2 l bioreactor

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.

TML3 was found to be the most potent antifungal producer and this was further cultured in bulk amounts to obtain the bioactive compounds for further assessments. The fungal hyphal blocks were transferred to 1.5 L of potato dextrose broth in a 2 L bioreactor (Eyela, Japan) and were fermented for 5 days under agitation at 120 rpm. The fungal culture broth was filtered using Whatman filter paper no 4. The cell-free culture extract was mixed with a double volume of ethyl acetate (EA) and stirred overnight using a magnetic stirrer. The EA part was separated using a separating funnel and was subjected to evaporation using a Rotary evaporator under low pressure and temperature. The remnants were dissolved in Dimethyl sulfoxide (DMSO) and stored for further bioactivity studies.