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DO diffusion profile in aerobic granule and its microbiological implications
Enzyme and Microbial Technology 43 (2008) 349–354
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
DO diffusion profile in aerobic granule and its microbiological implications Yong Li, Yu Liu ∗ , Liang Shen, Feng Chen Division of Environmental and Water Resources Engineering, School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore
a r t i c l e
i n f o
Article history: Received 19 February 2008 Received in revised form 14 April 2008 Accepted 16 April 2008 Keywords: Aerobic granule Dissolved oxygen Microelectrode Mass transfer Nitrification Denitrification PHB Aeration rate
a b s t r a c t By using a dissolved oxygen (DO) microelectrode, this study investigated the DO diffusion profiles in aerobic granules with different sizes under substrate-free and substrate-sufficient conditions. Results showed that DO only partially penetrated through 500 m from the granule surface under substratesufficient condition. On the contrary, no DO limitation was found in aerobic granules with a radius less than 2.2 mm under substrate-free condition, i.e., aerobic condition could be maintained in the entire aerobic granules. A one-dimensional model was also used to describe the DO diffusion in aerobic granules, and the model prediction was in good agreement with the measured DO profiles. It was shown that metabolism of aerobic granules involved conversion of external TOC to storage materials followed by microbial growth on storage materials and endogenous respiration. Because of the layered structure of aerobic granules, simultaneous nitrification–denitrification and reduced aeration during famine period can be expected in aerobic granular sludge SBR. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Aerobic granules are the aggregates of self-immobilized bacteria with a size up to several millimeters [1]. Compared with conventional activated sludge, aerobic granules have a regular, compact and strong structure and good settling properties, and they contain a high biomass and can handle high organic loading rates. Completely different from the floc-like sludge, the shape of aerobic granules is nearly spherical with a very clear outline. The average diameter of aerobic granules varied in the range of 0.3–5 mm, while the sludge volume index (SVI) of aerobic granules can be lower than 50 ml g−1 , indicating excellent settleability of aerobic granules and subsequently a fast settling and separation of the granular sludge from the treated effluent [1]. Since aerobic granules have large and compact structure, mass transfer limitation would be expected, which in turn affects the stability of aerobic granules, and further results in an overall reduction in the reactor treatment capacity. In general, substrate can penetrate into aerobic granules deeper than dissolved oxygen (DO). This implies that DO concentration would be limiting factor instead of substrate concentration. Using FISH technique, Tay et al. [2] found that a layer of dead microbial cells was located at a depth of 800–1000 m, while Li and Liu [3] simulated the oxygen diffusion
∗ Corresponding author. Tel.: +65 67905254; fax: +65 67910676. E-mail address: [email protected] (Y. Liu). 0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2008.04.005
in aerobic granules and reported a penetration depth of a few hundred micrometers for dissolved oxygen from the surface of aerobic granules. However, the direct measurements of DO profiles inside aerobic granules with different sizes have hardly been reported. The DO profiles inside the aerobic granule can further reflect the diffusion and reaction of oxygen within aerobic granules, which is crucial to the overall performance of the reactors. Thus, this study aimed to directly measure DO profiles in different-size aerobic granules by means of a DO microelectrode. The engineering implications of the findings were also discussed. It is expected that this work can offer useful information about the optimization of aerobic granular sludge SBR. 2. Materials and methods 2.1. Precultivation of aerobic granules Three sequencing batch reactors with a working volume of 2.5 l, a total height of 120 cm, and an internal diameter of 5 cm were used for cultivation of aerobic granules. The reactors were inoculated with activated sludge taken from a local municipal wastewater treatment plant. After 2 weeks of operation, aerobic granules were formed. The synthetic wastewater with the following composition was used for granule cultivation: sodium acetate 732.5 mg l−1 , NH4 Cl 22.0 mg l−1 , K2 HPO4 7.5 mg l−1 , CaCl2 ·2H2 O 9.5 mg l−1 , MgSO4 ·7H2 O, 6.25 mg l−1 , FeSO4 ·7H2 O 5 mg l−1 and microelement solution 1.0 ml l−1 . The microelement solution contained: H3 BO3 0.05 g l−1 , ZnCl2 0.05 g l−1 , CuCl2 0.03 g l−1 , MnSO4 ·H2 O 0.05 g l−1 , (NH4 )6 Mo7 O24 ·4H2 O 0.05 g l−1 , AlCl3 0.05 g l−1 , CoCl2 ·6H2 O 0.05 g l−1 , NiCl2 0.05 g l−1 . The reactors were operated sequentially with a cycle time of 4 h including feeding, aeration, settling and withdrawal of supernatant. The effluent was discharged
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Y. Li et al. / Enzyme and Microbial Technology 43 (2008) 349–354
Fig. 2. Wet and dry densities of aerobic granules with different sizes.
Fig. 1. Schematic of DO microelectrode: (1) microoxygen electrode; (2) position manipulator; (3) data collector; (4) computer.
from the middle port of the reactor. Fine air bubbles for aeration were supplied through a dispenser at the bottom with an airflow ratio of 2.5 l min−1 . The column reactors were operated at 25 ◦ C. 2.2. Analytic methods Mature aerobic granules were sorted according to their diameters in the range of 0.9–4.4 mm by the wet-sieving method developed by Pereboom [4]. The size of the sorted granule was further confirmed by an image analysis system (Image-Pro Plus, V4-0, Media Cybernetics, Silver Spring, MD, USA) with an Olympus SZX9 microscope (Olympus, Tokyo, Japan). The dry densities of the sorted aerobic granules were determined by standard water displacement method (APHA 1998). A fiber-optic oxygen meter (MICROX TX3) was employed to measure the dissolved oxygen profiles in aerobic granules (Fig. 1). The meter was equipped with a microoxygen probe (NTH-L2.5-NS40X40-TS-PST1-NOP), with a tip of 15 m. Microelectrode was first calibrated according to the method provided by the manufacturer (MICROX TX3). A three-dimensional micromanipulator was used to position the microelectrode on the granule with an accuracy of ±10 m. After the sharp tip of the microelectrode just touched on the granule surface, readings on the microsensor were taken at a depth interval of 50 m. The measurements were done in the substrate solution as described above as well as in tap water at 25 ◦ C, while dissolved oxygen concentration in the medium was kept at the saturation level of 8.6 mg l−1 by preaeration of the medium. Respirometric tests were also carried out to look into metabolic responses of aerobic granules to the availability of external organic substrate over culture time. For this purpose, automatic respirometer equipped with oxygen and carbon dioxide sensors (MicroOxymax, Columbus, USA) was used, and change in total organic carbon (TOC) concentration was also determined by TOC analyzer (Shimazu 5000, Japan).
3. Results
nearly saturation level of 8.6 mg l−1 . When getting near to the surface of the granule, the DO concentration dropped to 6.55 mg l−1 , indicating the existence of a boundary layer at the water–granule interface. It appears from Fig. 3 that the measured boundary layer is around 400 m for the aerobic granule with a radius of 2.5 mm, and the DO keeps dropping when it goes deeper into the aerobic granule. The DO concentration eventually becomes zero at the depth of 1.6 mm from the granule surface, i.e., DO could not penetrate the entire granule and oxygen diffusion limitation occurred beyond 1.6 mm from the granule surface. In fact, similar phenomenon has been reported in biofilm cultures, and the typical range of boundary layer for biofilms was reported to be in between 50 and 400 m [7]. 3.2.2. DO profiles in aerobic granules with different radiuses Fig. 4 shows the DO profiles measured in aerobic granules with various radiuses in the range of 0.45–2.2 mm. For aerobic granule with a radius of 0.45 mm, DO can diffuse into the entire aerobic granule under both substrate-free and substrate-sufficient conditions (Fig. 4), while the DO concentration under the substrate-free condition is always higher than that measured under the substratesufficient condition. For granule with a radius of 0.9 mm, DO can penetrate to the center of granule in water, while in the presence of substrate the DO concentration drops to zero at a depth of 0.5 mm from the granule surface beyond which anaerobic environment would appear and the aerobic activity in granule would be seriously suppressed. The similar phenomena were also observed in aerobic granules with a radius bigger than 0.9 mm (Fig. 4). It should be pointed out that the oxygen could penetrate into the entire
3.1. Density of aerobic granules The respective wet and dry densities of aerobic granules with different sizes were determined (Fig. 2). It was found that the wet density of aerobic granules tended to decrease from 1.03 to 1.018 g ml−1 , while the dry density also exhibited a decreasing trend from 44.9 to 35.1 g l−1 as the granule mean size increased from 1.5 to 4.0 mm. These show that smaller aerobic granules would have a more compact structure. Etterer and Wilderer [5] also found that the specific gravity of aerobic granules typically ranged from 1.004 to 1.065, while a dry density of 53 g-TSS l−1 was reported for the N-removal aerobic granules [6]. 3.2. DO profiles in aerobic granules 3.2.1. Boundary layer Fig. 3 shows change of the measured DO concentration from water to the inner of aerobic granule with the radius of 2.5 mm in tap water. In the water phase, the DO concentration remained at the
Fig. 3. Boundary layer of DO at the interface of aerobic granule and water.
Y. Li et al. / Enzyme and Microbial Technology 43 (2008) 349–354
351
Fig. 4. DO profiles in aerobic granules with different radiuses: (䊉) substrate-sufficient conditions; () substrate-free condition. Model simulation of the DO profiles under substrate-sufficient condition is shown by solid curve.
granule under the substrate-free condition when the granule radius is smaller than 2.2 mm. According to Li and Liu [3], a simplified equation of mass balance for steady-state diffusion of oxygen in a slice of a granule can be expressed as
Dfo
d2 Co 2 dCo + r dr dr 2
=
max Co x KCo + Co Yx/Co
(1)
where Dfo is the effective diffusivity for oxygen, Co is oxygen concentration within the granule, r is the one-dimensional coordinate, dr is the thickness of one layer, max and KCo are the maximum specific growth rate of aerobic granules and the half-saturation constant, respectively, x is biomass density and Yx/Co is the growth yield associated with oxygen. As Eq. (1) is a non-homogenous equation, a numerical method was developed to completely solve it using finite difference method (FDM) [3]. For this purpose, the
radius is divided into n grids, i.e.: Dfo
C
oi+1
C + Coi−1 − 2Coi − Coi−1 + oi+1 r r r 2
=
max Coi x KCo + Coi Yx/Co
(2)
where Coi+1 , Coi and Coi−1 represent the DO concentration in layer i + 1, i and i − 1, respectively. Values of the constants used are summarized in Table 1. The DO concentration within the granule can be obtained by numerically solving Eq. (2) in Matlab 7.0. Fig. 4 shows the numerical simulations of DO diffusion in aerobic granules under
Table 1 Values of constants used in Eq. (2) Constant
Value
Unit
Reference
Dfo max x Yx/Co
2 × 10−9
m2 s−1
Beyenal and Tanyolac [24] Yang et al. [25] This study Li and Liu [3]
2 10–60 1
day−1 g l−1 g g−1
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4. Discussion 4.1. Aerobic and anaerobic zones in aerobic granule
Fig. 5. Changes in OUR and TOC concentration during respirometric test of aerobic granules with mean size of 2.4 mm.
the substrate-sufficient condition, and the model predictions are in good agreement with the measured DO profiles. 3.3. Stored materials in aerobic granules with different sizes Fig. 5 shows changes in oxygen utilization rate (OUR) and substrate–TOC concentration during the respirometric experiment of aerobic granules with a mean size of 2.4 mm. Three metabolic phases can be differentiated in Fig. 5: (i) a quick reduction in the external TOC concentration was observed, and this TOC reduction was associated with a maximum OUR. In this phase, external organic carbon would be converted to storage materials first; (ii) after depletion of the external TOC, aerobic granules further grew on the stored materials derived from phase 1. As the result, a low OUR was recorded; (iii) at the end of phase 2, most stored materials were consumed, and microbial metabolism came into the endogenous respiration leading to a minimum OUR. Similar phenomena were also observed in the respirometric tests of aerobic granules with different sizes. Storage materials have been reported in activated sludge process, and most of the storage materials were found to be in the form of poly--hydroxybutyrate (PHB), especially in microbial cultures fed with acetate [8,9]. According to stoichiometric analyses, the storage materials synthesized by aerobic granules are indeed very close to the formula of PHB regardless of the sizes of aerobic granules (Table 2). It appears from Fig. 5 that the acetate–TOC is converted to PHB first, while the growth of aerobic granules would subsequently take place on the stored PHB at a low growth rate after depletion of external TOC. The accumulation of PHB was also found in the N-removal aerobic granules [10]. As can be seen in Figs. 4 and 5, DO can penetrate deeper into aerobic granule in the famine period, but the rapid degradation of external TOC in the feast period requires much more oxygen than that in the famine period. In fact, it has been found that PHB was stored in bacteria situated in deeper layers of aerobic granules [11]. Table 2 Composition of storage materials in aerobic granules with different sizes Mean size of aerobic granules (mm)
Composition
0.7 1.5 2.4 3.0 PHB
CH1.6 O0.4 CH1.6 O0.5 CH1.6 O0.3 CH1.5 O0.3 CH1.5 O0.5
For aerobic granules in substrate solution, when the granule radius becomes bigger than 0.9 mm, the DO diffusion limitation would occur. Li and Liu [3] developed one-dimensional model for simulation of substrate and DO diffusion in aerobic granules, and found that for aerobic granules with a radium larger than 0.5 mm, dissolved oxygen became a major limiting factor of metabolic activity of aerobic granule, while Jang et al. [12] also reported that the diffusion limitation of oxygen occurred when aerobic granules grew to a radius approximately 0.6–0.8 mm. Under the substrate-free condition, the DO diffusion limitation is not significant in aerobic granules with radius smaller than 2.0 mm (Fig. 4). These seem to imply that in big aerobic granules, aerobic and anaerobic zones would co-exist in a layered manner, i.e., the outer layer of granule would be aerobic, while its inner part would be subject to anaerobic condition. In fact, the presence of anaerobic bacteroides in aerobically grown microbial granules has been reported [13]. Li and Liu [3] found that in large aerobic granules, organic substrate was not a limiting factor. On the contrary, the microbial process would be governed by the availability of DO in aerobic granule. Toh et al. [14] detected a layer of dead microbial cells located at a depth of 800–1000 m below the granule surface where the aerobic activities were highly suppressed. Chiu et al. [15,16] studied DO diffusion in aerobic granules grown on by phenol and acetate as carbon source, respectively, and found that most DO was consumed by an active layer with a depth of less than 125–375 m from the granule surface, indicating that no aerobic oxidation would occur beneath this active layer. This and previous studies clearly show that small aerobic granules would be more effective for aerobic wastewater treatment as they have more live cells within a given volume of granules. Oxygen transfer limitation can influence the structure and stability of aerobic granules. The presence of anaerobic bacteria in the center of aerobic granules was likely to result in the production of organic acid and gases within the granules, which further diminished long-term stability of aerobic granules. Moreover, extracellular polysaccharide (EPS) in the centre of granule would be anaerobically degraded as potential energy source, and the biomass in the granule centre would undergo anaerobic decay. It has been shown that a significant portion of EPS produced by aerobic granules can be degraded by their own producers [17]. These in turn would lead to a porous and weak structure of aerobic granule. Granules would undergo breakup due to a low oxygen concentration, and it is even impossible to form stable granules from activated sludge at a low oxygen concentration [6]. Consequently, the directly measured DO profiles clearly reveal a layered aerobic and anaerobic structure in aerobic granules. 4.2. Variable DO demand in feast and famine phases Basically, a cycle of SBR operation consists of feast and famine phases [18,19]. The feast phase means that the exogenous substrate is available, while the famine phase represents a period in which there is no longer exogenous substrate. During the feast period, oxygen is quickly consumed for substrate oxidation to storage materials (Fig. 5). This indeed is in line with the activated sludge model no. 3. As shown in Fig. 4, under the substrate-sufficient condition, DO only partially penetrates into aerobic granules with a depth less than 500 m from the granule surface, while the oxygen can penetrate deeper in aerobic granules present in water (representing famine period) than in acetate solution (representing feast period). These imply that for aerobic granules in SBRs with a typical radius
Y. Li et al. / Enzyme and Microbial Technology 43 (2008) 349–354
Fig. 6. Schematic presentation of simultaneous nitrification–denitrification in aerobic granule.
from 0.5 to 1.0 mm, a low oxygen concentration might be sufficient for penetrating the entire granule in the famine period. This leads to a reasonable consideration that the oxygen demand in the famine period could be much lower than that in the feast period (Fig. 5), i.e., the oxygen supply by air aeration can be reduced substantially in the famine period. Obviously, a considerable amount of aerationassociated energy would be saved if a reduced aeration strategy is adopted in the famine phase of SBR operation. Previous study shows that the reduced aeration rate in famine period would not pose significant effect on granule size, SVI and effluent quality during a short-term operation [20]. MosqueraCorral et al. [6] also found the reduction of oxygen supply not only reduced the energy demand, but also increased the nitrogen removal efficiency. However, it should be realized that aerobic granules developed at the low oxygen concentration would not be stable, and filamentous growth would eventually encouraged [6,21]. Consequently, a long-term operation is strongly needed before any consolidate conclusion can be drawn with regard to the reduced aeration in the famine phase of aerobic granular sludge SBR. 4.3. Nitrification–denitrification in aerobic granule The nitrogen removal through conventional nitrification– denitrification pathway requires on alternative aerobic and anaerobic conditions. As discussed earlier, aerobic granule has a layered structure comprising aerobic and anaerobic zones from the surface to the center of aerobic granule. Thus, aerobic granule may have a great potential for simultaneous nitrification and denitrification even the DO concentration in the bulk solution is high (Figs. 4 and 6). Ammonia oxidizing bacteria were found to exist primarily in the upper and middle layers of the granule, and most of the nitrification is likely to occur from the surface to 300 m into aerobic granule, while nitrate produced seemed to be removed by denitrification in the granules [12]. Recently, Liu et al. [22] employed a microelectrode fabricated using photolithography to detect the DO profile in nitrifying granule and found that the active part of the nitrifying granule was situated at the upper layer of 150 m from the surface of aerobic granules. In conventional denitrification process, organic carbon is required as electron donor, i.e., COD is needed for denitrification in the anaerobic zone of aerobic granule. It has been reported that for a SBR dominated by aerobic granules bigger than 0. 5 mm, the DO was found to be the bottleneck which limits the utilization of organic substrate [3], i.e., the organic substrate could penetrate into deeper zone of aerobic granule than DO (Fig. 6). These imply that denitrification in the anaerobic zone of aerobic granule would be
353
possible. Previous study also showed that organic carbon oxidizing, nitrifying and denitrifying bacteria could co-exist in aerobic granules developed at different substrate N/COD ratios, and simultaneous aerobic COD removal, nitrification and denitrification were observed in an aerobic granular sludge reactor [23]. As discussed earlier, PHB storage is observed during rapid biodegradation of the external TOC. It has been reported that storage and subsequent degradation of PHB would benefit the denitrification, especially PHB was found to be stored in bacteria situated in deeper layers of aerobic granules [11]. Qin et al. [10] investigated the potential role of PHB for denitrification in aerobic granular sludge SBR, and found that (i) in case where both external organic carbon and nitrate were not available, PHB was degraded only for cell maintenance; (ii) with the addition of nitrate but no external organic carbon, stored PHB was utilized for both denitrification and cell maintenance. These imply that the stored PHB can be used as energy and carbon source for denitrification and cell maintenance when external carbon is no longer available. 5. Conclusion The density of aerobic granules was size-dependent, e.g., the smaller aerobic granule had a higher density and more compact structure. It was found that under the substrate-sufficient condition, the depth of the DO penetration could be up to about 500 m from the granule surface regardless of the granule radius, i.e., anaerobic zone would exist in aerobic granules with a radius bigger than 0.5 mm. On the contrary, in the substrate-free medium, no DO limitation was found in aerobic granules with a radius less than 2.2 mm, and aerobic condition could be maintained in the entire aerobic granules. It appears from this study that the preferable size of aerobic granule would be around 1.0 mm for aerobic oxidation of organics and nitrification in the outer layer of granule and an enhanced denitrification on the stored PHB in the deeper part of granule. The DO profiles determined under the substrate-free and substrate-sufficient conditions provided direct experimental support to the idea of reduced aeration rate in the famine period of SBR, but the long-term stability of aerobic granules would be of great concern. References [1] Liu Y, Tay JH. State of the art of biogranulation technology for wastewater treatment. Biotechnol Adv 2004;22:533–63. [2] Tay JH, Ivanov V, Pan S, Tay STL. Specific layers in aerobically grown microbial granules. Lett Appl Microbiol 2002;34:254–7. [3] Li Y, Liu Y. Diffusion of substrate and oxygen in aerobic granule. Biochem Eng J 2005;27:45–52. [4] Pereboom JHF. Size distribution model for methanogenic granules from full scale UASB and IC reactors. Water Sci Technol 1994;30:211–21. [5] Etterer T, Wilderer PA. Generation and properties of aerobic granular sludge. Water Sci Technol 2001;43:19–26. [6] Mosquera-Corral A, de Kreuk MK, Heijnen JJ, van Loosdrecht MCM. Effects of oxygen concentration on N-removal in an aerobic granular sludge reactor. Water Res 2005;39:2676–86. [7] Gantzer CJ, Rittmann BE, Herricks EE. Mass transport to streambed biofilms. Water Res 1988;22:709–22. [8] Beccari M, Majone M, Massanisso P, Ramadori R. A bulking sludge with high storage response selected under intermittent feeding. Water Res 1998;32:3403–14. [9] Dircks K, Pind PE, Mosbaek H, Henze M. Yield determination by respirometry—the possible influence of storage under aerobic conditions in activated sludge. Water SA 1999;25:69–74. [10] Qin L, Liu Y, Tay JH. Denitrification on poly--hydroxybutyrate in microbial granular sludge sequencing batch reactor. Water Res 2005;39:1503–10. [11] Beun JJ, Heijnen JJ, van Loosdrecht MCM. N-removal in a granular sludge sequencing batch airlift reactor. Biotechnol Bioeng 2001;75:82–92. [12] Jang A, Yoon YH, Kim IS, Kim KS, Bishop PL. Character and evaluation of aerobic granules in sequencing batch reactor. J Biotechnol 2003;105:71–82. [13] Tay STL, Ivanov V, Yi S, Zhuang WQ, Tay JH. Presence of anaerobic bacteroides in aerobically grown microbial granules. Microb Ecol 2002;44:278–85.
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[14] Toh SK, Tay JH, Moy BYP, Ivanov V, Tay STL. Size-effect on the physical characteristics of the aerobic granule in a SBR. Appl Microbiol Biotechnol 2003;60:687–95. [15] Chiu ZC, Chen MY, Lee DJ, Wang CH, Lai JY. Oxygen diffusion and consumption in active aerobic granules of heterogeneous structure. Appl Microbiol Biotechnol 2007;75:685–91. [16] Chiu ZC, Chen MY, Lee DJ, Wang CH, Lai JY. Oxygen diffusion in active layer of aerobic granule with step change in surrounding oxygen levels. Water Res 2007;41:884–92. [17] Wang ZW, Liu Y, Tay JH. Biodegradability of extracellular polymeric substances produced by aerobic granules. Appl Microbiol Biotechnol 2007;74:462–6. [18] Tay JH, Liu QS, Liu Y. Microscopic observation of aerobic granulation in sequential aerobic sludge blanket reactor. J Appl Microbiol 2001;91:168–75. [19] Liu QS. Aerobic granulation in sequencing batch reactor. PhD thesis. Singapore: Nanyang Technological University; 2003.
[20] Liu YQ, Tay JH. Variable aeration in sequencing batch reactor with aerobic granular sludge. J Biotechnol 2006;124:338–46. [21] Liu Y, Liu QS. Causes and control of filamentous growth in aerobic granular sludge SBR. Biotechnol Adv 2006;24:115–27. [22] Liu SY, Liu G, Tian YC, Chen YP, Yu HQ, Fang F. An innovative microelectrode fabricated using photolithography for measuring dissolved oxygen distributions in aerobic granules. Environ Sci Technol 2007;41:5447–52. [23] Yang SF. Development of aerobic granules for simultaneous organic carbon and nitrogen removal. PhD thesis. Singapore: Nanyang Technological University; 2005. [24] Beyenal H, Tanyolac A. The calculation of simultaneous effective diffusion coefficients of the substrates in a fluidized bed biofilm reactor. Water Sci Technol 1994;29:463–70. [25] Yang SF, Liu QS, Tay JH, Liu Y. Growth kinetics of aerobic granules developed in sequencing batch reactors. Lett Appl Microbiol 2004;38:106–12.
Contents lists available at ScienceDirect
Enzyme and Microbial Technology journal homepage: www.elsevier.com/locate/emt
DO diffusion profile in aerobic granule and its microbiological implications Yong Li, Yu Liu ∗ , Liang Shen, Feng Chen Division of Environmental and Water Resources Engineering, School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore
a r t i c l e
i n f o
Article history: Received 19 February 2008 Received in revised form 14 April 2008 Accepted 16 April 2008 Keywords: Aerobic granule Dissolved oxygen Microelectrode Mass transfer Nitrification Denitrification PHB Aeration rate
a b s t r a c t By using a dissolved oxygen (DO) microelectrode, this study investigated the DO diffusion profiles in aerobic granules with different sizes under substrate-free and substrate-sufficient conditions. Results showed that DO only partially penetrated through 500 m from the granule surface under substratesufficient condition. On the contrary, no DO limitation was found in aerobic granules with a radius less than 2.2 mm under substrate-free condition, i.e., aerobic condition could be maintained in the entire aerobic granules. A one-dimensional model was also used to describe the DO diffusion in aerobic granules, and the model prediction was in good agreement with the measured DO profiles. It was shown that metabolism of aerobic granules involved conversion of external TOC to storage materials followed by microbial growth on storage materials and endogenous respiration. Because of the layered structure of aerobic granules, simultaneous nitrification–denitrification and reduced aeration during famine period can be expected in aerobic granular sludge SBR. © 2008 Elsevier Inc. All rights reserved.
1. Introduction Aerobic granules are the aggregates of self-immobilized bacteria with a size up to several millimeters [1]. Compared with conventional activated sludge, aerobic granules have a regular, compact and strong structure and good settling properties, and they contain a high biomass and can handle high organic loading rates. Completely different from the floc-like sludge, the shape of aerobic granules is nearly spherical with a very clear outline. The average diameter of aerobic granules varied in the range of 0.3–5 mm, while the sludge volume index (SVI) of aerobic granules can be lower than 50 ml g−1 , indicating excellent settleability of aerobic granules and subsequently a fast settling and separation of the granular sludge from the treated effluent [1]. Since aerobic granules have large and compact structure, mass transfer limitation would be expected, which in turn affects the stability of aerobic granules, and further results in an overall reduction in the reactor treatment capacity. In general, substrate can penetrate into aerobic granules deeper than dissolved oxygen (DO). This implies that DO concentration would be limiting factor instead of substrate concentration. Using FISH technique, Tay et al. [2] found that a layer of dead microbial cells was located at a depth of 800–1000 m, while Li and Liu [3] simulated the oxygen diffusion
∗ Corresponding author. Tel.: +65 67905254; fax: +65 67910676. E-mail address: [email protected] (Y. Liu). 0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2008.04.005
in aerobic granules and reported a penetration depth of a few hundred micrometers for dissolved oxygen from the surface of aerobic granules. However, the direct measurements of DO profiles inside aerobic granules with different sizes have hardly been reported. The DO profiles inside the aerobic granule can further reflect the diffusion and reaction of oxygen within aerobic granules, which is crucial to the overall performance of the reactors. Thus, this study aimed to directly measure DO profiles in different-size aerobic granules by means of a DO microelectrode. The engineering implications of the findings were also discussed. It is expected that this work can offer useful information about the optimization of aerobic granular sludge SBR. 2. Materials and methods 2.1. Precultivation of aerobic granules Three sequencing batch reactors with a working volume of 2.5 l, a total height of 120 cm, and an internal diameter of 5 cm were used for cultivation of aerobic granules. The reactors were inoculated with activated sludge taken from a local municipal wastewater treatment plant. After 2 weeks of operation, aerobic granules were formed. The synthetic wastewater with the following composition was used for granule cultivation: sodium acetate 732.5 mg l−1 , NH4 Cl 22.0 mg l−1 , K2 HPO4 7.5 mg l−1 , CaCl2 ·2H2 O 9.5 mg l−1 , MgSO4 ·7H2 O, 6.25 mg l−1 , FeSO4 ·7H2 O 5 mg l−1 and microelement solution 1.0 ml l−1 . The microelement solution contained: H3 BO3 0.05 g l−1 , ZnCl2 0.05 g l−1 , CuCl2 0.03 g l−1 , MnSO4 ·H2 O 0.05 g l−1 , (NH4 )6 Mo7 O24 ·4H2 O 0.05 g l−1 , AlCl3 0.05 g l−1 , CoCl2 ·6H2 O 0.05 g l−1 , NiCl2 0.05 g l−1 . The reactors were operated sequentially with a cycle time of 4 h including feeding, aeration, settling and withdrawal of supernatant. The effluent was discharged
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Y. Li et al. / Enzyme and Microbial Technology 43 (2008) 349–354
Fig. 2. Wet and dry densities of aerobic granules with different sizes.
Fig. 1. Schematic of DO microelectrode: (1) microoxygen electrode; (2) position manipulator; (3) data collector; (4) computer.
from the middle port of the reactor. Fine air bubbles for aeration were supplied through a dispenser at the bottom with an airflow ratio of 2.5 l min−1 . The column reactors were operated at 25 ◦ C. 2.2. Analytic methods Mature aerobic granules were sorted according to their diameters in the range of 0.9–4.4 mm by the wet-sieving method developed by Pereboom [4]. The size of the sorted granule was further confirmed by an image analysis system (Image-Pro Plus, V4-0, Media Cybernetics, Silver Spring, MD, USA) with an Olympus SZX9 microscope (Olympus, Tokyo, Japan). The dry densities of the sorted aerobic granules were determined by standard water displacement method (APHA 1998). A fiber-optic oxygen meter (MICROX TX3) was employed to measure the dissolved oxygen profiles in aerobic granules (Fig. 1). The meter was equipped with a microoxygen probe (NTH-L2.5-NS40X40-TS-PST1-NOP), with a tip of 15 m. Microelectrode was first calibrated according to the method provided by the manufacturer (MICROX TX3). A three-dimensional micromanipulator was used to position the microelectrode on the granule with an accuracy of ±10 m. After the sharp tip of the microelectrode just touched on the granule surface, readings on the microsensor were taken at a depth interval of 50 m. The measurements were done in the substrate solution as described above as well as in tap water at 25 ◦ C, while dissolved oxygen concentration in the medium was kept at the saturation level of 8.6 mg l−1 by preaeration of the medium. Respirometric tests were also carried out to look into metabolic responses of aerobic granules to the availability of external organic substrate over culture time. For this purpose, automatic respirometer equipped with oxygen and carbon dioxide sensors (MicroOxymax, Columbus, USA) was used, and change in total organic carbon (TOC) concentration was also determined by TOC analyzer (Shimazu 5000, Japan).
3. Results
nearly saturation level of 8.6 mg l−1 . When getting near to the surface of the granule, the DO concentration dropped to 6.55 mg l−1 , indicating the existence of a boundary layer at the water–granule interface. It appears from Fig. 3 that the measured boundary layer is around 400 m for the aerobic granule with a radius of 2.5 mm, and the DO keeps dropping when it goes deeper into the aerobic granule. The DO concentration eventually becomes zero at the depth of 1.6 mm from the granule surface, i.e., DO could not penetrate the entire granule and oxygen diffusion limitation occurred beyond 1.6 mm from the granule surface. In fact, similar phenomenon has been reported in biofilm cultures, and the typical range of boundary layer for biofilms was reported to be in between 50 and 400 m [7]. 3.2.2. DO profiles in aerobic granules with different radiuses Fig. 4 shows the DO profiles measured in aerobic granules with various radiuses in the range of 0.45–2.2 mm. For aerobic granule with a radius of 0.45 mm, DO can diffuse into the entire aerobic granule under both substrate-free and substrate-sufficient conditions (Fig. 4), while the DO concentration under the substrate-free condition is always higher than that measured under the substratesufficient condition. For granule with a radius of 0.9 mm, DO can penetrate to the center of granule in water, while in the presence of substrate the DO concentration drops to zero at a depth of 0.5 mm from the granule surface beyond which anaerobic environment would appear and the aerobic activity in granule would be seriously suppressed. The similar phenomena were also observed in aerobic granules with a radius bigger than 0.9 mm (Fig. 4). It should be pointed out that the oxygen could penetrate into the entire
3.1. Density of aerobic granules The respective wet and dry densities of aerobic granules with different sizes were determined (Fig. 2). It was found that the wet density of aerobic granules tended to decrease from 1.03 to 1.018 g ml−1 , while the dry density also exhibited a decreasing trend from 44.9 to 35.1 g l−1 as the granule mean size increased from 1.5 to 4.0 mm. These show that smaller aerobic granules would have a more compact structure. Etterer and Wilderer [5] also found that the specific gravity of aerobic granules typically ranged from 1.004 to 1.065, while a dry density of 53 g-TSS l−1 was reported for the N-removal aerobic granules [6]. 3.2. DO profiles in aerobic granules 3.2.1. Boundary layer Fig. 3 shows change of the measured DO concentration from water to the inner of aerobic granule with the radius of 2.5 mm in tap water. In the water phase, the DO concentration remained at the
Fig. 3. Boundary layer of DO at the interface of aerobic granule and water.
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Fig. 4. DO profiles in aerobic granules with different radiuses: (䊉) substrate-sufficient conditions; () substrate-free condition. Model simulation of the DO profiles under substrate-sufficient condition is shown by solid curve.
granule under the substrate-free condition when the granule radius is smaller than 2.2 mm. According to Li and Liu [3], a simplified equation of mass balance for steady-state diffusion of oxygen in a slice of a granule can be expressed as
Dfo
d2 Co 2 dCo + r dr dr 2
=
max Co x KCo + Co Yx/Co
(1)
where Dfo is the effective diffusivity for oxygen, Co is oxygen concentration within the granule, r is the one-dimensional coordinate, dr is the thickness of one layer, max and KCo are the maximum specific growth rate of aerobic granules and the half-saturation constant, respectively, x is biomass density and Yx/Co is the growth yield associated with oxygen. As Eq. (1) is a non-homogenous equation, a numerical method was developed to completely solve it using finite difference method (FDM) [3]. For this purpose, the
radius is divided into n grids, i.e.: Dfo
C
oi+1
C + Coi−1 − 2Coi − Coi−1 + oi+1 r r r 2
=
max Coi x KCo + Coi Yx/Co
(2)
where Coi+1 , Coi and Coi−1 represent the DO concentration in layer i + 1, i and i − 1, respectively. Values of the constants used are summarized in Table 1. The DO concentration within the granule can be obtained by numerically solving Eq. (2) in Matlab 7.0. Fig. 4 shows the numerical simulations of DO diffusion in aerobic granules under
Table 1 Values of constants used in Eq. (2) Constant
Value
Unit
Reference
Dfo max x Yx/Co
2 × 10−9
m2 s−1
Beyenal and Tanyolac [24] Yang et al. [25] This study Li and Liu [3]
2 10–60 1
day−1 g l−1 g g−1
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4. Discussion 4.1. Aerobic and anaerobic zones in aerobic granule
Fig. 5. Changes in OUR and TOC concentration during respirometric test of aerobic granules with mean size of 2.4 mm.
the substrate-sufficient condition, and the model predictions are in good agreement with the measured DO profiles. 3.3. Stored materials in aerobic granules with different sizes Fig. 5 shows changes in oxygen utilization rate (OUR) and substrate–TOC concentration during the respirometric experiment of aerobic granules with a mean size of 2.4 mm. Three metabolic phases can be differentiated in Fig. 5: (i) a quick reduction in the external TOC concentration was observed, and this TOC reduction was associated with a maximum OUR. In this phase, external organic carbon would be converted to storage materials first; (ii) after depletion of the external TOC, aerobic granules further grew on the stored materials derived from phase 1. As the result, a low OUR was recorded; (iii) at the end of phase 2, most stored materials were consumed, and microbial metabolism came into the endogenous respiration leading to a minimum OUR. Similar phenomena were also observed in the respirometric tests of aerobic granules with different sizes. Storage materials have been reported in activated sludge process, and most of the storage materials were found to be in the form of poly--hydroxybutyrate (PHB), especially in microbial cultures fed with acetate [8,9]. According to stoichiometric analyses, the storage materials synthesized by aerobic granules are indeed very close to the formula of PHB regardless of the sizes of aerobic granules (Table 2). It appears from Fig. 5 that the acetate–TOC is converted to PHB first, while the growth of aerobic granules would subsequently take place on the stored PHB at a low growth rate after depletion of external TOC. The accumulation of PHB was also found in the N-removal aerobic granules [10]. As can be seen in Figs. 4 and 5, DO can penetrate deeper into aerobic granule in the famine period, but the rapid degradation of external TOC in the feast period requires much more oxygen than that in the famine period. In fact, it has been found that PHB was stored in bacteria situated in deeper layers of aerobic granules [11]. Table 2 Composition of storage materials in aerobic granules with different sizes Mean size of aerobic granules (mm)
Composition
0.7 1.5 2.4 3.0 PHB
CH1.6 O0.4 CH1.6 O0.5 CH1.6 O0.3 CH1.5 O0.3 CH1.5 O0.5
For aerobic granules in substrate solution, when the granule radius becomes bigger than 0.9 mm, the DO diffusion limitation would occur. Li and Liu [3] developed one-dimensional model for simulation of substrate and DO diffusion in aerobic granules, and found that for aerobic granules with a radium larger than 0.5 mm, dissolved oxygen became a major limiting factor of metabolic activity of aerobic granule, while Jang et al. [12] also reported that the diffusion limitation of oxygen occurred when aerobic granules grew to a radius approximately 0.6–0.8 mm. Under the substrate-free condition, the DO diffusion limitation is not significant in aerobic granules with radius smaller than 2.0 mm (Fig. 4). These seem to imply that in big aerobic granules, aerobic and anaerobic zones would co-exist in a layered manner, i.e., the outer layer of granule would be aerobic, while its inner part would be subject to anaerobic condition. In fact, the presence of anaerobic bacteroides in aerobically grown microbial granules has been reported [13]. Li and Liu [3] found that in large aerobic granules, organic substrate was not a limiting factor. On the contrary, the microbial process would be governed by the availability of DO in aerobic granule. Toh et al. [14] detected a layer of dead microbial cells located at a depth of 800–1000 m below the granule surface where the aerobic activities were highly suppressed. Chiu et al. [15,16] studied DO diffusion in aerobic granules grown on by phenol and acetate as carbon source, respectively, and found that most DO was consumed by an active layer with a depth of less than 125–375 m from the granule surface, indicating that no aerobic oxidation would occur beneath this active layer. This and previous studies clearly show that small aerobic granules would be more effective for aerobic wastewater treatment as they have more live cells within a given volume of granules. Oxygen transfer limitation can influence the structure and stability of aerobic granules. The presence of anaerobic bacteria in the center of aerobic granules was likely to result in the production of organic acid and gases within the granules, which further diminished long-term stability of aerobic granules. Moreover, extracellular polysaccharide (EPS) in the centre of granule would be anaerobically degraded as potential energy source, and the biomass in the granule centre would undergo anaerobic decay. It has been shown that a significant portion of EPS produced by aerobic granules can be degraded by their own producers [17]. These in turn would lead to a porous and weak structure of aerobic granule. Granules would undergo breakup due to a low oxygen concentration, and it is even impossible to form stable granules from activated sludge at a low oxygen concentration [6]. Consequently, the directly measured DO profiles clearly reveal a layered aerobic and anaerobic structure in aerobic granules. 4.2. Variable DO demand in feast and famine phases Basically, a cycle of SBR operation consists of feast and famine phases [18,19]. The feast phase means that the exogenous substrate is available, while the famine phase represents a period in which there is no longer exogenous substrate. During the feast period, oxygen is quickly consumed for substrate oxidation to storage materials (Fig. 5). This indeed is in line with the activated sludge model no. 3. As shown in Fig. 4, under the substrate-sufficient condition, DO only partially penetrates into aerobic granules with a depth less than 500 m from the granule surface, while the oxygen can penetrate deeper in aerobic granules present in water (representing famine period) than in acetate solution (representing feast period). These imply that for aerobic granules in SBRs with a typical radius
Y. Li et al. / Enzyme and Microbial Technology 43 (2008) 349–354
Fig. 6. Schematic presentation of simultaneous nitrification–denitrification in aerobic granule.
from 0.5 to 1.0 mm, a low oxygen concentration might be sufficient for penetrating the entire granule in the famine period. This leads to a reasonable consideration that the oxygen demand in the famine period could be much lower than that in the feast period (Fig. 5), i.e., the oxygen supply by air aeration can be reduced substantially in the famine period. Obviously, a considerable amount of aerationassociated energy would be saved if a reduced aeration strategy is adopted in the famine phase of SBR operation. Previous study shows that the reduced aeration rate in famine period would not pose significant effect on granule size, SVI and effluent quality during a short-term operation [20]. MosqueraCorral et al. [6] also found the reduction of oxygen supply not only reduced the energy demand, but also increased the nitrogen removal efficiency. However, it should be realized that aerobic granules developed at the low oxygen concentration would not be stable, and filamentous growth would eventually encouraged [6,21]. Consequently, a long-term operation is strongly needed before any consolidate conclusion can be drawn with regard to the reduced aeration in the famine phase of aerobic granular sludge SBR. 4.3. Nitrification–denitrification in aerobic granule The nitrogen removal through conventional nitrification– denitrification pathway requires on alternative aerobic and anaerobic conditions. As discussed earlier, aerobic granule has a layered structure comprising aerobic and anaerobic zones from the surface to the center of aerobic granule. Thus, aerobic granule may have a great potential for simultaneous nitrification and denitrification even the DO concentration in the bulk solution is high (Figs. 4 and 6). Ammonia oxidizing bacteria were found to exist primarily in the upper and middle layers of the granule, and most of the nitrification is likely to occur from the surface to 300 m into aerobic granule, while nitrate produced seemed to be removed by denitrification in the granules [12]. Recently, Liu et al. [22] employed a microelectrode fabricated using photolithography to detect the DO profile in nitrifying granule and found that the active part of the nitrifying granule was situated at the upper layer of 150 m from the surface of aerobic granules. In conventional denitrification process, organic carbon is required as electron donor, i.e., COD is needed for denitrification in the anaerobic zone of aerobic granule. It has been reported that for a SBR dominated by aerobic granules bigger than 0. 5 mm, the DO was found to be the bottleneck which limits the utilization of organic substrate [3], i.e., the organic substrate could penetrate into deeper zone of aerobic granule than DO (Fig. 6). These imply that denitrification in the anaerobic zone of aerobic granule would be
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possible. Previous study also showed that organic carbon oxidizing, nitrifying and denitrifying bacteria could co-exist in aerobic granules developed at different substrate N/COD ratios, and simultaneous aerobic COD removal, nitrification and denitrification were observed in an aerobic granular sludge reactor [23]. As discussed earlier, PHB storage is observed during rapid biodegradation of the external TOC. It has been reported that storage and subsequent degradation of PHB would benefit the denitrification, especially PHB was found to be stored in bacteria situated in deeper layers of aerobic granules [11]. Qin et al. [10] investigated the potential role of PHB for denitrification in aerobic granular sludge SBR, and found that (i) in case where both external organic carbon and nitrate were not available, PHB was degraded only for cell maintenance; (ii) with the addition of nitrate but no external organic carbon, stored PHB was utilized for both denitrification and cell maintenance. These imply that the stored PHB can be used as energy and carbon source for denitrification and cell maintenance when external carbon is no longer available. 5. Conclusion The density of aerobic granules was size-dependent, e.g., the smaller aerobic granule had a higher density and more compact structure. It was found that under the substrate-sufficient condition, the depth of the DO penetration could be up to about 500 m from the granule surface regardless of the granule radius, i.e., anaerobic zone would exist in aerobic granules with a radius bigger than 0.5 mm. On the contrary, in the substrate-free medium, no DO limitation was found in aerobic granules with a radius less than 2.2 mm, and aerobic condition could be maintained in the entire aerobic granules. It appears from this study that the preferable size of aerobic granule would be around 1.0 mm for aerobic oxidation of organics and nitrification in the outer layer of granule and an enhanced denitrification on the stored PHB in the deeper part of granule. The DO profiles determined under the substrate-free and substrate-sufficient conditions provided direct experimental support to the idea of reduced aeration rate in the famine period of SBR, but the long-term stability of aerobic granules would be of great concern. References [1] Liu Y, Tay JH. State of the art of biogranulation technology for wastewater treatment. Biotechnol Adv 2004;22:533–63. [2] Tay JH, Ivanov V, Pan S, Tay STL. Specific layers in aerobically grown microbial granules. Lett Appl Microbiol 2002;34:254–7. [3] Li Y, Liu Y. Diffusion of substrate and oxygen in aerobic granule. Biochem Eng J 2005;27:45–52. [4] Pereboom JHF. Size distribution model for methanogenic granules from full scale UASB and IC reactors. Water Sci Technol 1994;30:211–21. [5] Etterer T, Wilderer PA. Generation and properties of aerobic granular sludge. Water Sci Technol 2001;43:19–26. [6] Mosquera-Corral A, de Kreuk MK, Heijnen JJ, van Loosdrecht MCM. Effects of oxygen concentration on N-removal in an aerobic granular sludge reactor. Water Res 2005;39:2676–86. [7] Gantzer CJ, Rittmann BE, Herricks EE. Mass transport to streambed biofilms. Water Res 1988;22:709–22. [8] Beccari M, Majone M, Massanisso P, Ramadori R. A bulking sludge with high storage response selected under intermittent feeding. Water Res 1998;32:3403–14. [9] Dircks K, Pind PE, Mosbaek H, Henze M. Yield determination by respirometry—the possible influence of storage under aerobic conditions in activated sludge. Water SA 1999;25:69–74. [10] Qin L, Liu Y, Tay JH. Denitrification on poly--hydroxybutyrate in microbial granular sludge sequencing batch reactor. Water Res 2005;39:1503–10. [11] Beun JJ, Heijnen JJ, van Loosdrecht MCM. N-removal in a granular sludge sequencing batch airlift reactor. Biotechnol Bioeng 2001;75:82–92. [12] Jang A, Yoon YH, Kim IS, Kim KS, Bishop PL. Character and evaluation of aerobic granules in sequencing batch reactor. J Biotechnol 2003;105:71–82. [13] Tay STL, Ivanov V, Yi S, Zhuang WQ, Tay JH. Presence of anaerobic bacteroides in aerobically grown microbial granules. Microb Ecol 2002;44:278–85.
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