Master thesis weronika w%c3%b3jcik

CRACOW UNIVERSITY OF TECHNOLOGY

Krakw, Poland

in cooperation with

ROYAL INSTITUTE OF TECHNOLOGY

Stockholm, Sweden

TRITA LWR Degree Project

ISSN 1651-064X

LWR-EX-11-26

EVALUATION OF MICROBIOLOGICAL ACTIVITY DURING THE

DEAMMONIFICATION PROCESS FOR

NITROGEN REMOVAL

Weronika Wjcik

September 2011

Weronika Wjcik TRITA LWR Degree Project 11:26

ii

Weronika Wjcik 2011 Degree Project at Masters Level

Department of Land and Water Resources Engineering

Royal Institute of Technology (KTH)

SE-100 44 Stockholm, Sweden

Reference should be written as: Wjcik, W. (2011) Evaluation of microbiological activity during the deammonification process for nitrogen removal. TRITA LWR Degree Project, 11:26.

Evaluation of microbiological activity during the deammonification process for nitrogen removal

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SAMMANFATTNING

Detta examensarbete baseras p egna studier. En studie genomfrdes under en fyra mnadersperiod vid Hammarby Sjstadsverk, som r belgen i Stockholm. Enstegs teknik utvrderades fr deammonifikation fr tv systemutfranden i pilotskala.

Den teroretiska bakgrunden fr detta examensarbete presenteras i en frsta del och hrvid beskrivs negativa miljkonsekvenser av kvvefreningar liksom myndighetskrav fr renat avloppsvatten i

Europeiska Unionen (Polen och Sverige). I nsta del av examensarbetet beskrivs kvvecykeln med fokus p biologiska reaktioner fr kvveavskiljning. Speciellt behandlas nitrifikations-/denitrifikations- och anammoxprocesser med tonvikt p olika faktorer som pverkar anammoxprocessen samt fr- och nackdelar att anvnda denna process. Experimentella resultat fr fyramnadsstudien liksom utvrdering av mikrobiell aktivitet beskrivs i examensarbetets sista del.


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ACKNOWLEDGMENTS

This Master Thesis was carried out thanks to the Erasmus Program between the Department of Environmental Engineering at Cracow University of Technology (PK) in Krakw (Poland) and the Department of Land and Water Resources Engineering, Royal Institute of Technology (KTH) in Stockholm (Sweden).

At the beginning and primarily I would like to extend my thanks for my supervisor Professor Elbieta Paza for the opportunity to cooperate and opportunity to take part in research. Thank you for you help, advices, suggestions and your time!

I would also like to thank PhD Jerzy Mikosz for help, friendly cooperation, and engagement to "Erasmus Students" and support, which I can always count on!

Special thanks go to Jingjing Yang, PhD Student at KTH. Thank you for everything: .for excellent cooperation and helpful advices, suggestions, nice time in the lab, submitted knowledge and experience and friendship. Thanks to you, time spent in the lab was nice and interesting experience for me.

I would like to acknowledge PhD Jzef Trela, the leader of the Deammonification project for his help and knowledge.

I am also thankful PhD Christian Baresel and Lars Bengtsson from Swedish Environmental Research Institute IVL for their help, cooperation, friendliness and atmosphere which they create at the research station.

Thanks parents Marta and Bogusaw, and brothers Mateusz and ukasz, who supported me during all my stay in Stockholm, and who supported me in moments of weakness during the five years of study. Heartfelt thank you!

Finally, I want to thank my family and friends, for any help ... for that you are!

Thank you without You all this work could not have been accomplished!

Krakw, September 2011


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TABLE OF CONTENT

Sammanfattning ......................................................................................................................... iii Acknowledgments ........................................................................................................................ v Table of content ......................................................................................................................... vii Abbreviations and symboles ...................................................................................................... ix Abstract ........................................................................................................................................ 1 1. Introduction ....................................................................................................................... 1

1.1. Negative impact of nitrogen on the environment ..................................................... 1

1.2. Requirement for nitrogen removal from wastewater ................................................. 1 1.2.1. Polish standards ..................................................................................................... 1 1.2.2. Requirements in the European Union (Sweden) ..................................................... 1

1.3. Forms of nitrogen in the environment ....................................................................... 2

1.4. Nitrogen cycle .............................................................................................................. 2 2. Conventional process: Nitrification /Denitrification ..................................................... 4

2.1. General description of nitrification process ............................................................... 4

2.2. General description of denitrification process ........................................................... 4

2.3. Operational parameters ............................................................................................... 4 3. ANAMMOX process description .................................................................................. 5

3.1. Parameters affecting ANAMMOX process performance ...................................... 5 3.1.1. Dissolved Oxygen .................................................................................................. 5 3.1.2. Temperature .......................................................................................................... 5 3.1.3. pH and alkalinity .................................................................................................... 5 3.1.4. Organic matter ....................................................................................................... 5

3.2. Superiority of the ANAMMOX process .................................................................. 6 3.2.1. Deammonification ................................................................................................. 6

3.3. MBBR with deammonification in MBBR.................................................................. 6 4. Aim of the Study ................................................................................................................ 8 5. Methodology ...................................................................................................................... 8

5.1. Short description of the research station Hammarby Sjstadsverk ......................... 8

5.2. Description of experimental installation .................................................................... 9

5.3. Physical parameters measurements and chemical analysis ................................... 10 5.3.1. Physical parameters .............................................................................................. 10 5.3.2. Chemical analyses ................................................................................................ 10

5.4. Microbial activity tests............................................................................................... 11 5.4.1. Specific Anammox Activity (SAA) ....................................................................... 11 5.4.2. Oxygen Uptake Rate (OUR) ................................................................................ 13 5.4.3. Nitrate Uptake Rate (NUR) ................................................................................. 17

6. Results and discussions .................................................................................................. 20

6.1. Evaluation of microbiological activity...................................................................... 20 6.1.1. Specific Anammox Activity .................................................................................. 20 6.1.2. Oxygen Uptake Rate ............................................................................................ 21 6.1.3. Nitrogen Utilization Rate ..................................................................................... 22

6.2. Evaluation of microbiological activity for Short Term Test ................................... 23 7. Conclusions...................................................................................................................... 24 8. References ........................................................................................................................ 25


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Appendix I. Data from Nitrate Uptake Rate test for lab-scale reactors during February 1st 2011 may 31th 2011. ............................................................................. I

Appendix II. Data from Specific Anammox Activity test for lab-scale reactors during February 1st 2011 may 31th 2011 .......................................................................... VII

Appendix III. Comparison of the test results, according to frequency of samples control ............................................................................................................... XXXI

Appendix IV. Oxygen Uptake Rate test data ................................................................... XXXII Appendix V. Data necessary for OUR calculations ............................................................ XLII Appendix VI. Data, Results and calculations for SAA short term test, Pilot reactor 2 .... XLIV Appendix VII. Data necessary for OUR Short Term Test calculations ............................... LV Appendix VIII. Graphs necessary for OUR Short Term Test calculations ......................... LVI


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ABBREVIATIONS AND SYMBOLES

ATU Allylthiourea C4H8N2S

AOB Ammonium Oxidizing Bacteria

ANAMMOX ANaerobic AMMonium OXidation

BOD Biochemical Oxygen Demand

DO Dissolved Oxygen

HT Heterotrophs

kg Kilogram

MBBR Moving Bed Biofilm Reactor

NOB Nitrite Oxidize Bacteria

NH3 Free ammonia

NH4+-N Nitrogen in Ammonium form

NO2--N Nitrogen in Nitrite form

NO3--N Nitrogen in Nitrate form

NO Nitrous oxide

NO2 Nitric dioxide

NUR Nitrate Uptake Rate

OUR Oxygen Uptake Rate

ORP Oxidation Reduction Potential

p.e. Population equivalent

R1 Lab-scale Reactor 1

R2 Lab-scale Reactor 2

SAA Specific Anammox Activity

T Temperature

VSS Volatile Suspended Solids

WWTP Waste Water Treatment Plant


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ABSTRACT

This master thesis is based on own studies. A four-month study was performed at Hammarby Sjostad Research Station, which is located in Stockholm. One-stage deammonification process was evaluated in two different system configurations in pilot plant scale.

The theoretical background for this thesis works is presented in the first part and where is presented negative impacts of nitrogen compounds in environment and requirements for purified wastewater in European Union (Sweden and Poland). In the next part of the thesis the nitrogen cycle is described and with focus on biological reactions for nitrogen removal. Especially, nitrification/denitrification and anammox processes are described with special focus on parameters affecting the anammox process performance and its advantages and disadvantages of using this process. Experimental results from the four-month study and evaluation of the microbial activity are described in the last part.

Key words: Anammox, Moving Bed Biofilm Reactor, nitrogen removal, batch test, Specific Anammox Activity, Oxygen Uptake Rate, Nitrate Uptake Rate.

1. INTRODUCTION

1.1. Negative impact of nitrogen on the environment Nitrogen is a nutrient and it mean, that this substance allow and intensify biomass process growth. When there is a excessive amount of nitrogen in water, we can observed a disorder the natural balance in the tank. If it is delivered 1 kg Nitrogen (Nitrogen embedded in cells) we receive increase in biomass on 16 kg and it gives charge 20 kg O2 extra organic substances. This process is very unprofitable and we can observe it especially in the lakes. In the water receivers, where is constant supply of nutrients it is possible to observe vigorous plant growth and progressive overgrowing of the tank. The nitrogen is also nourishment for the algae. At the beginning it is only nourishment, but in the next steps we can observe expansion of the algae and water bloom. There are very

negative and dangerous situations. An increased volume of ammonia and nitrite in the water is very toxic (Dymaczewski, 1997).

1.2. Requirement for nitrogen removal from wastewater

1.2.1. Polish standards

Polish is a member of the European Union since 1 May 2004. Signing the Accession Treaty on 16 April 2003, the government committed itself to respect and implement EU legislation into national law. This is due to the transposition of EU directives into Polish legislation. It should be noted that although the Directives is the overarching legislation in relation to the law, they shall appoint only a goal to be achieved. Specific legislation is left to the legislature of the country concerned. In Poland, current legislation is Minister of Environment Regulation: Dz. U. 2006 nr 137 poz. 984. This regulations specify details for wastewater treatment for example references methods of wastewater sample analysis. Regulation also defines the standards for nitrogen in treated wastewater.

1.2.2. Requirements in the European Union (Sweden)

Council Directive 91/271/EEC concerning urban waste-water treatment was adopted on 21 May 1991. Its purposes to protect the environment from the adverse effects of urban waste water discharges and discharges from certain industrial sectors the collection, treatment and discharge


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domestic waste water, the mixture of sewage and wastewater from certain industrial sectors (Fig. 1).

1.3. Forms of nitrogen in the environment Nitrogen is one of the most important elements: it takes part in many processes, is utilization by plants and it is a component of many bio molecules such as amino acids, nucleotides and nucleic acids. In the environment there are two forms of nitrogen (Fig. 2): unoxidized and oxidized forms.

1.4. Nitrogen cycle This chapter shows only very short and general nitrogen cycle and Anammox process. All reactions, parameters and details will be presented in more detail in next chapters of this master thesis. The (Fig. 3) shows the microbial relationships between nitrification and denitrification, and shows Anammox process in this cycle.

The first step of traditional nitrogen cycle is microbial conversion of molecular nitrogen (N2) to ammonia (NH4). In the deamination process there is producing ammonia, because organic molecules containing nitrogen are deaminated during the decomposition of organic materials. Next step of nitrogen cycle is nitrification. This process is

Fig. 1. Review of Directive 91/271/EEC. (http:/ /ec.europa. eu/ environment/water/water-urbanwaste/index_en.html)

Table 1. The highest value of pollution indicators or minimum percentage reductions of contaminants for treated wastewater of domestic and communal, made to the water and to the ground (www.isap.sejm.gov.pl).

Nr Name of pollution indicator

Unit

The highest value of pollution indicators or minimum percentage reductions of contaminants depend on p. e.

100000

4. Total Nitrogen

mg N-l 301) 15

1) 15

1) 15 10

min. % red.

- - 352) 80 85

1) required only in the effluent entering the lakes and inflows and directly to the reservoirs located in the

flowing waters 2)

The minimum percentage reduction is not used in example when the wastewater is entering in to the lakes and lakes inflows directly to the artificial water reservoirs located in the flowing waters and to the ground.


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Fig. 2. Forms of nitrogen in the environment

a biological process of transformation ammonium form nitrogen to nitrate and nitrite form. At the beginning of nitrification bacteria of the

genus Nitrosomonas oxidize NH3 to nitrites (NO2), then bacteria of the

genus Nitrobacter oxidize the nitrites to nitrates (NO3). Last step of

traditional nitrogen cycle is denitrification. Denitrification is the process of biological reduction. During denitrification nitrate is reduced to dinitrogen gas (N2). The heterotrophic bacteria are responsible for quality of this process (Malovanyy, 2009).

The red line in the (Fig. 3) shows Anammox process. In this process

biological process, nitrite (NO2) and ammonium (NH4) are converted

directly to dinitrogen gas (N2).

Fig. 3. Nitrogen cycle with Anammox process (www.paques.nl).


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2. CONVENTIONAL PROCESS : NITRIFICATION /DENITRIFICATION

2.1. General description of nitrification process Nitrification is a first part of transformation ammonium to the dinitrogen gas, and it consists .of two different steps. The first step of nitrification process is called nitritation and it is transformation of ammonium to nitrite (Wiesmann, 1994). Nitritation is performed mainly by Nitrosomonas bacteria in aerobic conditions. The simplified equation of nitritation process is:

The second step of nitrification process is called nitratation and it is transformation of nitrite .to nitrate. Nitratation is performed by bacteria of genera Nitrobacter in aerobic conditions. The simplified equation of nitratation process is:

Both Nitrosomonasand Nitrobacter, that are responsible for nitrification process are autotrophic bacteria, called nitrifying bacteria (Malovanyy, 2009).

2.2. General description of denitrification process Second part of transformation ammonium to the dinitrogen gas is denitrification. During this process, nitrite and nitrate are reduced to dinitrogen gas, by heterotrophic bacteria mainly from gram-negative alpha and beta classes of Proteobacteria in anoxic conditions. Nitrate and nitrite are using oxygen as an electron acceptor. Heterotrophic bacteria in this process use external carbon as a carbon source for gaining energy and building cells. Usually easily degradable organic substances that come with wastewater are used as a carbon source but often an additional external carbon source (mainly methanol) has to be added in order to reduce all the nitrite and nitrate. The simplified equation of nitritation process is (Malovanyy, 2009):

2.3. Operational parameters Knowledge about nitrification and denitrification is very important .in wastewater technology. But by knowledge about processes very important is knowledge about operational parameters. It helps to achieve the maximum efficiency of process and minimal costs (Cema, 2009).

When we are talking about nitrification, we must know, that about efficiency of this process decide a lot of factors such as: temperature, pH, concentration of nitrogen in effluent, dissolved oxygen (DO), age of sludge, alkalinity and toxic substances. The optimal temperature for nitrification is above 20 degrees. When the temperature drops, the intensity of nitrification process drops too. Less than 5 degrees process

stops. The optimum pH ranges between 7.58.5, but process can be carried in different pH (for example 6.5) if value of pH is constant. Concentration of dissolved oxygen should be about 2 mg O2/l. When concentration of DO drops to 1 mgO2/l, nitrification becomes very slow, in the case, when DO rise above 2 mg O2/l, the efficiency of the nitrification is at the same level as efficiency in 2 mg O2/l. Nitrifying bacteria are very sensitive for toxic substances. Toxic substances are inhibitors for nitrification process. Denitrification runs most efficiently


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while keeping several conditions. First, in wastewater must be carbon compounds and nitrates. Then, as for nitrification, optimal temperature for denitrification is above 20 degrees. The optimal pH for

denitrification ranges between 6.57.5. Concentration of DO in denitrification chamber should be as low as it is possible and lower than 0.5 mg O2/l.

All these factors have strange influence for nitrification and denitrification. When one or more of these factors are not running correct, the nitrification or denitrification process have disturbed efficiency (Dymaczewski, 1997).

3. ANAMMOX PROCESS DESCRIPTION

Wastewater treatment, especially nitrogen removal is currently interest many of research groups. We are looking for a new way for nitrogen removal. One of this ways can be ANAMMOX. ANaerobic AMMonium Oxidation is biological oxidation of ammonia to nitrogen, and this process seems to be promising alternative for traditional nitrogen removal. During this process, under anoxic conditions, ammonium is directly oxidized to dinitrogen gas using nitrite as the electron acceptor (Jetten et al., 1999).

3.1. Parameters affecting ANAMMOX process performance

3.1.1. Dissolved Oxygen

Dissolved oxygen has an impact on efficiency of nitrogen removal by deammonification. On the one hand has a negative impact on the Anammox process, on the other hand, remember, that it is necessary for the nitritation process. For one-stage partial nitrification/anammox process, dissolved oxygen is parameter influencing the nitrogen removal rate in the system. DO concentration should stay at a certain level, to allow ammonium oxidizers to produce a sufficient amount of NO2--N for anammox reaction but also not to high NO2--N level to cause anammox inhibition effect or increasing Nitrite Oxidizers Bacteria growth (Cema et al., 2007).

3.1.2. Temperature

The correct temperature for ANAMMOX process is in the range of 20-43 degrees, and the optimal temperature is 40 degrees. Negative impact has in temperature change and situations, when temperature drops below 20 degrees, which was observed in Waste Water Treatment Plant in Hattingen. As a result of drops temperature observed decrease in efficiency of nitrogen removal in MBBR from 70-80% to 16-40% (ubrowska et al., 2010).

3.1.3. pH and alkalinity

pH is very important parameter, which has influence for anammox process. Different groups of researchers tried to provide anammox process in various pH. But in partial nitritation/anammox process, performance efficiency could be inhibited by free ammonia, when pH is above 8 and nitrous acid when pH is below 7.5.

3.1.4. Organic matter

In different studies were used different groups, kinds of organic matter, used as inhibitors. Among the most important inhibitors of the process anammox is oxygen and other compounds such us methanol and ethanol. But methanol and ethanol are irreversible inhibitors for anammox process (Ahn, 2004).


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Table 2. Factors influencing deammonification process (Hernando et al., 2010).

3.2. Superiority of the ANAMMOX process After analysis Anammox process and traditional nitrification/denitrification, we can observe a lot of advantages, and superiority ANAMMOX over the traditional methods of nitrogen removal for wastewater:

High nitrogen removal,

No external carbon source needed,

40% reduction in oxygen demand,

Reduced production of sludge,

Reduced nitrous oxide emission,

Reduced carbon dioxide emission,

Reduction of energy demand and power consumption up to 60-90%,

3.2.1. Deammonification

Deammonification process consists of two steps process: Partial Nitritation and Anammox reaction. During the first step of deammonification process - Partial Nitritation, about 50-60% of ammonium is oxidized to nitrite:

During the second step, anammox reaction, bacteria use ammonium and nitrite as substrates to produce nitrogen gas:

Deamonification process can be realised in two different strategies: in one-stage or two-stage strategy. In situation, when process is carried in two separate reactors, in the first reactor is carried a partial nitration, and in the second reactor Anammox stage. In one-stage reactor both partial nitritation and anammox process are realised in the same time. Both in one-stage and in two-stage strategies, nitritation and anammox are carried out by different microorganisms. In the first step, the ammonium is partially oxidized by aerobic autotrophic ammonia oxidizers (AOBs) and in the second stage the remaining ammonium is oxidized to nitrogen gas by anaerobic autotrophic ammonia oxidizers (Anammox) (Hernando et al., 2010).

The same as nitrification and denitrification, there are factors which have decisive for deammonification process (Tab. 2).

3.3. MBBR with deammonification in MBBR Moving Bed Biofilm Reactor is a high technology wastewater treatment, which becomes increasing recognition in the word.

Deammonification process

Partial nitritation Anammox process

Oxygen supply Oxygen-limited supply

Supernatant composition Nitrogen load in the inflow

Ratio nitrite/ammonium in the outflow Ratio nitrite/ammonium in the inflow

Hydraulic Retention Time Hydraulic Retention Time

pH decrease pH increase

Consumption of alkalinity Nitrite concentration inside the reactor

Free nitrous acid and free ammonia concentrations SAA of bacterial culture

Temperature Temperature


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Fig. 4. Samples of Kaldness carriers.

MBBR process is based on the principle of biological membrane: biofilm, which grows on specially designed elements (Fig. 4) of material immersed in the entire volume of the reactor.

MBBR elements were designed to present largest possible active surface area (from 200-1200 m2/m3) for biological membrane and optimal living conditions for different cultures of microorganisms.

Biofilm starts to grow within minutes/hours after start of the purification process. Microorganisms that are involved in the treatment process produces sticky substances attach themselves to the media and begin to create a high performance biofilm (Dosta et al., 2008).

In this technology, biofilm suspended on the tubular profiles is mixed .in the biological reactor chamber using: compressed air (aerobic reactors) and mixer (anaerobic reactors). In characterized pilot-plant we have two reactors: R1 based on aeration strategy and R2 based on temperature effects. Each of these reactors has a working volume 200 litres and Kaldness carriers 80 litres, which is about 40% reactors volume.

Biofilm, covering the surface of the fittings, has optimal conditions for development and provided an optimal supply of oxygen and organic matter to bacteria and higher microorganisms. Biofilm, located in the middle of Kaldness carrier consists of two very important and depend on each other zones. The zone, which directly adjacent to the Kaldness carriers anaerobic zone, and in this part of biofilm, we can observed anammox process. Then, in the second zone, which contacted with anaerobic zone and liquid we can observed nitritation process, and it is aerobic zone.

Conditions conductive to the growth of bacteria, high levels of biofilm and high concentration of oxygen in Moving Bed Biofilm Reactor technology cause the removed several times more pollution per day than in the traditional technology.

According to INWATEC Industrial Waste Technology (www.inwatec.pl), using movable bed guarantees:

stable wastewater treatment plant work,

possibility to adopt more pollutant loads,

approximately five times smaller bioreactors cubature,

BOD5 removal rate about 5000 BZT5/g/d m3 for 15 degrees,

nitrogen removal rate about 400 NH4-N/d m3, 670 NOx-N g/d m3 for 15 degrees,

no need to recycled sludge,

no clogging and self-cleaning,

high resistance for pH and temperature changes,

possibility of using technology to each shape of the reactor,

high durability carriers (up to 20 years),

reduction of excess sludge up to 50%.


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4. AIM OF THE STUDY

This Master Thesis is a part of Anammox technology nitrogen reduction project and it is focused on the one-step partial nitrification/Anammox process in the moving bed biofilm reactor (MBBR) with Kaldnes carriers.

The aim of this study was evaluation of microbiological activity during the deammonification process for nitrogen removal under different operation strategies (long term study):

Aeration strategy (DO 3.0 mg/l)

Temperature (190C)

The main goal of short term test was evaluate the influence of temperature on different groups bacteria:

Anammox Bacteria Activity: 35 - 50C, step: 3 degrees

Ammonium Oxidizing Bacteria Activity

Heterotrophic Activity 35 - 170C, step: 3 degrees

Nitrate Oxidizing Bacteria Activity

To achieve goals, during the research, was necessary:

Review literature and publications about nitrogen removal from wastewater;

Evaluation of the process performance by chemical analyses, physical parameters monitoring and biomass measurements;

Perform calibration and cleaning of the portable and on-line instruments;

Assess the Nitrate Uptake Rate (NUR) test by the biofilm and its evolution;

Monitor the evolution of Anammox bacteria activity though SAA tests;

Assess the evolution of Heterotrophic bacteria, Nitrosomonas and Nitrobacter bacteria activity in the biofilm though Oxygen Uptake Rate tests;

Try to find correlations between chemical analyses results and physical parameters.

5. METHODOLOGY

5.1. Short description of the research station Hammarby Sjstadsverk The Hammarby Sjstadsverk is located on the top of Henriksdal underground Waste Water Treatment Plant (Fig. 5) which is the biggest WWTP in Stockholm.

The Hammarby Sjstadsverk is one of the most popular institutions in Sweden, where are conducted research in the field of wastewater treatment. The objects which are located on the facilities were held in

October 2003 and they were constructed by Stockholm Water AB. Today it is a facility where are prepared projects of the Royal Institute of Technology (KTH) and IVL Swedish Environmental Research Institute. Furthermore, it is a place that gives the opportunity to develop for many Swedish and international students, PhD students and many scientists. Currently, the station carries the following major projects (www.sjostadsverket.se):


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Fig. 5. Location of research station (http://maps.google.com).

Reduce the greenhouse gas emissions from Swedish wastewater and sewage sludge management

Removal of pharmaceuticals from the wastewater

More efficient biogas production

Anammox technology: nitrogen reduction

Energy and resource management facility

Algae for water treatment and Biofuel production

5.2. Description of experimental installation One of the projects, which are realized in Hammarby Sjstadsverk is called Anammox technology: nitrogen reduction. And it is experimental installation (Fig. 6), which allowed the implementation of the experiments and research to support this work. The deammonification process is conducted by KTH since 1999. The experiments for this project are conducted by PhD students and Master Students, who are preparing their Master Thesis at KTH, under the leadership of Professor Elbieta Paza and PhD Jzef Trela from KTH (Bertino, 2010).

The technical-scale pilot plant reactor, which is the main element of installation, was designed as a continuous aerated and stirred Moving Bed Biofilm Reactor (MBBR) with Kaldnes carriers. The biocarriers, which is filled technical-scale pilot plant reactor were brought from Himmerfjrden Wastewater Treatment Plant (Bertino, 2010).

In addition to pilot reactor, in to the installation comes a lot of elements. First of them is storage tank, holding about 26 cubic meters, with reject water from Bromma Waste Water Treatment Plant. This tank is connected with smaller one, with holding about 1.3 cubic meters. From bigger to smaller tank, the reject water is regularly, twice per week, pumped.

Fig. 6. The one-stage pilot plant scale reactor for partial Nitratation/Anammox with equipment.

Hammarby Sjstadsverk


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Fig. 7. Fulfillment of pilot plant scale reactor.

From a small tank reject water is continuously pumped to the stirred and aerated reactor. The pilot reactor is filled by Kaldnes carriers (Fig. 7). At the beginning, where is reject water connected with reactor, there is conductivity and ORP measured. At the end of process, are measured: DO, pH, ORP, T and conductivity. All parameters are continuously recorded on the computer. Purified reject water is transmitted to the outflow tank (Bartino, 2010).

5.3. Physical parameters measurements and chemical analysis

5.3.1. Physical parameters

At the research station physical parameters are monitored every day. The physical parameters measurement such as:

Dissolved Oxygen

pH

Conductivity

Temperature

Redox Potential can be done off-line or on-line. On-line equipment provides a more efficient control of the process, with more reliable and accurate values than the manual measurements (Ridenoure, 2004). Off-line measurements are performed the verify on-line measurements.

5.3.2. Chemical analyses

Chemical analyses is another group of measured parameters. Chemical analyses are carried out in influent and effluent, regularly to follow up and monitor the process. The most important group is the analysis of nitrogen forms: Ammonium, Total Nitrogen, Nitrate, Nitrite, that allow processes evaluate quality. To accurately assess process in lab .is carried out COD and Alkalinity (or Acid Capacity) analysis. Samples taken from outflow and inflow are always filtrated with a 1.6 m pore size prefilter and 0.45 m pore size filter. For these analyses was applied spectrophotometric method (Fig. 8): Dr. Lange cuvettes and Dr. Lange Xion 500 spectrophotometer. To carried out the analysis were used cuvettes:

COD LCK 314 (15-150 mg/L O2) and LCK 514 (100-2000 mg/L O2)

Acid Capacity Ks 4.3 LCK 362 (0.5-8.0 mmol/l)

NH4-N LCK 303 (2-47 mg/L) and LCK 302 (47-130 mg/L)

NO3-N LCK 340 (5-35 mg/L)

NO2-N LCK 342 (0.6-6 mg/L)


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Fig. 8. Dr. Lange Xion 500 spectrophotometer and Dr. Lange cuvettes (LCK 340).

5.4. Microbial activity tests

5.4.1. Specific Anammox Activity (SAA)

To evaluate microbiological activity we are using batch tests, such as SAA. By Specific Anammox Activity test is measured Anammox Bacteria Activity. This batch test is based on the measurement of the increment of pressure inside a closed volume, proportional to the production of nitrogen gas by ANAMMOX bacteria which use nitrite and ammonium as their substrates (Strous et al., 1999).

At the beginning of each test is prepared equipment (Fig. 9) and test material. The rings were washed three times with phosphate buffer. (0.75 g/L K2HPO4 and 0.14 g/L KH2PO4) with a pH 7.8 manually prepared.

In the next step of experiment 15 Kaldnes rings with attached bacteria were put to the Pyrex vial with buffer solution until a volume of 24 mL was reached. For each reactor were prepared three vials. Then, the vials were closed and a needle connected to the nitrogen gas line. Nitrogen (N2) gas was inoculated for a few minutes (about 3 minutes) to remove all the oxygen inside the vial (Fig. 10).

Fig. 9. Equipment for SAA test.


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Fig. 10. SAA sample during the N2 gas inoculated.

When the samples for experiment were ready, they were put to the water bath, where the samples were kept during the whole experiment. Before substrates were dosed, the samples stayed in water bath (in 25 degrees) about 10-15 minutes until the desired stable temperature were reached. When sample reached 25 degrees the substrates 0.5 mL of NH4Cl and 0.5 mL of NaNO2 to fix initial concentrations of ammonium and nitrite inside the vials in 70 mg N/L, were added by needle and syringe. At the beginning of this part, the gas inside the vial was equalized to the atmospheric, and appropriate measurement was started at this time. The pressure was measured every 20-30 minutes with a pressure transducer. Transducer used in this experiment (produced by Centrepoint Electronic) displayed a value in mV. This value can be converted into mmHg multiplying by 2.65.

Below shows calculations used to estimate the SAA on the biocarriers:

N2 gas production rate:

SAA (Specific Anammox Activity):


.

slope of the pressure increase inside the vial plotted versus time (atm/min), VG volume of the gas phase (0.013 l), calculated by subtracting the volume of liquid with 15 biocarriers (25 ml) from the total volume of the vial (38 ml), R ideal gas constant 0.0820575 (atm l mol-1 K-1), T temperature (K) 28 molecular weight, of N2 (g N/mol), 60 and 24 unit conversion factors from min to days Sbiofilm surface area of 15 biocarriers = 7.0093510-3m2, calculated as a product of the specific area of Kaldnes media and the volume occupied by 15 rings (calculated by proportion on the base of the measurement that 107 rings occupy 100 ml), X grams of biomass attached on 15 rings. Calculations for the activated sludge have been calculated similarly:


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N2 gas production rate:


.

slope of the pressure increase inside the vial plotted versus time (atm/min), VG volume of the gas phase (0.013 l), calculated by subtracting the volume of liquid with 15 biocarriers (25 ml) from the total volume of the vial (38 ml), R ideal gas constant 0.0820575 (atm l mol-1 K-1), T temperature (K) 28 molecular weight of N2 (g N/mol), 60 and 24 unit conversion factors from min to days, X biomass concentration inside the vial (g VSS/l), VL volume of the liquid phase in the vial (approximately 18.97 ml). It has been calculated as difference between 25 ml and the equivalent volume occupied by carriers, based on the measurement that 4 l of rings occupy approximately a volume of 1.72 l.

5.4.2. Oxygen Uptake Rate (OUR)

The principle of OUR test is to monitor the rate of dissolved oxygen uptake by bacteria and selectively inhibit different bacterial populations during the test. During this test we measure three kinds of bacteria:

Measure Ammonium Oxidizing Bacteria Activity (AOB)

Measure Heterotrophic Bacteria Activity

Measure Nitrate Oxidizing Bacteria Activity (NOB) The tests and methodology were performed on the base of the methodology described by Gut et. Al. (2005).

At the beginning of this experiment it was necessary to prepare diluted with reject water approximately 1:10 in order to have a NH4+-N initial concentration of about 100 mg/l. This value was measured before starting the test (Fig. 11).

Fig. 11. Equipment for dilution concentration control.


14

Fig. 12. Equipment for OUR test.

The glass water with volume of 1.56 l with dilution was placed in the water bath until the temperature measured had reached about 25C. Than, water was supplied in the reject water to reach a DO concentration over 6.5 7 mg/l. Then in to the bottle was thrown magnetic stirrer and 107 Kaldnes carriers. A deal od magnetic stirrer is very important, because 107 Kaldnes carriers correspond to a volume of approximately 100 ml. At this stage of experiment, bottle was completely closed with rubber corks and parafilm, and the test was started. The dissolved oxygen was measured by YSI Model 57 Oxygen Meter with YSI 5905 BOD probe, and data were recorded every second by TESTO Comfort Software 2004 v 3.4.

During the first 5 minutes total oxygen uptake was measured (Fig. 13). After 5 minutes were added 4 ml of sodium chlorate (NaClO3, solution 470 g/L) in order inhibit NO2-N oxidation by Nitrite Oxidizing Bacteria. Dose and inhibitor concentration is determined by previous studies of the research group (Yang J., personal information, not published).

After 5 minutes, were added another inhibitor: 4 ml of Allylthiourea (ATU: C4H8N2S, solution 3.9 g/L). This inhibitor operates for Nitrate Oxidizing Bacteria and Ammonia Oxidizing Bacteria. The inhibitors

Fig. 13. First stage of OUR experiment.


15

Fig. 14. Dosing the first inhibitor.

were added by two needles in the rubber corks (Fig. 14). The temperature was around 25C during the whole test. The value in output from the recorder was in mV and it was converted to mg O2/L by the calibration done before starting that specific OUR test.

Below shows calculations used to estimate the OUR on the biocarriers:

Dissolved oxygen uptake rate:

OUR Nitrobacter:

.

Fig. 15. Inhibitors: NaClO3 and ATU.


16

Fig. 16. Test record on the computer.

OUR Nitrosomonas:r

OUR Nitrobacter:

slope of the dissolved oxygen concentration decrease inside the bottle plotted versus time (mg O2 l-1s-1). The values of the three slopes are the average of the three OUR tests performed, VL volume of the liquid phase (about 1.517 L) calculated by subtracting from the total volume of the bottle (1.56 L), the equivalent volume of liquid displaced by 107 Kaldnes biocarriers (calculated by a simple proportion, on the base of the measurement that 4 L of biocarriers occupy approximately an equivalent volume of water of 1.72 L). The volume of the liquid phase VL was slightly different during the three steps of the test because of the stepwise additions of inhibitors (4 ml). This was kept into account in the calculations and the volumes are approximately 1.509 L, 1.513 L and 1.517 L, Sbiofilm surface area of 107 biocarriers = 0.05 m2, calculated as the product of the specific area of Kaldnes media and the volume occupied by 107 rings, 60, 60 and 24 unit conversion factors from second to days, 1000 unit conversion factors from mg to g.

Calculations for the activated sludge have been calculated similarly:

Dissolved oxygen uptake rate:


17

Fig. 17. Equipment for NUR test.

OUR Nitrobacter:

OUR Nitrosomonas:

OUR Nitrobacter:

slope of the dissolved oxygen concentration decrease inside .the bottle plotted versus time (mg O2 l-1s-1). The values of the three slopes are the average of the three OUR tests performed, X biomass concentration inside the bottle (mg VSS/l), the biomass concentration inside the bottle was slightly different during the test because of the stepwise dilution, 60, 60 and 24 unit conversion factors from second to days.

5.4.3. Nitrate Uptake Rate (NUR)

The NUR test has the aim to assess the NO3-N removal rate from the liquor. The bacteria responsible for nitrate removal are essentially denitrifying bacteria. This test was carried out in a 1.5 L plastic contained (Fig. 17). To this test was used 1 L reject water diluted with tap water, with concentration about 350 450 mg NH4-N/L, and the process was performed in 25C.

Fig. 18. Another part of experiment: dropping Kaldness carriers to the plastic container.


18

Fig. 19. First part of NUR test.

In the first part of this experiment, nitrogen gas was supplied into the liquor to decrease the dissolved oxygen concentration under the 0.5 mg/L. The plastic container with liquor was all time covered by parafilm and the dissolved oxygen was measured by YSI Model 57 Oxygen Meter with YSI 5905 BOD probe.

When dissolved oxygen concentration dropped below 0.5 mg/L (Fig. 18) 400 ml of Kladnes carriers were cast into the plastic reactor.

The next step of this experiment were added 10 ml NaNO3 solution (6 g NaNO3/100ml). After this step waited about 1 minute, to be sure, that all solution was distributed through the all volume of the liquor and the first sample was taken. The sample was filtered with 0.45 and 1.6 mfilter, and inserted into the fridge in the special marked and closed by parafilm plastic bottle.

The samples were taken one each hour, for four hours. So after whole experiment were five bottles with samples. In the first and last samples were analysed COD, and in all five samples were analyses NO3-N.

Fig. 20. Dissolved Oxygen measurement.


19

Fig. 21. The first sample collection.

The dissolved nitrogen uptake rate was calculated by linear regression from the slope of the curve (straight line) of the nitrate uptake plotted versus time. The calculations for the biocarriers and activated sludge are similar, and look like this:

NUR (biocarriers):

NUR (activated sludge):

slope of the nitrate concentration consumption inside the container plotted versus time (mg NO3N l-1min-1), VL volume of the liquid phase equal to 1l, X biomass concentration inside the container (mg VSS/l), 60 and 24 unit conversion factors from minutes to days, 1000 deriving from conversion from mg to g and from ml to m3.

Fig. 22. Samples after the NUR experiment.


20

Fig. 23. Dr. Lange Cuvette and NUR samples ready for chemical analyses.

6. RESULTS AND DISCUSSIONS

6.1. Evaluation of microbiological activity

6.1.1. Specific Anammox Activity

Specific Anammox Activity tests were prepared for each reactor once per week. After three months we received eleven values, which define activity of Anammox Bacteria. As we see at (Fig. 24), the values of Anammox Bacteria for Pilot Reactor 1 is between 2.46 3.05 g N/dm2, and as we see at (Fig. 25), the values of Anammox Bacteria for Pilot Reactor 2 is between 2.23 2.87 g N/dm2. It means, that activity of this kind of bacteria is at similar level. But 20% difference between the lowest and highest value for Pilot Reactor 1, and 22% difference between the lowest and highest value for Pilot Reactor 2 evidence of disturbance. As we can noticed, the activity of Anammox Bacteria is higher in Pilot Reactor 1 in nearly all studies (only in 8/03/2011 the activity of Anammox bacteria was higher in Pilot Reactor 2).

At the beginning of this studies partial measurements were provided with frequency 30 minutes. The Anammox bacteria activity had a linear trend. At the end of March the frequency of checking pressure was changed from 30 minutes to 20 minutes. And during this experiment observed, that bacteria activity did not have linear trend. Consequently, research was conducted in one day, at the same time: 3 bottles with frequency 30 minutes, 3 bottles with 20 minutes. This experiment helped draw a conclusion, that during the Specific Anammox Activity test, it is important the frequency of sampling. The research was carried out at a constant temperature 25 degrees. We did not find a factors, which can proceed to the differences in the results.


21

Fig. 24. Specific Anammox Activity test results for Pilot Reactor 1.

Fig. 25. Specific Anammox Activity test results for Pilot Reactor 2.

6.1.2. Oxygen Uptake Rate

Oxygen Uptake Rate test allow for evaluation three groups of bacteria: Heterotrophic, Nitrosomonas and Nitrobacter. This kind of experiment was carried out once per two weeks for each reactor, so as a result we have a data for five experiment for each reactor. (Fig. 26 and 27) shows, that the largest group are bacteria Nitrosomonas. And for each reactors bacteria Nitrosomonas are at the similar level. The similar situation is for Heterotrophic bacteria. Heterotrophic bacteria in Pilot Reactor 1 are at the similar lever during the whole three months. And group of Nitrobacter bacteria in Pilot Reactor 1 is related to Heterotrophic. The Nitrobacter in Pilot Reactor 2 are at the lower level than in Pilot Reactor 1. The values of Heterotrophic bacteria .in Pilot Reactor 2 are totally irregular, and show problems: with Pilot Reactor or with experiment. The OUR experiment is very complicated, requires great accuracy and attention, and even small problems for example with mixing, or small bubble air under the cap have a strange influence for whole experiment, because finally values are based on seconds measurements. In summary, more stable situation exists in the Pilot Reactor 1.


22

Fig. 26. Oxygen Uptake Rate test results for Pilot Reactor 1.

Fig. 27. Oxygen Uptake Rate test results for Pilot Reactor 2.

6.1.3. Nitrogen Utilization Rate

These tests, like OUR, were carried out once per two weeks. Nitrogen Utilization Rate test is very sensitive of mixing: even small problems with mixing have a large impact for the final results. And during two experiments we had a problems with mixing. So two data are not representative unlucky both for the same reactor - Pilot Reactor 1. So it is a reason why we have four data for Pilot Reactor 1 and six data for Pilot Reactor 2 (Fig. 28 and 29). Nonetheless, we can notice that the nitrogen removal process was more stable for the Reactor 1. NUR result for this reactor is between 0.90 0.75 g N/m2d, and for Pilot Reactor 2 between 1.03 0.58 g N/m2d. Nearly 50% of the difference in value (in Pilot Reactor 2) probably shows a technical problem (for example not noticed problems with mixing), because other studies carried out in this period did not reveal any specific changes (disturbances).

0

1

2

3

4

5

2011-03-10 2011-04-07 2011-05-19

1.04

1.56

0.91 1.3 1.3

3.8 4.15

4.54 4.41

0.95

1.73

1.04 1.04 1.3 g

O2 /

m2

d

Oxygen Uptake Rate (OUR) - Pilot Reactor 1

OUR (Heterotrophic) OUR (Nitrosomonas)

0

1

2

3

4

5

2011-03-02 2011-03-30 2011-05-12

0.78

1.81

2.68

1.04

2.15

3.63 3.63 3.76

4.41

3.03

0.78 0.52

0.78 0.52

0.78

g O

2 /

m2

d

Oxygen Uptake Rate (OUR) - Pilot Reactor 2

OUR (Heterotrophic) OUR (Nitrosomonas)


23

Fig. 28. Nitrogen Utilization Rate test results for Pilot Reactor 1.

Fig. 29. Nitrogen Utilization Rate test results for Pilot Reactor 2.

6.2. Evaluation of microbiological activity for Short Term Test Next to the normal study, during May, was carried out Short Term Test for Specific Anammox Activity (Fig. 30) and Oxygen Uptake Rate (Fig. 31). The main goal of this experiment was checking the temperature effect on the results of the study. Specific Anammox Activity tests were carried out only for Pilot Reactor 2, for temperature between 35 to 5 degrees (with step 3 degrees). With decreasing temperature, was observed decreased of Anammox bacteria activity. Anammox bacteria activity dropped with linear trend from 2.10 g N/dm2 (for 35 degrees), to the 0.16 g N/dm2 (for 5 degrees). For temperatures close to zero was observed inhibition Anammox bacteria. Oxygen Uptake Rate tests were carried out for temperatures between 35 degrees to 17 degrees, with step 3 degrees (because in this sample were used a huge value of sample and inhibitors, having a negative impact on bacteria. It was observed, that in high temperature, in 35 degrees the activity of three groups of bacteria: Heterotrophic, Nitrosomonas and Nitrobacter were at the same level: close to 4.0 g O2/m2d. With decreasing temperature decreases the activity of bacteria. Bacterial activity decreases linearly, but with a different speed (Fig. 34). The Oxygen Uptake Rate Short Term Test was carried out the same like Specific Anammox Activity Short Term Test for Pilot Reactor 2.


24

Fig. 30. Specific Anammox Activity Short Term Test results for Pilot Reactor 2.

Fig. 31. Oxygen Uptake Rate Short Term Test results for Pilot Reactor 2.

7. CONCLUSIONS

The research for this master thesis were carried out on the Kaldness rings from pilot-plant scale reactor for a period of four months. All analyses are based on own research batch tests such as SAA, NUR and OUR carried out regularly every week. This allowed the conclusions drawn:

General:

Aeration conditions at pilot plants (mixing and aeration) affected the process performance.

Temperature has a strong influence on microbiological activity. The highest effects of temperature were observed for Anammox Bacteria.

The optimum temperature for high efficiency of the deammonification process should be higher than 25C.

Specific Anammox Activity:

Specific Anammox Activity test shows, that activity of Anammox Bacteria increases with the increase temperature.


25

During the Specific Anammox Activity test, it is important the frequency of sampling.

Short Term Test for Reactor 2 showed that after 8 degrees activity of Anammox Bacteria is close to zero.

Nitrogen Utilization Rate:

Denitrifies activity is more stable for Reactor 1 with intermittent aeration strategy.

Nitrogen Utilization Rate test is very sensitive of mixing: even small problems with mixing have a large impact for the final results.

Oxygen Uptake Rate:

Oxygen Uptake Rate test shows, that both in Reactor 1 and Reactor 2, the main role have Bacteria Nitrosomonas.

Short Term Test for Reactor 2 showed, that in high temperature (35C), activity of different groups of bacteria is similar (OUR).

Drop of temperature caused the significant decrease of Nitrate Oxidizing Bacteria activity (NOB).

8. REFERENCES

Ahn Y. H., Hwang I. S., Min K. S., 2004, ANAMMOX and partial denitritation in anaerobic nitrogen removal from piggery waste, Water Science and Technology. 49(5-60):145-53

Bertino, A. , 2010, Study on one-stage partial nitritation - anammox process in moving bed biofilm reactors: a sustainable nitrogen removal. Master Thesis, Department of Land, Environment and Geo-Engineering DITAG, Politecnico di Torino, Italy. 144 p.

Cema G., 2009, Comparative study on different Anammox systems, TRITA-LWR PhD 1053. Doctoral Thesis in Land and Water Resources Engineering, KTH Architecture and the Built environment, Stockholm. 72 p.

Cema G., Paza E., Surmacz-Grska J., 2007, Activated sludge and biofilm in the Anammox reactor Cooperation or competition?, Proceeding of Polish-Swedish seminars, Cracow March 17-18, 2005. Integration and optimization of urban sanitation systems. Eds: Plaza E., Levlin E., Report 13: 129-138

Dosta J., Fernandez I., Vazquez-Padin J., Mosquera-Corral A., Campos J. L., Mata-Alvarez J., Mendez R., 2008, Short- and long-term effects of temperature on the Anammox process, Journal of Hazardous Materials 154: 688-693

Dymaczewski Z., Oleszkiewicz J. A., Sozaski M. M., (red) 1997,Poradnik eksploatatora oczyszczalni ciekw. PZIiTS, Pozna, LEM s.c., Krakw. 223 p.

Dz. U. 2006 nr 137 poz. 984

Gut L., Paza E., Dugocka M., Hultman B., 2005, Partial nitritation process assessment, Vatten 61: 175-182, Lund

Hernando Z., Martinez S., 2010. Evaluation of Deammonification process performance for supernatant treatment, Master Thesis, Department of Land and Water Resources Engineering, Royal Institute of Technology, Sweden. TRITA LWR Degree Project,10-12, 79 p.


26

Jetten M.S.M., Strous M., van de Pas-Schoonen T., Schalk J., van Dongen U.G.J.M., van de Graaf A.A., Logemann S., Muyzer G., van Loosdrecht M.C.M., Kuenen J. G., 1999. The anaerobic oxidation of ammonium, FEMS Microbiology Reviews 22:421-437

Malovanyy A. 2009, Monitoring and application of anammox process in one stage deammonification system, Master Thesis, Department of Land and Water Resources Engineering, Royal Institute of Technology, Sweden. TRITA LWR Degree Project, 09-20, 74 p.

Malovanyy A., Paza E., Trela J., 2009, Evaluation of the factors influencing the specific Anammox activity (SAA) using surface modeling, Department of Land and Water Resources Engineering, Royal Institute of Technology (KTH). Research and application of the new technologies in wastewater treatment in Ukraine, Sweden and Poland (Polish-Ukrainian-Swedish seminar)

Ridenoure, J. 2004, Optimization of nitrogen removal from anaerobically pretreated swine wastewater (APTSW) in intermittent aeration reactors, Master of Since Thesis, North Carolina State University

Strous M., Kuenen J. G., Jetten M., 1999, Key physiological parameters of anaerobic ammonium oxidation, Applied Microbiology and Biotechnology, 65:3248-3250

Wiesmann U., 1994, Biological nitrogen removal from wastewater, Advance in Biochemical Engineering. Ed. Flatcher A. Berlin: Springer-Verlag. 113-154

ubrowska-Sudo M., Trela J., 2010, Proces Anammox jako alternatywna metoda intensyfikacji usuwania azotu ze ciekw, Gaz, Woda i Technika Sanitarna., September, 22-25

Other references:

Hammarby Sjstadsverk main website www.sjostadsverket.se AnoxKaldnes AB main website www.anoxkaldnes.com Paques bv main website www.paques.nl Internetowy System Aktw Prawnych www.isap.sejm.gov.pl INWATEC main website www.inwatec.pl

Yang, Jinjjing: PhD Student, Stockholm. Personal communication June 2011


I

APPENDIX I. DATA FROM NITRATE UPTAKE RATE TEST FOR LAB-SCALE REACTORS DURING FEBRUARY 1S T 2011 MAY 31T H 2011.

Table 3. NO3-N and COD results during the NUR test for Reactor 1.

10-0

3-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 102.0 91.6 88.1 80.2 74.0

COD (mg/l) filtr. 0.45 m - - - - -

24-0

3-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 100.8 92.0 82.4 77.6 73.2

COD (mg/l) filtr. 0.45 m 274 - - - 256

07-0

4-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 105.2 93.2 86.8 80.0 74.4

COD (mg/l) filtr. 0.45 m 309 - - - 280

05-0

5-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 89.6 81.2 90.0 86.0 82.4

COD (mg/l) filtr. 0.45 m 287 - - - 217

Technical problem with mixing, data are not reliable!

19-0

5-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 97.6 87.6 81.2 76.8 71.6

COD (mg/l) filtr. 0.45 m 268 - - - 246

Table 4. NO3-N and COD results during the NUR test for Reactor 2.

02-0

3-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 103.6 98.8 90.8 87.2 66.4

COD (mg/l) filtr. 0.45 m 314 - - - 289

17-0

3-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 95.2 91.6 85.2 79.2 68.8

COD (mg/l) filtr. 0.45 m 263 - - - 234

30-0

3-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 96.8 93.6 92.0 79.2 66.8

COD (mg/l) filtr. 0.45 m 293 - - - 254

14-0

4-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 100.0 92.0 87.6 86.4 78.8

COD (mg/l) filtr. 0.45 m - - - - -

28-0

4-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 99.6 92.8 79.2 81.6 68.4

COD (mg/l) filtr. 0.45 m 274 - - - 225

12-0

5-2

011

Time (min) 0 60 120 180 240

NO3-N (mg/l) 105.6 97.2 87.2 80.8 79.2

COD (mg/l) filtr. 0.45 m 293 - - - 240


II

Table 5. Nitrate Uptake Rate test results of 02-03-2011.

Pilot Reactor2, 02-03-2011

400 ml Kaldness

StartingDO

COD (mg/l/min)

COD (g COD/m2 d)

NO3-N (mg/l/min)

NO3-N (g N/m2 d)

0.4 mg/l 0.1042 0.75 0.1433 1.0320

Fig. 32. NUR results of 02-03-2011.


Pilot Reactor 1, 10-03-2011

400 ml Kaldness

StartingDO

COD (mg/l/min)

COD (g COD/m2 d)

NO3-N (mg/l/min)

NO3-N (g N/m2 d)

0.4 mg/l - - 0.1135 0.8172



III



400 ml Kaldness

StartingDO

COD (mg/l/min)

COD (g COD/m2 d)

NO3-N (mg/l/min)

NO3-N (g N/m2 d)

0.4 mg/l 0.1208 0.87 0.1087 0.7824




400 ml Kaldness

StartingDO

COD (mg/l/min)

COD (g COD/m2 d)

NO3-N (mg/l/min)

NO3-N (g N/m2 d)

0.4 mg/l 0.0750 0.54 0.1160 0.8352



IV



400 ml Kaldness

StartingDO

COD (mg/l/min)

COD (g COD/m2 d)

NO3-N (mg/l/min)

NO3-N (g N/m2 d)

0.4 mg/l 0.1750 1.26 0.1240 0.8928




400 ml Kaldness

StartingDO

COD (mg/l/min)

COD (g COD/m2 d)

NO3-N (mg/l/min)

NO3-N (g N/m2 d)

0.4 mg/l 0.1208 0.87 0.1247 0.8976



V



400 ml Kaldness

Starting.DO

COD (mg/l/min)

COD (g COD/m2 d)

NO3-N (mg/l/min)

NO3-N (g N/m2 d)

0.4 mg/l - - 0.0800 0.5760




400 ml Kaldness

StartingDO

COD (mg/l/min)

COD (g COD/m2 d)

NO3-N (mg/l/min)

NO3-N (g N/m2 d)

0.4 mg/l 0.2042 1.47 0.1227 0.8832



VI



400 ml Kaldness

StartingDO

COD (mg/l/min)

COD (g COD/m2 d)

NO3-N (mg/l/min)

NO3-N (g N/m2 d)

0.4 mg/l 0.2208 1.59 0.1153 0.8304




400 ml Kaldness

StartingDO

COD (mg/l/min)

COD (g COD/m2 d)

NO3-N (mg/l/min)

NO3-N (g N/m2 d)

0.4 mg/l 0.0967 0.66 0.1047 0.7536



VII

APPENDIX II. DATA FROM SPECIFIC ANAMMOX ACTIVITY TEST FOR LAB-SCALE REACTORS DURING FEBRUARY 1 S T 2011 MAY 31T H 2011

Table 15. Specific Anammox Activity test results for Reactor 1 and 2.

2011-03-08 R1 R2

1 2 3 4 5 6

0 10.5 13.0 12.0

12.4 18.4 6.0

20 14.1 16.7 15.4 16.6 21.5 9.2

40 18.3 20.2 18.5 20.6 24.6 12.8

60 22.0 23.4 21.4 24.0 27.1 14.2

80 23.8 26.2 23.1 25.7 29.2 16.6

100 27.0 28.2 25.3 29.6 32.4 19.6

120 29.0 30.5 27.2 30.7 34.1 20.3

2011-03-09 R1 R2

1 2 3 4 5 6

0 3.8 6.4 6.8

3.5 3.0 3.2

2 & 5 we add 0.5 ml methanol

20 8.9 12.3 12.9 7.1 7.6 6.5 3 & 6 we add 1 ml methanol

40 12.1 15.8 17.3 11.4 12.0 10.6

60 16.3 19.1 20.6 12.8 14.6 11.8

80 18.6 22.1 24.8 16.2 17.2 15.1

100 18.9 23.0 26.1 17.0 19.2 17.4

120 22.0 25.8 29.3 19.5 22.3 20.0

2011-03-15 R1 R2

1 2 3 4 5 6 7 8

0 3.2 3.2 3.1 3.6 6.2 5.0 3.6 4.8 3 & 7 we add 0,5 ml methanol

20 7.9 7.0 7.0 7.5 9.9 9.3 7.7 9.0 4 & 8 we add 1 ml methanol

40 12.9 10.2 10.8 10.8 12.9 12.0 10.8 11.7

60 16.7 13.0 13.7 13.9 16.3 14.8 13.9 14.2

80 20.8 15.9 17.3 17.0 20.4 19.0 18.2 17.0

100 24.6 18.5 19.4 19.7 23.4 21.9 20.8 19.4

120 25.3 19.2 20.5 20.4 25.2 23.2 22.3 20.9

2011-03-17 R1 R2

1 2 3

4 5 6

0 3.4 3.4 4.4 3.3 3.3 3.3 2 & 5 we add 0.5 ml methanol

20 10.2 9.3 11.3 9.6 8.5 10.0 3 & 6 we add 1 ml methanol

40 13.2 12.4 13.5 11.3 10.3 11.5

60 16.8 15.7 18.0 15.7 13.7 14.8

80 19.4 18.6 20.2 17.8 16.4 17.2

100 21.5 21.1 22.5 20.1 18.6 19.9

120 23.3 22.6 23.8 21.9 19.6 21.0

2011-03-22 R1 R2

1 2 3 4 5 6

0 4.2 3.8 4.4

3.2 3.8 3.3

20 8.2 7.3 8.3 9.6 8.7 6.7


VIII

40 11.7 12.3 11.9 13.6 14.1 10.3

60 14.1 14.8 16.6 18.0 15.9 11.4

80 17.7 18.0 19.7 19.2 16.4 13.6

100 18.5 18.3 20.7 23.4 19.2 15.1

120 19.9 20.1 22.4 25.9 22.0 16.9

2011-03-29 R1 R2

1 2 3 4 5 6

0 3.5 3.7 3.9

3.9 3.2 2.0

20 9.0 8.1 8.8 7.1 8.0 6.8

40 12.5 11.3 11.9 8.0 11.0 9.6

60 16.6 15.6 15.8 9.5 13.9 12.9

80 17.2 16.4 16.0 11.4 16.0 15.3

100 14.8 17.1 14.0 15.0 19.7 18.6

120 15.7 17.2 14.9 16.7 21.5 21.4

2011-04-05 R1 R2

1 2 3 4 5 6

0 2.8 3.3 3.3

2.6 2.4 2.0

20 7.5 5.8 7.7 6.2 5.5 5.8

40 12.8 10.4 14.4 12.4 11.7 12.2

60 14.3 12.9 17.6 14.0 13.3 13.0

80 15.6 13.2 15.4 16.8 16.0 16.5

100 13.4 14.2 16.5 17.0 16.7 16.8

120 15.1 15.9 18.4 17.5 18.6 17.1

2011-04-12 R1 R2

1 2 3 4 5 6

0 1.9 1.7 1.8

2.3 1.9 1.9

20 3.0 2.8 4.0 7.0 7.6 8.3

40 12.0 12.9 12.7 10.3 11.0 10.9

60 13.0 12.5 12.9 12.7 12.9 12.5

80 12.8 15.6 13.2 13.7 16.8 17.1

100 15.0 15.9 17.2 14.9 14.0 13.0

120 15.0 16.6 17.9 14.0 15.0 14.7

2011-04-19 R1 R2

1 2 3 4 5 6

0 2.7 2.6 2.7

2.8 2.9 3.0

30 8.9 8.2 8.5 6.9 7.0 6.9

60 15.7 14.7 15.2 9.8 10.5

90 18.7 17.8 18.5 12.4 13.4

120 22.2 21.2 22.2 17.3 10.4 16.9

2011-04-26 R1 R2

1 2 3 4 5 6

0 3.3 2.9 3.3

3.3 3.4 3.3

30 8.6 9.8 10.0 9.2 6.3 8.4

60 12.2 14.3 13.9 12.3 8.3 12.5

90 16.4 19.0 18.3 17.5 16.0 16.8


IX

120 20.0 21.8 25.0 21.0 16.8 19.8

2011-05-03 R1 R2

1 2 3 4 5 6

0 2.8 2.9 3.1

2.9 2.7 2.8

30 8.4 8.9 9.5 7.8 7.8 8.3

60 12.2 12.4 13.6 11.7 12.7 13.3

90 16.6 15.6 17.5 14.7 16.3 16.6

120 18.3 17.6 20.5 19.1 19.6 19.4

0

2.6 2.7 2.7

20 6.1 5.9 6.2

40 2.1 2.2 2.1

60 11.2 11.0 11.3

80 14.0 13.9 13.6

100 15.3 14.8 14.4

120 17.9 17.1 16.5

2011-05-17 R1 R2

1 2 3 4 5 6

0 4.6 4.0 4.0

3.5 3.8 3.1

30 9.4 8.1 8.9 5.3 5.4 6.0

60 14.3 13.2 14.5 9.2 9.3 10.3

90 19.7 17.7 19.7 12.8 12.0 13.0

120 22.0 20.2 23.1 16.4 15.4 16.1

2011-05-24 R1 R2

1 2 3 4 5 6

0 2.0 2.0 2.1

1.9 2.2 1.9

30 8.0 7.8 7.5 7.2 8.1 7.0

60 13.5 13.5 13.0 11.6 13.0 11.0

90 17.7 17.5 16.9 15.7 17.4 14.5

120 20.9 20.2 19.0 18.2 20.2 17.2


X

Table 16. Specific Anammox Activity test calculations, Pilot Reactor 1, March 8, 2011.

t (min)

P (mV)

Bottle 1 Bottle 2 Bottle 3

0 10.5 13.0 12.0

20 14.1 16.7 15.4

40 18.3 20.2 18.5

60 22.0 23.4 21.4

80 23.8 26.2 23.1

100 27.0 25.3

120

P(mV) / min 0.164714286 0.1655 0.132142857

P(mmHg) / min 0.436492857 0.438575 0.350178571

g N / min 8.545E-06 8.58576E-06 6.85527E-06

Biofilm area 0.007009346

SAA g N / dm2 1.755483872 1.763857819 1.408345691

SAA g N / dm2 1.642562461

Fig. 42. SAA results, Pilot Reactor 1, March 8, 2011.


XI


t (min)

P (mV)


0 6.2 3.6 4.8

20 9.9 7.7 9.0

40 12.9 10.8 11.7

60 16.3 13.9 14.2

80 20.4 18.2 17.0

100 23.4 20.8 19.4

120 25.2 22.3 20.9

P(mV) / min 0.163392857 0.160178571 0.132857143

P(mmHg) / min 0.432991071 0.424473214 0.352071429

g N / min 8.47644E-06 8.30969E-06 6.89232E-06


SAA g N / dm2 1.741400415 1.707143358 1.41595837

SAA g N / dm2 1.621500714



XII


t (min)

P (mV)


0 4.2 3.8 4.4

20 8.2 7.3 8.3

40 11.9

60 14.1 14.8 16.6

80 17.7 18.0 19.7

100 18.5

120

P(mV) / min 0.147616279 0.1795 0.1945

P(mmHg) / min 0.39118314 0.475675 0.515425

g N / min 7.65799E-06 9.31204E-06 1.00902E-05


SAA g N / dm2 1.573257572 1.913066336 2.072932603

SAA g N / dm2 1.853085504



XIII


t (min)

P (mV)


0 3.5 3.7 3.9

20 9.0 8.1 8.8

40 12.5 11.3 11.9

60 16.6 15.6 15.8

80 16.4

100

120

P(mV) / min 0.214 0.1645 0.194

P(mmHg) / min 0.5671 0.435925 0.5141

g N / min 1.11018E-05 8.53388E-06 1.00643E-05


SAA g N / dm2 2.280758751 1.753200068 2.067603728

SAA g N / dm2 2.033854182



XIV

Table 20. Specific Anammox Activity test calculations, Pilot Reactor 1, April 5, 2011.

t (min)

P (mV)


0 2.8 3.3 3.3

20 7.5 5.8 7.7

40 12.8 10.4 14.4

60 14.3 12.9 17.6

80 15.4

100

120

P(mV) / min 0.199 0.167 0.1705

P(mmHg) / min 0.52735 0.44255 0.451825

g N / min 1.03237E-05 8.66357E-06 8.84514E-06


SAA g N / dm2 2.120892484 1.779844446 1.817146575

SAA g N / dm2 1.905961168

Fig. 46. SAA results, Pilot Reactor 1, April 5, 2011.


XV


t (min)

P (mV)


0 1.9 1.7 1.8

20 3.0 4.0

40 12.0 12.9 12.7

60 13.0 12.5 12.9

80 12.8 15.6

100

120

P(mV) / min 0.159 0.169285714 0.21

P(mmHg) / min 0.42135 0.448607143 0.5565

g N / min 8.24855E-06 8.78215E-06 1.08943E-05


SAA g N / dm2 1.694582437 1.80420502 2.238127747

SAA g N / dm2 1.912305068



XVI


t (min)

P (mV)


0 2.7 2.6 2.7

30 8.9 8.2 8.5

60 15.7 14.7 15.2

90 18.7 17.8 18.5

120

P(mV) / min 0.182666667 0.173666667 0.180333333

P(mmHg) / min 0.484066667 0.460216667 0.477883333

g N / min 9.47632E-06 9.00942E-06 9.35528E-06


SAA g N / dm2 1.946815881 1.850896121 1.921947795

SAA g N / dm2 1.906553266



XVII


t (min)

P (mV)


0 3.3 2.9 3.3

30 9.8

60 12.2 14.3 13.9

90 16.4 19.0 18.3

120 20.0 25.0

P(mV) / min 0.140285714 0.176 0.177428571

P(mmHg) / min 0.371757143 0.4664 0.470185714

g N / min 7.2777E-06 9.13047E-06 9.20458E-06


SAA g N / dm2 1.495130236 1.875764207 1.890989566

SAA g N / dm2 1.753961336



XVIII

Table 24. Specific Anammox Activity test calculations, Pilot Reactor 1, May 3, 2011.

t (min)

P (mV)


0 2.8 2.9 3.1

30 8.4 9.5

60 12.2 12.4 13.6

90 15.6

120

P(mV) / min 0.156666667 0.143571429 0.175

P(mmHg) / min 0.415166667 0.380464286 0.46375

g N / min 8.1275E-06 7.44815E-06 9.07859E-06


SAA g N / dm2 1.669714351 1.530148561 1.865106456

SAA g N / dm2 1.688323123

Fig. 50. SAA results, Pilot Reactor 1, May 3, 2011.


XIX


t (min)

P (mV)


0 4.6 4.0 4.0

30 9.4 8.1 8.9

60 14.3 13.2 14.5

90 19.7 17.7 19.7

120

P(mV) / min 0.167333333 0.154 0.175666667

P(mmHg) / min 0.443433333 0.4081 0.465516667

g N / min 8.68087E-06 7.98916E-06 9.11318E-06


SAA g N / dm2 1.78339703 1.641293681 1.872211623

SAA g N / dm2 1.765634111



XX


t (min)

P (mV)


0 2.0 2.0 2.1

30 8.0 7.8 7.5

60 13.5 13.5 13.0

90 17.7 17.5 16.9

120

P(mV) / min 0.175333333 0.174 0.166333333

P(mmHg) / min 0.464633333 0.4611 0.440783333

g N / min 9.09589E-06 9.02672E-06 8.62899E-06


SAA g N / dm2 1.868659039 1.854448704 1.772739279

SAA g N / dm2 1.831949007



XXI


t (min)

P (mV)


0 12.4 18.4 6.0

20 16.6 9.2

40 20.6 24.6

60 24.0 27.1 14.2

80

100 32.4 19.6

120

P(mV) / min 0.194 0.139423077 0.134067797

P(mmHg) / min 0.5141 0.369471154 0.355279661

g N / min 1.00643E-05 7.23295E-06 6.95513E-06


SAA g N / dm2 2.067603728 1.485936462 1.428861217

SAA g N / dm2 1.660800469



XXII


t (min)

P (mV)


0 3.8 3.2 3.2

20 8.9 7.9 7.0

40 12.1 12.9 10.2

60 16.3 16.7 13.0

80 18.6 20.8 15.9

100

120

P(mV) / min 0.144821429 0.192142857 0.136964286

P(mmHg) / min 0.383776786 0.509178571 0.362955357

g N / min 7.513E-06 9.96793E-06 7.10539E-06


SAA g N / dm2 1.54347075 2.047810761 1.459731277

SAA g N / dm2 1.68367093


y = 0.185x + 4.54 R = 0.9857

y = 0.22x + 3.5 R = 0.9972

y = 0.157x + 3.58 R = 0.996

0.0

4.0

8.0

12.0

16.0

20.0

24.0

28.0

0 30 60 90 120

P (

atm

10-3

)

Time (min)


XXIII


t (min)

P (mV)


0 3.2 3.8 3.3

20 9.6 8.7 6.7

40 13.6 14.1 10.3

60 18.0 15.9 11.4

80 13.6

100 23.4 19.2

120

P(mV) / min 0.198108108 0.151554054 0.1265

P(mmHg) / min 0.524986486 0.401618243 0.335225

g N / min 1.02774E-05 7.86227E-06 6.56253E-06


SAA g N / dm2 2.111386922 1.615225398 1.348205524

SAA g N / dm2 1.691605948



XXIV


t (min)

P (mV)


0 3.9 3.2 2.0

20 7.1 6.8

40 8.0 11.0 9.6

60 13.9 12.9

80

100 15.0 19.7

120

P(mV) / min 0.1075 0.164230769 0.1775

P(mmHg) / min 0.284875 0.435211538 0.470375

g N / min 5.57685E-06 8.51991E-06 9.20829E-06


SAA g N / dm2 1.145708251 1.750330674 1.891750833

SAA g N / dm2 1.595929919



XXV

Table 31. Specific Anammox Activity test calculations, Pilot Reactor 2, April5, 2011.

t (min)

P (mV)


0 2.6 2.4 2.0

20 6.2 5.5 5.8

40 12.4 11.7 12.2

60 14.0 13.3 13.0

80

100

120

P(mV) / min 0.202 0.1945 0.197

P(mmHg) / min 0.5353 0.515425 0.52205

g N / min 1.04793E-05 1.00902E-05 1.02199E-05


SAA g N / dm2 2.152865737 2.072932603 2.099576981

SAA g N / dm2 2.108458441



XXVI


t (min)

P (mV)


0 2.3 1.9 1.9

20 7.0 7.6

40 10.3 11.0 10.9

60 12.7 12.9 12.5

80 17.1

100

120

P(mV) / min 0.1725 0.182 0.184571429

P(mmHg) / min 0.457125 0.4823 0.489114286

g N / min 8.9489E-06 9.44174E-06 9.57514E-06


SAA g N / dm2 1.838462078 1.939710714 1.96711636

SAA g N / dm2 1.915096384



XXVII


t (min)

P (mV)


0 3.3 3.4 3.3

30 8.4

60 12.3 8.3 12.5

90 17.5 16.0 16.8

120 21.0 16.8

P(mV) / min 0.15 0.120666667 0.148666667

P(mmHg) / min 0.3975 0.319766667 0.393966667

g N / min 7.78165E-06 6.25991E-06 7.71248E-06


SAA g N / dm2 1.598662676 1.286035308 1.584452341

SAA g N / dm2 1.489716775



XXVIII


t (min)

P (mV)


0 2.6 2.7 2.7

20 6.1 5.9 6.2

40

60 11.2 11.0 11.3

80 14.0 13.9 13.6

100

120

P(mV) / min 0.1395 0.1375 0.1345

P(mmHg) / min 0.369675 0.364375 0.356425

g N / min 7.23694E-06 7.13318E-06 6.97755E-06


SAA g N / dm2 1.486756289 1.465440786 1.433467533

SAA g N / dm2 1.461888203



XXIX


t (min)

P (mV)


0 3.1

30 5.3 5.4 6.0

60 9.2 9.3 10.3

90 12.8 12.0 13.0

120

P(mV) / min 0.125 0.11 0.113333333

P(mmHg) / min 0.33125 0.2915 0.300333333

g N / min 6.48471E-06 5.70655E-06 5.87947E-06


SAA g N / dm2 1.332218897 1.172352629 1.207878466

SAA g N / dm2 1.237483331



XXX


t (min)

P (mV)


0 1.9 2.2 1.9

30 7.2 7.0

60 11.6 13.0 11.0

90 17.4

120

P(mV) / min 0.161666667 0.17047619 0.151666667

P(mmHg) / min 0.428416667 0.451761905 0.401916667

g N / min 8.38689E-06 8.84391E-06 7.86812E-06


SAA g N / dm2 1.723003107 1.816892819 1.616425595

SAA g N / dm2 1.71877384



XXXI

APPENDIX III. COMPARISON OF THE TEST RESULTS , ACCORDING TO FREQUENCY OF SAMPLES CONTROL

Table37. Comparison of the results Specific Anammox Activity test according to frequency of samples control, Pilot Reactor 2.

NORMAL SAA, R2, every 20 minutes NORMAL SAA, R2, every 30 minutes

Time (min) 1 2 3 4 5 6

0 2.6 2.7 2.7 2.9 2.7 2.8

10

20 6.1 5.9 6.2

30 7.8 7.8 8.3

40 2.1 2.2 2.1

50

60 11.2 11.0 11.3 11.7 12.7 13.3

70

80 14.0 13.9 13.6

90 14.7 16.3 16.6

100 15.3 14.8 14.4

110

120 17.9 17.1 16.5 19.1 19.6 12.0

SAA = 1.461888203 SAA = 1.611688816


XXXII

APPENDIX IV. OXYGEN UPTAKE RATE TEST DATA

Fig. 63.OUR results, Pilot Reactor 2, March 2, 2011, Rep. I.

Fig. 64. OUR results, Pilot Reactor 2, March 2, 2011, Rep. II.

Fig. 65. OUR results, Pilot Reactor 2, March 2, 2011, Rep. III.


XXXIII

Fig. 66. OUR results, Pilot Reactor 1, March 10, 2011, Rep. I.

Fig. 67. OUR results, Pilot Reactor 1, March 10, 2011, Rep. II.

Fig. 68. OUR results, Pilot Reactor 1, March 10, 2011, Rep. III.

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