Supra 55

Five substrates were selected from our library and fabricated according to our previous protocol (Wang et al., 2016b (link)) (Figure 1A). Briefly, cSAPs #1 and #2 was composed of SiO₂ with 5 μm diameter and polystyrene (PS) with 200 or 400 nm diameter, #3, #4, and #5 was composed of SiO₂ with 2 μm diameter and PS with 65 nm diameter, carboxy-PS (PSC) with 50 or 100 nm diameter, and the cSAPs were fabricated within the 24-well tissue culture plates (TCPS, Falcon). TCPS was the flat control group in this study.
Scanning electron microscopy (SEM, ZEISS SUPRA ® 55, Carl Zeiss, Germany) and water contact angle (WCA) was used to characterize the surface structures and wettability of these cSAPs using an automated contact angle measurement device (PT-705B, Precise Test, China) at room temperature. Other detailed characterizations have been done previously (Wang et al., 2016b (link)).

Fibroblasts (14,000 cells/cm²) were seeded on cover slips in complete growth medium and underwent the same treatment as described above. Coverslips were decellularized and fixed in 2% PFA and 0.2% glutaraldehyde and prepared for SEM. Cover slips were washed with 0.1 M cacodylate buffer and incubated with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at room temperature. Then, cover slipes were washed with water and dehydrated with 30, 50 and 75% ethanol (EtOH), 15 min per each step, respectively, followed by dehydration in 100% EtOH for 30 min. To dry the samples, cover slips were first incubated in a 1:1 mixture of 100% EtOH and tetramethyl silane (TMS) for 10 min followed by 15 min incubation with 100% TMS after which the cover slips were air dried. Afterwards cover slips were gold coated using a sputter coater and cover slips were imaged using a Zeiss Supra55 at 3 KV with SE2 detector (Carl Zeiss, Breda, The Netherlands).

SEM/EDS-analysis (scanning electron microscopy and energy dispersive spectroscopy) was conducted to obtain morphology and elemental information. Cross-sections were analyzed using a LEO 1530 instrument (Zeiss, Oberkochen, Germany) with a Gemini column, upgraded to a Zeiss Supra 55 (Zeiss) and an EDS X-Max SDD (Silicon Drift Detector) 50 mm × 50 mm detector from Oxford Instruments (Oxford, UK). All analyses were performed by means of a FEI-XL 30 Series instrument (Oxford, UK), equipped with an EDS system (EDAX Phoenix) with an ultra-thin windows Si-Li detector. All images were obtained using backscattered electrons at an accelerating voltage of 15 kV. The data of EDS elemental composition was collected by Oxford Instruments INCA 5.04.

XRD patterns were taken using an X’Pert-Pro MPD diffractometer (Netherlands PANalytical) with a Cu Kα X-ray source (λ = 1.540598 Å). TEM images and TEM-EDS mapping were taken with an FEI Tecnai G2 F20 S-TWIN TMP microscope (200 kV). SEM image and SEM-EDS mapping were taken with a Zeiss scanning electron microscope (Zeiss Supra55). We note that three different regions with a size of ~5 μm × 5 μm for the samples were used for the EDS measurement; the O/F ratio was obtained by averaging these data, and determined to be (12.3 ± 2.0)%. XPS was performed on a Rigaku XPS-7000 spectrometer. The carbon peak at 284.6 eV was used as a reference to correct the charging effect.

The morphology the membranes before and after filtration was analyzed by scanning electron microscope (SEM, Zeiss supra-55, Carl Zeiss AG, Oberkochen, Germany). Before characterization, the sample with length of 10 cm was cut from the membrane module and soaked in deionized water for 2 days. Then, the residual water on the membranes was removed with the wiping paper, and the membranes were frozen in a low-temperature refrigerator at −20 °C for 40 min. After that, the samples were dried in a freeze dryer. Prior to the observation, the samples were sputtered with nano-gold.
During the experimental process, the flux of the membranes was tested as follows: the permeate water was collected for 30 min at a fixed time every day, and the flux was calculated by the Equation (1): $$J=\frac{V}{t\times A}$$
where J is the membrane flux (L/(m² h)), V is the permeate volume (L), t is the filtration time (h) and A is the effective area of the membrane (0.726 m²). Meanwhile, the transmembrane pressure difference (TMP) is recorded every 6 min to obtain five values TMP₁~TMP₅, and the average value was used as the TMP of this experiment.

In this study, SPs and SRCs were used as the reactor fillers. The surface morphology and microorganism morphology on the surface of the fillers were observed and analyzed by scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM-EDS) (Zeiss-Supra 55, Caise, Jena, Germany) [33 (link)]. And the specific procedure for the sample pretreatment is in the Supplementary Information.