By: Richard D. Cummings
In plants, glucose is produced from carbon dioxide (CO2) and water through photosynthesis, which generates oxygen (O2) as a byproduct. Without sunlight/photosynthesis, the Calvin Cycle, along with probably the most important enzyme in the world - RuBisCo (Ribulose-1,5-bisphosphate carboxylase/oxygenase) – life as we know it would not exist (1). But while plants require CO2, do humans also require CO2? While we do not use CO2 to synthesize organic molecules, the answer is yes, CO2 is essential for our life and used to regulate respiration and the pH of our blood. Amazingly, our brain monitors CO2 but not O2, which triggers our breathing reflex. This sensing involves a chemoreceptor reflex, and sensors in our brainstem and arteries, which help remove excess and keep our blood pH in the proper range of ~7.4.
CO2 occurs naturally in small amounts (about 0.04 percent) in our atmosphere. However, if you are familiar with culturing mammalian cells in the laboratory, such cultured cells in incubators require 5% CO2. You may ask why? Cells do not require CO2 per se to live, but they do produce CO2 through cellular respiration. However, the bicarbonate system, which occurs naturally in the human body, is used for in vitro cell culture to mimic animal physiology and the buffering capacity of carbonate, which reduces toxic side effects of chemical buffers that could be used to control pH (2,3).
In humans when CO2 enters the blood, it forms carbonic acid (H2CO3), when combined with the water. Carbonic acid is a relatively weak acid, which dissociates into hydrogen ions (H+) and bicarbonate ions (HCO3-). We also use carbonic anhydrase, which catalyzes interconversion of CO2 to bicarbonate (4). In incubators, the CO2 reacts with water in the culture media to form carbonate (H2CO3); the culture media, such as DMEM, also contains sodium bicarbonate NaHCO3. The dissolved H2CO3 and NaHCO3 dissociate to carbonic acid and H+ and sodium to create an equilibrium with the CO2 in the gas phase. Thus, carbonate helps to buffer the system to keep the pH near a physiological range. As cells metabolize glucose, they naturally produce lactic acid (lactate) even in aerobic conditions by their respiration (5). This can lead to a lowering of the pH of culture media, e.g., the phenol red indicator in the media may turn pink as a result, eventually requiring changing of the media. Phenol turns from yellow to red over a range of pH 6.6 to 8.0, and above pH 8.1 it is bright pink. So a yellow culture media means one should probably change the media as it is getting pretty acidic and the glucose has probably been largely consumed.
Interestingly, in cell culture D-glucose concentrations in media range from 1 g/L (5.5 mM) to as high as 10 g/L (55 mM). While ~5.5 mM glucose approximates normal blood sugar levels, levels above 10 mM can be toxic in several ways and more similar to those found in diabetic conditions. Hyperglycemia in cultured cells can lead to overproduction of hyaluronan (6,7), which in airway smooth muscle cells can alter their adhesive properties and could be associated with lung injury.
References
1. Sharkey, T. D. (2019) Discovery of the canonical Calvin-Benson cycle. Photosynth Res 140, 235-252
2. Maniarasu, R., and Kumar, M. R. (2022) A Mini-Review on CO2 Role in Cell Culture and Medicinal Applications. Journal of Cell Science & Therapy 13, 346-350
3. Patel, S., Miao, J. H., Yetiskul, E., Anokhin, A., and Majmundar, S. H. (2025) Physiology, Carbon Dioxide Retention. in StatPearls, Treasure Island (FL) ineligible companies. Disclosure: Julia Miao declares no relevant financial relationships with ineligible companies. Disclosure: Ekrem Yetiskul declares no relevant financial relationships with ineligible companies. Disclosure: Anya Anokhin declares no relevant financial relationships with ineligible companies. Disclosure: Sapan Majmundar declares no relevant financial relationships with ineligible companies. pp
4. Supuran, C. T. (2023) Carbonic anhydrase versatility: from pH regulation to CO(2) sensing and metabolism. Front Mol Biosci 10, 1326633
5. Chitwood, D. G., Uy, L., Fu, W., Klaubert, S. R., Harcum, S. W., and Saski, C. A. (2023) Dynamics of Amino Acid Metabolism, Gene Expression, and Circulomics in a Recombinant Chinese Hamster Ovary Cell Line Adapted to Moderate and High Levels of Extracellular Lactate. Genes (Basel) 14
6. Wang, A. J., Ren, J., Wang, A., and Hascall, V. C. (2023) Monocyte adhesive hyaluronan matrix induced by hyperglycemia in diabetic lung injuries. J Biol Chem 299, 104995
7. Wang, A., and Hascall, V. C. (2004) Hyaluronan structures synthesized by rat mesangial cells in response to hyperglycemia induce monocyte adhesion. J Biol Chem 279, 10279-10285