MOESM1 of Elimination of the flavodiiron electron sink facilitates long-term H2 photoproduction in green algae

Additional file 1: Figure S1. Schematic experimental setup of short-term pulse illumination experiments. In total, 19 ml of C. reinhardtii wt and flv mutant cultures grown in TP medium for 2 days in 50 µmol photons m−2 s−1 bubbling with 3% CO2 was withdrawn and subjected to less than 10-min darkness together with Ar purging before the 20-min pulse illumination experiments. Figure S2. Short-term H2 photoproduction under 1-s light/9-s dark pulse illumination protocol in two different knockout flv mutant lines. Cells were grown for 2 days at 50 µmol photons m−2 s−1 in TP medium bubbling with 3% CO2, transferred to a vial equipped with H2 and O2 sensors, flushed with Ar. The intensity of light pulses was around 250 µmol photons m−2 s−1. H2 yields during 10-min dark anaerobic adaptation phase, 20-min H2 photoproduction phase and 3-min dark H2 uptake phase in CC-4533, flv 208 and flv 791. Experiments have been performed in 3 independent replicates and are presented exemplary. Figure S3. Photosynthetic characteristics of C. reinhardtii wt and flv 208 mutant cultures grown in TP medium for 4 days in 50 µmol photons m−2 s−1 bubbling with 3% CO2. (a) Chl concentration (mg L−1) over a growth period of 4 days. The arrows show when the cultures were withdrawn for further treatments in the long- or short-term experiments. (b) Maximum quantum efficiency of PSII (FV/FM), (c) dark respiration and (d) effective yield of PSII (Y(II)) during 3 days of growth. Experiments have been performed in 5 independent replicates (± SD). (b, d) No statistical significance between wt CC-4533 and flv 208. (c) Statistical significance levels: *p < 0.05; ***p < 0.001. Figure S4. Short-term hydrogen photoproduction yield over 4 days of cultivation. H2 photoproduction is induced by the 1/9 pulse protocol in C. reinhardtii wt CC-4533 and the flv 208 mutant grown in TP medium for (a) 2 days, (b) 3 days and (c) 4 days under 50 µmol photons m−2 s−1 bubbling with 3% CO2. The curves depict H2 level during 10-min dark anaerobic phase, 20-min H2 photoproduction phase and 3-min dark H2 uptake phase. Experiments have been performed in 5 independent replicates and are presented exemplary. Figure S5. Short-term hydrogen photoproduction yield at different Chl concentrations. H2 photoproduction is induced by the 1/9 pulse illumination protocol in C. reinhardtii CC-4533 and the flv 208 mutant. (a) Two-day-old cultures (3 µg Chl ml−1) grown in TP medium under 50 µmol photons m−2 s−1 bubbling with 3% CO2 were concentrated (10 µg Chl ml−1) by centrifugation. (b) Four-day-old cultures (10 µg Chl ml−1) grown in TP medium in 50 µmol photons m−2 s−1 bubbling with 3% CO2 were diluted (3 µg Chl ml−1). Experiments have been performed in 3 independent replicates and are presented exemplary. Figure S6. H2 production rates in C. reinhardtii CC-4533 and flv 208 mutant. (a) Maximal H2 production rates under the 1/9 pulse illumination and 6/9 pulse illumination protocol. (b) H2 production rates during the 1/9 pulse illumination protocol. Cultures were grown 2 days under 50 µmol photons m−2 s−1 bubbling with 3% CO2. The maximal H2 production rates have been obtained within the last 5 min of pulse illumination for 1/9 pulse illumination and within the first 5 min of pulse illumination for 6/9 pulse illumination. Experiments have been performed in 4 independent replicates, and rates were calculated as mean of all replicates (± SD). Statistical significance level: **p < 0.01. Figure S7. Short-term H2 photoproduction under 6-s light/9-s dark pulse illumination protocol in C. reinhardtii CC-4533 and in the flv 208 mutant. The other experimental conditions are the same as in Fig. 1. (a and c) H2 yield in the absence and presence of 10 mM glycolaldehyde (GA). (b and d) Simultaneous monitoring of O2 yield in the absence and presence of GA. Experiments have been performed in 3 independent replicates, and exemplary measurements are presented. Figure S8. Immunoblot analysis of selected proteins from C. reinhardtii CC-4533 and flv 208 mutant grown under long-term 1/9 pulse illumination H2 photoproduction. The western blots shown here are representative of 3 biological replicates.