*3.6. Cultivation Conditions for Optimal Compound Production of Nine Indonesian Strains*

To determine optimal cultivation conditions in liquid culture, the nine Indonesian actinomycetes strains, SHP 22-7, DHE 17-7, DHE 7-1, BSE 7-9, BSE 7F, I3, I4, I5, and I6, were each cultivated in 50 mL inoculum medium (NL410) in 500-mL Erlenmeyer flasks (with steel springs) in an orbital shaker (180 rpm) at 28 ◦C. After 48 h, 10 mL of preculture was inoculated into 100 mL of twelve different production medium (SGG, YM, OM, R5, MS, TSG, NL19, NL300, NL330, NL500, NL550, and NL800 (Table S2) and cultivated for 48–168 h. Cell culture samples were harvested at different time points (48, 72, 96, and 168 h). In addition, 5 mL of each cell culture sample was extracted with the same volume of ethyl acetate (EtOAc) for 30 min at room temperature. The EtOAc was dried in a rotary evaporator and suspended in a total volume of 0.75 mL methanol. The methanolic extracts were used for bioassay experiments. The culture extract samples, which yielded the largest zone of inhibition in the bioassays against the test organisms, were used for further compound identification analysis. To determine optimal cultivation conditions on solid culture, the nine Indonesian strains were each spread on 100 mL agar plates consisting of the respective abovementioned cultivation media and then incubated for 7–10 days at 28 ◦C until spore formation was visible on agar plates. The overgrown agar was then used for bioassay experiments and further compound identification analysis.
