Strain performance without a neutralizing agent
Considering the promising result of the previous experiment, the LA2 strain was selected for further observation. Figure 3 shows that with and without the addition of calcium carbonate, the LA2 strain produced lactic acid in concentrations of 43.2 and 33.2 g·L-1, respectively. Even though the accumulation of lactic acid declined, the removal of calcium carbonate did not cause a significant decrease in the rate of glucose uptake because the glucose was completely consumed within only 9 h under both conditions. Nonetheless, as shown in Table 2 , these results were relatively competitive by comparison with those of the microbial hosts described in other studies (Ilmén et al., 2013; Ishida et al., 2005; Mitsui et al., 2020; Park et al., 2018). Despite its lower yield, the LA2 strain maintained the ability to utilize glucose rapidly and exhibited an even more stable level of productivity when the neutralizing agent was removed. With the addition of a neutralizing agent, the productivity was 4.80 g·L-1·h-1. Meanwhile, under a non-neutralized condition, the productivity remained high (3.68 g·L-1·h-1), although the pH of the medium dropped to 3.00 after 9 h of cultivation (∆pH = + 1.5, compared with a neutralized condition). Other studies included inTable 2 have exhibited a drop in productivity by as much as 50%, or more, when no neutralizing agent was supplemented into the medium.
Based on the result shown in Figure 3 , lactic acid generation was terminated because the glucose was completely consumed within 9 h. However, as depicted in Figure 4 , the LA2 strain could still accumulate lactic acid when glucose was fed to the medium even without supplementation with a neutralizing agent. After being fed with concentrated glucose three times at 6 h, 10 h and 14 h, the productivity of engineered strain stood at 3.45 g·L-1·h-1(C LA = 62.0 g·L-1) and 2.35 g·L-1·h-1(C LA = 42.2 g·L-1) under the neutralized and non-neutralized condition, respectively. Akin to the results from batch cultivation, this strain exhibited high productivity under both conditions, with a drop in value less than 35% when the neutralizing agent was removed from the medium.