0.22 mm sterilized nylon driven filters

Commercial cellulase (Celluclast 1.5 L, Novozymes, Denmark) was used for the saccharification of pretreated OPEFB into fermentable sugar. The saccharification was conducted at 1.5 L working volume in a 2-L bioreactor (EYELA, Japan) with an agitation speed of 150 rpm, the temperature at 35 °C for 96 h. The process was conducted by adding 5% of pretreated OPEFB into the 2-L bioreactor and autoclaved at 121 °C for 15 min. Approximately 1.5 L of sterilized 0.05 M acetate buffer, pH 5.5 was added into the bioreactor. Then, 15 FPU/mL of prepared cellulase solution sterilized via filtration using 0.22 mm sterilized nylon driven filters (Millipore, Denmark) was added into the bioreactor before sparged with nitrogen gas for about 1 h until no oxygen was detected. Samples were collected every 24 h and kept in the freezer of −20 °C for sugar analysis.

This SSF process was conducted based on the optimal conditions reported by Razali et al.^{17 (link)}. SSF was conducted by adding 5% of pretreated OPEFB into a 2-L bioreactor and autoclaved at 121 °C for 15 min. A 6 g/L of sterilized yeast extract solution and 0.05 M of acetate buffer pH 5.5 were added into the bioreactor. Then, 15 FPU/mL of prepared cellulase was sterilized via filtration using 0.22 mm sterilized nylon driven filters (Millipore, Denmark) before it was added into a bioreactor together with 2 mL of each P2 medium component. All the mixtures were sparged with nitrogen gas for about 1 h until no oxygen was detected. The SSF was initiated by inoculating 15% of prepared inoculum (with the OD₆₂₀ set at 1.0) into the bioreactor. The inoculated medium was incubated at 35 °C with an agitation speed of 150 rpm for 144 h. A 2 ml of the liquid sample from each fermentation bottle was withdrawn using a syringe and kept at −20 °C prior to sample analysis.