This study was conducted to investigate changes in intracellular microenvironment of Acetobacter pasteurianus CICIM B7003 and its high-yield mutant A.pasteurianus CICIM B7003-02 to unravel the acid-tolerant mechanisms of A. pasteurianus. The fermentation kinetics of parent and mutant strain during batch and semi-continuous fermentation in a 7.5 L fermenter under acidic stress, as well as changes in their intracellular microenvironment were compared. It was found that during batch fermentation, the average fermentation efficiency of the mutant strain reached 0.836 g/(L·h), which increased 29.6% compared against that of the parent strain. Moreover, the acid start-up time decreased 31 h during semi-continuous fermentation. Furthermore, enzymes that are directly related to acid production in the mutant strain, such as alcohol dehydrogenase and acetaldehyde dehydrogenase, increased their highest activities by 27.0% and 15.2%, respectively. Besides, the content of coenzyme Q9 increased by 69.5%. In addition, the mutant strain had a higher proportion of octadecenoic acid, which was 71.4% higher than that of the parent strain. The maximum intracellular ATP content of the mutant strain was 2.33-fold higher, and intracellular ATP was positively correlated with the specific growth rate. Also, intracellular glutamate and aspartic acid increased by 10.7% and 18.3%, respectively. In conclusion, the mutant strain was mainly relied on the synergistic effects of strengthening ethanol respiratory chain, ATP synthesis, and critical amino acid metabolism to improve its acid resistance.
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