Water Research 89 (2016) 132e141

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Influence of COD:N ratio on sludge properties and their role in
membrane fouling of a submerged membrane bioreactor
L. Hao a, S.N. Liss b, B.Q. Liao a, *
a
b

Department of Chemical Engineering, Lakehead University, 955 Oliver Road, Thunder Bay, ON P7B 5E1, Canada
School of Environmental Studies and Department of Chemical Engineering, Queen's University, Kingston, ON K7L 3N6, Canada

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 21 August 2015
Received in revised form
30 October 2015
Accepted 22 November 2015
Available online 2 December 2015

The effect of COD:N ratio on sludge properties and their role in membrane fouling were examined using a
well-controlled aerobic membrane bioreactor receiving a synthetic high strength wastewater containing
glucose. Membrane performance was improved with an increase in the COD/N ratio (100:5e100:1.8) (i.e.
reduced N dosage). Surface analysis of sludge by X-ray photoelectron spectroscopy (XPS) indicates significant differences in surface concentrations of elements C, O and N that were observed under different
COD/N ratios, implying changes in the composition of extracellular polymeric substances (EPS). Fourier
transform-infrared spectroscopy (FTIR) revealed a unique characteristic peak (C]O bonds) at 1735 cm1
under nitrogen limitation conditions. Total EPS decreased with an increase in COD/N ratio, corresponding
to a decrease in the proteins (PN) to carbohydrates (CH) ratio in EPS. There were no significant differences in the total soluble microbial products (SMPs) but the ratio of PN/CH in SMPs decreased with an
increase in COD/N ratios. The results suggest that EPS and SMP composition and the presence of a small
quantity of filamentous microorganisms played an important role in controlling membrane fouling.
© 2015 Elsevier Ltd. All rights reserved.

Keywords:
COD:N
Membrane bioreactor
Membrane fouling
Industrial wastewater
Extracellular polymeric substances
Sludge properties

1. Introduction
Integration of membrane technologies with conventional
biologically-based wastewater treatment has been widely applied
to the management of municipal and industrial wastewaters
providing a direct solideliquid separation by membrane filtration.
Compared to the conventional activated sludge process, membrane
bioreactors (MBR) offer several advantages including superior
effluent quality as well as a smaller footprint (Brindle and
Stephenson, 1996; Visvanathan et al., 2000; Rosenberger and
Kraume, 2003; Marrot et al., 2004). However, membrane fouling
which leads to poor membrane performance and high operational
costs (Kraume and Drews, 2010; Judd, 2011), continues to be a
major challenge that limits the further development and widespread application of MBRs.
In general, membrane fouling is caused by many factors,
including influent characteristics, solid retention time (SRT), hydraulic retention time (HRT), organic loading rate (OLR), and dissolved oxygen levels (Kraume and Drews, 2010). Among these
parameters, COD:N:P ratios remain one of the most important

* Corresponding author.
E-mail address: bliao@lakeheadu.ca (B.Q. Liao).
http://dx.doi.org/10.1016/j.watres.2015.11.052
0043-1354/© 2015 Elsevier Ltd. All rights reserved.

factors. This is largely because COD:N:P ratio can influence the
physiological properties of microorganisms and chemical compositions of biomass in MBRs. The COD:N:P ratio also can influence
the amount of extracellular polymeric substances (EPS) and the
composition of proteins (PN) and carbohydrates (CH) in EPS which
can affect membrane performance.
Considerable attention has been given to the effect of COD:N
ratio in conventional biological processes for municipal wastewater
treatment on sludge properties including settleability (Durmaz and
Sanin, 2003), filterability (Wu et al., 1982), and flocculation and
dewatering (Sanin et al., 2006). Other studies have focused on the
effect of COD:N ratios on nitrification and denitrification processes
as well as nutrient removal efficiency (McAdam and Judd, 2007;
Meng et al., 2008; Hwang et al., 2009). Studies found that EPS
production increased with an increase in the COD:N ratio in
municipal wastewater treatment (Durmaz and Sanin, 2001;
Miqueleto et al., 2010; Feng et al., 2012).
To date studies have largely focused on the effect of COD:N ratio
in conventional activated sludge processes, and mainly focused on
N removal in municipal wastewater treatment. There have only
been a few reports on the effect of COD:N:P ratio on biomass
properties and membrane fouling in submerged membrane bioreactors for municipal wastewater treatment with excess amount

Soluble microbial products (SMP) measurement SMP means the soluble EPS. Finer air aeration (2.49 1.26 0. Bound extracellular polymeric substances (EPS) extraction and measurement The bound EPS from sludge suspensions samples was extracted according to cations exchange resin (CER) (Dowex Marathon C. as shown in a previous publication (Hao and Liao. (Lowery et al. including C.. 7268 ± 289 and 6363 ± 181 mg/L under a COD:N:P ratio of 100:5:1. China). 1997.L. 2005).1.8 L/ min (LPM)) were installed under the membrane module for aeration as well as air scouring to limit membrane fouling. respectively (Hao and Liao.8:1) on sludge properties and their role in membrane fouling of an aerobic MBR for high strength industrial wastewater. On the other hand. The trans-membrane pressure (TMP) was monitored by a pressure gauge connected between the membrane module and the suction pump. Supernatant COD was determined after centrifuging the mixed liquor for 20 min at 18.2 by a pH regulation pump using 0.44 0. During the 133 Table 1 Composition and concentration of micronutrients in the feed. 100:2. Model S47030) located at the bottom of the reactor.4. The flat-sheet membranes were made of polyvinylidene fluoride (PVDF).2. 1998. stored at 4  C. The paper describes the effect of three different COD:N:P ratios (COD:N) (100:5:1100:2. The operational cycles were applied with 4 min suction followed by 1 min relaxation. N. The mixed liquor suspended solids (MLSS) concentration under steady-state operation was maintained at 8450 ± 325.700 g for 20 min at 4  C. treatment. 2011).. the reactor was fed with COD:N:P ratio of 100:2.. 100:1. 1996). Ltd. had a 6. microbial floc structure. USA). 2. simulating the food industry wastewaters. 100 mL sample of the sludge suspension was taken and centrifuged (IEC MultiRF. Needham Heights.25 0. nitrogen (NH4Cl) and phosphorus (KH2PO4) in a proportion of chemical oxygen demand (COD): N: P ¼ 100:5:1.000 Da. Thermo IEC. permeate and mixed liquor were sampled periodically from the system. The COD and MLSS were analyzed according to Standard Methods (APHA. Synthetic wastewater containing glucose has been widely as a receipt of laboratory-scale biological wastewater treatment to develop fundamental understanding of microbial kinetics. 2. Barnant Co.2.. Liao et al.45 mm membrane filters (Millipore) and used to estimate SMP. Ammary.1. Immersed coarse air bubble diffusers (3. Beverly. (DuBois et al. and O) on sludge surfaces. surface concentrations of elements (C.0 ± 0. Optimizing nutrient addition not only improves process efficiency but also saves chemical costs and reduces secondary pollution of treated effluent with added nutrients.. The sludge was then transferred to an extraction beaker filled with buffer and the CER (80 g/g-MLSS) and mixed for 2 h at 4  C. / Water Research 89 (2016) 132e141 of nutrients. 1951) and DuBois et al. Surface composition of sludge by X-ray photoelectron spectroscopy (XPS) The surface concentrations of elements. 1956).2. in Phase 2 (108e240 day). Mechanical mixing was achieved using a magnetic stirrer (Thermolyne Cimarec. The synthetic wastewater included glucose.5:1 and 100:1. 4 mM NaH2PO4.0 L working volume and contained flat-sheet microfiltration membranes (SINAP Membrane Science & Technology Co. USA) as well as a controller (Flowline. 2.2. extracellular polymeric substances (EPS). soluble microbial products (SMP) formed.6 LPM) was also used to provide additional oxygen to maintain satisfying dissolved oxygen (DO) level large than 2 mg/L during the aerobic period. in Phase 3 (240e380 day) the MBR was operated at nutrients ratio (COD:N:P) of 100:1. An instant membrane flux of 10 L/m2 h was applied in this study. The pH was monitored by a pH electrode (Thermo Scientific.07 2. USA). Measurements for PN and CH of SMP were as described above. O.2.41 operation of the bioreactor. Henze et al.. The total bound EPS was represented by adding the concentrations of bound CH and PN.31 0. a volume of 400 mL of sludge was discharged daily from the MBR for bulk sludge characterization and to maintain the SRT at 15 days. 9 mM NaCl and 1 mM KCl at pH 7. PA) method (Frølund et al.5:1. the MBR was fed at a COD:N:P ratio of 100:5:1. Stable operation was achieved after three SRTs operation following moving to each nutrient regime.. USA) at 18.8:1. the role of nutrients and COD:N ratios is most important for industrial wastewaters where nutrients (N and P) are added and required to support microbial growth and biodegradation (Tchobanoglous and Burton. 2.3 mm and molecular weight cut-off (MWCO) of 70. SigmaeAldrich. 2015).25 M NaOH.3. All chemicals were from SigmaeAldrich at analytical grade. 2003). and N were examined by XPS.8:1 and trace metals as summarized in Table 1. MA). 2015). like food industry wastewater. The study included examination of membrane filtration resistance. The bound EPS was determined as the sum of bound PN and CH and was measured colorimetrically by the methods of Lowery et al. respectively. and automatically adjusted to 7.5 g COD L1 day1.5:1. biomass separation.43 0. Bellefonte. The total SMP was the sum of the concentrations of soluble CH and PN. 100:2. Components Concentration (mg/L) MgSO4 ∙ 7H2O FeSO4 ∙ 7H2O Na2MoO4 ∙ 2H2O MnSO4 ∙ 4H2O CuSO4 ZnSO4 ∙ 7H2O NaCl(mg/L) CaSO4 ∙ 2H2O CoCl2 ∙ 6H2O 5. 2. Analytical methods 2. and membrane fouling in MBRs (Kovarova-Kovar and Egli. Water quality measurement The influent synthetic wastewater. The extracted supernatant recovered from mixed liquor following centrifugation as described above was filtered through 0. The wet sludge samples were freeze-dried . The reactor was operated at an HRT of approximately one day and an OLR of 2.8:1. The effluent was obtained by means of a suction pump connected to the membrane module. MA.25 0. The temperature of the bioreactor was maintained constant at 35 ± 1  C by means of water jacket. The system was continuously fed with a synthetic high strength wastewater containing glucose. The influent COD was approximately 2500 mg/L. The feed was pumped automatically by a peristaltic pump (Masterflex Model 7520-50. The operation of the reactor system included three phases: Phase 1 (0e107 day). and had a pore size of 0. and the abundance of filamentous microorganisms under different COD:N ratios.. Hong et al. 2002. Naþ form.700 g.. which was controlled by a liquid level sensor (Madison Co. Shanghai. The sludge pellets were re-suspended to their original volume using a buffer consisting of 2 mM Na3PO4. Hao et al. Material and methods 2.5:1. and 100:1. Experimental set-up and operating conditions The laboratory-scale submerged MBR system. 1991.2.

H) bonds) from lipids and amino acids side chains at a binding energy of 284. C1sC) were decomposed into four different bonds: (1) C bound only to C and H (C(C. Assessing the impact of nutrient conditions (i. N). Results The MBR was operated at three different COD:N:P ratios. 2008). and hemiacetal (CeOeC). T is the permeate temperature in  C. In order to prevent the structure and thickness of cake layer from change.05). 2. according to the classification of Jenkins et al. 3. and hT is the permeate dynamic viscosity (Pa s)..8:1.. 1 illustrates the typical morphology of sludge flocs under different COD:N ratios. including hydroxide (CeOH). The FTIR analysis was performed using absorbance mode. at a binding energy of 532. The student t-test also was applied to analyze the content of surface chemical composition of bulk sludge. Rf is fouling resistance due to irreversible adsorption and pore blocking (m1).0 eV (C1sC) arises from O]CeOH and O]C-OR. during the period close to the end of stable operation of each COD:N ratio. and O1sB) could be attributed to three bonds: OeC bond.2. COD removal efficiency of the MBR was only slightly decreased when N was reduced (COD removal ranged from 98. the C peaks (C1s. and amide. and (4) a weak peak at 289. 103. Morphology of sludge and textual of cross-section of fouled membranes Sludge samples were routinely taken out from the MBR and viewed for morphology under a conventional optical microscope (COM) (Olympus IX 51 Inverted Microscope. 2. Tokyo. at a binding energy of 286.5:1.8:1. Grinstead. in order to maintain the same membrane conditions for comparison and reproducibility of results in repeated membrane operation cycles at each nutrient (COD:N) ratio. USA) was employed to determine the major organic functional groups of freeze-dried sludge and to predict the chemical functional groups of the bulk sludge. Rt was calculated with the temperature corrected to 20  C to compensate for the dependence of viscosity on temperature.5:1. 100:2.. Surface characterization of sludge by XPS The surface chemical composition of bulk sludge at different COD:N:P ratios were analyzed by XPS. / Water Research 89 (2016) 132e141 at 35  C for one week.2. UK). NY) at a magnification of 40.2.7 eV. The samples for FTIR were from the same batches of samples for XPS.8:1 for over one year. (3) C bound to O making two single bonds or one double bond. Billerica. 1997. High resolution spectra were taken of C1s regions (PE25 eV).. When fouling occurred. 1996.2. 2008).3. and then the membrane was submerged in tap water for flux and TMP measurement. Fig. Hao et al. Sludge yield was reduced and effluent quality with respect to nutrient residuals improved indicating the feasibility of operating an aerobic MBR for high strength wastewater treatment with less nutrient addition while achieving good biological treatment (Hao and Liao. including bacteria.8 eV (C1s). and 100:5:1 and 100:1. The freeze-dried sludge samples were ground to a powder before being analyzed by a ThermoFisher Scientific K-Alpha XPS Spectrometer equipped with monochromatic AlK a X-ray source with a spot source of 400 mm (ThermoFisher. 2. O1sA. 100:5:1100:2.0 eV (C1sB). commonly found in carboxyl or ester groups (Badireddy et al. 2. on sludge properties and membrane fouling is equally important and was the subject of this study. carbonyl. carboxylate. Statistical analysis Statistical analysis was conducted using the Statistical Package for the Social Science (SPSS) 16. New membranes were used in each membrane operation cycle. 3. Rm is intrinsic membrane resistance and evaluated by the water flux of tap water. The paired p values were calculated for the differences between COD:N:P ratios of 100:5:1 and 100:2..6. Japan). Melville. and each peak corresponds to electrons with a characteristic binding energy from a particular element (Dengis and Rouxhet.8 (moderate level 3-4). and 100:1. Data sets were considered statistically different at a 95% confidence interval (p < 0.7. The position of the energy scale was adjusted to place the main C1s feature (CeC) at 285. C(O. membrane surfaces were wiped with a sponge and tap water.5:1 and 100:1. C1sA. E. Olympus America Inc. A survey spectrum was taken at low resolution (PE 150 eV).5%) (Hao and Liao.5. acetal. J represents the permeate flux (m3/m2h).0. carbonyl and amide at a binding .85% NaCl aqueous solution and then frozen at 22  C before being fixed on to a sample stage using optimal cutting temperature (O. C1sB.. The difference was considered statistically significant at a 95% confidence interval (p < 0.134 L. 3.0 eV except for those samples where the CeO peak was more dominant.T. A cross section of the cake layer was observed by a COM to investigate the physical structure of sludge cake layers formed on membrane surface. 2015). Ltd. Three sludge samples were collected at three different days. The experimental procedure to determine each resistance value is as follows: Rt is total membrane resistance (m1) and calculated from the filtration data at the end of operation.C) compound (Sakura Finetechnical Co. 2015). Badireddy et al. Floc size and morphology The morphology of sludge flocs was observed throughout the study using a COM. Dufrene et al. Sludge flocs at a COD:N ratio of 100:5 had a lower level (0-1) of filamentous microorganisms and were smaller in sizes than that at a COD:N ratio of 100:2. Membrane resistance The extent of fouling rate can be evaluated by the derivative of the filtration resistance: Rt ¼ DP ¼ Rm þ Rf þ Rc J  hT hT ¼ h20  C $e0:0239ðT20Þ (1) (2) where Rt is the total filtration resistance (m1). 2. COD:N) when added to achieve biological treatment. (2003). All data processing was performed using the software (Advantage) provided with the instrument. ester. Molecular composition of sludge by Fourier transforminfrared spectroscopy (FTIR) A Bruker Ten 37 FTIR Spectrometer (Bruker Optics Inc.5 and 100:1. alcohol. The O peaks (O1s. ester. at a binding energy of 288. and hemiacetal. under each singly COD:N ratio condition. Charge compensation was also provided. XPS has been used to study the surface functional groups of materials. acetal. C] O or OeCeO.05).4% to 99.3 eV (C1Sa). DP is the trans-membrane pressure difference (Pa). the cake layer was saturated with 0.1. and O]C in carboxylic acid. including amide. As shown in Fig. Rc is fouling layer resistance (m1). amine. carboxylate.e. MA. An analysis of variance (ANOVA) was employed to identify whether there is significant difference between treatment means when evaluating membrane fouling and EPS concentration under different COD:N ratios. The sludge samples were freeze-dried at 35  C for one week prior to analysis. (2) C singly bound to O or N. including ether.

P > 0. 100:2. SMP The soluble microbial products (SMPs) are defined as biopolymers released from bacteria into solution. 2006). which are biopolymers attached on bacteria surfaces. 5. P > 0.5 and (c) 100:1.10) mg/L at the COD:N:P ratio of 100:5:1.10 eV (N1sA). are reflecting CeH bonds in the alkenes class (Kim and Jang. 4.5:1 and 100:1.05). The total EPS were 35. The N peaks (N1s and N1sA) were attributed to the two different bonds: NeC bond in amide or amine at a binding energy of 400. (b) 100:2. Moreover.5(±3.5:1 and COD:N:P ¼ 100:1.8. PN.6 (±3. 3. The doublets at 2370 and 2343 cm1 attribute to carbon dioxide from atmosphere adsorbed on the sample surface.8:1. Surface concentrations of element C. 3(b)). The bound EPS contents at each COD:N:P ratio are shown in Fig. This corresponds to the change in PN/CH ratio in bound EPS as the COD:N ratio changed. Morphology of sludge flocs observed by COM under different COD:N ratios (a) 100:5. the general tendency of total N on sludge surface decreased with an increase in the COD:N ratio.5:1 and 100:1. 6 shows the changes of filtration resistance under different .8:1.05) at different COD:N:P ratios studied. p < 0. p < 0.05) reached to a peak of 32. respectively. The total SMP and soluble PN and CH concentrations at three COD:N:P ratios are shown in Fig.6. Furthermore. Membrane performance Fig. The PN/CH values (ANOVA. Statistical analysis using ANOVA also confirmed that differences in total EPS. p < 0. H). The asymmetrical stretching peak (C]O stretch) observed at 1735 cm1 was associated with O-acetyl ester bonds (Badireddy et al. All three sludge samples shown peaks at 3300 cm1 (overlapping of bands from the stretching vibrations of NeH and OeH) (Kumar et al. the content of PN and CH of bound EPS in sludge were measured.12 (±2. 3.8:1. In order to understand the role of bound EPS in membrane fouling under reduced N dosage in industrial wastewater treatment. although no statistically significance was observed (ANOVA.5. The amount of SMP (ANOVA. while the biomass produced similar CH level in SMP at COD:N of 100:5 and 100:2. p < 0. Peaks. N and O on sludge surfaces under different nutrients conditions (COD:N) were summarized in Table 2. p < 0.05).8.8:1.05).08). energy of 531. / Water Research 89 (2016) 132e141 135 Fig. But student t-test suggested significant difference in total N on sludge surface existed between the COD:N ratio of 100:5 and 100:1.3.5:1.. After decreasing nitrogen content in the feed. At the COD:N ratio of 100:1.4.05). Obviously. Bound EPS production and components 3. 2006).05). Although there was no significant difference in total C (Student t-test. C-(O.8:1 (Student t-test. significant differences in the quantity of C-(C. Hao et al. p < 0.L. the SMP production presented a decrease trend for other two conditions. N) and C]O were observed between COD:N:P of 100:5:1 and 100:2. the CH concentration in SMPs was almost twice of CH concentrations produced at the other two COD:N ratios. The FT-IR spectra under different nutrients (COD:N) conditions are shown in Fig. P < 0. the total O on sludge surface increased with an increase in the COD:N ratio (reduced N dosage) (Student t-test. CH were found as the predominant fraction and accounted for more than half of the SMP concentration at COD:N:P of 100:1. 2008). under a nitrogen shortage of COD:N:P ¼ 100:2.. CH were all statistically significant (ANOVA. 1. the PN content decreased with a decrease in the nitrogen content in feed.4 eV. 100:2.21 (±1.12 Ev (N1s) and NeH bonds in ammonia or protonated amine at a binding energy of 402. An increase in COD:N ratio led to a decrease in total bound EPS production but more carbohydrates in bound EPS. at wave numbers of 2926. 19. 3. FTIR analysis Bound EPS is defined as the biopolymers originally from cells attached on the surface of flocs or cells.8 (student t-test.05) showed the similar trend as shown in bound EPS in that PN/CH ratio decreased with an increase in the COD:N ratio. The PN/CH values (ANOVA. 3.05) decreased with an increase in the COD:N ratios (Fig. 100:5:1 and 100:1.5. as compared to bound EPS.04) and 15. 2847 and 1445 cm1.22) mg/g MLSS for COD:N:P ratios of 100:5:1.

66 7. value shown in parentheses. 347 and 352 for COD:N 100:1.328) Y (0.15 7.85 0.289) N (0.063) Y (0.005) N (0. During acclimation (day 1e14) (COD:N ¼ 100:5).125) Y (0. (c) N1s spectra.8). Table 2 Surface composition of sludge under different COD:N:P ratios determined by XPS: average atom fraction (%).80 0.003) Y (0. As illustrated in Fig.03 3.29 ± ± ± ± ± ± ± 0.090) Y (0.002) Y (0. (a) whole spectra.8:1 N (0. 6. The black region of TMP from day 115 to day 129 was caused by the overlap of TMP lines.09 29.59 64.8:1 100:5:1 and 100:1.H) Ce(O.51 24.113) Y (0. Similarly. Sample number n ¼ 3 for each condition.019) N (0.88 26. In order to recover membrane performance.00 100:5:1 a ± ± ± ± ± ± ± Significant differencea of COD:N:P ratio 100:2. (b) C1s spectra.46 6.8:1 62.001) Y (0.61 0.59 0. regimes of COD:N: P ratios studied.30 0.42 4.41 28. Day 218. The sudden increase in filtration resistance can lead to failure in membrane performance. and Day 339.88 1.42 1.52 0. / Water Research 89 (2016) 132e141 Fig.5 and 100:1.53 ± ± ± ± ± ± ± 1.044) Sig. TMP was unstable.54 0.33 4.53 0.03 25.89 33.044) N (0.5. significant deterioration of membrane performance at the early stage of COD:N of 100:2.96 0.73 0.99 26.31 1.5:1 and 100:1.98 1. A relatively stable and low filtration resistance was characteristic of an initial stage in the operation of the MBR and was followed by a second stage where there was a sudden increase in filtration resistance. At least three repeated membrane operation cycles were conducted under each COD:N ratio to check the reproducibility of the membrane performance.017) N (0.09 19.100) Y (0.5 was observed at days 115e129.N) C]O O]CeOH Total O Total N 64. (Sample number n ¼ 3 under each COD:N condition.74 6.039) N (0. a COD:N:P of 100:5:1 resulted in a more rapid membrane fouling rate or shorter period in stage 1 before a rise in filtration resistance when compared to other higher COD:N ratios tested (100:2.136 L. 224 and 229 for COD:N 100:2. and (d) O1s spectra.5:1 1.49 1.5:1 100:2. This result suggests that an increase in COD:N ratio (reduced N dosage) led to an improved membrane performance. XPS spectra of mesophilic sludge.09 100:1. physical cleaning or chemical cleaning is typically employed at the end of the filtration cycles.34 100:5:1 and 100:2.35 9. Hao et al.006) Y (0.141) Y (0.82 1.90 0.8).49 0. 87 and 90 for COD:N 100:5.28 4. sample dates: Day 74. due to TMP jumps of serious .000) Y (0. 2.027) N (0. Element component COD:N:P ratio Total C Ce(C.067) N (0.41 24.

224 and 229 for COD:N 100:2.8). Day 218. Fig. P) deficiency in influent would promote the growth of filamentous microorganisms in activated sludge (Jenkins et al. The purpose of measuring the filtration resistance was to identify the dominant fouling mechanism.7. Hao et al. sludge flocs had irregular shapes containing different level of filaments and changed in floc sizes. 347 and 352 for COD:N 100:1. and Day 339. 3. The cake layer thickness at COD:N 100:5 was usually thinner than that at COD:N 100:5 and 100:1. where there was likely better air scouring condition. p < 0. due to the fact that different ending TMP was used. sample dates: Day 74. 7 illustrates the thickness of wet cake layers formed on membrane surfaces There was variability with respect to the thickness of the cake layer over the surface of the membrane. 4..L. p < 0. A comparison of the filtration resistances at the end of an operation cycle among COD:N ratios studied is not reasonable. The cake layer thickness ranged from between 100 and 300 mm under the different nutrients (COD:N) conditions studied. Thus. membrane fouling. Fig.05. FTIR spectrum of bulk sludge under different COD:N:P ratios (sample number n ¼ 3 under each COD:N ratio.8 at the end of an operation cycle. number of measurements: n ¼ 5 for each condition). (a) SMP concentrations and (b) PN/CH in SMPs under different COD:N:P ratios (ANOVA. 3. 87 and 90 for COD:N 100:5. under different COD:N ratios could be explained by our general knowledge that nutrients (N. A thinner cake layer was usually observed at the center of membrane module. 1. Recovery cleaning was performed by physical cleaning using water with a wet sponge over the surface of the fouled membrane to reinstall membrane performance. number of measurements: n ¼ 5 for each condition). 5. / Water Research 89 (2016) 132e141 137 Fig.5. The changes in the abundances of filamentous microorganisms and floc sizes Fig. a moderate higher level (3-4) of filamentous microorganisms was observed under N deficiency . are shown in Table 3. Discussion As shown in Fig. 4. Comparison of (a) bound EPS of bulk sludge and (b) PN/CH in EPS under different COD:N:P ratios (ANOVA. obtained for each of the COD:N regimes studied.05. Fouling resistance Membrane filtration resistances evaluated at the end of an operation cycle. 2003). The value of Rc/(Rc þ Rf) (100%) indicates the formation of a cake layer was the dominant mechanism of membrane fouling in the MBRs.

Table 3 A series of resistances at the end of the study point of membrane cyclic filtration. Cross section of cake layer observed by COM under different COD:N ratios (a) 100:5. under the conditions of reduced growth due to N limitations. number of measurements: n ¼ 2 for each COD:N:P condition.8.07)  1012 (6. / Water Research 89 (2016) 132e141 Fig.10)  1012 (4.18)  1012 0 0 0 (8.5 and (c) 100:1.8:1 Resistances Cake resistance ratio (%) Rm Rc Rp Rt Rc/(Rc þ Rp) (1.71 ± 0. it is well-known that filamentous microorganisms could serve as a backbone for sludge floc formation and thus increase floc sizes (Jenkins et al.08)  1012 (6.53 ± 0. Furthermore.11)  1012 100 100 100 Sample average ± relative error.80 ± 0.01)  1012 (2..5:1 100:1.82 ± 0.02)  1012 (1.33 ± 0. 7. conditions.81 ± 0. (b) 100:2. 6.07)  1012 (1. a significant . Moreover.32 ± 0.52 ± 0.138 L. Hao et al. Membrane filtration resistance over time under different COD:N:P ratios (the recorded TMP pattern between days 115 and 129 is due to high membrane fouling rates observed in this time period).15 ± 0. 2003). Fig. Nutrients COD:N:P 100:5:1 100:2.01)  1012 (4.

This observation is consistent with previous findings (Durmaz and Sanin. The XPS results strongly suggest there are significant difference in bound EPS composition and the properties of the surface under different COD:N ratios... Thus. 2002). Thus. However. Wang et al.. 1997.. 2002). SMP might .. less membrane fouling was observed under N deficiency conditions.b). (2006b) in that the presence of a small quantity of filamentous microorganisms had a positive impact on membrane permeation (less fouling). Dufrene et al. A change in bound EPS composition and concentration would modify the interactions between floc surface and membrane surface or between floc surface and fouling layer surface and thus affect cake layer formation rate and structure. like C. 2008. Lin et al. Omoike and Chorover.8) might also explain the better membrane performance under these conditions. The higher content of Ce(C. It is the specific composition (like PN and CH) of bound EPS. as smaller flocs have a higher tendency to attach to the membrane surface and contributing to membrane fouling at a COD:N ratio of 100:5. a more broaden definition of EPS was introduced to include both bound EPS and soluble EPS (Laspidou and Rittmann. Membrane filtration performance of sludge is closely associated with sludge morphology and chemical/physical characteristics. The results from this study is consistent with the finding of Meng et al. polysaccharides. At the lowest COD:N:P ratio of 100:5:1. 2001. 2015).. (2000) found an increase in PN/CH in bound EPS correlated to a higher magnitude of negative surface charge and thus inhibited bioflocculation. Proteins in the activated sludge bound EPS are the primary source for the elemental nitrogen. It is well known that the bound EPS consist of proteins.. This observation is in consistent with previous studies (Lin et al. and the abundance of filamentous microorganism) is an important factor in controlling membrane fouling. The bound EPS extraction and chemical analysis results are consistent with the results of XPS as discussed above in that significant difference in bound EPS composition and concentration existed under different COD:N ratios. Another possible explanation would be based on the potentially significant difference in microbial communities under different COD:N ratios. 2013. Potential correlations between microbial communities and EPS/SMP production needs further studies using advanced molecular tools in the future to develop new insights in membrane fouling. Earlier XPS studies (Dengis and Rouxhet. a higher PN/CH ratio in bound EPS at COD:N 100:5 would enhance cake layer formation thus corresponding with greater membrane fouling. the cake layer porosity increased (Cao et al. 2006b). H) bonds might originate from lipids or from amino acid side chains. 2004). as reflected by the change in PN/CH ratio. floc size. that determines the specific interactions (like hydrophobic interaction. and sludge filtration performance directly affects the trans-membrane pressure (TMP). After reducing the nitrogen content in the feed (increased COD:N ratio). This is due to the fact that a large portion of feed COD was converted into storage compounds like CH in bound EPS under N deficiency conditions. 2003). / Water Research 89 (2016) 132e141 portion of feed COD would be converted into storage compounds as reflected by the higher level of CH in bound EPS. Badireddy et al. A possible explanation is that the filaments will serve as a net and form large floc and thus lead to a less dense or more porous cake layer (Cao et al. This might not be surprising that microorganisms responding to the stress of nutrient (N) deficient would produce different biopolymers (Omoike and Chorover. 2014). nucleic acids. Therefore. the results of XPS analysis and bound EPS extraction and chemical analysis are related. 2004. van der Waals interaction.8 (Fig.. EPS are generally considered as “extracellular polymeric substances of biological origin that participate in the formation of microbial aggregates” (Geesey. Although the cake layer thickness (100e150 mm) at COD:N 100:5 was thinner than that (200e300 mm) at COD:N 100:2. 2011) have reported that the C-(C.. Considering the argument in the literature on the role of bound EPS content in membrane fouling. Meng et al. which are located at or outside the cell surface. in addition to the differences in the abundances in filamentous microorganisms. 2006a. in a few nm thickness. H) and N and a lower 139 concentration of O in COD:N:P of 100:5:1 might correlate to a higher surface concentration of lipids and proteins and a lower surface concentration of CH in the sludge.5 and 100:1. cation bridging. The results from this study suggest that sludge floc structure (morphology.. with an increase in the COD:N ratio. At the lowest COD:N ratio of 100:5. SMP are equal to soluble EPS (Laspidou and Rittmann. Liao et al.L. 2008. 2009). the microorganisms are more likely to produce more EPS with the highest PN/CH ratio.5 and 100:1.. In recent years. Bound EPS is defined as the biopolymers attached on the surface of flocs or cells. In principle. while Geng and Hall (2007) found that the content of bound EPS was not directly associated with membrane fouling. which also resulted in the increase in floc sizes and changes in floc shape by enhanced bioflocculation. it is very likely the PN/CH ratio (or the bound EPS composition) played a more significant role than the quantity of bound EPS in controlling membrane fouling. the PN contents in bound EPS presented a rapid decrease.8 might suggest a looser structure of cake layer. This is supported by similar observations from direct chemical analysis of extracted EPS below. the CH concentration in the bound EPS increased. the longer operation period before TMP jump (less fouling) at COD:N 100:2. 2011). In this study. 7). Liu and Fang. a decrease in the PN/CH ratio in bound EPS correlated to a decrease in membrane fouling rate. As XPS detects the surface concentrations of elements.5 and 100:1. 2008. which are overlap with the bound EPS extracted from floc surfaces. Accordingly. but the presence of a small quantity of filamentous microorganisms did improve membrane filtration (Meng et al. N and O. Shin et al. 2015) and reduce membrane fouling.. as the sludge floc size increased. The XPS results suggest significant differences in the surface concentrations of elements N and O and C bonds (Table 2). SMP include biomass-associated products (BAP) and substrate-utilization-associated products (UAP). 1982). (2001) suggested that PN are more hydrophobic and stickier and have more tendencies to adhere on membrane surface and inducing membrane fouling. control the growth of filamentous microorganisms at a certain level is important in controlling membrane fouling. apparently. Lee et al. electrostatic interaction) between sludge flocs and membrane surfaces and thus affect cake formation. This could be attribute to the decreasing nitrogen level (increasing COD:N ratio) in the system. Some studies found filamentous bulking sludge caused more serve membrane fouling (Li et al. humic acids. Hao et al. Cetin and Erdincler (2004) found that sludge filterability and compactibility were improved considerably with a decrease in the PN/CH ratio in bound EPS. 1996. It is well-known that bound EPS play an important role in membrane fouling (Wang et al. the majority of carbon source was used in biomass synthesis instead of CH production. lipids. It is also believed that total amount of bound EPS was correlated to the membrane fouling as the increase in the bound EPS content could deteriorate membrane fouling (Al-Halbounia et al. The larger floc sizes and decreased MLSS concentration at a reduced nitrogen level (COD:N ¼ 100:2. A unified theory was developed by Laspidou and Rittmann (2002) to elaborate the interrelations between SMP and bound EPS.. Polysaccharides or CH contain hydroxide and acetal or hemiacetal building blocks. These results are parallel to the results by Durmaz and Sanin (2001). This is mainly because the nitrogen in the system was utilized for synthesis of PN and insufficient nitrogen was supplied for PN synthesis when COD:N ratio increased.

. J.. Van De Staey. The higher PN/CH ratio of SMP correlated well with the higher PN/CH ratio in bound EPS. a decrease in the length of membrane operation before TMP jump) (Figs.. Croue. suggesting there was no irreversible membrane fouling by adsorption and/or pore blocking. M. 2012). 2003).. A peak near 1100 cm1 (due to CeO stretching) was due to the functional group of carbohydrate (Croue et al. 5. and gel layer formation by SMP was likely the mechanism of SMP involved in membrane fouling. 2005.. * A unique characteristic peak corresponding to unique fat or saturated aliphatic aldehyde production at 1735 cm1 was observed by FTIR in sludge under N limitations. Washington. J. 2003. J. * The XPS results demonstrated significant differences in surface concentrations of elements O and N and C bonds under different COD:N ratios.. This result indicated that the large amount of fat or saturated aliphatic aldehyde could be produced when biomass grown under the stress of nutrients limitation (Hung et al. T. The unique FTIR peak at 1735 cm1 (due to C]O stretch) under N deficiency (COD:N of 100:2.A. due to its higher PN content in SMP. Spectroscopic characterization of extracellular polymeric substances from Escherichia coli and Serratia marcescens: suppression using sub-inhibitory concentrations of bismuth thiols. Correlation of EPS content in activated sludge at different sludge retention times with membrane fouling phenomena. while SMP were the major contributors to the self-accelerating fouling at stage 2.. A. The role of carbohydrate and protein parts of extracellular polymeric substances on the dewaterability of biological sludge. Furthermore. 1996)... D. Cao. SMP at a COD:N 100:5 would have a higher affinity to membrane surface. P.5. 49. Smets..5 and 100:1. Water Res. APHA. K. Engelhard. / Water Research 89 (2016) 132e141 have different role in membrane fouling. K. as observed by Li et al... S.F.. Dominant factors that determine cake layer formation would likely be the change in bound EPS and SMP composition (such as PN/CH ratio) and the presence a small quantity of filaments that led to increased floc size and looser cake layer structure. Water Sci. in order to check the reproducibility of membrane performance under each tested COD:N condition. Thus. Badireddy..8 than that under COD:N 100:2. Bioeng. 3079e3089.Y. as compared to bound EPS. for his help in XPS analysis. * The presence of a small quantity of filamentous microorganisms under N deficiency led to an increased floc sizes and improved membrane performance. References Al-Halbounia. Conclusions * An increase in COD:N ratio from 100:5 to 100:1. while bound EPS plays a more important role in cake layer formation (Meng et al. like PN/CH ratio. It is hypothesized that the high molecular weight SMP would be accumulated on the surface of membrane during filtration and thus would modify the surface properties of membrane and outer cake layer to enhance cake layer formation. Cetin. 2008. (2013). Increasing filtration resistance in the operation of MBRs is an important indicator of membrane fouling since it directly reflects deterioration in membrane permeability. Rana Sodhi at Surface Interface Ontario at the University of Toronto. 71 (7). Additionally. Hollenderb. Traberb. Thus. 2007). FTIR may be used as a tool to indirectly monitor the nutrients condition in MBRs. Special thanks go to Dr. This might suggest that it is the composition and PN/CH ratio but not the total SMP content that controls membrane fouling rate.. Water Sci.8 might suggest that more soluble CH were released from sludge flocs under the extreme N limitation condition. 50 (9). Melinc. J. considering the fact that glucose is a easily biodegradable substrate. FTIR can be used as a tool to indirectly monitor nutrients (N) conditions in MBRs. sludge with a higher PN/CH ratio in bound EPS at COD:N of 100:5 would have a higher affinity to the modified membrane surfaces with a higher PN/CH ratio in accumulated SMP and thus increase cake layer formation rate.8) suggest a unique compound in sludge produced... it could be concluded that pore clogging or adsorption caused by SMP did not occur. Benedetti. Standard Methods for the Examination of Water and Wastewater. Integrating activated sludge floc size information in MBR fouling modeling.. Technol.8 led to an improved membrane performance and a longer operation period before membrane cleaning. 2003). This study was financially supported by Natural Sciences and Engineering Council of Canada (NSERC) (RGPIN-2014-03727).. Environ. 2015. It is more likely SMP would cause pore blocking or gel-layer formation. T. A. The higher CH content of SMP at a COD:N of 100:1. 42. 601e610. D. (2013) found that bound EPS played a significant role in fouling development at stage 1. Stephenson. it can be concluded that the sludge under the nitrogen limitation conditions can generate a great amount of fat or saturated aliphatic aldehyde. played a more important role in controlling membrane fouling than the quantity of bound EPS and SMP. Dotta.140 L. Li et al. T. W.. A stronger intensity of the unique characteristic peak at 1735 cm1 correlated to a better membrane performance (e. The peaks of 1384 and 1244 cm1 suggest the presence of amide III (Jun et al. S. A.... the relative intensity of the peak at 1735 cm1 was stronger under COD:N 100:1. B. Violleau. American Public Health Association (APHA)/American Water Works Association (AWWA)/Water Environment Federation (WEF).R.8) may be used as an indicator of nutrients conditions in feed. Biomacromolecules 9. Characterization and copper binding of humic and nonhumic organic matter isolated from the South Platte River: evidence for the presence of nitrogenous binding site. Rosso. * Bound EPS/SMP composition. A. I. Ultrafiltration experiments (results not shown) with a membrane having a molecular weight cut-off (MWCO) of 1000 Da indicates that oligosaccharides were present in SMP. 2008. 4 and 6). g. 236e238.M. The application of membrane biological reactors for the treatment of wastewaters. Leenheer. J. Nutrients requirements in biological industrial wastewater treatment. Biotechnol. cake layer resistance (Rc) under each COD:N condition was equivalent to 100% of the total fouling resistance (Rt) (Table 3). Korpol. the higher PN/CH ratio in SMP at COD/N 100:5 could accelerate the self-accelerating fouling phenomena (increase cake layer formation rate) in stage 2. The characteristic peak at 1735 cm1 under nitrogen limitations (COD:N of 100:2... Janote. no irreversible membrane fouling likely developed and thus the cake layer was the only fouling mechanism.A.. Lykoc.5 and 100:1.. M.S. In such a short-term operation. T. Gassman. This might be due to the use of new membranes in each TMP cycle. 2003. . S. Erdincler. Technol. G. Lea. Brindle. Sci. Wu and Fane. Table 3 shows that cake layer formation was the mechanism of membrane fouling with no pore clogging or adsorption.g. B. which suggest significant difference in bound EPS composition.H. The total SMP content could not explain the improvement of membrane performance at an increase in the COD:N ratio but an increase in the PN/CH ratio is positively correlated to the membrane fouling rate (e... a lower membrane fouling rate or a longer operation time bore TMP jump). Acknowledgments We thank the anonymous reviewers and editors for their valuable comments and suggestions on revising and improving the work. Chellam. Biotechnol. Wintgensc.. Morel (1983) indicates that if nutrients (N and P) are present in very low concentrations.. 21st ed. ON. According to the FTIR spectra. Toronto. Tacked. Ammary.P. 2009. 1073e1080. SMP may be produced to scavenge the required nutrient. D. Hao et al. DC.R. 3. Afr.Y.L.. The peaks located at 1660 (stretching vibration of C]O and CeN amide I) and 1540 (NeH deformation and C]N stretching amide II) and 1245 cm1 are important as they indicate the presence of proteins (Croue et al. Importantly. 1475e1488. as carbohydrates with a MWCO less than 1000 Da were detected. 49e56.. 1996. 2004.

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