Stable Bioreactor Control Reveals Acidic pH–Driven Metabolic Reprogramming and Mitochondrial Dysfunction in Human Lymphoblastoid Cells

Human cells require pH regulation to maintain physiological function, yet the molecular consequences of acidic environments remain incompletely understood. Here, we employ a gas-only bioreactor to control pH, oxygen, and temperature. Integrated Omics analyses reveal that acidic pH induces a glycolytic metabolic shift, suppresses proliferation, and promotes accumulation of lactate and oncometabolites alongside mitochondrial dysfunction. Acidic conditions increase reactive oxygen species (ROS) and activate inflammatory and immune pathways, leading to heteroplasmic enrichment of a pathogenic mitochondrial mutation. Acidic pH depletes intracellular NAD⁺, partly driven by PARP1 activation. Restoring NAD⁺ through nicotinamide mononucleotide (NMN) supplementation partially rescues proliferation and stress-associated transcription, while elevating NAD+ levels by NMN or PARP1 inhibition reverses heteroplasmic enrichment of mutant mitochondrial DNA. These findings underscore the role of pH homeostasis in coordinating metabolism, redox balance, and immune signaling, and identify NAD⁺ metabolism as a mechanistic link between acidic microenvironments, mitochondrial genome instability, and immune–metabolic remodeling.