Stirred cell

The PNIPAm-functionalized membrane was placed in a stirred cell acquired from Millipore in order to study its temperature responsive flux behavior. The cell was filled with DIUF and temperature was maintained using electrical heating tape. The cell had a digital thermocouple that enabled continuous monitoring of the temperature of the DIUF inside the cell. UHP nitrogen was used to pressurize the cell, which had a maximum pressure limit of 5.5 bars. Whenever the pressure was varied, water flux through the membrane was allowed to reach steady state before any samples were taken. When the temperature was varied, samples were only taken once the permeated water temperature was equal to the cell’s internal temperature. Triplicate samples were taken to measure water flux by measuring permeated volume versus permeation time. Final runs were always conducted at conditions equal to the first run in order to test reversibility. Flux tests were performed using pure water at both 22 °C and at 35 °C with varying pressure in order to test the stability of the membrane, and can be found in the supporting information.. In order to further examine the temperature responsive nature of the PNIPAm functionalized membrane, the pressure was held constant and flux was measured as temperature was varied from 22 °C to 40 °C.

A. niger (ATCC11414) was maintained on complete agar medium, which is widely used for fungal growth (Bennette and Lasure, 1991 ). Conidia of spore inocula were grown on complete medium at 30 °C and harvested after 4 days. 1 × 10⁶ spores/ml were inoculated into 2 × 200 ml of modified minimal medium as described previously (Wang et al., 2011b (link)). The cultivation was performed at 30 °C in a 1-L baffled flask in the incubator shaker (New Brunswick Scientific) at 200 rpm. After 24 h of growth, the supernatant was collected by filtering the culture through two layers of sterile miracloth and then centrifuging it at 15,000g at 4 °C for 10 min to remove cell debris. About 400 ml of supernatant was concentrated to 20 ml in a stirred cell (Millipore, Billerica, Massachusetts, USA) with an ultrafiltration membrane (NMWL 3 kDa, Millipore, Billerica, Massachusetts, USA) overnight at 4 °C with continuous pressure of 40 psi N₂. Cold (−20 °C) acetone was mixed with the protein solution at a volume ratio of 5:1, and incubated at −20 °C for 1 h. The precipitated proteins (~30 mg) were harvested by centrifugation at 18, 000g for 15 min at 4 °C.