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Effects of temperature on tertiary nitrication in moving-bed biolm reactors

Roberta Salvetti, Arianna Azzellino, Roberto Canziani, Luca Bonomo
D.I.I.A.R. Environmental Engineering Department, Politecnico di Milano, Technical University of Milan, P.za Leonardo da Vinci, 32-20133 Milano, Italy

art i cle info

Article history: Received 31 August 2005 Received in revised form 4 May 2006 Accepted 9 May 2006 Available online 13 July 2006 Keywords: Nitrication Temperature Pure-oxygen moving-bed biolm reactor (PO-MBBR) Ammonia limitation Oxygen limitation Multivariate techniques

A B S T R A C T

The effect of wastewater temperature on the rate of nitrication was studied in two pureoxygen moving-bed biolm reactors, fed on secondary efuent from a municipal wastewater treatment plant. The rst Reactor (R1) was operated under ammonia-limiting conditions, while the second Reactor (R2) was operated under oxygen-limiting conditions. Quite surprisingly, the former showed a negligible inuence of thermal changes on nitrication rates, while the latter showed a much higher dependence. In this paper, a temperature coefcient y has been dened as the actual intrinsic biological temperature coefcient, similar to the corresponding coefcient that is usually adopted for the design of activated-sludge processes. In addition, an apparent coefcient ya has been quantied independently, which was calculated according to the actual values of nitrication rates at different temperatures. The actual biological temperature coefcient y, ranged between 1.086 and 1.109 (average value 1.098) under ammonia-limiting conditions, while under oxygen-limiting conditions was in the range 1.0231.081 (average value 1.058). The apparent value ya was near to unity (i.e. no temperature effect) under ammonia-limiting conditions, while only under oxygen-limiting conditions and at constant dissolved oxygen concentration ya coincided with y. An explanation was given that, under oxygen-limiting conditions, the specic biomass activity (i.e. the ratio of nitrication rate to biomass concentration) was strongly inuenced by the combined effects of oxygen penetration through the biolm and efuent temperature. & 2006 Elsevier Ltd. All rights reserved.

1.

Introduction

Moving-bed biolm processes have proved to be very reliable for tertiary nitrication because of the high volumetric loading rates that can be applied and the low solids buildup in the reactor. To treat a given volume of wastewater, the capacity of a moving-bed biolm reactor (MBBR) can be smaller than required for a conventional activated-sludge process and, usually, there is no need for a tertiary settling tank. Compared to xed-bed biolm reactors (biolters), MBBRs have much lower headlosses, lter-bed channelling
Corresponding author. Tel.: +39 02 23996431; fax: +39 02 23996499.

does not occur (i.e. all the bioreactor volume is used) and periodic backwashing is not needed. Moreover, existing concrete tanks can be equipped and adapted to a MBBR conguration with relatively minor modications. MBBRs are usually lled with low-density (slightly less than 1.0 g cm3) polyethylene KMTs biolm carriers. One such carrier consists of small cylindrical elements 10 mm in diameter and 8 mm in height, with small longitudinal ns that protrude on the outside surface and an internal cross member that divides each element into four circular sectors. The void ratio of the support media is as high as 0.95 with the

E-mail address: roberta.salvetti@polimi.it (R. Salvetti). 0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2006.05.013

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effect that, in a tank lled with water and carriers, the volume of water is 95%. The lling ratio is dened as the ratio between the volumes occupied by the carriers, considered as solid cylinders, and the total tank volume. Its maximum value for good mixing is 0.7. The theoretical specic surface area of the support media is dened as the amount of surface area per unit volume of biolm carrier, and for the media described is 700 m2 m3 (degaard and Rusten, 1993). Since the biolm grows in the protected internal faces of the media, the actual specic surface area can be assumed to be about 500 m2 m3 (Hem et al., 1994). In the experiments described in this paper, the actual lling ratio was 0.5, so that the surface area available for the biolm is 250 m2 m3 reactor. The use of pure oxygen instead of air enables higher dissolved oxygen (DO) concentrations to be maintained in the reaction vessel. As a consequence, greater transfer efciency can be achieved and oxygen can diffuse more deeply into the biolm. This produces higher nitrication rates, and, consequently, smaller reactor volumes. If pure oxygen is used in the rst stage, the process can be conveniently divided into two sequential stages, with aeration in the second stage. The rst stage could be operated with a high ammonia concentration and DO would become the rate-limiting substrate. Since zero-order intrinsic kinetics can be assumed for nitrication in biolms, zero-order kinetics can also be assumed with respect to the non-limiting substrate, i.e. ammonia. As far as DO is concerned, it has been found that oxygen can be the reaction-limiting substrate if the ratio of oxygen to ammonia is lower than 2 g O2 (g NH+4 N)1 (Hem et al., 1994) and this may happen even when DO concentration is high (510 mg L1). If the second stage had operated at low ammonia concentration, then the reaction would have shifted to ammonialimiting conditions, which would occur when the oxygen to ammonia ratio is higher than 5 g O2 (g NH+-N)1. Ratios of 4 25 g O2 (g NH+-N)1 should be avoided since transition to 4 ammonia limitation may occur and the process kinetics would then depend on biolm structure and thickness. In the second stage, the very low ammonia concentration in the efuent (0.51.0 mg L1) permits reasonably low DO concentrations to be maintained in completely mixed reactors (2.55 mg L1), so that aeration can also be performed by simple air sparging (Bonomo et al., 2000). The dependence of nitrication on temperature in MBBRs has been investigated in the past. degaard and Rusten (1993) analyzed the dependence of nitrication under oxygen-limiting conditions and did not nd a signicant increase of removal rates at different temperatures. This was apparently in contrast with many previous studies in which a marked effect of temperature on nitrication was described by an Arrhenius-like expression. However, the authors explained that the reason of this discrepancy is due to the fact that, at lower temperature, nitrication rate is certainly reduced, but at the same time the oxygen concentration that can be dissolved in water increases. Therefore, the temperature effect that they were able to observe was masked by the opposite effect due to the increased oxygen concentration. For suspended-growth systems, Painter and Loveless (1983) found a temperature coefcient, y, of 1.076, and similar values are reported by the USEPA (1975) and by Barnes and Bliss

(1983) in the temperature range of 530 1C. The dependence of the reaction rate on temperature was found to be lower than expected for nitrication in xed-lm biolters by Zhu and Chen (2002). In particular, the effect of temperature on the reaction rate was found to be even weaker under oxygen-limiting conditions compared with ammonia-limiting conditions. Popel and Fischer (1998) observed that the effect of temperature on nitrication in suspended-growth systems (namely, activated sludge processes) is often lower than expected from literature data, because other factors, such as reactor conguration, hydraulic residence time (HRT) and efuent concentration, may play an important role in reducing the observed inuence of temperature. This is because removal rates, either in suspended or in xed systems, depend also on the rate-limiting substrate concentration, which is usually a function of the above-cited factors. Hence, the inuence of factors other than temperature on the rate-limiting substrate concentration could mask the observed inuence of temperature on nitrication rates. In the same paper, Popel and Fischer (1998) proposed a distinction between the real temperature coefcient, that describes the dependence of the intrinsic biological process kinetics (y), and the apparent temperature coefcient (ya) that ts the actual operational reaction rates observed in the reactor. They showed also that the latter depends on the type of process and on the conguration of the reactor. In biolm processes diffusional resistances may also contribute to mask the effect of temperature on the intrinsic bacterial reaction rate. Therefore, the aim of the present work is to check whether Popel and Fischers theory can be extended to tertiary nitrication in MBBRs. Multivariate regression analysis was used to quantify the effect of temperature on nitrication rates independently from the operating conditions of the system.

2.

Materials and methods

Two stainless-steel pilot-scale reactors, 1 m3 volume each, have been used. They were fed with the secondary efuent of a wastewater treatment plant (WWTP) equipped with a pureoxygen-patented activated-sludge process (UNOXs); this process produces a low-COD, non-nitried settled secondary efuent (Table 1). The study of the effect of temperature on nitrication was quite convenient with non-nitried efuent from this WWT plant, because the efuent was used as cooling water in a nearby waste-to-energy facility. Throttle valves and owmeters enabled either heated or unheated efuent to be fed to each pilot-scale unit and, therefore, it was quite simple to vary the temperature of the efuent that was fed to the MBBR pilot plant up to 21.573.7 1C (Fig. 1). Under normal operations, Reactor 1 (R1) was fed with unheated efuent and Reactor 2 (R2) with heated efuent. However, the heated and unheated efuents were sometimes mixed and fed to R1 to obtain an intermediate efuent temperature that was of interest for the experimental analysis. Although R2 was usually fed with heated efuent, on some occasions the temperature decreased due either to

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pump failures in the circuit from the waste-to-energy plant or to cold weather. Both MBBRs were lled at a 0.5 lling ratio with KMTs carriers with an estimated available surface area for biomass growth of 250 m2 for each reactor. Temperature in the reactors was 23 1C lower than the temperature of the inuent, depending on the HRT. Complete mixing was ensured by means of a central, 2-blade double stirrer of 30-cm diameter and with blades placed at 24 and 70 cm below top-water level; the stirrer was driven by a 0.37 kW geared electric motor at a rotational speed of about 190 rpm. Pure oxygen was supplied from the bottom of each reactor through a 26-cm diameter diffuser made of an EPDM membrane.

DO was measured in R1 by a portable DO-meter and the ow-rate of oxygen was controlled by visual inspection of a ow-meter. In this way, the oxygen supply rate was adjusted in order to keep the concentration of DO fairly constant and high enough so that it was not limiting process kinetics. R2 was always operated at high ammonia loading rates, so that DO was the limiting substrate. Therefore, nitrication rates depended on DO, which was kept within the desired range of values by a DO-probe that controlled the ow-rate of oxygen. The pH was monitored by a pH-meter in both reactors. Other reactor characteristics and technical data are summarized in Table 2. Average daily inuent and efuent samples were collected hourly by a side-stream peristaltic pump that provided a 20-L

Table 1 Characteristics of the settled efuent from Bergamo WWTP fed to the pilot-scale moving-bed biolm reactors (MBBRs) Parameter (unit)
COD (mg L1) NH+-N (mg L1) 4 NO-N (mg L1) 2 NO-N (mg L1) 3

N
270 270 270 270

Average
34.8 12.04 0.26 2.21

Standard deviation
14.1 3.76 0.45 1.33

Coefcient of variation
0.41 0.31 1.71 0.6

Minimum
10 2.88 0.02 0.1

Maximum
121 24.7 2.29 8.21

ILLUSTRATIONS HE UNOX E P1 ambient T = 10 20C V1 R1 NE1 O2 T = 21.5 3.7C P2 V2 R2 NE2

Fig. 1 Scheme of the experimental arrangement. UNOXs: patented full-scale pure-oxygen activated-sludge process; E: nonnitried efuent; HE: heat exchanger (municipal solid waste incinerator economizer); O2: pure oxygen supply; R1 and R2: pure-oxygen moving-bed biolm pilot-plant reactors; NE1, NE2: nitried efuent.

Table 2 Description of the reactors used in the pilot-scale plants R1 and R2 Technical data (units)
Height (m) Length (m) Width (m) Volume (m3) Filling ratio (dimensionless) Total specic surface-area of carriers (m2 m3) Actual specic surface-area that can be colonized by biolm at a lling ratio of 100% (m2 m3) Actual surface-area that can be colonized by biolm at a lling ratio of 50% (m2) Flow rate (m3 h1) Hydraulic retention time (h)

Values
1.245 0.928 0.865 1.00 0.50 700 500 250 1.50 (R1)3.00 (R2) 0.33 (R2)0.67 (R1)

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24-h composite sample. During weekends, 50-L composite samples were collected over 3 d by adjusting the ow-rate of the sampling pump. Additional (26) grab samples of inuent and efuent were taken between 9:30 am and 3:00 pm for further analysis. Such grab samples were analyzed by means of quick-analysis kits (LCK cuvettes and CADAS 50S photometer by Dr. Lange) for COD, ammonia, nitrate and nitrite. Analyses obtained by kits were periodically checked against laboratory analyses performed according to Standard Methods (APHA, AWWA, WPCF, 1998). Ammonia was also determined potentiometrically by a specic-ion electrode (EA 940 ORION) that required daily calibration at the temperature of each experiment. Occasionally, when kits were not available, nitrate and nitrite were measured by means of ionic chromatography (DIONEX). Attached biomass was determined by the difference in weight of 100 carriers, before and after removing biomass by thorough washing. Washing was performed in a sodium hypochlorite solution (6% active chlorine) that was exposed to ultra-sound for 3 h with a rinse step every hour with the chlorinated solution and a nal rinse with de-ionized water. The result was expressed as g TS per m2 of attachable surface, assuming that the value of the attachable surface is only the inside portion of the carriers (Table 2). Biolm thickness was determined by means of electron microscope observations. Average pH value was between 7 and 7.4 and no alkalinity limitation was found.

of F statistics. It is also possible to mix and match categorical and continuous predictors to build models. These latter models are generally referred as Analysis of co-variance (ANCOVA) models. Traditionally, ANCOVA designs have referred more specically to designs in which the rst-order effects of one or more continuous predictor variables are taken into account when assessing the effects of one or more categorical predictor variables (Wildt and Olli, 1978). ANCOVA is in fact used to test both main and interaction effects of categorical variables on a continuous dependent variable, controlling for the effects of other selected continuous variables, which co-vary with the dependent variable. The control variable is called the co-variate. There may be more than one co-variate. ANCOVA uses a built-in regression, which, in turn, uses the co-variates to predict the dependent variable. Then it performs an Analysis of Variance (ANOVA) on the residuals (which are the differences between the predicted variables and the actual dependent variables) in order to see whether the factors are still signicantly related to the dependent variable after removing the variation due to the co-variates. The ANCOVA analysis for this study has been carried out by using a General Linear Modeling (GLM) approach (see Rutherford, 2001) Y i a b1 X1i b2 X2i b3 X3i bp Xpi Xpi i , (2)

2.1. 2.1.1.

Statistical analysis Non-linear bivariate regression analysis

For each combination of temperature and rate-limiting substrate concentration, the nitrication rate was studied by means of non-linear bivariate regression analysis. To run the regression analysis, the StatSoft STATISTICA software package was used. The loss function is dened as an ordinary leastsquares function, i.e. aimed at minimizing the sum of squared residuals around the regression curve. The QuasiNewton algorithm was chosen as the regression method. This method uses the rst-order and second-order derivatives to follow a path towards the minimum of the loss function. Signicance level, for coefcient estimates, was assessed by means of a Students t-test (A and Clark, 1996), where the null hypothesis was the independence of the response variable from the predictors and the test statistic was: tdf Bi 0 , SEBi (1)

where Yi is the observed value of the ith-dependent variable (i.e. nitrication rate, biomass activity, etc.); the population of Y is always continuous; a is the mean of the population Y when the value of Xpi is zero; p 1; 2; . . . ; n; Xpi are the values of the independent variables (Xpi can be continuous or discrete); p 1; 2; . . . ; n; bp are effect parameters (regression coefcients) that relate each Xpi with the dependent variable, Y; e is the error (or uncertainty).

3.
3.1.

Results and discussion

Theoretical model development

where Bi is the coefcient estimate, SE(Bi) is the standard error of the Bi estimate and the value of t was then compared with the tabled t percentiles with N2 degrees of freedom (df) to obtain the p-value.

2.1.2.

Analysis of co-variance by using a GLM approach

The statistical package SPSS 12.0 has been used to develop multivariate generalized linear models, GLMs. GLMs work with exible experimental designs and are able to estimate means and variances and to test and predict means in terms

The intrinsic reaction rate of biological nitrication is usually expressed by MichaelisMenten kinetics. However, for a biolm process, it is not possible to nd an analytical solution of the MichaelisMenten equation. Although numerical solutions are used (e.g. Bonomo et al., 2000), the simplifying assumption of an implicit zero-order kinetics very often provides an analytical solution which, while somewhat less accurate, is still acceptable for the design of biolm reactors. However, the most commonly used kinetic parameter is the overall reaction rate and it is equal to the ux of the substrate that diffuses into the biolm. When one of the substrates, that takes part in the reaction, is depleted at a certain depth inside the biolm, it can be said that the biolm is partially penetrated by that substrate. In this case, the order of the overall reaction rate is no longer zero because of the diffusive limitations due to the external liquid lm and the biolm itself. The general overall observed reaction rate, v, is given by v kca , (3)

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where k is the coefcient that depends on the biolm thickness (L) and on the diffusion coefcient (D) of the limiting substrate; D depends on temperature (Riemer, 1977; Hem et al., 1994; Christiansen et al., 1995), c is the ratelimiting substrate concentration, a is a coefcient with values between 1/2 and 1 that takes into account the diffusive limitations in the liquid lm and in the biolm (Hem et al., 1994; Pastorelli et al., 1997). The coefcient a depends mainly on the thickness of the liquid layer adjacent to the biolm. It is about 1/2 when the stagnant liquid layer is thin. This happens when local turbulence is high, the liquid layer is thin and the resistance to diffusion through this layer is negligible. The coefcient a tends to unity when local turbulence is low and the liquid layer is thick; under these conditions, diffusion is the ratelimiting step and follows the rst-order Ficks Law. Under steady-state conditions, it can be assumed that L and D (at the reference temperature) are constant; therefore, it can be assumed that k values depend only on temperature. The theory proposed by Popel and Fischer (1998) for nitrication in activated-sludge processes should still be valid for a biolm. Therefore, it can be assumed that the k value in Eq. (3) follows the Arrhenius-like relationship: k kR yTR , (4)

3.1.1.

Ammonia-limiting conditions

Under ammonia-limiting conditions, the value of the temperature coefcient y can be obtained from the following kinetic expression, obtained by combining Eqs. (3) and (4) vT kR yTR ce;T a , (8)

where ce,T is the ammonia concentration, which depends on temperature T and on the operational conditions of the reactor. A bi-variate regression analysis was used to nd the values of kR and a.

3.1.1.1. Apparent temperature coefcient under ammonia-limiting conditions. The mass balance for ammonia in a completely mixed nitrication reactor leads to an equation that describes the average overall ammonia conversion rate of the process, vproc vproc co;T ce;T , t (9)

where kR is the value of k at the reference temperature R, T is the temperature of the experiment y is the temperature coefcient that comes directly from the Arrhenius equation k A eEa =RT , (5)

where A is a constant (frequency factor, constant specic to a particular reaction), Ea is the activation energy (kJ mol1), R is the universal gas constant (8.314 103 kJ mol1 K1), T is the temperature (in Kelvin). Eq. (4) is obtained by the quotient of Eq. (5) written for temperature values equal to T and R. Since the derivative of an exponential function is given by dax ax ln a, dx

where co,T is the ammonia feed concentration at the temperature of the experiment (T), ce,T is the ammonia concentration in the efuent and in the bulk liquid (since completely mixed conditions have been assumed) at the temperature of the experiment (T), t is the HRT dened as the ratio of volume to ow rate (V/Q). According to Popel and Fischer (1998), vproc not only depends on temperature, but also on various operational parameters (i.e. reactor conguration, hydraulic retention time, efuent concentration, etc.). In biolm processes this concept should be even more marked, since operational conditions can affect diffusion processes as well as biological ones. The effect of both temperature and operational parameters on the overall kinetics is given by vproc;T vproc;R yTR , a (10)

(6)

the derivative of the function k(T) with respect to temperature in Eq. (4) gives dk=dT k lny kR yTR lny. (7)

where ya is the apparent temperature coefcient, which summarizes all temperature effects, vproc,R is the overall conversion rate of the process at the reference temperature, vproc,T is the overall conversion rate at the experimental temperature. Rearrangement of Eq. (10) leads to   vproc;T 1=TR . (11) ya vproc;R Substitution from Eq. (11) gives   co;T ce;T 1=TR . ya co;R ce;R

(12)

The intrinsic bacterial kinetics may depend on temperature in a way that may be different from that of the overall nitrication rate. In fact, this rate is the result of the combined effects of nitrication kinetics, diffusion of substrates from the bulk liquid through the biolm and backdiffusion of metabolites from the biolm to the bulk liquid. Diffusion processes through the biolm also depend on temperature, but in a way which may be different from that of bacterial kinetics. Therefore, in order to describe the dependence of the overall reaction rate on temperature, the apparent temperature coefcient (ya, according to the terminology introduced by Popel and Fischer, 1998) was considered.

The specic relationship between co,T and ce,T follows from the mass balance in a completely mixed reactor, in which the reaction rate is given by Eq. (3): V dce;T =dt Qco;T Qce;T VAS kce;T a , (13)

where AS is the specic surface area, dened as the ratio of the total biolm area to the reactor volume. At steady state, Eq. (13) simply reduces to ce;T co;T tAS kce;T a . (14)

Since the intrinsic nitrication reaction k(ce,T)a depends on temperature as described by Eq. (8), the relationship between the intrinsic, or real, temperature coefcient y and the

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apparent temperature coefcient ya is obtained by combining Eq. (12) with Eq. (14) and (8) !1=TR   tAS kR yTR ca co;T ce;T 1=TR e;T ya co;R ce;R tAS kR ca e;R  a=TR ce;T y . 15 ce;R

Therefore, ya is equal to y, indicating that biological kinetics are only inuenced by temperature and are independent from operational parameters, with the obvious exception of DO concentration.

4.
4.1.

Results and discussion

Ammonia-limiting conditions (Reactor R1)

3.1.2.

Oxygen-limiting conditions

Under oxygen-limiting conditions, the nitrication rate no longer depends on the efuent ammonia concentration but instead upon DO (O2). Therefore, the reaction rate can be written as vT kR yTR O2 a vR yTR . (16)

The regulation system of the pure-oxygen supply enabled a preset DO concentration to be maintained in the reactor, independently from temperature, so that it can be written dv=dT dk=dTO2 a kR yTR lnyO2 a . The ammonia mass balance is simply given by V dce =dt Qco Qce VvT , which at steady state, gives ce;T co;T tAS vT co;T tAS kR yTR O2 a . (19) (18) (17)

The derivative of the efuent ammonia concentration with respect to temperature is now described by dce dvT tAS tvT AS lny dT dT TR tAS kR y O2 a lny.

20

3.1.2.1. Apparent temperature coefcient under oxygen-limiting conditions. The development of an apparent temperature
coefcient is similar to that leading to Eq. (15) for the ammonia-limiting conditions and, in this case, can be written as: !1=TR   tAS kR yTR Oa co;T ce;T 1=TR 2;T ya co;R ce;R tAS kR Oa 2;R  a=TR O2;T y . 21 O2;R If DO is kept at a constant concentration in the reactor, then O2;R O2;T and Eq. (21) reduces to ya yTR 1=TR y. (22)

The Reactor R1 was operated for 7 months (JanuaryAugust 2001) under ammonia-limiting conditions. The R1 operating conditions are summarized in Table 3. Prior to January 2001, the biomass concentration was too low to achieve a high removal efciency as shown in Fig. 2. From January 2001 to March 2001, the increase of the reaction rates was due to the combination of the increase of the biomass in the reactor and to the seasonal temperature increase. Cross-sections of biolm, observed with an electron microscope, indicated a biolm thickness in R1 of about 200 mm; such a biolm thickness allows only a partial penetration of ammonia into the biolm (Zhang and Bishop, 1994). Such a biolm thickness was used to calculate the biomass density by dividing biomass weight (ranging from 10.8 to 13 g TS m2 ) by thickness. A biomass density of about 60 mg TS cm3 was determined and that is consistent with published data (e.g. Van Benthum et al., 1995). After July 2001, microorganisms such as rotifers and nematodes were found in the biolm. These metazoans caused a massive detachment of biolm from the support media and, consequently, the nitrication rate decreased. Therefore, for the purpose of this study, the system was considered close to steady-state conditions from April to the end of June. The specic operating conditions during this period are reported in Table 4. Removal rates during this period were correlated to the corresponding temperatures in the reactor. R1 showed a fairly constant efuent ammonia concentration so R1 data were pooled together to study nitrication as a function of temperature. The results showed that there was no signicant (p-level40.05) correlation between nitrication-rate and temperature. Since these results refer to the overall reaction rate, this means that the apparent temperature coefcient (ya) can be assumed to be equal to unity.

Table 3 Operating conditions in R1 (JanuaryAugust 2001; DO dissolved oxygen; O/N Oxygen over Nitrogen ratio in the reactor) Parameter (unit)
Temperature (1C) DO (mg L1) NH4-N (mg L1) Efciency (%) Loading rate (gNH4-N m2 d1) Removal rate (gNH4-N m2 d1) O/N (dimensionless)

N
319 319 319 319 319 319 319

Average
17.9 24.4 1.84 86.1 1.96 1.69 16.0

Standard deviation
3.0 3.7 0.83 4.9 0.68 0.61 7.8

Coefcient of variation
0.17 0.15 0.45 0.06 0.35 0.36 0.49

Minimum
10.2 13.0 0.59 62.9 0.56 0.43 4.93

Maximum
23.3 35.0 6.08 92.4 3.89 3.45 55.6

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However, the intrinsic reaction rate could depend on temperature according to the real temperature coefcient y, which could be higher than 1. Therefore, a bi-variate regression analysis was performed. All the data available with different ammonia concentrations and temperatures were considered and Eq. (8) was used in the bi-variate model. If a reference temperature of 20 1C is chosen, the equation becomes vT kR yT20 ca . e (23)

Table 5 shows the results of statistical analysis and the estimates of kR, y and a values as they were obtained from the bi-variate regression analysis. As it can be seen, y is in the range of 1.0861.109 (average 1.098), and the order of the reaction is very close to unity.

4.2.

Oxygen-limiting conditions (reactor R2)

As it has been already seen from Eq. (22), at steady-state and under oxygen-limiting conditions and at constant oxygen

Fig. 2 Biomass concentration on carriers in R1 and R2.

Table 4 Operating conditions in R1 (from April to end of June 2001; DO dissolved oxygen; O/N Oxygen over Nitrogen ratio in the reactor) Parameter (unit)
Temperature (1C) DO (mg L1) NH4-N (mg L1) Efciency (%) Loading rate (gNH4-N m2 d1) Removal rate (gNH4-N m2 d1) O/N (dimensionless)

N
101 101 101 101 101 101 101

Average
19.2 23.7 2.0 0.9 2.3 2.0 13.8

Standard deviation
2.10 3.57 0.82 0.03 0.68 0.59 6.31

Coefcient of variation
0.11 0.15 0.41 0.03 0.30 0.29 0.46

Minimum
13.4 17.0 0.9 0.8 1.0 0.9 5.5

Maximum
22.0 31.0 4.3 0.9 3.9 3.5 33.3

Table 5 Regression statistics under ammonia limiting conditions (R1) during the period AprilJune 2001 Multiple correlation coefcient (R) Coefcient of Determination (R2) 0.892 0.795 kR
Mean Standard error Range t (90) p-Level 1.163 0.0637 1.0991.226 18.256 o0.01

y
1.098 0.0096 1.0861.109 95.140 o0.01

a
0.918 0.0749 0.8430.993 12.254 o0.01

Level of signicance is also shown for all the regression estimates.

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concentration, the apparent temperature coefcient coincides with the biological temperature coefcient. Therefore, the intrinsic temperature coefcient (y) affecting the intrinsic biological process can be determined from the experimental data. As in R1, the biomass concentration in R2 varied during the experimental period. However, nematodes and rotifers colonized the biolm starting as early as in March 2001 and their grazing activity on the biolm also caused loss of biomass and nitrication rates decreased. While in reactor R1 this phenomenon started in summer and lasted two weeks only, the presence of metazoans in reactor R2 persisted until July 2001. The biolm in reactor R2 was thicker but looser and that, according to van Loosdrecht et al. (1995), can be related to the higher loading rates. Probably, such a thicker biolm was more suitable for the colonization of metazoans than the more compact biolm that developed in reactor R1, at much lower loading rates.

Operating conditions and experimental data for R2 are shown in Table 6. From June 2000 to February 2001, DO concentration in the reactor was varied in order to study nitrication kinetics under oxygen-limiting conditions. By examining the data from this period, two different patterns of experimental conditions were recognizable, before and after November 10th 2000, even though nitrication rates remained fairly constant (Fig. 3). After the data had been standardized, they were split into two different sets by means of a Hierarchical Cluster Analysis (Kaufman and Rousseeuv, 1990). Classication was based on biomass, temperature, DO and the log-transformed nitrication rate. The Euclidean Distance v u q uX 2 (24) d2 xi ; xj t xik xjk 2
k1

Table 6 R2 operating conditions (from June 2000 to August 2001); DO: dissolved oxygen; O/N: Oxygen divided by nitrogen ratio in the reactor) Parameter (unit)
Temperature (oC) DO (mg L1) NH4-N (mg L1) Efciency (%) Loading rate (gNH4-N m2 d1) Removal rate (gNH4-N m2 d1) O/N (dimensionless)

N
325 325 325 325 325 325 325

Average
21.1 7.86 11.8 50.6 6.59 3.17 0.92

Standard deviation
3.0 2.54 6.58 13.1 2.56 1.15 0.88

Coefcient of variation
0.10 0.32 0.56 0.26 0.39 0.36 0.96

Minimum
12.5 4.50 1.32 16.9 2.23 1.70 0.19

Maximum
28.1 22.8 37.1 86.3 14.3 10.4 3.05

Fig. 3 Reactor 2: time series of biomass levels and nitrication rates (upper graph); dissolved oxygen and temperature (lower graph). Data were splitted into two sets: before and after 10 November 2000 (thick vertical line).

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was used as distance measure, and Wards method was used as agglomerative criterion. By this means two separate groups of data were distinguishable. Since the biomass content of each group of data differed signicantly, (MannWhitney U test: U: 76, p-levelo0.001, N: 195) they were dened as low and high biomass sets (high biomass: median: 9.77, min: 7.11, max 10.71, N: 82; low biomass: median: 6.67, min: 4.58, max 7.71, N: 59). Table 7 summarizes the main characteristics of the two clusters of data. Moreover, a GLM ANCOVA was applied to these data. The GLM model was yij m b1 xi b2 aj b3 axij ij , (25)

The ANCOVA, applied to these two sets of data (referred to the period June 2000February 2001), showed that the nitrication rate was signicantly different (F: 91.7, df1: 2, df2: 143; p-levelo0.01) for low- and high-biomass content conditions. The high biomass trials showed a faster kinetics (i.e. higher nitrication rates corresponding to increasing DO levels) than the low biomass trials (Fig. 4). Therefore, the dependence on thermal changes has been studied for each of these two situations. Eq. (16) describes the relationship between nitrication rate and DO for the reference temperature of 20 1C: vT kR yT20 O2 a . (26)

where yij is the kth nitrication rate observation of the jth level of factor a Biomass content: high and low biomass corrected by the ith observation of co-variate x1 ln(dissolved oxygen); m is the true overall mean; x1 is the co-variate score ln(dissolved oxygen) for the ith subject in the jth condition; aj is the incremental effect of factor a level j (i.e. Biomass content: high and low biomass), such that aj mj m; mj is the true population mean for the jth level of factor A (i.e. Biomass content); (a x)ij is the interaction effect for the jth level of factor a and the ith observation of co-variate x, i.e. ln(dissolved oxygen); eij is the error; bi...;n are the regression coefcients.

As R1 data, R2 data have been also analyzed by means of bivariate regression analysis. The results are shown in Tables 8 and 9. As it can be seen, the intrinsic biological temperature coefcient y under oxygen-limiting conditions (R2) varied between 1.023 and 1.081 (with an average value of 1.056). The value of y under oxygen-limiting conditions appears to be somewhat lower than under ammonia-limiting conditions (R1), but the difference is not statistically signicant. On the other hand, the dependence of the overall nitrication rate on temperature is negligible for R1 (i.e. the value of ya is close

Table 7 Average characteristics of the two groups of data recognised from cluster analysis Clusters
Low biomass (June October 2000) Temperature (1C) Dissolved oxygen (mg L1) Biomass (g TS m2)

Mean

Median

Minimum

Maximum

Std. deviation

25.5 12.8 6.56

25.7 12 6.67

23.2 6 4.58

28.1 22.75 7.71

1.4 4.7 0.77

59 59 59

High biomass (November 2000 February 2001) Temperature ( 1C) 18.6 8.8 Dissolved oxygen (mg L1) 9.28 Biomass (g TS m2)

18.8 8 9.77

13.1 4.5 7.11

22.2 15 10.71

1.8 2.5 1.14

82 82 82

Fig. 4 Reactor R2, period from June 2000 to February 2001different kinetics as function of biomass levels (low vs. high). 95% Condence limits are also shown.

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Table 8 Regression statistics for the nitrication kinetics in the R2 reactor under oxygen-limiting conditions from June to October 2000 (i.e. variable dissolved oxygen concentrations) Multiple correlation coefcient (R) Coefcient of Determination (R2) 0.479 0.229 kR
Mean Standard error Range t (62) p-Level Data set with low biomass content. 0.994 0.4385 0.5551.432 2.266 0.0269

y
1.052 0.0289 1.0231.081 36.358 o0.01

a
0.454 0.1351 0.3180.589 3.358 o0.01

Table 9 Regression statistics for the nitrication kinetics in the R2 reactor under oxygen-limiting conditions from November 2000 to February 2001 (i.e. variable dissolved oxygen concentrations) Multiple correlation coefcient (R) Coefcient of determination (R2) 0.900 0.810 kR Mean
Standard error Range t (84) p-Level Data set with high biomass content

y 1.059
0.0103 1.0481.071 92.075 o0.01

a 0.686
0.0528 0.6330.739 12.991 o0.01

1.014
0.1248 0.8891.139 8.124 o0.01

to 1, so that the effect of temperature is hardly perceptible on the overall reaction rates). This result appears to be in contrast with the ndings of Zhu and Chen (2002). However, it seems reasonable that their conclusion depends on their specic experimental conditions. In their case air was sparged instead of pure oxygen and DO concentration was not kept constant, but it decreased as temperature increased. Therefore, the increase of nitrication rates, related to higher temperature values, was probably counterbalanced by the lower oxygen penetration depth in the biolm. It is noteworthy to observe that, even though the two clusters (high and low biomass) were at different temperatures (Students t-test: t: 24.5, df: 139, p-levelo0.001) and DO concentrations (MannWhitney U test: U: 1241, p-levelo0.001, N: 141), the difference observed between the nitrication rates are essentially due to the change in the biomass content. In fact, when considering the specic nitrifying activity (i.e. the ratio of nitrication rate to biomass content on the support media) there is evidence of a difference between the two biomass conditions. Despite the fact that Fig. 5 seems to suggest that there is no appreciable difference, ANCOVA, after removing the variation due to both temperature and DO concentration, clearly shows that the specic nitrication activity of the two biomass conditions is signicantly different (F: 8.8, df1: 1, df2: 137, p-levelo0.01).

In addition, when considering the lowest concentrations of DO (DOp8 mg/L), the specic nitrifying activity of the biomass appears to be limited by the oxygen penetration depth through the biolm. Comparing the average specic nitrication activity of the high biomass data set (0.36 g NNH4 g TS1 d1) with that of the low biomass data set (0.442 g N-NH4 g TS1 d1), in fact, a signicant difference between the two means can be found (Students t-test for unequal variances: t: 3.382, df: 39, p-levelo0.01). Under low DO conditions (DOo8 mg L1) the biolm thickness plays a major role in driving the kinetics and leads to a higher specic biomass activity when the biolm thickness is low. This effect was also suggested by Zhu and Chen (2002) when they observed a reduction of the temperature impact on xed lm nitrication rates due to the dominance of mass diffusion transport processes and by Hao et al. (2002), who found that biolm thickness and density were the most important parameters in determining the effect of DO on the observed N-concentrations. During the period from March to August 2001, DO concentration in reactor R2 was kept almost constant (at about 68 mg L1) in order to investigate further the effect of temperature on nitrication-rates. Also in this case, data were grouped into two different sets depending on their biomass content. Biomass contents of these two groups were, respectively, 8.9 g TS m2 (min: 7.6, max 11.0; std. deviation: 0.93, N: 124) for the high biomass data set and 7.7 g TS m2

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Fig. 5 Reactor R2, period from June 2000 to February 2001biomass nitrication activity (nitrication rate/biomass) vs. dissolved oxygen and temperature for low and high biomass content on the support media.

Table 10 Temperature coefcients y for nitrication kinetics in R2 reactor under oxygen-limiting conditions from March to August 2001 (i.e. constant dissolved oxygen concentrations) Temperature coefcient (y)
Mean Standard error Range R2 T p-Level

High biomass conditions

1.061 0.0086 1.0441.078 0.563 113.11 o0.01

Low biomass conditions

1.060 0.012532 1.0471.073 0.510 12.42 o0.01

(min: 6.87, max 10.58; std. deviation: 0.85, N: 73) for the low biomass data set. The difference in this case was smaller but still signicant (MannWhitney U test: U: 1219, p-levelo0.001, N: 198), consequently the two data sets were analyzed separately. The kinetics were described by Eq. (16) after the following linear transformation: lnvT lnvR T 20 lny. (27)

Fig. 6 Reactor R2, period from March to August 2001 specic nitrication activity (nitrication rate/biomass) vs. temperature in the two biomass groups. From the graph two linear relationships with temperature are visible.

Since oxygen concentration was kept nearly constant, vR was considered to be constant. Table 10 shows the results of the linear regression analysis. It is important to observe that the two estimates of y are similar in both situations and are also comparable with the estimates obtained in the previous analysis. Fig. 6 shows the relationship between specic biomass activity of Reactor R2 during the period from March to August 2001 and the temperature for the two biomass groups. From the graph it is evident that the range of temperatures was not exactly the same for each group. Nevertheless, examination of the graph indicates that for any temperature increase in the higher range (i.e. 2328 1C) the biomass increases its activity more than in the lower temperature range (i.e.

1822 1C). However, such a difference may be not only due to temperature, but to deeper penetration of DO into the biolm. In fact, diffusion was more efcient as temperature increased, since DO concentration was kept constant in the bulk liquid. In order to clarify the relationship between temperature and specic biomass activity, independently from oxygen penetration in the biolm, ANCOVA was applied to these data. The GLM model was yij m b1 xi;1 b2 aj;2 b3 ax3 ij ij , (28)

where yij is the kth nitrication activity observation of the jth level of factor a (Biomass level: high and low biomass) corrected by the ith observation of co-variate x1 (i.e. DO), m is the true

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overall mean, x1 is the co-variate score for the ith subject in the jth condition (i.e. DO), aj is the incremental effect of factor a level j (i.e. Biomass level: high and low biomass), such that aj mj m, mj is the true population mean for the jth level of factor A (i.e. Biomass level), x3 is the co-variate score for the ith subject in the jth condition (i.e. T20), (a x3)ij is the interaction effect for the jth level of factor a and the ith observation of co-variate x3 (i.e. Biomass level T20), eij is the error, bI;...;n are the regression coefcients. Even removing the effect of DO concentration, the specic nitrication activity showed a signicantly faster increase (F: 8.20 df1: 2, df2: 140; p-levelo0.01) in the higher temperature range (2328 1C) than in the lower temperature range (1822 1C). This can be easily explained by the combined effects of higher temperature and thinner biolm. In fact, as temperature increases, the resistance to diffusion decreases, a higher proportion of biomass is exposed to oxygen and, therefore, is active, resulting in a higher specic nitrication activity.

content (i.e. thin biolm), in the temperature range of 2328 1C, while this effect was less marked in the range of 2225 1C, though still statistically signicant.

These results could have a profound consequence on the design of MBBRs. Reactors that are designed to operate under ammonia-limiting conditions can be designed without taking into account the effect of temperature on overall nitrication conversion rates; on the contrary, if these reactors are operated under oxygen-limiting conditions, temperature effects have to be taken into account in designing the reactors.

Acknowledgments
The authors wish to thank SIAD S.p.A., which partially funded the experimental program and provided the pilot plants. In particular, the authors wish to thank Eleonora Pasinetti, SIAD laboratory supervisor, for providing help, support and for making data available to us. BAS S.p.A. provided logistic assistance for laboratory routine analyses. We would like also to thank Dr. Arthur Boon for the review of English grammar. This paper beneted also from the comments of two anonymous reviewers that signicantly improved the manuscript.
R E F E R E N C E S

5.

Conclusions

The aim of this work was the study of the effect of temperature on the rate of biological nitrication in pureoxygen moving-bed biolm reactors (MBBRs). This analysis showed that these effects can be lower than expected under certain operational conditions, as found by Popel and Fischer (1998) for activated-sludge systems. 1. The inuence of operating parameters (such as the hydraulic retention time, the inuent ammonia concentration, the specic available surface and the kinetic of the process) on the value of efuent concentration (and therefore on nitrication rate) has been analyzed either under ammonia or under oxygen-limiting conditions. 2. The intrinsic or real biological temperature coefcient y (characterizing the intrinsic biological nitrication process) has been quantied independently from the apparent temperature coefcient (ya) by means of bivariate regression analysis. 3. At steady-state and under ammonia-limiting conditions, the apparent temperature coefcient ya resulted to be very close to unity, so that the effect of temperature was negligible. 4. However, the real biological temperature coefcient y under ammonia-limiting conditions was found in the range 1.0861.109 (average 1.098), but it had no effect on the overall conversion rates. 5. At steady-state and under oxygen-limiting conditions, the biological temperature coefcient y was in the range 1.0231.081 (average value: 1.058). 6. The real biological temperature coefcient y coincided with ya when dissolved oxygen concentration is kept constant. 7. Finally, under oxygen-limiting conditions, it has been shown that the specic biomass activity (i.e. the ratio of nitrication rate to biomass content on the support media) was strongly inuenced by the combination of the deeper penetration of oxygen into the biolm at low biomass

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