Journal of Biotechnology 106 (2003) 7786

A novel granular sludge sequencing batch reactor for removal of organic and nitrogen from wastewater
Shu-Fang Yang, Joo-Hwa Tay, Yu Liu
Environmental Engineering Research Centre, School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore Received 3 June 2003; received in revised form 14 July 2003; accepted 24 July 2003

Abstract Microbial granules were developed at different substrate N/COD ratios in sequencing batch reactors (SBR). Results showed that heterotrophic, nitrifying, and denitrifying populations could peacefully co-exist in microbial granules, while increased substrate N/COD ratio led to signicant shifts among three populations in granules. Enhanced activities of nitrifying and denitrifying populations were obtained in microbial granules developed at high substrate N/COD ratios, however, heterotrophic populations in granules tended to decrease with the increase of substrate N/COD ratio. It was found that dissolved oxygen (DO) concentration had a pronounced effect on the efciency of denitrication by microbial granules, meanwhile the results also indicated that a certain mixing power would be provided to ensure mass transfer between liquid and granules during denitrication. It was demonstrated that complete organics and nitrogen removal can be achieved in single granule-based SBR with high efciency and stable performance. This is the rst study to show the capability of microbial granules in simultaneous removal of organic carbon and nitrogen from wastewater. 2003 Elsevier B.V. All rights reserved.
Keywords: Microbial granule; Substrate N/COD ratio; Organic removal; Nitrication; Denitrication

1. Introduction As stricter environmental regulations are imposed, advanced and cost effective techniques for nitrogen removal from wastewater become more and more important. Many modications and processes had been developed and implemented for nitrogen removal from wastewater. Basically, these processes for nitrogen removal can be classied as suspended sludge and xed-lm cultures. The suspended sludge systems

Corresponding author. E-mail address: cyliu@ntu.edu.sg (Y. Liu).

suffer from sludge bulking, large reactor volume required, sensitive to shock loading, while the xed-lm systems usually encounter biolm-associated clogging and sloughing problems and so on. Meanwhile, due to the sensitivity of nitrifying bacteria to environmental factors as well as their lower growth rates, it is difcult to obtain and maintain sufcient nitrifying biomass in conventional suspended or xed culture-based wastewater treatment systems (Moreau et al., 1994; Ballinger et al., 2002; Ochoa et al., 2002), while nitrication is the rst step towards denitrication that converts nitrate and nitrite to nitrogen gas.

0168-1656/$ see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2003.07.007

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Aerobic granulation is a recently described phenomenon and currently under active investigation as potential bioagents in wastewater treatment (Beun et al., 1999; Peng et al., 1999; Tay et al., 2001; Moy et al., 2002). Compared to conventional aerobic wastewater treatment systems, the granulation system offers several advantages, such as a denser and stronger microbial structure, good settleability, high biomass retention, and ability to withstand high organic loading rate. The aerobic granulation technology appears to have the potential to respond to the challenges of nitrogen removal from wastewater. Therefore, hybrid aerobic granules capable of simultaneously removing organic carbon and nitrogen are highly desired since wastewater often contains both organics and nitrogen. Complete nitrogen removal involves nitrication and denitrication. Nitrite and nitrate produced from nitrication is required to be reduced to gaseous nitrogen by denitriers. It is known that denitrication is an anoxic process that is affected by dissolved oxygen (DO). To date, there is very limited information on simultaneous organic and nitrogen removal by microbial granules. Thus, this study was focused on the development of aerobic granules at different substrate N/COD ratios, the feasibility of simultaneous organic and nitrogen removal in a single granule-based bioreactor as well as the effect DO and mixing on denitrication efciency by microbial granules.

In order to look into the denitrication capability of microbial granules developed at different substrate N/COD ratios, the SBR cycle time was increased to 6 h comprising 4 min of feeding, 230 min of aeration, 2 h of anaerobic or anoxic period, 2 min of settling, and 4 min of efuent withdrawal, from day 340 onwards. The following three series of experiments were then carried out: (i) The DO concentrations in all reactors were decreased to 0.8 mg l1 by lowering the air ow rate to 1.0 l min1 from day 342. (ii) Starting from day 350, the reactor DO was further lowered down to 0.5 mg l1 by reducing aeration rate to 0.5 l min1 . (iii) A DO-free condition was created in all reactors by stopping aeration from day 355 onwards. At the beginning of anaerobic or anoxic phase, ethanol as external carbon source for denitrication was fed to the reactors at a concentration of 600 mg COD l1 . 2.2. Media Reactors 14 were inoculated by 650 ml of fresh activated sludge (equivalent to 3000 mg l1 suspended solid) taken from a local municipal wastewater treatment plant. This gave an initial reactor biomass concentration of 2000 mg dry weight l1 . Synthetic substrate mainly consists of ethanol as sole carbon source, ammonium chloride, sodium bicarbonate, and other necessary nutrients. The ethanol chemical oxygen demand (COD) concentration was xed at 500 mg l1 , while the ammonium-nitrogen concentration varied from 25 to 150 mg l1 in R1 to R4, which gave a respective substrate N/COD ratio of 5/100 to 30/100. To satisfy the growth requirement of nitrifying bacteria, the ratio of bicarbonate to ammonium-nitrogen was kept constant at a value of 8.0 mg mg1 for all reactors. The micronutrients in the synthetic wastewater can be found elsewhere (Liu and Capdeville, 1996). The reactor pH fell into a range of 8.2 and 7.5. The experiments were conducted in a temperature control room of 25 C. 2.3. Analytical methods 2.3.1. Organic and nitrogen concentrations in solution Ammonium, nitrite, and nitrate concentrations were measured by using a ow injection analyzer (QuikChem Method 10-107-06-1-I, Lachat Instru-

2. Materials and methods 2.1. Reactor set-up and operation Four columns (80 cm in height and 6 cm in diameter) with a working volume of 2.4 l were used as sequencing batch reactors (SBR), and each reactor had the same geometrical conguration. The reactors were operated over 1 year. Before day 340, reactors 14 (R1 to R4) were supplied with an air ow rate of 4.0 l min1 , equivalent to a supercial upow air velocity of 2.4 cm s1 . During this time, DO concentration in reactors was above 2.0 mg l1 . All reactors were operated at a cycle time of 4 h, in a sequential mode: 4 min of feeding, 230 min of aeration, 2 min of settling, and 4 min of efuent withdrawal. Efuent was discharged from the middle port of the column reactor.

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ments, Inc.), while COD concentration was determined by standard method (APHA, 1998). 2.3.2. Specic oxygen utilization rate The specic oxygen utilization rate (SOUR)h by heterotrophic bacteria and specic ammonium and nitrite oxygen utilization rates, (SOUR)NH4 and (SOUR)NO2 by ammonium oxidizers and nitrite oxidizers, were measured using standard methods (APHA, 1998). A certain amount of granule sample was carefully washed with tap water, and was put in a pre-cleaned BOD bottle. Then, the BOD bottle was fully lled in with the pre-aerated nutrient and substrate solution, and the oxygen-sensing probe with stirring mechanism was immediately inserted into the BOD bottle. The decrease of DO was recorded at an interval of 15 s. Specic oxygen utilization rate was calculated according to the DO concentration recorded over time. The respective substrates used for determination of (SOUR)h , (SOUR)NH4 , and (SOUR)NO2 were ethanol, NH4 Cl, and NaNO2 , while the biomass, COD, NH4 -N, and NO2 -N concentrations were kept constant at 500, 400, 20, and 20 mg l1 , respectively. The SOUR tests were conducted at 25 C. 2.3.3. Physical characteristics of granule The size of granular sludge was obtained by using laser particle size analysis system (Malvern Mastersizer Series 2600) or image analyzer (Quantimnet 500 Image Analyzer, Lecia Cambridge Instruments). Suspended solid (SS) and volatile suspended solid (VSS) were determined by standard method (APHA, 1998).

when the substrate N/COD ratio increased from 5/100 to 30/100. Microscopic observation indicates that the aerobic granules developed in four reactors had compact structure with a clear spherical outer shape as compared to the seed sludge (Fig. 1). 3.2. COD and nitrication proles under aerobic conditions Fig. 2 shows COD and nitrication proles in R1 to R4 operated at a cycle time of 4 h. The salient points of the data are that (i) almost all inuent COD are removed in the rst 30 min; (ii) no nitrite and nitrate are produced in R1 run at a substrate N/COD ratio of 5/100, while typical nitrication proles are observed in R2 to R4 operated at a respective substrate N/COD ratio of 10/100, 20/100, and 30/100; (iii) a complete nitrication occurs after the COD removal in R2 to R4; (iv) ammonium-nitrogen removal in the rst 30 min of the cycle is the result of microbial growth requirement for nitrogen source instead of nitrication because neither nitrite or nitrate is produced in this period; (v) no lag nitrate production with respect to nitrite formation is observed. In fact, nitrication is mainly completed by two kinds of bacteria, ammonium oxidizers responsible for nitrite formation, and nitrite oxidizers for converting nitrite to nitrate. Under the normal culture conditions, at least two factors can inuence the nitrication proles, i.e. the relative specic growth rates of ammonium oxidizers to nitrite oxidizers in microbial community, and the relative mass ratio between these two species in the system (Liu and Tay, 2001). 3.3. Denitrication under DO-free condition without mixing The COD and denitrication proles in R2 to R4 under DO-free condition are shown in Fig. 3. It can be seen that slight denitrication occurred in the reactors. The total nitrogen removal efciency is 21, 24, and 26% in R2 to R4, respectively, while the COD removal efciency is also very low under this operation condition. Since aerobic granules which developed had much higher specic gravity as compared to water, they all settled down to the bottom of the reactor in the case where no sufcient mixing was provided. This in turn results in a poor contact between granules

3. Results 3.1. Aerobic granulation at different substrate N/COD ratios The seed sludge had a mean oc size of 90 m. After 20 days operation, aerobic granules formed in four reactors, and the sizes of aerobic granules gradually stabilized at a mean diameter of 1.9 mm in R1, 1.5 mm in R2, 0.5 mm in R3, and 0.4 mm in R4, respectively, at day 40. The biomass concentrations in the reactors increased to above 10 g SS l1 at steady state. The VSS/SS ratio declined from 0.94 to 0.79

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Fig. 1. Morphology of sludge. (a) Seed sludge; (b) aerobic granules developed at substrate N/COD of 5/100; (c) aerobic granules at substrate N/COD ratio of 10/100; (d) aerobic granules at substrate N/COD ratio of 20/100; (e) aerobic granules at substrate N/COD ratio of 30/100. Bar: 1 mm.

and substrate solution, i.e. mass transfer efciency is much lowered. Consequently, the mass transfer limitation due to the lack of mixing would be responsible for the observed low denitrication efciency (Fig. 3).

3.4. Denitrication at DO of 0.5 mg l1 with mixing In this phase, the reactor DO was maintained at 0.5 mg l1 by regulating the aeration rate, which in turn ensured solidliquid mixing. Fig. 4 shows COD

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R1
500 400 COD (mg l-1) 300 200 100 0 0 60 120 Time (min) 180 COD NH4 25 20 Nitrogen (mg l-1) 15 10 5 0 240 COD (mg l-1) 500 400 300 200 100 0 0 60

R2
50 40 30 COD NH4 NO2 NO3 120 Time (min) 180 20 10 Nitrogen (mg l-1) Nitrogen (mg l-1)

0 240

R3
500 400 COD (mg l-1) 300 200 100 0 0 60 120 Time (min) 180 COD NH4 NO2 NO3 100 80 60 40 20 500 400 COD (mg l-1) 300 200 100 0 0 60

R4
150 120 90 COD NH4 NO2 NO3 60 30 0 240

0 240

Nitrogen (mg l-1)

120 Time (min)

180

Fig. 2. COD and nitrication proles in R1 to R4.

and denitrication proles observed in R2 to R4. It appears that complete denitrication occurred in R2 to R4. All nitrate converted to gaseous nitrogen during 2-h anoxic period. The respective specic total nitrogen reduction rate was 0.42, 0.85, and 0.91 mg N g1 SS min1 in R2 to R4. These values are comparable to those obtained in conventional biological processes (Glass and Silverstein, 1998; Foglar and Briki, 2003). 3.5. Denitrication at DO of 0.8 mg l1 with mixing In order to investigate the effect of DO on denitrication by microbial granules, the DO concentrations in all reactors were increased to 0.8 mg l1 by

increasing aeration rate. COD and denitrication proles at DO concentration of 0.8 mg l1 in R2 to R4 are shown in Fig. 5. The nitrogen removal efciency was about 40% in R2 to R4, while nitrate concentration was still high in the efuent in all reactors, indicating a partial denitrication as compared to the results presented obtained at the DO concentration of 0.5 mg l1 (Fig. 4). Figs. 4 and 5 suggest that the activity of denitrifying populations in microbial granules can be inhibited by high DO concentration. It had been known that the DO seems to act as an inhibitor to the activity of the denitrifying reductases rather than as a repressor of their synthesis, while denitrication can be ignored when dissolved oxygen concentrations are greater than 1.0 mg l1

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R2
700 600 COD (mg l-1) 500 400 300 200 100 0 240 280 320 Time (min) COD NO2 NO3 15 10 5 0 360 25 Nitrogen (mg l-1) COD (mg l-1) 20 700 600 500 400 300 200 100 0 240

R2
30 Nitrogen (mg l-1) Nitrogen (mg l-1) Nitrogen (mg l-1) COD NO2 NO3 25 20 15 10 5 0 360

280 320 Time (min)

R3
700 600 COD (mg l-1) 500 400 300 200 100 0 240 280 320 Time (min) COD NO2 NO3 60 40 20 0 360 100 Nitrogen (mg l-1) COD (mg l-1) 80 700 600 500 400 300 200 100 0 240

R3
100 COD NO2 NO3 80 60 40 20 0 360

280 320 Time (min)

R4
700 600 COD (mg l-1) 500 400 300 200 100 0 240 280 320 Time (min) COD NO2 NO3 90 60 30 0 360 150 Nitrogen (mg l-1) COD (mg l-1) 120 700 600 500 400 300 200 100 0 240

R4
150 COD NO2 NO3 120 90 60 30 0 360

280 320 Time (min)

Fig. 3. COD and denitrication proles under DO-free condition without mixing.

Fig. 4. COD and denitrication proles at DO concentration of 0.5 mg l1 with mixing.

(Eckenfelder and Argaman, 1991; Kornaros and Lyberatos, 1998). 3.6. Activities of heterotrophic, nitrifying, and denitrifying populations The respective activity of ammonium oxidizers and nitrite oxidizers was described by the specic ammo-

nium oxygen utilization rate (SOUR)NH4 , and the specic nitrite oxygen utilization rate (SOUR)NO2 , while the activity of heterotrophic bacteria was quantied by its specic heterotrophic oxygen utilization rate (SOUR)h . (SOUR)NH4 , (SOUR)NO2 , and (SOUR)h of steady-state aerobic granules which developed at different substrate N/COD ratios are shown in Fig. 6. It can be seen that both (SOUR)NH4 and (SOUR)NO2 in-

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R2
700 600 COD (mg l -1) 500 400 300 200 100 0 240 280 320 Time (min) 15 10 5 0 360
COD NO2 NO3

30 Nitrogen (mg l -1) 25 20

trogen reduction rate (qobs ) was presented in Fig. 7. It appears from Fig. 7 that high DO concentration results in a lower denitrifying activity described by qobs , while denitrifying populations in granules seems to be proportionally related to the substrate N/COD ratio or nitrifying populations.

4. Discussion Fig. 1 shows that microbial granules can form in a wide range of substrate N/COD ratios from 5/100 to 30/100. Over 95% of inuent COD was removed during aerobic phase, while the ammonium-nitrogen was completely converted to nitrate (Fig. 2). The settling velocity of the aerobic granules cultivated in R1 to R4 was greater than 60 m h1 , while the biomass retention reached about 9 g VSS l1 in all reactors. The settling velocity of conventional activated sludge was generally less than 10 m h1 (Campos et al., 1999). Compared to the conventional bioocs, excellent settleability of aerobic granules can ensure easy and effective separation of biosolids from the efuent, while high biomass concentration implies that a compact and small aerobic granular sludge reactor could be developed. It should also be realized that aerobic granular sludge reactors can be started up in 24 weeks, as opposed to anaerobic granulation systems (e.g. UASB), which require at least 4 months of careful incubation for start-up. In this study, the aerobic granular sludge reactors had been run stably over a period of 1 year before the experiments were terminated. These results seem to indicate that the use of aerobic granules for upgrading of the existing wastewater treatment plants towards simultaneous organics removal and nitrication may be feasible and benecial. It appears from Figs. 26 that heterotrophic, nitrifying, and denitrifying populations can co-exist in microbial granules developed at different substrate N/COD ratios. In fact, most denitrifying populations are facultative bacteria that are distributed among a wide variety of physiological and taxonomic groups. Under aerobic conditions, they can use oxygen as terminal electron acceptor, and under anoxic conditions they use nitrate and nitrite as terminal electron acceptor. Figs. 35 reveal the effect of DO and mixing on denitrication efciency of microbial granules. Since microbial granules developed are very heavy,

R3
700 600 COD (mg l -1) 500 400 300 200 100 0 240 280 320 20 0 360
COD NO2 NO3

100 Nitrogen (mg l -1) Nitrogen (mg l -1) 80 60 40

Time (min)

R4
700 600 COD (mg l -1) 500 400 300 200 100 0 240 280 320 Time (min) 30 0 360
COD NO2 NO3

150 120 90 60

Fig. 5. COD and denitrication proles at DO concentration of 0.8 mg l1 with mixing.

crease signicantly with the increase of the substrate N/COD ratio, while the (SOUR)h decreases with the substrate N/COD ratio. These indicate enriched nitrifying bacteria in the granules developed at high substrate N/COD ratios. Similar phenomena had been observed in biolm reactors (Moreau et al., 1994; Ochoa et al., 2002). In addition, the activity of denitrifying populations in granules in terms of specic total ni-

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Fig. 6. Heterotrophic and nitrifying activities in microbial granules. (SOUR)NH4 : specic oxygen utilization rate by ammonia oxidizer; (SOUR)NO2 : specic oxygen utilization rate by nitrite oxidizer; (SOUR)h : specic oxygen utilization rate by heterotrophs.

and they all settled down to the bottom of the reactor in the case where no sufcient mixing power is provided. As a result, the contact between granules and soluble nitrate is extremely poor, and denitrication would not occur efciently as observed in Fig. 3. Thus, in order to achieve efcient denitrication in granules-based bioreactor, a certain mixing is required to ensure a good contact between granules and soluble nitrate. Under the mixing conditions, it can be seen in Figs. 4 and 5 that DO less than 0.5 mg l1 will be advantageous for denitrication, while denitrication by microbial granules would be hindered at a DO of
1.0 qobs (mg N g-1 SS min-1) 0.8 0.6 0.4 0.2 0.0 0.1 0.2
-1

0.5 mg DO l-1 0.8 mg DO l-1

0.3

Substrate N/COD ratio (mg mg )

Fig. 7. Denitrifying activity of microbial granules developed at different substrate N/COD ratios.

0.8 mg l1 . In fact, the denitrication enzymes, once synthesized, will be maintained by the cells under aerobic conditions but their function was inhibited by the high DO concentration. On the other hand, it has been widely reported that dissolved oxygen inhibited each step of the denitrication pathway to some extent (Wild et al., 1995; Kornaros and Lyberatos, 1998). Fig. 6 shows that the activities of ammonium and nitrite oxidizers signicantly increased with the increase of the substrate N/COD ratio, while the heterotrophic activity in the microbial granules tended to decrease markedly. These results seem to indicate that the fraction of nitrifying populations over heterotrophic populations in microbial granules increases with increasing substrate N/COD ratio, i.e. the heterotrophic populations became less and less dominant. Similar phenomena were also reported in the biolm reactors (Fdz-Polanco et al., 2000; Ballinger et al., 2002). Fig. 7 shows the effect of DO and substrate N/COD ratio on the overall activity of denitrifying populations in microbial granules. As discussed earlier, denitrifying bacteria are very sensitive to the DO concentration in bioreactor. At DO of 0.5 mg l1 , denitrifying bacteria showed higher activity than at DO of 0.8 mg l1 . The respective specic total nitrogen reduction rate by microbial granules cultivated in R2 to R4 was 0.42, 0.85, and 0.91 mg N g1 SS min1 , at DO concentration of 0.5 mg l1 , which are compara-

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ble with the activity data obtained from conventional denitrication processes (Glass and Silverstein, 1998; Foglar and Briki, 2003). It should be realized that qobs tends to increase with the increase of substrate N/COD ratio, while Fig. 6 reveals that the increased substrate N/COD ratio results in an enriched nitrifying population in microbial granules, i.e. the nitrate concentrations in the reactors also increases with the substrate N/COD ratio as shown in Fig. 2. Batchelor (1982) proposed Eq. (1) to describe the effects of DO and nitrate concentration on the activity of denitrifying bacteria: qNO3 = qNO3 ,max SNO3 Sc ko kNO3 + SNO3 kc + Sc ko +DO (1)

in which, qNO3 is the specic nitrate reduction rate ( mg N g1 SS min1 ), qNO3 ,max is the maximum specic nitrate reduction rate, SNO3 is NO3 -N concentration, mg l1 , Sc is concentration of organic substrate, mg l1 , kNO3 is half-saturation constant for NO3 -N, mg l1 , kc is half-saturation constant for organic substrate, mg l1 , and ko is half-saturation constant for oxygen, mg l1 . According to this model, the increase of nitrate concentration will lead to the increase of the specic nitrate reduction rate, while an increased DO decreases the specic nitrate reduction rate. The experimental data are in good agreement with the prediction of this model. Consequently, it appears from this study that heterotrphic, nitrifying and denitrifying populations can co-exist in microbial granules, and a novel high-efciency granules-based bioreactor could be expected for simultaneous organics and nitrogen removal.

ules over conventional activated sludge, i.e. different species may co-exist in the same microbial matrix, which provides a platform for bacteria to function synergically. In this case, a more compact bioreactor for carbon and nitrogen removal could be expected. It was demonstrated that DO concentration and mixing are two factors inuencing the efciency of denitrication by microbial granules. Complete denitrication was achieved at DO concentration of 0.5 mg l1 , while a certain mixing is necessary to ensure a sufcient contact between granules and soluble nitrate, otherwise denitrication by microbial granules would be slowed down markedly. This study opens a door for environmental engineers to further develop novel compact and high-efciency granules-based biological process for removing both organic and nitrogen from wastewater. References
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5. Conclusions Microbial granules cultivated at different substrate N/COD ratios in SBRs are capable of simultaneously removing organic and nitrogen. It was found that heterotrophic, nitrifying, and denitrifying populations can co-exist in the granules, and shifts in microbial population in granules were closely related to the substrate N/COD ratio. The microbial granules developed at high substrate N/COD ratios exhibited enhanced nitrifying and denitrifying activities, while the activity of heterotrophic bacteria in granules showed a decreasing trend. This is the advantage of microbial gran-

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