2.2 Instances of CBM applications in fermentation optimization
The culture medium is one of the most significant factors affecting cell
growth and productivity in bio-based products [4, 50, 51].
Optimization of culture medium and development of proper feeding
strategies has always been a critical factor in bioprocess development
[2, 52]. Since medium components, nutritional supplements, and
culture additives directly affect cellular metabolism [53], CBM can
be a suitable approach to analyze the effect of medium components on
cell growth and productivity. CBM-based methods have been extensively
used to analyze and optimize the culture medium and develop appropriate
feeding strategies to overproduce a wide range of products such as
recombinant proteins [54-58], biofuels [59-61], lipids [62,
63], chemicals [64-66], and foods [36]. For a more detailed
example, Swayambhu et al. used FBA and its derivatives to identify gene
deletion/overexpression targets and culture medium components to enhance
the production of siderophore compounds in recombinant Escherichia
coli . First, they used minimization of metabolic adjustment (MoMA) and
OptForce algorithms to identify the gene deletion and overexpression
targets, respectively. Then, they combined the FBA with Plackett Burman
(PB) in silico to identify the amino acids and carbon sources that had a
major impact on productivity. Also, it was observed that by changing the
rate of nutrient uptake, the priorities of some candidates for gene
deletion or overexpression have changed [67]. Fouladiha et al. used
a genome-scale metabolic model of the Chinese hamster ovary (CHO) to
design feeding strategies to increase the production of a monoclonal
antibody. In this study, CBM has been used as pre-step of the design of
experiment (DoE) methods to decrease the number of variables and
experiments. First, they set the objective function to maximize antibody
production and used flux variability scanning based on enforced
objective flux (FVSEOF) algorithm to detect reactions whose boundaries
had changed. Fifteen exchange reactions were identified related to
various media supplements, including three vitamins, seven amino acids,
and five other metabolites. PB was then applied to screen for these 15
metabolites, of which threonine and arachidonic acid were identified as
the most effective supplements for the culture medium. A more than
two-fold increase in protein production was observed through this
strategy [68]. In another study, Sarkandy et al. developed a
stoichiometric model to predict the most efficient amino acids for
enhancing the production of interleukin-2 in fed-batch culture of
recombinant Escherichia coli . The results showed that the mixture
of leucine, aspartic acid, and glycine improves protein productivity by
almost two-fold [69]. Recently, Shahidi et al. have comprehensively
reviewed the applications of systems biology approaches, including CBM,
in chemically defined media formulations for the overproduction of
recombinant proteins [70]. FBA has also been used to investigate the
effect of glucose, glycerol, and glucose-glycerol dual mixtures on the
internal carbon flux distribution in simultaneous ethanol and butanol
production [71].
Fermentation conditions also can directly affect the cellular metabolism
toward the growth and productivity. Therefore, CBM methods are a
convenient and low-cost approach to optimize fermentation conditions.
For example, Calic et al. used the MFA method to investigate the effect
of pH on the intracellular metabolic network of Bacillus
licheniformis , a β-lactamase-producing bacterium. In this study, the
values of metabolic fluxes related to cell growth, by-products, and
desired product production at pH = 6.5, 7, 7.5 were studied. The results
show that in the period of cell growth, the by-product fluxes have the
highest value at pH = 7 and the lowest value at pH = 7.5, while the
change in pH in this period does not have a significant effect on the
production of β-lactamase. On the other hand, it has been observed that
in the stationary phase and the product formation period, the flux value
of the desired product is maximum, and the flux values of by-products
are minimum at pH = 7. Finally, this article proposes a pH operation
strategy to improve β-lactamase production as follows: First, the
initial pH should be set to pH = 7.5 and allowed to decrease to pH = 7
during fermentation, and then kept constant at this value [8]. In
another study, Ivarsson et al. manipulated lactate consumption and
production by pH alteration during mammalian cell cultivation. They used
FBA to see how pH changes affect lactate metabolism. The results show
that by lowering the pH from the standard pH = 7.2 to pH = 6.8, lactate
consumption increases, the cell becomes more energy-efficient, and
antibody production increases. Moreover, they found that gluconeogenic
enzymes regulated the TCA cycle at undesirable pH levels [72]. FBA
method constrained by experimentally measured extracellular fluxes has
been developed by Sou et al. to investigate the effect of mild
hypothermia conditions on antibody glycosylation in the late exponential
of a CHO cell. Flux distribution values showed that during the
stationary phase at 32 °C, more energy and metabolites are expended to
increase cell productivity, which limits most of the resources necessary
for antibody glycosylation [73]. The overall overview of CBM
applications for fermentation parameters highlighted in this review is
presented in Table 1 .