensor (mg/l)

091013 NO₂-N

DO, NO₂-N (mg/l)

7

6

5

4

3

2

1

0

NO₂-N

biosensor

(mg/l)

DO (mg/l)

NO₂-N LCK

342 out

(mg/l)

NOx-N (mg/l)

5

4

4

3

3

2

2

1

1

0

NO₂-N in

(mg/l)

NO₂-N out

(mg/l)

0 20 40 60

0 20 40 60

Time (min)

Time (min)

Figure D32.

Figure D35.

091013 NH₄-N

091013 NO₃-N

300

35

250

30

NH₄-N (mg/l)

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

NOx-N (mg/l)

25

20

15

10

5

NO₃-N in

(mg/l)

NO₃-N out

(mg/l)

0

0

0 20 40 60

0 20 40 60

Time (min)

Time (min)

Figure D33.

Figure D36.

82


300

091014 NH₄-N

30

091014 NO₃-N

250

25

NH₄-N (mg/l)

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

NOx-N (mg/l)

20

15

10

5

NO₃-N in

(mg/l)

NO₃-N out

(mg/l)

0

0 20 40 60

0

0 20 40 60

Time (min)

Time (min)

Figure D37.

Figure D40.

NOx-N (mg/l)

35

30

25

20

15

10

5

0

091014 NOx-N

0 20 40 60

NOx-N in

(mg/l)

NOx-N out

(mg/l)

NH₄-N (mg/l)

350

300

250

200

150

100

50

0

091015 NH₄-N

0 20 40 60

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

Time (min)

Time (min)

Figure D38.

Figure D41.

NOx-N (mg/l)

5

4

4

3

3

2

2

1

1

0

091014 NO₂-N

0 20 40 60

NO₂-N in

(mg/l)

NO₂-N out

(mg/l)

NOx-N (mg/l)

70

60

50

40

30

20

10

0

091015 NOx-N

0 20 40 60

NOx-N in

(mg/l)

NOx-N out

(mg/l)

Time (min)

Time (min)

Figure D39.

Figure D42.

83


35

091015 NO₃-N

70

091016 NOx-N

30

60

NOx-N (mg/l)

25

20

15

10

5

NO₃-N in

(mg/l)

NO₃-N out

(mg/l)

NOx-N (mg/l)

50

40

30

20

10

NOx-N in

(mg/l)

NOx-N out

(mg/l)

0

0 20 40 60

0

0 20 40 60

Time (min)

Time (min)

Figure D43.

Figure D46.

NOx-N (mg/l)

12

10

8

6

4

2

0

091015 NO₂-N

0 20 40 60

NO₂-N in

(mg/l)

NO₂-N out

(mg/l)

DO, NO₂-N (mg/l)

12

10

8

6

4

2

0

091016 DO, NO₂-N biosensor (mg/l),

0 20 40 60

NO₂-N

biosensor

(mg/l)

DO (mg/l)

NO₂-N out

LCK 342

(mg/l)

Time (min)

Time (min)

Figure D44.

Figure D47.

350

091016 NH₄-N

35

091016 NO₃-N

300

30

NH₄-N (mg/l)

250

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

NOx-N (mg/l)

25

20

15

10

5

NO₃-N in

(mg/l)

NO₃-N out

(mg/l)

0

0 20 40 60

0

0 20 40 60

Time (min)

Time (min)

Figure D45.

Figure D48.

84


NOx-N (mg/l)

12

10

8

6

4

2

0

091016 NO₂-N

0 20 40 60

NO₂-N in

(mg/l)

NO₂-N out

(mg/l)

DO, NO₂-N (mg/l)

12

10

8

6

4

2

0

091017 DO, NO₂-N biosensor (mg/l),

0 20 40 60

NO₂-N

biosensor

(mg/l)

DO (mg/l)

NO₂-N out

(mg/l)

Time (min)

Time (min)

Figure D49.

Figure D52.

350

091017 NH₄-N

35

091017 NO₃-N

300

30

NH₄-N (mg/l)

250

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

NOx-N (mg/l)

25

20

15

10

5

NO₃-N in

(mg/l)

NO₃-N out

(mg/l)

0

0 20 40 60

0

0 20 40 60

Time (min)

Time (min)

Figure D50.

Figure D53.

70

091017 NOx-N

12

091017 NO₂-N

NOx-N (mg/l)

60

50

40

30

20

10

NOx-N in

(mg/l)

NOx-N out

(mg/l)

NOx-N (mg/l)

10

8

6

4

2

NO₂-N in

(mg/l)

NO₂-N out

(mg/l)

0

0 20 40 60

0

0 20 40 60

Time (min)

Time (min)

Figure D51.

Figure D 54

85


350

091018 NH₄-N

35

091018NO₃-N

300

30

NH₄-N (mg/l)

250

200

150

100

50

NH₄-N in

(mg/l)

NH₄-N out

(mg/l)

NOx-N (mg/l)

25

20

15

10

5

NO₃-N in

(mg/l)

NO₃-N out

(mg/l)

0

0 20 40 60

0

0 20 40 60

Time (min)

Time (min)

Figure D55.

Figure D58.

70

091018 NOx-N

12

091018 NO₂-N

NOx-N (mg/l)

60

50

40

30

20

10

NOx-N in

(mg/l)

NOx-N out

(mg/l)

NOx-N (mg/l)

10

8

6

4

2

NO₂-N in

(mg/l)

NO₂-N out

(mg/l)

0

0 20 40 60

0

0 20 40 60

Time (min)

Time (min)

Figure D56.

Figure D59.

091018 DO, NO₂-N biosensor (mg/l),

DO, NO₂-N (mg/l)

12

10

8

6

4

2

0

0 20 40 60

NO₂-N

biosensor

(mg/l)

DO (mg/l)

NO₂-N out

(mg/l)

Time (min)

Figure D57.

86


Appendix E Scientific Article

N2O production in a single stage nitritation/anammox MBBR process.

Sara Ekström

Water and Environmental Engineering Department of Chemical Engineering, Lund

University, Sweden.

Abstract. The nitrous oxide (N 2O) production from a laboratory nitritation/anammox MBBR reactor was

determined from N 2O measurements in the water phase with a Clark-type microsensor. The reactor was

operated at intermittent and continuous aeration to evaluate which operation mode that gives the highest

N 2O production. Different aeration rates were used during continuous operation to examine the influence

of dissolve oxygen (DO) on N 2O emissions. Measurements of N 2O production during prolonged unaerated

periods were performed to examine possible mechanisms of the N 2O production. The MBBR produces 6-

11% of removed inorganic nitrogen as N 2O during intermittent operation, whereas only 2-3% was

produced during continuous operation at low oxygen concentrations. Higher inorganic nitrogen removal

was achieved during continuous operation and better process performance is thought to be one

explanation of lower N 2O emissions during continuous operations of the laboratory MBBR.

Introduction

Nitrous oxide, a greenhouse gas with a global warming potential 320 times stronger

than that of CO2, is known to be produced during nitrification and denitrification

processes used to remove nitrogen from wastewaters (Jacob, 1999). Variable

temperature and loading rates of inorganic nitrogen compounds, low pH, alternating

aerobic and anaerobic conditions together with growth rate and microbial composition

are parameters that have great influence on N2O emissions from a wastewater treatment

plant (Kampschreur et al., 2008).

Wastewater treatment plants using biologic treatment processes for nutrient removal

are producing excessive sludge giving rise to ammonium rich effluent from the

anaerobic sludge digestion. This internal wastewater stream is recombined with the

influent of the treatment plant and corresponds to 15-20% of the total nitrogen load of

the wastewater treatment plant (Fux et al., 2003). In the early 1990s a new biological

treatment process for nitrogen removal through anaerobic ammonium oxidation

(anammox) with nitrite as electron acceptor was discovered by research teams in

Holland, Germany and Switzerland (Mulder et al., 1995, Hippen et al., 1997, Siegrist et

al., 1998). Total stoichiometry of the anammox process has been estimated by Strous et

al., (1998):

1NH


1.32NO 0.066HCO


0.13H

1.02 N 0.26NO


0.066CH2O . N . 2.03 H O.

Anammox has turned out to be suitable for treatment of reject waters and other

problematic wastewaters with a low COD/N ratio and high ammonium concentrations.

87


The bacteria performing the microbial conversion of nitrite into dinitrogen gas are strict

anaerobe autotrophs and the process has the potential to replace conventional

nitrification/denitrification of recirculated high strength ammonium streams within the

wastewater treatment plant (Strous et al., 1997). No additional carbon source is needed,

the oxygen demand is reduced by 50-60% in the nitrifying step and the aeration can

thereby be strongly reduced (Jetten et al., 2001, Fux et al., 2002). This means that the

process offers an opportunity to decrease the carbon footprint of the wastewater

treatment plant in terms of saving possibilities of both additional carbon source and

power consumption (Jetten et al., 2004). Further advantages with the anammox process

is that the production of surplus sludge is minimized and that high volumetric loading

rates can be obtained resulting in reduced operational and investment costs (Abma et

al., 2007). Indications that the process may produce significant amounts of N2O gas with

negative environmental impacts detracting the process advantages. The aim of this

study was to determine the amount of N2O produced in a nitritation/ anammox MBBR

process during different operation modes.

Materials and methods

MBBR system

A 7.5 litre laboratory MBBR (see Figure 1) fed with a synthetic medium was used to

determine the N2O emissions from a single stage nitritation/anammox system. The

reactor was originally started up in October 2008 with a carrier material with already

established biofilm taken from Himmerfjärdsverkets full scale DeAmmon ® reactor. The

used carrier was AnoxKaldnes carrier media type K1 with a protected surface area of

500 m 2 /m 3 . The total volume of carriers in the reactor was 3.5 litres which corresponds

to a total protected area of 1.7 m 2 and a filling degree of 46.7%.

Figure 1. The left part of the figure shows a photograph of the MBBR system, the schematic

drawing to the right shows the main features of the MBBR system.

88


Cycle studies

To examine what operation conditions that seem to produce the largest amounts of N2O

gas, the reactor was operated at different DO concentrations during intermittent and

constant aeration. A study where the anoxic phase was prolonged to two hours was

made to observe how the N2O production was influenced. Parameters monitored every

minute on-line in the reactor were; DO, pH, N2O and NO2-N. To examine the

concentration changes of NH4-N, NO2-N and NO3-N grab samples were taken in both

influent and effluent water.

Analytical methods

N2O concentrations were measured in the water phase with a Clark-type microelectrode

sensor developed by Unisense, Århus, Denmark.

Concentrations of NH4-N, NO2-N and NO3-N were determined with Dr Lange

spectrophotometry kit after filtration through Munktel 1.6 µm glass fibre filters. During

cycle studies NO2-N and N-tot were analyzed directly with Dr Lange’s method. Samples

were frozen and flow-injection analysis was used to determine NH4-N and NOx, the sum

of NO2-N and NO3-N. The NO3-N content was calculated by subtraction of NO2-N from the

sum of the two NOx species.

Dissolved oxygen and pH was measured with a portable meter HQ40d with mounted

oxygen and pH probe. Parameters analysed and method used are summarised in Table

15.

Table 15 Analysed parameters and methods.

Analysed parameter Method

N2O

Unisense N2O microsensor

NH4-N

LCK 303/FIA

NO2-N

LCK 342/341/biosensor

NO3-N LCK 339

NOx

FIA

N-tot LCK 238

DO

HQ40d

pH

HQ40d

Results and discussion

Intermittent aeration

The reactor was operated at a DO concentration of ~3 mg/l during the aeration phase.

One reactor cycle lasted for one hour with 40 minutes of aeration and 20 minutes of

mechanical mixing. Grab samples in the effluent were taken every 6 th minute. Only three

measurements of the influent medium was taken in one cycle ( 0, 36 and 66 minutes)

since it was considered that the variation of the influent medium during one hour should

not be significant.

89


Typical profiles of how N2O and DO changes during the cycle are shown in Figure . The

N2O concentration measured in the water phase varies with the aeration of the MBBR.

When aeration starts at the beginning of the cycle the airflow strips N2O out of the water

phase and the concentration decreases to a constant minimum level. As soon as the

aeration is shut of the N2O starts to accumulate in the water phase until aeration is

switched on again and the procedure starts over as shown in Figure 2.

N₂O (µmol/l)

12

10

8

6

4

2

0

4.5

3.75

2.25

1.5

0.75

0

0 20 40 60 80

Time (min)

3

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

Figure 2. Concentration profiles of N 2O and O 2, reactor is operated with intermittent aeration at a

DO concentration of~3mg/l in the aerated phase. The cycle study starts at the beginning of the

aerated period. N 2O gas is stripped from the water phase at the same time as the oxygen

concentration rises.

Initial N2O production varies between 6-11% of influent nitrogen concentration that is

converted into dinitrogen gas (here after referred to as removed inorganic N-

concentration), while the maximum production ranges from 11-30% of removed

inorganic nitrogen.

Prolonged study, intermittent aeration

A study of the effect of prolonged, intermittent aeration was made in order to observe

how the N2O production was influenced by a longer anoxic period, results are shown in

Figure 3. The reactor was operated in the same manners as above and the same

sampling procedure was applied. After the anoxic period of 20 minutes when the

aeration usually went on during a normal cycle the mechanical mixing proceeded for

another two hours.

90


12

10

4.5

3.75

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 50 100 150 200

Time (min)

Figure 3. Concentration profiles of N 2O and O 2 during prolonged unaerated period.

At first the N2O accumulation is rather linear, when DO decreases under 1 mg/l the

accumulation rate of N2O is reduced until a maximum concentration is reached at DO

concentrations close to 0 mg/l. The N2O concentration is constant under a period of 20-

50 minutes and then slowly starts to decrease as seen in Figure 3.

Continuous aeration

Measurements with continuous aeration were performed at DO concentrations of ~1.5

mg/l and ~1 mg/l. To be able to determine the production of N2O gas during these

operation conditions the aeration was turned off. The unaerated period was chosen to

20 minutes to be comparable with the measurements done during intermittent aeration.

Measurement proceeded 20 minutes after the aeration was switched on again. Two

measurements were performed at a constant aeration with a DO concentration of ~1.5

mg/l and ~1 mg/l respectively a typical profile of O2 and N2O concentrations are shown

in Figure 4.

91


12

10

091014 Continously operation, DO 1.0 (mg/l)

4.5

3.75

N₂O (µmol/l)

8

6

4

2

3

2.25

1.5

0.75

DO (mg/l)

N₂O

(µmol/l)

DO

(mg/l)

0

0

0 20 40 60 80

Time (min)

Figure 4. Concentration profiles of N 2O and O 2 during measurement at continuous aeration with

DO ~1.0 mg/l.

As seen in Figure 4 N2O is only accumulating for the first 5-6 minutes of the unaerated

period then there is a short time span when production and N2O flows leaving the

reactor are in equilibrium. The N2O concentration measured in the water phase is

decreasing before aerations starts again which have not been noticed in any of the

former cases. Initial N2O production is below 2% of reduced inorganic nitrogen and

maximum production is also very low.

Conclusions

Conclusions that can be made from the experiments are summarised below:

• The single stage nitritation/anammox system produced significant amounts of

N2O with a minimum production of 2% of removed inorganic nitrogen.

• Operating the MBBR at intermittent aeration with a DO of ~3 mg/l gave the

highest N2O production with initial and maximum productions of 6-11% and 10-

30% respectively.

• Smaller amounts of N2O were produced by the partial/nitritation anammox

system during continuous operation at DO in the interval 1-1.5 mg/l. The initial

N2O production was found to be 2-3% and the maximum N2O production

corresponded to 2-6%.

• When the MBBR was exposed to a longer period of anoxic conditions both

ammonium oxidation and N2O production ceased.

• The absolute number on overall N2O production for an operation mode (based on

the measurements of N2O accumulating during the anoxic phase) could be both

overestimated or underestimated and should therefore be used as a comparative

tool.

92


Acknowledgements

The experiments were carried out at AnoxKaldnes in Lund during the work with my

master thesis and I would like to thank my supervisor my supervisor Magnus

Christenson for all guidance, support, sharing off valuable knowledge and experiences,

also for giving me the opportunity to get to know the fascinating anammox process.

I would also like to thank my supervisor Professor Jes la Cour Jansen at Water and

Environmental Engineering Department of Chemical Engineering, Lund University for

scientific guidance and encouragement, for all your valuable aspects on my work and

always reminding me of looking into things from a wider perspective.

References

Abma W.R., Schultz C.E., Mulder J.W., van Loosdrecht M.C.M., van der Star W., Strous M.,

Tokutomi T., (2007). The advance of Anammox. Water 21, February 2007.

Fux C., Boehler M., Huber P., Brunner I., and Siegrist H., (2002). Biological treatment of

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