181 10 Essential Roles of Extracellular Polymeric Substances in Aerobic Granulation Yu Liu and Zhi-Wu Wang CONTENTS 10.1 Introduction 181 10.2 Main Composition of EPS in Aerobic Granules 181 10.3 Major Factors Inuencing EPS Production In Aerobic Granules 183 10.4 The Role of EPS in Aerobic Granulation 185 10.5 EPS-Enhanced Stability of Aerobic Granules 187 10.6 Conclusions 191 References 191 10.1 INTRODUCTION Extracellular polymeric substances (EPS) are sticky materials secreted by cells; extra- cellular polysaccharides (PS) are the important component of EPS and are highly involved in the formation of matrix structure and improvement of long-term stability of aerobic granules, as discussed in the preceding chapters. It is thought that EPS actasaneffectivebiogluetocross-linkbacteriaintoanaerobicgranule (gure10.1), and the EPS matrix of the aerobic granule can protect bacteria from harsh environ- mentalconditions.InviewoftheimportanceofPS,thischapterattemptstoprovide adeeperinsightintothefunctionsofEPSinaerobicgranulation. 10.2 MAIN COMPOSITION OF EPS IN AEROBIC GRANULES EPShavebeendetectedinsignicantquantitiesinbothaerobicandanaerobicgran- ules,andtheyformathree-dimensionalmatrixinwhichbacteriaandotherparticles are embedded. EPS are produced by microorganisms themselves during cultivation, whichareadvantageousinmanyrespectsfortheirsurvivalinvariouscircumstances. Intermsofmicrobiology,EPScanhelpstabilizemembranestructureandalsomay serveasaprotectivebarrier(Prescott,Harley,andKlein1999).EPSproducedin biogranules contain variable proportions of proteins, polysaccharides, nucleic acids, humics-likesubstances,lipids,andheteropolymers-likeglycoprotein(Goodwinand 53671_C010.indd 181 10/29/07 7:32:41 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 182 Wastewater Purification Forster1985;HoranandEccles1986;Grotenhuisetal.1991;Urbain,Block,and Manem1993;Jorandetal.1995;Frolundetal.1996).Itshouldbepointedoutthat polysaccharides are the only component that are synthesized extracellularly for a specic function with neutral and acidic polysaccharides, while proteins, lipids, and nucleic acids can exist in the extracellular polymer network due to the excretion of intracellularpolymersorasaresultofcelllysis(DurmazandSanin2001;Mahmoud etal.2003).EPShasbeenfoundinalmostallformsofmicrobialaggregates,includ - ing bioocs, biolm, anaerobic and aerobic granules. However, the content of EPS inaerobicgranuleswasfoundtobemuchhigherthanthoseinconventionalbioocs andbiolms(Tay,Liu,andLiu2001c). In the environmental engineering literature, there are contradictory reports onthecompositionofEPSinbiogranules,especiallytheratioofcarbohydratesto proteins.ProteinshavebeenreportedtobethepredominantcomponentofEPSin anaerobic granules (Fukuzaki, Nishio, and Nagai 1995), while other evidence shows that EPS were mainly composed of carbohydrates (Fang 2000). It seems that the quantity and the composition of EPS produced by bacteria depend on a number of factors, such as microbial species, the growth phase of the strain, the type of limiting substrate (carbon, nitrogen, and phosphorous), oxygen limitation, ionic strength,culturetemperature,shearforceandsoon(Nielsen,Jahn,andPalmgren 1997;Tay,Liu,andLiu2001a;Nicholsetal.2004;Qinetal.2004).Forexample,the totalamountofEPSandtheircompositionintermsoftheratioofcarbohydratesto proteins were inuenced by the magnitude of carbon and nitrogen sources (Sheng, Yu,andYue2006).Thismayimplythatthecompositionofextracellularpolymers varies and is related to microbial species, the physiological state of bacteria, and operatingconditionsunderwhichbiogranulesaredeveloped.Infact,ashiftinbac - terialspeciesduringbiogranulationhasbeenreported,andsuchamicrobialshift wouldaffecttheproductionandcompositionofEPS(Etchebehereetal.2003;Yiet al.2003).ItappearsthattheincomparableEPSresultsreportedintheliteratureresult partiallyfromthecomplexityofamixedmicrobialcultureunderdifferentoperation conditions. Another point that needs to be addressed is that the EPS mainly include boundandsolublepolymers,theratiobetweenwhichmaychangesubstantiallyeven withinthesamespeciesundervariousgrowthconditions.Thesolublepolymerscan Individual bacterium Polymeric chain of EPS Bridging FIGURE 10.1 A schematic interpretation of extracellular polysaccharides in aerobic granulation. 53671_C010.indd 182 10/29/07 7:32:42 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Essential Roles of Extracellular Polymeric Substances in Aerobic Granulation 183 be transferred to the supernatant after centrifugation, while the bound polymers are still tightly attached to cells and cannot be recovered from the supernatant (Nielsen, Jahn, and Palmgren 1997). Zhang, Bishop, and Kinkle (1999) compared different EPS extraction methods for a biolm sample, including the regular centrifugation, ethylenediamine tetraace - tic acid (EDTA) extraction, ultracentrifugation, steaming extraction, and regular centrifugation with formaldehyde (RCF), and found that the RCF method gave the highest yield ratio of carbohydrates to proteins of 13.7 for the aerobic/sulfate reduc - ingbiolmsandtheothermethodsgavearangeofratiosof1.54to2.23.Thisis indeedconsistentwiththendingbyAzeredo,Lazarova,andOliveira(1999)that manyEPSextractionmethodsdevelopedforbiolmswerenotefcient,andsome - howcouldpromoteleakageofintracellularmaterials.Sheng,Yu,andYu(2005a)also compared four extraction methods (EDTA, NaOH, H 2 SO 4 , heating-centrifugation) ofEPSproducedfromaphotosyntheticbacterium, Rhodopseudomonas acidophila, and they concluded that the EDTA extraction method was the most effective over theothers.Areviewbasedonover200publicationsrelatedtoEPSinactivated sludge reveals that reported composition and quantity of EPS strongly depend on the extractionmethodsemployed(LiuandFang2003).Althoughanumberofphysical andchemicalmethods,forexample,high-speedcentrifugation,boilingtreatmentin acidoralkali,andutilizationofsolventextractionorcationexchangeresinsarecur - rentlyavailableforextractingEPSfrombiogranules,noneofthemhasbeenadopted as a standard procedure yet. In addition to the effect of microbial species, the use of nonstandardized procedures causes incomparability of the EPS results available in the present literature. 10.3 MAJOR FACTORS INFLUENCING EPS PRODUCTION IN AEROBIC GRANULES There is no need for microorganisms to secrete excessive EPS under normal culture conditions.TheobservedenhancedproductionofEPSinbiogranulesisinducedby someso-calledstressfulcultureconditions(Nicholsetal.2004;Qinetal.2004).So far, it has been understood that a number of operating parameters, including reactor type, substrate composition, substrate loading rate, hydraulic retention time, hydro - dynamic shear force, settling time in sequencing batch reactors (SBRs), feast-famine regimeninSBRs,culturetemperature,etc.,maystimulatebacteriatosecretemore EPS.ThecompositionofEPSisalsorelatedtothecharacteristicsofthefeedwaste - water,forexampleEPSinanaerobicgranulesgrownonprotein-richwastewaterhad highproteinandDNAlevels,whereashighpolysaccharidescontentwasfoundin anaerobicgranulesfedbyothertypesoforganicwastewaters(BatstoneandKeller 2001).Inaddition,thedeciencyofnitrogenwasfoundtofavortheproductionof EPS, which in turn accelerated anaerobic granulation (Punal et al. 2003). Experimental evidence from aerobic granulation research shows that stressful operatingconditionsintermsofhighhydrodynamicshearforce,shortsettling time/hydraulic retention time, and periodic feast-famine periods would signicantly stimulate bacteria to produce more extracellular polysaccharides over proteins in SBRs, as shown in the preceding chapters. The EPS production seems to be positively 53671_C010.indd 183 10/29/07 7:32:43 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 184 Wastewater Purification relatedtothespecicoxygenutilizationrate(SOUR)ofaerobicgranulesdeveloped in SBRs. In fact, the catabolic activity of microorganisms is directly correlated with the electron transport system activity, which can be roughly described by the SOUR (Trevors1984;Lopez,Koopman,andBitton1986).Moreover,intheaerobicoxi - dation process the respiratory activity of cells couples to the proton translocation activityandaclearlinkageofoxygenreductiontoprotontranslocationhasbeen established (Babcock and Wikstrom 1992). This implies that aggregated bacteria canrespondtothestressfulcultureconditionsbyregulatingtheirenergymetabolism (Liu and Tay 2002). Chan et al. (2004) thought that the purpose of polymer production was to local - ize iron oxyhydroxide mineral precipitation in order to enhance metabolic energy generation. In general, the environmental factors inuencing EPS production and compositioncanbeattributedtochangesinenvironmentalconditionsthatcauseashift in the microbial community. Subsequently, the distribution of EPS-producing micro- organisms varies (increases or decreases) in the whole microbial consortium, and regulationofthemetabolicpathwayofEPSproductionoccursinresponsetochanges intheenvironmentalconditions.InastudyofEPSproductioninthepresenceoftoxic substances, addition of toxic substances, such as Cu 2+ ,Cr 6+ ,Cd 2+ ,and2,4-dichloro- phenol (2,4-DCP), was found to stimulate Rhodopseudomonas acidophila to secrete more EPS, for example, at respective concentrations of 30 mg L –1 Cu 2+ ,40mgL –1 Cr 6+ ,5mgL –1 Cd 2+ ,and100mgL –1 2,4-DCP, the EPS content in Rhodopseudomonas acidophila wasincreasedby5.5,2.5,4.0,and1.4timesthatofthecontrol(Sheng,Yu, andYue2005b).TheseresultsindicatethatEPScanprotectcellsagainsttoxiceffect.In fact, the strong and compact EPS-mediated structure of aerobic granules (gure 10.2) should provide adequate protection against exposure to chemical toxicity. It has not been demonstrated yet whether the genes for EPS production are expressed before or after bacterial granulation. There are two scenarios for EPS pro - duction in time sequence along microbial granulation: (1) microorganisms initially prepare EPS for subsequent self-attachment; (2) microbial attachment comes rst, followedbytheproductionofEPS.Inscenario1,EPSproductionoccurspriorto FIGURE 10.2 Extracellular (EPS) matrix structure observed in acetate-fed aerobic granules. 53671_C010.indd 184 10/29/07 7:32:44 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Essential Roles of Extracellular Polymeric Substances in Aerobic Granulation 185 granulation, and the appearance of EPS at the initial site of contact between micro- bialcellsmaybeduetothemigrationofpolymermoleculesalreadyexistingon thecellsurface.Inscenario2,EPSisproducedafterinitialmicrobialattachment, and bacterial attachment in this case may provide some physiological conditions necessary for EPS excretion. 10.4 THE ROLE OF EPS IN AEROBIC GRANULATION EPS facilitate cell-to-cell interaction and further strengthen microbial structure throughformingapolymericmatrix.InrecognitionoftheimportanceofEPS quantity in biogranulation process, the contribution of the EPS properties, such as hydrophobicity and charge, also need to be taken into account (Andreadakis 1993; Liaoetal.2001;Wang,Liu,andTay2005).SinceEPSmayaccumulateatthecell surface, it could alter cell surface properties, such as cell surface hydrophobicity, surfacechargedensity,bindingsite,andsurfacemorphology.Thesurfacechargehas long been believed to be important in controlling the stability of microbial aggre- gates.Itiswellknownthatbacteriacarrynetnegativesurfacechargewhencultivated at physiological pH values (Rouxhet and Mozes 1990). According to the well-known DLVOtheory,whenthetwosurfaceshaveachargeofthesamesign,repulsiveforce betweencellswillpreventtheapproachofonecelltoanother. Some results showed that EPS could decrease the negative charges of the cell surfaces,andtherebyfurtherbridgetwoneighboringcellsphysicallytoeachother (Shen, Kosaric, and Blaszczyk 1993; Schmidt and Ahring 1994). Using a colloid titra - tion technique, Morgan, Forster, and Evison (1990) reported that granular sludge was lessnegativelychargedthanactivatedsludge,whileTsunedaetal.(2003)employed asoftparticleelectrophoresistechniquetoinvestigatetheinuenceofEPSoncell surface electrokinetics, and found that EPS could increase the softness of the cell surface and further decrease the negative surface charge density surrounding the cellsurface,thatis,theEPSlayercouldholdalowernegativechargecomparedwith thoseofthenativecellsurface. It should be realized that electrostatic interaction between cells is closely associ - atedwiththeamountofEPSproducedaswell.Microbialattachmentontoasolid surface can be inhibited by electrostatic interaction when the EPS amount is small, whereascelladhesionisenhancedbypolymericinteractioniftheEPSamountis large(Tsunedaetal.2003).Wangetal.(2006)reportedthatalongwithaerobic granulationinanSBR,bothbiomassandEPSconcentrationstendedtoincrease, example,theEPScontentinthematureaerobicgranuleswasabout47mgg –1 MLSS (mixed liquor suspended solids) whereas it was only 17 mg g –1 MLSS in the seed activated sludge. This seems to imply that aerobic granulation would be associated with an increase in the EPS content. Cell surface hydrophobicity has been considered as a triggering force of bio- granulation(seechapter9).Microorganismswithdifferenthydrophobicitieswere detected in activated sludge (Singh and Vincent 1987; Jorand et al. 1995), and the highcellsurfacehydrophobicitywasusuallyassociatedwiththepresenceofbrillar structureoncellsurfaceandspeciccellwallproteins(McNabetal.1999;Singleton, 53671_C010.indd 185 10/29/07 7:32:44 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC andadecreasedsludgevolumeindex(SVI)wasfoundaccordingly(gure10.3),for 186 Wastewater Purification Masuoka,andHazen2001).Infact,thecellwallofbacteriainanaerobicgranules wassurroundedbyanEPSlayer(Forster1991;deBeeretal.1996;Veigaetal. 1997). This implies that cell surface hydrophobicity may be related to EPS. Some evidence suggests that proteins and amino acids are the hydrophobic components oftheEPS,whilepolysaccharidesarehydrophilic(Dignacetal.1998).Wangetal. (2006) studied the correlation between EPS and cell surface hydrophobicity of aerobic granules, and a positive correlation of cell surface hydrophobicity to total EPScontentwasobserved,asshowningure10.4. Jorand et al. (1998) studied hydrophobic and hydrophilic properties of extra- cellularpolymersproducedbyactivatedsludge,andfoundthatasignicantpropor - tion of the extracellular polymers was hydrophobic, that is, hydrophobic extracellular Relative Hydrophobicity (%) 45 50 55 60 65 70 75 PN, PS and Total EPS (mg g –1 MLSS) 0 10 20 30 40 50 FIGURE 10.4 Correlation of cell surface hydrophobicity to proteins ($) and polysaccharides ( c )andtotalEPS(D).(DatafromWang,Z.P.etal.2006.Chemosphere 63: 1728–1735.) Time (days) 02468101214161820 EPS (mg g –1 MLSS) 15 20 25 30 35 40 45 50 MLSS (g L –1 ) 0 2 4 6 8 10 SVI (mL g –1 ) 20 40 60 80 100 120 140 160 180 FIGURE 10.3 Changes in EPS (D), MLSS ($), and SVI ( c ) in the course of operation. (Data fromWang,Z.P.etal.2006.Chemosphere 63: 1728–1735.) 53671_C010.indd 186 10/29/07 7:32:46 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Essential Roles of Extracellular Polymeric Substances in Aerobic Granulation 187 polymersareinvolvedintheformationandorganizationofmicrobialaggregates. Due to the presence of hydrophobic and hydrophilic groups in EPS, the measured hydrophobicity of EPS indeed reects an average of the hydrophobicity of its com- ponents (Daffonchio, Thaveesri, and Verstraete 1995), whereas the cell surface hydrophobicity and charge is related to the production, composition, and physical characteristicsofEPS(Liaoetal.2001),butnospecicevidenceshowsthequantita- tive contribution of hydrophobic components of EPS to the overall hydrophobicity of biogranulessofar.Thereisevidencethatthereductionofsurfacechargewouldnot bearequirementfortheformationofactivatedsludge,thatis,thechargeneutraliza- tionwouldnotbethemainmicrobialocculationmechanism(Pavonim,Tenney, and Echelberger 1972; Strand, Varum, and Ostgaard 2003), whereas hydrophobic interaction may be fundamental in biogranulation (see chapter 9). It appears from thestudyofbiolmsthatfewbacteriawerede facto in contact with the hydrophilic surface,butmorewithhydrophobicsurfacesofothercells(Ghigo2003). Sofar,differentviewsexistwithregardtotheroleofEPSinaerobicgranulation. Di Iaconi et al. (2006) reported that the main component of EPS in aerobic granular biomass in a sequencing batch biolter reactor was made of proteins, and protein- richEPSwouldimprovethestabilityofgranularbiomassstructure.Asthemain componentofproteinsisaminoacids,whichoftencarrynegativecharges,theywill contribute more than carbohydrates to electrostatic bonds, with consequent increase of biomass structure stability (Di Iaconi et al. 2006). Zheng, Yu, and Sheng (2005) studied the physical and chemical characteristics of aerobic granular sludge from a sequencing batch airlift reactor, and they found that the contents of proteins and carbohydrateinEPSdecreasedfrom126.6and15.3mgg –1 volatile suspended solids (VSS)inseedsludgeto51.4and5.9mgg –1 VSS in aerobic granular sludge. These resultsseemtoimplythatEPSarenotinvolvedinaerobicgranulationinthisspecial case,butthereasonbehindthisisnotyetclear.Itmaybeduetodifferentbiodegrada- tion kinetics of carbohydrates and proteins under specic operation conditions. 10.5 EPS-ENHANCED STABILITY OF AEROBIC GRANULES The accumulation of EPS has been found to correlate with biological aggregation, whereasthemetabolicblockingofthesynthesisofextracellularpolysaccharides prevents microbial aggregation (Cammarota and Sant’Anna 1998; Yang, Tay, and Liu2004).EPSingranuleshavebeenoftenhypothesizedtobridgetwoneighboring bacterial cells physically to each other as well as with other inert particulate matter, andsettleoutasaggregates(Ross1984;Shen,Kosaric,andBlaszczyk1993;Schmidt andAhring1994;Tay,Liu,andLiu2001b).Inthebiogranulationprocess,EPSpro- vide an extensive surface area for bacterial binding. Furthermore, EPS matrixes surrounding aggregated bacteria can provide sites available for attraction of organic andinorganicmaterials(Yu,Tay,andFang2001;Sponza2002).Thetotalconcen - trationsofelectrostaticbindingsitesonEPSwerefoundtobe20-to30-foldhigher than those reported for bacterial cell surfaces (Liu and Fang 2002). This seems to indicate that EPS of neighboring microbial cells may form a cross-linked network by attraction of organics or inorganics, such as cationic bridging, and further strengthen the structural integrity of a biogranule (Yu, Tay, and Fang 2001). 53671_C010.indd 187 10/29/07 7:32:47 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 188 Wastewater Purification Microscopic observation shows that EPS with a lamentous structure is present withinandaroundthestructureofbiogranules(Forster1991;deBeeretal.1996; Veigaetal.1997;Tay,Liu,andLiu2001c),whileEPSalsocanllintheintercellular spaces in the microcolonies present in aerobic granules (Jiang, Tay and Tay 2002; McSwainetal.2005).ItismostlikelythatEPSplaysanimportantroleinmaintain - ingthestructuralandfunctionalintegrityofaerobicgranules(gure10.5). Tay,Liu,andLiu(2001b)investigatedtheroleofEPSintheformationand stabilityofaerobicgranules.Itwasfoundthattheformationofaerobicgranuleswas coupled with a signicant increase in EPS expressed as the ratio of polysaccharides (PS) to proteins (PN), while disappearance of aerobic granules, indicated by a signicant decrease in bioparticle size, was associated with a simultaneous decrease in extracellular polysaccharides (gure 10.6 and gure 10.7). This indicates that extracellular polysaccharides play a crucial role in the formation as well as in main - tainingthestabilityofaerobicgranules. It is apparent that extracellular polysaccharides excreted by cells can assist in bacterium-to-bacterium self-aggregation by bridging bacterial cells, which induces the formation of initial microbial aggregates. In fact, in the study of anaerobic granulation, Harada et al. (1988) observed that the extracellular polymers excreted by acidogenic bacteria appeared to enhance the strength and structural stability of anaerobic granules. A similar phenomenon was also reported in biolm systems (Vandevivere and Kirchman 1993). Extracellular polysaccharides indeed play a crucialroleinthebuildingarchitectureofaerobicgranules,andsubsequentlya certain content of extracellular polysaccharides is required in order to maintain the stabilityofthemicrobialstructure.Ithasbeenproposedthatextracellularpolymers can change the surface negative charge of bacteria, and thereby bridge two neigh - boring bacterial cells physically to each other as well as other inert particulate mat - ters, and nally settle out as occus aggregates (Shen, Kosaric, and Blaszczyk 1993; Schmidt and Ahring 1994). It appears from gure 10.6 that the content of cellular EHT = 20.00 kV WD = 11 mm Photo No. = 1348 Detector = SE1 Mag = 2.50 K X 1 µm FIGURE 10.5 EPS matrix structure observed in aerobic granule with nitrication capability. 53671_C010.indd 188 10/29/07 7:32:49 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Essential Roles of Extracellular Polymeric Substances in Aerobic Granulation 189 polysaccharides is much higher than the content of cellular proteins in both occi andaerobicgranules.Asimilarphenomenonwasalsoreportedinabiolmsystem (Vandevivere and Kirchman 1993). It may imply that cellular proteins contribute less totheformation,structure,andstabilityofgranulesandbiolms. Sofar,notmuchinformationisavailableontheEPSdistributionintheearlier stageofbiogranulation,whereasthespatialdistributionofEPSinmaturegranules has been reported. By using the calcouor staining method and uorescent micro- scopyorconfocalscanningelectronmicroscopy,itwasrevealedthatapproximately Operation Time (cycles) 0 20 40 60 80 100 120 140 160 Bioparticle Mean Size (mm) 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 FIGURE 10.6 Change in microbial aggregate size in the course of SBR cycle operation at different specic upow air velocities of 0.3 (D), 1.2 ($), and 2.4 ( d )cms –1 .(Datafrom Tay,J.H.,Liu,Q.S.,andLiu,Y.2001b.Lett Appl Microbiol 33: 222–226.) Operation Time (cycles) 0 20 40 60 80 100 120 140 160 PS/PN (mg mg –1 ) 2 4 6 8 10 12 14 16 18 FIGURE 10.7 ChangeinPS/PNratiointhecourseofSBRcycleoperationatdifferent specicupowairvelocitiesof0.3(D), 1.2 ($), and 2.4 ( d )cms –1 .(DatafromTay,J.H., Liu, Q. S., and Liu, Y. 2001b. Lett Appl Microbiol 33: 222–226.) 53671_C010.indd 189 10/29/07 7:32:51 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 190 Wastewater Purification 50%ofthetotalamountofEPSwaspresentinatop40µmthickzonefromthe surfaceofanaerobicgranules,andtherestoftheEPSwasrandomlyallocatedinthe deeperpartsofthegranules(deBeeretal.1996).Itshouldbepointedoutthatthe EPSlayeronthesurfaceofbiogranuleswasnotfoundinconventionalbioocs. Studyofanaerobicandaerobicgranulationindifferenttypesofbioreactors under a wide spectrum of operating conditions shows that the total amount of EPS producedisnotadecisivefactorintheformationandmaintainingthestabilityof biogranules. Instead, the distribution and composition of EPS plays a crucial role in biogranulation(Tay,Liu,andLiu2001b;Punaletal.2003;Wang,Liu,andTay2005). It has been reported that the content of extracellular polysaccharides in anaerobic granules was almost three times higher than that in anaerobic bioocs (de Beer et al. 1996),whiletheformationofaerobicgranuleswasfoundtobeaccompaniedwith a sharp increase of cellular polysaccharides normalized to cellular proteins (Tay, Liu, and Liu 2001c). The ratio of extracellular polysaccharides over proteins by weightinaerobicgranulesfellintoarangeof2to16asdiscussedinthepreceding chapters, which seems to be higher than that reported for the anaerobic granulation process. It seems that the characteristics of biogranules are related to the ratio of polysaccharides to proteins. Previous research showed that anaerobic granules and aerobicbioocswithahigherproteinstopolysaccharidesratiohadalowershear strength and a poorer settleability (Batstone and Keller 2001; Martinez et al. 2004), while Quarmby and Forster (1995) thought that the extracellular polysaccharides could contribute highly to the strength and stability of anaerobic granules. Similar resultswerealsoobtainedinaerobicgranulation,showingthatthespecicgravity andmechanicalstrengthofaerobicgranulesincreasedsignicantlywithincreasein theratioofpolysaccharidestoproteins(Tay,Liu,andLiu2001a,2002). Theformationofbiogranulesindeedisamicrobialevolutioninsteadofaran - dom aggregation of suspended bacteria. It should be a reasonable hypothesis that the spatial distribution of EPS in biogranules would be correlated to microbial evolution and distribution along with granulation. The investigation on the spatial distribution ofEPSinheterotrophicbiolmsshowedthattheproductionyieldofEPStendedto decreasewiththebiolmdepth(ZhangandBishop2003).Thisisprobablyduetothe fact that viable biomass loses its ability to produce EPS at the deeper sections of bio - lmsbecauseofitslowermicrobialactivityresultingfromlowernutrientavailabil- ity.Inaddition,theEPSproducedbybacteriacanbeutilizedassecondarysubstrate in the deeper layers or zones of biolms and biogranules where readily degradable substrates are not available or are limiting. Without doubt, the spatial distribution of EPSinbiogranulesandbiolmsplaysanessentialroleinstabilizingthestructure and maintaining the strength of microbial aggregates. This is also supported by the ndingthatEPSneartheedgeofgranuleshadagreatereffectonshearstrength thaninthecenterofthegranule(BatstoneandKeller2001).Finally,itshouldbe stressed that literature information on the spatial distribution of EPS in biogranules is very limited, and some questions still remain unanswered, for example, whether thespatialdistributionofEPSinbiogranulesisrelatedtooperatingconditionsoris correlated to microbial distribution and activity in biogranules. Obviously, further research on this aspect is needed. 53671_C010.indd 190 10/29/07 7:32:52 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC [...]...Essential Roles of Extracellular Polymeric Substances in Aerobic Granulation 191 10. 6 CONCLUSIONS This chapter shows the essential roles of EPS in the formation and maintaining structural stability of aerobic granules It becomes clear that the composition and the content of EPS in aerobic granules affect the matrix structure and the integrity of aerobic granules However, it should be realized that there... Klein, D A 1999 Microbiology Singapore, Indonesia: McGraw-Hill Punal, A., Brauchi, S., Reyes, J G., and Chamy, R 2003 Dynamics of extracellular polymeric substances in UASB and EGSB reactors treating medium and low concentrated wastewaters Water Sci Technol 48: 41–49 Qin, L., Liu, Q S., Yang, S F., Tay, J H., and Liu, Y 2004 Stressful conditions-induced production of extracellular polysaccharides in aerobic. .. culture-independent approach for studying microbial diversity in aerobic granules Water Sci Technol 47: 283–290 Yu, H Q., Tay, J H., and Fang, H H P 2001 The roles of calcium in sludge granulation during UASB reactor start-up Water Res 35: 105 2 106 0 Zhang, X Q and Bishop, P L 2003 Biodegradability of biofilm extracellular polymeric substances Chemosphere 50: 63–69 Zhang, X Q., Bishop, P L., and Kinkle,... Distribution of EPS and cell surface hydrophobicity in aerobic granules Appl Microbiol Biotechnol 69, 469–473 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 Yang, S F., Tay, J H., and Liu, Y 2004 Inhibition of free ammonia to the formation of aerobic granules Biochem Eng J 17: 41–48 Yi,... force in the formation of biofilm and granular sludge Water Res 36, 1653–1665 Lopez, J M., Koopman, B., and Bitton, G 1986 INT-dehydrogenase test for activated sludge process control Biotechnol Bioeng 28: 108 0 108 5 Mahmoud, N., Zeeman, G., Gijzen, H., and Lettinga, G 2003 Solids removal in upflow anaerobic reactors: A review Bioresource Technol 90: 1–9 Martinez, F., Lema, J., Mendez, R., Cuervo-Lopez,... and Muxi, L 2003 Evolution of the bacterial community during granules formation in denitrifying reactors followed by molecular, culture-independent techniques Water Sci Technol 48: 75–79 Fang, H H P 2000 Microbial distribution in UASB granules and its resulting effects Water Sci Technol 42: 201–208 Forster, C F 1991 Anaerobic upflow sludge blanket reactors: Aspects of their microbiology and their chemistry... Group, LLC © 2008 by Taylor 193 53671_C 010. indd & Francis Group, LLC 10/ 29/07 7:32:53 AM 194 Wastewater Purification Tay, J H., Liu, Q S., and Liu, Y 2002 Characteristics of aerobic granules grown on glucose and acetate in sequential aerobic sludge blanket reactors Environ Technol 23: 931–936 Trevors, J T 1984 The measurement of electron transport system (ETS) activity in freshwater sediment Water Res 18:... of granular methanogenic sludges in upflow anaerobic sludge blanket reactors fed with various defined substrates J Ferment Bioeng 79: 354–359 Ghigo, J M 2003 Are there biofilm-specific physiological pathways beyond a reasonable doubt? Res Microbiol 154: 1–8 Goodwin, J A S and Forster, C F 1985 A further examination into the composition of activated sludge surfaces in relation to their settlement characteristics... sludge cells into aerobically grown microbial granules for the aerobic biodegradation of phenol Lett Appl Microbiol 35: 439–445 Jorand, F., Zartarian, F., Thomas, F., Block, J C., Bottero, J Y., Villemin, G., Urbain, V., and Manem, J 1995 Chemical and structural (2D) linkage between bacteria within activated sludge flocs Water Res 29: 1639–1647 Jorand, F., Boue-Bigne, F., Block, J C., and Urbain, V 1998... 221–231 Frolund, B., Palmgren, R., Keiding, K., and Nielsen, P H 1996 Extraction of extracellular polymers from activated sludge using a cation exchange resin Water Res 30: 1749–1758 © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor 191 53671_C 010. indd & Francis Group, LLC 10/ 29/07 7:32:52 AM 192 Wastewater Purification Fukuzaki, S., Nishio, N., and Nagai, S 1995 High-rate performance and characterization . Factors In uencing EPS Production In Aerobic Granules 183 10. 4 The Role of EPS in Aerobic Granulation 185 10. 5 EPS-Enhanced Stability of Aerobic Granules 187 10. 6 Conclusions 191 References 191 10. 1. hydrophilic surface,butmorewithhydrophobicsurfacesofothercells(Ghigo2003). Sofar,differentviewsexistwithregardtotheroleofEPSinaerobicgranulation. Di Iaconi et al. (2006) reported that the main component of EPS in aerobic granular biomass in a sequencing batch biolter reactor was made of proteins, and protein- richEPSwouldimprovethestabilityofgranularbiomassstructure.Asthemain componentofproteinsisaminoacids,whichoftencarrynegativecharges,theywill contribute. 181 10 Essential Roles of Extracellular Polymeric Substances in Aerobic Granulation Yu Liu and Zhi-Wu Wang CONTENTS 10. 1 Introduction 181 10. 2 Main Composition of EPS in Aerobic Granules 181 10. 3