Equal amounts of total protein were loaded in each lane. at the proximity of the stromal face of the thylakoid membrane. Furthermore, we found that in transition to high light, the hydrogen production rate is significantly enhanced for a short duration (100 s), thus indicating that [FeFe]-hydrogenase functions as an immediate sink for surplus electrons in aerobic as well as in anaerobic environments. In summary, we show that an anaerobic locality in the chloroplast preserves [FeFe]-hydrogenase activity and supports continuous hydrogen production in air-grown microalgal cells. Photosynthetic hydrogen production is limited to microbial photosynthetic organisms, such as is usually absent in air-grown cultures, and its accumulation is induced only upon anoxia, with a maximal accumulation observed after 3 h of nitrogen sparging (Happe et al., 1994) or 72 h of sulfur deprivation (Winkler et al., 2002). Accordingly, atmospheric levels of oxygen completely inactivate the catalytic site of [FeFe]-hydrogenase within a few seconds (Ghirardi et al., 2007). In contrast, Kosourov and Seibert (Kosourov and Seibert, 2009) observed continuous hydrogen production from cultures embedded in films under ambient atmosphere, although the oxygen levels within the films were not decided. In further support of this, recent reports have suggested that [FeFe]-hydrogenase is usually capable of aerobic activity in strains of could maintain hydrogen production under atmospheres of 21% (Hwang et al., 2014) or 15% (Chader et al., 2009) oxygen. The active pool of hydrogenase was estimated at 30 models per mg of dry weight (Hwang et al., 2014). Last, in a recent paper by Godaux et al. (Godaux et al., 2015), the authors claim that in a transition from dark anoxia to light, high rate of hydrogen production decreased to lower rate, before the onset of oxygen accumulation. Hence, they conclude that a competition for electrons with downstream processes such as FNR and CEF govern the hydrogen production rate rather than oxygen concentration. All of GSK2656157 these findings contradict the common view since the oxygen sensitivity of [FeFe]-hydrogenase is usually well documented (Ghirardi et al., 1997). Furthermore, it is unclear why the transcripts of hydrogenase and its maturases, Hyd EF and G, are present under aerobiosis and increase under anaerobiosis induced by sulfur deprivation by roughly 20%, as we analyzed from published RNAseq data (Gonzlez-Ballester et al., 2010). The mechanism underlying [FeFe]-hydrogenase activity in air-grown cultures is yet to be resolved. We have therefore used the model organism (strain CC-124) at 2.5 g (chl)/mL were cultivated in aerated 100-mL flasks (Fig. 2A, inset) with constant stirring and under three light intensities (77, 155, and 600 E m?2 s?1; hereafter E). To ensure GSK2656157 full aerobiosis in these conditions, we quantified the concentration of dissolved oxygen using oxygen electrode and Winkler reaction (Winkler, 1888). Furthermore, we analyzed the headspace gas using a gas chromatograph. Growth at all irradiances showed constant atmospheric levels of oxygen in the headspace of the growth vessels as well as 250 m dissolved oxygen in the growth media (Supplemental Fig. S1); the same oxygen concentration was measured for the positive controla cell-free aerobic growth media. Open in a separate window Physique 2. Photosynthetic activity and quantification of [FeFe]-hydrogenase in air-cultivated cells. A, Steady-state hydrogen production rates measured by MIMS in wild-type cultures cultivated under three light intensities: 77, 155, and 600 E. Inset, Cells were produced in flasks stoppered with a sponge completely permeable to air, with constant light and stirring. B, LEF from PSII to hydrogen recorded upon transition from 77 E to 1200 E of either wild-type cells in the presence (broken green line) or absence (solid green line) of the PSII donor side inhibitor DCMU. The mutant was used as unfavorable control (black). C, Inhibition of hydrogenase by the inhibitor CO shuts down hydrogen production (green bar) in air-grown cells upon transition from 77 E to 1200 E at the onset of the recording. Photosynthetic oxygen production was not affected by the GSK2656157 addition of CO (blue bars). D, Quantification of [FeFe]-hydrogenase in cells cultivated aerobically under 77, 155, and 600 E. Top, An immunoblot performed using anti-HydA antibody. Purified [FeFe]-hydrogenase (5 ng of HydA) was used as marker and reference for band intensity quantification. Equal amounts of total protein were loaded in each lane. [FeFe]-hydrogenase quantities were normalized either to ng HydA per g total protein (brown), shown in the left axis, or to ng HydA per 1 million cells (orange), shown in the right axis. The cultivation light intensities are shown at the bottom of each lane. All experiments were carried out in triplicates. Hydrogen production and oxygen production/uptake rates were investigated using a membrane inlet mass spectrometer, which monitors multiple gas traces in real time and thus can.2A) under varying irradiance. s), thus indicating that [FeFe]-hydrogenase functions as an immediate sink for surplus electrons in aerobic as well as in anaerobic environments. In summary, we show that an anaerobic locality in the chloroplast preserves [FeFe]-hydrogenase activity and supports continuous hydrogen production in air-grown microalgal cells. Photosynthetic hydrogen production is limited to microbial photosynthetic organisms, such GSK2656157 as is usually absent in air-grown cultures, and its accumulation is induced only upon anoxia, with a maximal accumulation observed after 3 h of nitrogen sparging (Happe et al., 1994) or 72 h of sulfur deprivation (Winkler et al., 2002). Accordingly, atmospheric levels of oxygen completely inactivate the catalytic site of [FeFe]-hydrogenase within a few seconds (Ghirardi et al., 2007). In contrast, Kosourov and Seibert (Kosourov and Seibert, 2009) observed continuous hydrogen production from cultures embedded in films under ambient atmosphere, although the oxygen levels within the films were not decided. In further support of this, recent reports have suggested that [FeFe]-hydrogenase is usually capable of aerobic activity in strains of could maintain hydrogen production under atmospheres of 21% (Hwang et al., 2014) or 15% (Chader et al., 2009) oxygen. The active pool of hydrogenase was estimated at 30 models per mg of dry weight (Hwang et al., 2014). Last, in a recent paper by Godaux et al. (Godaux et al., 2015), the authors claim that in a transition from dark anoxia to light, high rate of hydrogen production decreased to lower rate, before the onset of oxygen accumulation. Hence, they conclude that a competition for electrons with downstream processes such as FNR and CEF govern the hydrogen production rate rather than oxygen concentration. All of these findings contradict the common view since the oxygen sensitivity of [FeFe]-hydrogenase is usually well documented (Ghirardi et al., 1997). Furthermore, it is unclear why the transcripts of hydrogenase and its maturases, Hyd EF and G, are present under aerobiosis and increase under anaerobiosis induced by sulfur deprivation by roughly 20%, as we analyzed from published RNAseq data (Gonzlez-Ballester et al., 2010). The mechanism underlying [FeFe]-hydrogenase activity in air-grown cultures is yet to be resolved. We have therefore used the model organism (strain CC-124) at 2.5 g (chl)/mL were cultivated in aerated 100-mL flasks (Fig. 2A, inset) with constant stirring and under three light intensities (77, 155, and 600 E m?2 s?1; hereafter E). To ensure full aerobiosis in these conditions, we quantified the concentration of dissolved oxygen using oxygen electrode and Winkler reaction (Winkler, 1888). Furthermore, we analyzed the headspace gas using a gas chromatograph. Growth at all irradiances showed constant atmospheric levels of oxygen in the headspace of the growth vessels as well as 250 m dissolved oxygen in the growth media (Supplemental Fig. S1); the same oxygen concentration was measured for the positive controla cell-free aerobic growth media. Open in a separate window Physique 2. Photosynthetic activity and quantification of [FeFe]-hydrogenase in air-cultivated cells. A, Steady-state hydrogen production rates measured by MIMS in wild-type cultures cultivated under three light intensities: 77, 155, and 600 E. Inset, Cells were produced in flasks stoppered with a sponge completely permeable to air, with constant light and stirring. B, LEF from PSII to hydrogen recorded upon transition from 77 E to 1200 E of either wild-type cells in the presence (broken green line) or absence (solid green line) of the PSII donor side inhibitor DCMU. The mutant was used as unfavorable control (black). C, Inhibition of hydrogenase GSK2656157 by the inhibitor CO shuts down hydrogen production (green bar) in air-grown cells LAMC2 upon transition from 77 E to 1200 E at the onset of the recording. Photosynthetic oxygen production was not affected by the addition of CO (blue bars). D, Quantification of [FeFe]-hydrogenase in cells cultivated aerobically under 77, 155, and 600 E. Top, An immunoblot performed using anti-HydA antibody. Purified [FeFe]-hydrogenase (5 ng of HydA) was used as marker and reference for band intensity quantification. Equal amounts of total protein were loaded in each lane. [FeFe]-hydrogenase quantities were normalized either to ng HydA per g total protein (brown), shown in the left axis, or even to ng HydA per 1 million cells (orange), demonstrated in the proper axis. The cultivation light intensities are demonstrated in the bottom of each street. All experiments had been completed in triplicates. Hydrogen creation and air creation/uptake rates had been investigated utilizing a membrane inlet mass spectrometer, which screens multiple gas traces instantly and may discriminate between therefore.