3 RESULTS AND DISCUSSION
3.1
Achievement of high cell concentration with functional cellular
skeletons during glycerol feeding phase by the periodic DO control
strategy
The environment of “high DO-low glycerol concentration” based on
DO-stat control mode (strategy A) was firstly implemented to achieve
high cell-density culture during the cell growth phase. As shown
in Fig1A, when using this strategy, DO was controlled at 20-80% and
glycerol concentration naturally stayed at 0-1 g L-1during the glycerol feeding phase. However, severe reactive oxygen
species (ROS) accumulation occurred when cells were exposed to the
oxygen-enriched (high DO) environment for long time, leading to the loss
of cell growth or even cell death (Fig.1D). More concretely, ROS
concentration rapidly rised to the highest level of 152.95 Fluorescence
intensity g-DCW-1 at 18 h when implementing the
environment of “high DO-low glycerol concentration”. After that, cells
growth almost stopped. Meanwhile, about 17.09% of the cells cannot
tolerate such a ROS concentration at the end of glycerol feeding phase
when using this control strategy (Fig.1G). The environment of “low
DO-high glycerol concentration” (strategy B) was also implemented by
controlling glycerol concentration at 2-5 g L-1 and DO
naturally reduced to almost zero level during most of the period in the
glycerol feeding phase (Fig.1B). When using this strategy, maximum ROS
concentrations declined to a very low level of 28.25 Fluorescence
intensity g-DCW-1 (Fig.1E), which could be due to
decrease in cell metabolic activity at oxygen limitation (low DO)
environments. However, with this strategy, final cell concentration
finished at a very low level of 46.25 g-DCW L-1 and
cell mortality was at the highest level of 26.08% (Fig.1E and 1H),
which were far beyond the expectation. In this case, the shortage of
oxygen (low DO) could not stratify the lowest requirement for cell
growth and maintenance metabolism, a lower ROS level cannot achieve high
cell-density cultivation.
Aiming to compromise the positive effects of above glycerol feeding
strategies, a novel periodic DO control strategy (strategy C) was
implemented via periodically switching the cultivation environment from
“high DO-low glycerol concentration” (DO 20-80%, glycerol
concentration 0-1 g L-1; T1≈5 h) to
“low DO-high glycerol concentration” (DO ~0%,
glycerol concentration 2-5 g L-1; T2≈1
h) for 4 cycles (Fig.1C). As shown in Fig.1F, the highest cell
concentration of 118.96 g-DCW L-1 was achieved at the
end of glycerol feeding phase with this periodic control strategy.
Meanwhile, maximum ROS concentration declined to a lower level of 48.35
Fluorescence intensity g-DCW-1 and cell mortality was
controlled below 10% throughout the entire glycerol feeding phase
(Fig.1F and 1I).