U3000

GA3 was analyzed by high-performance liquid chromatography (Dionex U3000) equipped with a Venusil MPC18 column (5 μm, Agela Technologies). The pretreated samples were separated using a mobile phase composed of methanol/water/phosphoric acid (68:32:0.05) at a flow rate of 0.7 mL/min. The detection wavelength was 210 nm, and the injection volume was 10 μL. The retention time of GA3 was 24.12 min. The standard curve was prepared by diluting the 0.1 g GA3 standard with 10 ml methanol to yield solutions of 50, 100, 200, 400, 600, and 800 mg/L. Then, the standard solutions were filtered through a 0.22 μm pore-size organic membrane for liquid phase analysis. The standard curve was made, and the formula was as follows:
Y = 0.1256*X-0.2476 (R² = 0.9991),
where Y is the peak area and X represents the concentration of GA3.

The LC system consisted of a U3000-Dionex apparatus with an injector comprising a 1 µL loop and a UV detector at 280 nm. The analytical column used was an Acclaim mixed-mode HILIC-1 (Thermo Scientific, Bellefonte, PA, USA) ID 2.1 mm column (150 mm × 5 µm × 120 Å) and eluted at a flow rate of 200 µL/min using a gradient ranging from 2% solvent B to 25% solvent B in a time span of 26 min and 30 s. Solvent A consisted of pure methanol and solvent B consisted of 10 mM ammonium formate in water at pH 6.8. The Electrospray Ionization—High Resolution Mass Spectrometry (ESI-HRMS) was a micrOTOF_Q^TM apparatus (Bruker Daltonics, Bruker, Bremen, Germany).
The GC system consisted to QP2010-Shimadzu equipment (Shimadzu, Kyoto, Japan) operating in the EI mode at 70 eV. An SLB5 column DB-5 ms (30 m, 0.25 mm film thickness) was employed with a 36 min temperature program of 60–320 at 10 °C/min followed by a 10 min hold at 320 °C. The injector temperature was 250 °C; the flow rate of the carrier gas (helium) was 1 mL/min; and the split ratio was 1:50. The interval of the scan m/z was between 35 and 900 and the identification of the compounds was based on an individual spectrum comparison of each compound in the Shimadzu NIST08 data. The retention index was calculated using the mixture of alkanes standard from C10 to C40 (Merck KGaA, Darmstadt, Germany).

Samples were withdrawn from the fermenter (Erlenmeyer flask) for analysis at regular intervals, and 50 mL of suspensions was collected by centrifugation (10,000 × g, 5 min), which was carried out in triplicate. The kind of oligosaccharides in the supernatant was identified by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC; Dionex, U-3000, United States) using a PA-100 anion exchange chromatography column (250 mm × 4 mm, CarboPac, Dionex, United States). The mobile phase was 100 mM NaOH and 150 mM NaAc. The mobile phase was filtered with a 0.22-μm microfiltration membrane before use. The sample injection volume was set to be 25 μL, and the column was eluted at 25°C with a flow rate of 0.25 mL/min using a pulse ampere detector (PAD).
Pure neoagarobiose, agarotriose, neoagarotetraose, agaropentaose, neoagarohexaose, agaroheptaose, neoagarooctaose, agarononaose, neoagarodecaose, agaroundecaose, and neoagarododecaose (Qingdao BZ Oligo Biotech Co., Ltd., China) were used as reference standards in the TLC experiment. Pure neoagarobiose, agarotriose, neoagarotetraose, agaropentaose, and neoagarohexaose (Qingdao BZ Oligo Biotech Co., Ltd., China) were used as reference standards in HPLC.

Surfactin samples were obtained from fermentation broths and then flocculated with chitosan (0.5 g/L), and sodium alginate (0.3 g/L), pH = 5.0, before being dissolved in 100% ethanol and freeze-dried. High performance liquid chromatography (HPLC) (U-3000, Dionex, Sunnyvale, CA, USA) was carried out equipped with an Agilent C18 column (4.5 mm × 250 mm, Agilent, Palo Alto, CA, USA) and a UV detector was used to identify the surfactin sample [49 (link)]. In brief, the surfactin sample was injected into the column and then eluted with acetonitrile with 0.1% TFA at a flow rate of 0.84 mL/min. Eluent absorbance was monitored at 210 nm. The purity of surfactin was approximately 88.6% (the result is shown in the Figure S5) after purification [13 (link)], and the collected surfactin dry powder was stored at 4 °C for following tests.