Mechanism of CO₂ acquisition in an acid-tolerant Chlamydomonas

The acid-tolerant green alga Chlamydomonas (UTCC 121) grows in media ranging in pH from 2.5 to 7.0. Determination of the overall internal pH of the cells, using ¹⁴C-benzoic acid (BA) or [2-¹⁴C]-5,5-dimethyloxazolidine-2,4-dione (DMO), showed that the cells maintain a neutral pH (6.6 to 7.2) over an external pH range of 3.0-7.0. The cells express an external carbonic anhydrase (CA) when grown in media above pH 5.5, and CA increases to a maximum at pH 7.0. Removal of external CA by trypsin digestion or by acetazolamide (AZA) inhibition indicated that CA was essential for photosynthesis at pH 7.0 and that the cells had no capacity for direct bicarbonate uptake. Monitoring of CO₂ uptake and O₂ evolution by mass spectrometry during photosynthesis did not provide any evidence of active CO₂ uptake. The CO₂ compensation concentration of the cells ranged from 9.4 μM at pH 4.5 to 16.2 μM at pH 7.0. An examination of the kinetics of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco), in homogenates of cells grown at pH 7.0, showed that the Km (CO₂) was 16.3 μM. These data indicate that the pH between the cell interior and the external medium was large enough at acid pH to allow the accumulation of inorganic carbon (Ci) by the diffusive uptake of CO₂, and the expression of external CA at neutral pH values would maintain an equilibrium CO₂ concentration at the cell surface. This species does not possess a CO₂-concentrating mechanism because the whole cell affinity for Ci appears to be determined by the low Km (CO₂) Rubisco of the alga.