Abstract
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 multi-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.
Data availability
Raw bulk RNA-seq and ATAC-seq sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) repository. The bulk RNA-seq datasets are available under accession numbers GSE254130 and GSE317794, and the bulk ATAC-seq dataset is available under accession number GSE254130. Processed data used for downstream analyses, including RNA-seq gene expression matrices, ATAC-seq accessibility matrices, NMR data, and source data underlying the graphs presented in the main figures, are provided as Supplementary Data 1–5. Uncropped and unedited Western blot images are provided in Supplementary Fig. 6. All other data are available from the corresponding author (or other sources, as applicable) on reasonable request.
Materials availability
Materials related to the bioreactor system will be available upon request. Information related to cell culture and reagents is present in the methods found in this study.
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Acknowledgements
This work was supported by the KAUST Office of Sponsored Research (OSR), under award number BAS/1/1080-01 (ML) and BAS/1/1084-01 (LJ); and by KAUST Center of Excellence for Smart Health (KCSH), under award number 5932 (ML) and 5297 (LJ). We thank the members of the Li lab, Jinna Xu, Xuan Zhou, Yeteng Tian, and KAUST Imaging and Characterization Core Lab and Bioscience Core Lab for their help in this research.
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S.A. conducted cell culture experiments and related assays, including RNA-seq, ATAC-seq, Sanger sequencing, viability assay, and ddPCR, for the acidic pH experiments. S.A. and M.L. drafted and revised the initial manuscript and performed data integration and analysis. S.A., Y.Z. and M.L. led the revision of the manuscript and performed data integration and analysis. U.S. and K.S. conducted all NMR-related work. Y.Q. conducted cell culture experiments and related assays for restoration of NAD⁺ levels, including RNA-seq and viability assay. Y.Z. performed the bioinformatic analysis of bulk RNA-seq and ATAC-seq data, and performed RT-qPCR and ddPCR for mtDNA variant analysis. N.S. conducted all Seahorse assays. S.G.K. analyzed the bioreactor gas flow data. B.Y. carried out NANOLIVE imaging and data analysis. C.B. designed the ddPCR primers. C.B. and Y.Z. performed mitochondrial mutation analysis. S.F. performed western blot experiments and data analysis. S.A., Y.Z., M.L., U.S., L.J., C.M.D., S.G.K. and M.W. revised the manuscript. J.C.I.B., P.J.M., C.M.D., L.J. and M.L. conceived the study. M.L. supervised the study.
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An application based on methods described in this paper has been filed by King Abdullah University of Science and Technology. The authors declare no other competing interests. M.L. is an Editorial Board Member for Communications Biology, but was not involved in the editorial review of, nor the decision to publish this article.
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Alsolami, S., Zhang, Y., Singh, U. et al. Stable bioreactor control reveals acidic pH–driven metabolic reprogramming and mitochondrial dysfunction in human lymphoblastoid cells. Commun Biol (2026). https://doi.org/10.1038/s42003-026-10013-5
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DOI: https://doi.org/10.1038/s42003-026-10013-5
