The surfactin production was quantified according to a published HPLC method (Zhu et al., 2014 ). Briefly, 1 ml samples were extracted from the shake flasks, and a cell‐free supernatant was obtained by centrifugation at 10,000 g for 10 min. An aliquot comprising 300 µl of the supernatant was withdrawn, diluted fivefold with methanol, and shaken for 1 min. Then, the precipitate of samples was removed through centrifugation for 2 hr at 10,000 g under 4°C.
Subsequently, the supernatant needed to be further filtered by a 0.22 μm nylon membrane remove impurities. The final sample was analyzed by HPLC on a U‐3000 system (Thermo Fisher Scientific, USA) equipped with a Synchronis C18 column (4.6 × 250 mm, 5 μm; Thermo Fisher Scientific) and a UV detector (Thermo Fisher Scientific). The analytes were detected at 214 nm, using 90% (v/v) methanol, 10% (v/v) water, and 0.05% trifluoroacetic acid as mobile phase with a flow rate of 0.8 ml/min. The authentic surfactin (98%) reference standard was purchased from Sigma‐Aldrich, USA. The proportions of surfactin isoforms were analyzed by comparing the peak areas observed by HPLC to the sum of areas of all surfactin peaks.
Subsequently, the supernatant needed to be further filtered by a 0.22 μm nylon membrane remove impurities. The final sample was analyzed by HPLC on a U‐3000 system (Thermo Fisher Scientific, USA) equipped with a Synchronis C18 column (4.6 × 250 mm, 5 μm; Thermo Fisher Scientific) and a UV detector (Thermo Fisher Scientific). The analytes were detected at 214 nm, using 90% (v/v) methanol, 10% (v/v) water, and 0.05% trifluoroacetic acid as mobile phase with a flow rate of 0.8 ml/min. The authentic surfactin (98%) reference standard was purchased from Sigma‐Aldrich, USA. The proportions of surfactin isoforms were analyzed by comparing the peak areas observed by HPLC to the sum of areas of all surfactin peaks.