Aromatic lactic acid production by <i>Bifidobacterium longum</i> subsp. <i>infantis</i> is determined by cell density, substrate availability, and pH <i>in vitro</i>

<i>Bifidobacterium longum</i> subsp. <i>infantis (B. infantis</i>) is an important colonizer of the infant gut. This species is known to produce a variety of health-beneficial metabolites, including aromatic lactic acids (ALAs), supporting early-life immune development. However, the regulation of ALA production in <i>B. infantis</i> remains poorly understood. In this study, we investigated how environmental factors, such as substrate availability and pH, affect <i>B. infantis</i> DSM20088 growth and ALA production <i>in vitro</i>. Bacterial batch cultivations supplemented with different amino acid concentrations revealed a linear relationship between indole-3-lactic acid (ILA), 4-hydroxyphenyllactic acid (OH-PLA), and phenyllactic acid (PLA) production, and exogenous concentrations of tryptophan, tyrosine, and phenylalanine, respectively. Furthermore, chemostat cultivations at physiologically relevant pH levels (4.5, 5.5, and 6.5) showed that an acidic pH slowed growth and shifted ALA production by <i>B. infantis</i>. Overall, ALA concentrations correlated strongly with bacterial abundance across and within pH levels, showing that cell density is a major determinant of ALA production. After considering the growth-limiting effects of lower environmental pH, it had a limited impact on the total production of ALAs, but the relative production of PLA was stepwise enhanced at more acidic pH at the expense of ILA, independent of cell density. In conclusion, this study shows that cell density, substrate availability, and environmental pH determine aromatic lactic acid production by <i>B. infantis</i>.IMPORTANCE<i>Bifidobacterium longum</i> subsp. <i>infantis</i> can be an abundant bacterial species in the infant gut microbiota, where it supports immune development, e.g., through the production of aromatic lactic acids. In this study, we cultured <i>Bifidobacterium longum</i> subsp. <i>infantis</i> at different concentrations of aromatic amino acids and pH levels to investigate the effect of these environmental factors on aromatic lactic acid production. We show that there is a linear relationship between aromatic lactic acid production and the exogenous aromatic amino acid concentration, that the production strongly follows cell density, and that pH regulates the preference toward producing different aromatic lactic acids. This knowledge may help inform strategies to enhance or direct beneficial aromatic lactic acid (ALA) production in the infant gut.