Impact of synthetic or real urban wastewater on membrane bioreactor (MBR) performances and membrane fouling under stable conditions

Bioresource Technology 155 (2014) 235–244

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Impact of synthetic or real urban wastewater on membrane bioreactor (MBR) performances and membrane fouling under stable conditions Maud Villain a,⇑, Isabelle Bourven b, Gilles Guibaud b, Benoît Marrot a a b

Aix-Marseille Université, CNRS, M2P2 UMR 7340, 13545 Aix en Provence, France Université de Limoges, GRESE EA 4330, 123 avenue Albert Thomas, 87060 Limoges, France

h i g h l i g h t s  Impact of substrate type (real or synthetic) on MBR performances was assessed.  Autotrophic biomass showed better acclimation in MBR fed with real wastewater.  Molecular weight distribution of SMP protein and humic-like contents were measured.  Protein with high molecular weight (>600 kDa) was responsible for removable fouling.  MBR has to be fed preferentially with real wastewater.

a r t i c l e

i n f o

Article history: Received 25 September 2013 Received in revised form 12 December 2013 Accepted 14 December 2013 Available online 24 December 2013 Keywords: Membrane bioreactor Substrate type Fouling Biomass activity Soluble microbial products

a b s t r a c t Influence of substrate type (synthetic (SWW) or real wastewater (RWW)) on lab scale MBR performances (e.g. COD and N-NHþ 4 removal rates and bioactivities) was assessed. Membrane fouling was related to MBR biological medium characteristics. With RWW, autotrophic biomass was better acclimated with complete ammonium removal. MBR biological medium was characterized by main soluble microbial products (SMP) (proteins, polysaccharides and humic-like substances) quantification and molecular weights (MW) distribution determination. The biological medium of SWW acclimation contained 60 mg L1 more of SMP, mainly composed of proteins and polysaccharides. A protein fraction having high MW (>600 kDa) could be responsible for higher removable fouling fraction in that case. SMP of RWW experiment were mainly composed of small proteic and humic-like fractions, poorly retained by the membrane and resulting in a weak augmentation of irremovable and irreversible fouling fractions compared to SWW acclimation. Therefore RWW utilization is preferable to approach real operating MBR. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction MBR process is a promising technology in the field of wastewater treatment. Indeed MBR technology offers many advantages such as a complete retention of biomass, a high treated water quality and stability, a low footprint and a totally independence between hydraulic retention time (HRT) and sludge retention time (SRT). However, as several parameters control the operating cost of MBR process, such as power requirements, costs of power, membrane cleaning, membrane life and replacement, there are still limitations to use MBR for wastewater treatment. The major obstacle for the application of MBR is the rapid decline of permeate flux over time as a result of membrane fouling. Many studies were performed at lab scales over the past two decades to find optimal ⇑ Corresponding author. Tel.: +33 4 67 14 33 62; fax: +33 4 67 14 49 90. E-mail address: [email protected] (M. Villain). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.12.063

operating conditions of MBR systems and limit membrane fouling. At this scale, the feeding to the MBR can be supplied with both types of effluents: synthetic (SWW) or real (RWW). SWW can be used because it offers a stable and repeatable composition (Ng and Hermanowicz, 2005). Others studies performed their experiments with RWW (Trussell et al., 2006), which can be renewed every day or stocked for several days with or without addition of chemical products to standardize its composition. This choice of substrate type is not harmless and has consequences on biological MBR medium characteristics. In the case of SWW, organic carbon source used was reported to have an influence on main biological mixed liquor characteristics like floc size and structure. Mcadam et al. (2007) pointed out that biomass fed with acetic acid showed large flocs with a loosely structure sensitive to high shearing whereas with ethanol, flocs were more compact and more resistant to high shearing. Exopolymeric substances (EPS) (bound part of polymer matrix) and soluble microbial products (SMP) (free part of polymer matrix), are currently considered as the predominant

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cause of fouling in MBR (Meng et al., 2009). As these polymers whose main compounds are proteins, humic-like substances and polysaccharides, can be degraded as carbon and energy sources when food to micro-organism (F/M) ratio from the substrate is limited. The nutrient quantity has a significant effect on polymer production and composition (Sheng et al., 2010). Polymer content increases with an augmentation of F/M ratio in MBR system (Jang et al., 2007). The substrate composition (in term of biodegradability, relative proportion of carbon, nitrogen and phosphorus sources) has a proved effect on the microbial community present in the sludge as well as on the bacterial metabolism and consequently on the polymer secretion mechanism (Fishman et al., 1997). COD=N-NHþ 4 ratio also has a considerable effect on the polymer composition. For a limitation of carbon substrate ðCOD=N-NHþ 4 < 20), the protein proportion increases as the ratio decreases (Bura et al., 1998). For a limitation of nitrogen source ðCOD=N-NHþ 4 > 20Þ, the relative quantity of proteins decreases. Li et al. (2008) showed that bacteria from activated sludge (AS) fed with acetate had a higher polymer production than bacteria fed with glucose. As a consequence, the substrate choice for experiments influences the fouling nature and its establishment. Another well-known effect of substrate type is the influence on biomass yield. Salhi (2003) noticed that with an increase in inert components (e.g. non biodegradable part of organic matter) of the effluent, biomass yield rises. But no work was performed with real or synthetic substrate in MBR set with the same operating parameters to observe the impact of inert effluent content on biomass yield. So far, no study was reported to compare the effects of synthetic or real substrate on removal performances, heterotrophic and autotrophic bacteria bioactivities and characteristics of the biological medium related to membrane fouling in MBR with the same characteristics. However for about 10 years, about half of the scientific community supplied MBR with SWW (Ng and Hermanowicz, 2005; Wu and Lee, 2011) and the other half used RWW (Drews et al., 2006; Sun et al., 2008). This assessment points out the relevance of such a study which compares MBR characteristics with real or synthetic substrate. This type of study could allow for obtaining answers to understand the large range of results in this field depending on the type of substrate used. Accordingly, the present study aims at comparing the impact of substrate type (real or synthetic) on the characteristics of the biological medium (e.g. floc size, SMP main components concentrations), removal performances and heterotrophic and autotrophic bacteria related to membrane fouling in MBR with the same characteristics. Fig. 1 presents a schematic of the process flow to highlight the objectives and background of this study. A lab scale MBR was successively fed with a RWW and a SWW with glucose as carbon source. Glucose was chosen rather than acetate to avoid any risk of additional fouling involved by the release of various small particles reported by Holakoo et al. (2007). Before the different measurements to assess characteristics of MBR with real or synthetic wastewaters, MBR were stabilized at a chosen SRT of 50 days (d). This choice was made first according to the result of Meng et al (2009) who indicated that a SRT between 20 d and 50 d needed to be set in order to control EPS concentration and thus membrane fouling within the same time by keeping high removal rates of COD and N-NHþ 4 . Secondly 50 d was preferably used because Villain and Marrot (2013) demonstrated the interest in working at this SRT. Indeed heterotrophic bacteria adapted themselves well to the MBR with faster substrate biodegradation than at 20 d. Autotrophic bacteria showed an easier development with N-NHþ 4 with a relatively high removal rate (78%). Finally with a F/M ratio increase, the exogenous activities of both characteristic micro-organism types present in AS (e.g. autotrophic and heterotrophic bacteria) tend to rise (Huang et al., 2001; Han et al., 2005). Therefore a comparison of bioactivities depending

on substrate type has to be realized at the same F/M ratio of 1 1 ammonium and COD. F/M ratios of 0:15 kgN-NHþ kgMLVSS d and 4 1 1 0:2 kgCOD kgMLVSS d were chosen to be near wastewater treatment plant (WWTP) F/M ratios. An original method was used to obtain information about the influence of SMP on membrane fouling. This method coupled the analysis of three dimensional excitation-emission matrices (3D-EEM) generated by SMP samples to size exclusion chromatography (SEC) with a fluorescence spectrometer as detector. This method uses the fluorescent characteristic of proteins and humic-like substances, two of the main SMP components with polysaccharides. With this method, specific MW distribution of protein and humic-like substances content of SMP extracted from both effluents, MBR mixed liquor and permeate can be determined. 2. Methods 2.1. The MBR setup and biomass acclimations to the MBR process The external MBR (Polymem, France) was composed of a bioreactor equipped with a cooling coil to maintain biomass at 25 ± 1 °C (Fig. 2). The membrane system used was a tubular ceramic membrane (ultrafiltration, Novasep-Orelis, France) made with ZrO2–TiO2. Membranes were characterized by a 150 kDa cut off, a 0.02 m2 filtration area and an initial water permeability of 110 L h1 m2 bar1. Ceramic membrane was preferred to polymeric membrane for experiments at lab scale because it is more convenient to clean and to maintain a specific flow rate. Biological medium aeration was provided by small bubbles in a sequential mode (2 h with air/1 h without air) to allow nitrification and denitrification reactions. Crossflow filtration was operated with a centrifugal pump that recycled sludge back to the membrane. The influent was provided to the bioreactor with a feed pump connected to a level regulator. Table 1 summarizes parameters set for both acclimations. During both acclimations in the external MBR, the mixed liquor volatile suspended solid (MLVSS) was stabilized at 7.0 ± 0.7 gMLVSS L1 during acclimation with SWW and at 5.9 ± 0.7 gMLVSS L1 for acclimation performed with RWW. In both cases, biomass came from the same municipal wastewater treatment plant which is a submerged membrane bioreactor (Le Rousset, France, 12,000 inhabitant equiv1 1 alent, 1800 m3 d1, organic load 0:1 kgBOD5 kgMLVSS d ). The initial MLVSS was around 6 g L1. Samples were taken from the anoxic tank and then transported either to the MBR in the laboratory without aeration (1 h) for the experiments with SWW or to the MBR which was located on the municipal wastewater plant (5 min) for experiments with RWW. The crossflow velocity was held constant at 4 m s1 to limit membrane fouling. The constant flow rate mode of operation was employed. Transmembrane pressure (TMP) was monitored with manometers set at the inlet and outlet of the membrane module. The COD and N-NHþ 4 F/M ratios were maintained constant all along experiments to allow the comparison of autotrophic and heterotrophic biomass activities. Unfortunately, as real wastewater was less concentrated in COD and 1 N-NHþ 4 , the choice to increase the permeate flow from 0.76 L h to 1.64 L h1 was done during experiments with real wastewater to reach the required F/M ratio. Therefore, hydraulic retention time decreased from 24 h to 9 h. The SRT was fixed at 50 d in both acclimations to control the amount of waste sludge. The amount of waste sludge is inversely proportional to the wanted sludge age (Eq. (1)):

V waste ¼ V R =SRT

ð1Þ

with Vwaste: the volume of waste sludge per waste event (L), VR: the total volume of MBR (L) and SRT: the desired sludge retention time (d).

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Fig. 1. Schematic representation of the objectives.

Electromagnetic flowmeter

V5

P2

Inlet

Bioreactor LSH

PI

PI

Water

LSL

Permeate

Water

V4

NaHCO3

pH controler

pHc

V3 Air

V2 V1

P1 Fig. 2. Experimental set up of membrane bioreactor.

AS was wasted every day from the drain of the bioreactor. In order to reduce the effect of discontinuous sludge wasting on sludge analysis, all sludge samples were collected just before sludge wasting. The daily production of sludge (Px) was calculated with the following mass balance (Eq. (2)):

Px ¼ Q P  MLVSSR þ V R D  MLVSSR =Dt

ð2Þ

With Px: Daily production of sludge (gMLVSS d1), Qp: Flow of sludge wasted (L d1), MLVSSR: MLVSS concentration in the bioreactor (gMLVSS L1), MLVSSR: Variation of the MLVSS concentration in the bioreactor (gMLVSS L1) and t: Time variation (d). Process was considered as stable as soon as the accumulated daily production of sludge ðP x accumulated Þ was linear (Delgado 2009) (Eq. (3)):

Px

accumulated

¼

n X Pxi

ð3Þ

i¼1

2.2. Wastewater types: synthetic or real A balanced synthetic wastewater (C/N/P ratio = 100/10/2) was prepared with mass ratios of 2.1 C6H12O6, 1.0 (NH4)2SO4, 0.2 KH2PO4, 0.4 NaHCO3, 0.1 MgSO4 and 0.02 CaCl2 and was used to perform experiments with synthetic substrate. Heterotrophic biomass biodegrades organic carbon contained into glucose to grow. Autotrophic micro-organisms use ammonium contained into (NH4)2SO4 and inorganic carbon which came from NaHCO3 for their growth. Effluent NaHCO3 was also used to set the pH at 7 during experiments with synthetic effluent.

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Table 1 Operating conditions of acclimations performed with RWW and SWW. Substrate type Substrate N-NH4 F/M (food to microorganisms ratio) 1

1

ðkgN-NHþ kgMLVSS d 4

SWW

RWW

0.016 ± 0.003

0.014 ± 0.008

Þ

1

1

COD F/M ðkgCOD kgMLVSS d Þ Mixed liquor suspended solids (MLSS) (gMLSS L1) Mixed liquor volatile suspended solids (MLVSS) (%) Process Volume (L) Hydraulic retention time (HRT) (h) Permeate flow (L h1) Permeate flux at 20 °C (L h1 m2) Recirculation rate (m s1) Transmembrane pressure (TMP) (bar)

0.21 ± 0.02

0.19 ± 0.02

9.1 ± 1.2

7.6 ± 0.9

82 ± 1

78 ± 2

18 24 ± 1 0.76 ± 0.02 32.6 ± 2.5 3.5–4 1.3 ± 0.1

15 9±3 1.64 ± 0.4 70.1 ± 16 3.5–4 1.2 ± 0.2

Real wastewater (RWW) was sampled after the different pre treatments performed in the wastewater treatment plant of Le Rousset. Wastewater was submitted to an additional filtration (cut off of 200 lm) to avoid MBR pump breakage before storage in a tank at room temperature. The main characteristics of RWW were assessed during 4 months and are reported in Table 2. No pH regulation was necessary in that case as bioreactor pH was constant at 7.0 ± 0.1. 2.3. Analytical methods AS was regularly sampled in the bioreactor (30 mL) and samples were centrifuged for 15 min at 16,000g to separate the dissolved matrix from the suspended solids. COD and ammonium were analyzed in MBR inlet and permeate to determine their removal rates. Nitrates were quantified in the bioreactor supernatant. As for ammonium, nitrates were evaluated by spectrophotometry (Spectro Aquamate, Thermo spectronic, UK) with reagent kits obtained from Merk (Germany). Reagent kits purchased from Aqua Lytic (Germany) were used for the COD. Like the major components of SMP contents, polysaccharides (Dubois et al., 1956), protein and humic-like substances (Frolund et al., 1995) were quantified with colorimetric methods in the substrate, the bioreactor supernatant and in the permeate to evaluate the fraction of SMP resulting from bacterial metabolism and the fraction retained by the membrane after extraction by centrifugation (4000g, 20 min, 4 °C). Glucose (Sigma–Aldrich, 99.5%) was used, bovine serum albumin (Sigma–Aldrich, 98%) and humic acids (Sigma–Aldrich) were used respectively as a standard for polysaccharides, proteins and humic-like substances contents. The fluorescent properties of proteins and humic-like substances enable the determination of MW distribution of both compounds by a recent method, which couples a 3D-EEM spectra analysis with SEC with fluorescence detection (Bhatia et al, 2013). To obtain 3D-EEM spectra, SMP samples were filtered with a 0.22 lm acetate filter and diluted with a 50 mM phosphate buffer

Table 2 RWW main characteristics. Parameters 1

MLSS (mg L ) COD (mg L1) BOD5 (mg L1) 1 N-NHþ 4 (mg L ) Global ammonium (mg L1) Total phosphorous (mg L1)

1 (Month)

2 (Month)

3 (Month)

4 (Month)

270 ± 20 665 ± 78 255 ± 35 51 ± 4 67 ± 6 8±1

283 ± 35 767 ± 212 279 ± 92 44 ± 11 62 ± 15 7±1

323 ± 64 626 ± 14 157 ± 85 29 ± 15 47 ± 12 5±2

284 ± 42 720 ± 95 282 ± 28 52 ± 4 71 ± 4 8±1

at pH 7.0 ± 0.1 to obtain a solution ranging from 4 to 50 mg L1 of SMP. SMP with the highest protein content, corresponding to strong fluorescence intensities in both areas I and II (Chen et al., 2003) are more diluted. 3D-EEM spectra were measured at 22 ± 1 °C using a Shimadzu RF-5301 PC spectrofluorophotometer. Emission was scanned from 300 to 500 nm after excitation ranging from 220 to 400 nm using 10 nm increments. The fluorescence data were processed with the software Panorama Fluorescence 3.1 (LabCognition, Japan). For MW determination, the separation of SMP was carried out with a Merck Hitachi LA Chrom chromatograph equipped with a L7200 autosampler, a L7100 quaternary pump, a D7000 interface, fluorescence detector (L7485) and a diode array UV detector (L7455). Separation was performed with a high molecular weight (HMW) Amersham Biosciences column, the superdex 200 10/ 300 GL and low molecular weight (LMW) Agilent Bio SEC 100 Å column with high MW resolution range. The total permeation volume of HMW and LMW columns was estimated to 23.7 mL and 10.3 mL respectively, with a sodium nitrate solution at 60 mg mL1. A solution containing 150 mM NaCl and 50 mM phosphate buffer (25 mM Na2HPO4 and 25 mM NaH2PO4, pH 7.0 ± 0.1 with a constant flow rate of 1 mL min1 for both columns was used as mobile phase. 100 lL of filtered samples (0.2 lm) were injected for each analysis. Excitation and emission wavelengths were selected for the fluorescence detector from 3D EEM fluorescence spectra of SMP samples: 221/350 nm for protein-like substances and 350/440 nm for humic-like substances. The HMW column was calibrated using four proteins with MW ranges from 440 to 13.7 kDa: Ferritine – 440 kDa; Immunoglobulin G from human serum-155 kDa; Bovine serum albumin – 69 kDa and Ribonuclease A – 13.7 kDa. The LMW column was calibrated for both proteins and humic-like substances. For proteins, the following molecules were used: Bovine serum albumin; Ovalbumin – 45 kDa; Ribonuclease-A and Glucagon – 3.8 kDa. The humic-like substance calibration curve was realized with four synthetic polymers of polystyrene sulfonate with MW ranging from 0.21 to 13 kDa. For mass calibration curves, the logarithm of molecular mass (log(MW)) is plotted vs retention volume (mL) (Table S1 available on-line as additional material). Particle size distribution of AS was measured with a laser granulometer Mastersizer S (Malvern Instruments), which measures particle sizes from 0.1 lm to 900 lm. The median diameter (D50) was recorded for AS of both experiments. The filtrate of the mixed liquor recovered after AS filtration was used to dilute 100 times the AS samples in order to obtain an acceptable obscuration to measure the particle size distribution. Main divalent cation concentrations (Mg2+ and Ca2+) were measured by a French certified laboratory with an ionic exchange chromatography in supernatant of both bioreactors. 2.4. Respirometry tests The biological activity was followed by respirometry tests to measure autotrophic and heterotrophic activities thanks to a differentiation of endogenous and exogenous activities. The endogenous activity is reached when no external substrate (e.g., an organic carbon source for heterotrophic bacteria and an ammonium source for autotrophic biomass) is available. Bacteria are forced to biodegrade their EPS as substrate to provide the energy necessary for cell maintenance and their respiration is minimal. The exogenous activity is obtained after substrate addition; bacteria use the substrate available to supply their energy demand, and the resulting respiration is at its highest value. A 1 L reactor was filled with AS from MBR and maintained aerated during 4 h to guarantee endogenous respiration of the biomass. After this period 50 mL of AS were sampled every 2 min and injected into another

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mum activity of autotrophic micro-organisms (ammonium removal was confirmed to be correlated with nitrate production). Thirdly, autotrophic micro-organisms were inhibited with allylthiourea (inhibitor of the nitrifying ammonium monooxygenase enzyme) to isolate heterotrophic endogenous respiration. A concentration of 0:1 gallylthiourea g1 MLVSS was shown to be sufficient to inhibit the respirometry activity of autotrophic micro-organisms, and nitrates were not produced after ammonium addition ð0:02 gN-NHþ g1 MLVSS Þ, which validated the autotrophic inhibition. 4

Lastly, the maximum activity of heterotrophic micro-organisms was measured after glucose addition (0:3 gCOD g1 MLVSS ).

0.8

12 0.7 10

MLVSS (gMLVSS.L-1)

4

A

0.6 0.5

8

0.4

6

0.3 4 0.2 2 II

I

III

0.1

IV

0

0.0 0

20

40

60

80

100

Time (d)

B

0.3

16 14

Stabilisation

0.2

MLVSS (gMLVSS.L-1)

10

3.1. Effects of substrate type on MBR performances The biomass development was divided into four steps for both acclimations (Fig. 3A and B). For acclimation performed with SWW (Fig. 3A), the F/M ratio was initially fixed at 1

1

1

0:1 kgCOD kgMLVSS d due to rainy days at that time as well as the application of a smaller permeate flow and a too long HRT to sat1 kgCOD kgMLVSS

1

d . It explained isfy a F/M ratio at a value of 0:2 the reason why permeate flow and HRT initially fixed at the same values as SWW experiment ones 0.75 L h1 and 24 h were changed after 8 days to reach 1.64 L h1 and 9 h and as a consequence a va1

1

lue of F/M ratio around 0:2 kgCOD kgMLVSS d . Process stability was confirmed for both MBRs at the end of the transient period whereas the accumulated daily production of sludge (Pxaccumulated) was linear (Fig. 3C). At the end of both acclimations, a slightly lower MLVSS concentration (5.9 ± 0.7 gMLVSS L1) was reached for MBR fed with RWW compared to the campaign realized with SWW (MLVSS concentration = 7.0 ± 0.7 gMLVSS L1). The MLVSS/MLSS ratio was constant at 0.8 ± 0.02 during both experiments indicating that no mineralization occurred over time. This difference in MLVSS concentration was explained by the F/M ratio slightly higher during the 1

1

experiment performed with SWW ð0:21  0:02 kgCOD kgMLVSS d Þ compared to the one performed with RWW ð0:19  0:01 1

1

kgCOD kgMLVSS d Þ. MBR global performances realized with SWW and RWW are presented in Table 3. The use of the same MBR set up with identical operating parameters as the conservation of COD and N-NHþ 4 F/M ratios constant allowed the comparison of the results of both campaigns. For a high COD removal in both cases, exogenous heterotrophic activity during experiments with SWW was more than five times

6

0.1

4 2

I

IV

III

II

0

0.0 0

C

10

20

RWW

SWW

30 40 Time (d)

50

60

70

200 180

Px accumulated (gMLVSS.d-1)

1

8

F/M (kgCOD.kgMLVSS-1.d-1)

12

3. Results and discussion

0:2 kgCOD kgMLVSS d to approximate WWTP conditions. The MLVSS concentration decreased during a first step (I) followed by a lag phase that was typical of an adaptation period of micro-organisms to the synthetic wastewater and the MBR process. Then, the biomass growth defined the second step (II), where the nutrient concentration had to be increased. The third (III) and fourth (IV) steps were controlled to stabilize the bioprocess. During step III, nutrient concentration was voluntarily reduced until MLVSS concentrations were maintained at a constant value (step IV). For MBR fed with RWW, the initial F/M ratio was low at

F/M (kgCOD.kgMLVSS-1.d-1)

reactor without oxygenation to measure the specific oxygen uptake rate (SOUR) with continuous dissolved oxygen probe (HQ 40d, Hach LDO, Germany). Then specific nutrients or inhibitor were added to the aerated reactor according to four different steps. Endogenous respirations of autotrophic and heterotrophic microorganisms were first measured over a period of 1 h. Secondly, ammonium was added ð0:02 gN-NHþ g1 MLVSS Þ to measure the maxi-

160 140 120 100 80 60 40 20 0 0

20

40

60

80

100

Time (d)

Fig. 3. Biomass development during acclimations. (A) MLVSS and F/M ratio in the external MBR fed with SWW and (B) RWW. (C) Accumulated sludge production in both MBR experiments.

higher than for RWW campaign. Although COD F/M ratios were similar in both experiments, SWW was only composed of glucose, which is easily biodegradable, as organic carbon source. It was not the case for RWW because even if this effluent was considered as biodegradable (COD/BOD5 = 2.9), around 65% of COD was slowly biodegradable. As a consequence, if F/M ratios were calculated considering BOD5 concentrations, F/M ratio for SWW campaign 1

1

ð0:21  0:02 kgBOD5 kgMLVSS d Þ became approximately twice that 1

1

obtained with RWW ð0:11  0:02 kgBOD5 kgMLVSS d Þ. The differences in exogenous heterotrophic activities observed during the treatment of both effluents came partly from the double BOD5 F/M ratio noticed for SWW. Furthermore, the endogenous respiration of heterotrophic bacteria obtained for the campaign carried out

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M. Villain et al. / Bioresource Technology 155 (2014) 235–244

with SWW was around twice that observed for heterotrophic bacteria with RWW. The double BOD5 F/M ratio allowed a better development of heterotrophic micro-organisms. This difference was due to the limits of the MBR process parameters which did not allow a sufficient increase in the permeate flow rate to have 1

1

the same BOD5 F/M ratio value of 0:20 kgBOD5 kgMLVSS d during the experiment performed with RWW. However, the biomass yield 1

obtained during campaign with RWW ð0:17 kgMLVSS kgCOD Þ and 1

SWW ð0:14 kgMLVSS kgCOD Þ were similar. The slight difference could have two origins. The first one was linked to the inert fraction of COD present in the biomass which came from the RWW itself and also from the non biodegradable fraction of bacterial lysis (Massé, 2004). The second one could be due to a variation of biomass decay rate as well as SMP hydrolysis rate. The impact of particular COD fraction considered as inert in RWW on biomass yield was low and at a SRT of 50 d, most of COD was not inert but slowly biodegradable. As a conclusion, most of COD contained in RWW seemed to be biodegradable at a SRT of 50 d. The evolution of bacterial metabolism with increasing substrate concentration is well known in the literature (Lobos et al., 2008). Oxygen needs for substrate biodegradation are proportional to the substrate concentration. First, without exogenous substrate presence in the medium, oxygen is only required for oxidation of organic matter which provides the energy necessary for the cell maintenance and synthesis (endogenous conditions). Then, with the increase in substrate concentration, oxygen requirements increase (e.g. increase of respiration) as well as biomass activity to allow the development of active biomass. Concerning the influence of substrate type on autotrophic bacteria and their ammonium removal rate, the study of Table 3 reveals a better autotrophic biomass development (exogenous 1 autotrophic activity at 4:5 mgO2 g 1 with a 99% ammonium MLVSS h removal rate for experiments realized with RWW while during the stable state of campaign with SWW, only 77% of ammonium were eliminated with a low autotrophic exogenous activity equal 1 to 0:9 mgO2 g1 MLVSS h . A nitrification limitation due to a lack of inorganic carbon (IC) was rarely taken into account in the literature because IC was usually in excess in wastewaters and CO2 produced by heterotrophic bacteria during COD oxidation brought the eventual required complement to reach a total nitrification (Guisasola et al., 2007). At a neutral pH, most of the total IC present in the system was in 1 the hydrogenocarbonate form ðHCO of HCO 3 Þ. 8.7 mg L 3 were 1 required to oxidize 1 mg L of ammonium into nitrates. 480 mgHCO3 L1 and 30 mgN-NH4 L1 were the mean concentrations measured in RWW used in this study. Therefore, IC concentration of RWW allowed a complete nitrification. It was one of the reasons why, with a MBR working under low F/M ratio, ammonium removal was almost total (Huang et al., 2001; Pollice et al., 2008; Braclow et al., 2010). Table 3 Influence of substrate type on global MBR performances. SWW Removal rates (%) COD N-NHþ 4

96 ± 3 77 ± 4 1

Biological activities ðmgO2 g1 MLVSS h Heterotrophic: Exogenous Endogenous Autotrophic: Exogenous Endogenous 1

Biomass yield ðkgMLVSS kgCOD Þ

RWW 97 ± 3 99 ± 1

Þ 49.3 ± 3.6 1.7 ± 0.4 0.9 ± 0.2 2.1 ± 0.5

9.1 ± 1.2 1 ± 0.2 4.5 ± 0.3 0.4 ± 0.1

0.14 ± 0.1

0.17 ± 0.2

In many MBR studies (Han et al., 2005; Liang et al., 2010; Feng et al., 2012), the effluent used was a synthetic one, which was composed of tap water mixed with an organic carbon source, ammonium, phosphate sources and different mineral salts. The mean hydrogenocarbonate concentration was 181 mg L1 for the tap water used to prepare SWW. This concentration was insufficient to allow a complete ammonium nitrification if the ammonium concentration was higher than 21 mgN-NH4 L1 . In these studies, IC requirements in SWW for nitrification needs were therefore neglected. In many publications, when an IC supply was mentioned, it was only to regulate pH at 7, either with a pH controller or by direct addition in SWW as in the present study. NaHCO3 can be used (Guo et al., 2007; Clouzot et al., 2011) and Na2CO3 or CaCO3 (Fu et al., 2009). In the present work, a low IC concentration was voluntarily used to regulate the pH at 7.0 ± 0.1. The purpose was to be in the same situation as many studies where nitrification was limited by a lack of IC. SWW was concentrated at 326 mgHCO3 L1 which was a low concentration compared to the 1000 mgHCO3 L1 theoretically necessary to achieve complete nitrification of the mean value of 115 mgHCO3 L1 contained in SWW. At a SRT of 50 d with SWW, 77% of ammonium were degraded with a low exogenous autotrophic activity. Heterotrophic microorganisms showed a high exogenous activity and the CO2 release resulting from this activity (e.g. via COD oxidation) exhibited an influence on the exogenous autotrophic activity and their ammonium elimination rate (Fig. 4). However released CO2 by heterotrophic bacteria was not enough to obtain complete nitrification. Studies which mention neither pH control nor IC addition to the synthetic substrate reach ammonium removal rates slightly lower than in the present work. Liang et al. (2010) and Feng et al. (2012) obtained respectively a 69% and 75% ammonium removal rate. It can be assumed that CO2 released by heterotrophic bacteria was used by autotrophic micro-organisms but also reacted with the H3O+ released during the medium acidification due to nitrification reaction. In contrast, studies which have maintained medium pH at 7 and where F/M ratio was comprised between 0.1 and 1

1

0:2 kgCOD kgMLVSS d reached better ammonium removal rates. Han et al. (2005) were at 94% of ammonium removal rate at a SRT of 50 days. Fu et al. (2009) as Clouzot et al. (2011) found also better removal rates at 84 and 85% respectively. The lack of IC clearly influenced autotrophic exogenous activity. Indeed for experiments performed with RWW, it was 1

which was in the same range as values ob4:5 mgO2 g1 MLVSS h tained in other studies with RWW (Huang et al., 2001; Pollice et al., 2008) whereas under IC limitation for SWW, their activity 1

decreased at 0:9 mgO2 g1 MLVSS h

which represents a drop by 80%.

3.2. Influence of the type of wastewater on the characteristics of the biological medium and consequences on membrane fouling 3.2.1. Characteristics of the biological medium Mean concentrations of the main components of SMP contained in both substrates and biomass during stable state of acclimations performed with RWW and SWW are presented in Fig. 5. The mean polysaccharide concentration measured in SMP content extracted from SWW is not shown voluntarily. Indeed, as organic carbon used to prepare SWW was glucose, the main part of polysaccharide concentration observed in the SWW came from glucose. Polysaccharide content in SWW linked to micro-organism metabolism was assumed low compared to the glucose concentration. The total concentration of biopolymers from SWW was low (40 mg L1) and represented only around 25% of biopolymer content from RWW (155 mg L1). Polymers from RWW were mainly composed of humic-like substances (65%) whereas SMP from

M. Villain et al. / Bioresource Technology 155 (2014) 235–244

2 Waste sludge events beginning

Exogenous specific autotrophic activity (mgO2.gMLVSS-1.h-1)

1.8 1.6

Process stabilized at SRT of 50 d

1.4 1.2 1 0.8 0.6 0.4 0.2 0 15

20 25 30 35 40 45 50 55 Exogenous specific heterotrophic activity (mgO2.gMLVSS-1.h-1)

60

Fig. 4. Influence of heterotrophic exogenous activity on autotrophic exogenous activity before and after stabilization during experiment with SWW.

SWW contained in their major part proteins (63%). The high humic-like substances content of RWW was explained easily by the fact that real water was in contact with soil and wastes where organic matter was biodegraded. SMP biomass contents presented different proportions of biopolymers compared to those measured in the substrates and also different proportions among them. The main components of SMP extracted from biomass acclimated with SWW were polysaccharides at 46% (58 mg L1) and proteins at 38% (50 mg L1). Protein concentration was higher in SMP extracted from biomass than from SWW. This result implies that bacterial metabolism was responsible for a soluble protein production in the MBR. As glucose can be considered as an easily biodegradable substrate, the main part of polysaccharide content measured in the sludge supernatant of biomass acclimatized with SWW can be related to a microbial origin. For humic-like substances, their concentration was 40% higher in SMP extracted from bioreactor fed with RWW (22 mg L1) than for SMP extracted from MBR supplied with SWW (13 mg L1). Unlike acclimation carried out with SWW where total SMP concentration increased from the effluent to the MBR, the total SMP concentration observed in bioreactor fed with RWW decreased compared to the one measured in the real effluent. In SMP extracted from MBR acclimated with RWW, there was no polysaccharide and proteins and humic-like substances

180 Humic-like substances Polysaccharides Proteins

Biopolymer concentration (mg.L-1)

160 140 120 100 80 60 40 20 0 RWW

MBR

P

SWW

MBR

P

Fig. 5. Evolution of biopolymers concentrations (proteins, polysaccharides and humic-like substances) of substrate, MBR and permeate for both experiments.

241

concentrations have respectively decreased from 8 to 53 mg L1. Polysaccharides from RWW were probably totally adsorbed on the bioflocs as polysaccharides were not present from bioreactor permeate. Polysaccharides from RWW had MW higher than the 150 kDa membrane cut off. The humic-like substances content decrease between the effluent and the MBR. This could be the result of various phenomena such as their sorption on bioflocs, their participation to bound EPS content, their elimination by daily sludge waste event and their transfer trough the membranes due to their low MW (<5 kDa) (Table 4). As far as proteins are concerned, a biomass active secretion could occur but it was probably hidden because a major part of protein content was not retained by the membrane. Indeed, the measurement of protein concentration in MBR and permeate (Fig. 5) made protein retention on the membrane surface accessible. Only 13% of proteins were retained which means that 87% had smaller MW than the membrane cut off. Table 4 gives the MW distribution of humic-like substances and proteins from SMP extracted from both effluents and AS. SWW was specifically made up with only small proteins (<5 kDa) whereas RWW contained a protein fraction with high MW (>600 kDa) considered until now as present only in MBR, a fraction of 50 kDa and a variety of small ones. RWW was a complex medium compared with SWW. It contained higher humic-like substances and main divalent cation concentrations (Ca2+ (100 ± 10 mg L1) and Mg2+ (13 ± 3 mg L1) concentrations in RWW were higher than those observed in SWW set at 4 mgCa2þ L1 and 13 mgMg2þ L1 ). The latter can participate to the formation of big structures in which proteins can be embedded. However, this distribution was in total contradiction with the one observed in MBR where proteins extracted from AS fed with SWW were composed of three high MW fractions (>600 kDa, 330 kDa, 41 kDa) whereas the only one for MBR acclimated with RWW was the one with MW higher than 600 kDa. The appearance of those big proteins with high MW in MBR fed with SWW could be due not to simple protein structure but to a combination of proteins with polysaccharides with loosely attached links in MBR system. A larger number of small proteic fractions (<2.5 kDa) was measured in SMP extracted from MBR fed with RWW (8 fractions) compared to proteins extracted from SMP of SWW acclimation (5 fractions). Humic-like substances extracted from SWW were composed of numerous small fractions (7 fractions) whereas a bimodal distribution was observed for humic-like substances from RWW. If these fractions were more numerous and smaller in MBR fed with SWW, the ones related to the MBR acclimated with RWW were composed of two fractions with approximately the same MW as those observed in the RWW. They were also composed of two fractions with higher MW which did not exist in the RWW. Both new fractions of MW respectively of 4.4 and 2.6 kDa were then generated in MBR process and certainly related to the sorption phenomenon of humic-like substances. Furthermore, divalent cation concentrations (e.g. Mg2+ and Ca2+) as previously mentioned were higher in RWW than in SWW. However, humic-like substances can form stable complex with these cations. Therefore, the presence of a higher amount of these divalent cations could make humic-like substances conglomeration easier. At stable state, the mean diameter of flocs obtained during acclimation with RWW was 35 ± 4 lm whereas it was 78 ± 6 lm when SWW fed MBR. This result contradicted the higher divalent cation (Ca2+ and Mg2+) concentrations measured in RWW. Indeed higher amounts of Ca2+ and Mg2+ promoted flocs aggregation due to links created with negative charges on functional sites of SMP. One hypothesis to explain the smaller size of flocs coming from MBR fed with RWW was the SMP structure and the number of

242

M. Villain et al. / Bioresource Technology 155 (2014) 235–244 Table 4 MWD of proteins and humic-like substances extracted from SMP of RWW and SWW and SMP from bioreactors. Effluent type

Protein MW distribution

Substrate SWW RWW

4.6 kDa; 0.8 kDa; <0.4 kDa (6 peaks) >600 kDa; 50 kDa; 2.5 kDa; 1.1 kDa; <0.4 kDa (9 peaks)

Bioreactor SWW RWW

>600 kDa; 330 kDa; 41 kDa; 2.5 kDa; 1.3 kDa; 1.1 kDa; <0.4 kDa (2 peaks) >600 kDa; 2.5 kDa; 1.6kDa; 1.1 kDa; 1 kDa; 0.8 kDa; 0.6 kDa; <0.4 kDa (2 peaks)

Effluent type

Humic-like substances MW distribution

Substrate SWW RWW

2.6 kDa; 2.1 kDa; 1.2 kDa; <0.9 kDa (4 peaks) 2.4 kDa; 1.2 kDa

Bioreactor SWW RWW

1.9 kDa; 1.6 kDa; 1.2 kDa; <0.9 kDa (6 peaks) 4.4 kDa; 2.6 kDa; 2.1 kDa; 1.2 kDa

functional sites negatively charged allowing the creation of links between microbial aggregates. Furthermore the SMP total concentration was lower in MBR fed with RWW with lower protein content and a total absence of polysaccharides. Therefore flocs of MBR fed with RWW cannot be bigger because of a lack of SMP and as a consequence of functional sites with negative charges. A last possibility to explain this smaller size of flocs from MBR fed with RWW was the important presence of monovalent cations such as Na+ and K+. These cations could link functional sites with negative charges with SMP without the possibility to form bridges between bacterial aggregates. Other substrate and characteristics of the biological medium could explain this phenomenon. Polysaccharides were totally absent under the soluble form in the MBR fed with RWW. However these polymers could act as flocculation agents and induce an increase in the floc size if they were in large quantity. Massé (2004) pointed out that an increase in MLSS concentration from 5.5 to 7.8 g L1, D50 increased from 80 to 95 lm. MLSS concentration of MBR fed with RWW was 7.4 g L1 and 8.8 g L1 for acclimation with SWW. MLSS variation between both experiments certainly influence floc mean diameter. Structural characteristics of organic carbon source used as substrate revealed an impact on the floc size. Gabinska-Loniewska (1991) showed that AS fed with weak acids presented large flocs, whereas AS fed with alcohol exhibited the same size as flocs from conventional wastewater treatment plants. Mcadam et al. (2007) confirmed and added that biomass fed with acetic acid showed large flocs with a loosely structure sensitive to high shearing whereas with ethanol flocs were more compact and more resistant to high shearing. As a consequence, authors as Holakoo et al. (2007) preferably used glucose to feed biomass of their MBR rather than acetate to avoid any risk of additional fouling resulting from various small particles release. As a conclusion, organic carbon source can be a better explanation of the smaller size of flocs from RWW campaign than from SWW acclimation.

3.2.2. Consequences on membrane fouling After 30 d of filtration at stable state for both experiments, ceramic membranes were submitted to different cleanings in order to assess the fouling reversibility. With the use of the three different types of fouling defined by Meng et al. (2009), the fouling obtained during stable conditions for both effluents was characterized. These authors differentiate removable, irremovable and irreversible fouling. The first can be removed by hydraulic cleaning. Irremovable fouling cannot be removed by simple hydraulic cleaning and requires intensive chemical cleaning. Irreversible fouling cannot be removed by any approach.

To distinguish the three types of fouling, a succession of cleanings was applied. First a simple cleaning with distilled water was performed to remove removable fouling (15 min at 1.5 bar). After chemical cleanings (with NaOH 40 g L1 (1 h at 1.5 bar) followed by HNO3 concentrated at 68% (30 min at 1.5 bar)) were performed. Between each cleaning step, the membrane permeability was assessed. Hydraulic resistances resulting from each fouling type previously defined are calculated with Eq. (4) and their specific contributions obtained. Membrane hydraulic resistance was previously measured and is equal to 3.3  1012 m1:

Lp ¼

1

l  ðRm þ Rf Þ

ð4Þ

with Lp: membrane permeability (m3 m2 s1 Pa1), Rf: hydraulic resistance to the fouling (m1), Rm: membrane hydraulic resistance (m1) and l: dynamic viscosity of the permeate at 20 °C (Pa s).Hydraulic resistances called R0, R1 and R2 were calculated and corresponded respectively to the hydraulic resistance due to removable, irremovable and irreversible fouling (Table 5). Calculations of hydraulic resistances revealed that the major part of fouling can be attributed to irremovable fouling. Indeed it represents 92% and 94% of total fouling in the experiment performed respectively with SWW and with RWW. A low part of fouling can be considered as removable. 7% of total fouling in the case of campaign with SWW and 4% for RWW experiment. For irreversible fouling, it corresponds only to 1% of total fouling for SWW experiment and 2% for RWW campaign. Therefore, the most part of fouling can be considered as removable and chemical cleanings are necessary to remove it. Different mechanisms are implied in fouling establishment. Removable fouling is caused by the creation of a cake layer at the membrane surface. This type of fouling is due to particles with sizes much higher than the membrane cut off like for example bioflocs, colloids or biopolymers. Chu and Li (2005) and Sun et al. (2008) pointed out that the deposited biopolymers near the membrane surface allowed easier and faster bacterial adhesion. Furthermore, biopolymers held the flocs more tightly on the membrane and increased the difficulty to remove the cake by physical cleaning. Removable fouling was 3% higher on the membrane used for SWW experiment. However in SWW, a higher content of proteic compounds with high MW (between 40 and 600 kDa) was observed (Table 4). Sun et al. (2008) announced that biopolymers acted as a gel deposition which made impermeable cake development at the membrane surface. Therefore the additional 3% of removable fouling obtained during SWW experiment could be caused by the higher amount and diversity (e.g. size diversity) of biopolymers.

M. Villain et al. / Bioresource Technology 155 (2014) 235–244

However the fouling part due to removable fouling remained relatively low compared to the part obtained for irremovable fouling which was higher than 90% in both experiments. The rapid recirculation rate applied on membrane surroundings (4 m s1) limited the removable fouling establishment. Meng et al. (2009) said that irremovable fouling is caused by membrane pore blocking. This mechanism occurred for long term filtration and was related to bacterial flocs and SMP which piled up because they were bigger than membrane pores or because they were adsorbed on fouling matter already in presence. As soon as they were attached to the membrane surface, bacteria multiplication could occur and micro-organisms could develop their biopolymer network to form a fouling layer tightly attached on the membrane surface. Even if irremovable fouling proportion was almost the same for both campaigns, the fouling rate seven times higher for RWW study can be attributed to irremovable fouling. Although TMP were the same during stable state period for both experiments, permeate flux was double for RWW campaign. Therefore a bigger amount of particles (flocs, SMP, colloids) were brought to the membrane surface. Furthermore for RWW experiment, activated sludge flocs exhibited smaller sizes than the ones obtained with SWW. However Massé (2004) proved that membrane filtration realized with flocs with smaller dimensions led to a deposit with higher capacity to compress and so to an augmentation of fouling rate when pressure increased. SMP presence and especially proteins in deposit structure were demonstrated by Shiau et al. (2003). They brought the evidence that during filtration of protein solution, the rapid increase in fouling resistance was the result of deposit compression. This augmentation of fouling resistance occurred sooner when either applied pressure or protein concentration was higher. This result showed that a protein deposit is compressible and that deposit compressibility could be modified depending on the applied pressure or the protein concentration. Irreversible fouling is permanent and cannot be removed. The penetration inside membrane pores of particles with sizes smaller than membrane pores and their adsorption on membrane material constituted the main mechanism of irreversible fouling. It represented the lower part of fouling. It was of 1% and 2% for experiments performed respectively with SWW and RWW. The main part of proteins extracted from SMP was smaller (62.5 kDa) and the humic-like amount was higher during experiment with RWW. These results could be related to the slightly increase in irreversible fouling during RWW campaign. Thanks to cleaning steps with sodium hydroxide and nitric acid, the determination of organic and inorganic fouling repartition was possible. Organic fouling represented 87% of total fouling for acclimation achieved with SWW whereas it was 72% when MBR was fed with RWW. Organic fouling corresponded to the entire organic matter in MBR which can contribute to membrane fouling. Therefore organic fouling included EPS (mainly proteins, polysaccharides and humic-like substances) coming from effluent, bacterial metabolism, bioflocs and organic colloids. MBR working with SWW possessed a SMP content 63% higher than for RWW acclimation,

Table 5 Hydraulic resistances related to membrane fouling of campaigns realized with SWW and RWW. Hydraulic resistance

R0 (removable fouling) R1 (irremovable fouling) R2 (irreversible fouling)

Campaign SWW

Campaign RWW

(m1)

(%)

(m1)

(%)

3.6  1011 4.4  1012 2.1  1010

7 92 1

3.2  1011 6.7  1012 4.3  1010

4 94 2

243

with a strong presence of proteic biopolymers, a higher biomass concentration of 1.5 g L1. It seemed reasonable to find a higher organic fouling proportion in case of SWW experiment. Inorganic fouling during RWW study was twice (28% of total fouling) that observed for SWW experiment (13% of total fouling). Inorganic fouling could be under two forms: chemical or biological precipitation. In mixed liquor of MBR, various kinds of cationic and anionic species can be present as Ca2+, Mg2+, Al3+, Fe3+, 2 3  CO2 3 ; SO4 ; PO4 , OH , etc. 4. Conclusion This study allowed to identify some of the main consequences related to the initial effluent choice (SWW or RWW) on MBR performances and on the characteristics of biological media. This choice is not harmless and differences observed in term of characteristics of biological media were responsible for various fouling establishment in both cases. Therefore at lab scale, the best option to approach real MBR characteristics was like in this study to install a pilot near a wastewater treatment plant to receive RWW. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013.12. 063. References Bhatia, D., Bourven, I., Bordas, F., Simon, S., van Hullebusch, E., Rossano, S., Lens, P., Guibaud, G., 2013. Fluorescence detection to determine proteins and humic-like substances fingerprints of exopolymeric substances (EPS) from biological sludges performed by size exclusion chromatography (SEC). Bioresour. Technol. 131, 159–165. Braclow, U., Drews, A., Gnirss, R., Klamm, S., Lesjean, B., Stüber, J., Barjenbruch, M., Kraume, M., 2010. Influence of sludge loading and types of substrates on nutrients removal in MBRs. Desalination 250, 734–739. Bura, R., Cheung, M., Liao, B., Finlayson, J., Lee, B., Droppo, I., 1998. Composition of extracellular polymeric substances in the activated sludge floc matrix. Water Sci. Technol. 37 (4–5), 325–333. Chen, W., Wersterhoff, P., Leenheer, J., Booksh, K., 2003. Fluorescence excitationemission matrix regional interaction to quantify spectra for dissolved organic matter. Environ. Sci. Technol. 37, 5701–5710. Chu, H., Li, X., 2005. Membrane fouling in membrane bioreactor: sludge cake formation and fouling characteristics. Biotechnol. Bioeng. 90 (3), 323–331. Clouzot, L., Roche, N., Marrot, B., 2011. Effect of membrane bioreactor configurations on sludge structure and microbial activity. Bioresour. Technol. 102 (2), 975–981. Delgado, F.D., 2009. Bioréacteur à membrane externe pour le traitement d’effluents contenant des médicaments anticancéreux: Elimination et influence du cyclophosphamide et de ses principaux métabolites sur le procédé (Ph.D. thesis). INP Toulouse, France. Drews, A., Lee, C.-H., Kraume, M., 2006. Membrane fouling – a review on the role of EPS. Desalination 200 (1–3), 186–188. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350–356. Feng, S., Zhang, N., Liu, H., Du, X., Liu, Y., Lin, H., 2012. The effect of COD/N ratio on process performance and membrane fouling in a submerged bioreactor. Desalination 285, 232–238. Fishman, M.L., Cescutti, P., Fett, W.F., Osman, S.F., Hoagland, P.D., Chau, H.K., 1997. Screening the physical properties of novel Pseudomonas exopolysaccharides by HPSEC with muli-angle light scattering and viscosity detection. Carbohydr. Polym. 32, 213–221. Frolund, B., Griebe, T., Nielsen, P.H., 1995. Enzymatic activity in the activated-sludge floc matrix. Appl. Microbiol. Biotechnol. 43 (4), 755–761. Fu, Z., Yang, F., Zhou, F., Xue, Y., 2009. Control of COD/N ratio for nutrient removal in a modified membrane bioreactor (MBR) treating high strength wastewater. Bioresour. Technol. 100 (1), 136–141. Gabinska-Loniewska, A., 1991. Denitrification unit biocenosis. Water Res. 25 (12), 1565–1573. Guisasola, A., Petzet, S., Baeza, J.A., Carrera, J., Lafuente, J., 2007. Inorganic carbon limitations on nitrification: experimental assessment and modelling. Water Res. 41, 277–286. Guo, W.S., Vigneswaran, S., Ngo, H.H., Xing, W., 2007. Experimental investigation on acclimatized wastewater for membrane bioreactors. Desalination 207 (1–3), 383–391.

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