Figure Legends
Figure 1 – Scheme for all three processes, batch, fed-batch and perfusion-batch with cell-recycling of S. pneumoniae RM200 in a 10 L bioreactor, including the microfiltration system used in downstream process for cell separation, washing and concentration. The feeding flask (number 3) was not used for batch. The microfiltration system (numbers 6 and 7) was applied only for downstream processing (cell separation, washing and concentration) in batch and fed-batch processes. For perfusion-batch, microfiltration system (numbers 6 and 7) was used during cultivation for cell-recycling and for downstream processing. Feeding flow-rates: 0.5 L/h, measured in flask 3 for fed-batch, and 7.3 L/h, measured in flasks 3 and 7, for perfusion-batch.
Figure 2 Time profile for batch (n = 6 runs, top), fed-batch (n = 7 runs, middle) and perfusion-batch (n = 3 runs, bottom) fermentation of S. pneumoniae RM200 in a 10 L bioreactor: residual glucose (), residual lactate () and residual acetate () measured by HPLC; biomass measured by optical density at 600 nm () and dry cell weight (). Each point represents mean of values and bars represent standard deviation. Dot line indicates the start of the fed-batch mode (middle) or the perfusion mode with feeding medium 1 (bottom). Dash line indicates the start of the perfusion with feeding medium 2 (bottom).
Figure 3 – Comparison of 3 fermentation protocols to total glucose consumption, lactate production, biomass production measured by dry cell weight and optical density at 600 nm: batch (), fed-batch () and perfusion-batch (). Each point represents mean of values and bars represent standard deviation.
Figure 4 – The total cell biomass (dry cell weight) produced per run (each point represents a run of batch (), fed-batch () and perfusion-batch ()) versus the time for running each lot, which was calculated according to Table 3. The horizontal dot line shows when batch and fed-batch produce the same amount of biomass obtained in perfusion-batch.
Figure 5 – Western blot analysis of different PWCV preparations probed with specific antibodies indicated in the left corner of the figure. Antibodies were produced against purified pneumococcal proteins or anti-PWCV polyclonal serum was used. Lane 1: Engineering lot 007/09 produced at 60 L bioreactor was used as positive control (Gonçalves et al., 2014); Lane 2: Batch fermentation performed at 10 L bioreactor as Engineering lot and cells harvested at similar OD (6.5); Lane 3: Batch fermentation with cells harvested at the highest OD (10); Lane 4: Fed-batch fermentation with cells harvested at OD 6; Lane 5: Fed-batch fermentation with cells harvested at the highest OD (14); Lane 6: Perfusion-batch fermentation with cells harvested at the highest OD (30). Numbers at right indicate the molecular weight in kDa.