Rotilabo syringe filter

Consumption of the carbon source methanol and production of lactate was monitored using high-performance liquid chromatography (HPLC-UV-RI). To this end, 1 mL of culture was centrifuged at maximum speed and the supernatant was filtered with Rotilabo syringe filters (Carl Roth, pore size 0.2 µm). The filtered supernatants were subsequently analyzed with a DIONEX UltiMate 3000 HPLC System (Thermo Fisher Scientific) equipped with a Metab-AAC column (300 × 7.8 mm, ISERA, Düren, Germany). For elution 5 mM H₂SO₄ at a flow rate of 0.6 mL/min and a temperature of 60 °C was used. Detection was performed with a SHODEX RI-101 detector (Showa Denko Europe GmbH, München, Germany) and a DIONEX UltiMate 3000 Variable Wavelength Detector (Thermo Fisher Scientific) set to 210 nm.

After 4 days of cultivation the cultures were homogenized using an Ultra-Turrax at 16000 rpm (T25 basic IKA Werke, Germany) until complete homogenization was obtained (after approximately 20 seconds). 100 mL of ethyl acetate were added and a second homogenization for approximately 20 seconds was performed. The mixture was transferred to a separation funnel. The solvent phase was given into a round bottom flask. The evaporation of the solvent was carried out using a rotary evaporator. The dry extracts were then resolved in 1 mL of methanol and filtered through a 0.2 µm PTFE filter (Rotilabo-syringe filters, ROTH) via a syringe.

To facilitate homogenous hydrogel solutions, dried ADA with a degree of oxidation (%DO) of 13% was dissolved in Dulbecco’s phosphate buffered saline (DPBS) and stirred overnight, yielding a 5% (w/v) solution. Further, solid PEG-diol (molecular weight = 1–8 kDa) as well as PEG-diamine (molecular weight = 1–8 kDa) were dissolved in DPBS yielding PEG solutions. The concentration of each solution was determined so that all aldehyde groups present in ADA were sufficiently intercepted by possible amine groups present in PEG-diamines. Values for PEG-diols were derived from the corresponding PEG-diamine ones. In Table S1, detailed information about the used concentrations can be found. Then, all PEG solutions were combined with ADA solutions in a ratio of 1:1 yielding 4 mL inks and stirred for 10 min at 37 °C, respectively, yielding either a hydrogel system with (+) or without (-) Schiff’s base formation, namely, ADA-PEG(+) or ADA-PEG(-) (Table 2). For cell experiments, all hydrogels were additionally sterile filtered using Millipore filters (Rotilabo-syringe filters, PDVDF, Carl Roth, Germany) with pore diameters of either 0.22 µm (for PEG solutions) or 0.45 µm (for ADA solutions).