Modeling kinetics of ammonium oxidation and nitrite oxidation under simultaneous inhibition by free ammonia and free nitrous acid

Process Biochemistry 44 (2009) 631–640

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Modeling kinetics of ammonium oxidation and nitrite oxidation under simultaneous inhibition by free ammonia and free nitrous acid Seongjun Park a,1,2, Wookeun Bae b,* a b

Center for Environmental Biotechnology, Biodesign Institute at Arizona State University, 1001 South McAllister Avenue, Tempe, AZ 85287-5701, USA Department of Civil & Environmental Engineering, Hanyang University, Sa 1-Dong, Ansan, Gyunggi-Do 425-791, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 November 2008 Received in revised form 12 January 2009 Accepted 3 February 2009

Inhibition of ammonium oxidation and nitrite oxidation by free ammonia (FA) and free nitrous acid (FNA) was studied using three different sludges. An uncompetitive inhibition model fit the experimental data well when the reactions were under FA inhibition, whereas a noncompetitive model fit well under FNA inhibition. The estimates of the inhibition constant (KI) of nitrite oxidation were 46 mM for FA and 1.7–6.8 mM for FNA, each of which was significantly smaller than that of ammonium oxidation, which were 290–1600 mM for FA and 12 mM for FNA. The much smaller values of KI for nitrite oxidation reflected the susceptibility of that reaction to inhibition by FA and FNA, which could lead to accumulation of nitrite during nitrification. A kinetic model for simultaneous inhibition by FA and FNA was derived. The model predicted that nitrite oxidation should be affected more seriously than ammonium oxidation by the simultaneous inhibition, which would accelerate the accumulation of nitrite in a strong nitrogenous wastewater treatment. It also indicated that a complete removal of ammonia could be achieved with high accumulation of nitrite in a sequencing batch reactor, which is impossible in a continuous-flow reactor. ß 2009 Elsevier Ltd. All rights reserved.

Keywords: Partial nitrification Nitrite oxidation Free ammonia Free nitrous acid Inhibition kinetics Modeling

1. Introduction Partial nitrification that oxidizes ammonium (NH4+) to nitrite (NO2) is useful for novel nitrogen-removal technologies. For instance, the anaerobic ammonium oxidation (ANAMMOX) process converts ammonium to nitrogen gas, using nitrite as an electron acceptor [1,2]. Another example is the shortcut biological nitrogen removal (SBNR) or the single reactor system for high activity ammonium removal over nitrite (SHARON) process, in which nitrite is accumulated and, then, denitrified in the presence of organic matters [3–7]. Nitrite denitrification is more economical than conventional nitrate denitrification, since it consumes 40% less carbon source and is faster (as much as 4.3 times) [5]. To ensure the practicality of partial nitrification, it is necessary to minimize the rate of nitrite oxidation, while maximizing that of ammonium oxidation. Previous studies identified factors that affected the rate of ammonium/nitrite oxidation, including substrate concentration, pH, dissolved oxygen, temperature, and

* Corresponding author. Tel.: +82 31 400 5148; fax: +82 31 417 8139. E-mail addresses: [email protected] (S. Park), [email protected] (W. Bae). 1 Tel.: +1 480 965 7495; fax: +1 480 727 0889. 2 At the time of work, he was a graduated student at Hanyang University, Republic of Korea. 1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2009.02.002

sludge retention time [3,8–10]. Among them, the effects of substrate concentration are strong and complicated due to its inhibitory effects when unionized. Anthonisen et al. [9] observed that both ammonium and nitrite oxidations are inhibited by unionized free ammonia (NH3, FA); inhibition of nitrite oxidation began at a concentration of 0.1–1.0 mg FA/L, while ammonium oxidation became inhibited at 10–150 mg FA/L, allowing selective inhibition of nitrite oxidation at a range of FA concentration of 1.0– 10 mg/L. Supporting this observation, Bae et al. [11] reported that nitrite accumulation occurred at an initial FA concentration of around 4.7 mg/L, giving a high NO2/NOX ratio (up to 77%) in a batch reactor. Chung et al. [3] accomplished a long-term accumulation of nitrite in a continuous-flow reactor by maintaining the FA concentration in the reactor around 20 mg/L. Chung et al. [4], however, found that a FA concentration of 5–10 mg/L was most efficient in inhibiting nitrite oxidation without slowing down the rate of ammonium oxidation. On the other hand, the works of Vadivelu et al. [12,13] indicated that nitrite oxidation could be selectively inhibited by free nitrous acid (FNA). The inhibition on Nitrosomonas growth started at approximately 0.10 mg HNO2-N/L and completely stopped at 0.40 mg HNO2-N/L, while the inhibition on Nitrobacter growth started at approximately 0.011 mg HNO2-N/ L and completely stopped at approximately 0.023 mg HNO2-N/L. It indicates that the Nitrobacter growth can be selectively inhibited in a range of FNA of approximately 0.011–0.10 mg HNO2-N/L.

S. Park, W. Bae / Process Biochemistry 44 (2009) 631–640

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To optimize the design and operation of a partial nitrification process, it is necessary to have appropriate kinetic expressions for both ammonium oxidation and nitrite oxidation under inhibition. Though mathematical models were suggested or used previously, many of them were not verified with proper experimental data (for instance, by performing experiments under changing pH, or simply borrowing kinetic models from previous studies), or contradict each other. Further, no model is available to describe the oxidation reactions under simultaneous inhibition by FA and FNA. This paper suggests and validates kinetic models for ammonium oxidation and nitrite oxidation focusing on inhibition by FA and FNA. The parameters such as KI and KS are estimated from experimental results with mixed cultures. A model of simultaneous inhibition by FA and FNA was also derived. The differences between the effects of the single and simultaneous inhibition on the nitrification reactions are illustrated through model simulations.

f(pH)S: q¼ ¼

qˆ f ðpHÞ S K FA þ f ðpHÞ Sð1 þ SFA =K I Þ ˆ qS ðK FA = f ðpHÞÞ þ Sð1 þ SFA =K I Þ

in which f(pH) =(17/14)(10pH/[exp(6334/(273 + 8C)) + 10pH]) (from Anthonisen et al. [9]). Eq. (3b) resembles Eq. (2), but the half-maxrate concentration is now a variable that changes with pH. If the S in Eq. (2) is the concentration of NH3 rather than that of TAN (NHX), the half-max-rate concentration, KFA/f(pH), will change according to pH as shown in Eq. (3b). In this study, the traditional form of substrate for S is adopted, as rewritten in Eq. (4), which forms an uncompetitive inhibition model: qAOB ¼

2. Modeling approach Four kinetic models for ammonium oxidation inhibited by (1) FA or (2) FNA and nitrite oxidation inhibited by (3) FA or (4) FNA were derived from enzyme inhibition models. The types of inhibition considered were competitive, uncompetitive (or, self), and noncompetitive. When the inhibitor concentration does not depend on the substrate concentration (e.g., cases 2 and 3), the ‘direct linear plot method’ [14] was adopted to determine the type of inhibition from experimental data. When the inhibitor concentration depends on the substrate concentration (cases 1 and 4) such that a direct linear plot method is inapplicable, then a most probable type of inhibition was selected to test its suitability against experimental data. 2.1. Inhibition of ammonium oxidation by FA A classical kinetic model of ammonium oxidation is the selfinhibition model by Andrews [15]: ˆ qS q¼ ¼ S þ K S þ S2 =K I K S þ Sð1 þ S=K I Þ

(1)

in which q and qˆ are a specific substrate utilization rate and its maximum value (mg S/mg VSS-day), respectively. S is the substrate concentration, KS is the half-max-rate concentration, and KI is the inhibition constant. Andrews [15] and several other researchers [16–18] regarded all the S in Eq. (1) to be the concentration of total ammonium nitrogen (TAN, NH4+–N + NH3–N), which is quite acceptable when the pH is invariant. Since ammonium oxidation is inhibited by FA [9,19,20] and the concentration of FA is pH dependent, it would be more reasonable to substitute the inhibition term S/KI in Eq. (1) with SFA/KI, in which SFA is the concentration of free ammonia (NH3): q¼

ˆ qS K S þ Sð1 þ SFA =K I Þ

qˆ AOB SNHX K S;AOB þ SNHX ð1 þ SFA =K I;FA;AOB Þ

(4)

in which qAOB and qˆ AOB are the specific and the maximum specific TAN utilization rates (mg N/mg VSS-d), respectively, SNHX is TAN, and KI,FA,AOB is the FA inhibition constant. The subscript ‘AOB’ indicates that the equation is for ammonium oxidizing bacteria (AOB). Although the equation for uncompetitive inhibition was adopted as the most probable kinetic model for ammonium oxidation, the competitive and noncompetitive models are also examined using the experimental data, from which the best fitting kinetic equation is derived. 2.2. Inhibition of ammonium oxidation by FNA To determine the type of an unknown inhibition and its kinetic parameters from experimental data with errors, the ‘direct linear plot method’ is very effective [14]. The method is based on a Monod-type substrate utilization kinetic model: q¼

ˆ qS

(3b)

qˆ eff S K S;eff þ S

(5)

in which qˆ eff and KS,eff are the effective qˆ and effective KS, respectively, which reflect the effects of inhibition. Rearranging Eq. (5) gives Eq. (6): qˆ eff ¼

q K þq S S;eff

(6)

Eq. (6) gives a straight line with a slope of q/S and an intercept of q at a given S–q pair (Fig. 1). Different S–q pairs will generate different straight lines, but all lines will pass through the coordinate ðK S;eff ; qˆ eff Þ because it is the common coordinate that

(2)

There are two thoughts on the concentration of substrate, S, in Eq. (2). Traditionally, S has been considered as the concentration of TAN. Recently, however, some researchers adopted free ammonia for S [13,20–22]: q¼

ˆ FA qS K FA þ SFA ð1 þ SFA =K I Þ

(3a)

in which KFA is the half-max-rate concentration when the substrate is SFA. Since SFA is a function of pH, it can be substituted with

Fig. 1. Direct linear plot method and types of inhibition. qˆ eff : maximum effective substrate utilization rate (mg S/mg VSS-day); KS,eff: effective half-max-rate concentration; Sinitial: initial substrate concentration in a batch experiment.

S. Park, W. Bae / Process Biochemistry 44 (2009) 631–640

the lines possess. Though the lines never intersect at one point due to errors, this method is known to give the most reliable estimate for KS,eff and qˆ eff among the graphical methods because it prevents the effect of the outliers by taking the median for the intersects [14,23]. When a reaction is inhibited, the intersecting coordinate will move a certain direction according to the type of inhibition as illustrated in Fig. 1. This is because a competitive inhibition ˆ and an increases KS, a noncompetitive inhibition decreases q, uncompetitive inhibition decreases both. Thus, the type of inhibition of ammonium oxidation by FNA is found from direct linear plot analysis of experimental data. 2.3. Inhibition of nitrite oxidation by FA The FA inhibition on nitrite oxidation seems to be the most critical factor for nitrite accumulation [4,9,11,17,24–26]. There are two different suggestions regarding the effect of FA: uncompetitive inhibition [26] and noncompetitive inhibition [19,24]. In this study, direct linear plot analysis, illustrated in Fig. 1, will be utilized to distinguish which inhibition occurs. 2.4. Inhibition of nitrite oxidation by FNA An earlier model that was used to explain the inhibition of nitrite oxidation is the noncompetitive inhibition model [27], as shown in Eq. (7) in which SI is the inhibitor concentration (mg N/L): q¼

ˆ qS ðK S þ SÞð1 þ ðSI =K I ÞÞ

(7)

Boon and Laudelout [27] suggested to use NO2 as S and HNO2 as SI. More recently, Gee et al. [26] substituted both S and SI with NO2–N, while others [13,20–22] substituted the both with HNO2. Since there was experimental evidence that the inhibitor is FNA (HNO2) rather than nitrite (NO2) [22], the latter, NO2, was no longer considered for SI in this study. If the direct substrate is FNA as suggested by Hellinga et al. [21] and Magri et al. [19], then KS in Eq. (7) will become, like in Eq. (3b), a function of pH, KS/f(pH), in which f(pH) = 47/14 (1/[exp(2300/(273 + 8C))  10pH + 1]) (from Anthonisen et al. [9]). In this study, the model by Boon and Laudelout [27], which is rewritten in Eq. (8), was chosen for the most probable in describing FNA inhibition of nitrite oxidation: qNOB ¼

qˆ NOB SNO2 ðK S;NOB þ SNO2 Þð1 þ SFNA =K I;FNA;NOB Þ

addition, the competitive and uncompetitive models will be also tested. 3. Materials and methods 3.1. Organisms Microorganisms were obtained from three different sources that had different feed concentrations and pH. A sludge from a conventional wastewater (TAN = 40 mg/L) treatment plant (WWTP) in Ansan, Korea was used directly or after acclimation to ammonium (100 mg N/L) in a sequencing batch reactor (SBR). The pH in the SBR varied from app. 8.0 right after the ammonium feeding to as low as app. 6.3 after the aerobic phase. Cells from a lab-scale SBNR reactor were also used [3]. The SBNR reactor was a continuous-flow, mixed-culture reactor that treated high concentrations of ammonium (1000 mg N/L) and COD (2000 mg/L) at a pH between 8.0 and 8.5. The volatile suspended solid (VSS) of the sludges were in the range of 1000–1600 mg/L. 3.2. Batch tests Kinetic experiments were performed in a batch reactor that consisted of a 500mL Erlenmeyer flask, a stirring device with temperature control, an air diffuser, a pH meter, and a DO meter. The feed solution contained (NH4)2SO4 and NaNO2 as the electron donor and/or inhibitor. The composition of the mineral medium was (in mg/mg N-L): K2HPO4 3.9, MgSO47H2O 1, FeSO47H2O 0.04, CaCl2 0.08, MnSO4H2O 0.1, NaHCO3 10.2, and KCl 0.14. A sufficient amount of oxygen was supplied and the temperature of the reactor was kept at around 27  2 8C, such that its effects upon kinetics could be precluded. The pH in the reactor was maintained near 7 or 8 as designed (within 0.2) by injecting a sodium hydroxide solution, as alkalinity is consumed with ammonium oxidation. To test the inhibition of ammonium oxidation by FA, ammonium solutions of various initial concentrations (5–450 mg-TAN/L) were prepared (Table 1). The reactor pH was adjusted to 7 or 8 in order to vary the concentration of FA at a given concentration of TAN. Cells obtained from all of the sources were used. To test the inhibition of ammonium oxidation by FNA, various initial concentrations of TAN (20–250 mg N/L) and FNA (0.0, 0.2 and 0.4 mg/L) were used. The pH in the reactor was maintained at 7, such that the effects of FA inhibition could be insignificant. The cells taken from the SBR were used. Inhibition of nitrite oxidation by FA or FNA was also tested in a similar manner, as summarized in Table 1. To block oxidation of FA by AOB, 5 mg/L of ATU (allylthiourea) was added. 3.3. Samplings and analysis Samples were taken from the batch reactors and immediately filtered through a glass–fiber filter (Whatman, GF/C; pore diameter app. 1 mm). The TAN, nitrite and nitrate concentrations were determined by the Phenate Method 4500-NH3 F, and Methods 4500-NO2 B and 4500-NO3 B, respectively, in Standard Methods [28]. The concentrations of FA and FNA were calculated using Eqs. (9a) and (9b), respectively, which are modified from Anthonisen et al. [9]:

FA ðmgNH3 =LÞ ¼

17 TAN  10pH 14 ½expð6334=ð273 þ CÞÞ þ 10pH 

FNA ðmgHNO2 =LÞ ¼

(8)

in which SNO2 is the total nitrite nitrogen concentration (TNN, NO2–N + HNO2–N) and SFNA is the concentration of free nitrous acid (HNO2). The appropriateness of Eq. (8) will be evaluated with experimental data obtained at two different pH conditions. In

633

(9a)

47 TNN 14 ½expð2300=ð273 þ CÞÞ  10pH  þ 1

(9b)

The VSS was measured following the Standard Methods. The temperature/pH (Orion, 720A) and DO (Orion, 850) were detected using electrodes. The initial degradation rate of substrate per unit biomass in each batch test was considered as the q for that initial concentration of substrate (So), generating a set of So–q pairs under the given inhibitory conditions. The kinetic parameters were estimated from each So–q data set by nonlinear least-square regression unless specified otherwise.

Table 1 Batch tests for inhibition upon AOB and NOB by FA and FNA. Reaction to test

Ammonium oxidation

Initial concentrations (mg/L) of substrate/inhibitor pH Source of cells Temperature DO

NHX–N: 5–450; FNA: 0 7 and 8 WWTP, SBR and SBNR 27 8C Above 5 mg/L

Nitrite oxidation NHX–N: 20–250; FNA: 0, 0.2, 0.4 7 SBR

Note: 5 mg/L of allylthiourea (ATU) was used to block oxidation of ammonium (and FA).

NO2–N: 10–500; FA: 0, 2, 4, 5 mg/L ATU (see note) 8 SBR

NO2–N: 10–250; FA: 0 7 and 8 WWTP, SBR and SBNR

634

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4. Results and discussion 4.1. Inhibition of ammonium oxidation by FA The initial degradation rate of ammonium was linear in all batch tests with the three different sludges (data not shown). The initial slope (DS/DtimeX) is equivalent to the specific substrate utilization rate (q) at the given initial substrate concentration (So). ˆ was estimated The maximum specific substrate utilization rate, q, by nonlinear least-square regression of a set of So and q pairs obtained from each sludge based on a proposed equation. The q was normalized with qˆ for better comparison, of which results are shown in Fig. 2. The solid lines are the best-fit curves using Eq. (4). It is obvious from Fig. 2 that the trend of the curves for pH 8 was different from that for pH 7. The self-inhibition was clearly apparent at pH 8, giving decreasing q values at higher substrate concentrations (e.g., above 100 mg N/L). The difference between

the two curves at the two pH values is probably due to inhibition by free ammonia rather than total ammonia. At pH 8 and 27 8C, approximately 7% of TAN is present in the form of FA, which is almost 10-times higher than that at pH 7. If the inhibitor were total ammonia, the curves for the two pH conditions should coincide. The observation in Fig. 2 is a confirmation that ammonium oxidation is inhibited by FA rather than total ammonia [9,19]. Fig. 2 also indicates that the inhibition is not competitive, because competitive inhibition by SFA does not reduce q at a high S, as shown in Eq. (10) in which f(pH) is constant at given pH and temperature: q¼ ¼

ˆ ˆ qS qS ¼ K S ð1 þ SFA =K I Þ þ S K S ð1 þ f ðpHÞS=K I Þ þ S ˆ qS K S þ Sð1 þ f ðpHÞ=K I Þ

(10)

The results of nonlinear least-square regression with the data in Fig. 2 are given in Table 2. The KS values are quite similar among the experimental conditions. From Eq. (3b), the KS value should decrease by 9.4 times with the pH rise from 7 to 8, if the substrate were FA. There is no such decrease in Table 2 although there are slight differences in KS among the test conditions, which are most probably caused by experimental errors. Therefore, it is concluded that the substrate for the kinetic equation of ammonium oxidation, given in Eq. (4) should be total ammonia. The excellent fit of the solid lines to the data supports the appropriateness of the model. The inhibition constant (KI,FA) should be invariant with pH. Table 2 shows relatively similar KI,FA values in each sludge. A significant increase in the KI,FA value with the SBR or SBNR sludge compared to the WWTP sludge might be a result of acclimation of the cells to high concentrations of FA [3,29]. The qˆ value is subject to the relative concentration of AOB in the sludge, which varied with substrate conditions and was unknown. Therefore, the values of qˆ in Table 2 are pseudo maximum specific substrate utilization rates, which have limited meaning. Nevertheless, the observation that qˆ increases with pH is in agreement with previous reports, which show the optimal pH for AOB around 7.5– 8.5 [11,30,31]. Particularly, qˆ almost quadrupled with the pH increase in the SBNR sludge, probably due to its growth environment; the SBNR reactor was operated at a pH around 8– 8.5, while the WWTP was operated near neutral pH and the SBR between 8 and 6.3. A noncompetitive model for FA inhibition was also evaluated by estimating the kinetic parameters. The residual sum of the squares P ðRSS; ðqdata  qmodel Þ2 Þ were almost same for the two models with the SBR or SBNR sludge. However, with the WWTP sludge, the RSS doubled indicating that the noncompetitive model was slightly inferior in fitting the data. Therefore, it is concluded that the classical self-inhibition model (uncompetitive) is appropriate to describe the kinetics of ammonia oxidation only if total ammonia is the substrate and free ammonia is the inhibitor. 4.2. Inhibition of ammonium oxidation by FNA

Fig. 2. Experimental results of ammonium oxidation at pH 7 and 8 with WWTP sludge (a), SBR sludge (b), and SBNR sludge (c). The solid lines are the best-fit curves using Eq. (4) (uncompetitive inhibition).

Table 3 was obtained by the direct linear plot method to determine the type of FNA inhibition on AOB. The effective qˆ and effective KS, respectively, represent the ordinate and abscissa of the median for intersects (refer to Fig. 1). The effective qˆ value decreased by 40% with an increase in the FNA concentration from 0.0 to 0.4 mg/L, while the effective KS value varied only slightly and non-systematically. The coefficient of variation for the KS values was 5.5%, which might be considered insignificant. Therefore, the ˆ with an increase in FNA downward movement of the ‘‘effective q’’ inhibition indicated that the inhibition of nitrite oxidation by FNA

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Table 2 Estimated kinetic constants for ammonium oxidation under uncompetitive FA inhibition. Source of sludge

WWTP

pH qˆ (mg NHX-N/mg VSSd) KS (mg NHX-N/L) KI (mg FA/L)

7 1.1  0.15 51.3  11.27 5.2  1.48

SBR 8 2.1  0.39 37.3  9.94 5.0  1.49

7 0.9  0.108 37.2  8.34 22.3  36.12

is noncompetitive, as given in Eq. (11): qAOB ¼

qˆ AOB SNHX ðK S;AOB þ SNHX Þð1 þ ðSFNA =K I;FNA;AOB ÞÞ

(11)

The FNA inhibition constant (KI,FNA) could be calculated using Eq. (12): qˆ eff ¼

qˆ 1 þ ðSFNA =K I;FNA Þ

or

1 1 1 ¼ SFNA þ ˆ I;FNA qˆ eff qK qˆ

(12)

A linear regression of the variables (SFNA and 1=qˆ eff from Table 3) estimates KI,FNA to be 0.57 mg FNA/L (R2 = 0.97). 4.3. Inhibition of nitrite oxidation by FA Table 4 was obtained from direct linear plot [32] analyses to determine the type of FA inhibition on NOB. Both qˆ e f f and KS,eff decreased significantly as FA increased from 0 to 4.0 mg FA/L, indicating that the inhibition was uncompetitive. A kinetic model for uncompetitive inhibition is expressed as Eq. (13): qNOB ¼

qˆ NOB SNO2 K S;NOB þ SNO2 ð1 þ ðSFA =K I;FA;NOB ÞÞ

(13)

A decrease in KS,eff attenuates the loss in the maximum specific ˆ However, under nitrite accumulating conditions reaction rate, q. (i.e. when SNO2 is high), the influence of KS,eff on the reaction rate is ˆ insignificant allowing a net decrease in q. The FA inhibition constant (KI,FA) could be calculated by definition of the effective parameter in uncompetitive inhibition, ˆ such as qˆ eff ¼ q=ð1 þ SFA =K I;FA Þ and KS,eff = KS/(1 + SFA/KI,FA). These equations can be rewritten in linear forms: 1 1 1 ¼ SFA þ ˆ I;FA qˆ eff qK qˆ

(14a)

1 1 1 ¼ SFA þ K S;eff K S K I;FA KS

(14b)

Table 3 Estimated kinetic constants of ammonium oxidation under FNA inhibition with the SBR sludge. FNA (mg FNA/L) pH qˆ eff (mg NHX-N/mg VSSd) KS,eff (mg NHX-N/L) KI (mg FNA/L) a

0.0 7 0.96 57.1 0.57a

0.2 7 0.77 51.2 0.57a

0.4 7 0.57 53.7 0.57a

KI value was estimated from Eq. (12).

Table 4 Estimated kinetic constants of nitrite oxidation under FA inhibition with the SBR sludge. Inhibition constant KI (mg FA/L) FA (mg FA/L) pH qˆ eff (mg NO2-N/mg VSSd) KS,eff (mg NO2-N/L) a

0.0 8 1.73 11.7

2.0 8 0.56 1.8

4.0 8 0.31 1.3

KI value was estimated from Eqs. (14a) and (14b).

8 0.81a 0.75a

SBNR 8 2.4  0.68 23.7  12.76 15.1  10.82

7 0.9  0.03 24.5  2.74 27.3  2.82

8 3.4  0.14 32.7  3.68 22.0  4.85

The KI,FA was obtained by linear regression of the SFA, 1=qˆ eff data set (0.81 mg FA/L, R2 = 1.0), or the SFA, 1/KS,eff data set (0.75 mg FA/L, R2 = 0.96) from Table 4. The similar KI,FA values supports that the model is proper. 4.4. Inhibition of nitrite oxidation by FNA The normalized specific oxidation rates of nitrite at various experimental conditions are shown in Fig. 3. The solid lines are the best-fit curves by Eq. (8). When the pH in the reactor was lowered from 8 to 7, the FNA concentration went up 10 times at a given nitrite concentration, and the q=qˆ ratio decreased significantly at high nitrite concentrations. This confirms that FNA, rather than nitrite, causes inhibition [22,27,33]; if nitrite were the inhibitor, the two curves should coincide. Fig. 3 also indicates that the inhibition was not competitive, because competitive inhibition does not reduce q at a high S, as seen in Eq. (10). The kinetic constants in Eq. (8) were estimated by nonlinear least-square regression of the data in Fig. 3, and shown in Table 5. The KS values are quite close to each other, although it is somewhat higher with the SBNR sludge that was exposed to continuously high concentrations of nitrite (above 200 mg N/L). Particularly, the KS value for each sludge is almost invariant with the pH change, which indicates that the substrate was nitrite. If the substrate were FNA, the KS value obtained at pH 8 should be one-tenth of that at pH 7. The excellent fitting of the solid lines to data in Fig. 3 supports that the model in Eq. (8) is proper. The inhibition constant at pH 7 was 0.08 mg FNA/L for WWTP sludge, 0.19 mg FNA/L for SBR sludge, and 0.32 mg FNA/L for SBNR sludge. The difference is most probably due to the varying degree of acclimation: the sludge from the WWTP was grown in a FNA-free environment, while the SBR and SBNR sludges were acclimated to FNA as high as 0.008 mg/L and above 0.015 mg/L, respectively. At pH 8, a similar trend is unapparent probably because the estimates were less accurate due to the very weak inhibition at that pH. The qˆ estimates varied significantly among the sludges, reflecting different nitrite-oxidizing activity of the sludges. The low qˆ value with the SBNR sludge indicated that NOB were scarce in the reactor due to severe inhibition. The qˆ value increased in the SBR and SBNR sludges when the pH increased from 7 to 8 because the cells were well acclimated to the higher pH, which is in accord with previous observations by Bae et al. [11] and Park et al. [30]. Again, the possibility of uncompetitive inhibition by FNA was tested. The RSS values of the model predictions were virtually the same for the two models (noncompetitive and uncompetitive) with the SBR, SBNR, and WWTP sludge; even the parameter estimates by the two models differed only slightly (data not shown). This observation was in contrast to the results with a competitive model, in which the KS and KI estimates were always negative values (data not shown). These results indicate that nitrite oxidation by NOB could be expressed either by a noncompetitive model or uncompetitive model. In this study, the noncompetitive model originally suggested by Boon and Laudelout [27] is selected, in which the substrate is total nitrite and the inhibitor is free nitrous acid.

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the substrate for ammonia monooxygenase (AMO) is unionized ammonia [35,36], the cells appear to possess a mechanism to utilize ammonium, which is the dominant form of ammonia in most environments. In addition, the claim that the substrate for NOB is FNA [12,20–22,34] was not supported in this study either. Boon and Laudelout [27] found that the hydroxyl ion (OH) competitively inhibits the oxidation of nitrite by nitrite oxidase, which indicates that the substrate for nitrite oxidase is in an anionic form. The observation in this study was in agreement with the finding of Boon and Laudelout [27]. Although our results showed that TAN and TNN are the actual substrates, further studies may be needed with various cultures, pure and mixed, to thoroughly understand why contradictory results have been observed in other studies [19–21]. It was confirmed that FA and FNA were the inhibitors of nitrification reactions from Figs. 2 and 3, respectively. Though some researchers reported that free hydroxylamine (NH2OH) is responsible for the inhibition [37,38], our experiment performed in the presence of FA and in the absence of free hydroxylamine, inhibited nitrite oxidation (see Tables 1 and 4). The type of inhibition by FA was uncompetitive and that by FNA was close to noncompetitive for both AOB and NOB. Having the same type of inhibition for two groups of nitrifiers that possess different key catabolic enzymes was rather unexpected. One explanation is that inhibition in a bacterial cell could take place over an array of catabolic and anabolic reactions, not just on a single enzyme. Recent reports by Vadivelu et al. [12,22] with Nitrosomonas and Nitrobacter cultures, in which inhibition was exhibited both on growth and on substrate oxidation, supports this explanation in part. The poor specificity of the inhibitors might be the reason why FNA inhibition of NOB could be expressed by either noncompetitive or uncompetitive inhibition kinetics. The estimates of KS and KI are shown in the last two columns of Table 6. The qˆ values are not presented since it depends on the fraction of AOB or NOB in the sludge, and the fraction varies with substrate conditions in a mixed culture. Although the parameters are estimated from a limited number (mostly four) of data points, the estimates are acceptable because they give similar values from a number of measurements. For example, KS for a given sludge is invariable. In Tables 2 and 5, the KS estimates for each sludge sample are quite similar between the two pH conditions. The KS values obtained from our experiments for AOB (1.7–4.1 mM) and NOB (0.6–2.0 mM) are comparable to those in the literature as shown in Table 7: 0.79–5.3 mM for AOB and 0.11–1.6 mM for NOB. The similarity of the values is surprising considering the differences in the adopted model, the cells, and culture conditions. The estimates of KI of NOB are much smaller than those of AOB for each inhibitor. This confirms that NOB are more susceptible to inhibition by FA or FNA [3,9,39,40]. The KI estimates for AOB in this study (290–1600 mM FA and 12 mM FNA) lie near the lower range of those in the literature, 527–4500 mM FA and 15–146 mM FNA. The same is true for NOB: 46 mM FA and 1.7–6.8 mM FNA in this study, while 1400 mM FA and 4–165 mM FNA in the literature. The significantly higher KI values reported indicate that the cells could acclimate to FA and FNA to different degrees. Also, the estimates of KI in this study are comparable to the threshold concentrations

Fig. 3. Experimental results of nitrite oxidation at pH 7 and 8 with WWTP sludge (a), SBR sludge (b), and SBNR sludge (c). The solid lines are the best-fit curves using Eq. (8) (noncompetitive inhibition).

4.5. Summary of the results The key results obtained from the modeling and experimental analyses are summarized in Table 6. It shows the species of substrate and inhibitor, kinetic model, and coefficient values. Using FA as the substrate for AOB in the kinetic model [12,20–22,34] is not supported by the experimental results of this study. Although Table 5 Estimated kinetic constants for nitrite oxidation under noncompetitive FNA inhibition. Source of sludge

WWTP

pH qˆ (mg NO2-N/mg VSSd) KS (mg NO2-N/L) KI (mg FNA/L)

7 0.30  0.08 7.8  4.88 0.09  0.04

SBR 8 0.20  0.04 10.2  2.19 0.11  0.07

7 1.27  0.01 11.9  0.28 0.19  0.01

SBNR 8 1.76  0.03 11.6  0.57 0.97  1.22

7 0.07  0.01 23.3  8.73 0.32  0.01

8 0.23  0.03 28.3  9.98 0.10  0.08

S. Park, W. Bae / Process Biochemistry 44 (2009) 631–640

637

Table 6 Summary of the experimental results and analyses (temperature = 278C).

KS (mM)

KI (mM)

qˆ AOB SNHX K S;AOB þ SNHX ð1 þ ðSFA =K I;FA;AOB ÞÞ

1.7–3.7

290–1600

qAOB ¼

qˆ AOB SNHX ðK S;AOB þ SNHX Þð1 þ ðSFNA =K I;FNA;AOB ÞÞ

4.1a

12a

Uncompetitive

qNOB ¼

qˆ NOB SNO2 K S;NOB þ SNO2 ð1 þ ðSFA =K I;FA;NOB ÞÞ

0.84a

46a

Noncompetitive

qNOB ¼

qˆ NOB SNO2 ðK S;NOB þ SNO2 Þð1 þ ðSFNA =K I;FNA;NOB ÞÞ

0.6–2.0

1.7–6.8

Organism

Substrate

Inhibitor

Type of inhibition

Kinetic model

AOB

TAN

FA

Uncompetitive

qAOB ¼

(NH4+ + NH3)

FNA

Noncompetitive

TNN

FA

(NO2 + HNO2)

FNA

NOB

a

The coefficient values obtained with the SBR sludge; all the other values are from three different sources of sludge.

Table 7 Kinetic parameter values in the literature. Substrate

Inhibitor

Inhibition Model

Kinetic parametera

References

KS (mM) (as TAN or TNN) AOBb FA

KI (mM) (as FA or FNA)

– FNA

– Noncompetitive

3.3

– 15

FA

FA FNA

Self Noncompetitive

4.5

3300 17

FA

FA FNA

Noncompetitive Noncompetitive

5.3

– 146

TAN

TAN –

Self –

0.79

4500 –

Carvallo et al. [16]

TAN

TAN –

Self –

0.90

527 –

Carrera et al. [33]

– FNA

– Self

0.15

– 19

FNA

FA FNA

Noncompetitive Self

0.48

1400 165

TNN

– TNN

– Self

0.29

– 25

TNN

– TNN

– Self

0.11

– 4

TNN

– FNA

– Noncompetitive

1.6

NOBb FNA

a b

– 13.5

Hellinga et al. [21]

Magri et al. [19]

Van Hulle et al. [20]

Hellinga et al. [21]

Magri et al. [19]

Carvallo et al. [16]

Carrera et al. [33]

Boon and Laudelout [27]

The reported values were converted to molar concentrations of TAN or TNN for KS, and FA or FNA for KI. Mixed cultures, except for Boon and Laudelout, who used Nitrobacter winogradskyi.

causing inhibition in the literature: for AOB, 590 mM FA [9], 7 mM FNA [12]; for NOB, 6 mM FA [9], and 0.8 mM FNA [22].

derived based on enzyme kinetics (derivation is given in Appendix A):

4.6. Simultaneous inhibition of ammonium and nitrite oxidations by FA and FNA

qAOB ¼

In a partial nitrification process, nitrite is accumulated during ammonium oxidation under high concentration of FA. Therefore, simultaneous inhibition by both FA and FNA is possible. In enzymatic reactions, an uncompetitive inhibitor (e.g., FA in nitrification) binds the enzyme–substrate complex, whereas a noncompetitive inhibitor (e.g., FNA) binds both the enzyme and enzyme–substrate complex. If we extend the analogy between the Monod equation for cells and the Michaelis–Menten equation for enzymes, kinetic equations for AOB and NOB which are under simultaneous inhibition by FA and FNA, can be

qˆ AOB SNHX ðK S;AOB ð1 þ ðSFNA =K I;FNA;AOB ÞÞ þ SNHX ð1 þ ðSFNA =K I;FNA;AOB Þ þ ðSFA =K I;FA;AOB ÞÞ (15a)

qNOB ¼

qˆ NOB SNO2 ðK S;NOB ð1 þ ðSFNA =K I;FNA;NOB ÞÞ þ SNO2 ð1 þ ðSFNA =K I;FNA;NOB Þ þ ðSFA =K I;FA;NOB ÞÞ (15b)

The effect of FA inhibition becomes insignificant in an acidic environment (e.g., pH 6) unless the NHX concentration is extremely

638

S. Park, W. Bae / Process Biochemistry 44 (2009) 631–640

Fig. 4. Simulation of Eq. (15a) (AOB) at pH 7, assuming KI,FA = 10 mg/L (588 mM) and KI,FNA = 0.5 mg/L (10.6 mM) (all concentrations in the in-set are in mg/L; numbers in parentheses on abscissa are the FA concentrations). (a) Relative change of the maximum specific substrate utilization rate by FA and FNA inhibition; (b) relative change of the half-max-rate concentration by FA and FNA inhibition.

high (e.g., over 10,000 mg N/L for AOB or over 2000 for NOB), and Eqs. (15a) and (15b) can be simplified to noncompetitive inhibition by FNA. Similarly, the influence of FNA inhibition becomes negligible at an alkaline pH (e.g., pH 8) and the equations can be simplified to an uncompetitive form. At near neutral pH and high concentrations of ammonium and nitrite, however, both FA and FNA affect the reaction rate significantly, as illustrated with Figs. 4 and 5. The inhibition constants used in the figures are similar to or within the range of those found in this study. In Fig. 4a, the qˆ eff of AOB slightly decreased with an increase in the concentration of substrate (and FA) or FNA. On the other hand, the KS,eff was mainly affected by the increase of FA. In Fig. 5 (for NOB), the effective qˆ value decreased significantly as the concentration of ammonium (thus, FA) or nitrite (thus, FNA) increased. The rate-reduction was compounded significantly when the reaction was inhibited by the both inhibitors simultaneously; the ratio qˆ eff =qˆ was 0.31 when it was inhibited by FNA (0.222 mg/L) alone, but became 0.17 when inhibited simultaneously by FNA (0.222 mg/L) and FA (2 mg/L). The effective KS value also changed due to FA and FNA inhibitions, but in opposite directions (Fig. 5b). Overall, the direction of the change of KS,eff was to compensate for the reduction of the reaction rate to some degree. In a batch reaction, FA will be the primary inhibitor in the beginning of the reaction, but FNA will be predominant later, maintaining the inhibition power throughout the reaction. Fig. 6 was drawn with the data from the sequencing batch reactor that was operated to obtain the SBR sludge. At the initial stage when the concentration of FA was high, the qˆ eff =qˆ ratio for NOB was less than

Fig. 5. Simulation of Eq. (15b) (NOB) at pH 7, assuming KI,FA = 0.75 mg/L (44 mM) and KI,FNA = 0.1 mg/L (2.1 mM) (all concentrations in the in-set are in mg/L; numbers in parentheses on abscissa are the FNA concentrations). (a) Relative change of the maximum specific substrate utilization rate by FA and FNA inhibition; (b) relative change of the half-max-rate concentration by FA and FNA inhibition.

0.1. It increased gradually with time as FA removed, and reached about 0.6 by 100 min. Then, the ratio went down slightly to reach a plateau value around 0.45 by an increase in FNA. A decreasing pH (from 8.3 to 6.8) with nitrification helped to increase the FNA fraction in the solution. The nitrite accumulation ratio, NO2/(NO2 + NO3), was 95% even at the end of the reaction when no ammonium or FA was present. To a much lesser degree, dual inhibition also affected

Fig. 6. Inhibition effects by FA and FNA in a batch reaction.

S. Park, W. Bae / Process Biochemistry 44 (2009) 631–640

AOB. The observation in Fig. 6 indicates that a complete removal of ammonia with a high accumulation of nitrite is possible in a SBR, which is not possible in a continuous-flow reactor.

In which Kn1 = Kn following the definition of noncompetitive inhibition: ES þ Iu $ ESIu : K u ¼

5. Conclusions The inhibitory effects by FA and FNA on ammonium oxidation and nitrite oxidation were studied using three different sludges. The key conclusions are:

½E ¼

Appendix A. Enzyme reaction kinetics under simultaneous inhibition by FA (uncompetitive) and FNA (noncompetitive)

½E½In  K S ½ES ½In  ¼ from Eqs: ðA3Þ and ðA6Þ Kn ½S K n

(A5)

(A6)

(A7)

½ESIn  ¼

½ES½In  from Eq: ðA4Þ Kn

(A8)

½ESIu  ¼

½ES½Iu  from Eq: ðA5Þ Ku

(A9)

Rewriting Eq. (A1) with Eqs. (A6)–(A9), ½Et ¼

K S ½ES K S ½ES ½In  ½ES½In  ½ES½Iu  þ ½ES þ þ þ ½S ½S K n Kn Ku

(A10)

Rearranging for [ES], ½Et ðK S =½SÞ þ 1 þ ðK S =½SÞð½In =K n Þ þ ð½In =K n Þ þ ð½Iu =K u Þ ½Et ½S ¼ K S ð1 þ ð½In =K n ÞÞ þ Sð1 þ ð½In =K n Þ þ ð½Iu =K u ÞÞ

½ES ¼

Acknowledgements This work was supported by grant No. R01-2001-00437 from the Korea Science & Engineering Foundation (KOSEF). We thank Mr. Gihyun Bae and Ms. Eunyoung Hong for their experimental helps, and Dr. Steven Van Ginkel for the invaluable comments on the manuscript.

½ES½Iu  ½ESIu 

K S ½ES from Eq: ðA2Þ ½S

½EIn  ¼  An uncompetitive inhibition model fits the data well for FA inhibition, while a noncompetitive model fits for FNA inhibition.  The estimates of the inhibition constant (KI) for nitrite oxidation were 46 mM for FA and 1.7–6.8 mM for FNA, which were significantly smaller than those of ammonium oxidation, 290– 1600 mM for FA and 12 mM for FNA. The much smaller values of KI reflect the susceptibility of the nitrite oxidizing bacteria to inhibition by FA and FNA.  A model of simultaneous inhibition by FA and FNA predicts that the effective maximum specific substrate utilization rate ðqˆ eff Þ of the nitrite oxidizing bacteria will be reduced significantly by compounding inhibition by the unionized substrate and product. It also indicates that a complete removal of ammonia could be achieved with high accumulation of nitrite in a SBR, which is impossible in a continuous-flow reactor.

639

(A11)

Since d[S]/dt = q = k2[ES], q¼

k2 ½Et ½S K S ð1 þ ð½In =K n ÞÞ þ Sð1 þ ð½In =K n Þ þ ð½Iu =K u ÞÞ

(A12)

By definition, k2 ½Et ¼ qˆ q¼

ˆ q½S K S ð1 þ ð½In =K n ÞÞ þ Sð1 þ ð½In =K n Þ þ ð½Iu =K u ÞÞ

(A13)

References

E = enzyme concentration per unit biomass In = HNO2 Iu = NH3 Kn = noncompetitive inhibition constant Ku = uncompeitive inhibition constant ½Et ¼ ½E þ ½ES þ ½EIn  þ ½ESIn  þ ½ESIu  E þ S $ ES : K S ¼

½E½S ½ES

E þ In $ EIn : K n ¼

½E½In  ½EIn 

ES þ In $ ESIn : K n1 ¼

½ES½In  ½ESIn 

(A1) (A2)

(A3) (A4)

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