Biostat b plus bioreactor

To study the effect of VHb on M. alpina, especially under the oxygen-restricted conditions, we compared the DCW, total lipid content, and arachidonic acid production between the transformant VHb-20 and the wild-type strain in shake flasks and a bioreactor under dO₂ limiting and non-limiting conditions. First, the VHb-20 transformants and wild type were inoculated into six 250 mL shake flasks with baffles. Three shake flasks contained 50 mL medium (normal, dO2 non-limiting condition), and three contained 150 mL medium (dO2 restriction). They were shaken at 25 °C and 200 rpm for 7 days. Meanwhile, bioreactor cultures were grown in 5-L Biostat B plus bioreactors (Sartorius Stedim Biotech, Germany) with 3 L of medium. The rate of ventilation and agitation during growth in the bioreactor was set to non-limiting oxygen (1.7 vvm, 200 rpm) and limiting oxygen (0.8 vvm, 100 rpm) conditions. The transformant and wild-type cultures were grown for 7 days at 25 °C, pH 6.5. The glucose concentration was maintained at approximately 20 g L⁻¹ by feeding the 80% (w / v) glucose solution, which was measured with a SBA-40D Biosensor (Institute of Biology, Shandong Academy of Sciences, China). Twenty milliliter samples were taken at 24 h intervals in all experiments. The entire fermentation process used bioprocess-automated software for monitoring and control.

The precultures were prepared as described above ("Growth and media used in the experiments"). The main culture was grown on 50 mL of YNB medium (C/N 60) containing the following: galactose 60.0 g; YNB 1.7 g; NH₄Cl 1.5 g; 0.7 g KH₂PO₄, and 1.0 g MgSO₄ × 7H₂O in 1 L. The pH was kept at 6.8 using 0.05 M phosphate buffer. Tap water was used as a source of microelements. To increase the C/N ratio of the medium to 100, 1.25 g/L of NH₄Cl was used. Culture conditions were as described above ("Growth and media used in the experiments").
Lipid biosynthesis was also evaluated using batch cultures (BC) that were kept in 5-L stirred-tank BIO-STAT B-PLUS bioreactors (Sartorius, Frankfurt, Germany) for 96 h under the following conditions: 2-L working volume, 28 °C, 800 rpm of agitation, and 3.5-L/min aeration rate. The production medium was prepared as described above. The pH was kept at 6.8 using a 40 % (w/v) NaOH solution. The cultures were grown in 0.2 L of YPD medium in 0.5-L flasks at 170 rpm, at 28 °C for 48 h. The volume of the inocula added to the bioreactor cultures was equal to 10 % of the total working volume.

Chemostat cultivations were performed in duplicate, all in 2 L Biostat B plus Bioreactors (Sartorius Stedim, Goettingen, Germany) according to García Ortega et al. [37 (link)]. Batch and chemostat media compositions are stoichiometrically identical to the detailed in the reference [37 (link)]; however, the concentrations were reduced by half.
Cultivation conditions were monitored and controlled at the following set points: pH, 5.0 with addition of 15% (v/v) ammonium hydroxide; temperature, 25 °C; stirring rate, 700 rpm; air flow, 0.8 vvm and pO₂ values were variable depending on the dilution rate. pO₂ values were above 20% in all conditions tested. An exhaust gas condenser with cooling water at 4 °C minimized mass loses by water evaporation and other possible volatile compounds.
A broad range of dilution rates were covered for the three expression systems tested. Taking into consideration that 0.19 h⁻¹ was the P. pastoris µ_max of GAP-C at 25 °C (data not shown), the following dilution rates were used: 0.05 h⁻¹, 0.10 h⁻¹ and 0.15 h⁻¹ (dilution rates were tested as low, middle and high growth rate conditions, respectively). In order to ensure that the steady state was reached, the stability of the parameters of interest were monitored from the third residence time until the fifth one, where samples were taken.