Strategies for high cell density cultivation of Akkermansia muciniphila and its potential metabolism

ABSTRACT Akkermansia muciniphila ( A. muciniphila ) has sparked widespread interest as a potential probiotic bacterium with many physiological functions that colonizes the human intestinal tract. The development of its in vitro culture is a promising and urgent research direction. Therefore, culture conditions were first optimized, and a significant improvement of cell density of A. muciniphila was achieved in a shake flask with 15.0 g/L of glucose, 37.0 g/L of tryptone, and an initial pH of 7.8. A high OD 600 (optical density at 600 nm) value of 13.03 (1.03 × 10 10 CFU/mL) was reached in a 5-L bioreactor by stage pH controlling, which is the highest reported value by use of a sole carbon source (glucose). Analysis of cell characteristics and protein expression showed that the optimized culture did not affect cellular morphology and the expression of the special outer membrane functional protein (Amuc_1100), while remarkably improving cell hydrophobicity, which is beneficial for bacterial colonization of the gut. The pattern of supernatant metabolites indicated that the optimized medium may promote cell reproduction by inducing cells to produce dethiobiotin and strengthen the metabolic pathway of glycerol 3-phosphate by shifting more glyceraldehyde 3-phosphate dehydrogenase toward secretion at the cell surface, thereby improving cell surface hydrophobicity and adhesion to mucin. This study accomplished the high cell density culture of A. muciniphila without affecting its biological function, which also provides a more conducive fermentation strategy to enhance cell adhesion and facilitates its colonization in the gut. IMPORTANCE Currently, there is significant interest in Akkermansia muciniphila as a promising next-generation probiotic, making it a hot topic in scientific research. However, to achieve efficient industrial production, there is an urgent need to develop an in vitro culture method to achieve high biomass using low-cost carbon sources such as glucose. This study aims to explore the high-density fermentation strategy of A. muciniphila by optimizing the culture process. This study also employs techniques such as LC-MS and RNA-Seq to explain the possible regulatory mechanism of high-density cell growth and increased cell surface hydrophobicity facilitating cell colonization of the gut in vitro culture. Overall, this research sheds light on the potential of A. muciniphila as a probiotic and provides valuable insights for future industrial production.