3.1. Evaluation of modified genome-scale metabolic models at different pH levels
Figure 3 demonstrates the effect of proton exchange flux on the growth rate of Z. mobilis , S. cerevisiae, and E. coli . As the steady-state condition is assumed by FBA, the amount of charge entering the cell has to be equivalent to the leaving amount; thus, protons are consumed or produced for the charge balance. It can be inferred from Figure 3 that Z. mobilis is more sensitive to pH rather than S. cerevisiae and the yeast is more regulated by pH rather than E. coli . E. coli is a neutralophilic bacterium that can maintain its cytoplasmic pH in a narrow range of ~7.5–7.7 and can grow at extracellular pH values from ~5.5–9.0 (Martinez et al., 2012). However, the influence of extracellular pH on intracellular value is significantly more for acidophilic microorganisms Z. mobilis and S. cerevisiae . By decreasing the external pH from 7.0 to 2.2, a progressive reduction of the internal pH from 7.1 to 5.1 was observed in exponentially grown cells (Valli et al., 2005). Experimental data (Kalnenieks et al., 1987) showed that the reduction of extracellular pH of Z. mobilis from 5.6 to 3.5 would change the intracellular pH from 6.4 to 5.75.
A comparison of the results shows that Z. mobilis is the most sensitive microorganism to proton exchange and Figure 3a indicates that this bacterium is only capable of producing protons. The charge balance of this microorganism is merely possible to the proton production rate of approximately 6 mmolgDCW-1h-1. This range is small in comparison with two other microbes and indicates the less flexibility of Z. mobilis metabolism to pH changes. Furthermore, the proton exchange range for optimal growth of Z. mobilis is in the limited amount of 0.5 to 1 mmolgDCW-1h-1. Z. mobilis is an ethanologenic bacterium that prefers the maintenance of intracellular pH rather than more growth. Motamedian et al.(Kalnenieks et al., 1987; E Motamedian et al., 2016) expressed that more proton exchange leads to further growth only when growth and energy are coupled. So, the low flexibility of Z. mobilis metabolism to pH changes and its tendency for the maintenance of intracellular pH can justify its energy-uncoupled growth.
Comparison of Figures 3b and 3c indicates that the metabolic sensitivity of S. cerevisiae is more than E. coli . Similar to Z. mobilis , S. cerevisiae also mostly produces protons and the optimal growth of S. cerevisiae is accompanied by proton production in the limited range of proton exchange rates, which is near to zero. It is evident that as the internal pH drops to acidic condiction, charges of metabolites would lead to positive numbers (Figure S1). Given that the steady-state assumption, it is expected that proton is produced in optimal growth condition, which can be seen in the robustness analysis of S. cerevisiae and Z. mobilis since glucose as the carbon source has no charge and ammonium with positive charge can be the determinant substrate for charges entering the cell. However, E. coli has the opposite behavior by consuming protons, as the internal pH drops and at pH=5, the optimal proton consumption rate for the growth of E. coli is equivalent to -10 mmolgDCW-1h-1 the biomass charge will be more positive in acidic pH (Figure S2). This effect of the biomass charge can also be seen in pH=7 for Z. mobilis andS. cerevisiae , which biomass charge has a noticeable difference with other pH levels (Figure S2).
Figures S3-8 demonstrate another metabolic similarity of S. cerevisiae and Z. mobilis in which optimal growth rate is associated with maximum ethanol at all pH levels while the results forE. coli is not monotonic (Figure S9-11). In this regard, S. cerevisiae and Z. mobilis have more in common as they are known as industrial ethanologenic organisms with an ethanol yield of 0.41 and 0.5 g/g (Table S5). However, Maximal ethanol production under optimal growth for E. coli is obtained for pH=6 and this maximal value for pH=7 is more than that for pH=5. The experimental data also confirm that the optimal pH for ethanol production by E. coli is 6.5. Besides, the lowest yield of ethanol production is reported for E. coli 0.09 g/g, while this bacterium has the highest growth yield, according to Table S5. Bekers et al.(Bekers, Heijnen, & Van Gulik, 2015) experimentally evaluated the amount of NAD: NADH ratio assuming a decrease in intracellular pH would increase this ratio, which means more NADH consumption and higher ethanol production. Z. mobilis andS. cerevisiae are the acidophilic microorganisms and capable of more ethanol production than E. coli , which is the neutrophilic bacteria. In fact, cells with the capability of higher ethanol production showed tightly regulated metabolism and responsiveness to pH changes due to the imbalance of NAD: NADH ratio. Consequently, Z. mobilis , which converts 95% of its carbon source into ethanol and CO2, then S. cerevisiae and E. coli are more regulated by pH, respectively.
Despite the similarity of the metabolism of S. cerevisiae andZ. mobilis , S. cerevisiae is more flexible and its sensitivity to proton exchange rate reduces as the internal pH decreases (Figures 3a and 3b). The metabolic models predict that pH=5 is the most appropriate level for growth and ethanol production (Figures S6-8). Sensitivity to proton production rate at this pH level disappears and maximal ethanol production in the wide range of proton production rates under optimal growth rate is possible. Experimental data confirm the predicted optimal pH for S. cerevisiae and Narendranath et al. (Narendranath & Power, 2005) stated that the ideal pH for S. cerevisiae is at the optimum enzyme activity of that product, which is 5-5.5 for the production of ethanol and yeast growth. Furthermore, the growth yield (Table S5), growth rate (Figures 3a and 3b) and tolerance to environmental stresses of S. cerevisiae are significantly higher than Z. mobilis . These characteristics have made S. cerevisiae the main strain for ethanol production in the industry.