Chemicals
All chemicals applied in the current study were of analytical grade and purchased from Carl Roth GmbH (Karlsruhe, Germany). The Surfactin standards for HPLC analysis were obtained from Sigma-Aldrich Laborchemikalien GmbH (Seelze, Germany).
Microorganism and strain maintenance
The wildtype strain Bacillus subtilis DSM 10T was used for all experiments during this study. The microorganism was obtained from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) and stored as glycerol stocks, prepared from a culture in Lysogeny Broth (Bertani 1951) from the exponential growth phase, at −80°C.
Culture conditions
Media
The employed mineral salt medium was based on the fermentation medium of Cooper (Cooper et al. 1981): 8.0 × 10−4 M MgSO4, 7.0 × 10−6 M CaCl2, 4.0 × 10−6 M FeSO4, 4.0 × 10−6 M Na2EDTA, 1 × 10−6 M MnSO4. In contrast to the original medium (40 g/L glucose) the concentration of glucose was altered to 2.5 g/L, 5 g/L, 7.5 g/L and 10 g/L, during various cultivations. Furthermore, the former nitrogen source 0.05 M NH4NO3 was replaced with 0.1 M NH4Cl and 0.1177 M NaNO3. The deployed concentration of the phosphate buffer demanded slight changes depending on its usage for inoculum cultures or fermentation processes. For the cultivation in serum bottles the original 0.07 M phosphate buffer (0.03 M KH2PO4 and 0.04 M Na2HPO4) was used, whereas for the cultivation in benchtop bioreactors a 0.01 M phosphate buffer was employed (4.29 × 10−3 M KH2PO4 and 5.71 × 10−3 M Na2HPO4).
The preparation of medium for the cultivation in serum bottles demanded a different approach compared to the preparation of medium for the cultivation in benchtop bioreactors. Four different stock solutions were prepared for the cultivation in serum bottles. One stock solution contained the salt compounds (NH4Cl, NaNO3, KH2PO4, Na2HPO4) and was later completed to the final volume of 50 or 100 mL, respectively. The second stock solution included a 5.56-fold glucose solution of the final glucose concentration. In comparison, the third and fourth stock solution contained a 50-fold MgSO4 solution and a 1000-fold solution of the trace elements (CaCl2, FeSO4, Na2EDTA, MnSO4). All solutions were filled into separate serum bottles and anaerobic conditions were adjusted by 20 alternating cycles of purging with gas (20 vol.-% CO2 in N2, 45 s) and evacuating (70 mbar, 45 s). Subsequently the bottles were autoclaved and the salt stock solution was completed under anaerobic conditions to receive the final concentrations of glucose, MgSO4 and trace elements.
For the preparation of the bioreactor medium four stock solutions were prepared in a similar fashion. However, the first stock solution (NH4Cl, NaNO3, KH2PO4, Na2HPO4) was autoclaved inside the bioreactor. Whereas the glucose, MgSO4 and trace elements stock solutions were prepared and autoclaved in separate vessels. The medium was completed inside the bioreactor after sterilization and thereafter anaerobic conditions were reached by purging the bioreactor with N2 (4 Lpm, 1050 rpm, 20 min, Figure 2: valve 1 open).
Preparation of inoculum cultures
For the preparation of the first seed culture a loop of B. subtilis DSM 10T from the glycerol stock solution was inoculated in 20 mL of Lysogeny Broth (inside a 100 mL baffled shake flask) and incubated in a shake incubator chamber (Multitron II, HT Infors, Bottmingen, Switzerland) at 30°C and 120 rpm for 24 h. The second seed culture was inoculated with a resulting OD600 of 0.05 under anaerobic conditions in prepared serum bottles with 50 or 100 mL of mineral salt medium, respectively. The serum bottles were incubated in a horizontal position but otherwise in the same manner as the first seed culture. After 24 h of incubation approximately 200 mL of the second seed culture were used to inoculate the aqueous phase of the bioreactor (Figure 1 A). The initial OD600 inside the bioreactor fluctuated between 0.03 and 0.07, depending on bacterial growth of the second seed culture.
Cultivation in a 2.5 L benchtop bioreactor
All cultivations were carried out in 2.5 L benchtop bioreactors (Minifors, HT Infors, Bottmingen, Switzerland) with 1.0 L mineral salt medium. The bioreactors were equipped with pH (Mettler-Toledo International Inc., Greifensee, Switzerland) and pO2 electrodes (Oxyferm, Hamilton Bonaduz AG, Bonaduz, Switzerland), a temperature sensor and Rushton turbines. The temperature was adjusted to 30°C and the pH was controlled to a value of 7.0 by the addition of 4 M NaOH or 4 M H3PO4 (Figure 2). The stirrer was adjusted to 300 rpm the entire time of cultivation. The medium was not exposed to gas flow throughout the whole fermentation process to guarantee an absolutely foam-free cultivation. However, to avoid reflux of air through the exhaust cooler and to allow the measurement of CO2 through the exhaust gas analysis, a constant N2 gas flow through the headspace of the bioreactor with 0.1 Lpm (1.5 L headspace volume) was adjusted (Figure 2: valve 2 open).
The fermentation process was started with 1.0 L of the described mineral salt medium and the additional volume of the inoculated seed culture (200 mL). Since the bioreactor cultivation was realized as a batch cultivation, no further medium components were added. During the cultivation pH, pO2, CO2 exhaust, temperature, stirrer speed and addition of acid and base were consistently monitored (Figure 2). Samples were taken from the cultivation broth (4 mL) without allowing any air flow inside the bioreactor. All fermentations were performed as duplicates.