Rapid start-up and improvement of granulation in SBR
© Jalali et al.; licensee BioMed Central. 2015
Received: 16 September 2014
Accepted: 8 April 2015
Published: 25 April 2015
The aim of this study is to accelerate and improve aerobic granulation within a Sequencing Batch Reactor (SBR) by cationic polymer addition.
To identify whether the polymer additive is capable of enhancing granule formation, two SBRs (R1 and R2, each 0.15 m in diameter and 2 m in height) are used by feeding synthetic wastewater. The cationic polymer with concentration of 30 to 2 ppm is added to R2, while no cationic polymer is added to R1.
Results show that the cationic polymer addition causes faster granule formation and consequently shorter reactor start-up period. The polymer-amended reactor contains higher concentration of biomass with better settling ability (23% reduction in SVI15) and larger and denser granules (112% increase of granular diameter). In addition, the results demonstrate that the cationic polymer improve the sludge granulation process by 31% increase in Extracellular Polymer Substance(EPS) concentration, 7% increase in Specific Oxygen Uptake Rate(SOUR), 18% increase in hydrophobicity, and 17% reduction in effluent Mixed Liquor Suspended Solid(MLSS) concentration.
Concludingly, it is found that using the cationic polymer to an aerobic granular system has the potential to enhance the sludge granulation process.
KeywordsAerobic granulation Cationic polymer Rapid granulation
Aerobic granulation is a biological wastewater treatment which has been defined as a self-immobilization process to transform loose sludge flocs into dense granules . Compared to activated sludge, aerobic granules have many advantages such as dense construction, excellent settling ability, simultaneous nutrient removal capability, high ability in repulsion of shock loading, and excellent tolerance for toxic substances . These characteristics have led to numerous studies for identification of many factors which affect granule formation. The factors which have been studied include substrate composition [3,4], organic loading rate , shear force , dissolved oxygen concentration , settling time , substrate starvation , food to microorganism ratio , hydraulic retention time , volumetric exchange ratio , aspect ratio , pH  and oxygen concentration . However, the mechanism of granulation is not yet clear . Furthermore, instability during the start-up and operation of granulation in SBR have been observed [17,18].
The formation and stability of the aerobic granules are important for operation to succeed. An innovative method for improvement of granules’ stability is to apply a chemical additive. Some studies have investigated the effect of divalent metal ions on granule formation. In these works, it is reported that divalent metal ions, such as Ca2+ and Mg2+ could accelerate the granulation process through bridging between negatively charged groups on cell surfaces and linking extracellular polymers [19-21]. The results of these studies also showed that divalent metal ions indeed significantly decrease the time of granulation and result in better physical characteristics of granules and settling ability of biomass.
Researchers have suggested that granulation could be started by bacterial adsorption and bacterial adhesion to inert materials through physicochemical interactions. In addition, extracellular polymers excreted by bacteria, can strengthen the initial granules , so chemical additives whose behaviour is like extracellular polymers, can be more effective than divalent ions.
The key contribution of this paper is that a cationic polymer is used to enhance speed of granule formation and to improve granule structure. The cationic polymer is adsorbed to the cell surfaces which are typically negatively charged. Therefore their interactions can neutralize the cell surface charge and thus help the biomass aggregation like extracellular polymers. For implementing this idea, two bioreactors under the same operational conditions, but one without polymer addition (R1) and the other one with polymer addition (R2) are operated. Then, we measure operational parameters such as Chemical Oxygen Demand (COD), Mixed Liquor Suspended Solid (MLSS), Volatile Suspended Solid (VSS), Specific Oxygen Uptake Rate (SOUR), and Sludge Volume Index in 15 minutes (SVI15). Furthermore, we measure granule structure parameters such as Extracellular Polymer Substance (EPS), cell hydrophobicity and morphological parameters.
Geometry of reactors
Operation of reactors
Operating cycle consisted of four steps. These include aeration, sedimentation, filling and discharge. Air flowrate is set to 0.6 liters per second to maintain superficial upflow air velocity at 3.6 cm per second . Optimum operation cycle is 6 hours , in this research, the operation cycle is chosen 12 hours because of lab limitations. Optimum settling time for aerobic granulation is five minutes . For this study, the settling time is considered ten minutes at the beginning of reactor start-up. Then by improving settling ability, it gradually decreases to five minutes. The optimal discharge time is five minutes  which is considered in this research Filling time is chosen five minutes. Seed sludge was obtained from the wastewater treatment plant of southern Tehran. Initial biomass concentration was considered 3000 mg/L .
Method of analysis
The analyses of COD, MLSS, VSS, SOUR and SVI15 are done according to standard methods . EPS are extracted by Li’s method . The polysaccharide (PS) content is determined by phenol-sulfuric acid method  and the protein (PN) content is measured by Lowery method . Consequently, EPS value is obtained by adding PS value to PN value. Cell hydrophobicity is determined by Rosenberg et al.’s method  with using Hexadecane phase. The hydrophobicity is defined as the percentage of cells adhering to the hexadecane phase after 15 minutes in which water phase and hexadecane phase assumed to be in equilibrium in decantor.
Morphological parameters are determined by image processing techniques. This technique consist of taking pictures and then analysis them by Image J Software. For morphological analysis, two parameters are required: (i) Feret diameter, which is the maximum distance between two points on the perimeter of a granule’s picture, and (ii) aspect ratio, which is the ratio of maximum elliptical diameter to its minimum of granule’s picture . Furthermore, granule diameter distribution is determined by sieving and image analysis.
Synthetic wastewater is made by diluting sugar cane molasses with water to achieve COD concentration of about 1500 mg/L. Ammonium Phosphate is used to adjust values of carbon, nitrogen and Phosphorus to the 100:5:1 ratio, respectively. The pH is adjusted to 7.0 by the addition of Potassium Dihydrogen Phosphates and Potassium Hydrogen Phosphates. Cationic polymer (Reifock Flockungshilfsmittel RP3) was obtained from the Reiflock Company.
Results and discussion
In low polymer concentration values up to 30 ppm, as shown in Figure 2, a significant effect on SVI15 can be observed, but in the moderate to high concentration values, SVI15 is only slightly affected. When pH is between six and eight, similar to our case, microorganisms have negative surface charge . Thus, cationic polymer can easily neutralize the surface charge of cells which aggregates microorganisms to make flocs. Spatial structure of flocs is described by fractals; a mathematical concept, which is defined for irregular shape of objects that possess the property of self-similarity  and consists of three components: microorganisms, extra cellular polymers and water gaps. Cationic polymer probably changes fractal structure and hence decreases water gaps in flocs. With addition of polymer, the water content of flocs decreases and hence their density increases. As a result of this behaviour, settling ability of granules improves. In case of moderate to high values of polymer concentration, due to accumulation of polymer layers on surface of the flocs, repulsion forces make flocs to disintegrate. As a result, the settling ability of biomass decreases and also the supernatant liquid becomes turbid.
The zeta potential of fine particles during the aerobic granulation process gradually decreased. The decrease of zeta potential might be a necessary condition for the formation and stability of aerobic granules . Adding cationic polymer can decrease zeta potential during the operation of reactor.
Effluent suspended solid
Volatile suspended solid
During the reactors’ operation, VSS/TSS slightly decreases in both reactors. This pattern shows that inorganic compounds increase in the granules. Inorganic compounds are dominantly polyvalent ions such as calcium, magnesium and iron which make coherent structure with extra cellular polymers and hence change granules into more durable and denser [33,34,12].
The settling ability of granules
EPS content and SOUR of granules
Cell surface hydrophobicity
Physical properties of granules
It seems that the addition of cationic polymer improves the floc formation ability of some microorganisms which could not have ability to make granules. Furthermore, this behaviour has been observed for poly aluminum chloride .
Formation and stability of aerobic granules is one of the most important parameters in operation and startup of aerobic granule sludge. Using chemicals for improving these characteristics has been done, but none of them is effective. In this study, we used cationic polymer as a strong flocculent agent to improve aerobic granulation. Experimental results illustrated that cationic polymer addition to the system results in fast granulation, less washout from reactor, increasing activity of microbes, more durable granules and improvement of SVI15.
Chemical oxygen demand
Effluent suspended solid
Extracellular polymer substance
Mixed liquor suspended solid
Part per million
Reactor without polymer
Reactor with polymer
Sequencing batch reactor
- SVI15 :
Sludge volume index
Specific oxygen uptake rate
Total suspended solid
Volatile suspended solid
Financial support by the Tehran Province Water and Wastewater organization is gratefully acknowledged. The authors thank the anonymous referees for comments and suggestions which led to great improvement of the quality of the paper.
- Lv Y, Wan C, Lee D-J, Liu X, Tay J-H. Microbial communities of aerobic granules: granulation mechanisms. Bioresour Technol. 2014;169:344–51.View ArticleGoogle Scholar
- Rosman NH, Nor Anuar A, Chelliapan S, Md Din MF, Ujang Z. Characteristics and performance of aerobic granular sludge treating rubber wastewater at different hydraulic retention time. Bioresour Technol. 2014;161:155.View ArticleGoogle Scholar
- Tay JH, Liu QS, Liu Y. Microscopic observation of aerobic granulation in sequential aerobic sludge blanket reactor. J Appl Microbiol. 2001;91:168–75.View ArticleGoogle Scholar
- Yang S, Tay J, Liu Y. Effect of substrate nitrogen/chemical oxygen demand ratio on the formation of aerobic granules. J Environ Eng. 2005;131:86–92.View ArticleGoogle Scholar
- Tay JH, Pan S, Tay ST, Ivanov V, Liu Y. The effect of organic loading rate on the aerobic granulation: the development of shear force theory. Water Sci Technol. 2003;47:235–40.Google Scholar
- Tay JH, Liu QS, Liu Y. The effects of shear force on the formation, structure and metabolism of aerobic granules. Appl Microbiol Biotechnol. 2001;57:227–33.View ArticleGoogle Scholar
- Li Y, Liu Y. Diffusion of substrate and oxygen in aerobic granule. Biochem Eng J. 2005;27:45–52.View ArticleGoogle Scholar
- Qin L, Liu Y, Tay J-H. Effect of settling time on aerobic granulation in sequencing batch reactor. Biochem Eng J. 2004;21:47–52.View ArticleGoogle Scholar
- Wang Z-W, Li Y, Zhou J-Q, Liu Y. The influence of short-term starvation on aerobic granules. Process Biochem. 2006;41:2373–8.View ArticleGoogle Scholar
- Li A-J, Li X-Y, Yu H-Q. Effect of the food-to-microorganism (F/M) ratio on the formation and size of aerobic sludge granules. Process Biochem. 2011;46:11.Google Scholar
- Pan S, Tay JH, He YX, Tay ST. The effect of hydraulic retention time on the stability of aerobically grown microbial granules. Lett Appl Microbiol. 2004;38:158–63.View ArticleGoogle Scholar
- Wang ZW, Liu Y, Tay JH. The role of SBR mixed liquor volume exchange ratio in aerobic granulation. Chemosphere. 2006;62:767–71.View ArticleGoogle Scholar
- Liu Y. Wastewater purification: aerobic granulation in sequencing batch reactors. Boca Raton: CRC Press; 2006.Google Scholar
- Yang SF, Li XY, Yu HQ. Formation and characterisation of fungal and bacterial granules under different feeding alkalinity and pH conditions. Process Biochem. 2008;43:8–14.View ArticleGoogle Scholar
- Dangcong P, Bernet N, Delgenes JP, Moletta R. Aerobic granular sludge-a case report. Water Res. 1999;33:890–3.View ArticleGoogle Scholar
- Khan MZ, Mondal PK, Sabir S. Aerobic granulation for wastewater bioremediation: a review. Can J Chem Eng. 2013;91:1045–58.View ArticleGoogle Scholar
- Wang XH, Zhang HM, Yang FL, Xia LP, Gao MM. Improved stability and performance of aerobic granules under stepwise increased selection pressure. Enzym Microb Technol. 2007;41:205–11.View ArticleGoogle Scholar
- Zheng Y-M, Yu H-Q, Liu S-J, Liu X-Z. Formation and instability of aerobic granules under high organic loading conditions. Chemosphere. 2006;63:1791–800.View ArticleGoogle Scholar
- Jiang HL, Tay JH, Liu Y, Tay ST. Ca2+ augmentation for enhancement of aerobically grown microbial granules in sludge blanket reactors. Biotechnol Lett. 2003;25:95–9.View ArticleGoogle Scholar
- Yu H, Fang H, Tay J. Effects of Fe2+ on sludge granulation in upflow anaerobic sludge blanket reactors. Water Sci Technol. 2000;41:199–205.Google Scholar
- Li X-M, Liu Q-Q, Yang Q, Guo L, Zeng G-M, Hu J-M, et al. Enhanced aerobic sludge granulation in sequencing batch reactor by Mg2+ augmentation. Bioresour Technol. 2009;100:64–7.View ArticleGoogle Scholar
- Geradi MH. Troubleshooting the sequencing batch reactor (wastewater microbiology). John Wiley & Sons, Inc: Hoboken, New Jersey; 2010.View ArticleGoogle Scholar
- Clesceri LS, Eaton AD, Greenberg AE, Association APH, Association AWW, Federation WE, et al. Standard methods for the examination of water and wastewater. 1998.Google Scholar
- Li XY, Yang SF. Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge. Water Res. 2007;41:1022–30.View ArticleGoogle Scholar
- DuBois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem. 1956;28:350–6.View ArticleGoogle Scholar
- Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–75.Google Scholar
- Rosenberg M, Gutnick D, Rosenberg E. Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiol Lett. 1980;9:29–33.View ArticleGoogle Scholar
- Soille P. Morphological image analysis: principles and applications. New York: Springer; 2010.Google Scholar
- Yuehuei H, An RJF. Handbook of bacterial adhesion: principles, methods, and applications. Canada: Humana Press; 2000.Google Scholar
- Li D-H, Ganczarczyk JJ. Structure of activated sludge floes. Biotechnol Bioeng. 1990;35:57–65.View ArticleGoogle Scholar
- Su B, Qu Z, Song Y, Jia L, Zhu J. Investigation of measurement methods and characterization of zeta potential for aerobic granular sludge. J Chem Environ Eng. 2014;2:1142–7.View ArticleGoogle Scholar
- Schwarzenbeck N, Borges JM, Wilderer PA. Treatment of dairy effluents in an aerobic granular sludge sequencing batch reactor. Appl Microbiol Biotechnol. 2005;66:711–8.View ArticleGoogle Scholar
- Wang Z-W, Li Y, Liu Y. Mechanism of calcium accumulation in acetate-fed aerobic granule. Appl Microbiol Biotechnol. 2007;74:467–73.View ArticleGoogle Scholar
- Sobeck DC, Higgins MJ. Examination of three theories for mechanisms of cation-induced bioflocculation. Water Res. 2002;36:527–38.View ArticleGoogle Scholar
- Wang Y, Show K-Y, Tay J-H, Sim K-H. Effects of cationic polymer on start-up and granulation in upflow anaerobic sludge blanket reactors. J Chem Technol Biotechnol. 2004;79:219–28.View ArticleGoogle Scholar
- Xiong Y, Liu Y. Importance of extracellular proteins in maintaining structural integrity of aerobic granules. Colloids Surf B: Biointerfaces. 2013;112:435–40.View ArticleGoogle Scholar
- Liu YQ, Liu Y, Tay JH. The effects of extracellular polymeric substances on the formation and stability of biogranules. Appl Microbiol Biotechnol. 2004;65:143–8.Google Scholar
- Costerton JW. The role of bacterial exopolysaccharides in nature and disease. J Ind Microbiol Biotech. 1999;26(22):551–63.View ArticleGoogle Scholar
- Luo YL, Yang ZH, Xu ZY, Zhou LJ, Zeng GM, Huang J, et al. Effect of trace amounts of polyacrylamide (PAM) on long-term performance of activated sludge. J Hazard Mater. 2011;189:69–75.View ArticleGoogle Scholar
- Liu Y, Yang SF, Liu QS, Tay JH. The role of cell hydrophobicity in the formation of aerobic granules. Curr Microbiol. 2003;46:270–4.View ArticleGoogle Scholar
- Goldberg S, Doyle RJ, Rosenberg M. Mechanism of enhancement of microbial cell hydrophobicity by cationic polymers. J Bacteriol. 1990;172:5650–4.Google Scholar
- Liu Z, Liu Y, Zhang A, Zhang C, Wang X. Study on the process of aerobic granule sludge rapid formation by using the poly aluminum chloride (PAC). Chem Eng J. 2014;250:319–25.View ArticleGoogle Scholar
- Verawaty M, Tait S, Pijuan M, Yuan Z, Bond PL. Breakage and growth towards a stable aerobic granule size during the treatment of wastewater. Water Res. 2013;47:5338–49.View ArticleGoogle Scholar
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