Dasgip parallel bioreactor system

Experiments were performed in a DASGIP parallel bioreactor system (Eppendorf, Jülich, Germany) with a working volume of 1 L. Temperature, pH, agitation speed, and air flow were set at $30{}^{\circ }\text{C}$ , 6.8, 500 rpm, and 1 vvm, respectively. Dissolved oxygen was not actively controlled, but never dropped below 30%. Antifoam (Y-30 emulsion, Sigma-Aldrich, Overijse, Belgium) was added to avoid foam formation (500  $\mathrm{\mu }$ L/L initially, and further supplemented when required). For fluxomics, duplicate reactors were inoculated from the same preculture. One reactor contained medium with labeled glucose and the other reactor contained medium with unlabeled glucose. The parallel experiment with unlabeled glucose was necessary to obtain representative values for the off-gas analysis, as labeled carbon gave rise to erroneous ${\text{CO}}_2$ concentration values in the infrared off-gas analysis. Parallel runs inoculated from the same preculture are here referred to as technical replicates, while repeat experiments started from different precultures are further referred to as biological replicates. Samples for transcriptome analysis were collected under the same conditions as those of the fluxomics experiments, in either the DASGIP parallel bioreactor system, or in a Bioflo 3000 bioreactor (New Brunswick, New Jersey, USA) containing 3 L medium.

Strains were cultivated in 200 mL CDM with 10 g L⁻¹ sucrose in pH-controlled batch fermentations in a 250-mL DASGIP Parallel Bioreactor System, controlled using Dasware Control version 5.5.1 (Eppendorf). Cysteine-HCl and menaquinone-4 were added prior to inoculation at final concentrations of 0.5 g L⁻¹ and 0.001 g L⁻¹, respectively. Precultured cells were harvested by centrifugation at 8,000 rpm and 4°C for 4 min and used for inoculation to an initial OD₆₀₀ of 0.05. The medium was cold-inoculated at 5°C. To initiate the fermentation, the temperature was increased to 37°C at a specific time, selected based on the lag phase duration of each strain. During cultivation the temperature was maintained at 37°C. The pH was maintained at 6.5 through automatic titration with 13.5% NaOH. To maintain anaerobic conditions, the headspace was continuously flushed with 80% N₂ and 20% CO₂ at a flow rate of 3 L h⁻¹. Agitation was set to 250 rpm using one Rushton impeller and one pitched-blade impeller.

Hen egg white lysozyme (HEWL) was purchased from Afilact^® (Geinberg, Germany). All other chemicals were purchased in the analysis quality from AppliChem (Darmstadt, Germany), Carl Roth (Karlsruhe, Germany), Sigma-Aldrich (United Kingdom), or Honeywell Fluka™ chemicals (München, Germany) and used without further purification if not specified. All buffer solutions were filtered using a 0.45 µm cellulose acetate filter (Sartorius, Göttingen, Germany) and those for chromatographic applications were further degassed in an ultrasonic chamber (5510E-DTH, Bransonic, Missouri, United States).
Humira was produced in a recombinant monoclonal Chinese hamster ovary K1 (CHO_K1) cell line using Geneticin G-418 Sulphate (Gibco, United Kingdom) as the selection system. Fed-batch fermentations with chemically defined ActiPro™ medium (HyClone™, Austria) supplemented to 8 mM L-Glutamine were realized in DASGIP^® Parallel Bioreactor System (Eppendorf, Germany). Fermentation parameters were set to 60 rpm stirrer speed, 37°C, 60% dissolved oxygen, and 3 s L/h gas flow rate, and pH 7.2 was controlled by CO₂ and 7.5% sodium bicarbonate addition. A glucose concentration of at least 2 g/L was maintained by a 10% glucose feed. Harvesting of the fermentation broth was performed on day 7 with maximum cell densities of 11 × 10⁶ cells/mL.

For proteomic analysis, C. glutamicum ATCC13032 was cultivated in a four-fold parallel bioreactor setup (DASGIP^® Parallel Bioreactor System, Eppendorf SE, Germany) with a filling volume of 1 L each. Bacteria were grown in CGXII-U medium without MOPS and pH was maintained at defined levels (pH = 5 or 8) using 4 M KOH or 6 M H₃PO₄. Ample oxygen supply was guaranteed by using an oxygen controller set to 30% dissolved oxygen and using a cascade of stirrer speed, gas flow and oxygen concentration in inlet air. The bioreactors were inoculated to an OD₆₀₀ of 1.5. Samples for proteome analysis were taken in exponential and stationary growth phase in technical triplicates. For each replicate, 1.6 ml culture broth were samples to a proteome vial and centrifuged for 5 min at 4°C and 21,500 × g. The supernatant was discarded, and pellets were frozen in liquid nitrogen and stored at −80°C. Proteomic analysis was carried out as described elsewhere (Unrean et al., 2018 (link)). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022 (link)) partner repository with the dataset identifier PXD046499.

For the study of the thermotolerance capacity, Chlorella sp. QUCCCM3 was scaled up gradually, as previously described (Section 2.2), to 1 L in bioreactors (DASGIP parallel bioreactor system, Eppendorf Inc., Ocala, FL, USA), with an initial OD_750nm of 0.2. The strain was subjected to four culture temperatures, nameley 30, 35, 40, and 45 °C, with a mixing set to 200 rpm, using a pitch blade impeller. The illumination was provided by 3 internal DASGIP LED sticks having 3 channel emission spectrum (channel A, 660,780 nm; channel B, 572,625,640 nm; channel C, 453 nm). Set points were 2.00, 1.244, and 2.00 μmol photons·s−1 for channels A, B, and C, respectively, fixed for a photon flux density of approximately 200 µmol photons m⁻² s⁻¹, which is considered to be optimum for the standard cultivation of microalgae, at 12 h:12 h dark:light cycles. A daily assessment of the OD_750nm was performed to be able to compare the growth performance of the strain under the different temperatures tested.

The implementation of a perfusion process utilizing the Xeno™ medium but a different MDCK cell line was reported previously (Wu et al. 2021 (link)). In the present study, the STR (DASGIP® Parallel Bioreactor System, Eppendorf AG, 76DG04CCBB, working volume 700 mL) used for DIP production was equipped with one inclined blade impeller (three blades, 30° angle, 50-mm diameter) and two spargers (one macro- and one micro-sparger). The micro-sparger was only used for additional gassing, when the VCC increased to concentrations where gassing with the macro-sparger alone was not sufficient anymore to maintain a pO₂ above 40%. Please note that the additional gassing with the micro-sparger did not increase the air volume per liquid volume per minute (VVM), as the gas flow rate of the macro-sparger was reduced accordingly by the controller unit. Before inoculation, cells grown in shake flasks (as described before) were centrifuged (300 × g, 5 min, room temperature) and the medium exchanged. The STR was inoculated with a VCC of 1.0E + 6 cells/mL and operated at 37 °C, pO₂ ≥ 40%, pH 7.5, and 150 rpm. Before perfusion was started, the bioreactor was operated for 24 h in batch mode.