223 13 Calcium Accumulation in Acetate-Fed Aerobic Granules Zhi-Wu Wang, Yong Li, and Yu Liu CONTENTS 13.1 Introduction 223 13.2 Effect of Calcium on Aerobic Granulation 224 13.3 Calcium Accumulation in Acetate-Fed Aerobic Granules 225 13.4 Chemical form of calcium in acetate-fed aerobic granules 226 13.5 Calcium Distribution in Acetate-Fed Aerobic Granules 227 13.6 Granule Size-Dependent CaCO 3 Formation in Acetate-Fed Aerobic Granules 227 13.7 Mechanism of Calcium Accumulation in Acetate-Fed Aerobic Granules 229 13.7.1 Ionic Equilibrium of Carbonate Ion 230 13.7.2 Diffusion Kinetics in Aerobic Granules 231 13.7.3 Distribution of pH and CO 3 2- in Acetate-Fed Aerobic Granules 233 13.7.4 Size-Associated Formation of CaCO 3 in Acetate-Fed Aerobic Granules 234 13.8 Conclusions 235 Symbols 236 References 236 13.1 INTRODUCTION Ahighcalciumcontenthasbeenreportedinacetate-fedaerobicgranuleseven though the calcium concentration in the synthetic wastewater was very low (Qin, Liu, and Tay 2004; Wang, Liu, and Tay 2005). Extensive accumulation of calcium wasalsofoundinbiolmsandanaerobicgranules(Batstoneetal.2002;Kemner et al. 2004). Two hypotheses have been put forward to explain the calcium accumu- lation: (1) calcium links with extracellular polymeric substances (EPS) and forms an EPS-Ca 2+ -EPScross-linkage;and(2)calciumispresentintheformofCaCO 3 (Yu, Tay, and Fang 2001; Wloka et al. 2004). This chapter thus explores the mechanism behind the accumulation, chemical form, and spatial distribution of calcium ion in acetate-fed aerobic granules. 53671_C013.indd 223 10/29/07 7:34:46 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 224 Wastewater Purification 13.2 EFFECT OF CALCIUM ON AEROBIC GRANULATION Ca 2+ has been reported to enhance the formation of anaerobic granules and acido- genicbiolms(HuangandPinder1995;Yu,Tay,andFang2001).Jiangetal.(2003) studied the effect of calcium on aerobic granulation in sequencing batch reactors (SBRs).Forthispurpose,twoSBRswereoperatedattherespectiveCa 2+ concentra- tionsofzeroand100mgL –1 .Itwasfoundthataerobicgranuleswereformedinboth SBRs,andgranulesizeswerestabilizedataround2mmand2.8mminthecalcium- freeandcalcium-addedSBRs,respectively,after2monthsofoperation(gure13.1). These results indicate that aerobic granulation may not depend on calcium ion, that is,calciumionisnotessentialforaerobicgranulationinSBRs.Mahoneyetal.(1987) investigated anaerobic granulation in two upow anaerobic sludge blanket (UASB) reactors fed with aero and 100 mg Ca 2+ L –1 ,respectively.Similartotheresultsshown in gure 13.1, successful anaerobic granulation was achieved in both reactors, indi - catingthatcalciumisnotanessentialelementforanaerobicgranulationeither. Compared to aerobic granules grown on calcium-free medium, aerobic granules cultivated with addition of calcium showed better settleability andhigherstrength (gure 13.1). It is thought that the Ca 2+ ionshouldbindtonegativelychargedgroups A B FIGURE 13.1 Aerobicgranulescultivatedatdifferentcalciumconcentrations,0mg Ca 2+ L –1 (a) and 100 mg Ca 2+ L –1 (b).(FromJiang,H.L.etal.2003.Biotechnol Lett 25: 95–99. With permission.) 53671_C013.indd 224 10/29/07 7:34:48 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Calcium Accumulation in Acetate-Fed Aerobic Granules 225 of extracellular polysaccharides present on bacterial surfaces, and act as a bridge to interconnect these components, so as to promote bacterial aggregation and further enhance the structural stability of aerobic granules, anaerobic granules, and bio - lms (Costerton et al. 1987; van Loosdrecht et al. 1987; Bruus, Nielsen, and Keiding 1992).Itshouldbepointedoutthatsuchaviewisstilldebatable. 13.3 CALCIUM ACCUMULATION IN ACETATE-FED AEROBIC GRANULES Wang,Li,andLiu(2007)systematicallyinvestigatedthecalciumaccumulationin acetate-fedaerobicgranulesharvestedfromacolumnSBRafter2monthsofopera - tion, while calcium concentration in inuent was as low as 4.65 mg L –1 .Itwasfound that acetate-fed aerobic granules had a high calcium content of 225 mg Ca 2+ mg g –1 , contributingto37%ofgranuleashcontent.Comparedtoacetate-fedaerobicgranules, aerobicgranulesgrownonethanolshowedverylowcalciumandashcontents (gure 13.2). This seems to suggest that calcium accumulation is a phenomenon closely associated with the substrate applied. Ca 2+ (mg g –1 SS) 0 50 100 150 200 250 Acetate Ethanol Ash Content (g g –1 SS) 0.0 0.1 0.2 0.3 FIGURE 13.2 Calcium and ash contents in ethanol- and acetate-fed aerobic granules. (Data onethanolfromLiu,Yang,andTay2003andonacetatefromWang,Li,andLiu2007.) 53671_C013.indd 225 10/29/07 7:34:49 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 226 Wastewater Purification 13.4 CHEMICAL FORM OF CALCIUM IN ACETATE-FED AEROBIC GRANULES To investigate the chemical form of calcium ion accumulated in acetate-fed aerobic granules,Wang,Li,andLiu(2007)quantiedtheelementalcomposition(Ca,Mg,P, Fe, Al) of acetate-fed aerobic granules. The amount of carbonate ion in the acetate- fedaerobicgranuleswasalsoanalyzed.Forthispurpose,3mlof1Mhydrochloric acidsolutionwasaddedto50mlof2gsolublesolids(SS)L –1 acetate-fed aerobic granules,andthecarbondioxidegasproducedwasonlinemeasuredbythecarbon dioxidesensorequippedwiththerespirometer(gure13.3).Changesininorganic carbon in the liquid phase were determined by total organic carbon analyzer before andaftertheexperiment(Wang,Li,andLiu2007).Thus,thecontentofcarbonatein acetate-fedaerobicgranuleswascalculatedfromthesumofproducedcarbondioxide gasandincreasedinorganiccarbonintheliquidphase.Figure13.4showsthemajor inorganic components of acetate-fed aerobic granules. As can be seen, both Ca 2+ and CO 3 2- aredominantovertheotherinorganiccomponents,suchasMg,P,Fe,and 4°C 6 4 5 2 1 3 FIGURE 13.3 Respirometersystemforanalysisofcarbonateintheacetate-fedaerobic granule:1.computerfordatacollection;2.respirometer;3.fridge;4.shaker;5.acidcontaining vial;6.reactionbottle.(FromWang,Z W.,Li,Y.,andLiu,Y.2007.Appl Microbiol Biotechnol 74:467–473.Withpermission.) CO 3 2– mmol ion g –1 SS 0.0 0.3 0.6 0.9 1.2 1.5 1.8 Al Mg P Fe Ca FIGURE 13.4 Ioniccompositionofacetate-fedaerobicgranules.(DatafromWang,Z W., Li,Y.,andLiu,Y.2007.Appl Microbiol Biotechnol 74: 467–473.) 53671_C013.indd 226 10/29/07 7:34:50 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Calcium Accumulation in Acetate-Fed Aerobic Granules 227 calcium to carbonate was estimated as 1:1.16, indicating that most calcium ions in aerobicgranulesexistintheformofcalciumcarbonate.Intermsofchemistry,this also implies that the concentration product of Ca 2+ and CO 3 2- in acetate-fed aerobic granules should be larger than the solubility product constant of calcium carbonate. 13.5 CALCIUM DISTRIBUTION IN ACETATE-FED AEROBIC GRANULES The calcium distribution in acetate-fed aerobic granules was investigated using a scan- ning electron microscope (SEM); meanwhile, energy dispersive x-ray spectroscopy (EDX) was also employed for mapping of calcium distribution (Wang, Li, and Liu 2007). The carbonate localization was determined by chemical titration method, that is,1Mhydrochloricacidsolutionwasdroppedonaslicedgranulecrosssection,and theoriginofbubbleswasvisualizedbyimageanalysistechnique(Wang,Li,andLiu 2007).Freshacetate-fedaerobicgranuleswithaspecicoxygenuptakerate(SOUR) of 64 mg O 2 g –1 volatile solids (VS) h –1 ;sludgevolumeindex(SVI)of52mLg –1 ,and oftheaerobicgranule,whilethegranuleshellwasnearlycalciumfree.Theimage analysis further showed white deposits localized at 300 µm beneath the granule sur - (gure 13.5c), gas bubbles were immediately generated (gure 13.5d). The gas phase analysis conrmed that the bubbles generated were carbon dioxide (gure 13.5d). These results clearly indicate that both calcium and carbon ions coexist in the same zoneofacetate-fedaerobicgranules,thatis,calciumexistsmainlyintheformof CaCO 3 intheacetate-fedaerobicgranules,whichisingoodagreementwiththe stoichiometric analysis (gure 13.4). Theaccumulationofcalciumwasobservedinbiolmsandanaerobicgranules, and Ca 2+ has been often considered to bridge negatively charged sites on extracellular biopolymers, thus enhancing the matrix stability of attached microbial communi - ties (Bruus, Nielsen, and Keiding 1992; Korstgens et al. 2001; Batstone et al. 2002; Kemneretal.2004;Wlokaetal.2004).Accordingtosuchahypothesis,excessive calciumhasoftenbeenintroducedintothemediumforenhancedformationofbio - lmandanaerobicgranules(HuangandPinder1995;Yu,Tay,andFang2001). However,itappearsfromgures13.4and13.5thatcalciumdetectedinacetate-fed aerobic granules was mainly in the form of calcium carbonate rather than in associa - tion with extracellular polymeric substances. 13.6 GRANULE SIZE-DEPENDENT CACO 3 FORMATION IN ACETATE-FED AEROBIC GRANULES ItshouldbepointedoutthattheaccumulationofcalciumintheformofCaCO 3 in acetate-fed aerobic granules was found to be granule size-dependent (Wang, Li, and Liu2007).Ascanbeseeningure13.6,thecalciumcontentofacetate-fedaerobic granuleswasproportionallyrelatedtothegranulesize,forexample,thecalcium 53671_C013.indd 227 10/29/07 7:34:51 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Al,whichareindeedmarginal.Accordingtogure13.4,themolarratioofgranule ameandiameterof1.4mmwereusedfortheabove-mentionedanalyses (gure13.5). face(gure13.5c).Afterhydrochloricacidwasaddedtothezoneofwhitedeposits Figure 13.5a and b clearly show that calcium was mainly accumulated in the core part 228 Wastewater Purification Sludge Radius Range (mm) 0.1-0.2 0.3-0.4 0.5-0.6 0.7-0.8 0.8-1.0 1.4-2.0 Ca 2+ (mg g –1 SS) 0 50 100 150 200 250 FIGURE 13.6 Calcium contents in aerobic granules with different radius. (Data from Wang, Z W.,Li,Y.,andLiu,Y.2007.Appl Microbiol Biotechnol 74: 467– 473.) (a) (b) (c) (d) FIGURE 13.5 (a) Cross-section view of the acetate-fed aerobic granule by SEM; (b) the EDX mapping for calcium indicated by white color; bar: 100 µm; (c) image analysis cross- section view of the acetate-fed aerobic granule; (d) generation of gas bubbles during the acid-granulereaction;scalebar:200µm.(FromWang,Z W.,Li,Y.,andLiu,Y.2007.Appl Microbiol Biotechnol 74:467–473.Withpermission.) 53671_C013.indd 228 10/29/07 7:34:53 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Calcium Accumulation in Acetate-Fed Aerobic Granules 229 contentinbigaerobicgranuleswithradiusof1.4to2.0mmwasnearlytentimes higherthanthatinsmallaerobicgranuleswithradiusof0.1to0.2mm(gure13.6). Inthecourseofaerobicgranulation,itwasfoundthattheashcontentwasverylowat theinitialstageofaerobicgranulation,butitsharplyincreasedontheeighthdayin responsetoasignicantincreaseingranulesize,andgraduallystabilizedatthelevel ofabout0.4gg –1 SSafter40daysofoperation(gure13.7).Thisimpliesthatthecon- tent of CaCO 3 or so-called ash content was indeed very low in small aerobic granules, butittendedtoincreasewiththegrowthinsizeofacetate-fedaerobicgranules. 13.7 MECHANISM OF CALCIUM ACCUMULATION IN ACETATE-FED AEROBIC GRANULES As discussed earlier, calcium ion is not an essential element necessary for success- ful aerobic granulation (gure 13.1), and the extensive accumulation of calcium was Ash Content (g g –1 SS) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Time (days) 0 10203040 Sludge Mean Radius (mm) 0.0 0.3 0.6 0.9 1.2 1.5 FIGURE 13.7 Ashcontentandcorrespondingmeanradiusofacetate-fedaerobicgranules inthecourseofaerobicgranulation.(DatafromWang,Z W.,Li,Y.,andLiu,Y.2007.Appl Microbiol Biotechnol 74: 467–473.) 53671_C013.indd 229 10/29/07 7:34:54 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 230 Wastewater Purification onlyfoundinaerobicgranulesgrownonacetate(gure13.2).Furthermore,most accumulated calcium actually existed in the form of CaCO 3 ,anditwasmainly centralizedinthecorepartofacetate-fedaerobicgranules(gures13.4and13.5). One necessary condition for CaCO 3 formation at the low calcium ion concentration of 4.65 mg Ca 2+ L –1 is the presence of high CO 3 2- concentration at the core of the acetate-fedaerobicgranulesothattheionicproductofCa 2+ and CO 3 2- canbehigher than the solubility product constant of calcium carbonate. 13.7.1 IONIC EQUILIBRIUM OF CARBONATE ION Intermsofchemistry,theCaCO 3 formationisdeterminedbyitsionicconcentra- tion product: [][ ] , Ca CO K sp CaCO 2 3 2 3   (13.1) where K sp CaCO, 3 is the CaCO 3 solubility product constant. Calcium carbonate will form only when the concentration product of calcium and carbonate is greater than K sp CaCO, 3 .Acetatecanbeoxidizedinawaysuchthat: CH COO O H CO H O 32 22 22  m  (13.2) Dissolutionofcarbondioxidecanbeexpressedasfollows: CO H O H HCO 22 3 j  (13.3) and K HCO H CO a1 3 2   [][] [] (13.4) HCO H CO 33 2  j (13.5) and K CO H HCO a2 3 2 3    [][] [] (13.6) The overall reaction for carbonate can be expressed as: CO H O CO H 22 3 2 2j   (13.7) It should be pointed out that CO 2 produced in equation 13.2 can be dissolved into liquid phase according to Henry’s law: 53671_C013.indd 230 10/29/07 7:34:59 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC Calcium Accumulation in Acetate-Fed Aerobic Granules 231 PKCO CO h CO 22 2  , [] (13.8) where P CO 2 is the partial pressure of CO 2 in gas phase, [CO 2 ] is molar concentration of CO 2 in the liquid phase, and K hCO, 2 is the Henry’s constant for CO 2 . 13.7.2 DIFFUSION KINETICS IN AEROBIC GRANULES Itwasassumedinchapter8that(1)anaerobicgranuleisisotopicinphysical, chemical, and biological properties; (2) an aerobic granule is ideally spherical; (3) no anaerobic reaction occurs in the process; (4) aerobic granule responses to the change of bulk substrate concentration occur so quickly that the response time can be ignored. As presented in chapter 8, the mass balance equations for a substance betweenthetwolayersingranulewhoseradiusesare,respectively,randr + dr can be written as: D ds dr r ds dr R ss 2 2 2  ¥ § ¦ ´ ¶ µ  (13.9) where D s and R s are, respectively, the diffusion coefcient and mass conversion rate of the substance.Accordingtoequation13.9,Wang,Li,andLiu(2007)proposedthefollowing mass diffusion balance equations for O 2 ,H + ,HCO 3 - ,andCO 3 2- in aerobic granules: D dC dr r dC dr R O OO O 2 22 2 2 2 2  ¥ § ¦ ´ ¶ µ  (13.10) D dC dr r dC dr R H HH H     ¥ § ¦ ´ ¶ µ  2 2 2 (13.11) D dC dr r dC dr R HCO HCO HCO HCO 3 33 3 2 2 2    ¥ § ¦ ¦ ´ ¶ µ µ   (13.12) D dC dr r dC dr R CO CO CO CO 3 2 3 2 3 2 3 2 2 2 2    ¥ § ¦ ¦ ´ ¶ µ µ   (13.13) Li and Liu (2005) showed that dissolved oxygen would be a rate-limiting factor inthegrowthofaerobicgranules,andtheoxygenutilizationratecanbedescribed by the Monod equation: R Y C KC O x xO O OO 2 2 2 22   R M / max (13.14) 53671_C013.indd 231 10/29/07 7:35:04 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC 232 Wastewater Purification in which R x is biomass density, Y xO/ 2 is the dissolved oxygen-based growth yield, K O 2 is the dissolved oxygen-associated half-rate constant, and µ max is the maximum specic growth rate. Accordingtoequation13.2,theoxygenutilizationrateandtheH + consumption rateareinterrelatedbyequation13.15,thatis: RR O H consumption 2 2  () (13.15) Similarly,thefollowingrelationshipcanbeobtainedfromequations13.3and 13.7 for H + ,HCO 3 - ,andCO 3 2- : RRR H production HCO CO   () 33 2 2(13.16) Thus,thenetconsumptionrateofH + ,namely R H  in equation 13.11, is given by equation 13.17: RR R H H consumption H production    ()() (13.17) The dissolved oxygen (DO) concentration at the granule surface can be reason- ablyassumedtobeequaltoitsbulkconcentrationanditsrateofchangeinthegran- ulecenterwouldbeclosetozeroinconsiderationoftheDOsymmetricaldistribution inthegranulecenter(LiandLiu2005),thatis: CC O r R bulk O 22   , (13.18) dC dr O r 2 0 0   (13.19) Likewise, C H  atthegranulesurfaceisassumedtobeequaltothebulkH + con- centration, and the derivative of C H  atthecenterofthegranuleiszero(Wang,Li, and Liu 2007): CC H r R bulk H    , (13.20) dC dr H r    0 0 (13.21) Equations13.10to13.13weresolvednumericallybyMatlab™7.0basedonthe nite differentiation principle as described in chapter 8. 53671_C013.indd 232 10/29/07 7:35:11 AM © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor & Francis Group, LLC [...]... Accumulation in Acetate-Fed Aerobic Granules 8.56 8.55 pH 8.54 8.53 8.52 8.51 8.50 2.9e-5 CO32–(M) 2.8e-5 RCaCO3 2.7e-5 2.6e-5 2.5e-5 2.4e-5 0.0 0.2 0.4 0.6 r(mm) FIGURE 13. 8 Simulated profiles of pH and CO3 2- in acetate-fed aerobic granules (Data from Wang, Z.-W., Li, Y., and Liu, Y 2007 Appl Microbiol Biotechnol 74: 467–473.) 13. 7.3 DISTRIBUTION OF PH AND CO3 2- IN ACETATE-FED AEROBIC GRANULES According to... aerobic granulation In fact, increasing evidence shows that microbial granulation is a cell-to-cell self-immobilization process driven mainly by selection pressures (see chapter 6), that is, calcium may play a very minor role in the microbial granulation process, and aerobic as well as anaerobic granulation is indeed calcium independent (Mahoney et al 1987; Jiang et al 2003) This point is also supported... why calcium is not accumulated in the ethanol-fed aerobic granules but only in those fed with acetate (figure 13. 2) 13. 7.4 SIZE-ASSOCIATED FORMATION OF CACO3 IN ACETATE-FED AEROBIC GRANULES The volume fraction of the CaCO3 formable region in acetate-fed aerobic granules, namely ( RCaCO3 R)3 , was simulated at various granule radiuses in the range of 0 to 2 mm (figure 13. 9) It appears that the formation... observation, as shown in figure 13. 5 It can be concluded from figure 13. 8 that the calcium accumulation in acetate-fed aerobic granules is mainly due to the fact that alkalinity in the form of hydroxide ion was produced during the biological oxidation of acetate, as illustrated in equation 13. 2 In contrast, no hydroxide ion actually can be generated in the biological oxidation of ethanol This explains why calcium... in aerobic granules (figure 13. 9) Batstone et al (2002) has put forward a hypothesis that preformation of a crystal CaCO3 core is a prerequisite of anaerobic granulation However, both experimental and theoretical evidence in figures 13. 6, 13. 7, and 13. 9 point to the fact that calcium accumulation occurs only after aerobic granulation, and a crystal CaCO3 core is indeed not required for aerobic granulation. .. mol L –1 In view of the CO3 2- profile in figure 13. 8, it is reasonable to consider that CaCO3 will be formed mainly in the region with a depth below 300 µm from the granule surface, that is, r < RCaCO3 zone in figure 13. 8 Such a theoretical prediction is in good agreement © 2008 by Taylor & Francis Group, LLC © 2008 by Taylor 233 53671_C 013. indd & Francis Group, LLC 10/29/07 7:35 :13 AM 234 Wastewater. .. in aerobic granules smaller than 0.5 mm in radius, and calcium carbonate starts to form only in aerobic granules larger than 0.5 mm in radius This in turn provides a plausible explanation for the observed size-dependent CaCO3 formation in aerobic granules (figures 13. 6 and 13. 7) Such theoretical estimation is pretty consistent with the experimental results measured as the calcium and ash contents in. .. fact that CaCO3 is only formed in aerobic granules with a radius larger than 0.5 mm (figures 13. 6 and 13. 7) indicates that the formation of calcium carbonate in the acetate-fed aerobic granules is actually granule size dependent, which can be explained by the diffusion limitation described by equations 13. 10 to 13. 13 This is because the microbial metabolites, such as alkalinity and carbon dioxide produced... Technol 19: 249–260 Qin, L., Liu, Y., and Tay, J H 2004 Effect of settling time on aerobic granulation in a sequencing batch reactor Biochem Eng J 21: 47–52 Thiele, J H., Wu, W M., Jain, M K., and Zeikus, J G 1990 Ecoengineering high rate anaerobic digestion systems Analysis of improved syntrophic biomethanation catalysts Biotechnol Bioeng 35: 990–999 Van Langerak, E P A., Gonzalez-Gil, G., Van Aelst,... previous studies on aerobic and anaerobic granulation (Guiot et al 1988; Thiele et al 1990; El-Mamouni et al 1995; Van Langerak et al 1998; Jiang et al 2003) It appears from microscopic observation and chemical analysis that the accumulation of calcium is in the form of CaCO3, and those accumulations were centralized in the core part of acetate-fed aerobic granules (figures 13. 4 and 13. 5) The fact that . Accumulation in Acetate-Fed Aerobic Granules 225 13. 4 Chemical form of calcium in acetate-fed aerobic granules 226 13. 5 Calcium Distribution in Acetate-Fed Aerobic Granules 227 13. 6 Granule Size-Dependent. Accumulation in Acetate-Fed Aerobic Granules 233 13. 7.3 DISTRIBUTION OF PH AND CO 3 2- IN ACETATE-FED AEROBIC GRANULES According to the models presented above, the pH and CO 3 2- proles in aerobic gran- ules. Ca 2+ L –1 ,respectively.Similartotheresultsshown in gure 13. 1, successful anaerobic granulation was achieved in both reactors, indi - catingthatcalciumisnotanessentialelementforanaerobicgranulationeither. Compared to aerobic granules