Pretreatment and Purification of Pulp Waste

The PW was processed according to Kubisch and Ochsenreither [47 (link)] prior to cultivation. Potassium hydroxide was used to adjust the pH of 500 mL PW to 6.5–7. Afterwards, the PW was centrifuged at 4000 g for 30 min. The supernatant was filtered with a funnel filter. The filtered PW was vacuum concentrated with a rotary evaporator ‘Hei-VAP Core’ (Heidolph Instruments GmbH & Co. KG; Kelheim, Germany) at 80 °C. The pressure was reduced every 30 min from 400 to 200 mbar and maintained at that pressure for 4 h. The remaining solid was resuspended in 500 mL deionized water and sonicated for 30 min. The pH was further increased to 13 by adding solid potassium hydroxide and the PW was boiled in a closed bottle at 100 °C for 2 h. After cooling to RT, the processed PW was filtered with a funnel filter and neutralized by addition of 96% (w/v) H₂SO₄. After repeated filtration, 10% (w/v) activated carbon was mixed with PW on a magnetic stirrer and incubated for 10 min at RT. The activated carbon was separated from the PW by centrifugation at 4000 g for 30 min at RT. The supernatant was filtered as described above and sterile filtered with a 0.2 µm PES membrane filtration cup (VWR; Darmstadt, Germany). The processed PW was stored at 4 °C until further use.