Effect of heterotrophic growth on autotrophic nitrogen removal in a granular sludge reactor

Transcription

1 Faculty of Bioscience Engineering Centre for Environmental Sanitation Academic Year: Effect of heterotrophic growth on autotrophic nitrogen removal in a granular sludge reactor Md. Salatul Islam Mozumder Promotor: Prof. dr. ir. Eveline I. P. Volcke Master s dissertation submitted in partial fulfillment of the requirements for the degree of Master of Environmental Sanitation

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3 Acknowledgment This thesis is the result of one year of work during which I have been accompanied and supported by many people. It is a pleasant aspect that I hereby have the opportunity to express my gratitude for all of them. First of all I would like to thank my promoter, prof. dr. ir. Eveline Volcke. I have been in her laboratory in the department of Biosystems Engineering since August 21. During this period I have known prof. Eveline as a motivated, patient and principle centered person. Her extreme enthusiasm and integral view on research and her mission for providing only high quality work have made a deep impression on me. I owe her a great debt of gratitude for showing me the way of research. I will never forget that she helped me to build confidence and collect experience. These will affect me in my future life. My sincere gratitude also goes to prof. dr. ir. Mark van Loosdrecht and dr. ir. Cristian Picioreanu, Department of Biotechnology, Delft University of Technology, The Netherlands for their valuable advice and constructive comments that inspired me to work more effectively. I also send my cordial thanks to Matthijs Daelman for his support during my research stay at Delft University of Technology. I am also grateful to the fellow students I met while studying in Belgium during the past two years. They are too numerous to mention but I want to thank all of them for being such good colleagues and friends. Finally, I am forever indebted to my parents and wife who supported me in a lot of things that really matter in life. Without their wholehearted help and understanding, I could not have accomplished my master study. Md. Salatul Islam Mozumder Ghent, 211

4 Notation index List of abbreviations AOB Ammonium oxidizing bacteria NOB Nitrite oxidizing bacteria Anammox Anaerobic ammonium oxidation COD Chemical oxygen demand List of symbols Soluble compounds (S) S s Concentration of organic substrate g.m -3 S NH Ammonium concentration g.m -3 S NO2 Nitrite concentration g.m -3 S NO3 Nitrate concentration g.m -3 S N2 Nitrogen concentration g.m -3 S N2A Nitrogen concentration produced by autotrophs g.m -3 S N2H Nitrogen concentration produced by heterotrophs g.m -3 Particulate compounds (X) X AOB Ammonium oxidizing bacteria gcod.m -3 X NOB Nitrite oxidizing bacteria gcod.m -3 X AN Anammox bacteria gcod.m -3 X H Heterotrophic bacteria gcod.m -3 X H,A Aerobic heterotrophs gcod.m -3 X H,NO2 Anoxic heterotrophs on nitrite gcod.m -3 X H,NO3 Anoxic heterotrophs on nitrate gcod.m -3 X I Inert biomass gcod.m -3 Process ρ G,AOB Growth rate of X AOB gcod.m -3.d -1 ρ G,NOB Growth rate of X NOB gcod.m -3.d -1 ρ G,AN Growth rate of X AN gcod.m -3.d -1 i

5 Notation index ρ G,H Growth rate of X H,A gcod.m -3.d -1 NO2 ρ AG,H NO3 ρ AG,H Growth rate of X H,NO2 gcod.m -3.d -1 Growth rate of X H,NO3 gcod.m -3.d -1 ρ D,AOB Decay rate of X AOB gcod.m -3.d -1 ρ D,NOB Decay rate of X NOB gcod.m -3.d -1 ρ D,AN Decay rate of X AN gcod.m -3.d -1 ρ D,H Decay rate of X H gcod.m -3.d -1 Stoichiometric parameters Y AOB Yield of ammonium oxidizers on ammonia gcod.g -1 N Y NOB Yield of nitrite oxidizers on nitrite gcod.g -1 N Y AN Yield of Anammox bacteria on ammomium gcod.g -1 N Y H Yield of aerobic heterotrophic bacteria gcod.g -1 N Y H,NO2 Yield of anoxic heterotrophic bacteria on nitrite gcod.g -1 N Y H,NO3 Yield of anoxic heterotrophic bacteria on nitrate gcod.g -1 N i NXB Nitrogen content in active biomass gn.g -1 COD i NXI Nitrogen content in X I gn.g -1 COD i NSS Nitrogen content in organic substrate gn.g -1 COD f I Inert content in biomass gcod.g -1 COD inetic parameters AOB max Maximum growth rate of X AOB d -1 NOB max Maximum growth rate of X NOB d -1 AN max Maximum growth rate of X AN d -1 H max Maximum growth rate of X H d -1 AOB NH Affinity constant of X AOB for ammonium gn.m -3 NOB NO2 Affinity constant of X NOB for nitrite gn.m -3 AN NH Affinity constant of X AN for ammonium gn.m -3 AN NO2 Affinity constant of X AN for nitrite gn.m -3 H NO H NO 2 Affinity constant of X H for nitrite gn.m -3 3 Affinity constant of X H for nitrate gn.m -3 H S Affinity constant of X H for organic substrate gcod.m -3 ii

6 Notation index AOB O 2 Affinity constant of X AOB for oxygen go 2.m -3 NOB O 2 Affinity constant of X NOB for oxygen go 2.m -3 AN O 2 Affinity constant of X AN for oxygen go 2.m -3 H O 2 Affinity constant of X H for oxygen go 2.m -3 b AOB Decay constant of X AOB d -1 b NOB Decay constant of X NOB d -1 b AN Decay constant of X AN d -1 b H Decay constant of X H d -1 η NO2 Anoxic reduction factor for X HNO2 - η NO3 Anoxic reduction factor for X HNO3 - Physical parameters D NH4 Ammonium diffusion coefficient in water m 2.d -1 D NO2 Nitrite diffusion coefficient in water m 2.d -1 D NO3 Nitrate diffusion coefficient in water m 2.d -1 D O2 Oxygen diffusion coefficient in water m 2.d -1 D N2 Nitrogen diffusion coefficient in water m 2.d -1 D S Organic substrate diffusion coefficient in water m 2.d -1 ρ A Density of autotrophic and particulate inert biomass gcod.m -3 ρ H Density of heterotrophic biomass gcod.m -3 r p Granule radius mm T Temperature T ref Reference temperature R Gas constant J.mole AOB E a NOB E a AN E a Activation energy of X AOB kj.mole -1 Activation energy of X NOB kj.mole -1 Activation energy of X AN kj.mole -1 iii

7 Summary This study deals with the influence of heterotrophic growth on autotrophic nitrogen removal in a granular sludge reactor. Autotrophic nitrogen removal is an innovative technique for biological nitrogen removal from wastewater during which ammonium is nitrified to nitrite by ammonium oxidizing bacteria followed by subsequent oxidation of ammonium and reduction of nitrite to nitrogen gas by anammox bacteria. In this process, nitrification of nitrite to nitrate needs to be prevented by outcompeting nitrite oxidizing bacteria, which can be achieved at relatively low oxygen level. The abovementioned biomass groups concern autotrophic species 1. Heterotrophic organism, are a priori not expected when treating influent wastewater stream which contain only nitrogen and no organic carbon. However, heterotrophic bacteria can grow on microbial decay products and their presence will affect the process performance. The relation between autotrophic and heterotrophic species is the subject of this dissertation. In this research work, a mathematical model was constructed to describe the effect of heterotrophic growth on completely autotrophic nitrogen removal. The developed model considered both autotrophic and heterotrophic growth, besides decay of all. Subsequently, simulation studies were performed in which autotrophic nitrogen removal with and without heterotrophic growth would be compared. With respect to the modeling assumption, the sensitivity of the density of heterotrophs was evaluated and found to be insensitive to the simulation results. The biomass profile in a granule revealed that heterotrophs were present at the outer layer just below the ammonium oxidizing and nitrite oxidizing bacteria that consume oxygen. Anammox bacteria grew in the inner anoxic parts of the granules where they consume ammonia and nitrite and produce nitrogen gas. The nitrogen removal was significantly higher when heterotrophic growths were considered in the model. The optimum bulk oxygen concentration levels corresponding with maximum nitrogen removal were related to the process variables such as granular size, possible presence of organic substrate in influent, ammonium surface load and temperature. Higher granular size, organic substrate load and ammonium surface load needed higher bulk oxygen concentration for maximum nitrogen 1 Use CO 2 as a carbon source. iv

8 Summary removal. Similar maximum nitrogen removal efficiency was found in a range of temperatures. This study considers a wastewater influent stream containing only nitrogen, as well as a wastewater stream that contains organic substrate. Heterotrophic growth that increased nitrogen removal, was facilitated by the presence of organic substrate in the influent. This study clearly demonstrates the influence of heterotrophs on the performance of autotrophic nitrogen removal in a granular sludge reactor even if little or no organics is present in the wastewater stream. The insight gained on the interaction between heterotrophic and autotrophic bacteria becomes ever more important at lower temperature and will thus gain importance for the operation of granular sludge reactor in future energy-positive wastewater treatment plants. v

9 Table of Contents Chapter I: Introduction... 1 Chapter II: Literature review Introduction Nitrogen removal pathways Conventional nitrification-denitrification over nitrate Nitrification-denitrification over nitrite Anaerobic ammonium oxidation (Anammox) Partial nitritation combined with anaerobic ammonium oxidation Reactor conditions affecting biological nitrogen removal Oxygen concentration Temperature ph Relation between influent organic carbon (COD/N ratio) and biological nitrogen removal Nitrification-denitrification over nitrate Anammox process Partial nitritation-anammox Conclusions Chapter III: Model development Process stoichiometry and kinetics Reactor configuration, simulation parameters and initial conditions Chapter IV: Results and discussion Role of heterotrophic growth on nitrogen removal Active biomass composition Competition among active biomass Comparison of nitrogen removal performance Biomass dynamics and steady state Biomass dynamics in a granule Influence of initial conditions on the time needed to reach steady state Influence of operational parameters on the reactor performance Influence of the oxygen concentration Dynamics of nitrogen removal and steady state biomass profile vi

10 Table of contents Steady state performance and biomass composition Sensitivity analysis for the density of heterotrophs Influence of the granule size Dynamics of nitrogen compounds and steady state biomass profile Steady state reactor performance and biomass composition Interaction between granule size and oxygen concentration Role of temperature Effect of temperature at fixed oxygen level Interaction of bulk oxygen with temperature Effect of ammonium surface load Influence of influent organic substrate on reactor performance Effect of organic substrate at fixed oxygen level Effect of oxygen concentration at fixed influent organic substrate Interaction between organic substrate and oxygen concentration Effect of organic substrate on dynamics of nitrogen removal... 5 Chapter V: Conclusions and perspectives Steady state and dynamic model behaviour Influence of operational parameters and influent organic substrate Future works References vii

11 Chapter I: Introduction Nowadays, nitrogen removal is very important in terms of water pollution control. The nitrogen pollutants in wastewater are either ammonium (NH + 4 ) or organic nitrogen compounds which are ultimately converted to ammonium through hydrolysis. Traditional biological nitrogen removal from wastewater is performed through nitrificationdenitrification over nitrate. This pathway requires a significant amount of aeration energy for biological nitrification and an external carbon source for denitrification. Partial nitritationanammox process is a promising alternative biological nitrogen removal pathway. During partial nitritation, about 5% of the ammonium present in the wastewater is converted to nitrite. In the subsequent anammox reaction, ammonium and nitrite are combined to form nitrogen gas. The resulting process requires up to 63% less oxygen (low aeration cost) and causes less carbon-dioxide emissions and a lower sludge production compared to conventional nitrification-denitrification over nitrate. A key issue in the partial nitritation - anammox process is partial nitrite production and prevention of further oxidation of nitrite to nitrate. One process option to achieve partially nitrite formation is by limiting oxygen in a biofilm reactor such as the granular sludge reactor. The effectiveness of nitrogen removal process does not only depend on the applied treatment technology but also the process conditions. The principles of biological nitrogen removal processes and the parameters affecting their operation are reviewed in Chapter II. In most previous studies on partial nitritation on anammox processes, only autotrophic organism were considered to be present (och et al., 2; Matsumoto et al., 21; Okabe et al., 25; van de Graaf et al., 1996). However, some authors showed that in autotrophic biofilms, heterotrophic biomass can grow on microbial decay products (indaichi et al., 24; Lackner et al., 28; Okabe et al., 25). However, until now this has not been studied for autotrophic nitrogen removal in granular sludge reactors. In Chapter IV (section 1), the performance of autotrophic nitrogen removal in a granular sludge reactor together with biomass profile in a granule with and without heterotrophic growth are compared. The nitrogen compounds in bulk are also examined while identifying the effects of heterotrophs. A biofilm granule is a complex microbial system containing different types of bacteria. In a granular sludge reactor, ammonium oxidizers are active in the outer layer of the granules and 1

12 Chapter I: Introduction produce sufficient amount of nitrite for anammox bacteria active in the inner part of the granules. The dynamics of the microbial profile in a granule and the time required to reach steady state are evaluated in Chapter IV (section 2). One of the most important process variables for establishing nitrite formation in a biofilm reactor is dissolved oxygen. The dissolved oxygen concentration has a large influence on both ammonium oxidizers and nitrite oxidizers. On the other hand, the anammox bacteria are strictly anaerobic and inhibited by the dissolved oxygen. The partial nitritation - anammox process can be well controlled by regulating dissolved oxygen concentration. The limited oxygen concentration allows for the partial oxidation of ammonium to nitrite while preventing nitrite oxidation. Under anoxic conditions the unconverted ammonium and nitrite are utilized by anammox bacteria to remove the nitrogen from aqueous system as nitrogen gas. Chapter IV (section 3.1) analyzes the effect of bulk oxygen concentration on the nitrogen removal performance through partial nitritation-anammox process in a granular sludge reactor. The granule size in a granular sludge reactor determines the surface to volume ratio and affects the nitrogen removal performance. Chapter IV (section 3, sub-section 3.2) addresses changing granular size and evaluates its effects on the performance of granular sludge reactor in terms of nitrogen removal. The process temperature plays a very important role in the partial nitritation-anammox process. It is not only responsible for the microbial growth rate but also for all kinds of interactions within the system, such as decay rate, microbial activities, equilibrium relation etc. A higher temperature increases the growth rate, decay rate and microbial activities but at too high temperature the microbial community is destroyed. Chapter IV (section 3.3) discusses the temperature effects on partial nitritation - anammox process. The presence of organic substrates in the influent significantly influences the microbial community composition in an autotrophic granule. When organic carbon is present heterotrophic growth increases. Nitrate can be reduced by these denitrifiers to nitrite that can be utilized by anammox for the oxidation of ammonium (umar and Lin, 21). To investigate the effect of heterotrophic bacteria, growth on influent organic substrate is addressed in Chapter IV (section 4). 2

13 Chapter II: Literature review 1. Introduction Generally, nitrogen is present in wastewater in the form of ammonium (NH + 4 ). Several human activities such as agriculture, industrial processes, and household activities produce nitrogen containing wastewater. High strength nitrogen containing wastewater originates from manure (Luo et al., 22; Qiao et al., 21), landfill leachate (Cema et al., 27), several organic chemicals; plastics and synthetic fibers industries (Love et al., 1999) and sludge digester supernatants (Fux et al., 22; van Loosdrecht and Salem, 26). Uncontrolled disposal of wastewater containing high ammonium concentration causes a huge damage to the environment. It is prime factor for the eutrofication of the receiving aquatic system. Besides, dissolved ammonium is considered as a harmful agent for the aquatic life (Effler et al., 199). For this reason, nitrogen removal from wastewater has become an important issue. Due to increasingly stringent environmental regulations, advanced and cost effective techniques for the nitrogen removal from wastewater are required. In conventional wastewater treatment plants (WWTPs), ammonium is removed by biological nitrification-denitrification over nitrate. New approaches that are based on (partial)nitritation and/or the anaerobic ammonium oxidation (Anammox) process (Mulder et al., 1995) to remove the nitrogen are more cost-effective, environmentally friendly, efficient and sustainable. The combined partial nitritation-anammox process is a completely autotrophic process which can be performed either in one stage or in two stages (reactors). A number of research groups worked on autotrophic nitrogen removal processes resulting in a variety of process configurations and various names such as Oxygen-Limited Autotrophic Nitrification/Denitrification (OLAND) (uai et al., 1998), Completely Autotrophic Nitrogen removal Over Nitrite (CANON) (Third et al., 25), Sustainable High rate Ammonium Removal Over Nitrite (SHARON) (Hellinga et al., 1998), Deammonification (Hippen et al., 1997), ph controlled Deammonification (DEMON) (Wett, 26) etc. This literature review gives a short introduction on the principle pathways involved in biological nitrogen removal followed by an overview of the reactor conditions affecting the process. This knowledge is important to increase the effectiveness of biological nitrogen processes. 3

14 Chapter II: Literature review To establish autotrophic nitrogen removal, oxygen is the most important process variable. In first place oxygen is needed to establish partial nitritation but the oxygen level should be low enough in order not to inhibit anaerobic denitrification. Another critical parameter that has a large effect on nitrogen removal is the organic (carbon) load which can be expressed as COD to nitrogen ratio. The partial nitritation-anammox process is an autotrophic process. Nevertheless, upto half of the biomass in autotrophic biofilms can be heterotrophic, growing on the microbial decay products(indaichi et al., 24; Okabe et al., 25). Heterotrophic growth reduces the nitrate production. It utilizes the organic substrate in both aerobic and anoxic conditions and produces carbon dioxide that ultimately decreases the ph of the system. Therefore heterotrophic activity is an important factor that affects autotrophic nitrogen removal. 2. Nitrogen removal pathways 2.1. Conventional nitrification-denitrification over nitrate Biologically nitrification and denitrification are two individual processes that are carried out by distinct groups of bacteria. During nitrification, ammonium is oxidized to nitrate (Eq. 1). The ammonium oxidizing bacteria (X AOB ) convert ammonium to nitrite (NO - 2 )(Eq. 1a), which can be further oxidized to nitrate (NO - 3 ) (Eq. 1b) by nitrite oxidizing bacteria (X NOB ). During denitrification, nitrate is transformed to nitrogen gas (Eq. 2). C (in Eq. 2) denotes the carbon source; for autotrophic denitrification it is carbon dioxide (CO 2 ) and for heterotrophic it is organic carbon. Nitrification: NH O 2 NO 2 + H 2 O + H + (1a) NO O 2 NO 3 - (1b) Overall: NH O 2 NO 3 + H 2 O + 2H + (1) Denitrification: - NO 3 + C + 2H + CO 2 +.5N 2 + H 2 O (2) Heterotrophic denitrification is a four step reduction processes in which nitrogen gas (N 2 ) is formed from nitrate (NO - 3 ) over nitrite (NO - 2 ), nitric oxide (NO) and nitrous oxide (N 2 O). Each reduction step is catalyzed by different enzymes (Baumann et al. 1996). If for any 4

15 Chapter II: Literature review reason, one or more individual reduction steps become slower, the intermediate products may accumulate in the system, ultimately reducing nitrogen removal (Udert et al., 28). Biological nitrification-denitrification over nitrate is considered as an efficient process characterized by a relatively easy operation and moderate costs (Metcalf and Eddy, 23). It is generally used for the treatment of wastewater containing low nitrogen concentration (<1mgNL -1 ). This conventional biological nitrification and denitrification process is considered as more favorable than the chemical nitrogen removal by magnesium-ammoniumphosphate (MAP) precipitation or by air stripping (Siegrist, 1996) for the removal of ammonium nitrogen from the wastewater Nitrification-denitrification over nitrite Ammonium in a concentrated stream is oxidized to nitrite only (Eq. 1a) by controlling the aeration, saving up to 25% aeration cost. The denitrification of nitrite to nitrogen gas is based on external carbon source (Eq. 3). NO C + H + N 2 + CO 2 + H 2 O (3) Nitrification-denitrification over nitrite needs less external carbon source, saving 4% cost for external carbon. Moreover the process emits less carbon dioxide (CO 2 ) and produces less sludge compared to conventional nitrification-denitrification over nitrate Anaerobic ammonium oxidation (Anammox) Until the early 199s, it was believed that the oxidation of ammonium could only proceed under aerobic conditions. This thinking was changed by the discovery of the anaerobic ammonium oxidation process by Mulder et al. (1995). At that time the scientific community was greatly surprised by the proof of a biological process in which nitrite and ammonium are directly converted into dinitrogen gas. The overall reaction is: NH NO H + 1.2N NO H 2 O (4) Hydrazine and hydroxylamine are produced as intermediates during the anammox process (Sinninghe-Damste et al., 22; van de Graaf et al., 1997). Strous et al. (26) found NO as an intermediate product of the anammox process. As the process is anoxic, anammox bacteria do not need oxygen which results in decreased aeration costs. Furthermore anammox 5

16 Chapter II: Literature review bacteria use CO 2 as a carbon source and hence they do not require the addition of organic compounds. They grow relatively slowly, leading to a low sludge production Partial nitritation combined with anaerobic ammonium oxidation The application of the anammox process for the removal of ammonium from wastewater requires a proceeding step in which nitrite is produced. About half of the ammonium needs to be converted to nitrite, a process that is known as partial nitritation (Eq. 1a) and is carried out by ammonium oxidizing bacteria. The resulting nitrite and unconverted ammonium are converted to nitrogen gas in the anammox process (Eq. 4). The overall process stoichiometry becomes (Eq. 1a+Eq. 4). NH O 2 + HCO - 3.5N 2 + CO H 2 O (5) The combined partial nitritation-anammox process requires 63% less oxygen and no additional organic carbon source compared to conventional nitrification-denitrification over nitrate. The partial nitritation and anammox processes can take place in a single reactor in which ammonium oxidizing bacteria and anammox coexist in a biofilm or form compact granules. Batch experiments and microbial analysis showed that nitrite is at the outer biofilm layer under aerobic conditions. The remaining ammonium and nitrite diffuse into the deeper part of the biofilm where anoxic conditions are maintained and nitrite acts as an electron acceptor, reacting with the remaining ammonium to form nitrogen gas (och et al., 2). The success of partial nitritation-anammox process depends first of all on the continuous suppression of nitrite oxidizers and, secondly on the produced nitrite to ammonium ratio, which should be about 1.32 (stoichiometric ratio, see Eq.4). The increase of either the ammonium or nitrite concentration has an adverse influence on the anammox activity. Dapena-Mora et al. (27) found that higher ammonium and nitrite concentration reduced the performance of anammox bacteria. Jung et al. (27) described a decrease of anammox bacterial activities with increasing free ammonium concentration. 6

17 Chapter II: Literature review 3. Reactor conditions affecting biological nitrogen removal This section consist effect of oxygen concentration (subsection 2.1), temperature (subsection 2.2) and ph (subsection 2.3) on biological nitrogen removal through various pathways for biological nitrogen removal Oxygen concentration The dissolved oxygen concentration is very important for both ammonium and nitrite oxidation. It becomes a limiting factor for nitrification when it is lower than 2 mgo 2 L -1 (Beccari et al., 1992). Due to higher oxygen affinity, at low oxygen concentration level the ammonium oxidizers are more vigorous then the nitrite oxidizers. In other words, oxygen deficiency influences the performance of nitrite oxidizers more significantly than the ammonium oxidizers (Philips et al., 22). This is explained by the oxygen half saturation constant. Hunik et al. (1994) found that the half saturation constant for dissolved oxygen was.16 mgo 2 L -1 for ammonium oxidizers and.54 mgo 2 L -1 for nitrite oxidizers. In the activated sludge processes, oxygen half saturation constants of ammonium oxidizers and nitrite oxidizers were.25.5 and mgo 2 L -1 respectively (Barnes and Bliss, 1983). A reason for this variability is that the oxygen concentration inside the sludge matrix and in the bulk liquid is not same. As a result, the half saturation constant depends on a number of parameters such as biomass density, the size of sludge matrix, the mixing intensity and the rate of diffusion of oxygen into the sludge matrix (Munch et al., 1996; Manser et al., 25). It is possible to remove nitrogen through nitrification-denitrification over nitrite by controlling the dissolved oxygen concentration. High oxygen levels favor nitrite oxidizers, resulting in nitrate formation. In oxygen limiting conditions nitrite oxidizers are outcompeted and nitrite accumulates. This is demonstrated by Peng et al. (24) in a sequencing batch reactor and by Jubany et al. (29) in an activated sludge system. Nitrogen removal over nitrite can be established by turning off aeration at the point where the ammonium oxidation has completed. Hidaka et al. (22) reported an aeration pattern to control ammonium to nitrite. By frequently changing between aerobic and anoxic in an activated sludge system, the nitrate formation can also effectively be prevented (Yoo et al., 1999). The aeration was turned off before all the ammonium was consumed and nitrite started to be converted to nitrate. 7

18 Chapter II: Literature review The anammox process is a strictly anaerobic process and is inhibited by oxygen concentration. The anammox metabolism is reversible at low oxygen concentration (.25-2% air saturation) but irreversible at high concentration (higher than 18% air saturation) (Egli et al., 21). Bulk oxygen concentration is a very important controlling variable for partial nitritationanammox process. In partial nitritation, oxygen is needed for converting half of the ammonium to nitrite but the conversion to nitrogen gas from unconverted ammonium and nitrite through anammox process is completely anaerobic. At high oxygen concentration nitrate formation prevails. Volcke et al. (21) demonstrated that in partial nitritationanammox process nitrite was converted to nitrogen gas at low bulk oxygen concentration. The same was observed by Hoa et al. (22) Temperature Temperature affects the nitrification process directly as well as indirectly. A higher temperature increases the microbial growth rate according to the Arrhenius law, which is valid up to a certain critical temperature, above which biological activity starts to decrease. Grunditz and Dalhammar (21) found an optimum temperature of 35 C for ammonium oxidizers and 38 C for nitrite oxidizers. Van Hulle et al. (27) reported a maximum oxygen uptake rate by ammonium oxidizing bacteria in the temperature range between 35 and 45 C. Hellinga et al. (1998) mentioned that above 25 C the specific growth rate of ammonium oxidizing bacteria become higher than that of nitrite oxidizing bacteria in a SHARON process. The optimal temperature for anammox bacteria is reported between 3-4 C (Strous et al., 1999; Egli et al., 21). Dosta et al., (28) indicated that temperature of 45 C or higher causes irreversible loss of efficiency of anammox bacteria. On the other hand the anammox process can be successfully operated at temperature as low as 2 C (Cema et al., 27; Isaka et al., 27). In this case slow adaptation of anammox bacteria to low temperature is very important. Temperature makes an indirect effect on biological nitrogen removal process by participating in free ammonium and nitrous acid accumulation. Anthonisen et al. (1976) made mathematical expressions (Eq. 6 and 7) for calculating the amount of free ammonia and 8

19 Chapter II: Literature review nitrous acid based on total ammonium (TAN) and total nitrite (TNO 2 ) and incorporating with temperature (T) and ph: (6) (7) According to these equations, the amount of free ammonia increases with increasing temperature while the amount of nitrous acid decreases. The effect of temperature on biological nitrogen removal from wastewater was examined by omorowska- aufman et al. (26) in the temperature range from 7.8 to 21 C. They related influence of temperature on a nitrification-denitrification to sludge age. Temperatures above 15 C are favorable for nitrification even when the sludge age was very short. For a temperature below 15 C and sludge age lower than 2 days, the nitrification process became unstable and the removal efficiency varied between 61.7 to 99.3%. They also found that the unfavorable effect of low temperature (below 15 C) was reduced and stabilized nitrification process was achieved when the sludge age was more than 2 days. Yamamoto et al. (26) performed partial nitritation in a swim-bed reactor. In this study, a stable efficiency was maintained between 15 to 3 C but the performance suddenly deteriorated below 15 C ph During the conversion of one mole of ammonium to one mole of nitrogen through nitrification-denitrification over nitrate one mole of H + is produced. As a result, sufficient alkalinity is required for buffering the produced protons in wastewater. The optimum ph for both ammonium oxidizers and nitrite oxidizers lies between 7 and 8 (van Hulle et al., 21). The ammonium oxidizers prefer a slightly alkaline environment as these organisms use ammonia (NH 3 ) as substrate (Suzuki et al., 2974). It maintains the inorganic carbon (HCO - 3 ) that is important for metabolism of nitrifying bacteria. Hellinga et al. (1998) detected that the growth rate of nitrite oxidizers were decreased by a factor 8 for the ph change from 8 to 7 whereas the change of the growth rate of the ammonium oxidizer were negligible. 9

20 Chapter II: Literature review Anammox bacteria can grow in a ph range from 6.7 to 8.3. Strous et al. (1999) mentioned an optimum ph of 8.. Jung et al. (27) reported that it is important to keep free ammonia below 2 mgn.l -1 and free nitrite nitrogen below 35 mgn.l -1 for continuous growth of anammox bacteria. Below these levels the anammox activity increases gradually in an anaerobic condition. The ph also influences the concentration of free ammonia (NH 3 ) and free nitrous acid (HNO 2 ), which are the actual substrates for ammonium oxidation and nitrite oxidation respectively and also inhibit nitrification (Anthonisen et al., 1976). In general nitrite oxidizing bacteria are more sensitive to free ammonia and nitrous acid inhibition ammonium oxidation. According to eq. 6 and 7, ph has influence on NH /NH 3 and HNO 2 /NO 2 equilibrium. The amount of nitrite (NO - 2 ) increases with increasing ph. At high ph (>8), free ammonia becomes the main inhibitor for the nitrification process; at low ph (<7.5) nitrous acid is the main inhibitor. Nitrite plays a very critical role in biological nitrogen removal process as it may cause severe substrate limitation for nitrite oxidizing bacteria at low concentration. High nitrite concentration inhibits anammox activities. Inhibition starts at nitrite concentrations higher than 1 mgn.l -1 (Strous et al., 1999) and microbial activities are completely lost at or above 185 mgn.l -1 (Egli et al., 21). The optimum ph for nitrification is 8 and the nitrification rate abruptly decreases below a ph of 6.5 (Shammas 1986). The ph interval for anammox process is whereas ph 8. is considered as optimum. 4. Relation between influent organic carbon (COD/N ratio) and biological nitrogen removal In systems for biological nitrogen removal from wastewater, autotrophic and heterotrophic bacteria coexist. In case of conventional nitrogen removal through nitrification-denitrification over nitrate, nitrification is autotrophic but denitrification is heterotrophic and requires external organic carbon. In case of completely autotrophic nitrogen removal through partial nitritation-anammox, no organic carbon source is required. However, even if the influent does not contain organic carbon, heterotrophic growth is possible on organic material 1

21 Chapter II: Literature review generated from biomass decay (Lackner et al., 28) and/or on excretion of the living cells (Rittmann et al., 22). Matsumoto et al. (21) studied an autotrophic biofilm process for ammonium oxidation to nitrite. The process behavior was without any external carbon source and heterotrophic growth was based on decay of nitrifying bacteria. The resulting biomass distribution profile in a nitrifying granule (Figure 1) reveals that 22% of the microbial community is heterotrophs and 68% nitrifying bacteria (ammonium oxidizing and nitrite oxidizing). Figure 1. Microbial community composition for the nitrifying granule as determined by quantitative FISH (Matsumoto et al., 21). Figure 2. Effect of influent COD concentration on the concentration of the heterotrophic biomass in nitrification-denitrification over nitrate system (Moussa et al., 25). 11

22 Chapter II: Literature review Heterotrophic bacteria in the treatment system do not only consume COD but also generate some COD by decay. Moussa et al. (25) examined the simultaneous effect of influent COD and sludge retention time (SRT) on the heterotrophic biomass fraction in a nitrifying SBR system (Figure 2). They mention that the influent COD yields about 4% of the total heterotrophic biomass and the remaining 6% results from decay for 1 mg.l -1 influent COD and 3 days SRT. They also found that the heterotrophic biomass increased by 11% with increasing SRT from 3 to 1 days resulting from increasing decay product with SRT Nitrification-denitrification over nitrate The influent COD/N ratio is a very important factor for the biological nitrogen removal through conventional nitrification-denitrification over nitrate. It affects both nitrifying and denitrifying bacterial population growth in the system. Yang et al. (24) observed that in a granular sludge reactor the performance of both ammonium oxidizing bacteria and nitrite oxidizing bacteria significantly increased with a decreasing influent COD/N ratio from 2 to 3.3. They also found that the specific oxygen utilization rate of nitrifying bacteria increased with decreasing COD/N ratio level whereas the specific heterotrophic oxygen utilization rate tended to decrease. It implied that higher COD/N ratio is favorable for heterotrophic population. At high organic carbon, heterotrophic bacteria grew excessively and competed with ammonium oxidizing bacteria for oxygen. This reduced the nitrification process and ultimately increased the ammonium concentration in the effluent. Moreover high concentration of organic compounds also stimulated the biofilm growth as well as increased the diffusion resistance of ammonium into the biofilm. This also reduced the nitrification. At high nitrogen levels the nitrifying bacteria were competitive with heterotrophs for oxygen and the nitrifying bacteria became an important component of the aerobic granules. Carrera et al., (24) estimated the effect of COD/N ratio on the nitrification rate in a process of nitrification-denitrification over nitrate. They found an exponential decrease of nitrification rate with changing the COD/N ration from.71 to 3.4 and the relation defined by an exponential mathematical expression (Eq. 8) r nitrification = e (- 1.66(COD/N)) (8) 12

23 Chapter II: Literature review The influent COD/N ratio not only affect the nitrification rate but also the nitrification capacity. The nitrifying biomass fraction in a biofilm was increase with decreasing COD/N ratio (Rittmann et al., 1999). Harremoes et al. (1995) evaluated the autotrophic biomass fraction for an activated sludge system with biological nitrogen removal and found the autotrophic biomass fraction increase by 1.5 to 2% with decreased COD/N ratio from 3.4 to 2.6 gcod.gn -1. There is also a relationship between fraction of nitrifying bacteria and the relationship between biological oxygen demand (BOD 5 ) and total jeldahl nitrogen (TN) in the influent (EPA., 1975). Carrera et al. (24) developed a mathematical expression (Eq. 9) based on obtained experimental data from a pilot scale biological nitrogen removal system, relating the fraction of nitrifying bacteria with BOD 5 and TN as: Nitrifiers fraction = e (-2.39(BOD 5 /TN)) +.21e (-.43(BOD 5 /TN)) (9) According to the eq. 9, the nitrifying biomass decreases with increasing BOD5/TN ratio. Therefore low organic carbon is required for nitrification. For heterotrophic denitrification, organic carbon is required. Most types of wastewater contain some COD that may be used for the nitrogen production. Carrera et al. (24) reported that the nitrification rate remained constant (.32 gn.gvss -1 per day) at COD/N ratio higher than 4 gcod.gn -1 even though a ratio of at least 7.1 was required to achieve complete denitrification. They also found that the denitrification percentage had a linear relation with the COD/N ratio when it was below 7.1. Hsieh et al. (23) experimentally revealed that the nitrification and denitrification efficiency decreased with increasing influent ammonium loading from 2. to 11.5 gnm -2 d -1 in a biofilm reactor which could have resulted from limited surface area of the biofilm causing insufficient reaction site. But nitrification and denitrification rates increased to a peak value and then decreased at the highest ammonium loading. At highest ammonium loading, some part of it transformed into free ammonia, which is toxic to most microorganisms and decreased the nitrification and denitrification rates. They also showed that the nitrification efficiency also decrease with COD concentration while the denitrification efficiency increased. Vrtovšek and Roš (26) performed an experiment in which ground water was treated in a biofilm reactor; they found minimal nitrite, nitrate and residual COD concentrations in the effluent for an influent COD/N ratio 3.7. A higher influent COD/N ratio 13

24 Chapter II: Literature review led to higher residual COD concentration in the effluent, while a lower influent COD/N ratio caused incomplete denitrification. For the simultaneous removal of organic compounds and nitrogen from wastewater, the membrane aerated biofilm reactor (MABR) was considered as an advanced technology (Lackner et al., 28; Satoh et al., 24; Semmens et al., 23). In a MABR, the biofilm grows on a membrane through which oxygen is supplied, while substrate diffuses from the bulk liquid through the other side of the biofilm. The satisfactory removal of COD and nitrogen largely depends on the oxygen concentration in the gas stream and the influent COD/N ratio. Liu et al. (21) described the effect of substrate COD/N ratio on denitrification for membrane aerated biofilm reactor and found 96% ammonium removal for the ratio 3. The effluent nitrate (NO - 3 ) sharply decrease with increasing the COD/N ratio to 5 whereas other substances remained same as ratio 3 and COD removal, nitrification and denitrification efficiency reached 85, 93 and 92% respectively. When the COD/N ratio was further increased to 6, the effluent ammonium concentration increased very rapidly Anammox process The anammox process does not require organic carbon source. A number of studies report that the presence of organic matter has a negative effect on the anammox processes (Chamchoi et al., 28; Guvan et al., 25; Jianlong and Jing, 25; Sabumon, 27; Tang et al., 21). If certain amounts of organic carbon are present the growth rate of denitrifiers is higher than the one of anammox bacteria (Strous et al., 1999), such that anammox bacteria cannot compete with denitrifiers. Lowering the influent COD/N ratio can control denitrifiers and results in higher nitrogen removal through anammox process Partial nitritation-anammox Lackner et al. (28) performed a simulation study regarding the effect of heterotrophic growth on autotrophic nitrogen removal through partial nitritation-anammox process. In their simulations they found that by including the heterotrophic growth on decay biomass only the nitrogen removal efficiency decreased for the counter diffusion biofilm model but no significant difference was found for co-diffusion. In the counter diffusion model, anammox denitrification dominates at COD/N ratio of but at the COD/N ratio equal or higher than 2 14

25 Chapter II: Literature review the autotrophic denitrification disappears completely. Under increasing COD load anammox bacteria are outcompeted by denitrifying heterotrophic bacteria and nitrogen removal is due to heterotrophic denitrification. In the co-diffusion system the anammox microbial fraction is almost constant but the heterotrophic bacteria slightly increase and ammonium oxidizing bacteria decrease with COD/N ratio. 5. Conclusions Biological nitrogen removal techniques are widely applied to treat nitrogen containing wastewaters. Among different treatment options, partial nitritation-anammox process is more sustainable than conventional nitrification-denitrification over nitrate. The success of operation of partial nitritation-anammox and also nitrification-denitrification process depends on influent characteristics and operating parameters of the biological nitrogen removal process such as COD/N ratio and organic carbon concentration, oxygen concentration, temperature, ph etc. The highest effectiveness of nitrogen removal for partial nitritation-anammox process is achieved at lower COD/N ratio. The anammox bacteria are anaerobic bacteria and are inhibited by dissolved oxygen. A lower bulk oxygen concentration is important for the oxidation of half of the ammonium in partial nitritation-nammox process. For successful biological nitrogen removal it is required to maintain the temperature within a certain range. The nitrogen removal is relatively higher in the presence of heterotrophic bacteria. Heterotrophic bacteria can grow on influent COD. But the success of heterotrophic nitrogen removal is observed up to a certain value of COD/N ratio. There are also possibilities to inhibit the ammonium oxidizing bacteria by increasing nitrite concentration by the heterotrophic bacteria at low ph. So an optimum ph level must be maintained for getting the best performance. 15

26 Chapter III: Model development In this study, an existing model for autotrophic nitrogen removal in a granular sludge reactor was extended to include the influence of heterotrophs on the reactor performance. The granular sludge reactor model was based on a previous model (Volcke et al., 21), in which the heterotrophic growth was neglected. 1. Process stoichiometry and kinetics The model of Volcke et al. (21) was extended to evaluate the effect of heterotrophic activities on autotrophic nitrogen removal through partial nitritation-anammox process in a granular sludge reactor. Four different groups of bacteria were considered: ammonium oxidizing bacteria (X AOB ), nitrite oxidizing bacteria (X NOB ), anammox bacteria (X AN ) and heterotrophic bacteria (X H ). Nitrification is described as a two-step process: ammonium oxidation to nitrite by X AOB followed by nitrite oxidation to nitrate by X NOB. Anammox bacteria convert ammonium and nitrite to nitrogen gas (S N2A ). Growth of heterotrophic - bacteria takes place under aerobic as well as anoxic (in presence of NO 2 and/or NO - 3 ) conditions. Heterotrophic growth relies on organic carbon, which is either present in the reactor influent or results from biomass decay. In case of no readily biodegradable suspended or particulate organics or soluble organic substrate (S s ) in the influent, heterotrophic growth only results from decay material (dead biomass). In absence of dissolved oxygen, nitrite (NO - 2 ) or nitrate (NO - 3 ) is used as an electron acceptor in heterotrophic growth. Therefore three types of heterotrophic bacteria were considered: aerobic heterotrophs (X H,A ) on soluble - organic substrate (S S ), anoxic heterotrophs (X H,NO2 ) on S S and NO 2 and anoxic heterotrophs (X H,NO3 ) on S S and NO - 3. In heterotrophic processes X H,NO3 reduce nitrate to nitrite whereas X H,NO2 nitrite to nitrogen gas (S N2H ). Biomass decay has been modeled according to the death-regeneration concept instead of the endogenous respiration approach followed by Volcke et al. (21). The death-regeneration concept includes a transition of living cells to substrate together with a fraction of inert material by decay of microorganism and/or hydrolysis (van Loosdrecht and Henze, 1999). All decay processes follow first order kinetics and convert biomass to inert and particulate organics. Hydrolysis of particulate organics makes soluble organic substrate that is utilized 16

27 Chapter III: Model description by the heterotrophic bacteria. Within the steps of decay and hydrolysis, decay rather than hydrolysis is the rate limiting step (personal communication with Mark van Loosdrecht on November 21). Moreover hydrolysis compile with decay in death-regeneration concept. Therefore, in this model hydrolysis is not considered and soluble organic substrate is generated directly from the decay of biomass. The stoichiometric matrix format is outlined in Tables 1 and Table 2 gives the process rate expressions. The values for kinetic and stoichiometric parameters were based on literature and are summarized in Tables 3 and 4. Ten processes are included in the model. The autotrophic process comprises growth of X AOB, X NOB and anammox and decay of them and heterotrophic process includes growth and decay of X HA, X H,NO2 and X H,NO3. The growth of X AOB, X NOB and anammox were based on Hao et al. (22) and heterotrophs was based on ASM1 (Gujer 1999). Like ASM1, decay of X AOB, X NOB, anammox and heterotrophs were expressed as a death-regeneration concept (Henze et al. 2). The model stoichiometry and kinetics were based on the ones from och et al. (2) and Hao et al. (22). 17

28 Chapter III: Model description Table 1. Stoichiometric matrix A ij A ij i component j process growth 1. growth of X AOB S S [gcod. m -3 ] S NH [gn.m -3 ] -1/Y AOB - i NXB S NO2 [gn.m -3 ] 1/Y AOB S NO3 [gn.m -3 ] 2. growth of X NOB -i NXB -1/Y NOB 1/Y NOB 3. growth of anammox 4. aerobic growth of heterotrophs 6. anoxic (on NO 2 - ) growth of heterotrophs 7. anoxic (on NO - 3 ) growth of heterotrophs decay -1/Y H -1/Y AN - i NXB -i NXB +1/ Y H. i NSS -(1/Y AN )- (1/1.14) 1 YH, NO 2 1 -i NXB +1/ Y H. Y 1.71Y NO H,NO2 2 i H, NSS 1 -i NXB +1/ Y H. Y H,NO3 i NSS 1 Y 1.14Y H,NO3 H,NO3 S O2 [go 2.m -3 ] /Y AOB /Y NOB S N2A [gn.m -3 ] S N2 [gn.m -3 ] S N2H [gn m -3 ] X AOB [gcod. m -3 ] 1 X NOB [gcod. m -3 ] 1 X AN [gcod. m -3 ] 1/1.14 2/Y AN 1 1 Y 1.14Y H,NO3 H,NO3 X H,A [gcod. m -3 ] 1-1/Y H 1 1 Y Y H,NO2 H,NO 2 X H [gcod.m -3 ] 8. decay of X AOB 1-f I i NXB - f I i NXI (1-f I ) i NSS -1 f I 9. decay of X NOB 1-f I i NXB - f I i NXI (1-f I ) i NSS -1 f I 1. decay of X AN 1-f I i NXB - f I i NXI (1-f I ) i NSS -1 f I 11. decay of X H 1-f I i NXB - f I i NXI (1-f I ) i NSS -1 f I composition matrix gcod/unit comp gn/unit comp i NSS i NXB i NXB i NXB i NXB i NXI X H,NO2 [gcod. m -3 ] X H,NO3 [gcod. m -3 ] 1 X I [gcod. m -3 ] 18

29 Chapter III: Model description Table 2. inetic rate expressions j process 1. growth of X AOB SO2 SNH G,AOB = AOB max X AOB AOB AOB S S 2. growth of X NOB 3. growth of anammox 4. growth of aerobic heterotrophs 5. anoxic growth (on NO 2 - ) of heterotrophs 6. anoxic growth (on NO 3 - ) of heterotrophs G,NOB = G,AN = G,H = AG,H NO2 = NOB max AN max H max AN O2 O2 S O2 NOB O2 AN O2 S H S S O2 S O2 S O2 NH S NH AN NH O2 H O2 S NH NO2 NOB NO2 S NH O2 S S S. S S S NO2 S AN NO2 S NO2 S NH NOBH NH S NH NOBH NH S NO2 NH X X S AN H NH. X H H O2 NO2 NO2 S max ηno2. H H H O2 SO2 NO2 S NO2 S NO2 S NO3 S SS AG,H NO3 = 7. decay of X AOB D,AOB = b AOB X AOB 8. decay of X NOB D,NOB = bnob X NOB 9. decay of anammox 1. decay of heterotrophs S H H O2 NO3 NO3 S max ηno3. H H H O2 SO2 NO3 S NO3 S NO2 S NO3 S SS D,AN = D,HA = b AN X AN bh X H S S S S NOB S NH NOBH NH S NH NOBH NH S S NH NH X X H H 19

30 Chapter III: Model description Table 3. Stoichiometric and kinetics parameters values parameter value Unit Stoichiometric parameters Y AOB.2 g COD.g -1 N Wiesmann, 1994 (1) Y NOB.57 g COD.g -1 N Wiesmann, 1994 (1) Y AN.17 g COD.g -1 N Strous et al (1998) (2) Y H.67 g COD.g -1 COD Henze et al (2) (ASM1) Y H,NO2.53 g COD.g -1 COD Muller et al (23) Y H,NO3.53 g COD.g -1 COD Muller et al (23) i NXB.7 g N.g -1 COD Assumed in this study i NXI.7 g N.g -1 COD Assumed ame as i NXB i NSS.3 g N.g -1 COD Henze et al (2) (ASM3) f I.8 g COD.g -1 COD Henze et al (2) (ASM1) kinetic (at 3 C) AOB max 1.36 d -1 Hellinga et al (1999) (3) NOB max.79 d -1 Hellinga et al (1999) (3) AN max.52 d -1 Strous et al (1998) (3) H max 12 d -1 Henze et al (2) (ASM1) (4) AOB NH 1.1 g N.m -3 Wiesmann (1994) (5) NOB NO2.51 g N.m -3 Wiesmann (1994) (5) AN NH.3 g N.m -3 Assumed, such that ratio AN 2 NO AOB NH : AN NH is about the same as in Hao et al (22).5 g N.m -3 Assumed, such that ratio H NO H NO NOB NO2 : AN NO2 is about the same as in Hao et al (22) 2.3 g N.m -3 Alpkvist et al (26) 3.3 g N.m -3 Alpkvist et al (26) H S 2 g COD.m -3 Henze et al (2) (ASM1) AOB O 2.3 g O 2.m -3 Wiesmann (1994) NOB O g O 2.m -3 Wiesmann (1994) AN O 2.5 g O 2.m -3 Assumed in this study H O 2.2 g O 2.m -3 Henze et al.(2) (ASM1) 2

31 Chapter III: Model description b AOB.68 d -1 Assumed, set such that b AOB AOB : max = b H H : b NOB.4 d -1 Assumed, set such that b NOB NOB : = b H H : max b AN.26 d -1 Assumed, set such that b AN AN : = b H H : max b H.6 d -1 Assumed H / 2 for this study max η NO2 =η NO3.8 - Henze et al. (2) (ASM1) mass transfer D NH4 1.5x1-4 m 2.d -1 Williamson and McCarty P.L. (1976) D NO2 1.4x1-4 m 2.d -1 Williamson and McCarty P.L. (1976) D NO3 1.4x1-4 m 2.d -1 Williamson and McCarty P.L. (1976) D O2 2.2x1-4 m 2.d -1 Picioreanu et al. (1997) D N2 2.2x1-4 m 2.d -1 Williamson and McCarty P.L. (1976) D S 1x1-4 m 2.d -1 Hao and van Loosdrecht (24) max max max (1) after unit conversion, using a typical biomass composition of CH 1.8 O.5 N.2, corresponding with g COD.g -1 (2) after unit conversion, using a anammox biomass composition of CH 2 O.5 N.15, (Strous et al., 1998) corresponding with 36.4 g COD.mole -1 or 1.51 g COD.g -1 (3) Conversion of values given by Hellinga et al. (1999) at 35 C and by Strous et al. (1998) at 32.5 C to 3 C using the relationship (written for X AOB, analogous for X NOB and X AN ) AOB1 max with ( T ) AOB1 max (T ref AOB E a =68 kj.mole -1 ; 1999); R=8.31 J.mole E ) exp NOB a AOB a T T R T T ref ref E =44 kj.mole -1 ; E AN a = 7 kj.mole -1 (Strous et al., (4) Conversion of ASM1-values given by Henze et al. (2) at 1 C and 2 C to 3 C using temperature relationship proposed by these authors (ASM3). (5) Calculated value at T=3 C and ph=7 from NOB HNO2 AOB NH3 =.28 g NH 3 -N.m -3 and from = 3.2x1-5 g HNO 2 -N.m -3 considering the T and ph dependency of the chemical equilibrium NH4 NH3 H and HNO2 NO2 H 21

32 Chapter III: Model description Table 4: Temperature dependent kinetic parameters Temperature Parameters AOB max (1) NOB (1) max AN (1) max H (2) max 1 C 15 C 2 C 25 C 35 C 4 C b AOB (3) b NOB (3) b AN (3) b H (4) (1) Conversion of values given by Hellinga et al. (1999) at 35 C and by Strous et al. (1998) at 32.5 C to different temperature using the relationship (written for X AOB, analogous for X NOB and X AN ) AOB1 max with ( T ) AOB1 max (T ref AOB E a =68 kj.mole -1 ; 1999); R=8.31 J.mole E ) exp NOB a AOB a T T R T T ref ref E =44 kj.mole -1 ; E AN a = 7 kj.mole -1 (Strous et al., (2) Conversion of ASM1-values given by Henze et al. (2) at 1 C and 2 C to different using temperature relationship proposed by these authors (ASM3). (3) Assumed, set such that b AOB AOB : max = b H H : (written for X AOB, analogous for X NOB and X AN ). (4) Assumed H / 2 for this study. max max 22

33 Chapter III: Model description 2. Reactor configuration, simulation parameters and initial conditions A one dimensional biofilm model, only considering radial gradients was set up to describe the autotrophic and heterotrophic interaction in a granular sludge reactor. The model was implemented in the Aquasim software (Reichert, 1994). The reactor had a fixed volume of 4 m 3. Spherical biomass particles (granules) were grown from an initial radius of.1 mm to a predefined steady state granule radius, r p (.75mm < r p < 2.75 mm) such that the reactor eventually contains 1 m 3 of particulate material, comprising both active biomass as well as inert material generated during growth and decay. Growth of the granules was associated with a decrease in the bulk liquid volume to 3 m 3. The oxygen level in the bulk liquid was controlled at a fixed value (between.1 and 4. go 2.m -3 ). The bulk liquid was assumed to be well-mixed, and external mass transfer limitation was neglected, which simplifies the evaluation of the simulation results. Biomass granules were typically quite dense with very small pores, in which no relevant motion of suspended solids takes place. The granule structure was further assumed to be rigid, meaning that particulate components were displaced only due to the expansion or shrinking of the biofilm solid matrix. Besides, the biofilm porosity had been assumed constant (ε W =.75); its value was determined by the initial fractions of particulate components (ε ini XAOB =.1; ε ini XNOB =ε ini XAN =ε ini XH =.5; ε ini XHA =ε ini XHNO2 =ε ini XHNO3 =ε ini XH /3; ε ini XI =). The density of autotrophic biomass and particulate inerts (ρ A ) in the granules were set to 6 g VSS.m -3 (van Benthum et al., 1995), corresponding to 8 g COD.m -3 (for a typical conversion factor of.75 g VSS.g -1 COD (Henze et al., 2)). The density of the heterotrophs (ρ H ) was 2 gvss.m -3 (van Benthum et al., 1995) which is equivalent to g COD.m -3. The reactor behavior had been simulated for an influent containing mainly ammonium, with a flow rate of 25 m 3.d -1. The ammonium concentration through the process was 3 g N.m -3, except when the ammonium concentrations were varied from 2 gn.m -3 to 9 gn.m -3 to find the effect of ammonium surface load on reactor performance (Chapter IV section 3.4). It was assumed that no nitrite or nitrate was present in the influent. Influent was assumed not to contain any readily degradable or particulate organic substrate except the part (Chapter IV section 4) where effects of the influent organic substrate on reactor performance were analyzed. To find out the effect of readily degradable organic substrate concentration on reactor performance, the concentration of organic substrate varied from to 1 gcod.m

34 Chapter III: Model description The initial concentrations of soluble compounds in the bulk liquid had been assumed equal to influent concentrations. The processes were operated at 3 C temperature. The temperature effect on nitrogen removal was analyzed (Chapter IV section 3, sub-section 3.3) where temperature was changed from 1 to 4 C. Simulations have been performed for several years of operation to assure steady state conditions. 24

35 Chapter IV: Results and discussion 1. Role of heterotrophic growth on nitrogen removal In order to compare the model with and without heterotrophic growth, the simulations without any influent organic substrate were run. For making the model without heterotrophic growth, the processes 4, 5, 6 and 1 in Table 2 are inactivated in the simulation. To investigate the effect of heterotrophic activities in the partial nitritation-anammox process, the reactor performance is evaluated in terms of nitrogen removal. The active biomasses and fraction of three types heterotrophic bacteria within heterotrophic biomass are shown in Figure 3, 4 and 5 and the dynamic results of the nitrogen removal and nitrogen compounds in bulk are summerized in Figure 6 and Active biomass composition Without heterotrophic activities, relatively higher ammonium oxidizing bacteria (X AOB ) and the Anammox bacteria are present (Figure 3). At the period of 2 to 6 days the anammox bacteria is higher in case of without heterotrophic growth that made higher nitrogen removal. The ammonium oxidizing bacteria (X AOB ) increase during first 2 days and then decrease; first rapidly (until 35 days) and then slowly. During first 6 days anammox is growing very fast but not enough to convert all the nitrite (NO - 2 ) to nitrogen gas. At steady state higher fraction of anammox and ammonium oxidizing bacteria are for without heterotrophic growth. Figure 4 is extracted from figure 3 where y-axis is extended to visualize the fraction of nitrite oxidizing bacteria (X NOB ) for both cases and found higher X NOB for without heterotrophic growth in the model. In the model an artificial distinction is made between three types of heterotrophic bacteria according to the substrate they grow on; aerobic heterotrophs (X H,A ) growing on organic substrate (S S ), anoxic heterotrophs on nitrite (X H,NO2 ) and anoxic heterotrophs on nitrate (X H,NO3 ). The evaluations of the fraction of these heterotrophic bacteria are shown in figure 5. At the beginning of the process, a higher amount of X H,NO2 are present in a granule and it decreases to zero at 12 days. A very low fraction of X H,NO3 is present during the first 1 days and then sharply increases. At steady state no X H,NO2 but X H,NO3 and X H,A present in the granules. 25

36 Active Biomass (gcodx1 5 ) fraction Active Biomass (gcodx1 5 ) Chapter IV: Results and discussion AOB (without heterotrophs) NOB (without Heterotrophs) Anammox (without Heterotrophs) AOB (with Heterotrophs) NOB (with Heterotrophs) Anammox (with Heterotrophs) Heterotrophic biomass Time (Day) Figure 3. Comparison of microbial community in a granule for the condition of considering heterotrophic growth and without considering heterotrophic growth (r p =.75mm, O 2 =.5 go 2.m -3, S S = gcod.m -3, NH 4(in) =3 gn.m -3, T=3 C) NOB (without Heterotrophs) NOB (with Heterotrophs) Heterotrophic biomass X H,A HA X H,NO2 HNO2 X H,NO3 HNO Time (Day) Figure 4. Comparison of nitrite oxidizing bacteria (NOB) in a granule in case of with and without heterotrophic growth and heterotrophic organism (extended from Figure 3) Figure 5. The dynamics of heterotrophic microbial Time [day] community fraction (r p =.75mm, O 2 =.5 go 2.m -3, S S = gcod.m -3, NH 4(in) =3 gn.m -3, T=3 C). 26

37 Chapter IV: Results and discussion 1.2. Competition among active biomass Different bacteria compete with each other for oxygen and substrate. This has an affect on nitrogen removal performance. Table 5 shows the microorganisms acting in partial nitritation-anammox process for both with and without heterotrophic growth in the model and their competition for oxygen and substrate. Table 5. Microorganism acting for both with and without heterotrophic growth in model and their competition for oxygen and substrate. Without heterotrophs With heterotrophs Substrate X AOB X NOB Anammox X AOB X NOB Anammox X H X H,A X H,NO2 X H,NO3 O NH NO NO When heterotrophic growth is not taken account (without heterotrophs), ammonium oxidizing bacteria (X AOB ) and nitrite oxidizing bacteria (X NOB ) compete with each other for oxygen. Anammox bacteria compete with X AOB for ammonium (NH + 4 ) and with X NOB for nitrite (NO - 2 ). But when the heterotrophic growth is taken in the model (with heterotrophs), aerobic heterotrophs (X H,A ) compete with X AOB and X NOB for oxygen and heterotrophs consuming nitrite (X H,NO2 ) compete with anammox and X NOB for nitrite. The heterotrophic bacteria have a very low competition with anammox and X AOB for ammonium. The heterotrophic bacteria based on nitrate (X H,NO3 ) do not compete strongly with any autotrophic bacteria, moreover it produces nitrite from nitrate without consuming any oxygen. 27

38 Chapter IV: Results and discussion 1.3. Comparison of nitrogen removal performance From Figure 6, it is found that first 2 days there is no significantly different on nitrogen removal performance between with and without heterotrophic growth. But at between 2 and 6 days better nitrogen removal for without heterotrophic growth whereas after that time the process with heterotrophs shows better nitrogen removal. The competitions of - heterotrophic bacteria with anammox for NO 2 reduce the anammox growth when heterotrophic growth is considered in the model. Therefore lower total nitrogen removal is observed in the period between 2 and 6 days, even though heterotrophic nitrogen removal is higher due to heterotrophic bacteria on nitrite (X H,NO2 ). But the amount of heterotrophic bacteria is very low;.32.14x1-5 gcod per granule between 2 and 6 days. On the other hand, during this period the difference between anammox bacteria in the simulation with and without heterotrophic growth condition varies between x1-5 gcod per granule. At steady state based on soluble compounds, that is after 12 days, the differences of anammox and ammonium oxidizing bacteria (X AOB ) between two conditions are very low. Despite the lower fraction of anammox and X AOB in the simulation with heterotrophic growth, higher nitrogen removal is observed due to lower nitrite oxidizing bacteria (X NOB ) and presence of heterotrophic bacteria on nitrate (X H,NO3 ). These heterotrophic bacteria convert the nitrate to nitrite and give advantages to anammox bacteria for higher nitrogen removal at steady state. In Figure 7 shows that first 1 days there is nitrite accumulation for both conditions. These nitrite accumulations are firstly increase upto 26 days and then decrease. The decreasing of nitrogen removal after 2 days (Figure 6) is due to this high concentration of nitrite in bulk that inhibits the anammox bacteria. Due to lower anammox bacteria, higher nitrite accumulation is in case of with heterotrophic growth. At the steady state, there is nitrate accumulation and lower accumulation in case of with heterotrophic growth. Lower nitrite oxidizing bacteria (X NOB ) and heterotrophic bacteria on nitrate (X H,NO3 ) in with heterotrophic growth are responsible for comparatively lower nitrate accumulation in with heterotrophic growth 28

39 Chapter IV: Results and discussion 25 2 gn.m Time (day) Total N removal without heterotrophs Total N removal with heterotrophs Autotrophic N removal with heterotrophs Only Heterotrophic N removal Figure 6. Comparison of nitrogen removal performance for with and without heterotrophic growth (r p =.75mm, O 2 =.5 go 2.m -3, S S = gcod.m -3, NH 4(in) =3 gn.m -3, T=3 C). gn.m -3 or gcod.m Without heterotrophs S_NH Without heterotrophs S_NO2 Without heterotrophs S_NO3 Without heterotrophs S_S With heterotrophs S_NH With heterotrophs S_NO2 With heterotrophs S_NO3 With heterotrophs S_S Time (day) Figure 7. The nitrogen compounds in bulk for with and without considering heterotrophic growth (r p =.75mm, O 2 =.5 go 2.m -3, S S = gcod.m -3, NH 4(in) =3 gn.m -3, T=3 C). In this section, it is found that at initial time (1 6 days) the nitrogen removal is higher for without considering heterotrophic growth compare to with considering heterotrophic growth. Anammox bacteria is the main responsible for this nitrogen removal. If the heterotrophic growth is considered, it reduce the initial anammox growth as well as nitrogen 29

40 Chapter IV: Results and discussion removal due to compition with heterotrophic bacteria for nitrite. But at steadystate higher nitrogen removal for the condition of heterotrophic growth. Lower amount of nitrite oxidizing bacteria (X NOB ) and heterotrophic bacteria on nitrite (X HNO2 ) give adventages to anammox bacteria to make better performance in terms of nitrogen removal. 2. Biomass dynamics and steady state In this biofilm model, granules are grown from an initial size to predefined steady state granule size. During this growing period, the composition of the active biomass and particulate inerts are changing together with their position in the granules. The duration of growing period depends on initial size of the granule and biomass composition in granules Biomass dynamics in a granule To evaluate the microbial community inside a granule in time, a simulation is performed in a model with.1 mm initial granule size and a predefined steady state granule size of.75 mm, an influent ammonium concentration of 3 gn.m -3 and a bulk oxygen concentration of 1 go 2.m -3. The results are displayed in Figure 8 to 1. Figure 8 describes that after 5 to 1 days of starting the process there are very small amounts of anammox present in the centre. More ammonium oxidizing bacteria (X AOB ) are observed from middle to surface of the granules. So within these 1 days, a large amount of nitrite is accumulated in bulk (Figure 9a). Moreover within these days the predefined granule size (.75mm) is not formed. It takes around 4 days to reach the steady state granular size (Figure 1). At the earlier time of steady state, anammox bacteria are formed in the centre of granules but this active biomass is move slowly towards surface of the granule. The nitrite oxidizing bacteria (X NOB ) starts to grow at around 4 days and as a consequenc nitrate starts to increase. The evaluation of the bulk nitrogen compounds and the biomass composition as well as the biomass fraction in a granule over time is shown in Figure 9. The amount of anammox bacteria and nitrogen removal increase up to 2 days and then anammox bacteria decrease at a very slow rate to reach the steady state level. The process takes almost 23 days to reach substrate steady state and 6 days to reach biomass steady state. At steady state, it is found that the active parts of the biomass are situated within.3 mm depth from the surface of the granule. 3

41 [kg COD.m -3 ] [kg COD.m -3 ] [kg COD.m -3 ] [kg COD.m -3 ] [kg COD.m -3 ] [kg COD.m -3 ] [kg COD.m -3 ] [kg COD.m -3 ] Chapter IV: Results and discussion 1 Time = 5 days 1 Time = 1 days X AOB X NOB 6 X AOB X NOB 4 X AN X I 4 X AN X I 2 X H X tot 2 X H X tot z [mm] Time = 5 days z [mm] Time = 1 days X AOB X NOB 6 X AOB X NOB 4 X AN X I 4 X AN X I 2 X H X tot 2 X H X tot z [mm] Time = 2 days z [mm] Time = 4 days X AOB X NOB 6 X AOB X NOB 4 X AN X I 4 X AN X I 2 X H X tot 2 X H X tot z [mm] Time = 6 days z [mm] Time = 1 days X AOB X NOB 6 X AOB X NOB 4 X AN X I 4 X AN X I 2 X H X tot 2 X H X tot z [mm] z [mm] Figure 8. The profile of biomass and particulate inerts in a granule over time (radius.75mm, bulk oxygen concentration 1. go 2.m -3, T=3 C). 31

42 Biofilm thickness [mm] [g N.m -3 or g COD.m -3 ] Chapter IV: Results and discussion (a) S O2 = S NH S NO2 S NO3 S N2 S S S Ntot (b) time [days] Figure 9. Evaluation of (a) bulk nitrogen compound and (b) biomass and inert fractions in a granule over time (radius.75mm, bulk oxygen concentration 1. go 2.m -3, T=3 C) Time [day] Figure 1. Evaluation of biofilm thickness over time (radius.75mm, bulk oxygen concentration 1. go 2.m -3, T=3 C) Influence of initial conditions on the time needed to reach steady state From the dynamic behavior of the active biomass and the bulk concentration, it is found that the process takes more than thousand days to reach steady state. But this time requirement depends on initial conditions of biomass matrix and bulk concentrations. It is observed that the change of initial biomass composition and initial granular size do not affect the steady state performance but changes the dynamic behavior and time to reach steady state. Table 6 describes the different initial conditions and time required to reach steady state of bulk 32