Desalination 231 (2008) 115–123

0011-9164/08/$– See front matter © 2008 Elsevier B.V. All rights reserved

Presented at The 4th IWA Conference on Membranes for Water and Wastewater TreatmentMay 15–17, 2007, Harrogate, UK

*Corresponding author.

Correlation of biofouling with the bio-cake architecturein an MBR

C.H. Leea*, P.K. Parka, W.N. Leea, B.K. Hwanga, S.H. Honga, K.M. Yeona,H.S. Oha, I.S. Changb

aSchool of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, KoreaTel. +82 (2) 880-7075; Fax +82 (2) 874-0896; email: leech@snu.ac.kr

bDepartment of Environmental Engineering, Hoseo University, Asan, 336-795, Korea

Received 17 May 2007; accepted 26 October 2007

Abstract

The experimental or theoretical method that can visualize or quantify the architecture of bio-cake formed on thesurface of membrane is of great interest in understanding biofouling mechanisms, also controlling biofouling in amembrane bioreactor (MBR). This paper summarizes the methods for determining the porosity of bio-cakeexperimentally or theoretically and also correlates the bio-cake architecture with membrane filterability in asubmerged MBR under various operating conditions.

Keywords: MBR; Biofouling; Biofilm; Bio-cake; CLSM; Image analysis; Cake layer; Porosity; EPS

1. Introduction

Biofilms are ubiquitous life forms. In someinstances their presence is desirable, such asbiofilms for bioremediation addressing treatmentof contaminated soils, and in some instancesbiofilms are not desirable, such as biofilms in anMBR [1,2]. Biofilms have been studied rigorouslyunder various perspectives according to the re-

searchers’ purposes and backgrounds. For ex-ample, molecular microbiologists place their re-search focus on the specific gene and its regula-tion mechanism during biofilm formation process[3]. Medical researchers investigate the virulencecaused by biofilm growth on wounds and medi-cal devices [4]. For the environmental engineers,main research topics have been on how to solveunwanted biofilm problems or how to take ad-vantage of biofilms to improve water quality [1,2].

doi:10.1016/j.desal.2007.10.026

116 C.H. Lee et al. / Desalination 231 (2008) 115–123

In spite of ubiquitous life forms of biofilms, fun-damental research themes on biofilms can be cat-egorized into three parts: i) development andphysiology [5], ii) structure, function and chem-istry of slime matrix [6,7] and iii) structural orarchitectural heterogeneity [8,9].

This paper discusses biofilms in an MBR whichcomprises mostly living organisms and microbialextracellular polymeric substances (EPS), particu-larly focusing on the architecture of biofilmsformed under various operating conditions. Un-derstanding the architecture and structure ofbiofilms can help the designers and operators ofMBR to select optimum operating conditions andrational cleaning procedures.

2. Definition of the term “bio-cake”

The biofouling layer formed on the membranein an MBR mostly consists of microbial flocs de-posited from the bulk phase. These microbial flocsare very heterogeneous, irregular structures con-sisting of microorganisms, organic and inorganicadsorbed particles, extra polymeric substances(EPS) and organic fibers [10]. We often encoun-ter a question; what is an appropriate term to rep-resent this kind of fouling layer in an MBR? Twowords easily cross our mind; cake layer andbiofilm. The term “cake layer” is defined as a layercomprised of rejected particulate materials resid-ing on the upstream face of a membrane [11]. Theother term “biofilm” can be broadly defined ascommunities of microbes associated with a sur-face, typically encased in an extracellular matrix[12]. Although the biofouling layer on the mem-brane in MBR consists of rejected microbial flocs,they are active to excrete slimy, glue-like sub-stances (e.g., EPS) rather than inactive inorganicparticles. It is also probable that irreversibly at-tached biofloc residues and planktonic bacteriaact as the seed for biofilm growth on the mem-brane surface [13]. That is why it is not easy tofind an appropriate term to represent this kind ofbiofouling layer in an MBR. In this text, instead

of two terms mentioned above, “bio-cake” whichcould represent both rejected active particulatematerials (e.g., microbial flocs) and growing mi-croorganisms on the membrane surface in an MBRwill be used.

3. Role of bio-cake porosity in an MBR

It would be of interest to compare the differ-ences in transport mechanisms through two dif-ferent biofilms. In a conventional biofilm process,in which a biofilm grows on a non-permeable sub-stratum, the liquid flows only in one direction,parallel to the biofilm, whereas additional liquidflows should exist in the form of a permeate fluxperpendicular to the bio-cake in the MBR pro-cess (Table 1). This can be attributed to the pres-ence of a permeate flux (J) through a porous sub-stratum (membrane) in MBR. Owing to the pres-ence of permeate flux in the MBR process, themajor solute transport mechanism should be “con-vection” whereas that of a biofilm grown on anon-permeable substratum in a conventional biofilmprocess is “diffusion.” As a result, in the MBR pro-cess, organic substrates, nutrients, and dissolvedoxygen that are required for living microorgan-isms are automatically supplied by the convectivemovement of bulk solution to the microbialcommunities in the bio-cake. In addition to solutetransport through the bio-cake layer, the permeate(water) flux is greatly dependent on the porosityand size of particulates in the bio-cake accordingto the well known Carman–Kozeny equation.

Needless to say the porosity of biocake is oneof key factors affecting directly the water flux oflow pressure membranes, which in turn deter-mines the operating cost of the MBR plant. Un-fortunately we can hardly find any experimentalanalytical method to directly measure the poros-ity of a bio-cake formed on the membrane in theMBR. It may be one of the reasons why we stillsuffer from biofouling problem although tremen-dous works have been devoted to solve membranebiofouling.

C.H. Lee et al. / Desalination 231 (2008) 115–123 117

Table 1Comparison between a biofilm grown on a non-permeable support and a bio-cake deposited on a membrane in MBR [14]

Biofilms grown on a non-permeable

support

Biofilms grown on a membrane in

MBR

Liquid flow

( : flow direction)

Bulk phase biofilm non-permeable support

Bulk phase biofilm membrane

Flux, J

Substrate

(–: concentration profile)

Bulk phase

CR

CS=0

biofilm non-permeable support

biofilmBulk phase membrane

CR

?

CP 0==CP 0==

Major solute transport mechanism Diffusion by concentration difference Convection by membrane flux

4. Experimental and theoretical determinationof bio-cake porosity in an MBR

4.1. Experimental method for cake porosity

Recently an experimental analytical methodfor the direct measurement of cake porosity wasdeveloped using a CLSM and image analysis tech-nique [15]. They prepared a suspension of 0.1-μmof fluorescent polystyrene latex beads and thenmicrofiltration of the suspension was carried outto form a cake layer whose porosity was mea-sured using CLSM and an image analysis tech-nique. To validate this method, measured porosi-ties (εCLSM) under three different transmembranepressures (5, 20, 40 kPa) were compared withthose (εα) calculated from specific cake resistancesusing Carman–Kozeny equation (Table 2). Al-though εCLSM seems to be consistent with εα, t-testwas run on these data set to statistically examine

whether the two methods give the same valuewithin the random error. They concluded that thetwo methods are not significantly different at the99.9% confidence level and the CLSM analysisis suitable for the measurement of cake porosityat least in the pressure range of 0–40 kPa whichis typical operating condition in a submergedMBR.

4.2. Theoretical model for cake porosity

Park et al. [16] developed a theoretical modelincorporating the effect of cake collapse to esti-mate the porosity of a cake formed by the deposi-tion of fractal aggregates during submergedmicrofiltration. Using several parameters — in-cluding the floc size, fractal dimension, and trans-membrane pressure — they predicted the porosi-ties within (εintra

c) and between aggregates (εinter)

118 C.H. Lee et al. / Desalination 231 (2008) 115–123

Table 2Comparison of porosities determined from specific cake resistances and CLSM analysis

εα: cake porosity calculated from specific cake resistanceεCLSM : cake porosity determined by CLSM analysisn: compressibility

εα εCLSM Run No.

5 kPa 20 kPa 40 kPa n 5 kPa 20 kPa 40 kPa

A 0.921 (±0.041)

0.876 (±0.048)

0.852 (±0.062)

0.43 0.923 (±0.017)

0.868 (±0.026)

0.851 (±0.035)

B 0.941 (±0.034)

0.904 (±0.042)

0.873 (±0.060)

0.50 0.870 (±0.034)

0.898 (±0.031)

0.851 (±0.040)

C 0.930 (±0.026)

0.889 (±0.046)

0.865 (±0.045)

0.45 0.932 (±0.027)

0.892 (±0.022)

0.834 (±0.042)

D 0.827 (±0.035)

0.807 (±0.059)

0.727 (±0.020)

0.38 0.807 (±0.055)

0.807 (±0.017)

0.704 (±0.036)

E 0.904 (±0.039)

0.872 (±0.050)

0.864 (±0.054)

0.26 0.878 (±0.020)

0.878 (±0.008)

0.876 (±0.031)

F 0.920 (±0.025)

0.893 (±0.047)

0.878 (±0.052)

0.30 0.902 (±0.032)

0.893 (±0.009)

0.869 (±0.051)

G 0.902 (±0.019)

0.870 (±0.038)

0.849 (±0.038)

0.31 0.851 (±0.045)

0.839 (±0.014)

0.749 (±0.079)

to estimate the porosity of the collapsed cake un-der pressure. The model’s predictions were veri-fied experimentally and used to investigate how(a) the size and fractal dimensions of the aggre-gates in suspension and (b) the suction filtrationpressure affect the cake porosity (Fig. 1).

Simulations based on this model tell us that asthe floc size (da) in the cake layer becomes largerand larger, not only the cake porosity becomesgreater but also the cake layer becomes less com-pressible. It suggests that making microbial flocsas large as possible is required to get a bio-cakelayer with higher porosity even under compres-sion in the MBR.

4.3. Fossil architecture of bio-cake

Depending on the operating conditions for anMBR, a bio-cake on the membrane surface has

historical characteristics along its depth like a fos-sil. Thus each bio-cake layer could reflect the tem-poral changes in the broth environment. Hong etal. [17] investigated the membrane filtration char-acteristics in a submerged MBR with intermittentaeration under constant flux, where the same mi-crobial community is repeatedly exposed to al-ternating aerobic and anoxic conditions. Variousbio-cakes were formed under different oxygenconditions and each bio-cake was analyzed interms of porosity, and then compared for spatialand temporal changes in the architecture (Fig. 2).

As shown in Fig. 5b, the porosity profile ofbio-cake 4 is shifted left compared to that of bio-cake 2 throughout the depth. It is because bio-cake 4 was exposed to a greater TMP for an elon-gated duration compared with bio-cake 2. Com-paring bio-cakes 3 and 5 under the same dura-tion, but with a reversed DO regime, there was

C.H. Lee et al. / Desalination 231 (2008) 115–123 119

Fig. 1. Estimated porosities in cakes as functions of da and ΔP: (a) εinter; (b) εintrac (ΔP = 20, 40, 60, 80, 100 kPa; Df = 2.4;

dp = 0.1 μm; c = 0.25). da aggregate diameter, c packing coefficient.

0

0.1

0.2

0.3

0.4

0.5

0.6

0 100 200 300 400

εinter (drel =1)

εinter (estimated) ΔP=20 kPa

ΔP=100 kPa

da (μm)

ε

0

0.1

0.2

0.3

0.4

0.5

0.6

0 100 200 300 400

εinter (drel =1)

εinter (estimated) ΔP=20 kPa

ΔP=100 kPa

da (μm)

ε

(b)

(a)

little difference in the porosity (Fig. 2c). This sug-gests that the sequence of the DO regime is not socritical for the bio-cake architecture, i.e., the orderof the DO condition has little impact on that. Con-sequently, they concluded that compression is themost important factor determining temporal andspatial variation in the bio-cake architecture.

4.4. Temporal variation in bio-cake architecture

Subcritical flux operation is preferred to main-tain sustainable flux and to avoid severe mem-

brane fouling in an MBR. However, extended fil-tration even at the subcritical flux usually givesrise to a two-stage increase in TMP, i.e., a slowand gradual TMP rise followed by a rapid TMPrise (Fig. 3). Several mechanisms have been pos-tulated for the rapid TMP rise at constant fluxoperation [18]. However, most of mechanisms donot seem to account for biological effect, but seemto rely on speculation and not scientific measure-ments, when the ratios of live to dead cells weremonitored along the depth of the bio-cake as afunction of operating time, however, the ratios be-

0.2

0.4

0.6

0.8

1

0 100 200 300 400

εintra

εintrac (estimated) ΔP=20 kPa

ΔP=100 kPa

da (μm)

ε

0.2

0.4

0.6

0.8

1

0 100 200 300 400

εintra

εintrac (estimated) ΔP=20 kPa

ΔP=100 kPa

da (μm)

ε

120 C.H. Lee et al. / Desalination 231 (2008) 115–123

Fig. 2. Porosity profiles along the bio-cake depth. Bio-cake 1 – formed only under aerobic conditions for 36 h, Bio-cake 2— formed only under anoxic conditions for 36 h; Bio-cake 3 — formed under aerobic conditions for 36 h and anoxicconditions for another 36 h (total 72 h); Bio-cake 4 — formed only under anoxic conditions for 72 h; Bio-cake 5 —formed under anoxic conditions for 36 h and aerobic conditions for another 36 h (total 72 h).

0

10

20

30

40

50

60

0.3 0.4 0.5 0.6 0.7 0.8 0.9 10

10

20

30

40

50

60

0.3 0.4 0.5 0.6 0.7 0.8 0.9 10

10

20

30

40

50

60

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1Porosity Porosity

Cake 2Cake 1

Cake 3

(n = 8)

Cake 2Cake 4

(a) (b)

Cake 3Cake 5

Porosity(c)

0

10

20

30

40

50

60

0.3 0.4 0.5 0.6 0.7 0.8 0.9 10

10

20

30

40

50

60

0.3 0.4 0.5 0.6 0.7 0.8 0.9 10

10

20

30

40

50

60

0.3 0.4 0.5 0.6 0.7 0.8 0.9 10

10

20

30

40

50

60

0.3 0.4 0.5 0.6 0.7 0.8 0.9 10

10

20

30

40

50

60

0.3 0.4 0.5 0.6 0.7 0.8 0.9 10

10

20

30

40

50

60

0.3 0.4 0.5 0.6 0.7 0.8 0.9 1Porosity Porosity

Cake 2Cake 1

Cake 3

(n = 8)

Cake 2Cake 4

(a) (b)

Cake 3Cake 5

Porosity(c)

came smaller, specially at a lower layer of bio-cakes corresponding to points 3 and 4 than at thoseto points 1 and 2 (Fig. 4).

As the bio-cake accumulates on the surface ofthe membrane, endogenous decay or cell-lysis atthe bottom layer would be expected to occur due

Fig. 3. Profile of TMP rise-up at constant flux in an MBR.

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30 35 40Time (days)

TMP

(kPa

)

Point 2 Point 2

Point 3 Point 3

Point 4 Point 4

Point 1Point 1

Flux = 13 l/mFlux = 13 l/m22hh

0

10

20

30

40

50

60

70

80

0 5 10 15 20 25 30 35 40Time (days)

TMP

(kPa

)

Point 2 Point 2

Point 3 Point 3 Point 3 Point 3

Point 4 Point 4

Point 1Point 1

Flux = 13 l/mFlux = 13 l/m22hh

to poor oxygen and substrate transfer. It gave riseto the excretion of EPS (polysaccharides and pro-teins) which could reduce the porosity of the bio-cake layer in terms of polysaccharide and protein(data not shown). Jumping of their concentrationsin the bio-cake layer between point 2 and 3 coin-

Bio

-cak

e th

ickn

ess (

μm)

C.H. Lee et al. / Desalination 231 (2008) 115–123 121

Fig. 4. Distribution of live/dead ratio along the depth of the bio-cake.

0 5 1015

1015202530354045

Dep

th o

f bio

cake

[μm

]

50

0 5 1015

101520253035404550Point 1Point 1

TMP 6TMP 6(8 days)(8 days)

0 5 1015

101520253035404550

0 5 1015

101520253035404550Point 2Point 2

TMP 8TMP 8(26 days)(26 days)

Point 3Point 3TMP 20TMP 20

(32 days)(32 days)

Point 4Point 4TMP 70TMP 70

(38 days)(38 days)

Live/dead ratio Live/dead ratio Live/dead ratio Live/dead ratio0 5 10

15

1015202530354045

Dep

th o

f bio

cake

[μm

]

50

0 5 1015

101520253035404550Point 1Point 1

TMP 6TMP 6(8 days)(8 days)

0 5 1015

101520253035404550

0 5 1015

101520253035404550Point 2Point 2

TMP 8TMP 8(26 days)(26 days)

Point 3Point 3TMP 20TMP 20

(32 days)(32 days)

Point 4Point 4TMP 70TMP 70

(38 days)(38 days)

Live/dead ratio Live/dead ratio Live/dead ratio Live/dead ratio

cided with the live/dead ratio. Such temporalchanges in biological properties and architecturesat the bottom layer of the biocake should be inclose association with the rapid TMP rise in Fig. 3.

4.5. Spatial variation in the bio-cake architecture

The bio-cake architecture would be greatlychanged along the length of submerged hollowfibers because transmembrane pressures along thefiber length would be different. It is important toinvestigate the spatial variation of the bio-cakearchitecture because it could provide informationon how to design a membrane module or air-scour-ing system to improve flux. Chemical additiveshave been used to reduce membrane biofouling[19,20]. Hwang et al. [21] and Lee et al. [22] re-ported how membrane fouling reducer (MFR)mitigated membrane biofouling in an MBR witha particular focus on changes that occurred in thebio-cake architecture. They ran two MBRs withand without MFR, respectively, and measuredlocal porosities of bio-cakes along the length ofthe membrane fibers. Three fibers were cut fromthe used hollow fiber modules and each fiber wasagain cut into six parts having the same length of2.5 cm (Fig. 5) for staining and CLSM. The po-

rosity of the MFR bio-cake was higher than thatof the control bio-cake at all locations (Fig. 6). Inboth reactors, bio-cake porosities near the pottedends (S1 and S2) were lower than those near thefree-moving ends (S3–S6) because the bio-cakeformed on the potted ends was more easily com-pressed by direct suction pressure. With the addi-tion of MFR, not only the porosities were in-creased but also the spatial distributions of po-rosities became more uniform along the length ofthe fibers.

5. Conclusion

Formation of a bio-cake on the membrane inan MBR has been known to be one of the keyobstacles to be solved to enhance the performanceof the MBR. It has still remained as a black-boxdue to its complexity and heterogeneity and thusit seems nearly impossible to completely preventor eliminate the bio-cake at this time. Using mod-ern molecular biology and instrumental analyti-cal methods, however, the black-box of the bio-cake could be revealed little by little. More thor-ough and ceaseless investigation from the molecu-lar biological point of view is needed to elucidate

122 C.H. Lee et al. / Desalination 231 (2008) 115–123

Fig. 5. Specification of the hollow fiber module and sampling points for bio-cake specimens (3 fibers × 6 specimens/fiber× 1 image/specimen).

Potted end

Cut

15 cm 2.5 cm

S1

S2

S3

S4

S5

S6

Moving end

Potted

Moving

Aeration

Permeate

Fig. 6. Local changes in the bio-cake porosity along themembrane fiber length in an MBR.

0.6 0.7 0.8 0.9

Porosity

Posi

tion

on m

embr

ane

ControlMFR

S6

S5

S4

S3

S2

S1

its formation mechanisms and ultimately to findefficient control methods.

Acknowledgments

This was supported by the Korea Science andEngineering Foundation (KOSEF) grant fundedby the government (MOST) (No. RoA-2007-000-20073-0). The authors thank the National Instru-mentation Center for Environment Management(NICEM) for the use of the confocal laser scan-ning microscope system.

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