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.