Additional file 1 of Novel plasmid-free Gluconobacter oxydans strains for production of the natural sweetener 5-ketofructose

Additional file 1: Table S1. Offline data for cultivation of the indicated G. oxydans strains with 80 g/L fructose in a RAMOS device shown in Fig. S1. Table S2. Offline date for the cultivation of G. oxydans IK003.1-igr3::fdhSCL in a RAMOS device with different fructose concentrations shown in Fig. S5. Table S3. Oligonucleotides used in this study. Figure S1. Cultivation of the indicated G. oxydans strains with 80 g/L fructose in a RAMOS device with online monitoring. Depicted are (a) the oxygen transfer rate (OTR) (b) the carbon dioxide transfer rate (CTR) (c) the respiratory quotient (RQ shown for OTR values above 5 mmol/L/h), (d) the total oxygen consumption (TOC) and (e) the total carbon dioxide evolution (TCE). The strains were cultivated in complex medium with 80 g/L fructose at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm. Shown are mean values of duplicates. Figure S2. Scale-up of batch fermentation of G. oxydans IK003.1-igr3::fdhSCL from shake flasks (RAMOS device) to a 2 L fermenter with 80 g/L fructose and 150 mM MES. Depicted are (a) oxygen transfer rate (OTR) and carbon dioxide transfer rate (CTR), (b) growth as OD600 and pH, (c) dissolved oxygen tension (DOT), agitation speed during fermentation, addition of antifoam agent (AF), and period of DOT control (indicated by arrows) and (d) fructose and 5-ketofructose concentration as determined by HPLC (method B). Cultivations were performed in complex medium with 80 g/L fructose prepared in the fermenter. The shake flask experiment was started with a sterile sample from the fermenter at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm using the RAMOS system. Fermentation was performed with 1 L filling volume in a L fermenter, DOT was kept ≥ 30% by variation of the agitation speed (500–1250 rpm), aeration rate (Q) = 1 L/min, 30 °C. Figure S3. Cultivation of G. oxydans IK003.1-igr3::fdhSCL in a 2 L fermenter with 100 mM MES and 80 g/L fructose. Depicted is (a) the oxygen and carbon dioxide transfer rates (OTR and CTR), (b) optical density (OD600) and pH, (c) dissolved oxygen tension (DOT) and agitation speed, and (d) fructose and 5-ketofructose concentrations. The cultivation was performed with 1 L filling volume, DOT ≥ 30% controlled by agitation speed (500–1500 rpm), aeration rate (Q) = 1 L/min, T = 30 °C. Figure S4. Cultivation of G. oxydans IK003.1-igr3::fdhSCL in a 2 L fermenter with 80 g/L fructose and pH control. Depicted is (a) the oxygen and carbon dioxide transfer rates (OTR and CTR), (b) optical density (OD600) and pH, (c) dissolved oxygen tension (DOT) and agitation speed, and (d) fructose and 5-ketofructose concentrations. The cultivation was performed with 1 L filling volume, DOT ≥ 30% controlled by agitation speed (500–1500 rpm), aeration rate (Q) = 1 L/min, T = 30 °C. Figure S5. Cultivation of G. oxydans IK003.1-igr3::fdhSCL in a RAMOS device with different fructose concentrations and 150 mM MES (initial pH of 6). Depicted is the oxygen transfer rate during growth with the indicated concentrations of fructose in complex medium at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm. Shown are mean values of duplicates. Figure S6. Cultivation of the indicated G. oxydans strains in a RAMOS device with 80 g/L fructose and 150 mM MES (initial pH of 6). Depicted is (a) the 5-ketofructose concentration (HPLC method B), the yield g/g, the OD600 and the final pH after 29 h and (b) the oxygen transfer rate (OTR). Cultivations were performed in complex medium with 80 g/L fructose and 150 mM MES buffer at 30 °C, 350 rpm, VL = 10 mL in 250 mL flasks, pHstart = 6 and a shaking diameter of 50 mm. Shown are mean values of duplicates.