Alliance series 2695 system

Purification of viscosin was performed as described previously by Bak et al. (2015) (link). Briefly, P. fluorescens SBW25 was cultivated on King's B agar plates (King et al., 1954 (link)) in darkness at 28 °C for 1 day before being transferred to 20 °C and incubated for another 3 days. Colony material was suspended in demineralized water (MilliQ; Millipore) and homogenized by shaking. Cells and supernatant were separated twice by centrifugation at 4700 r.p.m. for 20 min at 4 °C in a Sigma 3-18K centrifuge (Sciquip). The supernatant was acidified to pH 2.0 with 1 M HCl and left overnight on ice for a precipitate to form. The solution including the precipitate was centrifuged for 27 min at 7000 r.p.m. and 4 °C in a Sigma 3-18K centrifuge. The supernatant was discarded and the precipitate was washed four times with MilliQ water at pH 2.0. The precipitate was dissolved in MilliQ water and pH was adjusted to 8.0 with 1 M NaOH to fully dissolve the precipitate. The solution was lyophilized and the purity of the lipopeptide preparations was verified by HPLC. HPLC analysis was carried out using a Waters Alliance series 2695 system and a Waters model 996 photodiode array detector. The procedure was carried out as described previously by Bak et al. (2015) (link). The same HPLC protocol was used for quantification of viscosin produced in liquid cultures of P. fluorescens SBW25.

High-performance liquid chromatography (HPLC) analysis was carried out using a Waters Alliance series 2695 system and a Waters model 996 photodiode array detector (www.waters.com). Chromatographic analysis of the lipopeptides followed the protocol by Nielsen and Sørensen (2003 (link)) with minor modifications. Briefly, a Hypersil BDS C₁₈ column (100 × 4.6 mm and 3 μm particle size) (www.thermoscientific.com) was used for separation of the lipopeptides. Solvents were HPLC-grade acetonitrile (solvent A) and 0.1 % o-phosphoric acid (solvent B), mixed in a linear gradient of 15 to 100 % solvent A from 0 to 40 min, and of 100 to 15 % solvent A between 40 and 44 min. The flow rate was 1 ml/min, and the column temperature was 40 °C. The injected sample volume was 10 μl. The lipopeptides were monitored at wavelengths of 190 to 250 nm. For quantification of viscosin in cultures of the diesel degrading consortium, a wavelength of 220 nm was applied and viscosin concentrations were calculated using purified viscosin as a standard. Handling of chromatographic data was performed using Waters Empower 2 software.