195 11 Internal Structure of Aerobic Granules Zhi-Wu Wang and Yu Liu CONTENTS 11.1 Introduction 195 11.2 Internal Structure of Aerobic Granules 195 11.2.1 Heterogeneous Structure of Aerobic Granules 195 11.2.2 Porosity of Aerobic Granules 196 11.2.3 Size-Dependent Internal Structure of Aerobic Granules 197 11.2.4 Structure Change of Aerobic Granules during Starvation 198 11.3 Biomass Distribution in Aerobic Granules 199 11.4 PS Distribution in Aerobic Granules 201 11.5 Distribution of Cell Surface Hydrophobicity in Aerobic Granules 205 11.6 Diffusion-Related Structure of Aerobic Granules 206 11.7 Conclusions 207 References 207 11.1 INTRODUCTION Theuniquefeaturesofaerobicgranules,asdifferentfrombiooc,aretheirdense and spherical three-dimensional structure. A good perception into the conformation of this granular structure, in comparison with that of bioocs, will certainly help deepencurrentunderstandingofthemechanismofaerobicgranulation,aswellasits structural stability. As presented in chapter 10, an aerobic granule is mainly build up by microbial cells embedded in their excreted extracellular polysaccharides (PS), that is,PSplayacementingroleinconnectingindividualcellsintothethree-dimensional structure of an aerobic granule. Moreover, the PS characteristics also inuence the surfacepropertyofmicrobialcells(seechapter9).Itseemscertainthatthestructure of an aerobic granule is essentially determined by the distributions and properties of its construction blocks, namely the microbial cells and PS. Thus, this chapter offers up-to-date information about the internal structure of aerobic granules in terms of the distributions of the microbial cells, PS, and cell surface hydrophobicity. 11.2 INTERNAL STRUCTURE OF AEROBIC GRANULES 11.2.1 H ETEROGENEOUS STRUCTURE OF AEROBIC GRANULES Anaerobicgranulecultivatedinanacetate-fedsequencingbatchreactor(SBR)was sliced and its internal structure was visualized by imagine analysis technique (Wang, 53671_C011.indd 195 10/29/07 7:33:51 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 196 Wastewater Purification Liu,andTay2005).Itwasfound,asshowningure11.1,thattheinternalstructure of an aerobic granule consisted basically of an opaque outer layer and a relatively transparentinnercore.Theopaqueouterlayerhadadepthofabout800µmfromthe granulesurfacedownwards,andthegranulecenterlookedtransparent. 11.2.2 POROSITY OF AEROBIC GRANULES Porosity of biolm or anaerobic granules can facilitate nutrient transfer (Alphenaar etal.1992;ZhangandBishop1994).J.H.Tayetal.(2003)used0.1-μmuorescence beadstostudytheporosityofaerobicgranules,andfoundthattheporosityexisted throughouttheaerobicgranulestructure,butitpeakedat150and200μmbeneath thesurfaceofaerobicgranuleswithsizesof0.55and1.0mm,respectively.Never - theless,thetotalporouszonesdecreasedwithincreasinggranulediameter,onaunit volumebasis(J.H.Tayetal.2003). Thezigzagporechannelwasfoundtowindthroughthegranulematrixmadeup by PS, that is, the porosity should be correlated to the richness of PS (Zheng and Yu 2007). A study by size-exclusion chromatography method revealed that the PS con - tent increased, but the porosity decreased with the granule diameter, for example the poresizeofanaerobicgranulewithasizeof0.2to0.6mmwasnearlyseventimes Thepossiblecloggingcausedbyover-producedPSwasthusconsideredtoberespon - sible for the reduced porosity in large-sized aerobic granule. In addition, Chiu et al. (2006)alsoreportedthatalargegranulewouldhaveahighporosity,evidencedby an enhanced oxygen diffusivity, with an increase of granule size, for example, diffu - sion coefcients of oxygen were measured as 1.24 × 10 –9 to2.28×10 –9 m 2 s –1 as the sizeofacetate-fedaerobicgranulesincreasedfrom1.28to2.50mm,andasimilar phenomenon was also observed in phenol-fed aerobic granules. Based on these (a) (b) FIGURE 11.1 Cross-sectionview(400μmthickness)oftheaerobicgranuleinbrighteld (a) and dark eld (b) visualization modes. Scale bar, 500 μm. (From Wang, Z W., Liu, Y., and Tay, J H. 2005. Appl Microbiol Biotechnol 60:687–695.Withpermission.) 53671_C011.indd 196 10/29/07 7:33:53 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC biggerthanthatofaerobicgranuleswithalargersizeof0.9to1.5mm (gure11.2). Internal Structure of Aerobic Granules 197 controversialndings,itisdifculttoconcludethatgranuleporosityisdependent on its particle size. 11.2.3 SIZE-DEPENDENT INTERNAL STRUCTURE OF AEROBIC GRANULES Toinvestigatetheinternalstructureofaerobicgranuleswithvarioussizes,mature aerobicgranuleswithameandiameterof0.8to3.0mmwereslicedandfurthervisu- alized by image analyzer (Wang, Liu, and Tay 2005). The image analysis revealed thatthesmallaerobicgranulewithadiameterof0.8mmhadanearlyhomogenous structure,whereaslargeraerobicgranuleswithadiameterof3.0mmexhibiteda layeredinternalstructureinwhichaclearshellandcorecouldbedistinguished (gure 11.3). Furthermore, the granule structure seems to evolve with the growth of theaerobicgranuleinsize,thatis,atransitionfromahomogenoustoheterogeneous structure was observed with increase in the granule size (gure 11.3). As can be seen in gure 11.3, this is also evidenced by the gradually brightened transparent space fromthegranuleshelltoitscenterwithincreasedgranulesize. Asdiscussedinchapter8,theoccurrenceofdiffusionlimitationisassociated withthesizeoftheaerobicgranule.Themodelsimulationshowsthatdissolved oxygen(DO)wouldbecomealimitingfactorformicrobialgrowthatbulkCOD concentration greater than 465 mg L –1 ,andthesolubleCODcanpenetratethrough- outtheaerobicgranulewithadiametersmallerthan0.8mm,whichexhibited limitation would be encountered in large-sized aerobic granules of 1.0 to 1.5 mm (chapter 8). These results seem to indicate that the observed layered structure of large-sizedaerobicgranuleswouldresultfromdiffusionlimitationbecauseonly thosemicroorganismslivingintheshelloftheaerobicgranuleareaccessibleto DO and substrate. Range of Granule Size (mm) 0.2-0.6 0.6-0.9 0.9-1.5 Excluded Molecular Mass (Da) 0 20 × 10 3 40 × 10 3 60 × 10 3 80 × 10 3 100 × 10 3 120 × 10 3 140 × 10 3 160 × 10 3 FIGURE 11.2 The penetrable molecular mass for different sized aerobic granules. (Data from Zheng, Y M. and Yu, H Q. 2007. Water Res 41: 39–46.) 53671_C011.indd 197 10/29/07 7:33:54 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC ahomogeneousstructure(gure11.1a).Ontheotherhand,severeDOdiffusion 198 Wastewater Purification 11.2.4 STRUCTURE CHANGE OF AEROBIC GRANULES DURING STARVATION Fresh aerobic granules were starved under aerobic condition without addition of carbonandnutrientsourcesfor20days.Changesingranulestructurebeforeandafter the20-daystarvationareshowningure11.4.Comparedtothefreshaerobicgranule (gure 11.4a), the starved granule became more transparent. A transmittance analysis acrosstheintactgranuleindicatesthattheopaquecoreofthefreshaerobicgranulehad become highly light permeable (gure 11.5), and the sliced, starved granule clearly (a) (b) FIGURE 11.4 Aviewofanaerobicgranulebefore(a)andafter(b)long-termstarvation; scalebar:300μm.(FromWang,Z W.,Liu,Y.,andTay,J H.2005.Appl Microbiol Biotechnol 60:687–695.Withpermission.) (a) (b) (c) (d) FIGURE 11.3 Internalstructureofslicedaerobicgranuleswithdiametersof0.8mm(a), 1.3mm(b),2.0mm(c),and3.0mm(d);scalebars:0.5mm. 53671_C011.indd 198 10/29/07 7:33:56 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Internal Structure of Aerobic Granules 199 showedahollowstructureeventhoughitsoutershellstillremainedintact (gure11.6). These observations seem to suggest that the biomass present in the granule shell would not be taken up by bacteria over starvation, while the biomass located in the core of the aerobic granule can be biodegraded under the starvation condition. 11.3 BIOMASS DISTRIBUTION IN AEROBIC GRANULES The heterogeneous structure of aerobic granules indicates an uneven distribution of biomass.Chen,Lee,andTay(2007)useduorescentdyestovisualizethemicrobial                FIGURE 11.5 Light transmittal proles across intact aerobic granule before (black) and after (gray) long-term starvation (arrow indicates granule center). (From Wang, Z W., Liu, Y., and Tay, J H. 2005. Appl Microbiol Biotechnol 60:687–695.Withpermission.) FIGURE 11.6 The hollow structure of the starved aerobic granule. (From Wang, Z W., Liu, Y., and Tay, J H. 2005. Appl Microbiol Biotechnol 60:687–695.Withpermission.) 53671_C011.indd 199 10/29/07 7:33:58 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 200 Wastewater Purification cellsdistributionbymeansofconfocallaserscanningmicroscopy(CLSM),and foundthatlivecellswereconcentratedinthegranuleshell,indicatedbyared uorescence emitted from Syto 63 that stained nucleic acid (gure 11.7). In contrast, the uorescence from the granule core is rather weak, indicating a limiting number oflivebacteriainthecorepartoftheaerobicgranule.Similarobservationwasalso reportedbyTohetal.(2003)andMcSwainetal.(2005).InthestudybyMcSwainet al.(2005),theSyto63uorescencepeakedatadepthof100μmbeneaththegranule surfaceandthegranulecorepartwasalmostuorescencefree.Detaileddistribution ofliveanddeadcellsinsidetheaerobicgranulewasinvestigatedbyJ.H.Tayetal. Itwasdemonstratedthatmostlivebacteria,includingnitriers,onlyexistedinthe granule outer shell layer where they were within the reach of mass diffusion, while deadcellsandanaerobesweremainlydetectedatthecoreoftheaerobicgranule, indicating an uneven microbial distribution in aerobic granules that should result from diffusion limitation (gure 11.7). Optical density (OD) has been commonly used to quantify the biomass concen - tration,thatis,ahighODiscorrelatedtoahighbiomassconcentrationordensity in suspended and biolm cultures (Gaudy and Gaudy 1980). Figure 11.8 exhibits the ODprolemeasuredacrossthecrosssectionofanaerobicgranule.Itwasfoundthat theODinthegranulecenterwasclosetozero,indicatingaverylowbiomassdensity or a loose microbial structure at the core. In contrast, the peak OD was observed intheouterlayeroftheaerobicgranule,whichwouldresultfromahighbiomass density or a compact microbial structure (Wang, Liu, and Tay 2005). To conrm theseobservations,veaerobicgranules,namelyNo.1to5,wereslicedandthe respectivemassdensityoftheoutershelllayerandtheinnercorepartwasmeasured. It was found that the mass density of the outer layer of the granule was indeed much higher than that of the core part (gure 11.9). In fact, J. H. Tay et al. (2002) reported asimilarbiomassdistributioninaerobicgranules.Asdiscussedearlier,massdiffu - sionlimitationwouldberesponsiblefortheobserveddensesurfacelayerandloose FIGURE 11.7 Cell distribution in aerobic granules, Syto 63 (red)-stained nucleic acid. (FromChen,M.Y.,Lee,D.J.,andTay,J.H.2007.Appl Microbiol Biotechnol 73: 1463–1469. With permission.) 53671_C011.indd 200 10/29/07 7:33:59 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Internal Structure of Aerobic Granules 201 inner core of aerobic granules, that is, the unbalanced biomass distribution is due to thediffusionlimitationinsidetheaerobicgranule. 11.4 PS DISTRIBUTION IN AEROBIC GRANULES Calcouorwhiteisacommonlyuseduorescentdyeforlabelingbeta-linkedpoly- saccharides (PS) (deBeer et al. 1996). The beta-linked polysaccharides are believed toserveasthebackboneofthebiolmstructure(Sutherland2001).Tolocalizebeta- linked polysaccharides in an aerobic granule, the aerobic granule was sliced and its         "   !   FIGURE 11.8 TheODprolethroughthegranulecrosssection(arrowindicatesgranule center).(DatafromWang,Z W.,Liu,Y.,andTay,J H.2005.Appl Microbiol Biotechnol 60: 687–695.) Mass Density Ratio of Shell to Core 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 Granule No. 1 Granule No. 2 Granule No. 3 Granule No. 4 Granule No. 5 FIGURE 11.9 Biomassdensityratiosofshelllayertocorepartofaerobicgranules.(Data fromWang,Z W.,Liu,Y.,andTay,J H.2005.Appl Microbiol Biotechnol 60: 687–695.) 53671_C011.indd 201 10/29/07 7:34:01 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 202 Wastewater Purification crosssectionwasstainedwithcalcouor(Wang,Liu,andTay2005).Inafreshgranule, theuorescentdyewasattachedmainlytotheoutershellofthegranule,whilevery weak uorescence was detected at the center of the aerobic granule (gure 11.10b). Theuorescenceintensityprolemeasuredalongthedirectionofthegranuleradius furthershowedthatmostcalcouorwhite-stainedPSwassituatedintheoutershell ofthegranule,withadepthof400µmbelowthegranulesurface(gure11.11).These ndingsimplythatthebeta-linkedPSarelocatedmainlyintheoutershellofthe granule.Infact,asimilardistributionofbeta-linkedPSwasalsoobservedinanaerobic granules;themajorityofthecalcouorwhite-stainedPSwasfoundinthetop40μm fromthesurfaceoftheanaerobicgranule(deBeeretal.1996). Chen, Lee, and Tay (2007) used three different uorescence dyes, namely ConA andcalcouorwhiteandFITC,tolabelthealpha-,beta-linkedPSandalsoprotein (a) (b) FIGURE 11.10 Microscopic view of sectioned aerobic granule cross section before (a) and after(b)calcouorwhitestaining;scale:100μm.(FromWang,Z W.,Liu,Y.,andTay,J H. 2005. Appl Microbiol Biotechnol 60:687–695.Withpermission.)              FIGURE 11.11 Proleoftheuorescenceintensityfromthesurfacetothecenterofan aerobicgranule.(DatafromWang,Z W.,Liu,Y.,andTay,J H.2005.Appl Microbiol Biotechnol 60: 687–695.) 53671_C011.indd 202 10/29/07 7:34:04 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Internal Structure of Aerobic Granules 203 (PN). The observations by CLSM revealed that the alpha-linked PS was distributed mainly on the granule shell, while the granule core was almost alpha-linked PS free (gure 11.12a). A similar distribution of alpha-linked PS was also reported by McSwainetal.(2005).However,thestudybyChen,Lee,andTay(2007)showeda differentdistributionofthebeta-linkedPSfromwhatwasfoundingure11.10,that is, the beta-linked PS not only appeared on the granule shell, but also was concen - tratedinthegranulecore.Moreover,auorescentemptylayerwasfoundinbetween the granule shell and core (gure 11.12b). As for PN, a random distribution pattern wasfoundalongthegranuleradiumdirection(gure11.12c). ToquantifythePSdistributioninthelayeredaerobicgranule,Wang,Liu,and Tay(2005)measuredthePScontentsinthegranuleshellaswellasinthegranule core, for example, the PS present in the granule shell only accounts for one-fth of the PS found in the granule core. Such a nding implies that those gel-like substances observed in the granule center (gure 11.1) could be attributed to PS. As discussed (a) (b) (c) FIGURE 11.12 FluorescenceviewedongranulecrosssectionbystainingwithConAfor alpha-linkedPS(a);calcouorwhiteforbeta-linkedPS(b);andFITCforprotein(c).Scale bar:200μm.(FromChen,M.Y.,Lee,D.J.,andTay,J.H.2007.Appl Microbiol Biotechnol 73:1463–1469.Withpermission.) 53671_C011.indd 203 10/29/07 7:34:06 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC core(gure11.13).MostPSintheaerobicgranulewascentralizedatthegranule 53671_C011.indd 204 10/29/07 7:34:07 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 204 Wastewater Purification earlier, the PS present in the granule core is basically biodegradable. In view of the  total amount of PS determined in the aerobic granule, it appears that the alpha- or  beta-linked PS may not be the dominate constitution of EPS in aerobic granules. EPS  is  the  extracellular  products  synthesized  by  microbial  cells.  As  shown  earlier, the cell distribution would be granule size-dependent due to diffusion limita - tion. Hence, the distribution of PS would also be related to the size of the aerobic  granule. McSwain et al. (2005) investigated the PS distribution in small and large  aerobic granules with a respective size of 350 μm and 800 μm. The observation by  CLSM revealed that in the small bioparticle, both PS and PN were concentrated at  the core (gure 11.14a). For the large-sized bioparticle, PS and microbial cells turned  out to be only centralized on the granule outer shell, with a random distribution of  PN (gure 11.14b). Similar EPS distributions have been reported in anaerobic bio - oc and granule, that is, calcouor-stained PS was mainly distributed on the outer  Shell Core PS Content (mg cm –3 ) 0 50 100 150 200 250 fIGure 11.13 Distribution  of  PS  in  granule  shell  and  core.  (Data  from  Wang,  Z W.,  Liu, Y., and Tay, J H. 2005. Appl Microbiol Biotechnol 60: 687–695.) (a) (b) 100 µm fIGure 11.14 Fluorescence by Syto 63 for cells (bright), ConA for polysaccharides (gray),  and FITC for protein ( white) in biooc (a) and aerobic granule (b). (From McSwain, B. S.  et al. 2005. Appl Environ Microbiol 71: 1051–1057. With permission.) [...]... role in maintaining the structural stability of the aerobic granule Instead, the shellassociated nonbiodegradable and hydrophobic EPS is the key towards the long-term stability of the aerobic granule In this regard, hydrolysis or disappearance of the core-associated EPS would inevitably result in the disintegration of the aerobic granule This point is indirectly confirmed by a study of biofilm in which... more hydrophobic EPS The alpha- and beta-linked EPS have been found on the granule shell layer (figures 11. 10, 11. 12, and 11. 14) In fact, increased production of alpha-linked EPS can improve cell surface hydrophobicity (Lawman and Bleiweis 1991), and insoluble beta-linked EPS was also reported to serve as the backbone of biofilm structure (Sutherland 2001) For the granule core-associated EPS, the hydrophobic... and Jones1999) 11. 6 DIFFUSION-RELATED STRUCTURE OF AEROBIC GRANULES The size-associated structure of aerobic granules was discussed earlier, that is, a small aerobic granule has a homogeneous structure, whereas a heterogeneous structure is found in big aerobic granules (figure 11. 3) It appears that mass diffusion is a decisive factor influencing the structure shift of aerobic granules In the cycle operation... (figure 11. 13) 11. 5 DISTRIBUTION OF CELL SURFACE HYDROPHOBICITY IN AEROBIC GRANULES Cell surface hydrophobicity has been regarded as a trigger of aerobic granulation (chapter 9) Basically, cell hydrophobicity helps reduce the surface energy of individual cells so as to overcome the dispersive polar force from water, and further promote cell-to-cell co-aggregation After the formation of the aerobic. .. necessary As shown in figures 11. 4 to 11. 6, after a 20-day aerobic starvation, most of the granule-core-associated EPS disappeared, that is, those less hydrophobic EPS are highly biodegradable as compared to the shell EPS In general, only soluble EPS is biodegradable, while insoluble or bound EPS should be nonbiodegradable (Laspidou and Rittmann 2002) In fact, the hydrophilic EPS in the aerobic granule... hydrophobicity may continue to play a part in the stability of the aerobic granule structure Unfortunately, little information is presently available about the distribution of cell surface hydrophobicity in mature aerobic granules To give some insights into the cell hydrophobicity distribution in aerobic granules, Wang, Liu, and Tay (2005) separated the granule outer shell from its inner core, and their... properties of EPS (chapter 10) In the three-dimensional structure of aerobic Cell Surface Hydrophobicity (%) 70 60 50 40 30 20 10 0 Sell Core FIGURE 11. 15 Cell hydrophobicity distributions in the shell and core parts of an aerobic granule (Data from Wang, Z.-W., Liu, Y., and Tay, J.-H 2005 Appl Microbiol Biotechnol 60: 687–695.) © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor 205 53671_C 011. indd & Francis... cycle operation of an aerobic granular sludge SBR, almost all influent COD can be removed in the first hour of aeration, and aerobic granules are thus subjected to substrate starvation in the rest of the cycle time (see chapter 1) As a result, a periodic shift from substrate feast to famine exists in aerobic granular sludge SBRs Meanwhile, mass diffusion limitation was encountered in aerobic granules with... to out compete the fast-growing microorganisms located at the granule shell Such an unbalanced growth between the two parts of the aerobic granule naturally results in an uneven biomass distribution, as observed in figures 11. 7 to 11. 9 It has been recognized that the partial anaerobic condition can trigger the production of EPS (Gamar-Nourani et al 1998) As pointed out earlier, aerobic granules with... mm often have an anaerobic or partially anaerobic core Under such a circumstance, the overproduction of EPS at the granule core (figure 11. 13) would be reasonably explained On the other hand, the weak fluorescence intensity at the granule core (11. 7) and the distribution of live and dead cells in aerobic granules also point to the fact that a substantial portion of microbial cells in the granule core . 11. 10b). Theuorescenceintensityprolemeasuredalongthedirectionofthegranuleradius furthershowedthatmostcalcouorwhite-stainedPSwassituatedintheoutershell ofthegranule,withadepthof400µmbelowthegranulesurface(gure11 .11) .These ndingsimplythatthebeta-linkedPSarelocatedmainlyintheoutershellofthe granule.Infact,asimilardistributionofbeta-linkedPSwasalsoobservedinanaerobic granules;themajorityofthecalcouorwhite-stainedPSwasfoundinthetop40μm fromthesurfaceoftheanaerobicgranule(deBeeretal.1996). Chen,. Distribution in Aerobic Granules 199 11. 4 PS Distribution in Aerobic Granules 201 11. 5 Distribution of Cell Surface Hydrophobicity in Aerobic Granules 205 11. 6 Diffusion-Related Structure of Aerobic. to thediffusionlimitationinsidetheaerobicgranule. 11. 4 PS DISTRIBUTION IN AEROBIC GRANULES Calcouorwhiteisacommonlyuseduorescentdyeforlabelingbeta-linkedpoly- saccharides (PS) (deBeer et al. 1996). The beta-linked