To scale-up the olivetolic acid (OA) generating bioprocess, the recently reported D. discoideum pChR7 strain expressing a novel biosynthetic pathway for OA production was used .
Dictyostelium discoideum AX2 cells were grown in HL5 complex medium (Formedium) supplemented with 10 g/L glucose at 22 °C in petri dishes, shake flasks at 140 rpm or stirred tank bioreactors (STRs) at indicated stirrer speeds. Selection of recombinant amoebae was performed by adding 20 µg/mL G418 (InvivoGen). To induce the expression of the OA biosynthetic genes, main cultures were supplied with 20 µg/mL doxycycline (AppliChem).
Pre-cultures in shake flasks
In order to circumvent the necessity of cell number determination, we investigated if the metabolic activity can be used as a transfer criterion. The respiration activity was measured by using the Kuhner TOM (transfer-rate online measurement) system equipped with oxygen partial pressure sensors and infrared sensors to calculate the oxygen transfer rate (OTR) and the carbon dioxide transfer rate (CTR) . D. discoideum was grown at an initial density of 1 × 106 cells/mL in 1-L TOM flasks with a filling volume of 200 mL HL5 medium supplemented with 10 g/L glucose and 20 µg/mL G418. When the pre-cultures reached a cell concentration of 1 × 107 cells/mL and a CTR of 1.3 mmol/L/h (Additional file 1: Fig. S2), 100 mL were used for inoculation of the 1.6-L bioreactor.
Main cultures in stirred tank bioreactors (STRs)
All main cultures were inoculated with 10% filling volume to reach a starting concentration of 1 × 106 cells/mL and performed as batch cultures in STRs (1.6-L and 7-L: Sartorius, 75-L: bbi-biotech, 300-L: Sartorius/Frings) with parameters indicated in Table 2. To ensure turbulent conditions, Reynolds numbers were always over 10,000. The pH was adjusted to 6.5 by addition of sulfuric acid, and antifoam was added if necessary. The batch cultures were grown at a constant temperature of 22 °C and an aeration rate of 0.3 vvm.
Small scale STR cultivation for evaluation of ε
To determine the maximum tolerable hydromechanical stress level of the D. discoideum pChR7 strain, six 1.6-L scale batch fermentations were performed with different stress conditions by applying stirrer tip speeds of 0.35, 0.55, 1, 1.5, 2, and 2.5 m/s. After inoculation with a starting cell density of 1 × 106 cells/mL, the stirrer tip speed was kept constant at 0.35 m/s for 52 h to allow the amoeba to adopt to the altered conditions in the STR. After the lag phase, a stirrer profile was applied to the afore mentioned tip speeds within 6 min. To define a suitable scale-up criterion based on the optimal growth profile, the tip speeds were used to calculate the maximum local energy dissipation rate (εmax) as described below.
STR cultivation for scale-up
The scale-up of the OA production process to 300-L was performed using three seed bioreactors (1.6-L, 7-L and 75-L) based on εmax. In the first 1.6-L scale, the stirrer tip speed was kept constant at 0.35 m/s for 40 h as the uninduced amoebae exhibit a faster growth rate. Then, a stirrer profile to the determined εmax within 6 min followed and cultivation was continued for 8 h or until reaching final cell density. For the 7-L and 75-L seed bioreactors, εmax was constant from the beginning (Table 2), since the amoebae were adopted to the hydromechanical stress conditions in the first seed bioreactor. The transfer of the seed culture to the next scale was performed at a cell density of 1 × 107 cells/mL. For comparison, a small scale 7-L STR was carried out starting from a 1.6-L STR seed culture.
For all STR experiments, the dissolved oxygen tension (DOT), temperature and pH were measured online. For 7-L, 75-L and 300-L scales, the O2 and CO2 concentrations were measured using a Rosemount NGA 2000 off-gas analyzer (Emerson) with a paramagnetic sensor and an infrared analyzer. For the 1.6-L scale, the O2 and CO2 concentrations were measured using the BlueVary off-gas analyzer (BlueSens gas sensor). The oxygen transfer rate (OTR) carbon dioxide transfer rate and CTR were calculated, as previously reported . The value of the respiratory quotient was always “1”, since OTR and CTR were equal. Due to higher signal quality, only trends of the CTR are depicted.
During fermentation, samples were taken at regular time intervals in order to get offline data of the running process. At the indicated time points, the cell concentration and viability were determined at a size range of 7.6 to 17.6 µm using a CASY Cell Counter and the respective analyzer system (model TT, equipped with a 60 µm capillary, OLS Bio). The cell dry weight (CDW) was determined by centrifugation of 10 mL culture broth at 4 °C and 800 g for 5 min. While the supernatant was stored for further analysis, the cell pellet was washed with 5 mL of phosphate buffer (5 mM Na2HPO4, 5 mM KH2PO4, 1 mM CaCl2, 2 mM MgCl2), lyophilized overnight and weighed afterwards. For determination of the glucose concentration, 1 mL of the supernatant was measured using the YSI 2950 Analyzer (Kreienbaum). Moreover, 10 mL of culture broth were harvested for OA extraction and quantification using high performance liquid chromatography (HPLC) coupled with high resolution mass spectrometry (HRMS) as recently reported .