Fig. 1: Probing lumen acidification using NPQ during anaerobic treatment.

Chlorophyll fluorescence and oxygen concentration were simultaneously recorded on Chlamydomonas mutants stt7-9 (a), npq4 stt7-9 (b), and on Chlamydomonas eustigma NIES-2499 in aerobic condition (c), and NIES-2499 in anaerobic condition (d). The cells used for the experiments shown in (a, b, d) were grown under high light and resuspended in fresh HSM-ficoll (10%) (a, b) or M-Allen -ficoll (d) in a cuvette and sealed to induce anoxia. The ficoll is used to keep the cells in suspension. Fm’, indicated by the red dots, was recorded every 3 min. The addition of KOH completely relaxed the quenching in the stt7-9, but hardly affected the fluorescence in the double mutant npq4 stt7-9 and NIES-2499. For the experiment shown in (c), cells were first exposed to actinic light (AL 1500 μmol photons m⁻² s⁻¹) to induce chlorophyll quenching (20 s–80 s) in aerobic environment. After NPQ was fully released (80 s–200 s), a second round of fluorescence quenching was induced by adding 4.5 mM HAc (the pH of medium ~3.3), and after that, the addition of KOH (the pH of medium ~5.5) fully abolished the fluorescence quenching. The red bars represent actinic light illumination and the black bars dark treatment. The relationship between fermentation products formic acid (e) and acetic acid (f) accumulated in the medium of Chlamydomonas cultures and NPQ in stt7-9. Cells were kept in anaerobiosis in the dark for up to 4.5 h. Samples were taken at the indicated time points (0, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 4.5 h), centrifuged, and filtered, and the medium was analyzed by HPLC. Data are taken from triplicate samples derived from three independent experiments. Error bars represent SD.