Highly efficient fermentation of 5-keto-D-fructose with Gluconobacter oxydans at different scales

Abstract

Background: The global market for sweeteners is increasing, and the food industry is constantly looking for new low-caloric sweeteners. The natural sweetener 5-keto-D-fructose is one such candidate. 5-Keto-D-fructose has a similar sweet taste quality as fructose. Developing a highly efficient 5-keto-D-fructose production process is key to being competitive with established sweeteners. Hence, the 5-keto-D-fructose production process was optimised regarding titre, yield, and productivity.

Results: For production of 5-keto-D-fructose with G. oxydans 621H ΔhsdR pBBR1-p264-fdhSCL-ST an extended-batch fermentation was conducted. During fructose feeding, a decreasing respiratory activity occurred, despite sufficient carbon supply. Oxygen and second substrate limitation could be excluded as reasons for the decreasing respiration. It was demonstrated that a short period of oxygen limitation has no significant influence on 5-keto-D-fructose production, showing the robustness of this process. Increasing the medium concentration increased initial biomass formation. Applying a fructose feeding solution with a concentration of approx. 1200 g/L, a titre of 545 g/L 5-keto-D-fructose was reached. The yield was with 0.98 g_{5-keto-d-fructose}/g_fructose close to the theoretical maximum. A 1200 g/L fructose solution has a viscosity of 450 mPa∙s at a temperature of 55 °C. Hence, the solution itself and the whole peripheral feeding system need to be heated, to apply such a highly concentrated feeding solution. Thermal treatment of highly concentrated fructose solutions led to the formation of 5-hydroxymethylfurfural, which inhibited the 5-keto-D-fructose production. Therefore, fructose solutions were only heated to about 100 °C for approx. 10 min. An alternative feeding strategy was investigated using solid fructose cubes, reaching the highest productivities above 10 g_{5-keto-d-fructose}/L/h during feeding. Moreover, the scale-up of the 5-keto-D-fructose production to a 150 L pressurised fermenter was successfully demonstrated using liquid fructose solutions (745 g/L).

Conclusion: We optimised the 5-keto-D-fructose production process and successfully increased titre, yield and productivity. By using solid fructose, we presented a second feeding strategy, which can be of great interest for further scale-up experiments. A first scale-up of this process was performed, showing the possibility for an industrial production of 5-keto-D-fructose.

Keywords: 5-Ketofructose; Extended batch fermentation; Fructose dehydrogenase; Gluconobacter oxydans; Scale-up.

Fig. 1

Extended-batch-cultivation of G. oxydans 621H ΔhsdR pBBR1p264-fdhSCL-ST in a 2 L Visual Safety Fermenter (VSF, Bioengineering) with constant feeding of fructose (1035 g/L) between 19 and 61 h. Depicted is A the oxygen transfer rate (OTR, light blue), carbon dioxide transfer rate (CTR, orange) and respiratory quotient (RQ, dark red), B pH (pink), aeration rate (green) and filling volume (grey), C the dissolved oxygen tension (DOT, dark blue) and agitation rate (light green), D the optical density OD₆₀₀ (purple) and osmolality (black), E fructose (light blue) and 5-ketofructose concentration (red). Cultivation was performed in complex medium with 150 g/L initial fructose at 30 °C, initial pH value 6, pH control at 5.5 from 19–61 h and 5 from 61 h with 3 M KOH, V_L,start = 0.8 L in a 2 L fermenter. DOT was kept ≥ 30% by variation of agitation speed (500–2050 rpm), absolute aeration rate Q_g = 1 SL/min. Feeding solution: 1035 $\pm$ 10 g_fructose/L, heat pretreatment: 121 °C, 21 min. Feed rate: 14.3 g_fructose/h. Fructose feeding solution and peripheral feeding system were heated to ~ 55 °C. RQ-values are only shown, when OTR-values are above 5 mmol/L/h

Fig. 2

Correlation of viscosity and temperature of fructose solutions with different concentrations. Depicted is the correlation of viscosity and temperature for fructose solutions with concentrations of 800 g_fructose/L (green), 1000 g_fructose/L (pink) and 1200 g_fructose/L (light blue). Fructose solutions were preheated before viscosity measurement. Viscosity was measured using a MCR 301 rheometer (Anton Paar, Stuttgart, Germany) equipped with a cone (CP50-0.5/TG, cone truncation 55 µM, cone angle 0.467°) within the shear rate range of 100–5000 s⁻¹. The viscosity can be assumed to be Newtonian within the measured shear range

Fig. 3

Schematic experimental fermenter setup for concentrated liquid and solid fructose feeding. Extended batch fermentations were performed with A concentrated liquid fructose solutions and B solid fructose cubes. A: Fructose feed solutions were constantly stirred and heated to ~ 55 °C in a bottle using a heating magnetic stirrer. The peripheral feeding system was heated to ~ 55 °C using hot water or an electrical heating band with insulation (indicated with dark red lines). B: Fructose cubes were stored in a bottle attached to a silicon tube with a 2 cm diameter. The silicon tube was sealed with three hose clamps, to prevent humidity entering the storage bottle from the fermenter. For feeding, desired numbers of cubes were manually transferred from the bottle to the tube and into the fermenter. Schematic representation adapted from [91]. Parts of the figure were drawn by using pictures form Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license

Fig. 4

Extended-batch-cultivation of G. oxydans 621H ΔhsdR pBBR1p264-fdhSCL-ST in a 2 L fermenter (Sartorius) with constant feeding of fructose (1180 g/L) between 18 and 44 h. Depicted is A the oxygen transfer rate (OTR, light blue), carbon dioxide transfer rate (CTR, orange) and respiratory quotient (RQ, dark red), B pH (pink), aeration rate (green) and filling volume (grey), C the dissolved oxygen tension (DOT, dark blue) and agitation rate (light green), D the optical density OD₆₀₀ (purple) and osmolality (black), E fructose (light blue) and 5-ketofructose concentration (red). A. Cultivation was performed in complex medium (concentrated 1.6×) with 150 g/L initial fructose at 30 °C, initial pH value 6, pH control at 5 from 18 h with 3 M KOH, V_L,start = 1 L in a 2 L fermenter. DOT was kept ≥ 30% by variation of agitation speed (500–1500 rpm), absolute aeration rate Q_g = 1—2.5 SL/min. Feeding solution: 1180 $\pm$ 2 g_fructose/L, heat pretreatment: 100 °C, 10 min. Feed rate: 27.3 g_fructose/h. Fructose feeding solution and peripheral feeding system were heated to ~ 55 °C. RQ-values are only shown, when OTR-values are above 5 mmol/L/h. Sampling for further experiments Fig. 5) are indicated by time stamps t₁–t₄ in A. A drift in the oxygen sensor was corrected by linear regression. For original data please refer to Additional file 1: Fig. S3

Fig. 5

Cultivation of G. oxydans 621H ΔhsdR pBBR1p264-fdhSCL-ST in a RAMOS device with cells sampled at different time points (t₁–t₄) from the extended batch fermentation displayed in Fig. 4. Depicted is the oxygen transfer rate (OTR). The samples were taken during the extended batch fermentation (Fig. 4) after 19 h, 25 h, 32 h and 43 h, centrifugated and used for inoculation at an OD₆₀₀ of 0.1. Cultivations were performed at 30 °C, 350 rpm, V_L = 10 mL in 250 mL shake flasks, initial pH value 6 and a shaking diameter of 50 mm in complex medium with 80 g/L fructose

Fig. 6

Extended-batch-cultivation of G. oxydans 621H ΔhsdR pBBR1p264-fdhSCL-ST in a 2 L fermenter (Sartorius) with solid fructose feed between 20 and 43 h. Depicted is A the oxygen transfer rate (OTR, light blue), carbon dioxide transfer rate (CTR, orange) and respiratory quotient (RQ, dark red), B pH (pink), aeration rate (green) and filling volume (grey), C the dissolved oxygen tension (DOT, dark blue) and agitation rate (light green), D the optical density OD₆₀₀ (purple) and osmolality (black), E fructose (light blue) and 5-ketofructose concentration (red). Cultivation was performed in complex medium (concentrated 1.6×) with 150 g/L initial fructose at 30 °C, initial pH value 6, pH control at 5 from 20 h with 10 M KOH, V_L,start = 1 L in a 2 L fermenter. DOT was kept ≥ 30% by variation of agitation speed (500–1500 rpm), absolute aeration rate Q_g = 1–2.5 SL/min. Feeding: solid fructose cubes. RQ-values are only shown, when OTR-values are above 5 mmol/L/h

Fig. 7

Extended-batch-cultivation of G. oxydans 621H ΔhsdR pBBR1p264-fdhSCL-ST in a 150 L pressurised fermenter (Frings) with constant feeding of fructose (745 g/L) between 22 and 52 h. Depicted is A the oxygen transfer rate (OTR, light blue), carbon dioxide transfer rate (CTR, orange) and respiratory quotient (RQ, dark red), B the dissolved oxygen tension (DOT, dark blue) and aeration rate (light green), C pH (pink), headspace overpressure (green) and filling volume (grey), D the optical density OD₆₀₀ (purple) and osmolality (black), E fructose (light blue) and 5-ketofructose concentration (red). Cultivation was performed in complex medium (concentrated 1.6×) with 150 g/L initial fructose at 30 °C, initial pH value 6, pH control at 5 from 22 h with 3 M KOH, V_L,start = 50 L in a 150 L pressurised fermenter (Frings). DOT was kept ≥ 30% by variation of headspace overpressure (1–1.8 bar), agitation rate: 600 rpm, absolute aeration rate Q_g = 50–170 SL/min was increased linearly in parallel to the headspace overpressure. Feeding solution: 745 $\pm$ 25 g_fructose/L, heat pretreatment: 100 °C, 4 h. Feed rate: 900 g_fructose/h. Fructose feeding solution was heated to ~ 50 °C. RQ-values are only shown, when OTR-values are above 5 mmol/L/h. Due to technical problems between 22 and 26 h, 32 and 35 h, and 54 and 66 h, OTR, CTR and RQ data shown in dashed lines are not reliable and were approximated by straight lines. For raw data please refer to Additional file 1: Fig. S7

Fig. 8

Comparison of productivities, oxygen transfer rates and fructose concentrations for extended batch fermentations of G. oxydans 621H ΔhsdR pBBR1p264-fdhSCL-ST of this work, using different fermenters and fructose feed concentrations. Depicted are A productivities B oxygen transfer rates, C the fructose concentrations and D main information for cultivations displayed in Fig. 1 (black), Fig. 4 (blue), Fig. 6 (light blue), Fig. 7 (green), Additional file 1: Fig. S2 (orange) and Additional file 1: Fig. S5 (pink). For better visualisation of OTR curves during final batch phases, y-axis of B is only shown for OTR values between 0 and 30 mmol/L/h