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.