Correlating TMP Increases withMicrobial Characteristics in theBio-Cake on the Membrane Surfacein a Membrane BioreactorB Y U N G - K O O K H W A N G , †

W O O - N Y O U N G L E E , † K Y U N G - M I N Y E O N , †

P Y U N G - K Y U P A R K , † C H U N G - H A K L E E , * , †

I N - S O U N G C H A N G , ‡ A N J A D R E W S , § A N DM A T T H I A S K R A U M E §

School of Chemical and Biological Engineering, SeoulNational University, Seoul, Korea, Department ofEnvironmental Engineering, Hoseo University, Asan,Korea, and Department of Chemical Engineering,Technische Universita¨t Berlin, Berlin, Germany

Received November 29, 2007. Revised manuscript receivedMarch 14, 2008. Accepted March 25, 2008.

Subcritical flux operation is widely practiced in membranebioreactors (MBRs) to avoid severe membrane fouling and, thus,to maintain sustainable permeability. Filtration at a constantsubcritical flux, however, usually leads to a two-stage increasein the transmembrane pressure (TMP): initially slowly, thenabruptly. We have investigated the mechanism of this two-stage TMP increase through analyses of the structureand microbial characteristics of the bio-cake formed on themembrane. The MBR was operated under various subcriticaland supercritical flux conditions. Under subcritical conditions,we observed the typical two-stage TMP increase. When aconstantfluxaugmentedandreachedthesupercriticalconditions,however, the dual TMP change gradually transformed into asteeper,one-stageTMPincrease.ThesecondstageTMPincreaseunder the subcritical flux was closely related to the suddenincrease in the concentration of extra-cellular polymericsubstances (EPSs) at the bottom layer of the bio-cake; weattribute the one-stage TMP increase under the supercriticalconditions to the accumulation of microbial flocs and the reducedporosity of the bio-cake under compression. We explain thevariation of the EPS concentration in the bio-cake in terms ofthe spatial and temporal changes of the live-to-dead ratioalong the depth of the bio-cake.

IntroductionMembrane fouling remains a critical limiting factor affectingthe more widespread application of submerged membranebioreactors (MBRs) to wastewater treatment. To overcomethis fouling problem, submerged MBR systems are oftenoperated under subcritical flux conditions to maintain asustainable permeability, which usually leads to a two-stageincrease in transmembrane pressure (TMP), i.e., an initialslow and gradual increase followed by an abrupt rise in theTMP.

Many studies have focused on addressing the issue of thetwo-stage TMP increase under long-term subcritical fluxoperation. Ognier et al. reported that successive blocking ofmembrane pores by foulants reduces the working membranearea, resulting in an abrupt change in TMP (1). Nagaoka etal. noted that for a submerged MBR, a period of relativelylow fouling resistance was followed by a sudden rise inresistance; they suggested that this behavior was due to anincrease in the suspension viscosity caused by levels of extra-cellular polymeric substances (EPSs) in the feed (2). Cho etal. observed a similar phenomenon in a cross-flow micro-filtration system and concluded that it occurred because thelocal flux was higher than the critical flux (3). Zhang et al.divided the fouling profile for a submerged MBR into threestages and suggested a membrane fouling mechanism foreach one (4). Looking at the various studies that have beenperformed to address this issue, the explanations that Zhanget al. provided appear to be extensive and reasonable.However, they missed taking into account the temporalchange in the structure and microbial characteristics of thebio-cake that might occur during MBR operation.

The aim of this study was to elucidate mechanisms forthe various patterns of TMP increase in a submerged MBRunder various operating conditions. The three-dimensionalarchitecture of the bio-cake was analyzed and quantified,using confocal laser scanning microscopy (CLSM) and digitalimage analysis, to provide information relating to thetemporal change in the inner architecture of the bio-cake(5). Spatial and temporal changes in the microbial properties,such as the levels of the EPSs and the ratio of live and deadcells, were also investigated.

Materials and MethodsMBR System. The laboratory-scale submerged MBR con-sisted of interconnected anoxic (2 L) and aerobic (6 L) tanks(Supporting Information (SI) Figure S1). The syntheticwastewater whose composition was described in SI Table S1was first fed into the anoxic tank equipped with an agitator.The mixed liquor overflowed from the anoxic tank into theaerobic tank, in which four membrane modules wereimmersed. The aerobic tank was equipped with a coarsebubble diffuser connected to a blower that mixed the brothand maintained the dissolved oxygen concentration at 2.5-3mg/L. The rate of recirculation flow from the bottom of theaerobic tank to the anoxic tank was maintained at 1.2-1.5times the feed flow rate, Q. The concentrations of the mixedliquor-suspended solids (MLSS) were maintained at 7500((180) mg/L in the anoxic tank and 8200 ((300) mg/L in theaerobic tank. The hydraulic retention time (HRT) and solidretention time (SRT) were maintained at 8 h and 30 days,respectively. The both tanks were maintained at the tem-perature of 25-28 °C and a pH of 7.0-7.5.

The four identical membrane modules were mountedvertically within the aerobic tank. Polyethylene hollow fibermicrofiltration membranes (KMS, Korea) having a membranearea of 0.08 m2 per module were used. The nominalmembrane pore diameter was 0.4 µm. The internal andexternal fiber diameters were 410 and 650 µm, respectively.

Two sets of MBR operations were run under the sameconditions. For each set, the permeate was continuouslywithdrawn using a peristaltic pump under a constant flux of6, 13, 20, or 27 L/(m2 ·h). The filtration was stopped when theTMP reached 70 kPa without chemical cleaning. In the firstrun, four membrane modules were removed one by one afterspecific operation times and used for bio-cake analysis. Inthe second run, which was performed under operating

* Corresponding author phone: +82-2-880-7075; fax: +82-2-874-0896; e-mail: leech@snu.ac.kr.

† Seoul National University.‡ Hoseo University.§ Technische Universität Berlin.

Environ. Sci. Technol. 2008, 42, 3963–3968

10.1021/es7029784 CCC: $40.75 2008 American Chemical Society VOL. 42, NO. 11, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 3963Published on Web 04/29/2008

conditions identical to those of the first run, the fourmembrane modules were removed one by one after a specificoperation time (identical to that of the first run); thesemodules were subjected to quantitative analysis of theattached biomass and bound EPSs in the bio-cake formedon each membrane surface.

Analytical Methods. The bio-cake on the membranesurface was detached and resuspended in sterilized deionizedwater for analysis of the bound EPS (6), which was extractedfrom the resuspended bio-cake using heat treatment (7). Theextract was analyzed for total polysaccharides and proteins,which are the dominant components in the EPS. The sumof total polysaccharides and proteins was taken to be thetotal amount of EPS (8). The polysaccharides in the EPS weredetermined using the phenol/sulfuric acid method withglucose as the standard (9). The proteins were quantifiedusing a modified Lowry method and a commercial proteinassay kit (Sigma, Deisenhofen, Germany) with bovine serumalbumin (BSA) as the standard. COD, TP, and TN weremeasured spectrometrically (DR4000, Hach, U.S.) usingcorresponding reagent kits.

Bio-Cake Staining. The bio-cake was stained as describedpreviously for other relevant biofilm analyses using CLSM(10). A fraction of the membrane fibers located in the middlesection of membrane module were removed (length: 3 cm;SI Figure S1) and the bacterial cells and EPSs on the bio-cakewere stained using a mixture of fluorescent dyes that arespecific to each component. SYTO 9 (Molecular Probes,Eugene, U.S.; excitation (ex) ) 488 nm; emission (em) )515/30 nm), a cell-permeable nucleic acid dye, was used tovisualize all cells (10). Concanavalin A (ConA) lectin (Mo-lecular Probes, Eugene, OR; ex ) 568 nm; em ) 600/50 nm)conjugated with tetramethylrhodamine isothiocyanate(TRITC) was used to bind to the R-mannopyranosyl andR-glucopyranosyl sugar residues (polysaccharides) in the bio-cake (11). After incubating the stained bio-cake for 30 minin the dark at room temperature, the excess staining solutionwas removed by washing with phosphate-buffered saline(PBS) after each staining step. The live/dead distribution ofbacterial cells was determined by staining with the BacLightLive-Dead staining kit (Molecular Probes, Eugene, OR)according to the manufacturer’s instructions (12).

Acquisition of Bio-Cake Images Through CLSM. Speci-mens of the stained bio-cakes were immediately observedusing CLSM. A scanning confocal system (Bio-Rad Radiance2000), equipped with an argon-krypton laser and mountedon a microscope (Nikon TE 300), was used to obtain imagesof the bio-cakes nondestructively. The bio-cakes wereobserved with a 60 × 1.4 NA lens. A series of z-axis imageswas generated through optical sectioning at a slice thicknessof 1 µm. Each bio-cake specimen was scanned randomly atselected positions. Twelve confocal image stacks wereacquired for twelve cut fibers. The observed images coveredan area of 202 × 202 µm2 with a resolution of 512 × 512 pixels(256 gray-values). All images were stored and analyzed usingimage analysis programs.

Image Analysis. Two digital image analysis programs wereused to obtain information regarding the bio-cake archi-tecture. A commercial program for bio-cake analysis, ImageStructure Analyzer in three-dimensions (ISA-2), was used toanalyze the bio-cake architecture in terms of its porosity andaverage thickness based on the bio-cake images acquired byCLSM (13). ISA-2 was also used to determine the ratio of liveto dead cells in the confocal image stacks (12). Anotherprogram, IMARIS (v. 4.1.3, Bitplane AG, Zurich, Switzerland),was used to visualize the 3D structure of the bio-cake, i.e.,the CLSM images were reconstructed and presented as 3Dviews (11).

Results and DiscussionEffect of Bio-Cake Characteristics on TMP Profile. In thesuccessive MBR operations under all fluxes, the removalefficiencies of the COD, total nitrogen (TN), and totalphosphorus (TP) at the steady state did not change signifi-cantly, e.g., 94∼96% (COD), 67∼72% (TN), and 13∼19% (TP).The rate of increase of the TMP is an important factor affectingthe membrane filterability in a submerged MBR systembecause it is directly related to the extent of membranefouling. Figure 1 displays the TMP variations with respect tothe operating time for four separate modules evaluated atconstant fluxes of 6, 13, 20, and 27 L/(m2 ·h), respectively. Asexpected, the greater the flux, the steeper the increase in theTMP. The four TMP profiles in Figure 1 display similar trends:a slow, gradual increase in TMP followed by an abrupt, rapidincrease. A turning point around which distinct changes inTMP occurred was observed clearly for operating fluxes of13 and 20 L/(m2 ·h), but it was less distinct at the highest fluxof 27 L/(m2 ·h). Brooks et al. reported the similar observation(14). Thus, to elucidate the dependence of the pattern ofTMP increase on the flux, we used operating fluxes of 13 and27 L/(m2 ·h) to investigate the characteristics of the bio-cakeformed during each run. The bio-cake specimens from eachsubmerged membrane were sampled and analyzed at severalpoints (points 1-4 at a flux of 13 L/(m2 ·h); points 5-7 at aflux of 27 L/(m2 ·h); Figure 1) up to a TMP of 70 kPa.

To identify the main contributor to membrane fouling ateach point in Figure 1, we conducted hydraulic resistanceanalyses of the membranes at all points (points 1-7) usinga resistance-in-series model (Figure 2) (11). The intrinsicmembrane resistance (Rm) and the fouling resistance (Rf)appeared to be minor factors of the total resistance (Rt). The

FIGURE 1. TMP profiles plotted as a function of time fordifferent fluxes.

FIGURE 2. Resistances through the membrane and bio-cake atthe corresponding points in Figure 1.

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bio-cake resistance (Rc), however, was always a major portionof the total resistance. This is in agreement with Drews et al.(15) where a decreasing importance of polysaccharides athigher SRT was observed. Furthermore, the fraction of thebio-cake resistance (Rc) to the total resistance increasedsteadily as the TMP rose, e.g., from points 1 to 4 (flux ) 13L/(m2 ·h)) and from points 5 to 7 (flux ) 27 L/(m2 ·h)).Especially, the values of Rc at points 4 and 7, representingthe final stage of each operation, comprised more than 80%of the total resistance. This finding indicates that the valueof Rc caused by the formation of the bio-cake on themembrane surface was mainly responsible for the increasein TMP. Therefore, we investigated the bio-cake’s propertiesin more detail to determine its effect on the membranefilterability, which, in turn, is directly linked to the patternof TMP increase.

We determined two physical properties of bio-cakesamples removed from the used membranes correspondingto each point in Figure 1, namely the total amount of attached

biomass (TAB) on the membrane surface and the averagethickness of the bio-cake, obtained through CLSM and imageanalysis (Figure 3). The TAB profile coincided well with thatof the bio-cake thickness. As expected, the rate of TABaccumulation increase on the membrane surface (Figure 3a)increased upon increasing the flux from 13 to 27 L/(m2 ·h),as did the average thickness of the bio-cake (Figure 3b). Thesefindings are consistent with the TMP increase at a flux of 27L/(m2 ·h) being steeper than that at 13 L/(m2 ·h). It is worthnoting, however, that neither the TAB profile nor the averagethickness explains the different patterns in the TMP profiles,i.e., neither the one-stage TMP increase under a flux of 27L/(m2 ·h) and the two-stage TMP increase at 13 L/(m2 ·h) northe absolute values. At the same TMP (e.g., points 4 and 7),a bio-cake of almost the same thickness (cf. Figure. 3b) shouldcontain a similar amount of TAB if porosity is similar.However, the bio-cake at point 7 contains more TAB at thesame thickness, i.e., porosity has to be smaller and thus TMPshould be higher if the cells in the bio-cake were the onlyreason.

Bound EPS in the Bulk Phase and in the Bio-Cake Layer.There are three kinds of EPSs in the MBR system: (i) solubleEPSs in the bulk phase and (ii) bound EPSs that wereembedded in the microbial flocs in the bulk phase as wellas (iii) in the bio-cake layer. Because the bound EPSs providea highly hydrated gel matrix in which microorganisms areembedded, the EPSs and the microbial cells presumablypresent a significant hydraulic barrier to permeate flowthrough the bio-cake layer. Therefore, we performed aquantitative determination of the bound EPSs to elucidateits effect on membrane biofouling.

Figure 4 displays the variations of the bound EPSconcentration in the bulk phase (mg EPS/g MLSS) and in thebio-cake layers (mg EPS/g TAB) along the points 1-7. The

FIGURE 3. (a) Total amounts of attached biomass (TAB) and (b)average thicknesses of the bio-cake samples obtained at thecorresponding points in Figure 1.

FIGURE 4. Concentrations of bound EPSs in the bulk phasesand in the bio-cakes at the corresponding points in Figure 1.

FIGURE 5. Variations in porosities based on bacterial cells andpolysaccharides in the bio-cake at the corresponding points inFigure 1: (a) Flux ) 13 L/(m2 ·h); (b) flux ) 27 L/(m2 ·h).

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content of bound EPS in the bulk phase was ca. 95 mg EPS/gMLSS. At points 5-7, the total bound EPSs in the bio-cakeformed under the constant flux of 27 L/(m2 ·h) was similarto the total bound EPSs in the bulk phase. In contrast, thetotal bound EPSs in the bio-cakes formed under a flux of 13L/(m2 ·h) increased significantly from point 1 to 4. Inparticular, the increase in the bound EPSs between points2 and 3 (ca. 20 mg EPS/g MLSS) and between points 3 and4 (ca. 38 mg EPS/g MLSS) was much greater than that betweenpoints 1 and 2 (ca. 5 mg EPS/g MLSS). Recalling that theturning point of the TMP increase in Figure 1 lies betweenpoints 2 and 3, the change in the total EPSs coincides wellwith the pattern of TMP variation. This behavior provides uswith a clue to explain why the two-stage TMP increase wasaffected by the operating flux. The quantitative change inthe bound EPSs during the course of filtration seems to beclosely related to the change in the bio-cake architecture,such as the porosity, which in turn affects the TMP increase.In this context, we investigated the bio-cake architecture ingreater detail. Zhang et al. (4) listed five possible mechanismsreported to explain the two stage TMP increase and claimedthat more than one of the listed mechanisms could comeinto play simultaneously. However, all the five mechanismsmissed taking into account microbial characteristics of thebio-cake that might occur during MBR operation.

Porosities Based on Bacterial Cells and Polysaccharidesin the Bio-Cake. Figure 5 displays the variation in porositybased on bacterial cells and polysaccharides as functions ofoperating time under fluxes of 13 and 27 L/(m2 ·h), respec-tively. Under a flux of 13 L/(m2 ·h), the porosity based on thebacterial cells decreased in a constant manner upon in-creasing the operation time, whereas the porosity based onpolysaccharides decreased abruptly after point 2 and keptdecreasing to a value at point 4 of 0.85, which was the sameas the value of the porosity based on the bacterial cells (Figure

5a). This finding indicates that the content of polysaccharidesgenerated inside the bio-cake became increasingly importantupon increasing the operating time and thus TMP. Therefore,the abrupt decrease in the porosity beyond point 2 under aflux of 13 L/(m2 ·h) might be closely associated with the rapidincrease in the total EPS concentration in the bio-cake (Figure4) beyond point 2, which in turn might be related to thetwo-stage TMP increase observed in Figure 1.

At the higher flux of 27 L/(m2 ·h), however, the porositybased on the polysaccharides did not decrease abruptly;instead, it decreased almost linearly from point 5 to 7 (Figure5b). The porosity based on the cells decreased almost linearly,but the slope was steeper than that observed at a flux of 13L/(m2 ·h) (Figure 5a).

It is clear that the rate of the TMP increase was directlyrelated to both the porosity and the TAB of the bio-cake layerthrough which the permeate flow passed. From the porositiesbased on both bacterial cells and polysaccharides in Figure5, it appears that the bio-cake formed under a flux of 27L/(m2 ·h) was occupied mostly by bacterial cells, rather thanby polysaccharides. At an operating flux of 13 L/(m2 ·h),however, the volumetric portion of the bio-cake that wasoccupied by the cells increased linearly, whereas thatoccupied by the polysaccharides increased abruptly.

Because the EPS components are relatively sticky andhave gel-like intermingled structures, the EPSs could be agreater barrier to permeate flow than the bacterial cells, evenwhen the former occupy a much lower volume in the bio-cake than do the latter. In this context, the two-stage TMPincrease observed in Figure 1 under a flux of 13 L/(m2 ·h)seems to be closely related to the variation of the EPSsconcentration inside the bio-cake. On the other hand, thelinear increase in the TMP at a flux of 27 L/(m2 ·h) could beattributable to the monotonous sedimentation of bacterialflocs from the bulk phase to the membrane surface at the

FIGURE 6. Volumetric 3D reconstructed images of bacterial cells and polysaccharides present in the bio-cake at the correspondingpoints in Figure 1: (a) flux ) 13 L/(m2 ·h); (b) flux ) 27 L/(m2 ·h). Green color: bacterial cells; red color: polysaccharides; area of eachobserved image: 202 × 202 µm2.

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supercritical flux, causing a gradual increase in the TAB. Toconfirm this hypothesis, we visualized the spatial distributionof cells and EPS (polysaccharides).

Spatial Distributions of Bacterial Cells and EPS in theBio-Cake. Figure 6 displays reconstructed 3D images of thebio-cakes; the top and bottom views of the bio-cakescorresponding to each point in Figure 1 are presented interms of the cells and polysaccharides. These images providean insight into the temporal changes of the spatial distribu-tions of both components. Although we also obtained 3Dimages for proteins, we do not present them here becausethe spatial distributions of proteins were quite similar tothose for the polysaccharides.

As indicated in Figures 6a and b, the green colorsrepresen-ting microbial cellsswas dominant on the top of the bio-cake, regardless of the operating flux. At the bottom of thebio-cake, however, the red colorsrepresenting EPSs (polysac-charides)sincreased upon increasing the operating time, i.e.,from points 1 to 4 at a flux of 13 L/(m2 ·h) (Figure 6a). As wementioned above, the polysaccharide component of the EPSpresented a greater barrier to the permeate flow than did thebacterial cells. Therefore, it is likely that the development ofthe content of polysaccharides was directly linked to the rateof the TMP increase. In other words, the dramatic TMPincrease at a subcritical flux of 13 L/(m2 ·h) can be attributedto the rapid generation of polysaccharides resulting inreduced porosity.

In contrast, the development of the red color at the bottomof the bio-cakes was much less dramatic (cf. points 5-7) ata flux of 27 L/(m2 ·h) (Figure 6b) relative to that at 13 L/(m2 ·h).The slower rate of generation of polysaccharides at the higher

flux of 27 L/(m2 ·h) is consistent with the correspondingobserved gradual, linear TMP increase. Furthermore, weattribute the more rapid rate of increase of the TMP at thishigher flux mainly to the increased bio-cake thicknessresulting from the convective transport of microbial flocstoward the membrane as well as to the reduced porosity ofthe bio-cake based on microbial cells resulting from thegreater compaction under the higher TMP.

It is worth noting that polysaccharides were generatedand located predominantly at the lower parts of the bio-cakes. Unlike conventional biofilm processes, in which abiofilm grows on a nonpermeable substratum, in a sub-merged MBR process a liquid flow exists in the form of apermeate flux perpendicular to the bio-cake. As a result, inthe MBR process, substrates and oxygen that are requiredfor living organisms to survive are automatically supplied bythe membrane flux to the microbial communities in the bio-cake. When the bulk solution passes through the bio-cake,the living organisms will consume substrates as well asdissolved oxygen. Consequently, as the thickness of the bio-cake grows, an anoxic and endogenous environment woulddevelop in the lower parts of the bio-cake layer, ultimatelyleading to cell lysis and release of polysaccharides, decreasedpolysaccharide mineralization or even release of EPS by livingcells (16).

Spatial and Temporal Distributions of the Active andInactive Biomasses in Bio-Cakes. Our findings raised thefollowing question: Why was the generation of polysaccha-rides observed in Figure 6 less important at the higher fluxthan it was at the lower flux. To answer this question, thelive-to-dead ratiosdefined as the ratio of active to inactive

FIGURE 7. Vertical distribution of the live-to-dead cell ratio in the bio-cake at each corresponding point in Figure 1: (a) flux ) 13 L/(m2 ·h); (b) flux ) 27 L/(m2 ·h).

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biomass in the bio-cakessand its spatial and temporaldistributions were determined. Figures 7a and b display thetemporal changes of the live-to-dead ratio along the depthof the bio-cakes during periods of operation of 38 and 15days, respectively. We observe two interesting phenomena.First, over the operating time, the live-to-dead ratio changedcontinuously along the depth of the bio-cakes, regardless ofthe operating flux; e.g., the ratio at the lower layer of thebio-cakes became increasingly smaller, whereas the ratio atthe upper layer became increasingly larger. This behaviorindicates that the portion of living microorganisms in thelower layer of the bio-cakes decreased. As the bio-cakesaccumulated on the membrane surface, endogenous decayor cell lysis presumably occurred at the bottom layer as aresult of the poor transfer of oxygen and nutrients from thebulk solution. Therefore, the lower layer, i.e., near themembrane surface, was likely to become a stressful environ-ment for the microorganisms, thereby increasing the gen-eration of EPSs, as we observed in Figure 6. The reason whythe ratio was higher nearer the bulk solution is that the bio-cake closer to the surface consisted mostly of living micro-organisms because the nutritional conditions in the upperlayer were likely be similar to those in the bulk phase.

The second interesting phenomenon is that there was adifference in the spatial distribution of the live-to-dead ratiobetween the conditions of low flux and high flux. Thedistribution of the ratio at higher flux (points 5-7, Figure 7b)was relatively uniform along the depth of the bio-cakes,whereas that at lower flux (points 1-4, Figure 7a) changeddramatically along the depth. To elucidate the causes of thesephenomena, we investigated how long it took to arrive at thesame TMP for each rate of flux. As indicated in Figure 1, ittook ca. 15 days to arrive at a TMP of 70 kPa under a flux of27 L/(m2 ·h), whereas it took ca. 38 days to reach the samevalue at 13 L/(m2 ·h). Because the operation ended relativelyearlier at higher flux than at the lower flux, there wasinsufficient time to reach endogenous decay or cell lysis.Thus, the gap between the live-to-dead ratios at the top andbottom of the bio-cakes ranged from 0.5 to 4.5 at the lowerflux (point 4, Figure 7a), whereas it ranged from 1.5 to 4(point 7, Figure 7b) at higher flux. These values indicate thatthe portion of dead microorganisms near the surface of themembrane was much greater at lower flux, coinciding wellwith the intensity of the red colorsrepresenting polysac-charidessin Figure 6a, which ultimately gave rise to theabrupt increase in TMP in the second phase because thepolysaccharides provided a greater hydraulic barrier. Thisexplanation is consistent with the experimental resultspresented in Figure 4, where the porosities based onpolysaccharides are depicted for each corresponding pointin Figure 1. An additional cause could be the higher masstransfer at higher flux which provides more nutrients to thelower part of the bio-cake and thus prevents more cells fromdying.

In summary, during the operation of a submerged MBR,we observed a gradual increase in TMP followed by a rapidincrease at low flux and a rapid, one-phase TMP increase athigher flux. We believe that three factors affect the patternof the TMP increase: (i) the increasing thickness of the bio-cake resulting from the deposition of bacterial flocs from thebulk phase, (ii) the reduction of bio-cake porosity arisingfrom the compression effect exerted on the bio-cake by theincreasing external pressure (i.e., the TMP), and (iii) thegeneration of EPS like polysaccharides inside the bio-cakelayer. We conclude that the abrupt TMP increase at the secondstage under a subcritical flux of 13 L/(m2 ·h) is attributablemainly to the third factor, i.e., the substantial generation ofpolysaccharides in the lower layer of the bio-cake over longperiods of operation. In contrast, the first two factors seem

to provoke the more rapid and abrupt TMP increase at asupercritical flux of 27 L/(m2 ·h), because in this case theaccumulation of polysaccharides was negligible because ofthe relatively shorter operation time and the higher nutrienttransfer.

AcknowledgmentsThis work was supported by the Korea Science and Engi-neering Foundation (KOSEF) grant funded by the Koreangovernment (MOST). (no. R0A-2007-000-20073-0). We thankthe National Instrumentation Center for Environment Man-agement for allowing us to use the CLSM.

Supporting Information AvailableFigure S1 (schematic representation of the experimental setupfor the submerged MBR), and Table S1 (composition ofsynthetic wastewater). This material is available free of chargevia the Internet at http://pubs.acs.org.

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