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 .