evaluation of bacteria population dynamics in … of bacteria population dynamics in mainstream...

Water and Environmental EngineeringDepartment of Chemical EngineeringMaster Thesis 2014

Nelson Llano and Vadim Galkin

Evaluation of bacteria population dynamics in mainstream anammox

Postal address Visiting address Telephone P.O. Box 124 Getingevägen 60 +46 46-222 82 85 SE-221 00 Lund, Sweden +46 46-222 00 00 Web address Telefax www.vateknik.lth.se +46 46-222 45 26

Evaluation of bacteria population dynamics in mainstream anammox

by


Master Thesis number: 2014-07

Water and Environmental Engineering Department of Chemical Engineering

Lund University

June 2014

Supervisor: Professor Jes la Cour Jansen Co-supervisor: PhD David Gustavsson R& D Engineer, VA SYD Examiner: Associate professor Karin Jönsson

Picture on front page: Sjölunda WWTP. Photo from www.vasyd.se.

Abstract

Nitrogen removal is an important part of the wastewater treatment. Biological nitrogen removal process involves significant costs for its implementation at the wastewater treatment plants. It is due to the required hydraulic retention time that implicates basins with big areas and also for its daily operation due to amount of energy consumed by the aeration units and the required external addition of carbon sources such as methanol, to meet the wastewater quality standards. If the process efficiency does not fulfil the standard or if the process is not stable, the consequences can be dramatic for the recipient ecosystems. Now there is increasing pressure is in this area as the energy cost becomes higher, population is growing increasing the wastewater load and the effluent quality standards become stricter. To meet new challenges the wastewater treatment has to become more and more sustainable. Recently discovered in nature anammox bacteria give an idea of the way to reach the goal.

The ANAMMOX – ANaerobicAMMonium OXidation process involves simultaneous uptake of ammonium and nitrite in the wastewater and conversion of these compounds to inert atmospheric nitrogen gas by the anammox bacteria. This process requires less energy inputs in comparison with conventional nitrification-denitrification method of nitrogen removal. No external carbon source is needed as denitrifying heterotrophic bacteria do not participate in the process. These lead to a great saving and optimizing potential. Although the new process is already proved to be efficient in sidestream treatment systems (wastewater from digesters) the technology has few challenges to be solved before it can be implemented for mainstream (i.e. municipal) wastewater treatment.

This master thesis deals with one of the challenges – the optimization of ammonia and nitrite oxidising bacteria activities – to be able to precondition the inflow wastewater with a required proportion of ammonium and nitrite concentrations for the anammox bacteria activity. The thesis project work includes two parts. First is the development and adaptation of the method to be used for evaluation of the activities of the bacteria involved in the process. Second part is the application of the adapted method for evaluation of the behaviours of these bacteria at different conditions to find parameters which are important for the treatment process. As a result of the project work the oxygen uptake rate measurements method, further developed during the project, is evaluated and it demonstrated to be reliable, accurate and applicable for the needs of the project. The storage time of samples, dissolved oxygen and temperature conditions have been found to have the most significant influence on the bacteria activities. The impacts of different individual parameter variations are assessed and the real time bacteria population dynamics from the treatment process at Sjölunda pilot plant is evaluated by means of the method. One of the important conclusions of the present research is the demonstrated evidence of strong correlation between the adaptation conditions of the ammonia and nitrite oxidising bacteria and their activities and behaviours at varied conditions.

Keywords: Activity test, anammox, AOB, biofilm, endogenous respiration, heterotrophic bacteria, nitrogen removal, NOB.

Preface

This project was carried out at Water and Environmental Engineering, Department of Chemical Engineering at Lund University.

For this Master thesis project Jes la Cour Jansen from Water and Environmental Engineering and David Gustavsson from VA SYD were our supervisors. We would like to thank our supervisors for providing us with advice and all the necessary information and for their regular feedback.

All the samples used were taken from mainstream anammox pilot plant at Sjölunda Wastewater Treatment Plant with the help of our supervisor David Gustavsson.

The theoretical base for the current research is the previous work made by Marinette Hagman and Jes la Cour Jansen (Hagman and Jansen, 2007) regarding Oxygen Uptake Rate methodology, and the handbook “Wastewater Treatment. Biological and Chemical Processes” by Jes la Cour Jansen, Mogens Henze, Poul Harremoës and Erik Arvin (Henze et al., 1992) and the PhD thesis work made by Jes la Cour Jansen regarding biofilm kinetics principles (Jansen, 1983).

We want to thank SalarHaghighatafshar for sharing his experience and statistical data concerning the TS measurements method and Gertrud Persson at Water and Environmental Engineering, Department of Chemical Engineering at Lund University who helped us in the laboratory.

Vadim Galkin is a scholarship holder of Swedish Institute and he would like to acknowledge Swedish Institute as this master thesis has been produced during his scholarship period at Lund University thanks to financial support from Swedish Institute.

Finally we want to extend our warmest thanks to our families who have supported us during this challenging project.


Lund, June 2014

Abbreviations

Anammox Anaerobic ammonium oxidation

AOB Ammonium oxidising bacteria

ATU Allylthiourea

COD Chemical oxygen demand

DO Dissolved oxygen

HB Heterotrophic bacteria

Manammox Mainstream anaerobic ammonium oxidation

MBBR Moving bed biofilm reactor

NOB Nitrite oxidising bacteria

OUR Oxygen uptake rate

SOUR Specific oxygen uptake rate

Table of Contents 1 Introduction 1

1.1 Aim 1 1.2 Limitations 1

2 Background and Theory 3

2.1 The natural nitrogen cycle 3 2.2 The effects of additional anthropogenic release of different forms of nitrogen from wastewater treatment facilities to the environment 3 2.3 Conventional nitrogen removal 4 2.4 Anammox technology 5 2.5 Biofilm kinetics 5 2.6 Hydrolysis 8 2.7 Sjölunda Wastewater Treatment Plant 8

3 Materials and Methods 11

3.1 OUR experiments set-up, equipment and operating conditions 11 3.2 Experimental design 13

4 Results 25

4.1 Set of experiments 1. Influence of concentration of carriers in the reactor on bacteria activity 25 4.2 Set of experiments 2. Influence of HB activity on calculation of AOB and NOB activities 30 4.3 Set of experiments 3. Influence of the duration of the experiment on the bacteria activities 38 4.4 Set of experiments 4. Influence of substrates addition patterns, pH and storage time of samples on bacteria activities 41 4.5 Set of experiments 5. Influence of low temperature on bacteria activities 53 4.6 Set of experiments 6. Influence of the mixing of biofilm adapted to different conditions on the bacteria activities 59 4.7 Set of experiments 7. Influence of low DO concentrations on bacteria activities 61 4.8 Set of experiments 8. Influence of temperature stress and lower DO concentration conditions on bacteria activities 66 4.9 Set of experiments 9. Influence of storage time and temperature stress under 2 weeks storage time condition on bacteria activities 74 4.10 Set of experiments 10. Correlation between DO concentrations, thicknesses of oxygen penetration into the biofilm and bacteria activities 81

5 Discussion 87

6 Conclusions 91

7 Suggestions for further research 93

8 References 95

Appendix I

1

1 Introduction With regulations concerning residual nutrients in the effluent from wastewater treatment plants becoming stricter, incoming load to the WWTP trending to increase due to population growth and people’s diet variation and with the energy becoming more expensive, the nitrogen removal processes have to become more efficient and sustainable.

Recently discovered anaerobic ammonium oxidising (anammox) bacteria give an exciting opportunity to move towards energy-neutral or even energy-positive wastewater treatment. They are part of the natural nitrogen cycle and create a shortcut in deammonification process.

Anammox treatment technology requires less energy inputs in comparison with conventional nitrification-denitrification method due to less aeration and less recirculation. No external carbon source is needed as the process is fully autotrophic and, in addition, more organic matter in the raw wastewater can be separated and utilized for increased biogas production. These lead to a great saving and optimizing potential (Siegrist et al., 2008).

The Anammox method has been proved to be efficient by several full-scale installations treating ammonia-rich industrial wastewaters and sludge liquors, both of elevated temperatures (Lackner et al., 2014). The current challenge is to adjust the technology to optimize its use for municipal mainstream wastewater treatment, called Manammox (mainstream anammox).

1.1 Aim One of the issues for Manammox is the suppression of the activity of Nitrite Oxidising Bacteria (NOB) under conditions of the mainstream wastewater flow to obtain the required proportion of ammonium and nitritefor anammox. Thus thorough research of the nitrification process and the activities of bacteria involved are important.

The aim of this research project is to further develop, assess and apply the oxygen uptake rate measurements method to evaluate microorganisms behaviour and get more understanding of the dynamics of different groups of bacteria in a mixed culture, living in a form of the biofilm on the support medium (Moving Bed Biofilm Reactor, MBBR). Understanding of the influence of different operational conditions on the bacteria activities helps to develop reasonable and scientifically proven operational strategies for the pilot- and full scale applications of the Manammox technology.

1.2 Limitations Manammox process includes the activity of ammonium oxidising bacteria (AOB), NOB, heterotrophic bacteria (HB) and anammox bacteria in a mixed culture. This master thesis is focused on the activities of the aerobic groups of bacteria i.e. AOB, NOB and HB, but not the anammox bacteria. The study does not take into account also possible NOB activity in anoxic conditions – heterotrophic denitrification by NOB – with the presence of organic matter (Winkler et al., 2012).

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2 Background and Theory

2.1 The natural nitrogen cycle Nitrogen is an essential element for the biological life on the planet. Many oxidation and reduction reactions of consumption and production of different nitrogen compounds (see Figure 2.1), assisting the growth of all forms of life, from microorganisms to animals and humans (Bothe, Ferguson and Newton, 2006).

Figure 2.1. Schematic representation of the biological nitrogen cycle.

Organic nitrogen in the forms of amino acids and proteins is essential for the development of the higher living forms. After the death of the organism or from its excreta the organic nitrogen is released into the ecosystem and is then converted to ammonium by saprobiotic microorganisms. Nitrogen gas from the atmosphere is taken by microorganisms, plants and animals and is converted to ammonium by the process of nitrogen fixation (Bothe, Ferguson and Newton, 2006).

Further steps in the natural nitrogen cycle include the aerobic processes of nitrification (conversion of ammonium to nitrite and subsequently nitrite to nitrate), denitrification (conversion of nitrate to nitric oxide, nitrous oxide and subsequently nitrogen gas) and anaerobic ammonium oxidation (simultaneous uptake of ammonium and nitrite and conversion to nitrogen gas) (Bothe, Ferguson and Newton, 2006).

2.2 The effects of additional anthropogenic release of different forms of nitrogen from wastewater treatment facilities to the environment

Although nitrogen in different forms is required for living organisms on the planet, the extended excess of inorganic nitrogen in the ecosystems can lead to extremely adverse effects on the environment and the implicated species, including humans (Camargo and Alonso, 2006).

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The most relevant issues associated with long term high concentrations of inorganic nitrogen are: the acidification of freshwater ecosystems, eutrophication, the occurrence of toxic algae and the toxicity of nitrogen forms of ammonia, nitrite and nitrate (Camargo and Alonso, 2006).

Eutrophication of water bodies leads to low oxygen zones (anoxia zones) and create conditions for the formation of highly toxic substances such as hydrogen sulphide (H2S), which increase the negative impact of additional nutrients release (Camargo and Alonso, 2006).

In turn, all these effects put high pressure on public health, natural resources and economies. The direct losses occur with the reduction of fish and other aquatic organisms populations.

Either endogenic or other species with market or biological value can also accumulate the pollutants and toxins and can have adverse impact on human health. Drinking water polluted with inorganic nitrogen put high health risks. These lead to increased medical costs and work day’s losses due to treatment (Camargo and Alonso, 2006).

Additional treatment is required for drinking water preparation, which increases the price of tap water.

Decontamination and ecosystem restoration measures of high cost have to be undertaken.

2.3 Conventional nitrogen removal Following the natural nitrogen transformation processes the conventional biological nitrogen removal in Wastewater Treatment Plant (WWTP) is accomplished in two steps.

1) Nitrification – a) aerobic conversion of ammonium to nitrite by AOB and b) conversion of nitrite to nitrate by aerobic NOB, i.e. NH4

+Æ NO2-Æ NO3

-;

2) Denitrification – anaerobic conversion of nitrate to nitrogen gas by heterotrophic denitrifying bacteria, i.e. NO3

-Æ N2. As denitrifying bacteria are heterotrophic organisms they use organic matter as the source of energy.

In practice the treatment can be designed as post-denitrification or pre-denitrification process depending on the characteristics of the influent wastewater.

Post-denitrification process includes the nitrification step followed by denitrification step. Here the external carbon source has to be added at the second step to assist heterotrophic bacteria to reduce nitrate to nitrogen gas.

If the incoming wastewater contains high concentration of readily degradable organic matter pre-denitrification process can be applied with denitrification basin at the first place followed by nitrification basin. Here the requirement for external carbon is lower. However recirculation of the wastewater is necessary to send the nitrified wastewater back to denitrification step.

In conventional biological nitrogen removal process both the activated sludge and biofilm-based systems are used.

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2.4 Anammox technology ANAMMOX – ANaerobic AMMonium OXidation process involves simultaneous ammonium and nitrite uptake by the anammox bacteria that turn it directly to nitrogen gas, using a combination of these substrates as an energy source.

The optimal temperature for the activity of anammox bacteria varies depending on the prior long-term growth and adaptation temperature. For the bacteria adapted to 30ºC the maximum activity is at around 37ºC while for those adapted to 12ºC the maximum activity is at around 25ºC (Hu et al., 2013).

The dissolved oxygen and organic matter are two inhibitors of the activity of anammox bacteria. The activity of these bacteria is also suppressed by nitrite concentrations above 150 mg NO2

--N/L (Gustafsson, 2013).

The activity is independent of the pH in a range of 6.7 to 8.3 (Strous et al., 1999).

As anammox bacteria simultaneously consume ammonium and nitrite to convert it directly to nitrogen gas the wastewater has to be preconditioned during the process with a resulting 50% NH4

+ to 50% NO2- ratio suitable for anammox bacteria to be active (van Dongen et al., 2001).

Several treatment processes have been developed since the discovering of anammox bacteria. Sharon® technology of partial nitration followed by anammox process in two serial reactors (van Dongen et al., 2001) and Canon® reactor of simultaneous partial nitritation-anammox process has been implemented in full scale at several installations (van Dongen et al., 2001). The working conditions for both processes are mesophilic temperatures (25ºC to 40ºC) and high ammonium concentrations (van Dongen et al., 2001; Hu et al., 2013). Thus the existing techniques are used for treatment of sludge liquors from municipal wastewaters and suitable industrial wastewaters.

2.5 Biofilm kinetics The bacteria behaviour in the biofilms is analogous to that in suspended systems (activated sludge). The difference between two systems is that the density of the biofilms and concentration of bacteria in the biofilms is several times higher. For example, the concentration of the volatile suspended solids in the activated sludge lies within the range of 2 to 6 kg/m3, whereas in biofilms the range is from 10 to 60 kg/m3 (Henze et al., 1992).

General simplified picture of the processes inside the biofilm is shown in Figure 2.2 below.

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Figure 2.2. Simplified conceptual model for removal of soluble substrates focusing on phenomena inside the film. Figure taken with permission from Jansen (1983). Diffusion coefficient D is a new parameter introduced for biofilm systems compared to activated sludge systems. Existing methods for determination of this coefficient in biofilms are not highly reliable and the coefficient itself is variable depending on the conditions and other parameters of the biofilm. Thus for practical reasons it can be assumed equal to the molecular diffusion coefficient in water with the correction coefficient of 0.8 (Henze et al., 1992).

In turn molecular diffusion coefficient in the water depends on the water temperature and theoretical values can be found using correlation developed by Wilke and Chang (1955), which is based on the Stokes-Einstein equation:

𝐷𝑂2−𝐻2𝑂 = 7.4 ∗ 10−8 𝑇(𝜓𝐻2𝑂∗𝑀𝐻2𝑂)12

𝜇∗𝑉𝑂20.6 (1)

where:

T – absolute temperature, ºK;;

𝜓𝐻2𝑂 – association parameter for the solvent water = 2.26 (Reid et al., 1977);

𝑀𝐻2𝑂 – molecular weight of water = 18 g/mole;

µ - dynamic viscosity of water, centipoise (mPa*s);

𝑉𝑂2- the molar volume of oxygen = 25.6 cm3/g - mole (Welty et al., 1984).

From Henze et al. (1992), the growth rate of bacteria can be described by:

𝑟𝑣,𝑥𝑏 = 𝜇𝑚𝑎𝑥 ⋇𝑆

𝑆+𝐾𝑠⋇ 𝑋𝑏 (2)

where:

rv, xb– specific volumetric biomass production rate, kg/(m3*day) (v – volume inside the biofilm);

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𝜇𝑚𝑎𝑥 – maximum specific growth rate, day-1;

𝑆 – substrate concentration, kg/m3;

𝐾𝑠 – substrate saturation constant, kg/m3;

𝑋𝑏 – biomass concentration, kg/m3.

For calculations of volumetric uptake rate of substrate by the biofilm, density of the biofilm can be reasonably assumed equal to water density as the weight of dry biofilm in relation to the volume of wet biofilm is relatively very low (Hoehn and Ray, 1973).

The substrate uptake rate in the biofilm systems is dependent on the concentration of substrate at the surface of the biofilm, the diffusion coefficient of substrate and the biofilm thickness. Depending on these parameters the uptake rate change can follow first, ½ or zero order reaction (Henze et al., 1992).

In practice during substrate uptake rate experiments the reaction rate follows zero order reaction when the biofilm is fully penetrated by the substrate and ½ order reaction when the biofilm is partly penetrated (see Figure 3.3). The degree of penetration of the substrate into the biofilm is a function of the substrate concentration at the biofilm surface. Biofilm thickness L can be calculated with the formula shown in Figure 3.3 below, where Schange – substrate concentration at changing point; 𝑟𝑎 ,𝑚𝑎𝑥 – maximum surface uptake rate of substrate (Henze et al., 1992).

Figure 2.3. The change from 1/2 order to zero order reaction in the water. Figure taken with permission from Henze et al. (1992). For calculations of surface uptake rate of substrate by the biofilm, the effective area of the carriers, 70% filling of total protected area, should be found as this corresponds to real operating conditions of MBBR carriers without the detachment of the biofilm (Ødegaard, 1999).

From Henze et al. (1992), the substrate uptake rate reactions k0Vf, k1/2Vf , and k1Vf can be described by:

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Zero order reaction:

𝑘0𝑣𝑓 = 𝑟𝑣,𝑠 = 𝜇𝑚𝑎𝑥𝑌𝑚𝑎𝑥

⋇ 𝑋𝑏 (3)

½ order reaction:

𝑘1/2𝑣𝑓 = 2𝐷𝑘0𝑣𝑓

𝐿 (4)

First order reaction:

𝑘1𝑣𝑓 ∗ 𝑆 = 𝑟𝑣,𝑠 = 𝜇𝑚𝑎𝑥𝑌𝑚𝑎𝑥

𝑆𝐾𝑠

∗ 𝑋𝑏 (5)

where:

𝑟𝑣,𝑠 – volumetric substrate removal rate, kg/(m3*day);

𝑌𝑚𝑎𝑥 – maximum coefficient of biomass growth, kg biomass/kg substrate.

2.6 Hydrolysis Hydrolysis is a process of breakdown of the high molecule organic matters in smaller parts by addition of water (Morgenroth et al., 2002). Hydroxyl ion from water molecule is splitting the organic molecule in two parts and the hydrogen and hydroxyl ions are connecting with each part of the split molecule (Ellervik and Sterner, 2007).

The bacteria, including those bacteria groups that can be found in the wastewater and subsequently in the biofilm at WWTP, can release certain enzymes to the bulk water or attach enzymes to the bacterial membrane (Confer and Logan, 1998). The purpose is to speed up the hydrolysis process in order to obtain the organic particles in smaller size which are easier to consume (Dimock and Morgenroth, 2006). The size of the particles is of high importance for their biological availability and the attachment ability (Morgenroth et al., 2002).

2.7 Sjölunda Wastewater Treatment Plant The Sjölunda wastewater treatment plant (WWTP) is located in the northern part of Malmö, Sweden. It was built in 1963 and has been upgraded several times making the treatment process more environmentally friendly. Some examples are: the large storage facility for sludge dewatering erected in 1980 in order to implement sludge as a fertilizer on agriculture areas and the upgrading of the biological nitrogen removal process in 1999. Currently the design capacity of the plant is of 550 000 population equivalent (pe), which makes it one of the largest WWTP in Sweden. At the present time the plan runs with the load from circa 300 000 inhabitants. The wastewater comes mainly from Malmö and Burlöv municipalities, but also wastewaters from Lomma, Staffanstorp and Svedala are treated for nutrients removal at this WWTP.

VA SYD is a municipal organization operating in the southwest of Scania in Sweden; it is in charge of all pumping and treatment activities for the drinking water and wastewater in the region. VA SYD is responsible for the operation and the administration of Sjölunda WWTP.

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In 1972 a research station was built at Sjölunda facility, and then it has been upgraded between 1988 and 1995 for studying of nutrient removal at a pilot plant scale. In 2012 a project aiming to understand how the Anammox process can be implemented in the full mainstream scale started at the pilot plant at Sjölunda (Manammox pilot). VA SYD develops this project in close cooperation with Lund University. The present study was carried out at the Water and Environmental Engineering at the department of Chemical Engineering, Lund University.

The Manammox pilot plant consists of three main moving bed biofilm reactors, mainstream reactor 1 (MP1), mainstream reactor 2 (MP2) and sludge liquor reactor (RP). As influent MP1 receives the effluent water from the High Loaded Activated Sludge reactor (HLAS) mixed with the effluent from RP. Then the effluent from MP1 is used as influent to MP2. The RP receives the effluent from sludge liquor reactor as influent (see Figure 2.4 below) (Gustavsson et al., 2013).

Figure 2.4. Sjölunda pilot plant layout. Figure taken with permission from Gustavsson et al. (2013). Thus general conditions of the growth and adaptation of the biofilm from these reactors are following: mainstream flow temperature at MP1 and MP2 (depends on the season, 17 ºC on average), high concentrations of substrates at MP1 (depends on the conditions in HLAS reactor) and low concentrations of substrates at MP2; elevated temperature at RP (around 28ºC) and very high concentrations of substrates at RP (depends on the conditions in sludge liquor reactor) (Gustavsson et al., 2013). During normal operation pH varies between 7.6 and 7.7 pH units in the pilot plant effluent and the dissolved oxygen (DO) levels are kept at around 2 mg O2/L in MP1 and MP2 reactors and below 2 mg O2/L in RP reactor (Gustavsson et al., 2013).

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Every second working day biofilm carriers are exchanged between the mainstream reactors and the sludge liquor reactor at the pilot plant (Gustavsson et al., 2013). Around 3.15% and 11% of all carriers, in the mainstream and the sludge liquor pilot respectively, were moved at each occasion initially and these numbers were doubled after few month of operation of the plant (Gustavsson et al., 2013).

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3 Materials and Methods

3.1 OUR experiments set-up, equipment and operating conditions The OUR measurement approach described by Hagman and Jansen (2007) was used as a basis for developing a series of experiments for studying nitrifying bacteria’s activity in the biofilm attached to the carriers AnoxKaldnes type K1, sampled at Sjölunda Manammox pilot plant.

The idea of the OUR experiment is to find the oxygen consumption in the sample during a certain period of time. The respiration is associated with certain group or several groups of microorganism depending on the conditions of the experiment. Substrates and the inhibitor are used to control and differentiate between the activities of various bacteria groups in a mixed culture. The decline of the oxygen concentration usually follows a straight line within a certain timeframe and when the linear regression of this decline is related to time, the oxygen utilization rate can be found using the following formula:

b = ∑ (x-x)(y-y)∑ (x-x)2 (6)

where x is the means average of the time period and y is the means average of the oxygen concentrations.

The carriers sample is then re-aerated for a specific period of time in order to obtain the next decline and follow the experiment for longer period (Hagman and Jansen, 2007).

The oxygen utilization rate (OUR) can be related to the volatile solids (VS) to find the specific oxygen uptake rate (SOUR). However in this study the OUR was related to the total solids (TS) instead to eliminate errors due to the complexity of the method of VS analysis for the biofilm attached to plastic medium. The TS were measured by drying of 30 carriers sampled from the reactor after the experiments for 24 hours at 105°C, followed by weighting of these dried carriers. The weight of 30 empty dry carriers (without a biofilm) was subtracted from the obtained weight of 30 dried carriers with biofilm to find the weight of the biofilm sampled. After the calculations the total weight of biofilm per 1 liter of wastewater in the reactor was found.

A lab-scale batch reactor with 400 ml of tap water and 128 carriers was operated at 28 ± 0.5°C (see Figure 4.1). This number of carriers to the volume of water relation represents the actual concentration of carriers at the pilot plant. Temperature was kept constant by means of the water bath. The pH was stable during the experiments with a small increase due to CO2 stripping during aeration (Hagman and Jansen, 2007). pH adjustment to 7.5 through the buffer solution was implemented as its variations in preliminary experiments always were lying within the range of 7.5 to 8.2 and no effect on the process was noticed.

Another identical lab-scale reactor was operated in parallel without pH adjustment and without substrates addition, but keeping constant the remaining conditions to obtain the endogenous respiration pattern.

The reactors were continuously stirred with a magnetic stirring at around 150 rpm (Surmaz-Gorska et al., 2010). The water solution in the first reactor was sampled at a certain time for

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analyses of nitrogen in different forms. Sampling was done by a syringe with a volume of 10 ml. Filtered samples were then stored in sealed cuvettes (test-tubes) to ensure no further activity in the sample.

Figure 3.1. A: Schematic picture of the laboratory set-up for OUR- measurements B: Laboratory batch 1 liter. Figure taken and adapted with permission from Hagman and Jansen (2007). During all the performed experiments the oxygen concentration and temperature were measured in the liquid phase by means of dissolved oxygen probe HACH HQ 40d from HACH (Loveland, Colorado, USA). Oxygen concentration measurements were taken every 10 seconds and were stored on the USB memory device. The pH of the sample in the reactor was continuously measured with the WTW pH 320 meter. Waterproof magnetic stirring equipment was used to ensure proper mixing. The timer with connected air pump controlled intermittent aeration.

Samples taken out from the reactor at different times during the experiments were analyzed for nitrogen concentrations in forms of ammonium, nitrite and nitrate with HACH Lange DR 2800 spectrophotometer (Sköndal, Sweden) using HACH Lange cuvettes LCK303 (2.0-47.0 mg/L NH4

+-N) and LCK304 (0.015-2.0 mg/L NH4+-N) for ammonium, LCK341 (0.015-0.6

mg/L NO2--N) and LCK342 (0.6-6.0 mg/L NO2

--N) for nitrite, LCK339 (0.23-13.5 mg/L NO3

--N) and LCK340 (5.0-35.0 mg/L NO3--N) for nitrate.

All the OUR graphs with corresponded dissolved oxygen concentration decline lines regressions and nitrogen concentrations were plotted using Microsoft Excel.

In the present research a cycle of 5 minutes without aeration was followed by 5 minutes with aeration. Three cycles for each part of the experiment were used to obtain representative data cleared from the residual effects of the preceding part of the experiment with different conditions.

Ammonium sulfate (NH4)2SO4 in a buffer solution was used as a source of ammonium, added to reach the final concentration of 100 mg NH4

+/L in the reactor. Buffer solution included 23.6 g of (NH4)2SO4, 61.2 g of NaHCO3 and 4.4 g of KH2PO4 per 1 liter of distilled water. Sodium nitrite (NaNO2) in water solution (12.33 g of NaNO2 per 1 liter of distilled water) was added as a source of nitrite to reach the final concentration of 75 mg NO2

-/L in the reactor. Ammonium oxidation inhibitor allylthiourea or ATU (C4H8N2S) in water solution was used at a final concentration of 86 μM in the reactor (Ginestet et al., 1998). For some experiments sulfuric acid (H2SO4) in water solution with the concentration of 0.1 M was added after the substrates addition as an acid source to keep a pH value within the range of 7.6 to 7.7 pH

K1 carrier

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units in the reactor which corresponds to the actual pH at the pilot plant. All the substrates and inhibitor were injected into the reactor using pipettes 30 seconds before the end of the aeration phase for achieving better mixing.

Studied biofilm samples include mixed culture of bacteria, specifically AOB, NOB, HB and anammox bacteria. Without biodegradable organic matter and ammonium and nitrite ions in the reactor, endogenous respiration is present (Hagman and Jansen, 2007). The oxygen uptake by heterotrophic bacteria can be reasonably approximated to endogenous respiration rate for experiments carried out during present research. The respiration of AOB and NOB is developed after the individual addition of ammonium and nitrite ions respectively.

Samples of biofilm carries for the experiments were taken from the mainstream reactors MP1 and MP2 where bacteria were accommodated to the temperature of 14ºC between winter and spring and the sidestream reactor RP where bacteria were accommodated to the temperature of 28ºC between winter and spring from the pilot plant at Sjölunda wastewater treatment plant in Malmö, Sweden and transported to the laboratory. Samples were stored at 4ºC if extended storage was required. Total solids (TS), temperature and pH were determined. For all experiments the biofilm samples were preconditioned by a gentle rinsing with tap water avoiding stress temperature and high pressure, in order to minimize initial substrates concentrations contained in the original wastewater that can be retained on the biofilm surface and to remove weakly attached biofilm from the carriers and thus prevent parallel ammonification process in the bulk. Samples were warmed up or cooled down slowly to the test temperature and were aerated until a minimum initial dissolved oxygen level of 7.0 mg O2/L was reached.

3.2 Experimental design In order to use OUR experiment for analyzing the activity of nitrifying bacteria (sets of experiments 1 to 9), the inhibition properties of ATU and microorganisms (bacteria) activities were investigated first for all three streams (experiments 1-1 to 1-3). The ATU is expected to inhibit AOB activity without affecting other microorganisms activities and substrates concentrations are expected not to limit microorganisms activities (Ginestet et al., 1998).

A preliminary experiment was designed based on the literature study and previous experiments with activated sludge (Llano and Galkin, 2014). Then a study of effects of an individual parameter on activities of the microorganisms was carried out. Table 3.1 below shows the initial assessed conditions named reference 0 and its changes until the final reference 4 obtained through this study. The first three columns at the left contain the parameter’s description and their associated values. The selected parameter and its desired set point for evaluation are identified with an “x”. The upper part, from column 4 to column 25 contains experiments numbers and short comments regarding any particular conditions.

Sets of experiments 1 to 5 provide information about the influence of different parameters on the applicability, reliability and the sensitivity of the OUR method.

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Reference 0NO2-N

# of carriers50 and 100%

Pure End. Resp100% carriers

Pure End. Resp50% carriers

Reference 1with End. Resp

Timer set point

Pure NH4

Reference 2Two individual addition of substrates

+ H2SO4 at step 3

Control pH

Reference 3Control pH

Fresh carriers

Low -TempWithout pH control

Low -TempWith pH control

Mixing RP:MP1

min DO in

Low DO inFresh carriers

Reference 4no Temp stress Fesh carriers

No Temp stress Fesh carriers

No Temp stress Old carriers

no Temp stress Old carriers

Pure end. Respirationpure O 2-one cycle

No temp stress

Pure end. Respirationpure O 2-one cycle

No temp stress

1-1

1-1/3

1-4

1-4

2-1

3-1

4-1

4-2

4-3

4-4 /6

4-7 /9

5-1/3

5-4/6

6-1/3

7-1/2

8-2,5 & 9

8-1,4, & 8

8-3,6 & 7

9-1,4 & 8

9-3,6 & 7

10-1,2 & 4

10-3

Un

Value

Carriers

#128

xx

xx

xx

x

#64

xx

Substratesaddition

NA

togetherx

x

separatedx

xx

Aeration tim

e 5 : 5

xx

xx

2 : 2x

NH

4-Nm

g/L100

xx

xx

xx

0x

x

NO

2-Nm

g/L75

xA

TU+ N

O2

ATU + N

O2

x

mg/L

50x

m

g/L0

xx

x

pHU

of pH 7.5 - 8.1

x7.2

xx

x

7.6 - 7.7x

xx

xx

xx

Storage time

NA

oldx

xx

xx

freshx

xx

xx

xx

xx

Temperature

C28

xx

xx

xx

x

C14

xx

xx

x

DO

in -O2

mg/L

≥ 7.0x

xx

xx

x

mg/L

≤ 6.0

x

mg/L

(3-1.5)x

Mixing

%pilot plant

xx

xx

MP1 : RP

50 : 50x

MP1 : RP

20 : 80x

80 : 20x

Stiring Speedrpm

150x

xx

xx

Reactor Vol

L0.4

xx

xx

x

Assesm

ent of the Influence of

the parameter on

microorganism

's activities

Comments

Exp Design #

Table 3.1.Design experiment matrix.

15

3.2.1 Set of experiments 1. Experiments 1-1 to 1-3: Evaluation of the volumetric filling fraction percentage effect on microorganisms activity.

First, three cycles of endogenous respiration were recorded (step 1). Then ammonium sulfate ((NH4)2SO4) and sodium nitrite (NaNO2) were introduced (29.5 minute, step 2) and another three cycles of bacteria respiration in the presence of both substrates were followed (60 minutes in total). Finally, ATU was added (55.5 minute, step 3) and another three cycles of bacteria respiration in the presence of the remaining both substrates and the inhibitor were followed (90 minutes in total). Thus by subtracting the oxygen consumption of HB (step 1- endogenous respiration) from the NOB + HB oxygen consumption (step 3 after the addition of ATU) the computed NOB oxygen consumption can be found. Then by subtracting the NOB + HB oxygen consumptions obtained in the first slope of step 3- after the addition of ATU from the oxygen consumption of all three microorganisms groups HB, NOB and AOB (step 2- after the addition of (NH4)2SO4 and NaNO2) the oxygen utilization of AOB can be found (see Figure 3.2 and Table 3.2 below).

Figure 3.2. SOUR vs. time sketch for simultaneous substrate addition and later inhibitor addition of AOB activity. Each SOUR point represents each decline line of oxygen concentration (oxygen consumption). Table 3.2. Calculation of AOB and NOB activities based on Figure 3.2. Activities expressed in mg O2/gTS*h.

Step Microorganisms present in R1 R1

1 HB 1

2 AOB +NOB + HB 2

3 NOB + HB 3

Computed AOB = 2 - 3

Computed NOB = 3 - 1

16

Two reactors were run for each stream. Samples from the reactors were taken at minute 20 and minute 85 from the start of the experiment – at the lowest points of each third decline lines – before and after the addition of both substrates and before and after the ATU addition to check for substrates and ensure no (or negligible) AOB activity after the inhibitor addition.

Experiments with carriers sampled from 3 streams correspond to experiments 1-1 to 1-3.

3.2.2 Set of experiments 1. Experiment 1-4: Evaluation of the development of endogenous respiration without the presence of inhibitors and without substrates addition

Nine cycles of endogenous respiration were followed (90 minutes in total). Two lab-scale reactors were run in parallel with carriers from the same batch (MP2 stream). Reactor 1 was run with 64 carriers and reactor 2 was run with 128 carriers. The remaining conditions were kept the same as for previous experiments of set 1.

The objective of this experiment is to check the hypothesis that HB activity is independent of the number of carriers, making possible to keep this parameter constant.

No samples were taken during the experiment as variations on OUR during the endogenous respiration, mainly correspond to the conversion of carbon sources present in the biofilm into CO2 by HB. A very low presence of nitrogen sources is assumed due to the use of tap water and no substrates addition, thus the AOB and NOB activities can be assumed negligible under these conditions.

3.2.3 Set of experiments 2. Experiments 2-1 to 2-3: Evaluation of HB activity on AOB and NOB computed activities

The lab-scale reactor 1 was run according with the procedure described above in experiment 1. A second lab-scale reactor 2 was run in parallel with reactor 1, without pH control and without substrates addition, but keeping the remaining conditions set for reactor 1. All cycles of endogenous respiration were recorded simultaneously in both reactors. Thus by subtracting the oxygen consumption of HB obtained in reactor 2 in the last slope line from the correspondent NOB + HB oxygen consumption (step 3 after the addition of ATU) the computed NOB oxygen consumption can be found. Then by subtracting the computed NOB and the HB oxygen consumptions from the oxygen consumption of all three microorganisms HB, NOB and AOB (step 2- after the addition of (NH4)2SO4 and NaNO2) the oxygen utilization of AOB can be found (see Figure 4.3 and Table 4.2 below).

The objective of this experiment is to check the hypotheses that the starving point for HB (equal slopes) cannot be reached during the defined endogenous respiration cycles (90 minutes in total), making necessary the construction and use of the HB-SOUR vs. time curve to distinguish clearly the activities of AOB and NOB from the HB activity from experiment 1.

17

Figure 3.3. SOUR vs. Time, endogenous respiration sketch. Table 3.3. Calculation of AOB and NOB activities based on Figure 3.2 and Figure 3.3. Activities expressed in mg O2/gTS*h.

Step Microorganisms present in R1 R1 R2 (HB only)

1 HB 1 a

2 AOB +NOB + HB 2 b

3 NOB + HB 3 c

Computed AOB = 2 - [NOB] - b

Computed NOB = 3 - c = [NOB]


3.2.4 Set of experiments 3. Experiments 3-1 to 3-3: Evaluation of the aeration/no aeration cycle time effect on microorganisms activity

First, three cycles of endogenous respiration were recorded with an aeration/no aeration set point of 2 minutes/2 minutes respectively. Then ammonium sulphate (NH4)2SO4) and sodium nitrite (NaNO2) were introduced (11.5 minute, step 2) and another three cycles of endogenous respiration in the presence of the both substrates were followed (24 minutes in total).Finally, ATU was added (23.5 minute, step 3) and another three cycles of endogenous respiration in the present of the remaining both substrates and the inhibitor were followed (34 minutes in total) HB, NOB and AOB activities can be found according with the mathematical method described above in experiments 1 and 2.

Samples from the reactor were taken at 8 minute and 34 minute from the start of the experiment – at the lowest points of each third decline lines – before and after the addition of ATU to check for substrates and ensure no (or negligible) ammonium oxidising bacteria activity.

The objective of this experiment is to check the hypothesis that the aeration/no aeration cycle time can be reduced conserving same amount of endogenous respiration cycles. Thus the HB, NOB and AOB activities should not differ from those values obtained with the previous set point of 5 minutes/5 minutes.

18


3.2.5 Set of experiments 4. Experiment 4-1: Evaluation of initial NO2- external addition

influence on AOB/NOB competition The lab-scale reactor 1 was run according with the procedure described above in experiment 2. Only ammonium sulphate (NH4)2SO4) was added as substrate.

Here for the calculations of bacteria activities the new method was proposed (see the comparative description of two methods on Figures 3.4 and 3.5 and Tables 3.3 and 3.4). By subtracting the oxygen consumption of HB obtained in reactor 2 in the first slope line of the third step from the correspondent NOB + HB oxygen consumption (step 3 first slope after the addition of ATU) the computed NOB oxygen consumption can be found. Then by subtracting the computed NOB and the HB oxygen consumptions from the oxygen consumption of all three microorganisms HB, NOB and AOB (step 2- after the addition of (NH4)2SO4) the oxygen utilization of AOB can be found (see Table 3.4 below). The aim for introduction of the new method for calculation of the activities was to find the NOB activity at point 3, step 3 (see Figure 3.4) corresponding to the same conditions in terms of availability of NO2

- for NOB bacteria as the conditions during simultaneous AOB and NOB activity (step 2). For all further calculations it was decided to use the new proposed calculation method as more precise.

The objective of this experiment is to check the hypothesis that the NOB/AOB activity ratio is influenced by the early addition of NO2

- source, making it difficult to distinguish AOB activity from NOB activity under simultaneous substrates addition.

Figure 3.4. SOUR vs. time sketch for separate substrate and inhibitor addition. Each SOUR point represents each decline line of oxygen concentration (oxygen consumption).

19

Figure 3.5. SOUR vs. Time, endogenous respiration sketch. Table 3.4. Calculation of AOB and NOB activities based on Figure 3.4 and Figure 3.5. Activities expressed in mg O2/gTS*h.


1 HB 1 a

2 AOB +NOB + HB 2 b

3 NOB + HB 4 d


Computed NOB = 4 - d = [NOB]

Table 3.5. Calculation of AOB and NOB activities based on Figure 3.4 and Figure 3.5. Activities expressed in mg O2/gTS*h.


1 HB 1 a

2 AOB +NOB + HB 2 b

3 NOB + HB 3 c


Computed NOB = 3 - c = [NOB]

20

3.2.6 Set of experiments 4. Experiment 4-2: Evaluation of individual substrates addition influence on AOB/NOB competition

The lab-scale reactor 1 was run according with the procedure described above in experiment 2. Ammonium sulfate (NH4)2SO4) was added as substrate in step 2 and sodium nitrite (NaNO2) was introduced simultaneously with ATU in step 3 (see Figure 3.6). AOB and NOB activities can be found by following the same procedure described in experiment 4-1.

The objective of this experiment is to check the hypotheses that the individual additions of nitrogen sources help to distinguish clearly AOB activity from NOB activity.

Figure 3.6. SOUR vs. time sketch for separate substrates addition. Each SOUR point represents each decline line of oxygen concentration (oxygen consumption).

3.2.7 Set of experiments 4. Experiment 4-3: Evaluation of pH influence on NOB activity

The lab-scale reactor 1 run according with the procedure described above in experiment 2. Sodium nitrite (NaNO2) and ATU were introduced simultaneously at step 3 (see Figure 3.4), then sulfuric acid (H2SO4) 0.1M solution was used as acid source to control pH in the reactor within the range of 7.6-7.7. AOB and NOB activities can be found by following the same procedure described above in experiment 4.

The objective of this experiment is to check the hypothesis that pH control is not required in HB and NOB activities assessment by using the present OUR method for carriers.

3.2.8 Set of experiments 4. Experiments 4-4 to 4-6: Evaluation of pH influence on AOB activity

The lab-scale reactor 1 was run according with the procedure described above in experiment 4-3. Sulfuric acid (H2SO4) 0.1M solution was used as acid source to control pH in the reactor within the range of 7.6-7.7 with additions between step 2 and step 3. AOB and NOB activities can be found by following the same procedure described above in experiment 4.

The objective of this experiment is to check the hypothesis that pH control is not required in HB, AOB and NOB activities assessment by using the present OUR method for carriers.

21

Assessment of MP1, MP2 and RP streams can be found in experiments 4-4, 4-5 and 4-6 respectively.

3.2.9 Set of experiments 4. Experiments 4-7 to 4-9: Evaluation of storage time of carriers influence on AOB/NOB competition

The procedure described above in experiment 4-4 was followed here. Biofilm carriers were sampled in the morning and experiments were performed as soon as the samples arrived to the lab. AOB and NOB activities can be found by following the same procedure described in experiment 4.

The objective of this experiment is to check the hypothesis that a zero storage time influences AOB/NOB competition and HB activity assessments with the present method.

Assessment of MP1, MP2 and RP streams corresponds with experiments 4-7, 4-8 and 4-9 respectively.

3.2.10 Set of experiments 5. Experiments 5-1 to 5-3: Evaluation of low temperature influence on AOB/NOB competition with sample after storage

The procedure described above in experiment 4-4 was followed here, but temperature setting point was moved from 28°C to 14°C. Experiments were performed with carriers sampled 11 days before performing the experiments. AOB and NOB activities can be found by following the same procedure described in experiment 4.

The objective of this experiment is to check the hypothesis that a low temperature depresses AOB, NOB and HB activity.

Assessment of MP1, RP and MP2 streams correspond with experiments 5-1, 5-2 and 5-3 respectively.

3.2.11 Set of experiments 5. Experiments 5-4 to 5-6: Evaluation of low temperature influence on AOB/NOB competition with fresh sample

The procedure described above in experiment 4-4 was followed here, but temperature setting point moved from 28°C to 14°C. Experiments were performed with carriers sampled 1 day before performing the experiments. AOB and NOB activities can be found by following the same procedure described in experiment 4.

The objective of this experiment is to check for the influence of the storage of sample on the AOB, NOB and HB activity in the conditions of low temperature.

Assessment of MP1, MP2 and RP streams correspond with experiments 5-4, 5-5 and 5-6 respectively.

Following experiments (set of experiments 6 to set of experiments 10) are dedicated to evaluation of aerobic bacteria activities in the process at the pilot plant applying the method developed and adapted during the first part of the project.

3.2.12 Set of experiments 6. Experiments 6-1 to 6-3: Evaluation of RP/MP1 carriers ratio influence on AOB/NOB competition

The procedure described above in experiment 4-4 was followed here. Carriers from RP and MP1 streams were mixed in different percentiles of 50:50, 20:80 and 80:20 respectively to

22

generate three differently combined samples of 128 carriers. Experiments were performed with carriers sampled 2 days before performance of the experiments. Other changes included temperature setting point of 28°C and pH adjustment at 7.6-7.7. AOB and NOB activities can be found by following the same procedure described in experiment 4.

The objective of this experiment is to check the hypothesis that RP carriers enhance activities of the microorganisms.

Assessment of 80:20, 50:50 and 20:80 RP:MP1 carriers percent ratios corresponds to experiments 6-1, 6-2 and 6-3 respectively.

3.2.13 Set of experiments 7. Experiments 7-1 and 7-2: Evaluation of low dissolved oxygen (DO) concentration and temperature influence on AOB/NOB competition for MP1

The procedure described above in experiment 4-4 was followed here. Experiments were performed with carriers from MP1 stream only sampled 7 days before experiments performance. Other changes included DO concentration within (3.5 – 1.5) mg O2/L, pH adjustment at 7.6-7.7 and temperature setting point of 28°C and 14°C to simulate winter and summer temperatures at the pilot plant. AOB and NOB activities can be found by following the same procedure described in experiment 4.

The objective of this experiment is to check the hypothesis that high DO concentrations enhance high AOB/NOB activities ratios.

Assessment of 28°C and 14°C correspond with experiments 7-1 and 7-2 respectively.

3.2.14 Set of experiments 8. Experiments 8-1 to 8-3: Evaluation of AOB/NOB competition at zero storage time condition

The procedure described above in experiment 4-4 was followed here. All streams were assessed. Changes included DO concentration from 6.0 mg O2/L to saturation level, pH adjustment at 7.6 to 7.7 and temperature setting point for MP1 and MP2 streams of 14°C and 28°C for RP stream to simulate temperatures at pilot plant and avoid the temperature shock for bacteria. AOB and NOB activities can be found by following the same procedure described in experiment 4.

The objective of this experiment is to check the hypotheses that zero storage time prevents the depression of AOB and NOB activities.

Assessment of carriers from 3 streams correspond with experiments 8-1, 8-2 and 8-3.

3.2.15 Set of experiments 8. Experiments 8-4 to 8-6: Evaluation of the temperature shock influence on AOB/NOB competition at zero storage time condition

The procedure described above in experiment 4-4 was followed here. All streams were assessed. Changes included DO concentration from 6.0 mg O2/L to saturation level, pH adjustment at 7.6 to 7.7 and temperature setting point for MP1 and MP2 streams of 28°C and 14°C for RP stream to create shock temperature conditions for bacteria. AOB and NOB activities can be found by following the same procedure described in experiment 4.

23

The objectives of this experiment are to check the hypothesis that the shocking temperature depresses the AOB and NOB activities and to evaluate the activities at shock and zero storage time conditions.


3.2.16 Set of experiments 8. Experiments 8-7 to 8-9: Evaluation of lowered dissolved oxygen (DO) concentration influence on AOB/NOB competition at 14ºC and zero storage time conditions

The procedure described above in experiment 4-4 was followed here. All streams were assessed. Changes included DO concentration within (6.0 – 3.0) mg O2/L, pH adjustment at 7.6 to 7.7 and temperature setting point of 14°C. AOB and NOB activities can be found by following the same procedure described in experiment 4.

The objective of this experiment is to check the influence of consequent DO increase due to temperature decrease from 28 to 14ºC and check the hypotheses that higher DO concentrations enhance higher AOB/NOB activities ratios.


3.2.17 Set of experiments 9. Experiments 9-1 to 9-3: Evaluation of two weeks storage time influence on AOB/NOB competition

The procedure described above in experiment 4-4 was followed here. All streams were assessed. Other changes included DO concentration within (6.0 – 3.0) mg O2/L, pH adjustment around 7.5 and temperature setting point for MP1 and MP2 streams was of 14°C and 28°C for RP stream to simulate temperatures at pilot plant. AOB and NOB activities can be found by following the same procedure described in experiment 4.

The objective of this experiment is to check the hypotheses that two weeks storage time influences AOB and NOB activities.


3.2.18 Set of experiments 9. Experiments 9-4 to 9-6: Evaluation of the temperature shock influence on AOB/NOB competition at two weeks storage time condition

The procedure described above in experiment 4-4 was followed here. All streams were assessed. Changes included DO concentration from 6.0 mg O2/L to saturation level, pH adjustment at 7.6 to 7.7 and temperature setting point for MP1 and MP2 streams of 28°C and 14°C for RP stream to create shock temperature conditions for bacteria. AOB and NOB activities can be found by following the same procedure described in experiment 4.

The objectives of this experiment are to check the hypothesis that the shocking temperature and storage time depresses the AOB and NOB activities and to evaluate the activities at shock and two weeks storage time conditions.


24

3.2.19 Set of experiments 10. Experiments 10-1 to 10-3: Evaluation of dissolved oxygen concentration influence on AOB, NOB and HB kinetics

A lab-scale reactor containing 400 ml of tap water and 128 fresh carriers was stirred continuously at 150 rpm as in all previous experiments. After few minutes of initial aeration with pure oxygen a DO saturation level of 125% in the bulk was achieved.

Each experiment in this set was performed with 3 sub-experiments for each of three studied streams:

1. Ammonium sulfate and sodium nitrite were introduced and the oxygen consumption curve (changes of oxygen concentration with time) was recorded until zero oxygen uptake condition was reached (3.5 hours in total). Other conditions included pH adjustment at 7.6 to 7.7 and temperature setting point of 14°C for MP1 and MP2 streams and 28°C for RP stream to avoid temperature stress on microorganisms.

2. The procedure of the first experiment described above was applied, but now sodium nitrite and ATU were introduced to obtain NOB + HB activity. Temperature conditions remain the same as for first experiment avoiding the temperature stress.

3. Finally this procedure was applied one more time without substrates or inhibitor injections to obtain the reference for HB activity.

The objective of this experiment is to investigate the activities of microorganisms and find the effective thickness i.e. the thickness of oxygen penetration into the biofilm in presence of different bacteria groups. Nitrogen sources were added in abundance making O2 the limiting substrate. Shear and temperature stresses are assumed negligible.

Assessment of MP1 and MP2 streams at 14°C corresponds with experiments 10-1 and 10-2 respectively. Assessment of RP stream at 28°C corresponds with experiment 10-3.

25

4 Results For each experiment two types of data were generated, DO vs. time and SOUR vs. time. For experiments 7-1, 7-2, and the set of experiment 8, complementary substrates concentrations analyses as NH4

+-N, NO2--N and NO3

--N, vs. time as well as COD concentration vs. time were carried out. The corresponding charts for substrates concentrations can be found in the appendix, page 25III.

4.1 Set of experiments 1. Influence of concentration of carriers in the reactor on bacteria activity

4.1.1 Experiment 1-1: Evaluation of the volumetric filling fraction percentage effect on microorganisms activity, stream MP2

From Figure 4.1 and Figure 4.2 it is clear that the first step during the endogenous respiration (before substrates introduction), showed a decreasing trend for peaks and troughs of the DO concentrations. The second step (after substrates addition) showed also a decreasing trend for peaks and troughs of the DO concentrations, but with lowered minimum and maximum DO levels, which confirming NOB and AOB activities responses from the substrates injections. The third step (after ATU introduction) demonstrates that AOB activity was inhibited. The respiration here appeared to be higher compared with the previous pattern obtained during step 1, what is observed in both reactors with 64 and 128 carriers, meaning either that NOB or both NOB+HB activities are present.

Figure 4.1. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP. Reactor 1 with 64 carriers.

0123456789

10

0 0,5 1 1,5 2 2,5

DO

, mg/

l

Time, hr

NH4+ + NO2

- ATU

26

Figure 4.2. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP. Reactor 2 with 128 carriers.

Figure 4.3 and Figure 4.4 show the SOUR values obtained in each step for each reactor. The HB-SOUR (before substrates addition) pattern and values are almost the same in both reactors. The combined HB+NOB+AOB – SOUR (after substrates addition) pattern differ from each reactors. In reactor 1 (64 carriers) SOUR values are bigger than in reactor 2 and the pattern is also different. Finally during the third step (after ATU addition) the respirations look very similar in patterns and values.

Figure 4.3. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP2. Reactor 1 with 64 carriers.

0123456789

10

0 0,5 1 1,5 2 2,5

DO

, mg/

l

Time, hr

NH4+ + NO2

- ATU

0123456789

10

0 0,5 1 1,5 2 2,5

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ + NO2

- ATU

27

Figure 4.4. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP2. Reactor 2 with 128 carriers. Table 4.1 shows the computed values of HB, NOB and AOB SOURs found for MP2 stream with 64 and 124 carries, respectively. With these tables we can compare all activities since OURs are related to TS giving SOURs. According with HB results, its activities were almost equal and independent of the number of carriers. NOB activities were also very similar and lower than AOB activities in both cases. A bigger AOB activity was found in reactor 1 that contained 50% of the number of carriers employed in reactor 2. This initial result suggests that HB and NOB activities are independent of the carrier concentration and AOB activity is higher for a lower carrier concentration. One of the reasons for that can be higher DO concentrations for 64 carriers than for 128 carriers. It was confirmed for streams RP and MP1 at the same experimental conditions. Results can be found in Table 4.2 and Table 4.3, respectively.

Table 4.1. Computed AOB and NOB activities based on 64 carriers (R1) and 128 carriers (R2) expressed in mg O2/gTS*h. Stream MP2.

Step Microorganisms R1 R2

1 HB 2.3 2.4

2 AOB +NOB + HB 7.4 5.6

3 NOB + HB 3.9 3.6

Computed AOB 3.6 2.3

Computed NOB 1.6 1.3

0

1

2

3

4

5

6

7

8

9

10

0 0,5 1 1,5 2 2,5

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ + NO2

- ATU

28

Table 4.2 Computed AOB and NOB activities based on 64 carriers (R1) and 128 carriers (R2) expressed in mg O2/gTS*h. Stream RP.

Step Microorganisms present in R1 R1 R2

1 HB 2.9 2.7

2 AOB +NOB + HB 6.4 5.8

3 NOB + HB 3.2 3.3


Computed NOB 0.3 0.6

Table 4.3. Computed AOB and NOB activities based on 64 carriers (R1) and 128 carriers (R2) expressed in mg O2/gTS*h. Stream MP1.

Step Microorganisms present in R1 R1 R2

1 HB 5.8 6.1

2 AOB +NOB + HB 6.5 5.6

3 NOB + HB 4.0 4.6


Computed NOB - 1.7 - 1.5

Experiments 1-2 and 1-3 correspond to RP and MP1 streams respectively.

4.1.2 Experiment 1-4: Evaluation of the development of endogenous respiration without the presence of inhibitors and without substrates addition, stream MP2

Figure 4.5 and Figure 4.6 show the complete endogenous respiration (1.5 hours in total) for stream MP2 with 64 and 128 carriers, respectively. Reactor 2 (128 carriers) presents larger oxygen consumptions compared with reactor 1, at least during the first cycles, but later on the oxygen consumption seems to be almost equal in both reactors. However it is difficult to claim this from these charts.

29

Figure 4.5. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP2. Reactor 1 with 64 carriers.

Figure 4.6. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP2. Reactor 2 with 128 carriers. Due to the clear and stable patterns presented by stream 2 in the previous experiment, this stream was chosen to assess the continuous (absence of substrates and ATU) reduction of oxygen consumption through the time and the necessity of this reference for a better computed AOB/NOB competition analysis. Figure 4.7 shows the SOUR values obtained during the endogenous respiration for 64 and 128 carriers from stream MP2. Both patterns followed the same trend. SOUR values from reactor 2 (128 carriers) are low compared with those obtained in reactor 1 (64 carriers) during the first 45 minutes. Then both reactors showed the same SOUR values during the remaining time. The continuous variation of SOUR supports the necessity of this reference for future analysis and for generation of an accurately computed AOB/NOB activities. From Figure 4.7 is clear that the independency of HB SOUR activity from the number of carriers is achieved for this kind of experiments after approximately 50 minutes.

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

30

Figure 4.7. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP2. Both reactors. Based on the results presented above 128 carriers has been selected as a concentration of carriers in the reactors for the following experiments as the dependency of the bacteria activities on the number of carriers has been demonstrated.

4.2 Set of experiments 2. Influence of HB activity on calculation of AOB and NOB activities

4.2.1 Experiment 2-1: Evaluation of HB activity on AOB and NOB computed activities, stream MP2

Figure 4.8 in general demonstrates the same oxygen consumption pattern as was obtained during the previous experiments with MP2 stream for all three steps. Figure 4.9 shows the continuous oxygen consumption in reactor 2 for an endogenous respiration running at the same temperature and stirring condition as in reactor 1. The endogenous respiration here differs from previous experiments and oxygen uptake is lower and more constant.

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, mgO

2/gT

S*h

Time, hr

128 carriers

64 carriers

31

Figure 4.8. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP2. Reactor 1 with 128 carriers.

Figure 4.9. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP2. Reactor 2 with 128 carriers. From Figure 4.10 the SOUR values obtained during the whole experiment were lower compared with the previous values from experiment 1-1 with 128 carriers, suggesting a low initial amount of carbon source. During step 1 one extra slope was included. Figure 4.11 shows the continuous variation of the endogenous respiration through the time of the experiment.

0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ + NO2

- ATU

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

32


Figure 4.11. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP2. Reactor 2 with 128 carriers. Table 4.4 shows SOUR values from reactor 1 as well as from the endogenous respiration that is used as a reference for more accurate computed MP2-AOB and MP2-NOB activities. According to this table HB activities were very similar during step 1, making logic that HB activities are almost the same in both reactors. An interesting change after this new AOB/NOB computed activities method is that AOB activity is now lower than NOB activity. The computed NOB activity is now clear and seems to be the dominant microorganism during step 2. At this point the previous hypothesis that a better AOB/NOB competition can be obtain with a low volume filling percentage is unclear. Further studies in order to explain the reason for such a low HB activities is also needed. It should be taken into account that the sample of carriers from MP2 stream was stored for more than 72 hours prior to this experiment.

0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ + NO2

- ATU

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

33

Table 4.4. Computed MP2-AOB and MP2-NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration.


1 HB 1.3 1.2

2 AOB +NOB + HB 4.3 1.1

3 NOB + HB 3.2 1.0

Computed AOB 1.0

Computed NOB 2.2

4.2.2 Experiment 2-2: Evaluation of HB activity influence on AOB and NOB computed activities, stream MP1

From Figure 4.12 the whole patter looks unfamiliar. Two elements are new: A very slow reduction in oxygen consumption during step 1 and very small and slow oxygen consumption during step 2. One can doubt if substrates were injected, however the variation obtained during step 3 (after ATU addition) confirms the AOB inhibition and the previous response from AOB and NOB. Figure 4.13 shows the oxygen consumption in reactor 1 for endogenous respiration running at the same temperature and stirring condition as in reactor 2. This pattern confirms the extended oxygen consumption during the three first cycles suggesting a high initial amount of carbon source.


0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

NH4+ + NO2

- ATU

34

Figure 4.13. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP1. Reactor 2 with 128 carriers. From Figure 4.14 the presence of a high initial concentration of carbon source is confirmed by the high SOUR values during the first step. The oxygen consumption during the second step was relatively stable, meaning that NOB and AOB had substrates in abundance. The third step confirms the inhibition of AOB activity and the remaining combined activities of NOB and HB. Figure 4.15 shows the gradual reduction of organic carbon present during the endogenous respiration. The oxidation of organic matter dominates here during the whole experiment.


0123456789

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0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

0123456789

10

0 0,5 1 1,5 2

Spec

ific r

ate,

mgO

2/gT

S*h

Time, hr

NH4+ + NO2

- ATU

35

Figure 4.15. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1. Reactor 2 with 128 carriers. Table 4.5 shows SOUR values from reactor 1 as well as from the endogenous respiration that is used as a reference for more accurate computed MP2-AOB and MP2-NOB activities. The high values for HB activities confirm its dominant activity during this experiment and the presence of abnormal initial carbon sources. The low AOB/NOB activities can be associated with suppression of AOB and NOB by HB. The negative sign can be interpreted as a high HB activity in steps 2 and 3. A reason for such activities could be either hydrolysis generated during storage of carriers.

Table 4.5. Computed MP1-AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration


1 HB 5.0 4.8

2 AOB +NOB + HB 4.8 3.8

3 NOB + HB 3.7 2.8

Computed AOB 0.0

Computed NOB 0.9

4.2.3 Experiment 2-3: Evaluation of HB activity on AOB and NOB computed activities, stream RP

From Figure 4.16 slow reduction in oxygen consumption during step 1 is recognized. Then an expected much more pronounced oxygen consumption is demonstrated during step 2 due to AOB and NOB responses. Finally a clear AOB inhibition and the remaining NOB and HB activities can be found in step 3. Figure 4.17 shows a usual pattern for the endogenous respiration obtained with stream RP, but the reduction in peaks and troughs is more clear.

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10

0 0,5 1 1,5 2

Spec

ific r

ate,

mgO

2/gT

S*h

TIme, hr

36

Figure 4.16. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration and the influence of substrates and ATU on Stream RP. Reactor 1 with 128 carriers.

Figure 4.17. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP1. Reactor 2 with 128 carriers. From Figure 4.18 small SOUR values during the first step are related to low concentration of organic carbon. Oxygen consumption during the second step shows the peak for the injection point followed by a continuous reduction in microorganisms activities during the remaining two cycles. The third step confirms the inhibition of AOB activity and shows the remaining combined activities of NOB and HB. Figure 4.19 shows the gradual reduction of organic carbon present during the endogenous respiration. A low initial SOUR value suggests a low initial COD concentration.

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0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

NH4+ + NO2

- ATU

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

37

Figure 4.18. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream RP. Reactor 1 with 128 carriers.

Figure 4.19 The Specific Oxygen Uptake Rate of bacteria vs. time. Stream RP. Reactor 2 with 128 carriers. Table 4.6 shows SOUR values from reactor 1 as well as from the endogenous respiration that is used as a reference for more accurate computed RP-AOB and RP-NOB activities. Similar initial HB activities confirm similar conditions in both reactors. One more time the inverse pattern was obtained with this computed method. However AOB activity was found to be very similar to NOB activity.

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0 0,5 1 1,5 2

Spec

ific r

ate,

mgO

2/gT

S*h

Time, hr

NH4+ + NO2

- ATU

0123456789

10

0 0,5 1 1,5 2

Spec

ific r

ate,

mgO

2/gT

S*h

TIme, hr

38

Table 4.6 Computed MP1-AOB and MP2-NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration.


1 HB 1.6 1.7

2 AOB +NOB + HB 5.2 1.3

3 NOB + HB 3.3 1.2

Computed AOB 1.8

Computed NOB 2.1

Based on the results of the set of experiments 2 the reference for HB activity is included for further experiments as it has been demonstrated that the HB activity has significant influence on the accuracy of the method and it was shown that after 1 hour of aeration the starving point is not reached, although the change in the SOUR becomes relatively small.

4.3 Set of experiments 3. Influence of the duration of the experiment on the bacteria activities

During this set of experiments the aeration/no aeration cycle was set as 2 minutes/2 minutes holding the other parameters constant. DO concentrations vs. time and SOUR values vs. time can be found further down for one illustrative experiment. Tables with the computed AOB and NOB activities are presented below for all experiments.

4.3.1 Experiment 3-1: Evaluation of the aeration/no aeration cycle time effect on microorganisms activity, stream MP2

From Figure 4.20 the DO concentration span was almost constant in all three steps and was reduced to 1 mg O2/L. A constant lowered maximum DO value for peaks of ca 6.4 mg O2/L is observed during steps 1 and 2, meaning that the aeration rate and the oxygen consumption rate were almost equivalent, even after substrates addition, thus the computed SOUR were not representative at least for the assessment of AOB activity. Figure 4.21 shows the continuous oxygen consumption in reactor 2 for endogenous respiration running at the same temperature and stirring condition as in reactor 1.

39


Figure 4.21. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP2. Reactor 2 with 128 carriers. From Figure 4.22 the pattern observed during step 1 shows the opposite behaviour from experiment 2-1, high SOUR values suggesting high COD concentrations. However, since all carriers were sampled at the same day and experiments were carried out with one day in between, COD concentrations should be almost equal, meaning that also HB activity assessment was affected by the new aeration/no aeration set point. A comparison between Figure 4.23 and Figure 4.11 shows clearly how endogenous respiration depends on the defined aeration/no aeration set point. For the experiment with reduced cycle time bacteria were not able to develop the same activity as it was demonstrated for the experiment with longer cycle time.

The charts of oxygen consumption vs. time and SOUR vs. times for streams MP1 and RP (not shown here) also differ from those obtained with the previous aeration/no aeration set point.

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0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ + NO2

- ATU

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

40

Variations and associated causes are common in all cases, meaning that this parameter should be either kept constant as 5 minutes/5 minutes or the number of cycle with 2 minutes/2 minutes should be increase to compensate for the lack of oxidation cycles incurred with this reduction of time, in other words the total duration of the experiments cannot be shortened for the studied method.

Figure 4.22. The Specific Oxygen Uptake Rate of bacteria vs. time on stream MP2. Reactor 1 with 128 carriers.

Figure 4.23. The Specific Oxygen Uptake Rate of bacteria vs. time on stream MP2. Reactor 2 with 128 carriers. Tables 4.7 to 4.9 show the computed AOB/NOB activities for streams MP2, RP and MP1 respectively.

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0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ + NO2

- ATU

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

41

Table 4.7. Computed AOB and NOB for stream MP2 expressed in mg O2/gTS*h.


1 HB 5.1 4.2

2 AOB +NOB + HB 5.9 3.2

3 NOB + HB 4.5 2.3

Computed AOB 0.6

Computed NOB 2.2

Table 4.8. Computed AOB and NOB activities for stream RP expressed in mg O2/gTS*h.


1 HB 1.8 1.9

2 AOB +NOB + HB 4.6 1.6

3 NOB + HB 2.9 1.3

Computed AOB 1.4

Computed NOB 1.6

Table 4.9. Computed AOB and NOB activities for stream MP1 expressed in mg O2/gTS*h.


1 HB 4.8 5.5

2 AOB +NOB + HB 4.1 5.2

3 NOB + HB 3.2 5.1

Computed AOB 0.8

Computed NOB -1.9

Based on the results of the set of experiments 3 the aeration/no aeration cycle time has been selected to be 5 minutes/5 minutes for further experiments as it has been demonstrated that the cycle time is important for the development of bacteria activities during the experiments.

4.4 Set of experiments 4. Influence of substrates addition patterns, pH and storage time of samples on bacteria activities

An attempt to assess individual effect of pH, individual substrates injection and storage time on microorganisms activities was carried out here. DO concentrations vs. time, SOUR values vs. time and correspondent tables with computed AOB and NOB activities can be found further down. In some cases only one illustrative experiment is presented and the other experiments can be found in Appendix section.

42

4.4.1 Experiment 4-1: Evaluation of initial NO2- external addition influence on

AOB/NOB competition, stream MP1 From Figure 4.24 the oxygen concentration reduction pattern is the same as for previous experiments during steps 1 and 2, meaning that during step 2 AOB activity response was positive. However during step 3 a gradual reduction in the oxygen consumption is clear, suggesting that the NOB activity was very low or negligible during step 3. Figure 4.25 shows the continuous oxygen consumption in reactor 2 for endogenous respiration running at the same temperature and stirring conditions as in reactor 1. Patterns from step 1 and 3 are similar in both figures, but peak during step 1 from Figure 4.24 had bigger values, meaning that higher organic carbon concentration was present in reactor 1.

Figure 4.24. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP1. Reactor 1 without NO2

-

addition.

Figure 4.25. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP2. Reactor 2. From Figure 4.26 the pattern followed during step 1 shows a high HB activity compared with the combined HB and AOB activity obtained in step 2, meaning that HB activity is still

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0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

43

relevant in this step and that the amount of NH4+added did not limit the AOB activity. The

continuous reduction in SOUR values during step 3 in Figure 4.26 and the pattern obtained with the endogenous respiration in the same step (see Figure 4.27) confirm that NOB activity had been very low, as well as that AOB activity had been inhibited.

Figure 4.26. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1 Reactor 1 without NO2

- addition.

Figure 4.27. The Specific Oxygen Uptake Rate of bacteria vs. time for endogenous respiration. Stream MP1. Reactor 2. According to Table 4.10 and taking into account the fact that the initial HB activity in reactor 1 was higher than in reactor 2 NOB activity in reactor 2 can be considered negligible.

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10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

44

Table 4.10. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration for stream MP1, without addition of NO2

-.


1 HB 5 4.5

2 AOB +NOB + HB 5.6 3.7

3 NOB + HB 3.4 3.2

Computed AOB 1.7

Computed NOB 0.2

4.4.2 Experiment 4-2: Evaluation of individual substrates addition influence on AOB/NOB competition, stream MP1

Figure 4.28 shows that the carriers sample contained high concentrations of organic carbon, making HB activities relevant during all three steps. The oxygen consumption observed during step 3 after NO2

- addition is almost constant, meaning that the added amount of NO2-

did not limit the NOB activity. This can be confirmed by the SOUR values from Figure 4.30. It was proved by these charts that for this method an individual addition of substrates permits a clear identification of microorganisms activities.

Figure 4.28 The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP1. Reactor 1 with separated addition of NO2

-.

0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU +

NO2-

45

Figure 4.29. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP1. Reactor 2.

Figure 4.30. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1. Reactor 1.

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

46

Figure 4.31. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1. Reactor 2. According with Table 4.11 HB activity was high during all three steps and NOB activity was very low compared with AOB activity. Based on experiments 4-1 and 4-2, high HB activities through the whole three steps suggests the possible effect of hydrolysis during the storage time of 10 days, giving an explanation for the suppressed AOB/NOB activities.

Table 4.11. .Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream MP1.


1 HB 4.5 4.6

2 AOB +NOB + HB 5.1 3.6

3 NOB + HB 3.2 3.0

Computed AOB 1.3

Computed NOB 0.2

4.4.3 Experiment 4-3: Evaluation of pH influence on NOB activity, stream MP1 From Figure 4.32 the introduction of H2SO4 to control the pH between (7.6 - 7.7) during the step 3 is shown. The oxygen consumption during step 3 did not show significant difference from previously obtained results without pH control. From Figure 4.33 the SOUR values through step 3 does not show any considerable difference from previous results as well, meaning that NOB activity is not affected by pH values within (7.5 – 8.2).

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

47

Figure 4.32. The Dissolved Oxygen (DO) concentration changes with time and the influence of substrates and ATU on Stream MP1 with pH control. Reactor 2.

Figure 4.33. The Specific Oxygen Uptake Rate of bacteria vs. time and the influence of pH control. Stream MP1. Reactor 2.

4.4.4 Experiment 4-4: Evaluation of pH influence on AOB activity, stream MP1 Figure 4.34 shows the oxygen consumption through the time with the introduction of H2SO4 as an acid source to control the pH between (7.6 – 7.7) during step 2 and step 3 for stream MP1. The obtained patterns in all steps did not show evidence of pH effects on microorganisms activities. Figure 4.35 shows the endogenous respiration without injection of H2SO4 in reactor 2 and then the patterns obtained in both reactors during step 2 can be compared.

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

NH4+ ATU + NO2

- + H2SO4

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

- + H2SO4

48

Figure 4.34 The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP1. Reactor 1.

Figure 4.35. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP1. Reactor 2. From Figure 4.36 and Figure 4.37 the SOUR values and pattern followed those obtained without pH adjustments in experiment 4-2, suggesting that AOB activity is not affected by pH values within (7.5 – 8.2).

0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ + H2SO4 ATU + NO2

- + H2SO4

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

49


Figure 4.37. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1. Reactor 2. A comparison between Table 4.11 and Table 4.12 did not show the effects of pH within (7.5 – 8.2) on AOB activity. The negative sign of NOB activity in Table 4.12 is due to the high HB activity in all steps, but NOB activities in both experiments were comparable.

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0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr


- + H2SO4

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0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

50

Table 4.12. Computed MP1-AOB and MP1-NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration.


1 HB 4.3 4.4

2 AOB +NOB + HB 4.7 3.8

3 NOB + HB 3.1 3.3

Computed AOB 1.1

Computed NOB -0.2

The experiments 4-5 and 4-6 represent the same evaluation as experiment 4-4, but for two other streams. The results can be found in appendix, page I.

4.4.5 Experiment 4-7: Evaluation of storage time of carriers influence on AOB/NOB competition, stream MP1

From Figure 4.38 the dissolved oxygen concentration pattern shows some irregularities regarding the DO sensor in reactor 1, during step 2 and step 3. The basic cause for this issue could be associated with aeration currents from the diffuser that had been temporally in direct contact with the DO sensor. Fortunately, the affected slopes are not included in computing AOB/NOB activities. Figure 4.39 confirms that the equipment operated well during this experiment.

Figure 4.38. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP1. Reactor 1.

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10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr


- + H2SO4

51

Figure 4.39. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP1. Reactor 2. From Figure 4.40 a clear difference between SOUR values after each step can be seen. HB activity is low in all steps compared with those from previous experiments. From Figure 4.41 the SOUR values reduction tend to achieve equilibrium at the end of the third step.


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10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr


- + H2SO4

52

Figure 4.41. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1. Reactor 2. According to Table 4.13 computed AOB activity is higher than the computed NOB activity in stream MP1. However according to tables 4.14 and 4.15 AOB and NOB activities are almost the same in streams MP2 and RP. The low HB activity through all three steps for all streams with fresh carriers demonstrates the dependency of HB activity on the storage time of the sample. From these tables it is clear that stream RP contains a more concentrated carbon sources.

Table 4.13. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream MP1.


1 HB 2.1 2.1

2 AOB +NOB + HB 4.9 1.4

3 NOB + HB 2.8 1.3

Computed AOB 2.0

Computed NOB 1.5

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10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

53



1 HB 1.5 1.7

2 AOB +NOB + HB 5.2 1.5

3 NOB + HB 3.2 1.4

Computed AOB 1.9

Computed NOB 1.8

Table 4.15. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream RP.


1 HB 2.4 2.7

2 AOB +NOB + HB 6.1 1.2

3 NOB + HB 3.5 1.1

Computed AOB 2.5

Computed NOB 2.4

The experiments 4-8 and 4-9 represent the same evaluation as experiment 4-7, but for two other streams. The results can be found in appendix, page VI.

Based on the results of the set of experiments 4 separated addition pattern of NH4+ and NO2

- has been selected as more accurate determination of the bacteria activities is achieved. Although the influence of pH was not confirmed, adjustment of pH between 7.6 and 7.7 has been selected in order to simulate the conditions at the pilot plant.

4.5 Set of experiments 5. Influence of low temperature on bacteria activities

The following results are from stream MP1 (experiment 5-1). The results from streams MP2 an RP corresponding to experiments 5-2 and 5-3 can be found in appendix, page X.

4.5.1 Experiment 5-1: Evaluation of low temperature influence on AOB/NOB competition with sample after storage, stream MP1

From Figure 4.42 the HB activity seems to be high, it can be explained by the storage time of 13 days. Figure 4.43 shows a very low but gradual reduction of peaks and troughs after the first three cycles, meaning that the organic carbon consumption is present.

54

Figure 4.42. The Dissolved Oxygen (DO) concentration changes with time in the presence of substrates and ATU. Stream MP1. Reactor 1.

Figure 4.43. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP1. Reactor 2. From Figure 4.44 the SOUR values during step 2 are close to the last slope from step 1, but are a bit higher and almost constant. Thus AOB activity response was positive at 14°C. According with Figure 4.45 the HB activity remains almost constant after the first three slopes, confirming that the HB activity is limited by the amount of organic carbon.

0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

55


Figure 4.45. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1. Reactor 2. The AOB activity was low but much higher than NOB activity according to data from Table 4.16, suggesting a good condition in terms of AOB/NOB competition. It was also the case for stream MP2 (see Table 4.17), but for stream RP (see Table 4.18) the microorganisms competition was less clear.

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10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

56



1 HB 2.6 2.7

2 AOB +NOB + HB 3.4 2.9

3 NOB + HB 2.1 2.8

Computed AOB 1.2

Computed NOB -0.7



1 HB 3.4 3.0

2 AOB +NOB + HB 4.0 3.1

3 NOB + HB 2.3 3.0

Computed AOB 1.6

Computed NOB -0.7



1 HB 2.3 2.0

2 AOB +NOB + HB 3.8 1.3

3 NOB + HB 1.9 1.1

Computed AOB 1.7

Computed NOB 0.8

4.5.2 Experiment 5-4: Evaluation of low temperature influence on AOB/NOB competition with fresh sample, stream MP1

From Figure 4.46 and Figure 4.47 the oxygen consumption reduction during the endogenous respiration is gradual and very similar during step 1. The oxygen consumption reduction accelerates during step 2 as it was expected and during step 3 the oxygen consumption reduction is much more in reactor 1 than in reactor 2. Thus a fresh sample provides a better

57

definition in AOB/NOB activities assessment. This experiment was carried out without pH adjustment and the oxygen consumption patterns did not show irregularities that suggests no pH effects on microorganisms activities.


Figure 4.47. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP1. Reactor 2. From Figure 4.48 and 4.49 almost constant slopes are observed in each step. HB activity in reactor 2 is not constant during the whole experiment, confirming the importance of this reference to separate AOB, NOB and HB activities.

According to Table 4.19 the AOB/NOB activities ratio is low, meaning that at 14°C, there are good conditions for NH4

+ transformation into NO2- with low NO3

- generation. This behaviour was common for all streams, but a lower value for this ratio was observed for stream MP2.

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

NH4+

ATU + NO2-

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

58


Figure 4.49. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1. Reactor 2. Table 4.19. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream MP1.


1 HB 2.0 1.8

2 AOB +NOB + HB 3.7 1.5

3 NOB + HB 2.0 1.4

Computed AOB 1.6

Computed NOB 0.6

The experiments 5-5 and 5-6 represent the same evaluation as experiment 5-4, but for two other streams. The results can be found in appendix, page XIV.

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10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

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10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

59

The results of the set of experiments 5 demonstrate the effects of low temperature on AOB/NOB competition. It was shown that low temperature promotes the activity of AOB for both fresh sample and sample after storage time. The present experiments also confirm previously obtained results regarding storage time effects on bacteria activities to be relevant also for low temperature conditions. The influence of the temperature and storage time is investigated further in sets of experiments 8 and 9.

4.6 Set of experiments 6. Influence of the mixing of biofilm adapted to different conditions on the bacteria activities

4.6.1 Experiments 6-1 to 6-3: Evaluation of RP:MP1 carriers ratio influence on AOB/NOB competition

The computed activities of AOB and NOB for different RP:MP1 ratios are presented in Table 4.20 (50%:50%), Table 4.21 (80%:20%) and Table 4.22 (20%:80%). The resulting activities of AOB and NOB as well as AOB/NOB activities ratios from these tables are shown in Figure 4.60 for comparison.

These experiments demonstrate that sample with higher proportion of carriers from RP stream shows the highest specific activity of both groups of bacteria, AOB and NOB. Despite the temperature stress conditions for bacteria from MP1 stream (28ºC), the sample with highest proportion of carriers from MP1 demonstrated the highest AOB/NOB activities ratio which was not expected. The lower proportion of MP1 result in lower AOB/NOB activities ratios (see Figure 4.50).

Table 4.20. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. 50% RP and 50% MP1.


1 HB 1.8 1.8

2 AOB +NOB + HB 4.9 1.50

3 NOB + HB 2.7 1.4

Computed AOB 2.10

Computed NOB 1.3

60

Table 4.21. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. 80% RP and 20%:MP1.


1 HB 1.4 1.2

2 AOB +NOB + HB 5.6 1.0

3 NOB + HB 2.9 1.0

Computed AOB 2.7

Computed NOB 1.9

Table 4.22. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. 20% RP and 80%:MP1.


1 HB 2.6 2.5

2 AOB +NOB + HB 4.9 2.1

3 NOB + HB 3 2

Computed AOB 1.8

Computed NOB 1

Figure 4.50. AOB and NOB activities for different RP: MP1 mixing ratios of carriers. The results of the set of experiments 6 demonstrate a linear correlation between the proportion of carriers from RP and specific bacteria activities. The bigger part of RP carriers results in higher specific activities for both bacteria groups, AOB and NOB. At the same time linear

1,41,6

1,8

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

5,0

80 : 20 50 : 50 20 : 80

Spec

ific

Rat

e, m

gO2/

gTS*

h

AOB

NOB

AOB/NOB ratio

61

correlation between the proportion of MP1 carriers and AOB/NOB activities ratios was demonstrated. The bigger part of MP1 carriers results in higher AOB/NOB activities ratios.

4.7 Set of experiments 7. Influence of low DO concentrations on bacteria activities

The following results were obtained with carriers from MP1 for 28 °C (experiment 7-1) and 14 °C (experiment 7-2) by using a low DO concentration span.

4.7.1 Experiment 7-1: Evaluation of low dissolved oxygen (DO) concentration and temperature influence on AOB/NOB competition for MP1 at 28ºC

From Figure 4.51 and Figure 4.52 it can be confirmed that a low DO concentration span of (3 – 1.5) mg O2/L can be used for assessing microorganisms activities with this method and equipment.


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DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

62

Figure 4.52. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP1. Reactor 2. From Figure 4.53 the SOUR values remain almost constant during the first step, meaning that the oxygen concentration was enough for HB activity. On the contrary from the same figure gradual reduction of SOUR values is clear during step 2, suggesting that the greater the DO concentration, the higher AOB/NOB activities are. Also a combined effect of temperature stress and low DO could be a reason for this pattern in the second step, where suppressed AOB/NOB activity is presented. The SOUR pattern demonstrated during step 3 correlates with the previous results and shows a positive NOB response.

According to Table 4.23 the NOB activity had a negative sign, suggesting that HB activity is high during the whole experiment. However Figure 4.54 shows a continuous reduction of SOUR values and almost constant values during the last three cycles. Thus the activities of both groups, AOB and NOB are very low during the whole experiment and HB activity prevails. It should be taken into account that the sample was stored for 7 days prior to the experiment. It can explain elevated HB activities compared to what was expected.


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DO

, mg/

l

Time, hr

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0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU +

NO2-

63

Figure 4.54. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1. Reactor 2. Table 4.23. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream MP1 at 28°C and low DO span.


1 HB 1.4 1.4

2 AOB +NOB + HB 1.2 1.2

3 NOB + HB 1.0 1.1

Computed AOB 0.1

Computed NOB -0.1

4.7.2 Experiment 7-2: Evaluation of low dissolved oxygen (DO) concentration and temperature influence on AOB/NOB competition for MP1 at 14ºC

From Figures 4.55 and 4.56 the oxygen consumption patterns at 14 °C show irregular variations between aeration and no aeration slopes for both reactors, this can be explained by faster saturation rate of colder water and the equipment limitations in controlling the DO concentration span in the same range as at 28°C. However the obtained data is valid for the SOUR generation and microorganisms activity assessment.

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Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

64


Figure 4.56. The Dissolved Oxygen (DO) concentration changes with time for endogenous respiration on Stream MP1. Reactor 2. From Figure 4.57 the low SOUR values during the three steps confirm that the AOB/NOB activities have a direct and proportional dependency on the DO concentration level. It is also clear that AOB activity was bigger than NOB activity. From Figure 4.58 a low HB activity was present through the whole experiment, but it was not the case at 28 °C (see Figure 4.54), thus it is clear that HB activity is affected by a lower temperature.

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DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

65


Figure 4.58. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1. Reactor 2. According with Table 4.24 the NOB activity was negligible and AOB activity was high compared to AOB activity at 28°C, suggesting that 14°C is a favourable condition to convert NH4

+ into NO2- with a low production of NO3

- that is a desirable situation for Anammox process.

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Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

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0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

66

Table 4.24. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream MP1 at 14°C and low DO span.


1 HB 0.6 0.5

2 AOB +NOB + HB 0.9 0.3

3 NOB + HB 0.3 0.3

Computed AOB 0.6

Computed NOB 0.0

4.8 Set of experiments 8. Influence of temperature stress and lower DO concentration conditions on bacteria activities

After the method was proved to be valid for microorganisms activities assessment, a research and confirmation of the effects of storage time, temperature and lower DO concentration on microorganisms activities for all three streams was carried out with fresh carriers. All the results presented are for stream MP2. The results for streams MP1 and RP can be found in appendix, page XIX.

4.8.1 Experiment 8-1: Evaluation of AOB/NOB competition at zero storage time condition, stream MP2

From Figure 4.59 the oxygen consumption reduction at 14°C was low during the endogenous respiration (step 1); meaning that HB activity was low (it can be confirmed by Figure 4.60). During step 2 the oxygen uptake rate was high and in step 3 gradual reduction was present, meaning that AOB activity was high and NOB activity is present during step 3.


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DO

, m

g/l

Time, hr

NH4+ ATU +NO2

-

67

Figure 4.60. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration on Stream MP2. Reactor 2. According to Figure 4.61 and Figure 4.62 AOB/NOB competition is dominated by AOB activity.


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DO

, mg/

l

Time, hr

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Spec

ific

Rat

e, m

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gTS*

h

Time, hr

NH4+ ATU + NO2

-

68

Figure 4.62. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP2. Reactor 2. According to Table 4.25 low HB activity here is associated with fresh carriers and low temperatures as well as a dominant AOB activity and low NOB activity.



1 HB 0.5 0.6

2 AOB +NOB + HB 4.3 0.5

3 NOB + HB 1.6 0.5

Computed AOB 2.7

Computed NOB 1.1

Tables 4.25 and Table 4.26 present similar values for AOB and NOB activities. These experiments were performed at the same conditions with two fresh samples for the same stream but different batches sampled at different days. Thus the repeatability of the results is demonstrated.

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rate

, m

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gTS*

h

Time, hr

69

Table 4.26. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream MP2, Experiment 5-5.


1 HB 1.0 0.9

2 AOB +NOB + HB 4.9 0.6

3 NOB + HB 2.0 0.6

Computed AOB 2.9

Computed NOB 1.4

4.8.2 Experiment 8-4: Evaluation of the temperature shock influence on AOB/NOB competition at zero storage time condition, stream MP2

From Figure 4.63 two points with missed data can be identified, however the required slopes are not disturbed and the experiment is still valid. In general the patterns are well defined in both reactors (see Figure 4.64).

Figure 4.63. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP2. Reactor 1.

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DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

70

Figure 4.64. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration on Stream MP2. Reactor 2. A further inspection of these results in Figure 4.65 shows that SOUR values for the endogenous respiration (HB activity) was constant, but higher than those obtained at 14°C (see Figure 4.61). According to HB values from Table 4.25 and Table 4.27, the HB activity varies from 0.5 mg O2/gTS*h to 1.4 mg O2/gTS*h, by increasing the temperature from 14°C to 28°C. After the introduction of NH4

+ (step 2) the SOUR trend differs from the obtained at 14°C, where it was almost constant. According to the same tables the AOB activity decreased from 2.7 mg O2/gTS*h to 1.8 mg O2/gTS*h. These supressed microorganisms activities can be caused only by the temperature stress. From the same tables NOB activity varied during the step 3 from 1.1 mg O2/gTS*h to 1.0 mg O2/gTS*h, meaning that NOB activity is almost constant or was not significantly affected by this change of temperature in this stream. Similar analysis of data from streams RP and MP1, leads to the same conclusions about the temperature stress.


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DO

, mg/

l

Time, hr

71



1 HB 1.4 1.3

2 AOB +NOB + HB 4.1 1.3

3 NOB + HB 2.3 1.3

Computed AOB 1.8

Computed NOB 1.0

Tables 4.28 and Table 4.29 present similar values for AOB and NOB activities. These experiments were performed at the same conditions with two fresh samples for the same stream but different batches sampled at different days. Thus the repeatability of the results is demonstrated also for stream RP.



1 HB 1.7 1.5

2 AOB +NOB + HB 3.7 1.0

3 NOB + HB 1.3 0.8

Computed AOB 2.2

Computed NOB 0.5

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Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

72

Table 4.29. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream RP

Step Microorganisms present in R1 R1 R2 (HB only) 1 HB 0.8 0.8

2 AOB +NOB + HB 3.2 0.3

3 NOB + HB 1.1 0.3

Computed AOB 2.1

Computed NOB 0.8

4.8.3 Experiment 8-7: Evaluation of lowered dissolved oxygen (DO) concentration influence on AOB/NOB competition at 14ºC and zero storage time conditions, stream MP2

From Figure 4.67 the oxygen consumption reduction varies with almost flat slopes during the no aeration cycles for the endogenous respiration (step 1), meaning that the oxygen concentration was gradually increased due to a combination of low HB activity and a fast re-aeration rate in the bulk solution. Thus the DO concentration span is difficult to control at 14 °C with the equipment used. It was also confirmed by reactor 2 (see Figure 4.68). However, during step 2 and step 3 the oxygen consumption pattern was better defined, permitting the microorganisms activities assessment.


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0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

73

Figure 4.68. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration on Stream MP2. Reactor 2. From Figure 4.69 the SOUR values during all three steps have been lower than those obtained with higher DO concentration span (see Figure 4.61). From Figure 4.69 the SOUR values were also lower than those obtained with higher DO concentrations span (see Figure 4.62).


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0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

0

1

2

3

4

5

6

7

8

9

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

74

Figure 4.70. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP2. Reactor 2. According to Table 4.30 AOB activity was much higher than NOB activity (2.8 mg O2/gTS*h vs. 0.6 mg O2/gTS*h), meaning that AOB activity dominates at lower DO concentration span and HB activity is low and did not change during step 2 and step 3, meaning that it is possible to find a certain low DO concentration where AOB activity prevails. A comparison between Table 4.25 and Table 4.30 suggests that a better AOB/NOB competition (2.7/1.1 vs. 2.8/0.6) can be found with lower DO concentrations span. This observation was common for all streams.

The pH control carried out during this experiment showed low pH variation and pH values around 7.6. This can be explained by the decrease in stripping of CO2 from the bulk solution. Thus the pH can be controlled by manipulating the aeration rate if it is needed.



1 HB 0.2 0.0

2 AOB +NOB + HB 3.6 0.2

3 NOB + HB 0.8 0.2

Computed AOB 2.8

Computed NOB 0.6

The results of the set of experiments 8 showed that the temperature stress has significant impact on AOB and NOB activities as well as lower DO concentration.

4.9 Set of experiments 9. Influence of storage time and temperature stress under 2 weeks storage time condition on bacteria activities

This study continues with the assessment of storage and temperature effects on AOB/NOB competition. Carriers from the same batches have been stored at 4°C during two weeks. The

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rate

, m

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gTS*

h

Time, hr

75

presence and influence of temperature stress was evaluated at 14°C and 28°C. The comparison between these results for stream MP2 can be found below and the results for other two streams can be found in appendix, page XXXII.

Low DO effects on bacteria activities were not evaluated here.

4.9.1 Experiment 9-1: Evaluation of two weeks storage time influence on AOB/NOB competition, stream MP2

From Figure 4.71 and Figure 4.72 it is difficult to claim something about the storage time effects on bacteria activities, because the oxygen consumption demonstrates similar patterns as during previous experiments for each step in reactor 1, as well as for the endogenous respiration in reactor 2.


Figure 4.72. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration on Stream MP2. Reactor 2.

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0 0,5 1 1,5 2

DO

, m

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Time, hr

NH4+ ATU +NO2

-

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DO

, mg/

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Time, hr

76

A further inspection of SOUR values from Figure 4.73 and Figure 4.74 also shows similar patterns compared with the previous patterns obtained with fresh carriers. However during step 1, as well as during the first three cycles for the endogenous respiration, the SOUR values are bigger than those obtained with fresh carriers at 14°C. According to Table 4.25 (fresh carriers and 14°C) and Table 4.31, HB activity varies after the storage from 0.5 mg O2/gTS*h to 1.9 mg O2/gTS; meaning that possible hydrolysis process could occur during the storage. SOUR values form step 2 are lower than under fresh carries condition. According to these tables AOB varies after storage from 4.7 mg O2/gTS to 3.8 mg O2/gTS*h. The suppressed AOB activity due to temperature stress and/or hydrolysis process during storage are the most reasonable explanations for these behaviours. Finally during the last step, according to the same tables, the NOB activity varies after storage from 1.0 mg O2/gTS*h to 0.6. mg O2/gTS*h. It could be explained by the same arguments used here for explaining AOB behaviours. Thus the AOB/NOB competition conserves the pattern e.g. higher AOB and very low NOB at 14°C, but the reduction of AOB and NOB activities is clear, meaning that for this method it is proved that fresh carriers (storage time less than 72 hours) should be used for bacteria activities assessments.


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0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

77



2 AOB +NOB + HB 3.8 1.6

3 NOB + HB 2.1 1.5

Computed AOB 1.6

Computed NOB 0.6 The results from the other two streams corresponding to experiments 9-2 and 9-3 can be found in appendix, page XXXII.

4.9.2 Experiment 9-4: Evaluation of the temperature shock influence on AOB/NOB competition at two weeks storage time condition, stream MP2

From Figure 4.77 the endogenous respiration in the present experiment follows a reduction trend, meaning that the organic carbon concentration decreases gradually in step 1. It is confirmed by the endogenous respiration (Figure 4.78). From Table 4.27 the HB activity at 28°C for fresh carriers was 1.4 mg O2/gTS*h;; which is lower than the obtained here at 28°C after storage of 2 mg O2/gTS*h (see Table 4.32). The increment on HB activity could be explained by a probable hydrolysis process taking place during storage. Similarly the AOB activity was of 1.8 mg O2/gTS*h at 28°C with fresh carriers (see Table 4.27) and of 2.3 mg O2/gTS*h at 28°C after two weeks of storage time (see Table 4.32), meaning that different processes have taken place during the storage, presenting an inverse pattern compared with the previous temperature stress assessment obtained with fresh carriers, represented by experiment 8-4.

Finally a comparison in the same order for NOB activities result in 1 mg O2/gTS*h at 28°C and fresh carriers (see Table 4.27) vs. 1.4 mg O2/gTS*h at 28°C and two weeks storage time (see Table 4.32), that follows the same trend as the AOB activities.

78



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0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU +NO2

-

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0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

79




2 AOB +NOB + HB 5.1 1.4

3 NOB + HB 2.7 1.3

Computed AOB 2.3

Computed NOB 1.4

A comparison of the results described above is presented also in Figure 4.79, Figure 4.80 and Figure 4.81 as a summary of results from sets of experiments 8 and 9.

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0 0,5 1 1,5 2

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Rat

e, m

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gTS*

h

Time, hr

NH4+ ATU + NO2

-

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Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

80

Figure 4.79. Parameters effects on MP1 microorganisms activities.

Figure 4.80. Parameters effects on MP2 microorganisms activities.

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

5,0

Fresh Temperature stress

Lower DO Storage time Temperature & Storage

Spec

ific

Rat

e, m

gO2/

gTS*

h

AOB

NOB

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

5,0



Spec

ific

Rat

e, m

gO2/

gTS*

h

AOB

NOB

81

Figure 4.81. Parameters effects on RP microorganisms activities. Results of the set of experiments 9 confirm the significant impact of the storage time on the bacteria activities for all three streams. Samples after storage do not demonstrate the same reaction on the temperature stress conditions as fresh samples.

4.10 Set of experiments 10. Correlation between DO concentrations, thicknesses of oxygen penetration into the biofilm and bacteria activities

An attempt to understand how the efficiency of the biofilm can be improved was carried out with the specific objective of finding the effective biofilm thickness (thickness of oxygen penetration) as well as the microorganisms localization inside the biofilm. The following results are from stream MP1. Results from streams MP2 and RP can be found in appendix, page XLI.

From Figure 4.82 to Figure 4.85 the process of selection of the time interval for calculating the slopes from recorded DO curves is demonstrated. The criteria for the selection of time interval were based on the point from which towards higher DO concentrations all the slope points are equal or lower than this point. The point found in this way was assumed to represent the transition from the ½-order kinetics reaction to 0-order.

From these figures it is clear that the noise reduces with increased time interval. The graph built using selected time interval is shown on Figure 4.85.

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

4,0

4,5

5,0



Spec

ific

Rat

e, m

gO2/

gTS*

hAOB

NOB

82

Figure 4.82. The Specific Oxygen Uptake Rate of bacteria vs. DO. Stream MP1. The SOUR values present high noise due to a 30 seconds time interval in slope function.

Figure 4.83. The Specific Oxygen Uptake Rate of bacteria vs. DO. Stream MP1. The SOUR values present less noise due to a 40 seconds time interval used in slope function.

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0 5 10 15 20

Spec

ific

Rat

e, m

gO2/

gTS*

h

DO, mg/L

AOB + NOB + HB

NOB + HB

HB

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Spec

ific

Rat

e, m

gO2/

gTS*

h

DO, mg/L

AOB + NOB + HB

NOB + HB

HB

83

Figure 4.84. The Specific Oxygen Uptake Rate of bacteria vs. DO. Stream MP1. The SOUR values present less noise due to a 90 seconds time interval used in slope function.

Figure 4.85. The Specific Oxygen Uptake Rate of bacteria vs. DO. Stream MP1. Final adjustment of the selected interval of 120 seconds for minimising noise in the SOUR patterns.

4.10.1 Experiment 10-1: Evaluation of dissolved oxygen concentration influence on AOB, NOB and HB kinetics, stream MP1

At Figure 9.86 three lines are shown. Blue line represents the combined response from HB, AON and NOB activities under the presence of substrates for one cycle of no aeration The red line represents the combined response from HB and NOB activities due to the introduction of only NO2

- and ATU. Finally the green line represents the response from HB activity. From Figure 4.87 it is clear that all the microorganisms activities reduce following the reduction of the DO concentration through the time. Oxygen from the atmosphere was re-aerating the system since it was not isolated. A balance between the rate of this re-aeration and the oxygen uptake rate by the microorganisms, was found after 2.5 hour for the three lines. The flat slope patterns at the bottom of each line provide evidence of that, meaning that the system never reached zero oxygen concentration in the bulk.

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e, m

gO2/

gTS*

h

DO, mg/L

AOB + NOB + HB

NOB + HB

HB

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Spec

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Rat

e, m

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gTS*

h

DO, mg/L

AOB + NOB + HB

NOB + HB

HB

84

The green line had a lower initial oxygen concentration but still it was over 100% of the saturation level. The green line has a minimum DO concentration that is bigger than those obtained for blue and red lines. It could be explained by a combination of the presence of low organic carbon concentrations and a low oxygen concentration at the place where the HB is located in the inner part of the biofilm. Because of the limiting carbon and oxygen sources the effective thickness for presence of HB only cannot be defined in this study.

Figure 4.86. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP1 (blue and red lines respectively) and without external additions (green line). From Figures 4.87 and 4.88 the surface oxygen uptake rates vs. DO and DO1/2 for stream MP1 confirms ½-order reactions in all three lines. The intersection of lines with the x-axis shows that the oxygen uptake rates and re-aeration rates achieved sooner equilibrium in case of AOB inhibition.

Figure 4.87. The Specific Oxygen Uptake Rate of bacteria vs. DO. Stream MP1.

02468

101214161820

0 0,5 1 1,5 2 2,5 3

DO

, m

g/l

Time, hr

AOB + NOB + HB

NOB + HB

HB

050

100150200250300350400450500

0 5 10 15 20

Spec

ific

Rat

e, m

gO2/

m2*

h

DO,mg/L

AOB + NOB +HB

NOB +HB

HB

85

Figure 4.88 shows a convenient representation of the surface oxygen uptake rates vs. DO1/2

that permits the validation of 1/2-order and 0-order reactions as well as permits the determination of the effective thickness of the biofilm.

Figure 4.88. Transition from ½ - to 0 –order of reaction in the bulk water, in a convenient plot for determination of biofilm parameters for stream MP1. According to Table 4.33 the biofilm thickness is bigger than one from a trickling filter that could be of 117 µm (Henze, et al., 1992). It is also proved by the data in this table that MP1 and MP2 biofilms are almost equal in terms of effective thicknesses. For RP the values for effective thicknesses are not comparable with the ones for MP1 and MP2 because the experiments were carried out at different temperature conditions.

Table 4.33. Thickness of dissolved oxygen penetration into biofilm.

Thickness of oxygen penetration into biofilm from stream:

Bacteria active in the system

MP1 (µm)

MP2 (µm)

RP (µm)

AOB + NOB + HB 646 659 241

NOB + HB 1,071 1,108 661

Table 4.34 and Table 4.35 contain the volumetric and the surface oxygen uptake rates for stream MP1, MP2 and RP, respectively. Volumetric rates are calculated for 1 m3 of biofilm and the surface rates for 1 m2 biofilm. According to these tables the carriers from MP1 are more effective than those from MP2. These numbers could be used for biofilms efficiency comparisons. Carriers from RP stream cannot be compared with MP1 or MP2 carries as the conditions of the experiments were different in terms of temperature.

050

100150200250300350400450500

0 1 2 3 4 5

Spec

ific

Rat

e, m

gO2/

m2*

h

[DO,mg/L]1/2

AOB + NOB +HB

NOB +HB

HB

86

Table 4.34. Volumetric oxygen uptake rate in biofilm.

Volumetric oxygen uptake rate in the biofilm from stream:


MP1 (kgO2/(m3*day))

MP2 (kgO2/(m3*day))

RP (kgO2/(m3*day))

AOB + NOB + HB 94.6 86.4 161.3

NOB + HB 60.7 50.4 62.6

Table 4.35. Surface oxygen uptake rate in biofilm.

Surface oxygen uptake rate by the biofilm from stream:


MP1 (kgO2/(m2*day))

MP2 (kgO2/(m2*day))

RP (kgO2/(m2*day))

AOB + NOB + HB 0.0050 0.0043 0.0085

NOB + HB 0.0032 0.0027 0.0037

87

5 Discussion The main objectives of the present research include:

1) The development of the OUR measurements method, adaptation and assessment of the method for studying the activities and competition between AOB, NOB and HB in a mixed culture in the biofilm MBBR system;

2) The application of the developed method for investigation of the influence of different external conditions on the behaviour of the bacteria sampled at the Manammox pilot installation at Sjölunda WWTP.

Initial experiments (set of experiments 1 to set of experiments 5) provide information about the influence of different parameters on the applicability, reliability and the sensitivity of the OUR method.

Set of experiments 1 shows the role of the concentration of carriers in the wastewater. The results of these experiments demonstrate the importance of keeping the same concentration in the laboratory experiments as at the pilot plant. Due to the different movement pattern inside the reactor and higher DO concentrations at lower concentrations of carriers due to lower total oxygen consumption, the specific activity of the aerobic bacteria is higher at low concentration of carriers in the reactor. It was proved that the specific heterotrophic bacteria activity depends on the concentration of carriers, but after the first three aeration/no aeration cycles this dependency disappeared and bacteria activity reduces continuously, making necessary the generation of the individual HB activity or elimination of reduction of this activity through the time for the determination of the AOB and NOB activities with this method.

Set of experiments 2 provides information about the influence of heterotrophic bacteria activity on the quality of the method. The endogenous respiration pattern is changing through the time: during the whole experiment of 1.5 hours the respiration of HB reduces. By this experiment it was proved in all three streams that for higher precision of the method in differentiating between the activities of three groups of bacteria it is important to keep the “pure” respiration as a reference.

For practical reasons it can be recommended to start the experiment with initial continuous aeration of 1 hour instead of running the parallel reactor for the reference. The recommended time is based on all the experiments carried out during present research, which demonstrate relative stabilization of the HB respiration after 1 hour of aeration. It will eliminate the influence of HB respiration on bacteria activities after the addition of nitrogen sources as most of the residual substrates (COD, NH4

+ and NO2-) which could be released by the biofilm or

hydrolysed will be consumed during initial aeration period. However it only applies to fresh sample analyses which have been stored for not more than 72 hours prior to the experiment (based on assessment of fresh and stored carriers by means of OUR method). If the sample was stored for longer time, an extended time of initial aeration may be required. With this set of experiments it was proved that the defined substrates concentrations, i.e. concentrations of NH4

+ and NO2- guarantee the abundance of substrate condition during step 2 and step 3,

permitting the assessment of bacteria activities under full penetration conditions of the biofilm by these substrates.

88

Experiment 3 demonstrates the influence of the duration of the aeration/no aeration cycles on the reliability of the method. According to the results of the experiments the duration of cycles should not be decreased from the original duration of 10 minutes per cycle as shorter cycles do not give enough time for the clear response of the bacteria respiration to the addition of substrates. However the response to the addition of inhibitor (ATU) is clear showing that the penetration of the ATU into the biofilm is faster than penetration of the substrates. Increased duration of cycles has not been investigated within the present research.

With set of experiments 4 the best suitable substrates addition pattern was developed for the method. The influence of pH control was investigated. The activities calculation approach was developed based on the results of the first four sets of experiments. The influence of the storage of carriers on the reliability of the method was checked.

The simultaneous addition of both substrates, NH4+ and NO2

-demonstrated the promotion of higher activity of NOB while in the treatment process the bacteria first receive NH4

+ and only then receive NO2

- as a product of ammonium oxidation. From the results of these experiments it was concluded that the separate addition of NH4

+ and NO2- is necessary as NO2

- produced from oxidation of NH4

+ is consumed after the AOB inhibition almost instantaneously by NOB and anammox bacteria present in the system.

No noticeable influence of pH control between 7.6 – 7.7 pH units was demonstrated experimentally. Based on current results and previous research on activated sludge systems (Llano and Galkin, unpublished) it can be concluded that pH does not have noticeable impact on the nitrification process if it lies within the reasonable range for treatment process of 7.5 to 8.5 pH units. The bacteria activities calculation approach was changed for this set of experiments in order to increase precision and improve reliability of the method (see chapter 3.2). With a new method AOB and NOB bacteria activities are calculated at the points of experiment where similar conditions are provided.

Storage of samples prior to performing the experiments was demonstrated to have significant effect on the results of the experiments, i.e. on the bacteria activities. However developed method can be reliably used for evaluation of the bacteria competition in the sample after storage.

Set of experiments 5 demonstrates the sensitivity of the method, i.e. the ability to evaluate bacteria activities at low temperatures, when the activities are usually lower. The present method demonstrated high sensitivity thus can be used for evaluation of the process at low temperatures.

The second part of the current project (set of experiments 6 to set of experiments 10) was dedicated to evaluation of aerobic bacteria activities in the process at the pilot plant applying the method developed and adapted during the first part of the project.

Set of experiments 6 aims to assess the result of mixing of carriers from two streams, RP and MP1, with different process conditions at the pilot plant. The experiment confirmed the hypothesis that sample with higher proportion of carriers from RP stream would show the highest specific activity of both groups of bacteria, AOB and NOB. Interesting result is that despite the temperature stress conditions for bacteria from MP1 stream (28ºC), the sample with highest proportion of carriers from MP1 demonstrated the highest AOB/NOB activities

89

ratio which is a favorable condition for partial nitritation process. Differences in oxygen concentration conditions for all the experiments of this set were very small and were assumed negligible. It can be concluded that the biofilm adapted to colder water has higher AOB/NOB activities ratio than the biofilm adapted to warmer water even in experimental conditions different from the adaptation conditions (at higher temperature). The activities ratio demonstrated by experiment performed at temperature conditions similar to biofilm adaptation conditions is also higher for the cold water adapted biofilm, which is confirmed by several experiments described in chapter 4. However the specific activities of both groups of bacteria are higher (with smaller difference between two groups) at higher temperatures which is confirmed by set of experiments 6 and several experiments described in chapter 4 which are discussed below.

Set of experiments 7 demonstrates the influence of low dissolved oxygen level at adapted and stress temperature for sample from MP1 stream. The results of these experiments together with the results of experiments with the same conditions but with higher DO levels lead to conclusion that the DO concentration increment promotes the increase in activity of AOB more than in activity of NOB for this specific biofilm for both adapted and stress temperature conditions. This conclusion agrees with the research made by Regmi et al., (2014) at similar DO conditions.

Set of experiments 8 is aiming to perform a comprehensive study of the bacteria activities for all three streams at following conditions: adapted temperature, stress temperature and lowered DO concentrations at 14ºC temperature condition.

The results demonstrate that the temperature stress decrease the AOB activity for all streams, with most significant influence on MP1 and MP2 streams. Temperature stress promotes the activity of NOB for MP1 stream with the insignificant opposite effect on NOB for MP2 and RP streams. Interesting results were obtained at lower DO condition at 14ºC – with the opposite effect for MP1 and MP2 streams: for MP1 stream AOB activity was halved with constant NOB activity and for MP2 stream NOB activity was halved with constant AOB activity. Bacteria activities for RP stream were undetectable. It can be concluded that the biofilm structure and bacterial content differs in three streams and that both adapted temperature and adapted substrates concentrations (here – conditions at Sjölunda pilot plant) are important factors affecting the activities of AOB and NOB bacteria in the biofilm and their behaviour under various conditions.

Analyses of substrates concentrations, performed for few experiments in this set, support the observed results and conclusions made.

Set of experiments 9 aims to evaluate the two weeks storage time effect on bacteria activities for all three streams under adapted temperature and stress temperature conditions. Biofilm demonstrated significant decrease in the activities of both groups, AOB and NOB after 2 weeks of storage for samples from all three streams with simultaneous significant increase in the HB activity. One possible reason for the increased activity of HB is the hydrolysis of complex organic molecules during the storage time which makes more organic materials available for utilization by HB when oxygen is supplied. In general at higher temperature all bacteria activities were higher than for fresh samples despite the temperature stress for MP1 and MP2 streams. Simultaneously for these two streams with higher temperature the AOB/NOB activities ratio decreased meaning that stress temperature promotes the NOB activity for these samples. The opposite was demonstrated by carriers from RP stream: at their

90

adapted temperature of 28ºC the activities ratio was higher despite the higher temperature and their stress temperature of 14ºC, promoted the NOB activity.

Set of experiments 10 was accomplished using different OUR approach and the aim was to find the thickness of oxygen penetration into the biofilm (effective thickness) for three streams at adapted temperature conditions. Results show that the effective thicknesses are significantly higher in case of AOB inhibition for all streams as oxygen is not consumed by this group of bacteria while diffusing through the biofilm. The effective thickness is higher at lower temperature as the transition point appears at higher DO concentration. It can be concluded that the effective thicknesses are almost equal for MP1 and MP2 streams when the experiment is performed at the same temperatures. In order to confirm the results of this set of experiments microbiological investigations as fluorized in situ hybridization (FISH) and polymerase chain reaction (PCR) of bacteria distribution throughout the biofilm thickness is needed. Further experiments can be performed at the same temperature conditions for all three streams to check for the effective thickness of the biofilm from RP stream.

91

6 Conclusions OUR method was adapted and further developed for the application on studying the biofilm on the MBBR AnoxKaldnes carriers type K1® from the Mainstream Anammox process at the pilot plant at Sjölunda Wastewater Treatment Plant. Developed method demonstrated high reliability and precision in determination of relative activities of three aerobic bacteria groups present in the biofilm, namely AOB, NOB and HB, by measuring their respiration rate. Effectiveness and accuracy of the method can be proved by several repeated results for biofilm samples taken at different times when test was performed under the same conditions.

The results of the bacteria activities study using the described method can be summarized as follows:

x The proposed method can be used for evaluation of aerobic bacteria activities in the mixed culture in the biofilm-based treatment systems. The activity of each group of bacteria can be distinguished from the other bacteria groups in the mixed culture.

x Constant change (decrease) of HB respiration rate throughout the time of the experiment of 1.5 hours should be considered either with a parallel reactor with presence of only HB activity for reference or with initial pre-aeration of the reactor with presence of mixed groups activities.

x For obtaining reliable results with the present method the duration of the experiments cannot be reduced.

x The pH variations within the range of 7.5 to 8.5 pH units do not have any noticeable impact on AOB, NOB or HB activities at high concentrations of substrates used in current research.

x The storage time of more than 72 hours at 4ºC significantly decrease the activity of AOB and NOB and increase the HB activity. Storage time also affects differently the behaviour of AOB and NOB at stress temperature conditions.

x Biofilm adapted to colder water (14ºC) demonstrates higher AOB/NOB activities ratio than the biofilm adapted to warmer water (28ºC). This behaviour was demonstrated during experiments at both adapted water temperature and altered water temperature conditions.

x Specific activities of AOB, NOB and HB are higher at higher water temperatures, but AOB/NOB activities ratio is higher at lower water temperatures.

x DO concentration increment promotes the increase in activity of AOB more than in activity of NOB for studied biofilm at different temperatures.

x The behaviour of bacteria is different in studied samples from three different streams. This leads to conclusion that both adapted temperature and adapted substrates concentrations play very important role in the bacteria behaviour under various conditions.

x The oxygen uptake by AOB is an important factor affecting the maximum thickness of oxygen penetration into the biofilm.

x The thickness of oxygen penetration into the biofilm is equal (the difference is negligible) for samples from MP1 and MP2 streams at equal conditions, i.e. at the same temperatures and the same bacteria groups presence.

93

7 Suggestions for further research The activities of anaerobic anammox bacteria lies beyond the scope of the present research, thus investigations can be performed to further understanding of the influence of different external parameters on the behaviour of this group. Closer look can be taken on possible NOB activity in anoxic conditions – heterotrophic denitrification by NOB – with the presence of organic matter.

Microbiological investigation of bacteria distribution throughout the biofilm thickness for representative number of samples can be performed to confirm the conclusions made concerning the thickness of oxygen penetration for studied biofilm.

Further experiments can be performed at the same temperature conditions for samples from all three streams, MP1, MP2 and RP to check for the thickness of oxygen penetration into the biofilm from RP stream at 14ºC and for biofilm from MP1 and MP2 streams at 28ºC to make it possible to compare results for all three streams.

95

8 References Andersson, S, Nilsson, M. Dalhammar, G. and Rajarao,G.K., 2008.Assessment of carrier materials for biofilm formation and denitrification. VATTEN, [online] Available at: http://www.tidskriftenvatten.se/mag/tidskriftenvatten.se/dircode/docs/48_article_3539.pdf! [Accessed 11 February 20014].

Bolton, J., Tummala, Ch. K., Dandamudi, M. and Belovich, J.M., 2006. Procedure to quantify biofilm activity in carriers used in wastewater treatment systems. Journal of Environmental Engineering, [online] Available at: http://engagedscholarship.csuohio.edu/cgi/viewcontent.cgi?article=1029&context=encbe_facpub/! [Accessed 11 February 20014].

Bothe, H., Ferguson, S. and Newton, W. eds., 2007. Biology of the nitrogen cycle. Amsterdam: Elsevier.

Camargo, J. and Alonso, A., 2006. Ecological and toxicological effects of inorganic nitrogen pollution in aquatic ecosystems: a global assessment. Environment International, 32(6), pp. 831-849.

Confer, D. R. and Logan, B. E., 1998. Location of protein and polysaccharide hydrolytic activity in suspended and biofilm wastewater cultures. Water Research, 32(1), pp. 31 – 38.

Dimock, R. and Morgenroth, E., 2006. The influence of particle size on microbial hydrolysis of protein particles in activated sludge. Water Research, 40(10), pp. 2064 – 2074.

Ellervik, U. and Sterner, O., 2007. Organisk Kemi (2nd edition). Lund: Studentlitteratur.

Ginestet, P., Audic, J-M., Urbain, V. & Block J-C., 1998. Estimation of Nitrifying Bacteria Activities by Measuring Oxygen Uptake in the presence of the Methabolic inhibitors Allylthioureaand Azide.Applied and Environmental Microbial, 64(6), pp. 2266-2268.

Gustafsson, I., 2013. Methods for activity tests of anammox and ammonia oxidizing bacteria on carrier material, Uppsala: Uppsala University.

Gustavsson D. J. I., Aspegren H., Stålhandske L. and Jansen, Jes la Cour, 2013. Mainstream anammox at Sjölunda Wastewater Treatment Plant – pilot operation of sludge liquor treatment and start-up of the full process. Proceedings of 13th Nordic Wastewater Conference. Malmö, Sweden, October 8-10, 2013.

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Hagman, M. and Jansen, Jes la Cour, 2007. Oxygen uptake rate measurements for application at wastewater treatment plants. Water and Environmental Engineering, Volume 63, pp. 131-138.

Hoehn, R. and Ray, A., 1973. Effects of thickness on bacterial film. Water pollution control federation, 45(11), pp. 2302 – 2320.

Hu Z., Lotti T., de Kreuk M., Kleerebezem R., van Loosdrecht M., Kruit J., S. M. Jetten M. and Kartal B., 2013. Nitrogen removal by a nitritation-anammox bioreactor at low temperature. Applied and Environmental Microbiology. [online] Available at: <http://aem.asm.org/content/79/8/2807.full> [Accessed 08 04 2014].

Jansen, Jes la Cour, 1983. Fixed film kinetics – removal of soluble substrates in fixed films. Lyngby: Technical University of Denmark.

Henze, M., Harremoës, P., Jansen, Jes la Cour and Arvin, E., 1992. Wastewater Treatment: Biological and Chemical Process. 3rd ed. Berlin: Springer.

Kartal, B., Kuenen, J.G. and van Loosdrecht, M., 2010. Sewage treatment with Anammox, >online@ Available at: http://www.sciencemag.org/content/328/5979/702!>Accessed 11 February 2014@.

Lackner, S., Gilbert, E.M., Vlaeminck, S.E., Joss, A., Horn, H. and van Loosdrecht, M., 2014. Full-scale partial nitritation/anammox experiences - an application survey. Water Research, 55, pp. 292-303.

Levstek, M. and Plazl, I., 2009. Influence of carrier type on nitrification in the moving-bed biofilm process. Water Science & Technology-WST, [online] Available at: http://www.iwaponline.com/wst/05905/wst059050875.htm ! [Accessed 10 February 2014].

Llano, N. and Galkin, V., 2014. Evaluation of bacteria activities and inhibitor properties with the OUR measurements method for the activated sludge at Källby WWTP. Project course I VVA910.

Morgenroth, E., Kommedal, R. and Harremoës, P., 2002. Processes and modeling of hydrolysis of particulate organic matter in aerobic wastewater treatment – a review. Water Science and Technology, 45(6), pp. 25 – 41.

Regmi, P., Miller, M. W., Holgate, B., Bunce, R., Park, H., Chandran K., Wett, B., Murthy, S. and Bott, C. B., 2014. Control of aeration, aerobic SRT and COD input for mainstream nitritation/denitritation. Water Research, 57, pp. 162-171.

Reid, R., Praunsnitz J. and Sherwood T., 1977. The Properties of Gases and Liquids, Third Edition. New York: McGraw-Hill.

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Riemer, M. and Harremoës, P., 1978. Multi-component diffusion in denitrifying biofilms. Progress in Water Technology, 10(5/6), pp. 149-165.

Siegrist, H., Salzgeber, D., Eugster, J. and Joss, A., 2008. Anammox brings WWTP closer to energy autarky due to increased biogas production and reduced aeration energy for N-removal. Water Science and Technology, 57(3), 383-388.

Strous, M., Kuenen, J. G. and Jetten, M.S.M., 1999. Key Physiology of Anaerobic Ammonium Oxidation. Applied and Environmental Microbiology, pp. 3248-3250.

Surmaz-Gorska, J., Gernaey, K., Demuynck, P. and Vanrolleghem and Verstraete, W., 2010. Nitrification Process Control in Activated Sludge Oxygen Uptake Rate Measurements. Environmental Technology, 16(6), pp. 569-576.

Van Dongen, U., Jetten, M.S.M. and van Loosdrecht, M., 2001. The Sharon - Anammox process for treatment of ammonium rich wastewater. Water Science and Technology, [online] Available at: http://scholar.google.se/scholar?q=the+sharon-anammox+process+for+treatment+of+ammonium+rich+wastewater&hl=en&as_sdt=0&as_vis=1&oi=scholart&sa=X&ei=Spl7U_3QM66L4gSEyoD4Bg&ved=0CCcQgQMwAA! [Accessed 11 February 20014].

Welty, J., Wicks, C. and Wilson, R., 1984. Fundamentals of Momentum, Heat, and Mass Transfer, Third Edition. New York: John Wiley & Sons.

Wiesmann, U., Choi, I.S. and Dombrowski, E., 2007. Fundamentals of Biological Wastewater Treatment. Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA.

Wilke, C. and Chang, P., 1955. Correlation of diffusion coefficients in dilute solutions. A.E.CH.E. Journal, 1, pp. 264-270.

Winkler, M., Bassin, J., Kleerebezem, R., Sorokin, D., van Loosdrecht, M., 2012. Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge. Applied Microbiological Biotechnology, 94, pp. 1657 – 1666.

Ødegaard, H., 1999. The Moving Bed Biofilm Reactor. Water Environmental Engineering and Reuse of Water, pp. 250-305.

I

Appendix Experiment 4-3: Stream MP1

Figure 8.1.The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP1. Reactor 1.


0

1

2

3

4

5

6

7

8

9

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU + NO2

- + H2SO4 + NH4+

0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

- + H2SO4 + NH4+

II

Experiment 4-5: Stream MP2



0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr


- + H2SO4

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

III




1 HB 2.9 2.6

2 AOB +NOB + HB 5.7 1.6

3 NOB + HB 3.2 1.6

Computed AOB 2.4

Computed NOB 1.7

0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

ATU + NO2- + H2SO4NH4

+ + H2SO4

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

IV

Experiment 4-6: Stream RP

Figure 8.7. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration and the influence of substrates and ATU on Stream RP. Reactor 1.


0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr


- + H2SO4

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

V

Figure 8.9. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream RP. Reactor 1.

Figure 8.10. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP2. Reactor 2. Table 8.2. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream RP.


1 HB 2,4 2,2

2 AOB +NOB + HB 5,7 1,4

3 NOB + HB 3,2 1,3

Computed AOB 2,4

Computed NOB 1,9

0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

VI

Experiment 4-8: stream MP2



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10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ + H2SO4 ATU +NO2

- + H2SO4

0123456789

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0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

VII



0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

VIII

Experiment 4-9: stream RP


Figure 8.16. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration on Stream RP. Reactor 2.

0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

ATU +NO2- + H2SO4NH4

+ + H2SO4

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

IX



0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

X

Experiment 5-2: Stream RP



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10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU +NO2

-

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

XI



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10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU +NO2

-

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

XII

Experiment 5-3: Stream MP2



0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

XIII



0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

XIV

Experiment 5-5 MP2



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0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

XV


Figure 8.30. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP2. Reactor 2 Table 8.3 .Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream MP2.


1 HB 1.0 0.9

2 AOB +NOB + HB 4.9 0.6

3 NOB + HB 2.0 0.6

Computed AOB 2.9

Computed NOB 1.4

0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2-

-

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

XVI

Experiment 5-6 RP



0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

XVII


Figure 8.34. The Specific Oxygen Uptake Rate of bacteria vs. time. Stream RP. Reactor 2. Table 8.4. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream RP.


1 HB 1.7 1.5

2 AOB +NOB + HB 3.7 1.0

3 NOB + HB 1.3 0.8

Computed AOB 2.2

Computed NOB 0.5

0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

XVIII

Experiment 7-1.

Here and further down for all charts showing the concentration vs. time the current reported NH4, NO2 and NO3 should be understood as ions: NH4

+, NO2- and NO3

- respectively.

Figure 8.35. Substrates concentrations vs. time. Stream MP1. Experiment 7-2.

Figure 8.36.Substrates concentrations vs. time. Stream MP1.

0102030405060708090

100

0 0,5 1 1,5 2

Con

cent

ratio

n of

N, m

g/L

Time, hr

NH4-N

NO2-N

0

10

20

30

40

50

60

70

80

90

100

0 0,5 1 1,5 2

Con

cent

ratio

n of

N, m

g/L

Time, hr

NH4-N

NO2-N

XIX

Experiment 8-4, stream MP1.



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0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

0123456789

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0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

XX




1 HB 1.2 1.0

2 AOB +NOB + HB 3.6 0.7

3 NOB + HB 1.1 0.7

Computed AOB 2.5

Computed NOB 0.4

0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

XXI

Figure 8.41. Substrates concentrations vs. time. Stream MP1. Experiment 8-5, stream MP1.


0

10

20

30

40

50

60

70

80

90

100

0

10

20

30

40

50

60

70

80

90

100

0 0,5 1 1,5 2

Con

cent

ratio

n of

C, m

g/L

Con

cent

ratio

n of

N, m

g/L

Time, hr

NH4-N

NO2-N

NO3-N

COD

0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

XXII



0123456789

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0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

0123456789

10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

-

XXIII



1 HB 0.4 0.3

2 AOB +NOB + HB 1.8 0.2

3 NOB + HB 0.6 0.2

Computed AOB 1.2

Computed NOB 0.4

Figure 8.46. Substrates concentrations vs. time. Stream MP1.

0123456789

10

0 0,5 1 1,5 2

Spec

ific

rate

, m

gO2/

gTS*

h

Time, hr

0102030405060708090100

0102030405060708090

100

0,0 0,5 1,0 1,5 2,0

Con

cent

ratio

n of

C, m

g/L

Con

cent

ratio

n of

N, m

g/L

Time, hr

NH4-N

NO2-N

NO3-N

COD

XXIV

Experiment 8-6, MP1.



0123456789

10

0 0,5 1 1,5 2

DO

, m

g/l

Time, hr

NH4+ ATU + NO2

-

0123456789

10

0 0,5 1 1,5 2

DO

, mg/

l

Time, hr

XXV




1 HB 2.1 2.1

2 AOB +NOB + HB 3.7 1.8

3 NOB + HB 4.4 1.8

Computed AOB -0.7

Computed NOB 2.6

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10

0 0,5 1 1,5 2

Spec

ific

Rat

e, m

gO2/

gTS*

h

Time, hr

NH4+ ATU + NO2

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Figure 8.51. Substrates concentrations vs. time. Stream MP1. Experiment 8-7, stream RP.


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1 HB 1.1 1.1

2 AOB +NOB + HB 4.6 0.8

3 NOB + HB 2 0.8

Computed AOB 2.6

Computed NOB 1.2

Figure 8.56. Substrates concentrations vs. time. Stream RP.

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Experiment 8-8



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R2 (HB only)

1 HB 0.8 0.8

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Computed AOB 2.1

Computed NOB 0.8

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Figure 8.61. Substrates concentrations vs. time. Stream RP. Experiment 8-9 stream RP.


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NO3-N

COD

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, m

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Time, hr

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XXXII

Figure 8.63. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration on Stream MP1. Reactor 2. Experiment 9-2, stream MP1 at 14°C


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2 AOB +NOB + HB 2.5 1.8

3 NOB + HB 1.8 1.7

Computed AOB 0.6

Computed NOB 0.1 Experiment 9-3, stream RP


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1 HB 1.0 1.2

2 AOB +NOB + HB 3.6 1.0

3 NOB + HB 1.5 1.0

Computed AOB 2.1

Computed NOB 0.5

Experiment 9-5, stream MP1


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Figure 8.75 The Specific Oxygen Uptake Rate of bacteria vs. time. Stream MP1. Reactor 2. Table 8.12. Computed AOB and NOB activities expressed in mg O2/gTS*h and corrected with endogenous respiration. Stream MP1.


R2 (HB only)

1 HB 2.1 2.1

2 AOB +NOB + HB 3.7 1.8

3 NOB + HB 2.8 1.8

Computed AOB 0.9

Computed NOB 1 Experiment 9-6, stream RP


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1 HB 1.2 1.1

2 AOB +NOB + HB 2.4 0.9

3 NOB + HB 1.2 0.8

Computed AOB 1.1

Computed NOB 0.4

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Experiment 10-2 MP2

Figure 8.80. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration and the influence of substrates and ATU on Stream MP2 (blue and red lines) and without external additions (green line).

Figure 8.81. The Specific Oxygen Uptake Rate of bacteria vs. [DO]. Stream MP2.

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HB

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NOB + HB

HB

XLII

Figure 8.82. Transition from ½ - to 0 –order of reaction in the bulk water, in a convenient plot for determination of biofilm parameters for stream MP2. Experiment 10-3 RP.

Figure 8.83. The Dissolved Oxygen concentration (DO) changes with time for endogenous respiration and the influence of substrates and ATU on Stream RP (blue and red lines) and without external additions (green line).

050

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NOB + HB

HB

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, m

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Time, hr

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NOB + HB

HB

XLIII

Figure 8.84. The Specific Oxygen Uptake Rate of bacteria vs. [DO]. Stream RP.

Figure 8.85. Transition from ½ - to 0 –order of reaction in the bulk water, in a convenient plot for determination of biofilm parameters for stream RP.

050

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HB

Evaluation of bacteria population dynamics in Mainstream Anammox

Nelson Alberto Llano Alvarez and Vadim Galkin

Water and Environmental Engineering, Department of Chemical Engineering, Lund University, Sweden

June, 2014

Abstract

The behaviour and competition of aerobic bacteria in the biofilm at different conditions and the kinetic parameters of the biofilm were investigated using the adapted oxygen uptake rate method.

Biofilms from three reactors with different conditions sampled at Sjölunda pilot plant in Malmö, Sweden were studied. The results from the lowered dissolved oxygen concentration showed thatincrease of oxygen concentration promotes the activity of ammonium oxidising bacteria more than the activity of nitrite oxidising bacteria. The temperature and storage time of samples have been found to have the most significant influence on the bacteria activities. Evidence of strong correlation between the adaptation conditions of the ammonium and nitrite oxidising bacteria and their activities and behaviour at varied conditions was found.

Keywords: Activity Test, Anammox, AOB, Biofilm, Endogenous Respiration, Heterotrophic Bacteria, Nitrogen Removal, NOB

Introduction

Nitrogen removal is an important part of the wastewater treatment. Biological nitrogen removal process involves significant costs for its implementation at the wastewater treatment plants. It is due to the required sludge age that implicates basins with big areas and also for the daily operation due to amount of energy consumed by the aeration units and the required external addition of carbon source such as methanol, to meet the wastewater quality standards. If the process efficiency does not fulfil the standard or if the process is not stable, the consequences can be dramatic for the recipient ecosystems. Now there is increasing pressure in this area as the energy cost becomes higher, population is growing

increasing the wastewater load and the effluent quality standards become stricter. To meet new challenges the wastewater treatment has to become more and more sustainable.

Recently discovered anammox bacteria (ANAMMOX – Anaerobic AMMonium Oxidation) give an exciting opportunity to move towards energy-neutral or even energy-positive wastewater treatment. They are part of the natural nitrogen cycle and create a shortcut in the deammonification process.

Anammox treatment technology requires less energy inputs in comparison with conventional nitrification-denitrification method due to less aeration and no need for internal recirculation or addition of external carbon source as the process is fully autotrophic and, in addition, more organic matter in the raw wastewater can be separated

and utilized for increased biogas production. These lead to a great saving and optimizing potential [1]. The anammox method has been proved to be efficient by several full-scale installations treating ammonia-rich industrial wastewaters and sludge liquors, both of elevated temperatures [2]. The current challenge is to adjust the technology to optimize its use for municipal mainstream wastewater treatment. One of the issues for this process strategy is the suppression of the activity of Nitrite Oxidising Bacteria (NOB) under conditions of the mainstream wastewater flow to obtain the required proportion of ammonium and nitrite for anammox bacteria. Thus thorough research of the nitrification process and the activities of all bacteria involved are important.

The aim of this work is to use an adapted Oxygen Uptake Rate (OUR) measurements method to evaluate the behaviour of aerobic microorganisms and get more understanding of the dynamics of different groups of bacteria in a mixed culture, living in a form of the biofilm on the support medium. Understanding of the influence of different operational conditions on the bacteria activities helps to develop reasonable and scientifically proven operational strategies for the pilot- and full scale applications of this technology.

The introduction of the anammox process in an early stage involving the mainstream includes the activity of Ammonium Oxidising Bacteria (AOB), Ammonium Oxidising Achaea (AOA), Nitrite oxidizing Bacteria

NOB, heterotrophic bacteria (HB) and anammox bacteria in a mixed culture. AOB in this article should be considered as AOB and AOA microorganisms. The current study is focused on the activities of the aerobic groups of microorganisms, i.e. AOB, NOB and HB, but not the anammox bacteria. The study does not take into account also possible NOB activity in anoxic conditions – heterotrophic denitrification by NOB – with the presence of organic matter [3].

Materials and Methods

OUR method

The OUR measurement approach described by Hagman and Jansen [4] was further developed and used for studying nitrifying bacteria activities in the biofilm attached to the carriers AnoxKaldnes type K1, sampled at Sjölunda pilot plant [5]. The pilot plant consists of three main MBBR, mainstream reactor 1 (MP1), mainstream reactor 2 (MP2) and sludge liquor reactor (RP). The average operating temperature in MP1 and MP2 is 14°C and in RP is 28°C. As influent MP1 receives the effluent water from the High Loaded Activated Sludge reactor (HLAS) mixed with the effluent from RP. Then the effluent from MP1 is used as influent to MP2. The RP receives the effluent from sludge liquor reactor as influent (see Figure 1) [6].

The oxygen utilization rate (OUR) can be related to the volatile solids (VS) to find the

Figure 1. Sjölunda pilot plant layout.

specific oxygen uptake rate (SOUR). However in this study the OUR was related to the total solids (TS) instead to eliminate errors due to the complexity of the method of VS analysis for the biofilm attached to a plastic medium. The TS were measured by drying of 30 carriers sampled from the reactor after the experiments for 24 hours at 105°C, followed by weighting of these dried carriers. The weight of 30 empty dry carriers (without a biofilm) was subtracted from the obtained weight of 30 dried carriers with biofilm to find the weight of the biofilm sampled. After the calculations the total weight of biofilm per 1 litre of wastewater in the reactor was found.

A lab-scale batch reactor with 400 ml of tap water and 128 carriers was operated at 28 ± 0.5°C (see Figure 2). This number of carriers to the volume of water relation represents the actual concentration of carriers at the pilot plant. Temperature was kept constant by means of the water bath. The pH was stable during the experiments with a small increase due to CO2 stripping during aeration [4]. pH adjustment to 7.5 through the buffer solution was implemented as its variations in preliminary experiments always were lying within the range of 7.5 to 8.2 and no effect on the process was noticed.

Another identical lab-scale reactor was operated in parallel without pH adjustment and without substrates addition, but keeping constant the remaining conditions to obtain the endogenous respiration pattern.

The reactors were continuously stirred with a magnetic stirring at around 150 rpm [7]. The water solution in the first reactor was sampled at a certain time for analyses of nitrogen in different forms. Sampling was done by a syringe with a volume of 10 ml.

Figure 2. A: Schematic picture of the laboratory set-up for OUR- measurements B: Laboratory batch 1 litre, used here without the Expander Figure taken and adapted with permission from Hagman and Jansen (2007) [4]. Filtered samples were then stored in sealed cuvettes (test-tubes) to ensure no further activity in the sample and analyzed for nitrogen concentrations in forms of ammonium, nitrite and nitrate with HACH Lange DR 2800 spectrophotometer (Sköndal, Sweden) using HACH Lange cuvettes LCK303 (2.0-47.0 mg/L NH4

+-N) and LCK304 (0.015-2.0 mg/L NH4

+-N) for ammonium, LCK341 (0.015-0.6 mg/L NO2

--N) and LCK342 (0.6-6.0 mg/L NO2

--N) for nitrite, LCK339 (0.23-13.5 mg/L NO3

--N) and LCK340 (5.0-35.0 mg/L NO3

--N) for nitrate. During all the performed experiments the

oxygen concentration and temperature were measured in the liquid phase by means of dissolved oxygen probe HACH HQ 40d from HACH (Loveland, Colorado, USA). Oxygen concentration measurements were taken every 10 seconds and were stored on the USB memory device. The pH of the sample in the reactor was continuously measured with the WTW pH 320 meter. Waterproof magnetic stirring equipment was used to ensure proper mixing. The timer with connected air pump controlled intermittent aeration.

A cycle of 5 minutes without aeration was followed by 5 minutes with aeration. Three cycles for each part of the experiment were used to obtain representative data cleared from the residual effects of the preceding part of the experiment with different conditions. All the OUR graphs with corresponded dissolved oxygen concentration decline lines regressions

K1 carrier

and nitrogen concentrations were plotted using Microsoft Excel.

Ammonium sulphate (NH4)2SO4 in a buffer solution was used as a source of ammonium, added to reach the final concentration of 100 mg NH4

+/L in the reactor. Buffer solution included 23.6 g of (NH4)2SO4, 61.2 g of NaHCO3 and 4.4 g of KH2PO4 per 1 litre of distilled water. Sodium nitrite (NaNO2) in water solution (12.33 g of NaNO2 per 1 litre of distilled water) was added as a source of nitrite to reach the final concentration of 75 mg NO2

-/L in the reactor. Ammonium oxidation inhibitor allylthiourea or ATU (C4H8N2S) in water solution was used at a final concentration of 86 µM in the reactor [8]. For some experiments sulphuric acid (H2SO4) in water solution with the concentration of 0.1 M was added after the substrates addition as an acid source to keep a pH value within the range of 7.6 to 7.7 pH units in the reactor which corresponds to the actual pH at the pilot plant. All the substrates and the inhibitor were injected into the reactor using pipettes 30 seconds before the end of the aeration phase for achieving complete mixing.

The oxygen uptake by heterotrophic bacteria can be reasonably approximated to endogenous respiration rate for experiments carried out. The respiration of AOB and NOB is developed after the individual addition of ammonium and nitrite ions respectively.

Samples of biofilm carries for the experiments were taken from the mainstream reactors MP1 and MP2 where bacteria were accommodated to the temperature of 14ºC between winter and spring and the reactor RP where bacteria were accommodated to the temperature of 28ºC between winter and spring from the pilot plant. Samples were stored at 4ºC if extended storage was required. Total solids (TS), temperature and pH were determined. For all experiments the biofilm samples were preconditioned by a gentle rinsing with tap water avoiding stress temperature and high pressure, in order to minimize initial substrates concentrations contained in the original wastewater that can

be retained on the biofilm surface and to remove weakly attached biofilm from the carriers and thus prevent parallel nitrogen removing processes in the bulk. Samples were warmed up or cooled down slowly to the test temperature and were aerated until a minimum initial dissolved oxygen level of 7.0 mg O2/L was reached.

Biofilm kinetics

The bacteria behaviour in the biofilms is analogous to that in activated sludge systems. The difference between the two techniques is that the density of the biofilms and concentration of bacteria in the biofilms is several times higher [9].

General simplified picture of the processes inside the biofilm is shown in Figure 3.

Figure 3. Simplified conceptual model for removal of soluble substrates focusing on phenomena inside the film. Figure taken with permission from Jansen (1983) [14].

Diffusion coefficient D is a new parameter introduced for biofilm systems compared to activated sludge systems. Existing methods for determination of this coefficient in biofilms are not highly reliable and the coefficient itself is variable depending on the conditions and other parameters of the biofilm. Thus for practical reasons it can be assumed equal to the molecular diffusion coefficient in water with the correction coefficient of 0.8 [9].

In turn molecular diffusion coefficient in the water depends on the water temperature and theoretical values can be found using correlation developed by Wilke and Chang [10], which is based on the Stokes-Einstein equation:

!!!!!!! = 7.4 ∗ 10!! !(!!!!∗!!!!)!!

!∗!!!!.! (1)

where: T – absolute temperature, ºK; !!!! – association parameter for the

solvent water = 2.26 [10]; !!!! – molecular weight of water = 18

g/mole; µ - dynamic viscosity of water, centipoise

(mPa*s); !!!- The molar volume of oxygen = 25.6

cm3/g - mole [11]. For calculations of volumetric uptake rate

of substrate by the biofilm, density of the biofilm can be reasonably assumed equal to water density as the weight of dry biofilm in relation to the volume of wet biofilm is relatively low [12].

The substrate uptake rate in the biofilm systems is dependent on the concentration of substrate at the surface of the biofilm, the diffusion coefficient of substrate and the biofilm thickness. Depending on these parameters the uptake rate change can follow first-order, ½-order or 0-order reaction [9].

In practice during substrate uptake rate experiments the reaction rate follows 0-order reaction when the biofilm is fully penetrated by the substrate and ½-order reaction when the biofilm is partly penetrated (see Figure 4). The degree of penetration of the substrate into the biofilm is a function of the substrate concentration at the biofilm surface. Biofilm thickness L can be calculated with the formula shown in Figure 4 below, where Schange – substrate concentration at changing point; !!,!"# – maximum surface uptake rate of substrate [9].

For calculations of surface uptake rate of substrate by the biofilm, the effective area of the carriers, 70% filling of total protected area, should be found as this corresponds to real operating conditions of MBBR carriers without the detachment of the biofilm [13].

Figure 4. The change from 1/2 order to zero order reaction in the water. Figure taken with permission from Jansen et al. (1992) [9].

Evaluation of dissolved oxygen concentration influence on AOB, NOB and HB kinetics

A lab-scale reactor containing 400 ml of tap water and 128 fresh carriers was stirred continuously at 150 rpm. After few minutes of initial aeration with pure oxygen a DO saturation level of 125% in the bulk was achieved.

Each experiment in this set was performed with 3 sub-experiments for each of three studied streams:

Ammonium sulphate and sodium nitrite were introduced and the oxygen consumption curve (changes of oxygen concentration with time) was recorded until zero oxygen uptake condition was reached (3.5 hours in total).

Other conditions included pH adjustment at 7.6 to 7.7 and temperature setting point of 14°C for MP1 and MP2 streams and 28°C for RP stream to avoid temperature stress on microorganisms.

The procedure of the first experiment described above was applied, but now sodium nitrite and ATU were introduced to obtain NOB + HB activity. Temperature conditions remain the same as for first experiment avoiding the temperature stress.

Finally this procedure was applied one more time without substrates or inhibitor injections to obtain the reference for HB activity.

The objective of this experiment is to investigate the activities of microorganisms and find the effective thickness i.e. the thickness of oxygen penetration into the

biofilm in presence of different bacteria groups. Nitrogen sources were added in abundance making O2 the limiting substrate. Shear and temperature stresses are assumed negligible.

Results and discussion

Figure 5 shows the bacteria activities vs. the RP/MP1 carrier ratio. From this chart is clear that the greater the RP portion the highest the AOB and NOB activities are. It is also clear that the AOB/NOB activities ratio is better for the lowest RP portion suggesting that the adaptation to cold water stimulate AOB activity despite the temperature stress for biofilm from MP1 and MP2 streams as experiments were performed at 28°C.

Figure 5 AOB and NOB activities for different RP: MP1 mixing ratios of carriers.

Figures 6, 7 and 8 show the bacteria activities obtained at different conditions for MP1, MP2 and RP respectively.

Fresh means here that biofilm was sampled and assess with zero storage time holding the same temperatures as at the pilot plant. Temperature stress means that the cold streams MP1 and MP2 were assessed at 28°C instead of 14°C and the opposite for stream RP. Lower DO means that the DO span was reduced from (5-7) mg O2/L to (3-5) mg O2/L and storage time means that the biofilm from the same batch used in Fresh were stored for 14 days at 4°C before its assessment with the same temperatures as at the pilot plant. Finally the temperature stress and storage means that storage time influence was assessedtogether with temperature stress conditions.

Figure 6. Parameters effects on MP1 microorganisms activities.

Figure 7. Parameters effects on MP2 microorganisms activities.

Figure 8. Parameters effects on RP microorganisms activities.

The results demonstrate that the temperature stress decrease the AOB activity for all streams, with most significant influence on MP1 and MP2 streams. Temperature stress promotes the activity of NOB for MP1 stream with the insignificant opposite effect on NOB for MP2 and RP streams. Interesting results were obtained at lower DO condition at 14ºC – with the opposite effect for MP1 and MP2 streams: for MP1 stream AOB activity was halved with constant NOB activity and for MP2 stream NOB activity was halved with constant AOB activity. Bacteria activities for RP stream were undetectable. It can be concluded that the biofilm structure and bacterial content differs in the three streams and that both adapted temperature and adapted substrates concentrations (here – conditions at Sjölunda pilot plant) are important factors affecting the activities of AOB and NOB

bacteria in the biofilm and their behaviour under various conditions.

Biofilm demonstrated significant decrease in the activities of both groups, AOB and NOB after 2 weeks of storage for samples from all three streams with simultaneous significant increase in the HB activity. One possible reason for the increased activity of HB is the hydrolysis of complex organic molecules during the storage time which makes more organic materials available for utilization by HB when oxygen is supplied. In general at higher temperature all bacteria activities were higher than for fresh samples despite the temperature stress for MP1 and MP2 streams. Simultaneously for these two streams with higher temperature the AOB/NOB activities ratio decreased meaning that stress temperature promotes the NOB activity for these samples. The opposite was demonstrated by carriers from RP stream: at their adapted temperature of 28ºC the activities ratio was higher despite the higher temperature and their stress temperature of 14ºC, promoted the NOB activity.

From Figure 9 a plot of SOUR vs. DO1/2 shows the transition between ½-order and 0-order reaction for three different conditions for stream MP1. First including AOB, NOB and HB, second including NOB and HB and finally only for HB.

Results show that the effective thicknesses are significantly higher in case of AOB inhibition for all streams as oxygen is not consumed by this group of bacteria while diffusing through the biofilm (see Table 1). The effective thickness is higher at lower temperature as the transition point appears at higher DO concentration. It can be concluded that the effective thicknesses are almost equal for MP1 and MP2 streams when the experiment is performed at the same temperatures.

Figure 9. Transition from ½ - to 0 –order of reaction in the bulk water, in a convenient plot for determination of biofilm parameters for stream MP1.

In order to confirm the results of this set of experiments microbiological investigations as fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) of bacteria distribution throughout the biofilm thickness is needed. Further experiments can be performed at the same temperature conditions for all three streams to check for the effective thickness of the biofilm from RP stream.

Table 1. Thickness of dissolved oxygen penetration into biofilm.

Conclusions

The results of the bacteria activities study can be summarized as follows:

- DO concentration increment promotes the increase in activity of AOB more than in activity of NOB for studied biofilm at different temperatures.

- Biofilm adapted to colder water (14ºC) demonstrates higher AOB/NOB activities ratio than the biofilm adapted to warmer water (28ºC). This behaviour was demonstrated during experiments at both adapted water temperature and altered water temperature conditions.

- Specific activities of AOB, NOB and HB are higher at higher water temperatures, but

AOB/NOB activities ratio is higher at lower water temperatures. - The behaviour of bacteria is different in studied samples from three different streams. This leads to conclusion that both adapted temperature and adapted substrates concentrations play very important roles in the bacteria behaviour under various conditions.

- The storage time of more than 72 hours at 4ºC significantly decrease the activity of AOB and NOB and increase the HB activity. Storage time also affects differently the behaviour of AOB and NOB at stress temperature conditions.

- The thickness of oxygen penetration into the biofilm is equal (the difference is negligible) for samples from MP1 and MP2 streams at equal conditions, i.e. at the same temperatures and the same bacteria groups presence.

References

[1] Siegrist, H., Salzgeber, D., Eugster, J. and Joss, A., 2008. Anammox brings WWTP closer to energy autarky due to increased biogas production and reduced aeration energy for N-removal. Water Science and Technology, 57(3), 383-388. [2] Lackner, S., Gilbert, E.M., Vlaeminck, S.E., Joss, A., Horn, H. and van Loosdrecht, M., 2014. Full-scale partial nitritation/anammox experiences - an application survey. Water Research, 55, pp. 292-303. [3] Winkler, M., Bassin, J., Kleerebezem, R., Sorokin, D., van Loosdrecht, M., 2012. Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge. Applied Microbiological Biotechnology, 94, pp. 1657 – 1666. [4] Hagman, M. and Jansen, Jes la Cour, 2007. Oxygen uptake rate measurements for application at wastewater treatment plants. Water and Environmental Engineering, Volume 63, pp. 131-138. [5] Llano, N. and Galkin, V., 2014. Evaluation of bacteria population

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evaluation of bacteria population dynamics in … of bacteria population dynamics in mainstream...

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