ABSTRACTIn natural environments, nutrients are usually scarce, causing microorganisms to grow slowly while staying metabolically active. These natural conditions can be simulated using retentostat cultivations. The present study describes the physiological and proteome adaptations of the probioticBifidobacterium breveNRBB57 from high (0.4 h−1) to near-zero growth rates. Lactose-limited retentostat cultivations were carried out for 21 days in which the bacterial growth rate progressively reduced to 0.00092 h−1, leading to a 3.4-fold reduction of the maintenance energy requirement. Lactose was mainly converted into acetate, formate, and ethanol at high growth rates, while in the retentostat, lactate production increased. Interestingly, the consumption of several amino acids (serine, aspartic acid, and glutamine/arginine) and glycerol increased over time in the retentostat. Morphological changes and viable but nonculturable cells were also observed in the retentostat. Proteomes were compared for all growth rates, revealing a downregulation of ribosomal proteins at near-zero growth rates and an upregulation of proteins involved in the catabolism of alternative energy sources. Finally, we observed induction of the stringent response and stress defense systems. Retentostat cultivations were proven useful to study the physiology ofB. breve, mimicking the nutrient scarcity of its complex habitat, the human gut.IMPORTANCEIn natural environments, nutrients are usually scarce, causing microorganisms to grow slowly while staying metabolically active. In this study we used retentostat cultivation to investigate how the probioticBifidobacterium breveadapts its physiology and proteome under severe nutrient limitation resulting in near-zero growth rates (<0.001 h−1). We showed that the nutrient limitation induced a multifaceted response including stress defense and stringent response, metabolic shifts, and the activation of novel alternative energy-producing pathways.