Sodium ion stress accelerates cell lysis in Bacillus coagulans by exacerbating energy deficit during carbon source transition.

Abstract

The choice of pH neutralizer plays a critical role in determining the performance of industrial lactic acid fermentation. This study investigated the distinct phenotypic and metabolic responses of Bacillus coagulans during batch fermentation with mixed carbon sources (glucose and trehalose) when using sodium hydroxide versus calcium hydroxide for pH control. Compared to calcium hydroxide, sodium hydroxide resulted in a slightly lower lactic acid yield and induced significant cell lysis during the carbon source transition. Metal ion experiments confirmed that sodium ion stress is the primary factor responsible for extensive cell lysis. Omics analysis indicated that under sodium hydroxide conditions, cells require more energy to resist stress. To meet this demand, cells downregulated cellular growth and sodium ion uptake systems, while upregulating carbon source transport. However, during carbon source transition, cellular resource reallocation resulted in insufficient carbon source uptake, leading to stagnation of glycolysis and amino acid metabolism. The resulting energy deficit triggered upregulation of cellular hydrolases, ultimately causing population-wide cell lysis. In contrast, calcium hydroxide as a neutralizer does not impose sodium stress and contributes to maintaining cell wall stability under carbon source deficiency. This study contributes to the understanding of cellular regulatory mechanisms in response to different neutralizers, and provides insights for metabolic engineering and process optimization.IMPORTANCEpH neutralizer selection critically determines cell survival and process efficiency in industrial fermentation. Our study demonstrates that sodium hydroxide, although effective for pH control, imposes severe sodium ion stress that synergizes with the inherent energy vulnerability of cells during carbon source transition, triggering catastrophic population-wide lysis in Bacillus coagulans. Through integrated physiology, ion experiments, and multi-omics analyses, we reveal that the underlying cause is an energy crisis arising from the conflict between stress defense and metabolic reallocation. In contrast, calcium hydroxide not only avoids sodium stress but also confers structural and thermal stability to cells, thereby preserving cell density and improving carbon utilization. These findings shift the perspective on neutralizers from mere pH regulators to key determinants of cellular energy economy and survival. Our study thus offers a rational basis for neutralizer selection, strain engineering toward ion resistance, and the design of robust fermentation processes, with direct implications for the sustainable and cost-effective production of lactic acid and other microbial metabolites.