Wastewater Purification: Aerobic Granulation in Sequencing Batch Reactors - Chapter 10 doc

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 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 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
EPShavebeendetectedinsignicantquantitiesinbothaerobicandanaerobicgran-
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
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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
specic 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 bioocs, biolm, anaerobic and aerobic granules. However, the content of EPS
inaerobicgranuleswasfoundtobemuchhigherthanthoseinconventionalbioocs
andbiolms(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 inuenced 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.
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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 biolm 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
-
ingbiolmsandtheothermethodsgavearangeofratiosof1.54to2.23.Thisis

indeedconsistentwiththendingbyAzeredo,Lazarova,andOliveira(1999)that
manyEPSextractionmethodsdevelopedforbiolmswerenotefcient,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,thedeciencyofnitrogenwasfoundtofavortheproductionof
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 signicantly
stimulate bacteria to produce more extracellular polysaccharides over proteins in
SBRs, as shown in the preceding chapters. The EPS production seems to be positively
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184 Wastewater Purification
relatedtothespecicoxygenutilizationrate(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 inuencing 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.
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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
asoftparticleelectrophoresistechniquetoinvestigatetheinuenceofEPSoncell
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
highcellsurfacehydrophobicitywasusuallyassociatedwiththepresenceofbrillar
structureoncellsurfaceandspeciccellwallproteins(McNabetal.1999;Singleton,
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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,asshowningure10.4.
Jorand et al. (1998) studied hydrophobic and hydrophilic properties of extra-
cellularpolymersproducedbyactivatedsludge,andfoundthatasignicantpropor
-
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.)
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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 reects 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),butnospecicevidenceshowsthequantita-
tive contribution of hydrophobic components of EPS to the overall hydrophobicity of
biogranulessofar.Thereisevidencethatthereductionofsurfacechargewouldnot
bearequirementfortheformationofactivatedsludge,thatis,thechargeneutraliza-
tionwouldnotbethemainmicrobialocculationmechanism(Pavonim,Tenney,

and Echelberger 1972; Strand, Varum, and Ostgaard 2003), whereas hydrophobic
interaction may be fundamental in biogranulation (see chapter 9). It appears from
thestudyofbiolmsthatfewbacteriawerede 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 biolter 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 specic 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).
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188 Wastewater Purification
Microscopic observation shows that EPS with a lamentous structure is present
withinandaroundthestructureofbiogranules(Forster1991;deBeeretal.1996;
Veigaetal.1997;Tay,Liu,andLiu2001c),whileEPSalsocanllintheintercellular
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 signicant increase in EPS expressed as the ratio of polysaccharides
(PS) to proteins (PN), while disappearance of aerobic granules, indicated by a
signicant 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 biolm 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 nitrication capability.
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Essential Roles of Extracellular Polymeric Substances in Aerobic Granulation 189
polysaccharides is much higher than the content of cellular proteins in both occi
andaerobicgranules.Asimilarphenomenonwasalsoreportedinabiolmsystem
(Vandevivere and Kirchman 1993). It may imply that cellular proteins contribute less
totheformation,structure,andstabilityofgranulesandbiolms.
Sofar,notmuchinformationisavailableontheEPSdistributionintheearlier
stageofbiogranulation,whereasthespatialdistributionofEPSinmaturegranules

has been reported. By using the calcouor 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 specic upow 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
specicupowairvelocitiesof0.3(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.)
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190 Wastewater Purification
50%ofthetotalamountofEPSwaspresentinatop40µmthickzonefromthe
surfaceofanaerobicgranules,andtherestoftheEPSwasrandomlyallocatedinthe
deeperpartsofthegranules(deBeeretal.1996).Itshouldbepointedoutthatthe
EPSlayeronthesurfaceofbiogranuleswasnotfoundinconventionalbioocs.
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 bioocs (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
aerobicbioocswithahigherproteinstopolysaccharidesratiohadalowershear
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,showingthatthespecicgravity
andmechanicalstrengthofaerobicgranulesincreasedsignicantlywithincreasein
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
ofEPSinheterotrophicbiolmsshowedthattheproductionyieldofEPStendedto
decreasewiththebiolmdepth(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 biolms and biogranules where readily degradable
substrates are not available or are limiting. Without doubt, the spatial distribution of
EPSinbiogranulesandbiolmsplaysanessentialroleinstabilizingthestructure
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.
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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
thecontentofEPSinaerobicgranulesaffectthematrixstructureandtheintegrity
of aerobic granules. However, it should be realized that there are still a lot of con
-
tradictory research reports on the functional roles of EPS in aerobic granulation
process because of the complex composition of EPS, different culture conditions,
and analytical methods employed.
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