Continuous nitrifying granular sludge bioreactor: Influence of aeration and ammonium loading rate

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Continuous nitrifying granular sludge bioreactor: Influence of aeration and ammonium loading rate Maryam Hosseini a,b , Ali Baradar Khoshfetrat a,b,c,∗ , Eghbal Sahraei b , Sirous Ebrahimi a,b a b c

Biotechnology Research Center (BRC), Sahand University of Technology, Tabriz 51335-1996, Iran Department of Chemical Engineering, Sahand University of Technology, Tabriz 51335-1996, Iran Environmental Engineering Research Center (EERC), Sahand University of Technology, Tabriz 51335-1996, Iran

a b s t r a c t Granulation of nitrifying bacteria was investigated in a continuous bubble column bioreactor. Then, the combined effect of aeration and ammonium loading rates on dissolved oxygen (DO) concentration as well as nitrification process was evaluated in the system using an experimental design technique. After 120 days, stable nitrifying granules with average diameter of 1.4 mm and settling velocities of 55 m/h were obtained. The influence of increasing ammonium loading rate (ALR) was found to be more significant than decreasing aeration rate on the reduction of DO concentration inside the nitrifying bioreactor. The system could handle the ALR values of 0.48–1.92 gNH4 + -N/L d with the ammonium removal efficiency from 65% to nearly 100% at the tested airflow rates of 2.5 and 4.5 L/min. At the low aeration, the complete ammonium conversion to nitrate was replaced with nitrite when the ALR increased to 1.44 gNH4 + -N/L d. At the high aeration, however, almost complete nitrification was achieved except the high ALR in which the nitrite accumulation was observed up to 38%. The study demonstrated that the continuous bioreactor had a considerable performance for obtaining stable nitrifying granules to have nitrite accumulation under control with changing the ratio of aeration rate and ALR. © 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Nitrifying granules; Nitrite accumulation; Ammonium loading rate; Aeration rate; Continuous bubble column bioreactor

1.

Introduction

Treatment of high strength ammonium wastewater has become a matter of serious concern in recent years. Ammonium, abundant in many industrial and agricultural wastewaters, needs to be removed to prevent oxygen depletion and eutrophication of surface waters. Biological ammonium removal process is one of the most common processes for nitrogen removal from wastewater using nitrification–denitrification steps. Nitrification converts ammonium to nitrite by ammonium oxidizing bacteria (AOB) and then to nitrate by nitrite oxidizing bacteria (NOB), while denitrification is the biological reduction of nitrate into molecular nitrogen.

Because of low maximum specific growth rate and biomass yield of nitrifying organisms, retaining sufficient nitrifying bacteria in bioreactor is difficult to achieve. Artificial immobilization in gel beads (Hunik et al., 1994; Tanaka et al., 1994; Hayashi et al., 2002) and natural immobilization of nitrifiers as biofilm or granules can be considered the promising solutions to overcome the problem. Formation of nitrifying biofilms in turbulent bed bioreactor (Bernet et al., 2005) as well as biofilm airlift suspension bioreactor with an ammonium load of 5 gNH4 + -N/L d (Tijhuis et al., 1995; van Benthum et al., 1996; Garrido et al., 1997) has already been studied. Aerobic granular sludge is a new approach to the biofilm systems due to its excellent characteristics compared to

∗

Corresponding author at: Department of Chemical Engineering Sahand University of Technology, Tabriz, Iran. Tel.: +98 4113458180; fax: +98 4113444355. E-mail address: [email protected] (A.B. Khoshfetrat). Received 23 November 2012; Received in revised form 7 August 2013; Accepted 8 August 2013 0957-5820/$ – see front matter © 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.psep.2013.08.003 Please cite this article in press as: Hosseini, M., et al., Continuous nitrifying granular sludge bioreactor: Influence of aeration and ammonium loading rate. Process Safety and Environmental Protection (2013), http://dx.doi.org/10.1016/j.psep.2013.08.003

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conventional bioflocs; such as high biomass concentrations and volumetric loading rate and high sludge age. A review on the literature shows that the formation and operation of aerobic granules in organic wastewater treatment processes has been widely investigated by researchers in sequencing batch reactor (SBR) and to a lesser extend in continuous mode. However, fewer studies reported development of nitrifying granules in continuous mode using aerobic upflow fluidized bed (AUFB) and airlift bioreactors (Campos et al., 2000; Tsuneda et al., 2003, 2004; Jin et al., 2008; Kishida et al., 2010). Therefore, the formation study of nitrifying granules under continuous operation mode is still a subject of discussion and needs more challenge. Partial nitrification to nitrite instead of nitrate has gained interest since it results in saving of 25% oxygen and 40% carbon source needed for nitrification and denitrification processes, respectively (Garrido et al., 1997). Studies show DO concentration, temperature, free ammonia (FA) inhibition, ALR, solid residence time (SRT) are among parameters that affect the nitrification performance of bioreactor (Pollice et al., 2002; Ruiz et al., 2003; Chung et al., 2005; Kim et al., 2006). Studies also show that FA inhibition (Turk and Mavinic, 1989; Carrera et al., 2004; Hawkins et al., 2010) and DO control (Garrido et al., 1997; Bernet et al., 2001) are common approaches to achieve partial nitrification. Although FA inhibition can be used to obtain the partial nitrification, some researchers noted that nitrite oxidizers acclimate to high FA concentration. This limits the long term success of the strategy for the partial nitrification. For instance, some works have revealed the acclimation of NOB to a FA concentration of up to 22 mg/L, while the inhibitory concentration is started from 1 mg/L (Anthonisen et al., 1976; Turk and Mavinic, 1986). The influence of DO concentration on the partial nitrification has also been discussed by some researches and the stable nitrite accumulation up to 80% has been obtained in oxygen limited biofilm bioreactors (Bernet et al., 2001; Ruiz et al., 2006; Blackburne et al., 2008b). ALR and aeration rate are among the most important operational parameters affecting the DO concentration. Increasing ALR or decreasing aeration rate causes the lower DO concentration in the liquid bulk, leading to partial nitrification. Even though, the partial nitrification can be obtained in situations of oxygen limiting conditions, but under the oxygen limiting conditions, DO concentration becomes less sensitive. Consequently, the use of DO concentration as the only control parameter for partial nitrification can be problematic. Therefore, combined effects of ALR and aeration need to be studied in more detail. In this contribution, the formation of aerobic nitrifying granules under continuous operation mode was studied in a bubble column bioreactor. Following, the feasibility of partial nitrification as well as the variation of DO concentration with changing the operational parameters of aeration rate and ALR was investigated using an experimental design technique.

2.

Materials and methods

2.1.

Experimental set-up and operation

Experiments were carried out in a water-jacketed bubble column bioreactor (ID = 7 cm, H = 70 cm) equipped with threephase separator with working volume of 3.5 L. As shown in

Fig. 1, air was supplied at the bottom of the bioreactor to promote the formation of small bubbles and to guarantee the complete mixing by using air sparger under the continuous air supply. The superficial air velocity in the bioreactor was controlled by means of a mass flow controller system (5850E, Brooks Instrument BV, Veendaal, The Netherlands). The feed solution was introduced continuously by a peristaltic pump (Masterflex, Cole-Parmer, USA) at the bottom of the bioreactor. The pH of the bioreactor was controlled at 7.9 ± 0.1 by addition of 1 M HCl or 1 M NaOH, using a pH transmitter (2500, Mettler Toledo, Switzerland). Temperature was kept at 30 ± 1 ◦ C by using a water bath circulator with a heating system (UC4500, Sahandazar, Iran).

2.2.

System startup

The bioreactor was initially filled with a synthetic wastewater with the following composition: (NH4 )2 SO4 , 0.236–0.943 g/L; MgSO4 ·7H2 O, 0.3 g/L; NaHCO3 , 0.7–2.83 g/L; FeSO4 ·7H2 O, 0.005 g/L and trace elements (Vishniac and Santer, 1957), 1 mL/L. Subsequently, the bioreactor was inoculated with 300 mL seed sludge having the initial volatile suspended solid (VSS) concentration of around 10 g/L, provided from the municipal wastewater treatment plant of Tabriz city, located in the northwest of Iran. The bioreactor was operated on batch mode with constant aeration rate of 4.5 L/min, corresponding to a superficial air velocity of 1.9 cm/s, to keep high DO concentration in the bulk liquid. Since carbon source (sodium bicarbonate) could be limiting, particularly at high aeration rates, the amount of sodium bicarbonate was chosen three folds of the ammonium sulfate concentration. Following the achievement of 95% conversion of ammonium (on day 5), continuous feeding was initiated with an ammonium concentration of 50 mgNH4 + -N/L. The hydraulic retention time (HRT) was adjusted to 2.5 h in which the HRT was much shorter than the residence time corresponding with the washout dilution rate for nitrifying bacteria to stimulate granule formation.

2.3.

Experimental design

A two-factor factorial design with one replication was used to carry out the experiments. Operation period of each run was considered to be 10 days. The design was chosen due to the long length of each run which make it difficult repeat more than once. ALR and aeration rate were considered as two main factors and DO concentration in the bulk liquid, percentages of ammonium removal and produced nitrite and nitrate as the system response parameters. Four levels for the ALR (0.48, 0.96, 1.44, 1.92 gNH4 + -N/L d) and two levels for the airflow rate (2.5, 4.5 L/min) were selected. For the high airflow rate of 4.5 L/min, the ALR increased stepwise from 0.48 to 1.92 gNH4 + -N/L d and then decreased stepwise again to the initial value of 0.48 gNH4 + -N/L d, after the system startup. Subsequently, a decrease in the airflow rate to 2.5 L/min was followed by a stepwise increase in the ALR value from 0.48 to 1.92 gNH4 + -N/L d. The data obtained after 30 days was considered for the statistical analysis. The strategy and the restriction made on randomization were carried out to maintain and ensure the system stability throughout the experiment. The F-test analysis of variance with a 95% confidence interval (or a significance level of 0.05), was used to evaluate statistically the effect of main factors and their interaction on

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Fig. 1 – Schematic representation of experimental set-up. the average values obtained for the DO concentration, percentages of ammonium removal and produced nitrite and nitrate as typical outputs of the system. Therefore, main factors and interactions with a P-value less than 0.05 were considered as important and effective parameters on the output variable of the system. Due to only a single replicate, the two factor interaction effect and the experimental error cannot be separated in any obvious manner in the analysis of variance. A test developed by Tukey (1949) which partitions the residual sum of squares into a single degree of freedom component due to nonadditivity (interaction) and a component of error was used to analyze the data.

2.4.

Analytical methods

Samples were taken from the bioreactor, 20 cm above the bottom of the column using a 50 mL syringe with a large entry. Since most of the granules were at the one-third of the bioreactor bottom, aeration rate was increased to ensure complete mixing before sampling. Around 15 mL of the complete bioreactor contents was taken out as sample. To avoid settling of the heavier granules during sampling, which would lead to erroneous results, the liquid velocity in sampling tube was kept very high. Before sampling, the piston of the syringe was

pulled back and maintained in a maximal aspiration position to obtain maximal negative pressure. Taken samples were immediately filtered through a 0.2 ␮m filter (Whatman). The dry weight concentration was determined by drying the sample for at least 24 h at 105 ◦ C. The sludge volume index SVI8 was determined by reading the height of the settled bed in the bioreactor after 8 min settling and calculated from the settled bed volume and the dry weight concentration in the bioreactor. The density of the granules was determined using a procedure described elsewhere (Beun et al., 2002). Ammonium, nitrate and nitrite were determined with standard test kits (MERCK, ammonium test 14752, nitrate test 09713, nitrite test 14776) using UV/vis spectrophotometer (Pharo 300, MERCK, Germany). Concentration of inorganic carbon (IC) of the effluent filtered was measured using a TOC/TN analyzer (Shimadzu FormacsHT , The Netherlands) to make sure that carbon source is not limiting. Granule samples (200–300 granules) were taken regularly every week from the bioreactor to observe the changes in diameter of the granules using a microscope (IX71, Olympus, Japan). Particle size distribution analysis of the granules was then carried out using image J software (Version 1.24). DO, pH and temperature were monitored continuously and IC in the effluent, dry weight of the bioreactor content, ammonium, nitrite and nitrate were measured every two days.

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The overall oxygen transfer coefficient (kL a) can be calculated using the oxygen balance over the bioreactor (Eq. (1)): dCL,O = kL a.(C∗L,O − CL,O ) − rO2 dt

(1)

with: (dCL,O /dt) = 0 under quasi-stationary condition. The DO concentration in the bioreactor (CL,O ) was measured on line as a percentage of the saturation concentration (C∗L,O ) with a DO probe (12/220 type T, Mettler Toledo, Switzerland) connected to a dissolved oxygen transmitter (4300, Mettler Toledo, Switzerland). The oxygen conversion rate (rO2 ) was calculated from the nitrite (rNO2 − ) and nitrate (rNO3 − ) production rates: rO2 =

32 24 − + − r r 7 NO3 7 NO2

(2)

Nitrate and nitrite production rates were obtained from mass balances of nitrate and nitrite over the bioreactor assuming no accumulation in the system. The FA concentration was estimated using Eq. (3), as described elsewhere (Anthonisen et al., 1976). FA =

pH [NH+ 17 4 − N] × 10 × 14 Ka /Kw + 10pH

(3) ◦

in which: Ka /Kw = e6334/(273+T C) and [NH+ 4 − N] is ammonium concentration. Ka and Kw are ionization constants of ammonia and water, respectively. The percentages of ammonium removal and nitrate or nitrite production were calculated according to Eqs. (4) and (5), respectively. %removal [NH+ 4 − N] =

+ [NH+ 4 − N]i − [NH4 − N]o

%production [NO− x − N] =

[NH+ 4 − N]i

[NH+ 4

[NO− x − N]o

× 100

− N]i − [NH+ 4 − N]o

× 100

(4)

(5)

where subscripts of i and o refer to the influent and effluent concentrations, respectively. In the statistical data analysis, main factors and interactions with a P-value less than 0.05 were considered as important and effective parameters on the output variable of the system.

3.

Results and discussion

3.1. Bioreactor startup performance and nitrifying granules formation System startup and the formation of mature nitrifying granules lasted for four months. During the startup period, the bioreactor was operated with the inlet ammonium concentration of 50 mgNH4 + -N/L. Fig. 2 illustrates the time courses of ammonium, nitrite and nitrate concentrations during this period. During first month of the operation, the ammonium removal was negligible. After around 40 days, the biomass concentration started to increase because of granule formation and consequently a rapid increase of the ammonium conversion was observed. After almost two months, the obtained results revealed a complete conversion of ammonium. Initially nitrite formation rate was rather faster than nitrate formation, while after day 60, the nitrite accumulation leveled

Fig. 2 – Concentrations of ammonium, nitrite and nitrate during startup of the bioreactor. off. On day 68, the ammonium conversion to nitrate reached 54%, showing a higher conversion of ammonium to nitrate. Fig. 2 shows an almost complete conversion of ammonium to nitrate, achieving 96% on day 90. Due to no oxygen limitation during the startup period, the initial accumulation of nitrite can be related to the slower growth rate of NOB compared to AOB as well as the presence of FA in the system. The FA concentration was calculated to be in the range of 0.5–4.6 mg/L during the first 60-day of the startup period. It has been reported that the minimum inhibition concentrations of FA for AOB and NOB are 10 mg NH3 -N/L and 0.1–1.0 mg NH3 -N/L, respectively (Anthonisen et al., 1976). Other reports noted that the nitrite oxidizers can acclimate to the high FA concentration when an acclimated biomass is used. For instance, the FA levels as high as 40 mg/L did not appear to inhibit the nitrite oxidation step with the acclimated biomass, while in a non-acclimatized sludge at the FA concentration of 3.5 mg/L already nitrite oxidation inhibition occurred (Turk and Mavinic, 1989). The development of the nitrifying granules in the bioreactor was followed during the startup period. The high shear force and low HRT are among the main parameters which favors the development of aerobic granular sludge. Hydrodynamic shear force promotes production of extracellular polymer substance (EPS) which plays an important role in granulation (Tay et al., 2002). It was found that aerobic granules could be formed only above a threshold shear force value in terms of upflow air velocity above 1.2 cm/s (Liu and Tay, 2004). In the present study, granule formation was achieved as a result of operating the bioreactor under high shear forces (superfacial air veocity of 1.9 cm/s) and low HRT of 2.5 h. According to the literatures, biofilm formation can be stimulated if the dilution rate is higher than the maximum specific growth rate of the micro-organisms (Tijhuis et al., 1992). Under these conditions, suspended cells are washed out and the main substrate remains available for bacteria grown as granules. The maximum specific growth rate, ␮max , of pure cultures of nitrifying bacteria lies in the range of 0.014–0.064 h−1 (Prosser, 1990). Therefore, HRT was adjusted to 2.5 h which was much shorter than the residence time corresponding to the washout dilution rate for nitrifying bacteria (∼15–70 h), to stimulate granule formation. In the present study, the used seed sludge had initially irregular morphology. During first 2 days of operation, inoculated sludge was almost completely washed out as a result of the short HRT of 2.5 h. During first three weeks, granules were

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nearly absent, while after almost four weeks operation a large number of the small granules appeared in the system. After nearly 40 days, the average diameter of the granules increased and ranged between 0.2 and 0.4 mm. Microscopic observations showed no more flocs in the system. In general, granulation process consists of the self-immobilization of bacterial communities and a relatively long period is needed to form initial aggregates of the microorganisms or self aggregation. As soon as the first aggregates appeared in the system, quick growth of the granules and sudden increase of the granules diameter were observed. The installed three phase separator could act very efficient in retaining small granules inside the bioreactor. The superficial liquid velocity in the settler was about 0.5 m/h. Consequently, only flocs biomass pieces with settling velocity higher than 0.5 m/h could be retained in the bioreactor and large part of small-loose sludge flocs were washed out. The development of the granules was monitored by random sampling around 200–300 granules from the bioreactor every week and analysis of taken images. By day 45, most of the granules were smaller than 0.5 mm in diameter, while the granule size distribution shifted to the higher values over the operation time. The increase in average size of the granules was found to be around 0.8, 1.1, 1.3 and 1.4 mm on days 60, 80, 100 and 112, respectively. Size distribution of the granules on day 42 and day 112 are shown in Fig. 3. Despite a continued increase in the biomass concentration during the rest of experimental period, no significant differences in the granules size distributions as well as the average diameter of the granules were observed, revealing a considerable increase in the number of the granules in the bioreactor. The biomass concentration was increased up to 2.9 gVSS/L at the end of startup period. The obtained results showed that the mature granules were formed during four months with an average granules size of 1.4 mm. The mature granules had compact structure with high settling velocities of around 55 m/h, the specific gravity of 1.03 and SVI8 value of below 45 mL/g, which is significantly higher than that of activated sludge flocs. The granules had yellowbrownish color and no filamentous bacteria were observed on the surface of the granules. Fig 3c and d shows the typical appearance of the nitrifying granules after the two-month operation of the bioreactor and the mature granule, respectively. Although there are many studies for the formation of the nitrifying granules in SBRs, fewer attempts have been carried out on the granule formation in continuous bioreactors. For instance in SBRs, formation of nitrifying granules with an average diameter of 1.9–2.9 mm (settling velocities around 100 m/h) (Vázquez-Padín et al., 2010), 0.32 mm (after 120-day operation) (Shi et al., 2010), 0.9 mm (after 400 days of operation) (Belmonte et al., 2009) and 240 ␮m (on day 21) (Liu et al., 2008) has been already reported. In a continuous nitrifying AUFB bioreactor, elliptical nitrifying granules has been obtained with a diameter of 346 ␮m and no filamentous bacteria on the granules surface (Tsuneda et al., 2003). In another continuous work with airlift bioreactor, the nitrifying granules appeared with some filamentous bacteria on the surface of the granules having a mean diameter of 1.54 mm and settling velocities of 82 m/h (Jin et al., 2008). The characteristics of the granules obtained in the present study seem to coincide more with the cultivated granules observed in continuous-flow airlift bioreactor (Jin et al., 2008). In the present study, the aeration rate of 4.5 L/min (1.28 vvm) seems to provide sufficient hydrodynamic shear

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force for nitrifying granules formation. The result shows that stable mature nitrifying granules could be formed in a continuous bubble column bioreactor during four months which was longer than 75 days reported for continuous airlift bioreactor at aeration rate of 1.5 vvm (Jin et al., 2008) and shorter than 300 days in an aerobic upflow fluidized bed bioreactor (Tsuneda et al., 2003). The long HRT applied during start up period (5 days), the recycling of washed out sludge and low aeration rate (0.16 vvm) seems to be three main reasons for the prolonged startup period in the work of Tsuneda et al. (2003). However, it has been reported that using the aerobic granular sludge as seed, which contains considerable nitrifying bacteria, is very effective for the rapid start up of the nitrifying bioreactor (Kishida et al., 2012). Nevertheless, the long time required to achieve complete ammonium removal in the nitrifying granulation system is because of the lower growth rate of nitrifying microorganism compared to heterotrophic bacteria (0.033 h−1 and 0.3 h−1 at 20 ◦ C, respectively) (Wiesmann, 1994), as well as the lower growth yield of nitrifiers.

Effect of ammonium loading and aeration rate on 3.2. DO and nitrification Influence of the ALR and aeration rate on DO concentration as well as the nitrification process was studied in detail by manipulating the inlet ammonium concentration at two aeration rates of 4.5 and 2.5 L/min. The bioreactor was operated under a constant HRT of 2.5 h during the entire operation period.

3.2.1.

Biomass concentration

Fig. 4a illustrates the variation of the biomass and DO concentrations for the varied ALR values at the airflow rates of 2.5 and 4.5 L/min. Initial aggregates of microorganisms formed after four weeks operation of the bioreactor. After nearly 40 days, the granules diameter and consequently the biomass concentration and ammonium conversion started to increase. The biomass concentration reached to 2.9 gVSS/L after the four months operation of the bioreactor. At the high aeration rate, the biomass concentration increased over a 50-day operation period after the system startup from 2.9 to 13.8 gVSS/L with increasing the ALR values. The biomass concentration then remained approximately constant with a slight decrease between days of 180 and 190, due to the low ALR value of 0.48 gNH4 + -N/L d applied to the bioreactor. With reducing the aeration rate to 2.5 L/min, the biomass concentration showed a slight fluctuation around 12 gVSS/L for the ALR values less than 1.44 gNH4 + -N/L d with an increase to 16.5 gVSS/L for the higher ALR values at the remaining operation period until day 230. In biofilm airlift suspension bioreactor, a relatively high biomass concentration of 16 and 27 g/L has been reported after 110 and 150 days of operation, respectively, at ALR value of 5 gNH4 + -N/L d (Tijhuis et al., 1995). The biomass concentration of 30 g/L has also obtained after 100 days at the same ALR value of 5 gNH4 + -N/L d (van Benthum et al., 1996).

3.2.2.

DO concentration

DO concentration can be considered as one of the main factors effecting the nitrification process and performance of nitrifying bioreactor. ALR and aeration rate are two important parameters that affect the DO concentration inside bioreactors. Fig. 4a shows the time course of DO concentration during system startup period and under the different ALR values and aeration rates.

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Fig. 3 – Particle size distribution of the granules (a) on day 42 (start of the granulation), (b) on day 112 (a typical granules size distribution for the rest of the experimental period), (c) photograph illustrating appearance of the nitrifying granules after two months operation of the bioreactor and (d) a typical microscopy image of mature nitrifying granules.

Fig. 4 – (a) Variation of ALR, air flow rate and DO concentration during 230 days and biomass concentration after 40 days operation of the bioreactor and (b) variation of concentrations of ammonium, nitrite and nitrate after startup period due to change in influent ammonium concentration at air flow rates of 4.5 L/min (between days 120 and 190) and 2.5 L/min (between days 190 and 230) at the constant HRT of 2.5 h.

At the first four runs the DO concentration showed a considerable reduction from 7.1 to 2.3 mg/L, due to increasing the ALR value applied to the bioreactor (from 0.48 to 1.92 gNH4 + -N/L d). With a stepwise decrease of the ALR, the DO concentration increased from 2.3 to 6.3 mg/L. The differences observed in the DO values of the first four runs with the second ones, at the same ALR values, can be owing to the increase in the biomass concentrations. At biomass concentrations above 10 gVSS/L, the increase in biomass concentration (solid hold up) caused a decrease of the oxygen mass transfer coefficient. For instance, the kL a values were measured to be 1608, 1440 and 1248 1/d at the biomass concentrations of 10, 13.8 and 16.5 gVSS/L, respectively. As described elsewhere (Chisti, 1989) the increased solids content leads to dampening of the turbulence and bubble coalescing and as a result, the increasing solid hold up due to granules formation provokes a drop in the kL a values in the bioreactor. On day 190, with decreasing the airflow rate to 2.5 L/min, the DO concentration showed about 16% reduction (from 6.3 to 5.3 mg/L) at the lowest ALR value of 0.48 gNH4 + -N/L d. The DO concentration was then reduced to 2 mg/L when the ALR value increased to 0.96 gNH4 + -N/L d and reached to nearly 1 mg/L when the ALR values applied were above 1.44 gNH4 + -N/L d. Table 1 shows the analysis of variance for the DO values. According to this table, both main factors of the ammonium loading and aeration rates are significant (P < 0.05) with F0 values of 130.66 and 87.89, while there is no significant interaction between the two main factors. This means that the ALR is more important than the aeration rate for the DO concentration changes inside nitrifying bioreactor. Indeed, the effect of ALR on the DO values does not depend on the level chosen for the aeration rate in the range of tested values in this study.

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Table 1 – Analysis of variance for the DO concentration. Source of variation

Degrees of freedom

Sum of squares

3 1 1 2 7

21.92 4.91 0.10 0.11 27.04

ALR Aeration rate Interaction Error Total

Many researchers have studied the influence of DO concentration on the nitrification process at a constant ALR by manipulating aeration rate to have a defined DO concentration inside bioreactors (Garrido et al., 1997; Bernet et al., 2001; Bae et al., 2002). In the present study, the effect of simultaneous changes of aeration rate and ALR on the DO concentration was elucidated by using the experimental design technique. As shown in Fig. 4a, increasing ALR or decreasing aeration rate resulted in a reduction of DO concentration. The importance of both main factors of ALR and aeration rate and lack of their interactions for variation of DO concentration also coincide with our previous study in a submerged fixed film bioreactor at different organic loading and aeration rates (Baradar Khoshfetrat et al., 2011).

3.2.3.

Nitrification performance

Fig. 4b shows profiles for the ammonium, nitrite and nitrate concentrations versus operation time at the various inlets ALR and the airflow rates of 4.5 and 2.5 L/min. At the airflow rate of 4.5 L/min (days 120–190) and the ALR values less than 1.44 gNH4 + -N/L d, ammonium was completely converted to nitrate. Despite increasing or decreasing the ALR values, the new steady state conditions were reached within one day without any critical changes in the effluent ammonium concentration. However, at the ALR value of 1.92 gNH4 + -N/L d, incomplete conversion of ammonium to nitrate was observed (around 58% conversion to nitrate). The complete conversion of ammonium to nitrate at the lower ALR values can be because of the enough aeration rate of 4.5 L/min which provides a relatively high DO concentration in the bioreactor (i.e. >4.0 mg/L). When the ALR increased to the higher value of 1.92 gNH4 + -N/L d, the DO value was in the range of 2.3–3 mg/L and nitrite was accumulated in the system. Since the residual ammonium concentration in the bioreactor was low, the observed accumulation of nitrite cannot be due to the FA (less than 0.3 mg/L) inhibition. Therefore, conversion of ammonium to nitrate is limited by the oxygen concentration. Oxygen penetration depth was calculated to be in the range of 150–200 ␮m. When the granules size lies in the range of more than 200 ␮m, the inner parts suffer from oxygen limitation and nitrite production will be superior to nitrate production, due to a lower specific affinity of NOB for oxygen (Blackburne et al., 2008a). The half saturation constants of oxygen for AOB and NOB are 0.3 and 1.1 mg/L, respectively (Wiesmann, 1994). On day 190, the airflow rate was decreased to 2.5 L/min. For the ALR values until 0.96 gNH4 + -N/L d (influent ammonium concentration up to 100 mgNH4 + -N/L), ammonium was completely converted to nitrate while the nitrite accumulation was negligible. When the inlet ammonium concentration increased to 150 mgNH4 + -N/L, corresponding to the ALR value of 1.44 gNH4 + -N/L d, the nitrate formation went down dramatically accompanying with a rapid jump in the nitrite accumulation. Finally, almost complete ammonium conversion to nitrite was achieved during the last 4-day, in which the DO concentration was observed to be between 1.0 and

Mean square 7.31 4.91 0.10 0.05

F0 130.66 87.89 1.79

P-value 0.0014 0.0026 0.2732

1.2 mg/L (Fig. 4a). Nitrite accumulation at low DO concentration was also reported by other researchers in which more than 50% nitrite accumulation with a good ammonium conversion was obtained at the DO concentration around 1.5 mg/L and the ALR value of 5 gNH4 + -N/L d (influent ammonium concentration of 200 mgNH4 + -N/L) in a biofilm airlift suspension bioreactor, while at the DO value above 2.5 mg/L ammonium was completely converted to nitrate (Garrido et al., 1997). With further increasing the inlet ammonium concentration to 200 mgNH4 + -N/L (loading rate of 1.92 gNH4 + -N/L d), the ammonium removal was decreased to about 65% accompanied by a reduction in nitrite concentration to around 128 mgNO2 − -N/L. The DO concentration was observed at the low level of around 1 mg/L. The reduction in ammonium removal was mainly due to the oxygen transfer rate limitation. The oxygen mass transfer coefficient and oxygen penetration depth decreased due to an increase in biomass concentration and reduction in DO concentration in the liquid bulk, respectively. At biomass concentrations higher than 10 gVSS/L, oxygen mass transfer coefficient was observed to be more sensitive to the biomass concentration variations. For instance, at ALR value of 1.92 gNH4 + -N/L d, by increasing of the biomass concentration from 14.5 to 16.5 gVSS/L, the measured oxygen mass transfer coefficient value showed a reduction of around 8%. The results obtained showed that at the aeration arte of 4.5 L/min, the oxygen limiting condition begins at ALR value of 1.44 gNH4 + -N/L d. A further increase in the ALR value to 1.92 gNH4 + -N/L d, corresponding to DO concentration of 2.3–3 mg/L, resulted in nitrite accumulation. However, at aeration rate of 2.5 L/min, oxygen limiting condition was occurred at the lower ALR value of 0.96 gNH4 + -N/L d. It was also observed that increasing the ALR value resulted in nitrite accumulation and finally incomplete ammonium conversion, where the DO concentration was in the range of 1–1.2 mg/L. This means that high ammonium removal rate can be achieved in an oxygen non-limiting condition. For example, the ammonium removal rate of 16.7 gNH4 + -N/L d has been reported in an AUFB bioreactor when pure oxygen was supplied to the bioreactor (Tsuneda et al., 2006). Despite the changes in ALR and aeration rates and a continued increase in the biomass concentration, the average diameter of the granules did not change significantly and remained around 1.4 mm (days 120–230) in the present study. The SVI8 values were measured to be in the range of 35–48 mL/g at high aeration rate (days 120–190) and 42–54 mL/g at low aeration rate (days 190–230). The dense structure of the granules obtained in continuous mode may be mainly owing to the slow growth rate and low yield of the nitrifying bacteria. It has also been reported that feeding pattern, i.e., continuous and intermittent feeding, is not an important factor in aerobic granulation when completely inorganic wastewater is fed to the bioreactor (Kishida et al., 2010). The results of overall nitrogen balances after startup period are presented in Table 2. The nitrogen loss was calculated to be

Please cite this article in press as: Hosseini, M., et al., Continuous nitrifying granular sludge bioreactor: Influence of aeration and ammonium loading rate. Process Safety and Environmental Protection (2013), http://dx.doi.org/10.1016/j.psep.2013.08.003

Time-after startup period (day)

Inlet ammonium concentration (mgN/L)

0.48 0.96 1.44 1.92 1.44 0.96 0.48 0.48 0.96 1.44 1.92

Outlet ammonium concentration (mgN/L)

4.5 4.5 4.5 4.5 4.5 4.5 4.5 2.5 2.5 2.5 2.5

Nitrate concentration (mgN/L)

0 0 1 2 3 1 0 0 3 5 70

Nitrite concentration (mgN/L)

48 94 147 116 138 92 46 49 96 1 0

Nitrogen loss (%)

0 0 0 76 0 5 0 0 0 139 128

4 6 1.3 3 6 2 8 2 1 3.3 1

Table 3 – Analysis of variance for percentages of (a) ammonium removal, (b) nitrite production and (c) nitrate production. Source of variation

Degrees of freedom

Sum of squares

Mean square

F0

P-value

Sum of squares

Mean square

(a) ALR Aeration rate Interaction Error Total

3 1 1 2 7

449.98 132.84 368.42 4.76 956.01

149.99 132.84 368.42 2.38

F0

P-value

Sum of squares

Mean square

(b) 62.95 55.75 154.62

0.0042 0.0050 0.0011

6493.23 3321.12 2947.44 569.68 13331.48

2164.41 3321.12 2947.44 284.84

F0

P-value

(c) 7.59 11.65 10.34

0.0704 0.0420 0.0487

6594.16 3310.94 2565.43 454.76 12925.29

2198.05 3310.94 2565.43 227.38

9.67 14.56 11.28

0.0529 0.0317 0.0438

ARTICLE IN PRESS

50 100 150 200 150 100 50 50 100 150 200

Aeration rate (L/min)

Process Safety and Environmental Protection x x x ( 2 0 1 3 ) xxx–xxx

130 140 150 160 170 180 190 200 210 220 230

ALR (gNH4 + -N/L d)

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Table 2 – Performance of the nitrifying bioreactor at different inlet ammonium concentrations and airflow rates (after startup period).

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in the range of 1–8%, which could be partly because of denitrification. As the granule size increases, an anoxic zone is created in the inner part of the granules and consequently the denitrification relying on soluble microbial products would occur. The analysis of variance was performed to evaluate statistically the effect of ALR and aeration rate and their interaction on the performance of the nitrifying granular sludge system. The percentages of the ammonium removal and produced nitrite and nitrate were considered as the typical outputs of the system (Table 3). Because of smaller changes in the biomass concentration among days of 155–230 in comparison with the early period, the data obtained after around day 155 was used for the statistical analysis. According to Table 3a, the main factors of the ALR and aeration rate by considering the 95% confidence interval have impact on the ammonium removal, with the F0 values of 62.95 and 55.75 respectively. The statistical analysis also show that the interaction is more important than the both main factors with the F0 value of 154.62, meaning the increasing or decreasing effect of ALR on the ammonium removal is extremely dependent on the level chosen for aeration rate. Using the information given in Table 3b and c for the nitrite and nitrate formation, it can also be concluded that the aeration rate is the main factor with the F0 values of 11.65 (P < 0.05) and 14.56 (P < 0.05), respectively. The obtained F0 values of aeration rate for the nitrite and nitrate accumulation support the experimental results, in which NOB are more influenced by DO concentration than AOB, due to their lower affinity for oxygen. Interestingly, although the main factor of ALR showed a mild effect on the nitrite and nitrate formation at the used confidence interval, the interactions of the ALR-aeration rates were considerable with the F0 values of 10.34 (P < 0.05) and 11.28 (P < 0.05), respectively. The interactions indicate that the effect of ALR on the nitrite and nitrate formation varies with the aeration rate changes. The increase of ALR has smaller influence at higher aeration on the nitrite and nitrate accumulation in comparison with lower aeration. The experimental results as well as their statistical analysis indicated that the manipulation of ALR values and aeration rates as the operational parameters can be used to control the nitrite accumulation. To achieve nitrite accumulation, the system should be operated under oxygen limiting conditions. However, the DO concentration under oxygen limiting condition will not be sensitive enough to be considered as the only control parameter. Therefore a simple, practical strategy approach for partial nitrification can be performed as follows. First, the oxygen limiting condition should be reached either by increasing ALR or decreasing aeration rate. When oxygen limitation is attained, further increasing of ALR to aeration rate ratio will lead to nitrite accumulation. In practice, when ALR is fixed because of constant inlet flow rate and concentration, the reduction of the aeration rate is the only way to achieve the required nitrite accumulation. At lower aeration rates which shear force falls under the threshold value; procedures such as off-gas recycling can be applied to avoid disintegration of the formed granules. The results of the present study indicated that under the oxygen limiting conditions, the ratio of nitrite to nitrate production can be estimated based on the maximal oxygen transfer rate capacity of the system. It is apparent that further increase in the ALR to aeration rate ratio, results in incomplete conversion of ammonium.

4.

9

Conclusions

Nitrifying granules were successfully cultivated using a continuous bubble column bioreactor. After almost four months, stable nitrifying granules with average diameter of 1.4 mm and SVI8 value of below 45 mL/g were obtained. When the DO concentration was over 4 mg/L, ammonium was fully converted to nitrate regardless of the ALR value. Nitrite accumulation was observed for the aeration rates of 4.5 and 2.5 L/min, at the DO values of below 3 and 1.2 mg/L, respectively, corresponding to the ALR values of 1.92 and 1.44 gNH4 + -N/L d. Therefore, under oxygen limiting conditions, nitrite production prevails over nitrate. To promote the nitrite accumulation, the results showed the ALR to aeration rate ratio should be increased under oxygen limiting condition. The present study demonstrated the potential of the continuous bioreactor for obtaining stable nitrifying granules to remove nitrogen from wastewater with changing the ratio of operating parameters of aeration rate and ALR.

Acknowledgment The authors would like to thank Rural Water and Wastewater Organization of East Azarbijan Province for supporting in part of the project (No. 4.4884).

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Please cite this article in press as: Hosseini, M., et al., Continuous nitrifying granular sludge bioreactor: Influence of aeration and ammonium loading rate. Process Safety and Environmental Protection (2013), http://dx.doi.org/10.1016/j.psep.2013.08.003