Biostat b

The experimental work comprised nine cultivation runs (referred to as “ATSR1”, “ATSR2”, etc.). The cultivation runs differed with respect to the co-culture initation approach, medium composition and pH levels. Each of the runs involved three stirred tank bioreactors BIOSTAT^® B (Sartorius, Germany) operating in parallel (one of the bioreactors represented the “A. terreus vs. S. rimosus” co-culture, whereas the remaining two were employed for the A. terreus and S. rimosus monoculture controls, respectively). The initial working volume of the bioreactors was equal to 5.5 L. The dissolved oxygen level was controlled at 20% by the automatic adjustment of air flow rate and stirring speed. The minimum and maximum stirring speeds were set to 220 and 300 min⁻¹, respectively. The minimum air flow rate was equal to 1.5 L min⁻¹ whereas the maximum level was set to 5.5 L min⁻¹.

For shake flask RL production experiments, a seed culture of B. thailandensis E264 was grown in 100 ml NB+ 4 % glycerol at 30 °C with 200 rpm rotary shaking for 24 h. Ten millilitres of this seed culture was then added to 90 ml sterile NB+ 4 % glycerol to in a 1-l Erlenmeyer flask, and cultures were incubated at either 25 or 30 °C with 200 rpm rotary shaking. Cell growth was monitored by measuring the optical density (OD) of the culture at 600 nm throughout the fermentation. For each measurement, a 100-μl sample of the culture was taken and diluted 1:10 with sterile NB+ 4 % glycerol. All shake flask growth experiments were carried out in biological triplicate to ensure reproducibility.
For analysis under controlled fermentation conditions, B. thailandensis E264 was grown in the Biostat B bioreactor (Sartorius) at a working volume of 4 l. An incubation temperature of 25 °C and a dissolved oxygen (DO₂) level of 20 % were maintained throughout the fermentation. Stirrer speed was programmed to vary between 50 and 500 rpm in order to maintain DO₂ levels at 20 %. pH was monitored throughout the fermentation, and regular OD measurements were taken to track the biomass levels of B. thailandensis E264. Samples were taken at 24-h intervals to monitor RL production. All fermentations using the Biostat B fermenter system were carried out in duplicate to ensure reproducibility.

The strain VTT C-10880 and the HXK2 deletion strain were studied for 170 h in 1.5-l batch cultures in Biostat B bioreactor (Sartorius, Göttingen, Germany) at pH 5 and 30 °C using agitation at 200 rpm, under anaerobic conditions with 0.5 l/min nitrogen flushed to reactor headspace. The HXK2 deletion strain was cultivated in duplicate experiments. The inocula were prepared by transferring the cells from an YPD plate to 25 ml mineral medium in 100-ml Erlenmeyer flasks and incubated on a plate shaker (150 rpm, 30 °C) overnight. The inocula were further transferred into 75 ml mineral medium in 250-ml Erlenmeyer flasks and incubated (150 rpm, 30 °C) for 4-6 h. The inocula were centrifuged (2000 rpm, 4 °C, 5 min) and the cells were resuspended into the growth medium lacking the source of carbon. Initial cell dry weight in bioreactor was 0.15 g/l. The minimal mineral medium [56 (link)] contained 20 g glucose/l, 50 g xylose/l, 22 mg uracil/l, 5 g (NH₄)₂SO₄/l, 3 g KH₂PO₄/l, 0.5 g MgSO₄·7H₂O/l, 15 mg C₁₀H₁₄N₂Na₂O₈·2H₂O (EDTA)/l, 4.5 mg ZnSO₄·7H₂O/l, 0.84 mg MnCl₂·2H₂O/l, 0.3 mg CoCl₂·6H₂O/l, 0.3 mg CuSO₄·5H₂O/l, 0.4 mg Na₂MoO₄·2H₂O/l, 4.5 mg Na₂MoO₄·2H₂O/l, 3.0 mg FeSO₄·7H₂O/l, 1.0 mg H₃BO₃/l, 0.1 mg KI/l, 0.05 mg biotin/l, 1.0 mg Ca-Pantothenate/l, 5 mg nicotinic acid/l, 25 mg myo-inositol/l, 1.0 mg thiamine HCl/l, 1.0 mg pyridoxol HCl/l, and 0.2 mg p-aminobenzoic acid/l.