2.4. Batch fermentation
A loopful of pure seed culture taken from single colony on a
LB streak plate was inoculated into 50 ml sterilized Luria–Bertani
(LB) broth medium for 12–16 h overnight (O/N) preculture. Biomass
inoculated into 100 ml nitrogen-limited or phosphorus-limited MR
medium containing TES-I and TES-II and lauric acid for 72 h culture
at 30 ◦C, 200 rpm. Note that compared to other carbon sources
lauric acid was found to be more appropriate carbon source for PHA production (data not shown). After batch fermentation, cultured
broths were aseptically cooled to 4 ◦C in order to completely
solidify lauric acid (C12). Next, filter papers (ADVANTEC, NO. 5A,
7 mm) were used to separate biomass from solid particles of lauric
acid. To remove residual abiotic ash from biomass, biomass solutions
were three-times washed with distilled and deionized water,
centrifuged (10,000 rpm, 10 min), and then the supernatants were
discarded. The harvested biomass was then dried at 100 ◦C until
constant weight was achieved for PHA-content analysis afterwards.