A customized flat sheet type membrane module with an effective membrane area of 0.0045 m2 (0.09 m × 0.05 m) was submerged in a column type (polymethyl methacrylate) (PMMA) reactor (Fig. S2 ). The membrane module was manufactured by MemSis Turkey and employed a polysulfone (PS) ultrafiltration (UF) membrane (PHILOS, Korea) with 20 KDa of molecular weight cut-off (MWCO).
The system was operated in a gravity-driven filtration mode where the effluent (or permeate) was collected from the bottom of the tank, and more details can be found elsewhere24 (link), 25 (link). A synthetic secondary wastewater effluent (SSWE) was pumped continuously into a level regulator to keep the level of the feed water in the tank constant, resulting in a constant pressure head of 45 cm above the membrane (corresponding to a TMP of 4.5 kPa). Only a small quantity of activated sludge (4 mg of MLSS corrected from wastewater treatment plant at KAUST in Saudi Arabia) was initially added to the filtration tank to enhance the formation of a biofilm on the membrane surface. The whole reactor was covered with aluminum foil to elude the growth of algae by light exposure. The experiment was divided into two sets. The first set was conducted for 7 d with the aim to observe the initial stages of biofouling formation, and the second one was continued for 42 d to observe more long-term biofilm developments.
A SSWE was used as feed solution to grow the biofilm on the membrane. The detailed characteristics of feed water can be found elsewhere26 (link). The feed solution was refreshed every 7 d. Chemical oxygen demand (COD) of the SSWE was 7.5 mg/L for the first set of experiments. In order to enhance the biofilm formation, for the second experiment the COD concentration of the feed water was increased to 15 mg/L.
The permeate flow rate was measured using a flow meter (Sensirion). The permeate flux was calculated by dividing the permeate flow by the membrane area (0.0045 m2). As mentioned above, the GD-SMBR was operated under constant pressure by maintaining a constant water head as shown in Fig.S2 . A constant TMP of 4.5 kPa was applied to all experiments.
In this study, the OCT was employed to investigate the biofilm formation (or growth) on a membrane (submerged) in a GD-SMBR. The OCT (Thorlabs GANYMEDE spectral domain OCT system with a central wavelength of 930, Thorlabs, GmbH, Dachau, Germany) equipped with a 5X telecentric scan lens (Thorlabs LSM 03BB) was used.
The time-resolved OCT investigation was performed in three different periods of the filtration runs as given in TableS1 . In the first experiment (Experiment 1), a fixed position corresponding to 5.00 mm × 1.35 (width × depth) was monitored. The scan frequency was set to 10 min for the first 42 h (from 12 h to 42 h), and then 5 min from 84 h to 96 h. The second experiment (Experiment 2) was conducted for a period of 42 d, where OCT scans were acquired daily at a fixed position corresponding to 4.00 mm × 1.35 mm.
Serial static images (i.e. video) are commonly used to depict the dynamic process of fouling formation. In this study, three videos (Supplementary Videos1 –3 ) of the periods monitored (Table S1 ) are shown in supporting information. The preprocessed OCT scans were assembled into AVI digital movie format using Fiji software.
The system was operated in a gravity-driven filtration mode where the effluent (or permeate) was collected from the bottom of the tank, and more details can be found elsewhere24 (link), 25 (link). A synthetic secondary wastewater effluent (SSWE) was pumped continuously into a level regulator to keep the level of the feed water in the tank constant, resulting in a constant pressure head of 45 cm above the membrane (corresponding to a TMP of 4.5 kPa). Only a small quantity of activated sludge (4 mg of MLSS corrected from wastewater treatment plant at KAUST in Saudi Arabia) was initially added to the filtration tank to enhance the formation of a biofilm on the membrane surface. The whole reactor was covered with aluminum foil to elude the growth of algae by light exposure. The experiment was divided into two sets. The first set was conducted for 7 d with the aim to observe the initial stages of biofouling formation, and the second one was continued for 42 d to observe more long-term biofilm developments.
A SSWE was used as feed solution to grow the biofilm on the membrane. The detailed characteristics of feed water can be found elsewhere26 (link). The feed solution was refreshed every 7 d. Chemical oxygen demand (COD) of the SSWE was 7.5 mg/L for the first set of experiments. In order to enhance the biofilm formation, for the second experiment the COD concentration of the feed water was increased to 15 mg/L.
The permeate flow rate was measured using a flow meter (Sensirion). The permeate flux was calculated by dividing the permeate flow by the membrane area (0.0045 m2). As mentioned above, the GD-SMBR was operated under constant pressure by maintaining a constant water head as shown in Fig.
In this study, the OCT was employed to investigate the biofilm formation (or growth) on a membrane (submerged) in a GD-SMBR. The OCT (Thorlabs GANYMEDE spectral domain OCT system with a central wavelength of 930, Thorlabs, GmbH, Dachau, Germany) equipped with a 5X telecentric scan lens (Thorlabs LSM 03BB) was used.
The time-resolved OCT investigation was performed in three different periods of the filtration runs as given in Table
Serial static images (i.e. video) are commonly used to depict the dynamic process of fouling formation. In this study, three videos (Supplementary Videos
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