*3.7. Sample Preparation for Chemical Identification*

For chemical identification in the liquid sample, the nine Indonesian isolates were each cultivated in 50 mL of NL410 medium. After 48 h, 10 mL of the preculture was inoculated into 100 mL of optimal production medium. The 100 mL whole broth of each cell culture was extracted as described above. Then, the extracts were used for further experiment. For chemical identification from samples grown on solid medium, overgrown agar was cut into pieces and transferred to 50 mL Falcon tubes. The Falcon tubes were centrifuged at 13,000 rpm for 30 min at room temperature. The aqueous phase was concentrated to 1/5 of the original volume in the Genevac Centrifugal Evaporator EZ-2 Elite (SP Scientific). The concentrated aqueous phase was used for further chemical profiling.

The culture extract samples obtained from liquid medium extraction and the aqueous phase of the solid medium extraction were separated by solid-phase extraction (SPE) columns. The columns were washed twice with 2 mL methanol and 2 mL distilled water for activating the columns. The samples were prepared by adding 100% methanol to the culture extract samples and the aqueous phase until the samples were dissolved completely. The methanolic samples were applied onto the activated columns with a flow rate of 2 mL/min. The column was washed twice with distilled water. The column was eluted consecutively with 2 mL of 100% methanol, 50% methanol, and distilled water. Samples from the elution column were defined as fractions. The column was eluted with 100% methanol as the 100% fraction, with 50% methanol as the 50% fraction, and distilled water

as the distilled water fraction. The fractions were dried in the Genevac EZ-2 Elite (SP Scientific) and then dissolved with 0.5 mL methanol. The crude extracts and all fractions were analyzed with HPLC and high-resolution mass spectrometry (HRMS).
