2.1. Strain, Medium, and Inoculum
Recombinant Gram-negative bacteria of the Neisseriaceae family and OMV release were used for all studies reported in this publication. Bacterial growth was supported by employing a semi-defined medium free of animal components and containing essentially a phosphate buffer, lactic acid (carbon source), yeast extract and casamino acids, salts, trace elements, vitamins, and iron elements.
Although the bacterial strain and the medium components cannot be fully disclosed due to company restrictions related to intellectual property safeguards, the focus of this study was to demonstrate how to simplify a semi-defined medium. The medium in question initially contained high amounts of yeast extract (20 g/L) and casamino acids (10 g/L), employed for the growth of a fastidious Gram-negative microorganism of the Neisseriaceae family and to produce OMVs. We proved that, with the identification of essential key amino acids and the optimal concentrations of trace elements, vitamins, and iron citrate needed to grow this bacterium, it was possible to significantly reduce the quantity of yeast extract used, despite reaching the same level of growth obtained with a high yeast extract concentration. Due to this optimization, the analysis showed an increased quantity of produced OMVs and a noticeable reduction in unwanted contaminants, such as soluble proteins and nucleic acids, in the final purified OMV product.
All reagents used for this study were purchased from Merck, except for the yeast extract and casamino acids, which were bought from Becton Dickinson (BD).
For the inoculum cultivation media and the Ambr® 15F experiments, in addition to the undisclosed reagents, 7.5 g/L of lactic acid, 5 g/L of yeast extract, and 10 g/L of casamino acids without supplements (trace elements, vitamins, and iron citrate) were used. The media were sterilized using a 0.22 μm cut-off filter, Sartorius Sartolab® (Sartorius Biohit Liquid Handling Oy, Helsinki, Finland).
The 200-fold-concentrated trace element stock solutions contained the following substances: CuSO4 × 4H2O at 0.20 g/L, ZnSO4 × 7H2O at 0.15 g/L, H3BO3 at 0.5 g/L, and MnCl2 × 4H2O at 0.15 g/L.
The 100-fold-concentrated vitamin stock solutions consisted of 0.3 g/L thiamine chloride hydrochloride, 0.05 g/L nicotinic acid, 0.05 g/L pyridoxine hydrochloride, 0.2 g/L calcium pantothenate, and 0.2 g/L vitamin B12.
The 100-fold-concentrated iron citrate stock solution consisted of 4 g/L of C6H5FeO7.
For each amino acid, a 100 g/L concentrated stock solution was prepared.
The above concentrated solutions were sterilized using the 0.22 μm cut-off filter Sartorius Sartolab®.
Prior to this optimization study, the medium used for the 2 L benchtop bioreactor included a yeast extract (20 g/L) with a concentration higher than that of the inoculum, as well as 10 g/L of casamino acids. The medium was sterilized in an autoclave for 30 min at 121 °C. After being processed using the autoclave, the medium was supplemented with 7.5 g/L of lactic acid (carbon source), 5 g/L of the trace element stock solution, 10 g/L of the vitamin stock solution, and 1 g/L of the iron citrate stock solution.
The 24 different fermentation media used for the Ambr® 15F experiments were completed with trace element, vitamin, iron citrate, and amino acid stock solutions in accordance with the concentrations calculated using DOE software. The media were prepared using the Ambr® 15F’s liquid handler by programming the associated software.
At the end of this study, the optimized medium used was composed of 5 g/L of yeast extract and 10 g/L of casamino acids. Additionally, it was supplemented with 5 g/L of the trace element stock solution, 10 g/L of the vitamin stock solution, and 10 g/L of the iron citrate stock solution. Furthermore, the media were enriched with the following amino acids: arginine at 0.40 g/L, glutamate at 10.6 g/L, histidine at 0.58 g/L, proline at 3.49 g/L, and glutamine at 0.12 g/L.
The inoculum culture process started with the thawing of one frozen stock research cell bank (RCB) of the strain (1 mL of culture with 20% glycerol stored at −80 °C). The stock was used to inoculate 200 mL of medium (containing only 5 g/L of yeast extract and sterilized via 0.22 µm orthogonal filtration) in a 1000 mL baffled polycarbonate Erlenmeyer flask with a vent cap. The flask was incubated at 37 °C ± 1 on a rotary shaker (orbital diameter 25 mm) at 150 rpm for 8 h. At the end of growth, the culture was placed in an exponential phase and showed a final optical density (OD600nm) of 2.5, as measured using a photometer in 1 cm light path plastic cuvettes at 600 nm. The described inoculum procedure was used to initiate both the 2 L lab-scale benchtop bioreactor and the Ambr® 15F microbioreactors.
2.2. Cultivation in the Bioreactor Systems (Ambr® 15F and 2 L)
The fermentation runs reported in this article were performed using the Sartorius Ambr® 15F and the 2 L benchtop bioreactors (Sartorius UniVessel® Glass 2L, Biostat® B-DCU II MO. Sartorius Stedim Systems GmbH, Guxhagen, Germany). In both cases, the fermentations were conducted in batch mode.
The Ambr® 15F with 24 single-use microbioreactors was operated with an 8 mL working volume for the experiments in this study. The Ambr® 15F was set with an initial volume of 8 mL, and each microbioreactor was filled with the medium and additional solutions using the liquid handler according to the DOE conditions. To prevent foam formation, the medium contained polypropylene glycol 2000 (PPG) at a final concentration of 0.02% (v/v). The inoculum flask content was transferred to a 24-well plate to allow the Ambr® 15F liquid handler to initiate the inoculation phase for all microbioreactors, which were inoculated to an initial OD600nm of 0.2. The pH was measured online via the sensor’s patches embedded in each microbioreactor, and it was held at 7.20 using NaOH 1M or H3PO4 1M. Acid was added using the liquid handler, while the base was pumped into the microbioreactors via pumped liquid delivery. The offline pH reading was monitored using the analysis module (AM), which recalibrates the in-process pH sensor.
Dissolved oxygen (DO) was measured online using the sensor’s patches embedded in each microbioreactor, and it was set at 30%. To maintain this value, a cascade (air–oxygen–stirrer) was set as follows: air flow rate from 8 to 30 mL/min; oxygen enrichment flow rate from 0 to 30 mL/min; and stirrer from 750 to 3000 rpm.
The growth temperature of each bioreactor was set and maintained at 37 °C. Cell growth was monitored online using the Ambr® 15F’s sensor technology, consisting of cell scattering and an analysis of reflectance-associated signals. By applying user calibration (performed earlier with the same microorganism employed in this study), the reflectance value was converted into a measure of optical density (OD600nm). For the offline OD measurements at 600 nm, an external photometer in 1 cm light path plastic cuvettes was used.
The cultures were run in batch mode and ended when the growth stationary phase was reached. This was recognized when the online OD measurement graphical trend either remained constant or decreased.
The 2 L bioreactor contained 1.5 L of sterile medium, and it was assembled with baffles and two Rushton six-blade disc turbines (φ = 5.5 cm) used for mixing, one located 3.5 cm from the bioreactor bottom and the other 7 cm from the first turbine. The bioreactor operating conditions were as follows: temperature at 37.0 °C; dissolved oxygen (DO) at 30%; air flow rate from 1.5 to 3 L/min (1 to 2 vvm); and a stirrer from 200 to 1000 rpm. Air flow and agitation were set in cascade to maintain the DO value.
The pH was set at 7.2 and measured using an online sterilizable electrode (HAMILTON EasyFerm BIO HB Arc 225. Hamilton, Bonaduz, Switzerland). NaOH at 2M and H3PO4 at 1M solutions were used to maintain the pH setpoint. The DO concentration was detected using an online optical electrode (HAMILTON VISIFERM DO ECS 225 H0. Hamilton, Bonaduz, Switzerland). The 100% point was calibrated before the bioreactor inoculation with the same operative conditions.
The inoculum flask contents were used to inoculate the benchtop bioreactors. The volume of inoculum required for use was calculated to have an initial OD600nm of 0.2 units in the bioreactor. Foam was controlled by adding PPG in the sterile medium to a final concentration of 0.02% (v/v). The bioreactors were run in batch mode, and the OD of the cultures was monitored during growth using the photometer at 600 nm in 1 cm light path plastic cuvettes.
During the batch fermentation, microbial biomass and OMVs were formed. The fermentation ended when the growth stationary phase was reached. The culture stationary phase was recognized when the OD600nm, measured each hour, was equal to (or lower than) the previous measure. Furthermore, a DO spike associated with a stirrer drop and a reduction in the acid addition confirmed the stationary phase and the end of fermentation.