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).