Is ammonification the rate limiting step for nitrification kinetics?

Bioresource Technology 114 (2012) 117–125

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Case Study

Is ammonification the rate limiting step for nitrification kinetics? Tugce Katipoglu-Yazan a, Emine Ubay Cokgor a, Güçlü Insel a, Derin Orhon a,b,⇑ a b

Environmental Engineering Department, Faculty of Civil Engineering, Istanbul Technical University, 34469 Maslak, Istanbul, Turkey Turkish Academy of Sciences, Piyade Sokak, No. 27, 06550 Cankaya, Ankara, Turkey

a r t i c l e

i n f o

Article history: Received 26 December 2011 Received in revised form 2 March 2012 Accepted 5 March 2012 Available online 14 March 2012 Keywords: Ammonification Hydrolysis Nitrification Modeling Respirometry

a b s t r a c t This study investigated relative magnitude of hydrolysis and ammonification by separate analysis of ammonia release and nitrification mechanisms. A peptone mixture was used as substrate in two parallel experiments seeded with nitrifying biomass conducted with and without nitrification inhibitor. Results were evaluated by means of model analysis of the ammonia and the oxygen uptake rate (OUR) profiles. A dual hydrolysis mechanism with maximum rate coefficients of 6.3 and 0.5/day characterized the peptone mixture and a kinetic balance was established for the ammonia release mechanism with a corresponding ammonification rate of 0.08 m3/g COD day. The experiments also showed a low soluble ammonia nitrogen generation that was rapidly depleted, confirming the existence of ammonification. These rate coefficients were verified using model calibration of the OUR profile related to simultaneous carbon removal and nitrification. Results indicated that ammonification would not be rate limiting for wastewaters such as domestic sewage, with lower hydrolysis kinetics. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Effective control and removal of nitrogen in wastewaters require a thorough interpretation and manipulation of related biochemical processes: It essentially involves biological oxidation and reduction of inorganic nitrogen compounds – i.e. nitrification and denitrification. It should also take into account nitrogen utilization as part of microbial biosynthesis and its removal with excess sludge. Nitrification relies on the metabolic activities of a group of chemoautotrophic bacteria essentially utilizing ammonia nitrogen, SNH, and oxidizing it to nitrate nitrogen, SNO for energy. However, domestic sewage contains organic nitrogen of both particulate (XND) and soluble (SND) in nature (30–40% of the total nitrogen content) aside from ammonia nitrogen, which constitutes the major nitrogen fraction (Okutman Tas et al., 2009). Characteristics of industrial wastewaters are quite different from domestic sewage in terms of their nitrogen content: While some industrial effluents may even be deficient in nitrogen for effective organic matter removal, others – such as leather tanning effluents – are characterized by high nitrogen levels with a significant organic nitrogen fraction (Artan et al., 2004; Carucci et al., 1999; Murat et al., 2006). Therefore, ammonia release through microbial breakdown of organic nitrogen needs to be considered as a significant step for appropriate evaluation of nitrification and nitrogen removal efficiency; organic nitrogen should be converted to ⇑ Corresponding author. Tel./fax: +90 212 285 3793. E-mail addresses: [email protected] (T. Katipoglu-Yazan), [email protected] (E. Ubay Cokgor), [email protected] (G. Insel), [email protected] (D. Orhon). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2012.03.017

ammonia nitrogen for further utilization by autotrophic microorganisms for nitrification. In activated sludge models, two different approaches have been proposed for the conversion of organic nitrogen – specifically particulate organic nitrogen fraction, XND – into ammonia nitrogen. In Activated Sludge Model No.1 (ASM1), Henze et al. (1987) basically defined a two-step process: The first step involved the breakdown of XND to soluble organic nitrogen, SND through hydrolysis; in the second step SND was converted to SNH by means of a separate ammonification process. The latter – i.e. conversion of SND to SNH – was assumed to proceed much faster as compared to the hydrolysis rate of the particulate organic nitrogen and it was conveniently defined in terms of the following first-order reaction rate with a default value of 0.08 m3/g cell COD/day for the ammonification rate coefficient, ka:

dSND =dt ¼ ka SND X H where, XH is the concentration of active heterotrophic biomass. The second approach incorporated into ASM2d (Henze et al., 1995) simply disregarded the ammonification process and stipulated that ammonia nitrogen was directly released through the hydrolysis of slowly biodegradable organic matter into readily biodegradable substrate, also converting XND into SNH. This approach was approved and adopted for domestic sewage, mostly without the supporting experimental evidence. In suggested design procedures, the basic process stoichiometry was established on the basis of Total Kjeldahl Nitrogen (TKN) (organic + ammonia) with the presumption that ammonification was not rate limiting (Artan et al., 2004; Murat et al., 2006). A few studies yielded confirming results

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with the default value for ka suggested in ASM1: Lu et al. (2001) calibrated the ASM1 model and modified for soluble microbial product formation (SMP), for a membrane bioreactor (MBR) system operated for simultaneous carbon and nitrogen removal, for a ka value of 0.08 m3/g COD day. In a floc model with radial oxygen diffusion limitation a compatible ka value of 0.05 m3/g COD day was suggested to justify the experimental results (Wang et al., 2007). Similarly, an integrated ASM1-SMP model, also including cake layer attachment–detachment, fouling, transmembrane pressure (TMP) variation and resistance variation due to fouling was calibrated with an ammonification rate coefficient of 0.102 m3/g COD day. However, this range of an ammonification rate did not explain high levels of remaining organic nitrogen in strong industrial effluents. In the study of Gorgun et al. (2007), model calibration was used to evaluate organic carbon and nitrogen removal in a leather tanning wastewater treatment plant. Process performance of the activated sludge unit was evaluated by determining temperature dependent model parameters, governing nitrification, endogenous decay, hydrolysis and ammonification processes. In model calibration, process kinetics was calibrated using the monthly average data depending upon process temperature changes. As a result, the model was calibrated with an ammonification rate of 0.0003 m3/g COD day, which was far below the default value of 0.08 m3/g COD day adopted in ASM1. In a following study on the same treatment plant, the observed nitrogen removal efficiency was simulated with the calibrated activated sludge model, for three different process temperatures. The adopted model was calibrated with the ammonification rates of 0.003, 0.007 and 0.0082 m3/g COD day for different temperatures of 21 °C in January, 26 °C in November and 35 °C in August, respectively (Insel et al., 2009). Conflicting results necessitate a closer look at ammonia release from organic nitrogen, and especially on the ammonification process with a more valid experimental approach: In fact, the common deficiency of all previous studies was to evaluate complex systems performing simultaneous carbon and nitrogen removal, where ammonia release did not stand alone and was somewhat obscured by on-going simultaneous biochemical reactions. In this context, the objective of the study was to explore (i) the existence and (ii) the significance of ammonification by separate analysis of ammonia release and nitrification processes in parallel experimental systems. The impact of ammonia release on nitrification kinetics was evaluated by modeling all relevant parameters in experimental setups operated with and without nitrification.

2. Methods 2.1. Experimental setup The experimental setup involved (i) a laboratory-scale fill and draw reactor with a hydraulic retention time of one (1.0) day and sustained at a sludge age of 10 days at steady state under aerobic conditions, and (ii) a series of fully aerated batch reactors for the investigation of ammonia release, ammonification and nitrification mechanisms. The fill and draw reactor essentially provided the initial biomass seed – i.e. mixed microbial community with an active fraction of nitrifiers – for the batch tests. This approach ensured the consistency of the starting biomass seed with the same culture history (sludge age) in all the experiments (Katipoglu et al., 2010a). The experiments were conducted using a synthetic substrate composed of peptone, meat extract and urea – referred to as peptone mixture in the text, for simplicity – as the sole organic carbon and nitrogen source. The substrate was selected, mainly because (i) it approximated the composition and COD fractionation of domestic

sewage (Cokgor et al., 2011; Orhon et al., 2009); (ii) it was prescribed as a standard substrate for inhibition assessment based on respirometric measurements (Insel et al., 2006; ISO 8192, 1995); (iii) it could be adjusted to maintain the same characteristics in different phases of the study. The peptone mixture used as synthetic substrate was prepared with 16 g/L of peptone, 11 g/L of meat extract, 3 g/L of urea, 0.7 g/L of NaCl, 0.4/L of CaCl2. 2H2O, 0.2 g/L of MgSO47H2O and 2.8/L of K2HPO4 according to ISO 8192 (1995). Substrate feeding also included macro and micro nutrients: The stock solution for macro nutrients was prepared with 320 g/L K2HPO4, and 160 g/L KH2PO4; similarly, the stock solution for micro nutrients included 15 g/L MgSO47H2O, 0.5 g/L FeSO47H2O, 0.5 g/L ZnSO47H2O, 0.41 g/L MnSO4H2O, 2.65 g/L CaCl22H2O g/L. The feeding in the fill and draw reactor was adjusted to maintain around 450 mg COD/L and 50 mg N/L of total nitrogen. Biomass in the fill and draw reactor was acclimated to the synthetic substrate selected for the experiments within a start-up period before steady-state operation. At steady state, biomass concentration in the reactor, measured in terms of volatile suspended solids (VSS) stabilized around 2300 mg/L. pH was kept around 7.0–8.0 in the reactor. The temperature and oxygen concentration was maintained at 20 °C and 3 mg O2/L, respectively. 2.2. Ammonia release and OUR experiments Batch experiments were conducted in 2 L reactors and they were started with biomass taken from fill and draw reactor continuously operated at steady state. Biomass was adjusted to an initial food to microorganism (F/M) ratio of around 0.50–0.55 mg COD/ mg VSS, to approximate the F/M level in the fill and draw reactor. Ammonia concentration was monitored for 24 h to assess the amount of ammonia release by heterotrophic microorganisms in the presence of nitrification inhibitor. In parallel, ammonia release from peptone and meat extract - the two major organic components of the peptone mixture – were also monitored separately. For this purpose, feed solutions having only peptone or meat extract as carbon and nitrogen sources were prepared. Urea was not included in the feed solution because it would be easily converted to ammonia as a readily hydrolysable nitrogen compound. In all experiments micro and macro nutrients were supplied with feed solution. The overall characteristics of batch experiments are outlined in Table 1. The respirometric tests were started with biomass seeding alone to obtain the initial endogenous OUR (oxygen uptake rate) level. Changes in the OUR of the system were monitored for 14–15 h. In the first run nitrification inhibitor was used to observe only the response of heterotrophic activity associated with ammonia release. In the second run, respirometric experiments were repeated with peptone mixture adjusted to around 500 mg COD/L without nitrification inhibitor, to observe the OUR profile associated with carbon removal and nitrification. OUR measurements were performed with a Ra-Combo 1000 (Applitek Co., Nazareth, Belgium) continuous respirometer. COD, soluble TKN, nitrite and nitrate nitrogen were monitored in all sets. Parallel to OUR experiments, internal storage polymers i.e. polyhydroxyalkanoates (PHAs) including polyhydroxybutyrate (PHB), polyhydroxyvalerate (PHV), 3-hydroxy-2methylvalerate (3H2MV) and glycogen were also monitored. 2.3. Analytical procedures COD and soluble TKN samples were filtered through 0.45 lm Millipore membrane filters and preserved with H2SO4. COD analyses were performed according to ISO 6060 method (1986). Soluble TKN and ammonia nitrogen analyses were performed according to Standard Methods (2005). As a nitrification inhibitor sodium

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T. Katipoglu-Yazan et al. / Bioresource Technology 114 (2012) 117–125 Table 1 The overall characteristics of batch experiments. Runs

Sets

Substrate

Run 1

Set Set Set Set Set

455 mg 455 mg 280 mg 175 mg 502 mg

Run 2

1 2.1 2.2 2.3 3

COD/L COD/L COD/L COD/L COD/L

Peptone mixture Peptone mixture Peptone Meat extract Peptone mixture

sulfate and TCMP (2-chloro-6(trichloromethyl) pyridine) (Formula 2533, Hach Company) was used. TCMP inhibits ammonia monooxigenase enzyme which is responsible for ammonia oxidation to hydroxylamine by interfering with the cytochromes in the oxidation pathway (Hooper and Terry, 1973). 0.16 g nitrification inhibitor was added per 300 ml of sample as currently reported in similar studies (Lee et al., 2010). Nitrite and nitrate nitrogen were determined by Ion Chromatography (DIONEX ICS-1500 model). The biomass concentration was monitored by total suspended solids (TSS) and volatile suspended solids (VSS) measurements as described in Standard Methods (2005). PHA samples were taken from mixed liquor and preserved with formaldehyde in order to prevent biological activity. The liquid phase of mixed liquor was removed by centrifugation. Remaining biomass was washed with K-P buffer and the samples were freeze-dried. According to the method proposed by Beun et al. (2000), hydrochloric acid, 1-propanol, and dichloroethane mixture was used for extraction, hydrolysis and esterification of sample at 100 °C. After the removal of free acids by water extraction, the organic phase was analyzed by an Agilent 6890N gas chromatograph. The PHB, PHV and 3H2MV fractions of PHA were individually determined. Benzoic acid was used as the internal standard. For glycogen analysis, samples from mixed liqueur were acidified with 0.5 ml 6 M HCl to have a final volume of 5 ml. The samples were boiled at 100 °C for 5 h, centrifuged and the glucose content of supernatant was analyzed with (Agilent 1100 Series) HPLC in accordance with the method described by Smolders et al. (1994).

3. Results and discussion 3.1. Experimental rationale and design The rationale behind designing the experimental study involved a number of significant concerns, which basically defined the sequence of necessary experimental steps: First of all, evaluation of the existence and rate limitation likely to be imposed by the ammonification required accurate assessment of the hydrolysis mechanism – a heterotrophic process. Similarly, an experimental system which would allow nitrification together with heterotrophic organic carbon removal would naturally utilize ammonia nitrogen and not permit direct observation of the ammonia release mechanism. In this context, the first step of the experimental system was designed to observe and evaluate heterotrophic activity alone, without parallel nitrification. This step yielded the experimental data for (i) evaluating heterotrophic activity and mainly hydrolysis kinetics, and (ii) interpretation of the ammonia release mechanism. The kinetic information derived from this experimental system was also utilized for further exploring ammonia release by model simulation, which established the critical kinetic balance between hydrolysis and ammonification. In the next experimental step, which basically allowed simultaneous organic carbon removal and nitrification as they occur in biological treatment systems, modeling results were verified and confirmed. The sequence of related results are presented individually in separate sub-sections.

VSS (mg/L)

F/M (mg COD/mg VSS)

Nitrification

920 920 802 802 920

0.50 0.50 0.35 0.22 0.55

    +

Consequently, two different runs of batch experiments were carried out to evaluate the ammonification process: Nitrification was prohibited by means of selected inhibitor (TCMP) in the first run, and in the second, it was allowed to proceed. The first run included two parallel set of experiments (Set 1 and 2): The batch reactor system in the first set (Set 1) was used to monitor the oxygen uptake rate (OUR) profile, only reflecting reactions associated with the heterotrophic activity. In the second set, the release mechanism of ammonia was investigated by means of three parallel batch reactors fed with the peptone mixture, peptone alone and meat extract alone respectively (Set 2.1, 2.2., and 2.3). The second run only included a parallel batch reactor to obtain and evaluate a similar OUR profile generated by simultaneous organic carbon removal and nitrification (Set 3). A multi-component model was structured to provide the necessary kinetic information derived from the experimental results. It was calibrated with all relevant experimental data in different runs and sets so that a single set of model coefficients would equally apply to different experimental data: For example, the hydrolysis kinetics assessed in the OUR experiments were used to evaluate the ammonia release mechanism. 3.2. Rationale for model structure Model evaluation is now considered as an integral component of biological systems, as it yields numerical information about related process kinetics and this way, it provides a meaningful correlation between results derived from different experimental sets. The nature of problems investigated in this study necessitated a more complex model structure compared with the basic template of simpler models such as ASM1, for two main reasons (i) a more detailed COD fractionation for hydrolysis, better defining the selected synthetic substrate, and (ii) recognition and kinetic description of substrate storage. Breakdown of the slowly biodegradable substrate by hydrolysis and utilization of the generated readily biodegradable substrate for either direct growth or for intracellular storage are all interrelated heterotrophic processes affecting one another. In this context, ASM1 does not account for substrate storage. Disregarding storage would distort both hydrolysis and growth kinetics in model calibration and would give a wrong description of the hydrolysis process, one of the key components in this research. The model adopted in this study defined both growth and storage and therefore, its calibration included the experimental storage data monitored in the study. This way, the calibration could yield accurate values for the hydrolysis kinetics, a vital issue for the question investigated in the study. In this context, the model adopted for this study was structured to include model components and processes for: (i) organic carbon removal; (ii) ammonia release, and (iii) nitrification. Accordingly, the first-unit of the model, which constitutes the backbone of all similar activated sludge models, defined the organic substrate and the heterotrophic biomass. Previous evaluations indicated that the peptone mixture was quite similar to domestic sewage in terms of COD fractionation, mainly involving two slowly biodegradable COD components, SH1 and SH2 with markedly different

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hydrolysis rates and a small readily biodegradable COD (SS) fraction (Insel et al., 2003; Orhon et al., 2009). Consequently, major metabolic activities of the heterotrophic active biomass, XH were defined as growth on SS; hydrolysis of SH1 and SH2; and endogenous decay of XH. Dissolved oxygen, S0, was included as the significant model component in the OUR experiments. Generation of particulate residual microbial products, XP, should also be accounted for, as XP needs to be considered for nitrogen mass balance in the system. Studies also reported that substrate storage as polyhydroxyalkanoates (PHAs) occurred as an auxiliary biochemical process during the biodegradation of the peptone mixture in batch systems (Katipoglu et al., 2010b; Orhon et al., 2009). In fact, since experiments were carried out in batch systems involving initial/pulse substrate feeding, they were likely to induce substrate storage, which makes it necessary to also account for storage. Therefore, the sub-model was designed as a simultaneous growth-storage model, successfully implemented in many similar studies (Cokgor et al., 2011; Krishna and van Loosdrecht; 1999), also including storage products, XSTO, as model component and two new biochemical processes, namely storage and growth on stored PHAs. The second unit of the model mainly addressed the ammonia release mechanism as the major objective of the study. Its mechanistic structure was primarily based on the assumption that soluble organic nitrogen, SND was a fraction of the readily and slowly biodegradable COD, SH1 and SH2:

SND ¼iNSH1 SH1 þ iNSH2 SH2 where, iNSH1 and iNSH2 are the nitrogen fractions of SH1 and SH2 (mg N/mg COD), respectively. Consequently, generation of simpler (soluble) organic nitrogen, SND, could conveniently be defined in terms of hydrolysis:

dSND SH1 =X H SH2 =X H X H þ iNSH2 kh2 XH ¼ iNSH1 kh1 dt K X þ SH1 =X H K XX þ SH2 =X H Release of ammonia, SNH; was attributed to the breakdown of SND as suggested in similar activated sludge models (Lu et al., 2001; Wang et al., 2007):

dSNH dSND ¼ ¼ka SND X H dt dt where, ka is the ammonification coefficient (m3/g COD day). Accordingly, this sub-model added SND and SNH together with two processes, i.e. ammonification and hydrolysis modified with the iNSH1 and iNSH2 coefficients, to the main model structure. The third sub-model concerning nitrification obviously requires concentrations of ammonia, SNH, and nitrate, SNO3, with alkalinity and dissolved oxygen, SO. The complete description of the overall model is outlined in the usual matrix format in Tables 2 and 3

(Lu et al., 2001; Lubello et al., 2009; Wang et al., 2007). The AQUASIM simulation program was used in model calibration (Reichert et al., 1998). Kinetic and stoichiometric coefficients of the model were estimated based on the minimization of multi response objective function via SIMPLEX algorithm (Reichert et al., 1998) according to the procedure proposed by Insel et al. (2003). The UNCSIM module developed by Brun et al. (2001) was used for the assessment of parameter identifiability. A detailed description of the assessment process was already presented before (Cokgor et al., 2011).

3.3. Modeling heterotrophic activity Biodegradation characteristics of the peptone mixture utilized as the sole organic carbon source was evaluated primarily by model calibration of the OUR profile obtained from a batch reactor operated with the selected nitrification inhibitor; the batch reactor (Set 1) was started with an initial peptone mixture concentration of around 450 mg COD/L and a biomass concentration of around 1000 mg VSS/L, corresponding to a food to microorganism (F/M – SO/XO) ratio of 0.50 mg COD/mg VSS. As shown in Fig. 1, the resulting OUR profile included an initial OUR peak slightly higher than 100 mg O2/L.h, which decreased with sequential drops afterwards, reflecting the existence of three COD fractions with different biodegradation rates. The shape of the OUR curve was quite characteristic and similar to the one obtained in an experimental study also using the peptone mixture at the same sludge age of 10 days (Insel et al., 2006). The OUR profile dropped down to the initial endogenous respiration level after 0.4 days (9.6 h), where the soluble COD stabilized around 30 mg COD/L, a level corresponding to the generated soluble residual microbial products after total depletion of the available biodegradable substrate (Orhon et al., 1994a). Reliable modeling of substrate utilization also requires consideration of intracellular storage: It is well known that in biological reactors – such as batch reactors in this study - sustaining sequential feast (abundance of external substrate) and famine (absence of external substrate) conditions, a fraction of available substrate is likely to be diverted to storage (Krishna and van Loosdrecht, 1999). In this context, generation of the two storage products, i.e. polyhydroxyalkanoates (PHAs) and glycogen were also monitored, parallel to the OUR profiles. It should be mentioned that PHA assessment involved separate measurements of all relevant components, i.e. polyhydroxybutyrate (PHB); polyhydroxyvalerate (PHV) and 3-hydroxy-2-methylvalerate (3H2MV). As illustrated in Fig. 2, the results indicated a slight PHA generation of 20 mg COD/L, corresponding to around 4–5% of the initially available substrate and negligible glycogen storage, confirming similarly low levels of storage associated with peptone mixture at the same

Table 2 Matrix representation of represented model structure. Process

SO2

Ss

Growth of XH

HÞ  ð1Y YH

 Y1H

Growth of XA

AÞ  ð4:57Y YA

Growth on X STO by XH

HÞ  ð1Y YH

iNBM

1

Decay of XH

(1  fES  fEX)

fES

iNBM iNXPfEX  iNSPfES

1

Decay of XA

(1  fES  fEX)

fES

iNBM iNXPfEX  iNSPfES

Hydrolysis of SH1 Hydrolysis of SH2 Storage of PHA by XH Ammonification of SND COD (g COD) N (g N) Ionic charge (mole +)

(1  YSTO)

SP

SNH

1 1 1 1 –

SNOX

SH1

SH2

XH

XA

XSTO

XP

1

 Y1A  iNBM

Alk  iNBM 14

1

1 YA

2  iNBM 14  14Y A

 Y1H

 iNBM 14 fEX fEX

1 iNSH1 iNSH2

iNBM iNXP fEX iNSP fES 14 iNBM iNXP fEX iNSP fES 14

1 1 YSTO

1 –1 –

SND

iNBM

1 iNSP

1 1 14

1 14

1 1

4.57 1 1  14

1 iNSH1

1 iNSH2

1 iNBM

1 iNBM

1 –

1 iNXP

– – 1

T. Katipoglu-Yazan et al. / Bioresource Technology 114 (2012) 117–125 Table 3 Kinetic equations of adopted model structure. Process

Rate equation

Growth of XH

SO S l^ H K SSþS XH S K OH þSO S NH O l^ A K NH þSNH K OASþS XA O SO STO =X H l^ STO K STOXþX XH STO =X H K OH þSO

Growth of XA Growth on XSTO by XH Decay of XH Decay of XA Hydrolysis of SH1

bHXH bAXA

Hydrolysis of SH2

H2 =X H kh2 K XXSþS XH H2 =X H

Storage of XSTO by XH

SS kSTO SS þK XH S

Ammonification of SND

kaSNDXH

H1 =X H kh1 K XSþS XH H1 =X H

120

OUR (mg/L.h)

100 80 60 40 20 0 0

200

400

600

800

1000

Time (min) Experimental OUR data

Model simulations Total OUR Profile OUR - Heterotrophic activity, XH OUR - Autotrophic activity, XA OUR - Storage

Fig. 1. Model calibration of the OUR profile associated with heterotrophic activity.

121

was obtained with a heterotrophic yield, YH, value of 0.60 mg cell COD/mg COD and an endogenous decay coefficient, bH, of 0.1/day, which basically defined appropriate kinetics for microbial growth, hydrolysis and storage mechanism, adopting the default values for other stoichiometric coefficients defined in similar models (Beun et al., 2000; Koch et al., 2000; Krishna and van Loosdrecht, 1999; Lubello et al., 2009). It should be noted that the process kinetics defined in Table 4 are quite specific as the constituents of the peptone mixture are supplied as commercial grade chemicals with different characteristics in each supply. The main reason for the modeling of organic carbon utilization was the numerical assessment of hydrolysis kinetics, which would play a major role in the sequential release of the ammonia nitrogen controlling the extent of achievable nitrification. As mentioned above, slowly biodegradable COD that can only be utilized after hydrolysis, represented the major part – i.e. around 90% – of the total available COD in the peptone mixture, as in domestic sewage and most industrial wastewaters (Orhon et al., 1999). Therefore, the biodegradation characteristics of this fraction were better assessed by differentiating two hydrolysable components, SH1 and SH2. Accordingly, the maximum hydrolysis rate, kh1 associated with SH1 was defined as 6.3/day based on model calibration; the corresponding rate, kh2 for SH2 was a much lower value of 0.5/day. The same dual hydrolysis mechanism was also used to define substrate utilization for many wastewaters: Based on similar respirometric evaluation, the suggested kh1/kh2 values were 3.1/day and 1.2/day for domestic sewage and 1.1/day and 0.3/day for plain-settled tannery effluents; a kh1 range of 0.68–3.0/day and a kh2 range of 0.1– 1.0/day were calculated for different textile wastewaters (Orhon et al., 1998). These values provide a clear indication that the initial step of the ammonia release mechanism triggered by hydrolysis is bound to be quite variable depending on the characteristics of different wastewaters.

COD (mg/L)

3.4. Release mechanism of ammonia nitrogen 50 45 40 35 30 25 20 15 10 5 0 -200

0

200 400 600 800 1000 1200 1400 1600 Time (min) PHA components

PHA Data Seri 6 Model simulation - PHA

PHV data PHB data 3H2MV data

Fig. 2. Model calibration of the PHA profile.

sludge age (Cokgor et al., 2011). The area of the OUR curve above the endogenous respiration level, calculated as 191 mg O2/L, defined a heterotrophic yield coefficient, YH, of 0.60 mg cell COD/ mg COD, also accounting the small level of substrate COD diverted to storage. This yield value was adopted and justified by model calibration of the OUR profile. Model calibration yielded a close fit with the experimental OUR and PHA data for the set of stoichiometric and kinetic coefficients listed in Table 4; it also defined the relevant COD fractionation associated with the peptone mixture, which indeed included three components with different biodegradation characteristics, namely; a small readily biodegradable COD fraction, SS, of 43 mg/L (9.5%) together with a readily hydrolysable COD, SH1, of 254 mg/L (55.8%) and a slowly hydrolysable COD, SH2, of 158 mg/L (34.7%), in accordance with the shape of the OUR curve. Optimum calibration

The fate of nitrogen was investigated in batch reactors along the OUR experiments. As nitrification was inhibited, biodegradation of the peptone mixture also released ammonia nitrogen through sequential reactions that first hydrolysed organic nitrogen to simpler soluble organics and converted them to ammonia nitrogen through ammonification. The peptone mixture essentially contains two main components, peptone and meat extract, aside from urea. Release of ammonia was monitored on the peptone mixture (Set 2.1), as well as on the peptone (Set 2.2) and meat extract (Set 2.3) components of the substrate. The organic nitrogen content of the substrate mixture was bound within 412 mg/L of hydrolysable – slowly biodegradable COD. As there is no data to correctly distribute organic nitrogen between readily (SH1) and slowly (SH2) hydrolysable COD components, the stoichiometric model coefficients iNSH1 and iNSH2 were assumed to be the same and equal to 0.125 mg N/mg COD, based on the organic N/(SH1 + SH2) ratio, as outlined in Table 5. As shown in Fig. 3, the ammonia nitrogen profile for the peptone mixture started from 12 mg N/L originating from the hydrolysis of urea and rapidly increased to around 35–36 mg N/L within the first six hours (0.3 days) of the observation period, with a slight decline to 34 mg/L after 20 h (0.9 days). A similar trend of ammonia release was also monitored for the peptone and meat extract components of the mixture. Two significant observations should be underlined regarding the results obtained (i) There was a discrepancy of around 20 mg N/L between the initial and final nitrogen measurements in the reactor. Appropriate mass balance was first checked accounting for the nitrogen incorporated into biomass, NX , as part of microbial biosynthesis; NX could be calculated as 12 mg N/L based on process stoichiometry well defined in the

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Table 4 Results of model calibration for the biodegradation of peptone and nitrification (with/without nitrification inhibitor-Set 1 and Set 3). Model parameter & state

Unit

+



KS l^ A KNH bH bA KOH KOA kh1 KX kh2 KXX kSTO KSTO l^ STO ka YH YA YSTO iNBM iNSH1, iNSH2 iNXP iNSP fES fEX

1/day mg COD/L 1/day mg N/L 1/day 1/day mg O2/L mg O2/L 1/day g COD/g COD 1/day g COD/g COD 1/day mg COD/L 1/day m3/g COD day g COD/g COD g COD /g N g COD/g COD g N/g COD g N/g COD g N/g COD g N/g COD  

6.8 24 0.09 0.9 0.1 0.1 0.2 0.4 6.3 0.20 0.5 0.01 1.60 0.50 0.8 0.08 0.60 0.24 0.80 0.085 0.125 0.03 0.04 0.05 0.15

6.8 24 1 0.9 0.1 0.1 0.2 0.4 6.3 0.20 0.5 0.01 1.60 0.50 0.8 0.08 0.60 0.24 0.80 0.085 0.125 0.03 0.04 0.05 0.15

XH1 XA1 XSTO1 CS1 SS1 SH11 SH21 SNH1 SND1 SNOX1

mg mg mg mg mg mg mg mg mg mg

850 45 14 455 43 254 158 12 0 17

1100 60 14 502 48 280 174 14 0 21

l^ H

Maximum growth rate for XH Half saturation constant for growth of XH Maximum growth rate for XA Half saturation constant for growth of XA Endogenous decay rate for XH Endogenous decay rate for XA Half saturation coefficient of oxygen for XH Half saturation coefficient of oxygen for XA Maximum hydrolysis rate for SH1 Hydrolysis half saturation constant for SH1 Maximum hydrolysis rate for SH2 Hydrolysis half saturation constant for SH2 Maximum storage rate of XSTO by XH Half saturation constant for growth of XH on XSTO Maximum growth rate on XSTO for XH Ammonification rate of SND Yield coefficient for XH Yield coefficient for XA Storage yield of XSTO Fraction of nitrogen in biomass Fraction of nitrogen in SH1 and SH2 Fraction of nitrogen in particulate products from biomass Fraction of nitrogen in soluble products from biomass Fraction of biomass converted to SP Fraction of biomass converted to XP State variables Initial active heterotrophic biomass Initial active autotrophic biomass Initial amount of PHA Initial biodegradable peptone mixture COD Initial readily biodegradable peptone mixture COD Initial readily hydrolizable peptone mixture COD Initial slowly hydrolizable peptone mixture COD Initial ammonia nitrogen Initial soluble organic nitrogen Initial oxidized nitrogen

Table 5 COD and TKN components of peptone mixture fractions. Total COD Fractions mgCOD/ L

Slowly Biodegradable COD Fractions mgCOD/L

TKN Fractions mg N/L

Peptone Meat extract Urea Total (peptone mixture)

280 175 – 455

254 158 – 412

29 16 11 56

mg N/L

Constituents

40 35 30 25 20 15 10 5 0 -200

0

200 400 600 800 1000 1200 1400 Time (min)

Experimental data Ammonia nitrogen, SNH Soluble organic nitrogen, SND Oxidized nitrogen, NOX

Model simulations Ammonia nitrogen, SNH Soluble organic nitrogen, SND Oxidized nitrogen, NOX

Fig. 3. Model calibration of the ammonia release profile.

Nitrification inhibitor

COD/L COD/L COD/L COD/L COD/L COD/L COD/L N/L N/L N/L

literature (Artan et al., 2004). (ii) As nitrogen balance was still not established with NX additional analyses revealed partial nitrification despite inhibitor addition, which resulted in the accumulation of around 8 mg N/L of nitrate nitrogen; a nitrate pool of 17 mg N/L was initially imparted from the fill and draw reactor with biomass seeding and the pool increased to 25 mg N/L by the end of the observation period. Nitrite nitrogen formation was negligible. The fill end draw reactor, which supplied the microbial seed for batch experiments, obviously sustained a nitrifying environment since it was operated at a sludge age of 10 days. 3.5. Model evaluation of ammonia release – balance between hydrolysis and ammonification Accordingly, the calibration of the model with the ammonia release profile also included components and processes defining autotrophic activity as shown in Table 2 and 3, to account for the observed partial simultaneous nitrification. The calibration was started with the coefficients of dual hydrolysis kinetics associated with peptone biodegradation and yielded a good fit with the profile for an ammonification rate, ka of 0.08 m3/g COD day, which coincides with the default value suggested in activated sludge models and the range of 0.05–0.10 reported in the literature (Lu et al., 2001; Wang et al., 2007). The maximum growth rate for nitrifiers, l^ A , responsible for the observed limited nitrification was 0.09/day; as expected, this was a very low value that could possibly be sustained in the presence of the inhibitor; the corresponding half saturation coefficient for the growth of nitrifiers, KNH was found as 0.9 mg N/L. The calibration exercise involving the hydrolysis/ ammonification couple provided optimum interpretation of the

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experimental data showing only a slight accumulation of soluble organic nitrogen, SND of around 4–5 mg N/L, which was rapidly converted to ammonia nitrogen. The model simulation for the SND profile was also justified by experimental data indicating that hydrolysis only yielded SND as the first step in the ammonia release process. Modeling of the ammonia nitrogen profile also indicated a balance between the two sequential processes that seemed to proceed with the same rate so that the SND generated through hydrolysis was readily converted to SNH, justifying the general design assumption that all nitrogen, aside from the fraction incorporated into biomass would be potentially available for nitrification. Model simulations conducted with the stoichiometric and kinetic coefficients determined through calibration indeed indicated that either ammonification or hydrolysis would be rate limiting at lower rates: In fact, a lower ammonification rate ka of 0.008 m3/g COD day would lead to significant SND accumulation up to 20 mg N/L and it would give a totally different/lower SNH profile as shown in Fig. 4a. A similarly slower hydrolysis with kh1 = 1.0/day and kh2 = 0.1/day would significantly lower the available SNH level as a major nitrogen fraction would remain as soluble organics, SND (Fig. 4b). Conversely, a higher ammonification rate of 0.8 m3/g COD day would simply not affect the SNH profile, since it is no longer rate limiting (Fig. 5a), whereas a faster hydrolysis with kh1 = 8.0/day and kh2 = 6.0/day would distort the SNH curve with a much higher rate of increase, coupled with a narrow and higher SND peak (Fig. 5b). 3.6. Modeling simultaneous heterotrophic and autotrophic activities

40 35 30 25 20 15 10 5 0 -200

mg N/L

mg N/L

This section refers to batch experiment (Set 3) conducted without inhibitor addition, where organic carbon removal and nitrification are allowed to proceed together with no limitation.

The batch test was started with a slightly higher COD level of around 500 mg/L; the initial biomass level was adjusted to maintain the same food to microorganism ratio (S0/X0) of 0.55 mg COD/mg VSS, as in the previous test. The resulting OUR profile yielded, as expected, a higher peak and larger oxygen consumption due to nitrification simultaneously occurring with the biodegradation of the peptone mixture (Fig. 6a). Model simulation could separately identify OUR components related to organic carbon utilization and ammonia oxidation (Fig. 6b). Model calibration with the OUR profile verified and confirmed the same COD fractionation with 90% of total slowly biodegradable COD (SH1 and SH2) representing a total hydrolysable organic nitrogen potential, SND of around 50 mg N/L. The urea component of the peptone mixture also imparted an initial SNH concentration of 14 mg/L. There was again 21 mg N/L of nitrate nitrogen transport from the fill and draw reactor. The batch experiment secured full nitrification, where the level of nitrate nitrogen was increased to around 73 mg N/L with negligible nitrite generation and/or accumulation. It is well known that nitrification involves a sequence of two microbial mechanisms triggered with different microbial communities and disregarding nitrite generation is quite likely to distort nitrification kinetics. In fact, a similar study also provided experimental proof for the significant impact of nitrite accumulation on the assessment of readily biodegradable COD, correction factor for anoxic growth and denitrification rate (Sozen and Orhon, 1999). In this study, the possible impact of nitrite was taken into consideration and since its presence was almost null, nitrification was represented by means of an overall, single biomass (XA) process kinetics, as defined in the corresponding model structure in Tables 2 and 3. Model calibration of the OUR profile generated by means of simultaneous organic carbon removal and nitrification was

0

200 400 600 800 1000 1200 1400 Time (min) Model simulations

Experimental data Ammonia nitrogen, SNH Soluble organic nitrogen, SND Oxidized nitrogen, NOX

Model simulations

mg N/L

Experimental data

Model simulations

(a)

200 400 600 800 1000 1200 1400 Time (min)

Ammonia nitrogen, SNH Soluble organic nitrogen, SND Oxidized nitrogen, NOX

200 400 600 800 1000 1200 1400 Time (min)

Ammonia nitrogen, SNH Soluble organic nitrogen, SND Oxidized nitrogen, NOX

mg N/L

0

0

Experimental data

Ammonia nitrogen, SNH Soluble organic nitrogen, SND Oxidized nitrogen, NOX

(a) 40 35 30 25 20 15 10 5 0 -200

45 40 35 30 25 20 15 10 5 0 -200

Ammonia nitrogen, SNH Soluble organic nitrogen, SND Oxidized nitrogen, NOX

(b) Fig. 4. Simulated SNH and SND profiles for lower ammonification and hydrolysis rates: (a) ka = 0.008 m3/g COD day and (b) kh1 = 1.0/day and kh2 = 0.1/day.

45 40 35 30 25 20 15 10 5 0 -200

0

Ammonia nitrogen, SNH Soluble organic nitrogen, SND Oxidized nitrogen, NOX

200 400 600 800 1000 1200 1400 Time (min) Model simulations

Experimental data Ammonia nitrogen, SNH Soluble organic nitrogen, SND Oxidized nitrogen, NOX

Ammonia nitrogen, SNH Soluble organic nitrogen, SND Oxidized nitrogen, NOX

(b) Fig. 5. Simulated SNH and SND profiles for higher ammonification and hydrolysis rates: (a) ka = 0.8 m3/g COD day and (b) kh1 = 8.0/day and kh2 = 6.0/day.

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180 160

Experimental OUR data Total OUR Profile

OUR (mg/L.h)

140 120 100 80 60 40 20 0 0

200

400 600 Time (min)

800

1000

(a) 180 160

OUR (mg/L.h)

140 120 100 80 60 40 20 0 0

200

400 600 Time (min)

800

1000

Model simulations

Experimental OUR data

Total OUR profile OUR - Heterotrophic activity, XH OUR - Autotrophic activity, XA OUR - Storage

(b) Fig. 6. (a) Model calibration of the OUR profile with nitrification; (b) Simulated OUR profiles for organic carbon and nitrification.

started using model coefficients previously defined for the biodegradation of the peptone mixture and in particular, the same hydrolysis kinetics and the ammonification rate. This way, a good fit was obtained as shown in Fig. 6a and the kinetics of ammonia ^A release mechanism was verified with a maximum growth rate, l value of 1.0/day for nitrifiers and a corresponding half saturation ^ A value is quite coefficient, KNH of 0.9 mg N/L. The relatively high l justifiable in view of the fact that the experiment utilized a synthetic substrate environment with no ingredients with inhibitory effects. As a matter of fact, nitrification kinetics is quite sensitive ^ A is likely to asto different types of inhibition and therefore l sume different values depending on wastewater characteristics and experimental conditions: Henze et al. (1987) recommended ^ A value of 0.80/day for domestic sewage. Orhon et al. a default l ^ A level with (1994b) experimentally determined a much lower l an average value of around 0.50/day and a range of 0.35–0.62/ day for Istanbul wastewaters. Sarioglu et al. (2009a) reported a ^ A of 1.0/day for nitrification sustained at 20 °C in a memsimilar l brane bioreactor (MBR) treating domestic sewage with industrial wastewater interference. At later stages of the same MBR opera^ A was observed to drop down to 0.16/day due to significant tion l inhibition of industrial origin (Sarioglu et al., 2009b). The OUR curve obviously indicated a much higher oxygen consumption and OUR fractions corresponding to peptone biodegradation and nitrification have been accurately simulated by model simulation using calibrated model coefficients (Fig. 6b).

mental observations summarized above contributed to the understanding of this process and offered acceptable scientific explanations for major issues identified in the objectives of the study: (i) Observation of soluble organic nitrogen profile, SND, during ammonia release provided conclusive support for the existence of an ammonification process. Direct release of ammonia through hydrolysis, as stipulated in ASM2d, would not obviously generate SND. Under selected experimental conditions, the SND level remained quite low and it was rapidly depleted due to parallel ammonification; for wastewaters with different biodegradation characteristics however, SND accumulation may significantly impair the efficiency of nitrification, and therefore the corresponding nitrogen removal efficiency (Insel et al., 2009). (ii) Ammonification was not rate limiting for ammonia release: The results indicated a rate balance between the relative impact of hydrolysis and ammonification on the release mechanism. Indeed, a dual hydrolysis with kh1 = 6.3/ day and kh2 = 0.5/day and an ammonification rate, ka = 0.08 m3/g COD day yielded full oxidation of available ammonia to nitrate. It should be noted that the results obtained are quite specific for the experimental conditions and different rate limitations may apply for other wastewater characteristics. (iii) The validity of the results were confirmed through model evaluation of simultaneous heterotrophic and autotrophic activities, yielding the same process kinetics for hydrolysis and ammonification. (iv) The adopted experimental approach relied on full kinetic description of related biodegradation mechanisms; in this context, a kinetic fingerprint as defined in Table 4 described the nature of the selected synthetic substrate, allowing a basis for comparison with different wastewaters for practical applications. This way, it becomes possible to argue that the ammonification process would not be rate limiting for domestic sewage because it is characterized by much lower hydrolysis rates of around 2.0–3.0/day, as mentioned before (Okutman Tas et al., 2009; Orhon et al. 1998). Therefore, mass balance on total influent nitrogen generally used for process design remains justifiable as no SND accumulation would be expected to occur due to ammonification for domestic sewage. 4. Conclusions The study introduced an original experimental approach for identifying the relative impact of hydrolysis and ammonification on ammonia release. Conclusive experimental proof was provided for the existence of ammonification. A balance was established, where the ammonification rate, ka of 0.08 m3/g COD day was not rate limiting even for the high hydrolysis rates associated with the peptone mixture tested in the experiments. Model calibration with the OUR profile verified the kinetics of the ammonia release mechanism and full oxidation of available ammonia. Simultaneous heterotrophic and autotrophic activities confirmed the validity of results, yielding the same process kinetics for hydrolysis and ammonification. Acknowledgements This study was conducted as a part of the project ‘‘Evaluation of the Biodegradation Characteristics and Toxicity/Inhibition Effects of Xenobiotics on Nitrification Systems’’ and supported by the Joint Doctoral Degree Program of Turkish Academy of Sciences and the Scientific Research Fund of Istanbul Technical University. References

3.7. Overall evaluation of experimental results The ammonia release process is significant in assessing the extent of nitrification that can be achieved in the system. The experi-

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