Discussion

Oxygen plays a pivotal role in aerobic fermentation. Aerobic microorganisms generally require large amounts of oxygen to generate NAD(P)H or FADH2 and ATP for metabolism. Several studies also showed that the dissolved oxygen levels directly affect the transcription level of genes and synthesis of different enzymes and results in the changes of cell metabolism, product yield and productivity (Song et al., 2013) (Martinez, Bennett, & San, 2010).
The DO level was maintained at 10%-20% during DO-stat fed-batch fermentation in this study, and the DO level was significantly higher than that of fed-batch fermentation. We found that different dissolved oxygen level affects the metabolism of YL-C11. Low DO resulted in less ATP and the reducing power [NAD(P)H] that required for cell maintenance and growth in fed-batch fermentation. Using the DO-stat method revealed that biomass was enhanced than that of in fed-batch fermentation. The glycolytic, hexose monophosphate, and tricarboxylic acid cycle (TCA cycle) pathways not only are used as the hub to convert carbohydrates, proteins, and lipids but are the essential pathways to produce energy during the metabolism. In DO-stat fed-batch fermentation, the above three pathways were strengthened with adequate oxygen delivery. The oxidative phosphorylation and substrate-level phosphorylation was enhanced simultaneously. We set the oxygen level at 10%-20% for DO-stat fed-batch fermentation. That lead to more ATP and NADP+/DADPH were generated, thereby resulting in the boosted of biomass and β-carotene concentration. Several studies have confirmed that high oxygen levels could improve the metabolic flux of aerobic microorganisms; thus, more biomass and target product was obtained. (Kawaguchi et al., 2019b) (Xu et al., 2009) (Song et al., 2013). Moreover, the transcriptional level of related genes in the β-carotene biosynthesis pathway was higher in DO-stat fed-batch fermentation (Supporting Information, Figure 1).
The main aim of the present investigation was to achieve high cell concentration, thus increase β-carotene concentration. A series of the kinetic models were constructed to elaborate on the relationship among cell growth, substrates and products. The kinetic parameters were determined from different feeding strategies in this study. These simulations provided an insight into the operational protocol that may be implemented to obtain the best results.
The fed-batch fermentation produced of 73.5 g/L cell biomass and productivity of 0.61 g/L/h. In contrast, The DO-stat fed-batch fermentation produced 94g/L cell biomass and productivity of 0.78 g/L/h. The fermentative capacity was strongly affected by the specific growth rate of aerobic cultures (PIM VAN HOEK, 1998). The simulation results showed that the higher μm, Yx/s, and Yp/s values were achieved for DO-stat fed-batch fermentation. In addition, the DO-stat feeding strategy extended the logarithmic period, and more glucose was consumed in this period. As a result, more biomass was obtained for the DO-stat fed-batch fermentation. The improved growth can be attributed to that the Crabtree effect was blocked with DO-stat feeding strategy. The Crabtree effects are prevented from occurring during DO-stat fed-batch fermentation, which is one of the critical reasons that longer logarithmic phase, higher biomass, and glucose utilization were obtained. Thus, DO-stat fed-batch fermentation is an excellent approach to produce β-carotene. Although further research is needed to improve this kinetic model further, we could use this kinetic model to understand the relationship between cell growth, substrate consumption, and product synthesis for different feeding strategies.