3.3 Perfusion-batch with cell-recycling integrated to cell separation
To evaluate our hypothesis that high lactate production was responsible for the interruption of bacterial growth in the fed-batch process, perfusion-batch with cell recycling was performed in order to remove lactate, supply depleted nutrients and keep the cells inside the vessel. Moreover, the aim was to intensify the process and obtain the highest viable biomass; thus, this process was interrupted when the highest OD was reached (approximately after 9 h of cultivation), cells were then washed with the washing buffer and harvested to prepare the vaccine. For this reason, the perfusion was not operated as a continuous cultivation and the culture did not reach the steady-state, and the main advantage of this process over the others was the integration of up and downstream processing.
As presented in Figure 2, the batch phase was performed until 3 h, when the OD reached 4.0. Then, the feeding and removal of medium were initiated at the same flow-rate 7.3 L/h (D = 0.63 h-1). At OD 12, after 5 h of cultivation, a concentrated medium for glucose, soytone and yeast extract, was supplied at the same flow-rate (Medium 2, Table 1), until 9 h of cultivation, when the highest OD was reached 29.8 ± 4.1. This OD was 3 times higher than in the batch process performed in this study and 2 times higher than fed-batch. Moreover, it was 5 times higher than the OD reached in the cGMP lots (Gonçalves et al., 2014). When perfusion-batch was compared to continuous processes with cell-recycling of other Gram-positive lactate-producing bacteria, this OD was 6 times lower thanLactobacillus paracasei , using a similar D (0.6 h-1) (Xu et al., 2006). Dry cell weight (11.3 ± 1.4 g/L) was 2.5 fold higher than batch (4.15 ± 0.33 g/L) and fed-batch (4.43 ± 0.17 g/L). However, it was lower than other LAB cultivated in continuous process with cell-recycling, such as Lactobaccillus delbrueckii (118 g/L) and Lactococcus cremoris (88 g/L) (Chang et al., 1994), or Streptococcus cremoris (81.5 g/L) (Taniguchi et al., 1987). These differences can be explained by the fact that they are different bacteria, or because other media, dilution rates, and cell separation systems were applied. Moreover, S. pneumoniae RM200 might not have reached the maximum OD due to the accumulation of inhibitory metabolites in the vessel or in the absence of some nutrient.
Lactate production rose progressively during the cultivation until 21.9 ± 0.9 g/L at 8 h, when glucose concentration increased in the vessel. Acetate production increased until the end of the batch phase, then remained constant until 4 h, when the concentration started to raise again, reaching 5.52 ± 0.42 g/L at 7 h. After 7.5 h, acetate concentration decreased again, coincidently with the decline in lactate production. Around 8 h of cultivation, the decreased consumption of glucose and production of lactate and acetate indicated that the microorganism stopped growing, and we proceeded with cell washing and inactivation at 9 h cultivation.
It is worth to note that perfusion process could be further investigated in order to develop a continuous culture with cell-recycling. To this aim, longer fermentation runs, genetic stability, different dilution and bleeding rates should be evaluated. However, the most challenging for continuous whole-cell vaccine production would be to perform the downstream process for harvesting and washing the biomass at the same time as operating the cell-recycling during the continuous culture. In this case, there should be two different microfiltration systems: one for cell-recycling and another for cell separation, because the fermented broth with inhibitory metabolites has to be removed and cells have to be washed with lactate Ringer’s solution immediately after harvesting, before inactivation. This immediate downstream processing is very important to reach the vaccine quality for soluble protein content (Gonçalves et al. 2014). Moreover, a cost-effectiveness analysis should be done in order to verify the viability of a process with two microfiltration or other cell separation systems operating at the same time.