Wastewater Purification: Aerobic Granulation in Sequencing Batch Reactors - Chapter 14 doc

239 14 Influence of Starvation on Aerobic Granulation Yu Liu, Zhi-Wu Wang, and Qi-Shan Liu CONTENTS 14.1 Introduction 239 14.2 Positive Effect of Starvation on Aerobic Granulation 240 14.2.1 Observation of Aerobic Granulation in an SBR 240 14.2.2 Periodic Starvation in the SBR 241 14.2.3 Effect of Periodic Starvation on Cell Surface Hydrophobicity 242 14.3 Inuence of Short Starvation on Aerobic Granules 245 14.3.1 Inuence of Carbon and Nutrients Starvation on Cell Surface Property 246 14.3.2 Inuence of Carbon and Nutrients Starvation on EPS Content 250 14.3.3 Inuence of Carbon and Nutrients Starvation on Microbial Activity and Production 250 14.4 Conclusions 255 References 255 14.1 INTRODUCTION As discussed in the preceding chapters, the unique feature of a sequencing batch reactor (SBR) over the continuous activated sludge process is its cycle operation, whichinturnresultsinaperiodicstarvationphaseduringtheoperation.Suchperi- odical starvation has been thought to be important for aerobic granulation (Tay, Liu, andLiu2001a).Theresponsesofcellstostarvationhavebeenstudiedintensively. Nevertheless,controversialresultshavebeenwidelyreportedintheliterature.For instance, starvation has been thought to induce cell surface hydrophobicity, which facilitates microbial adhesion and aggregation (Bossier and Verstraete 1996; Y. Liu et al. 2004); however, the negative effect of starvation on cell surface hydrophobicity was also reported by Castellanos, Ascencio, and Bashan (2000). Moreover, constant cell surface hydrophobicity was observed during carbon starvation as well (Staffan andMalte1984;Sanin2003;Sanin,Sanin,andBryers2003),andsludgeoccula- tion capacity was found to decrease with prolonged starvation (Rhymes and Smart 1996; Coello Oviedo et al. 2003). In this case, this chapter especially discusses the potentialroleofstarvationinaerobicgranulation. 53671_C014.indd 239 10/2/07 2:18:33 PM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 240 Wastewater Purification 14.2 POSITIVE EFFECT OF STARVATION ON AEROBIC GRANULATION Q S.Liu(2003)investigatedtheroleofperiodicstarvationinaerobicgranulation, andconcludedthatithasapositiveeffectonaerobicgranulationinSBRs,aspre- sented below. 14.2.1 OBSERVATION OF AEROBIC GRANULATION IN AN SBR Theseedsludgehadanaveragesizeof0.07mm,andexhibitedatypicalmorphologyof conventional activated sludge ocs (gure 4.1). On day 23, aerobic granules appeared intheSBR,withaclear,compactphysicalstructure(gure14.2andgure14.3). FIGURE 14.1 Morphology of seed sludge. (From Liu, Q S. 2003. Ph.D. thesis, Nanyang Technological University, Singapore. With permission.) FIGURE 14.2 Morphology of aerobic granules at day 23 in an SBR. (From Liu, Q S. 2003. Ph.D.thesis,NanyangTechnologicalUniversity,Singapore.Withpermission.) 53671_C014.indd 240 10/2/07 2:18:34 PM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Influence of Starvation on Aerobic Granulation 241 14.2.2 PERIODIC STARVATIONINTHESBR Foraerobicgranulation,theSBRisoftenruninamodeoflling,aeration,settling, and withdrawal of the efuent. Figure 14.4 shows the substrate degradation time at different operation cycles in an SBR. At cycle 12, or the second day after the startup, acetate concentration in terms of COD dropped from 1000 mg L –1 to 50 mg L –1 within120minutes,whilethetimeperiodforthesameCODremovalwasreducedto 85minutesatcycle36,andfurtherto50minutesatcycle60.Theseresultsindicate thatthedegradationtimerequiredtoreducesubstrateconcentrationtoaminimum FIGURE 14.3 Morphologyofaerobicgranulesobservedbyscanningelectronicmicro- scopy. (From Liu, Q S. 2003. Ph.D. thesis, Nanyang Technological University, Singapore. With permission.) Time (min) 0 60 120 180 240 COD (mg L –1 ) 0 200 400 600 800 1000 FIGURE 14.4 The COD concentration at the 12th (D), 36th ($), and 60th ( )cyclesofan SBR. (From Liu, Q S. 2003. Ph.D. thesis, Nanyang Technological University, Singapore. With permission.) 53671_C014.indd 241 10/2/07 2:18:35 PM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 242 Wastewater Purification value was shortened markedly over the operation. Thus, for a given cycle length of 4 hours, a starvation phase would exist even at the beginning of the reactor operation (Q S. Liu 2003). It appears from gure 12.4 that the aeration period can be divided into two consecutive phases, a degradation phase, in which external substrate is depleted to a minimum concentration, followed by an aerobic starvation phase, in which the externalsubstrateisnolongeravailableformicrobialgrowth.Itwasfoundthatwith anincreaseinthenumberofcyclesintheSBR,thedegradationtimerequiredtobreak down the same amount of substrate became shorter (gure 14.4), that is, the starva - tiontimeisincreasedwiththenumberofoperationcycles.Buitron,Capdeville,and Horny(1994)studiedtherelationshipofdegradationtimetoSBRoperationcycles, andfoundthataftera10-cycleoperation,thedegradationtimewasreducedby80%, thatis,80%oftheaerationperiodwasinastateofaerobicstarvation.Foraxed cycletimeof4hours,theaerobicstarvationperiodwasfoundtobe105minutes atcycle12,140minutesatcycle36,and175minutesatcycle60(Q S.Liu2003). Figure 14.5 further exhibits the direct relationship between the cycle number and starvation time observed in the SBR. Similar results were also reported by Buitron, Capdeville, and Horny (1994). This seems to indicate that there is a periodic aerobic starvationphaseinthecycleoperationofSBR,butsuchaperiodicstarvationpattern doesnotexistinthecontinuousactivatedsludgeprocess. 14.2.3 EFFECT OF PERIODIC STARVATION ON CELL SURFACE HYDROPHOBICITY Figure14.6showschangesincellsurfacehydrophobicityandsubstratedegradation timeinthecourseoftheoperationofanSBRforaerobicgranulation.Ascanbe seen, the seed sludge had a surface hydrophobicity of 49%, while the cell surface hydrophobicitywasincreasedto70%atcycle36,andfurtherto80%atcycle60,and nally stabilized at 85% after the formation of aerobic granules. The cell surface Number of SBR Operation Cycles 12 36 60 90 130 Starvation Time (min) 0 30 60 90 120 150 180 FIGURE 14.5 The observed starvation time versus the number of cycles in the SBR. (Data fromLiu,Q S.2003.Ph.D.thesis,NanyangTechnologicalUniversity,Singapore.) 53671_C014.indd 242 10/2/07 2:18:36 PM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Influence of Starvation on Aerobic Granulation 243 hydrophobicity of aerobic granules was nearly two times higher than that of the seed sludge. As demonstrated in chapter 9, aerobic granulation is associated with an increase in cell surface hydrophobicity. Itcanbeseeningure14.7thatthecellsurfacehydrophobicitywasincreased from55%to85%whenthestarvationtimewasincreasedfrom105to190minutes. Apparently, the periodic starvation in the SBR improves the cell surface hydro- phobicity. However, in the continuous activated sludge reactor, no improvement in cell surface hydrophobicity was observed in the course of operation, for example, the cellsurfacehydrophobicityofsludgecultivatedinthecontinuousreactorwassimilar tothatoftheseedsludgeoverthewholeexperimentalperiod(Q S.Liu2003). Number of Reactor Operation Cycles 0 12 36 60 90 130 Cell Surface Hydrophobicity (%) 40 50 60 70 80 90 Degradation Time (min) 0 20 40 60 80 100 120 140 FIGURE 14.6 Changes in cell surface hydrophobicity and substrate degradation. Starvation Time (min) 105 140 175 191 192 194 Hydrophobicity (%) 50 60 70 80 90 FIGURE 14.7 Cell surface hydrophobicity versus starvation time in an SBR. (Data from Liu,Q S.2003.Ph.D.thesis,NanyangTechnologicalUniversity,Singapore). 53671_C014.indd 243 10/2/07 2:18:38 PM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 244 Wastewater Purification The response of bacteria to starvation has been widely reported (Kjelleberg and Hermansson 1984; Hantula and Bamford 1991; Bossier and Verstraete 1996). In asequencingbatchbiolterreactor,DiIaconietal.(2006)foundthatthestarva - tiontimeincreasedwiththebedheightaslessandlessamountofsubstratewould reachdeeperpartsofthelterbed;meanwhilecellsurfacehydrophobicitytended to increase along the depth of the lter bed (table 14.1). According to such results, Di Iaconi et al. (2006) concluded that starvation could improve cell surface hydro - phobicityandtheeffectofstarvationoncellsurfacehydrophobicitywouldbemore signicant than that of hydrodynamic shear force. Kjelleberg and Hermansson (1984) demonstrated that under starvation condi- tions, bacteria became more hydrophobic, which in turn facilitated microbial adhe - sion and aggregation. In fact, aggregation can be regarded as an effective strategy of cells against starvation. A similar phenomenon was also observed by Watanabe, Miyashita,andHarayama(2000),thatis,cellsshowedahighersurfacehydrophobic - ity when they were subject to starvation. It is believed that hydrophobic binding has aprimeimportanceforcellattachment,thatis,ahighercellsurfacehydrophobicity wouldresultinastrongercell-to-cellinteractionandfurtheradensestructure(see showingalowersludgevolumeindex(SVI)athighercellsurfacehydrophobicity. Inaparallelstudy,Q S.Liu(2003)foundthataerobicgranulationfailedinthe continuous activated sludge reactor, and aerobic granules were only developed in the SBR. These ndings imply that the periodic starvation-induced hydrophobicity is a governingfactorinaerobicgranulationintheSBR. Asshowninchapter9,cellsurfacehydrophobicityplaysacrucialroleinthe formation of biolm and biogranules. In a thermodynamic sense, increased cell surface hydrophobicity can result in a lowered surface Gibbs energy, which in turn favors cell-to-cell interaction. In addition, cells in starved colonies were found to form connecting brils, which in turn strengthened cell-to-cell interaction and com - munication(VaronandChoder2000).Apparently,suchstarvation-inducedchanges favortheformationofstrongmicrobialaggregates.Starvationhasbeenproposedto be a trigger in the microbial aggregation process (Bossier and Verstraete 1996). As discussedearlier,inanSBRmicroorganismsaresubjecttoaperiodicaerobicstar - vation.Tay,Liu,andLiu(2001a)thoughtthattheperiodicstarvationpresentinthe TABLE 14.1 Biomass Hydrophobicity at Different Filter Bed Depths Bed Depth (cm) Biomass Hydrophobicity (%) 0–10 25 10–20 35 20–30 40 30–40 60 Source: Data from Di Iaconi, C. et al. 2006. Biochem Eng J 30: 152–157. 53671_C014.indd 244 10/2/07 2:18:38 PM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC chapter9).Thispointindeedisconrmedbytheresultspresentedingure14.8, Influence of Starvation on Aerobic Granulation 245 SBRwouldbemoreeffectiveintriggeringchangesinthecellsurface,andfurther lead to a stronger microbial aggregate. BasedontheirstudyofaerobicgranulationinSBRs,Li,Kuba,andKusuda(2006) thoughtthat“aerobicgranulationisinitiatedbystarvationandcooperatedbyshear forceandanaerobicmetabolism,”andfurtherproposedanEPS-relatedpathwayof aerobic granulation, as illustrated in gure 14.9. According to the interpretation by Li, Kuba,andKusuda(2006),inthebeginning,starvationplaysanessentialroleinaerobic granulation, and subsequently the growth of the aerobic granule provides an anaerobic microenvironment inside the aerobic granule, which favors anaerobic metabolism of facultative microorganisms. Furthermore, both starvation and facultative microorgan - isms facilitate aerobic granulation. It appears from gure 14.9 that there are two pos - siblepathwaysleadingtoaerobicgranulation:(1)step1 n step 3 n step 4 n step 5, and this process is named starvation-driven granulation; (2) step 2 n step 3 n step 4 n step 5, called anaerobic granulation (Li, Kuba, and Kusuda 2006). So far, no solid evidence supports the mechanisms of aerobic granulation, as illustrated in gure 14.9, thus such interpretations of aerobic granulation are subject to further discussion. Consequently, the real role of starvation in aerobic granulation is still debatable and different views exist in the present literature. 14.3 INFLUENCE OF SHORT STARVATION ON AEROBIC GRANULES Toofferin-depthinsightsintotheroleofstarvationinaerobicgranulation,Z W. Wangetal.(2006)investigatedtheinuenceofshortstarvationonaerobicgranules as presented below. Hydrophobicity (%) 40 50 60 70 80 90 SVI (mL g –1 ) 40 60 80 100 120 140 160 180 200 FIGURE 14.8 Sludge volume index (SVI) versus cell surface hydrophobicity in an SBR. (From Liu, Q S. 2003. Ph.D. thesis, Nanyang Technological University, Singapore. With permission.) 53671_C014.indd 245 10/2/07 2:18:39 PM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 246 Wastewater Purification 14.3.1 INFLUENCE OF CARBON AND NUTRIENTS STARVATION ON C ELL SURFACE PROPERTY Figure 14.10 shows the effects of carbon, nitrogen, phosphorus, and potassium starva- tiononcellsurfacehydrophobicityofaerobicgranules.Thecellsurfacehydrophobicity tendedtodecreaseinthecourseoftheN,P,andKstarvationcultures,forexample, thecellsurfacehydrophobicitydecreasedfromtheinitialvalueof80%toabout60% after4hoursofNandPstarvation.Meanwhile,nosignicantchangeincellsurface hydrophobicityofaerobicgranuleswasfoundintheKstarvation,whereascellsur - facehydrophobicityexhibitedaslightincreaseby7%inthecourseofCstarvation culture.ChangesincellsurfacezetapotentialintheC,N,P,andKstarvationbatch cultureareshowningure14.11.Thecellsurfacezetapotentialofaerobicgranules undertherespectiveC,N,P,andKstarvationuctuatedaroundacertainvalue,that is, no signicant changes can be observed under these starvation conditions. The fundamental principle of charge interaction shows that oppositely charged objectswillexertanattractiveinuenceuponeachother,while,incontrasttothe attractiveforcebetweentwoobjectswithoppositecharges,twocellsthatareoflike chargewillrepeleachother(gure14.12).Itisobviousthatanegativelychargedcell AB E 1 2 5 D C 4 Water Negative Charge 3 FIGURE 14.9 Effect of EPS on aerobic granulation. (A) Seed sludge with low cell hydro- phobicity and high negative charge; (B) ocs or granules with low surface negative charge andhighcellhydrophobicity;(C)aggregatesofocsorgranules;(D)growthofgranule under given shear condition; (E) a reasonable amount of EPS. 1: Starvation-associated EPS consumption; 2: facultative microorganisms-associated EPS consumption and production; 3: EPS-caused modications of cell surface properties; 4: aggregation of ocs and growth of granules; 5: shear force-enhanced granule structure and detachment. (From Li, Z. H., Kuba, T., and Kusuda, T. 2006. Enzyme Microb Technol 38:670–674.Withpermission.) 53671_C014.indd 246 10/2/07 2:18:41 PM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Influence of Starvation on Aerobic Granulation 247 willexertarepulsiveforceuponasecondnegativelychargedcell,andthisrepulsive forcewillpushthetwocellsapart,andsubsequentlypreventmicrobialaggregation. Itisunderstandablethataweakrepulsiveforcecanbeexpectedatalow surface charge density, thus reduced surface charge density has been thought to promote microbial aggregation, which is a key step towards to successful aerobic granulationinSBRs.Furthermore,cellsurfacehydrophobicityseemstoinversely Hydrophobicity (%) 68 72 76 80 84 88 92 Hydrophobicity (%) 40 50 60 70 80 90 Hydrophobicity (%) 30 40 50 60 70 80 Time (hours) 012345 Hydrophobicity (%) 50 60 70 80 90 N-starvation C-starvation P-starvation K-starvation FIGURE 14.10 ChangesincellsurfacehydrophobicityinthecourseoftheC,N,P, and K starvation batch cultures. (Data from Wang, Z W. et al. 2006. Process Biochem 41: 2373–2378.) 53671_C014.indd 247 10/2/07 2:18:42 PM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 248 Wastewater Purification + – Oppositely-charged objects attract + + – – Objects with like charges repel FIGURE 14.12 Illustration of charge interaction. 53671_C014.indd 248 10/2/07 2:18:43 PM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Zeta Potential (mv) –80 –60 –40 –20 0 C-starvation Zeta Potential (mv) –60 –40 –20 0 N-starvation Zeta Potential (mv) –60 –40 –20 0 P-starvation Time (hours) 0 1 2 3 4 5 Zeta Potential (mv) –60 –40 –20 0 K-starvation fIGure 14.11 Changes in cell surface zeta potential in the course of the C, N, P, and K star- vation batch cultures. (Data from Wang, Z W. et al. 2006. Process Biochem 41: 2373–2378.) [...]... of aerobic granules This in turn results in instability of aerobic granular sludge In fact, potassium deficiency is commonly found in industrial wastewater (Murthy and Novak 1998) Similar to figures 14. 15 and 14. 16, Hueting, de Lange, and Tempest (1979) also observed a progressive increase in the respiration rate of cells, with a corresponding fall in the biomass production under K limitation Konings... 53671_C 014. indd & Francis Group, LLC 10/2/07 2:18:48 PM In uence of Starvation on Aerobic Granulation 253 As can be seen in figure 14. 14, the content of extracellular polysaccharides decreased slightly in the N- and P-starved aerobic granules, while about 53% and 65% of PS reduction was observed in aerobic granules deprived of carbon and potassium, respectively These results indicate that under the C- and... observed in SBRs would induce cell surface hydrophobicity which in turn facilitates aerobic granulation in the SBR It appears from figure 14. 10, however, that the cell surface hydrophobicity of aerobic granules subjected to N and P starvation shows a decreasing trend, while change in the cell surface hydrophobicity of the K-starved aerobic granules is insignificant and only the C-starved aerobic granules... 2000 Organization and cell-cell interaction in starved Saccharomyces cerevisiae colonies J Bacteriol 182: 3877–3880 Wang, Z P., Liu, L., Yao, J., and Cai, W 2006 Effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors Chemosphere 63: 1728–1735 Wang, Z.-W., Liu, Y., and Tay, J.-H 2005 Distribution of EPS and cell surface hydrophobicity in aerobic granules Appl Microbiol... the disintegration of aerobic granules was coupled with a sharp decrease in the PS content in aerobic granules (Tay, Liu, and Liu 2001b), that is, the stability of aerobic granules is tightly associated with the PS of aerobic granules which may serve as matrix materials to strengthen the spatial structure of aerobic granules One important engineering implication of figure 14. 14 is that when aerobic. .. content in aerobic granules In addition, Z P Wang, ZP et al (2006) also reported that extracellular polymeric substances (EPS) were produced mainly in the exponential phase, and served as carbon and energy sources in the starvation phase during the granulation process, and they thought that such a function of PS could regulate microbial growth in the interior and exterior of granules and help maintain the... indicate that under the C- and K-starvation conditions, aerobic granules prefer to metabolize their PS in order to provide energy for maintaining basic functions of the living cells This phenomenon has been observed in other cultures under C-starvation conditions (Patel and Gerson 1974; Boyd and Chakrabarty 1994; Ruijssenaars, Stingele, and Hartmans 2000; Zhang and Bishop 2003; Z.-W Wang, Liu, and Tay 2005)... turbidity was also measured in the course of the collective starvation (figure 14. 19) It was found that the bulk liquid became more and more turbid with the increase in the starvation time This may indicate a bacterial detachment from aerobic granules in response to the collective starvation In fact, a similar phenomenon was also found in the biofilm process under substrate- and nutrient-deficient conditions... of starvation in aerobic granulation Some results showed that starvation is essential in aerobic granulation, and can serve as a trigger for microbial aggregation However, it appears that aerobic C, P, N, and K starvation can alter the surface properties of aerobic granules and cause negative effects on the stability and activity of aerobic granules Therefore, discussion on starvation in aerobic granular... inorganic ions on protein degradation and ribonucleic acid synthesis in Escherichia coli J Bacteriol 143 : 1223–1233 Tay, J H., Liu, Q.-S., and Liu, Y 2001a Microscopic observation of aerobic granulation in sequential aerobic sludge blanket reactor J Appl Microbiol 91: 168–175 Tay, J H., Liu, Q.-S., and Liu, Y 2001b The role of cellular polysaccharides in the formation and stability of aerobic granules Lett . 239 14 In uence of Starvation on Aerobic Granulation Yu Liu, Zhi-Wu Wang, and Qi-Shan Liu CONTENTS 14. 1 Introduction 239 14. 2 Positive Effect of Starvation on Aerobic Granulation 240 14. 2.1. reactor, and aerobic granules were only developed in the SBR. These ndings imply that the periodic starvation-induced hydrophobicity is a governingfactorinaerobicgranulationintheSBR. Asshowninchapter9,cellsurfacehydrophobicityplaysacrucialroleinthe formation. starvation in aerobic granulation is still debatable and different views exist in the present literature. 14. 3 INFLUENCE OF SHORT STARVATION ON AEROBIC GRANULES Toofferin-depthinsightsintotheroleofstarvationinaerobicgranulation,Z