Samples were taken regularly from the individual four flasks to cover every other hour of the growth phase and samples were analyzed regarding the OD600, glucose and surfactin concentrations. The OD600 was determined using a spectrophotometer (Biochrom WPA CO8000, Biochrom Ltd., Cambridge, UK). Prior to further analysis, cells were removed by centrifuging for 10 min at 4700 rpm at 4 °C (Heraeus X3R, Thermo Fisher Scientific GmbH, Braunschweig, Germany).
Surfactin was analyzed using a HPTLC system (CAMAG, Muttenz, Switzerland) with a validated method as described previously (Geissler et al. 2017 (link)). In brief, a threefold extraction of 2 mL cell-free broth with each 2 mL chloroform/methanol 2:1 (v/v) was conducted. The pooled solvent layers obtained after each extraction were evaporated to dryness in a rotary evaporator (RVC2-25 Cdplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) at 10 mbar and 40 °C. For HPTLC analysis, samples were resuspended in 2 mL methanol and applied as 6 mm bands on HPTLC silica gel 60 plates from Merck (Darmstadt, Germany). A surfactin standard curve was applied in the range of 30–600 ng/band. The development was conducted using chloroform/methanol/water (65:25:4, v/v/v) over a migration distance of 60 mm. After the development, the plate was scanned at 195 nm to quantify surfactin.
Glucose concentrations were determined using a HPTLC method as well. Proper diluted cell-free supernatants were applied as 6 mm bands and the plate was developed with acetonitrile/H2O (85:15, v/v) over a migration distance of 70 mm. After development, the plate was dipped in the derivatization solution diphenylamine (DPA) for 3 s and the plate was heated for 20 min at 120 °C using the TLC plate heater. DPA reagent was prepared by first dissolving 2.4 g diphenylamine and 2.4 g aniline in 200 mL methanol and then adding 20 mL 85% phosphoric acid.
For further data analysis, the OD/cell dry weight (CDW) conversion factor was determined in a pre-liminary test. Therefore, the strains were cultivated as triplicates as described above for aerobic conditions until reaching the range of maximum OD600. 40 mL culture were filled in dried and pre-weighted falcons and centrifuged for 10 min at 4700 rpm and 4 °C. The supernatant was discarded, and the cell pellet was washed with saline solution prior to a second round of centrifugation. After discarding the supernatant, the weight of the cell pellets were determined after drying the loaded falcons at 110 °C for 24 h and the conversion factor was calculated. In this sense, the OD/CDW conversion factor for all strains used was determined as 3.76 ± 0.17 with a %RSD of 4.47%.
Surfactin was analyzed using a HPTLC system (CAMAG, Muttenz, Switzerland) with a validated method as described previously (Geissler et al. 2017 (link)). In brief, a threefold extraction of 2 mL cell-free broth with each 2 mL chloroform/methanol 2:1 (v/v) was conducted. The pooled solvent layers obtained after each extraction were evaporated to dryness in a rotary evaporator (RVC2-25 Cdplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode am Harz, Germany) at 10 mbar and 40 °C. For HPTLC analysis, samples were resuspended in 2 mL methanol and applied as 6 mm bands on HPTLC silica gel 60 plates from Merck (Darmstadt, Germany). A surfactin standard curve was applied in the range of 30–600 ng/band. The development was conducted using chloroform/methanol/water (65:25:4, v/v/v) over a migration distance of 60 mm. After the development, the plate was scanned at 195 nm to quantify surfactin.
Glucose concentrations were determined using a HPTLC method as well. Proper diluted cell-free supernatants were applied as 6 mm bands and the plate was developed with acetonitrile/H2O (85:15, v/v) over a migration distance of 70 mm. After development, the plate was dipped in the derivatization solution diphenylamine (DPA) for 3 s and the plate was heated for 20 min at 120 °C using the TLC plate heater. DPA reagent was prepared by first dissolving 2.4 g diphenylamine and 2.4 g aniline in 200 mL methanol and then adding 20 mL 85% phosphoric acid.
For further data analysis, the OD/cell dry weight (CDW) conversion factor was determined in a pre-liminary test. Therefore, the strains were cultivated as triplicates as described above for aerobic conditions until reaching the range of maximum OD600. 40 mL culture were filled in dried and pre-weighted falcons and centrifuged for 10 min at 4700 rpm and 4 °C. The supernatant was discarded, and the cell pellet was washed with saline solution prior to a second round of centrifugation. After discarding the supernatant, the weight of the cell pellets were determined after drying the loaded falcons at 110 °C for 24 h and the conversion factor was calculated. In this sense, the OD/CDW conversion factor for all strains used was determined as 3.76 ± 0.17 with a %RSD of 4.47%.
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