Optimizing algal hydrogen photoproduction: a simplified and efficient protocol for anoxic induction in a semi-autotrophic approach

Abstract

Green microalgae, such as Chlamydomonas reinhardtii, show great potential for producing green hydrogen using only water and sunlight, with no carbon emissions. However, sustainable hydrogen production requires addressing the hydrogenase sensitivity to oxygen and enhancing electron allocation to this enzyme. Previous methods for hydrogen photoproduction rely on a brief nitrogen flushing followed by a dark incubation phase to establish anoxia prior to exposure to high light. In this study, we present a straightforward protocol involving a mixotrophic growth phase followed by a semi-autotrophic hydrogen production phase. During the hydrogen production phase, extended nitrogen flushing induced anoxia in the liquid algae culture, even though oxygen was still present in the headspace. Anoxia was maintained under light at moderate intensity (120 μmol m^-2 s^-1) and a controlled temperature of 30°C, with an efficient mixing system. Throughout the hydrogen production phase, we monitored dissolved oxygen levels in the culture alongside traditional oxygen measurements in the headspace. Using our protocol with the pgr5 mutant of C. reinhardtii, we achieved a maximum specific rate of 72 μmol H₂ mg^-1 Chl h^-1 and an average rate of 30-35 μmol H₂ mg^-1 Chl h^-1 over 10 hours of illumination. Additional nitrogen flushing steps extended anoxia, resulting in a total hydrogen yield of 220 ± 20 mL L^-1 over 48 hours of illumination. This performance is attributed to maintaining the redox balance of the plastoquinone pool and minimizing photodamage to the photosystem II complex. Our protocol offers a significant advancement for scalable and sustainable green hydrogen production.

FIGURE 1

Graphical summary of our newly developed semi‐autotrophic protocol for initiating and maintaining H₂ photoproduction.

FIGURE 2

Changes in O₂ levels during H₂ production when using the two‐phase ambient protocol. Sealed cultures of pgr5, after a 3‐minute N₂ flushing, were dark‐incubated for approximately 30 minutes, followed by exposure to high light (350 μmol m⁻² s⁻¹) for up to 48 h. No additional N₂ flushing was applied during the light exposure. (A) Real‐time changes in dissolved O₂ levels in the pgr5 culture. (B) Zoomed‐in view of the changes in dissolved O₂ during the first hour. (C) The time course of O2 gas changes in the headspace (expressed as vol%) during the first hour, as measured by gas chromatography. Data are presented as the average ± SEM of 3 independent experiments with triplicates. The major steps of the two‐phase ambient protocol (N₂ flushing, dark incubation, and illumination) are shown as top bars in panels B and C.

FIGURE 3

Real‐time changes in dissolved O₂ levels in pgr5 cultures under two different incubation conditions. Sealed suspension cultures of the pgr5 mutant were subjected to either N₂ flushing or dark incubation, followed by exposure to constant light at intensities of 120 or 350 μmol m⁻² s⁻¹ at 30°C with continuous mechanical agitation. No additional N₂ flushing was performed after the initial treatment. (A) Changes in dissolved O₂ after 2 h dark incubation followed by light exposure. B) Changes in dissolved O₂ after 6–9 min of N₂ flushing without subsequent dark incubation, followed by light exposure. The presented data are average ± SEM of 3 independent experiments with triplicates.

FIGURE 4

Anoxia maintenance and H₂ production using our semi‐autotrophic protocol. Sealed cultures underwent 6–9 min of N₂ flushing, followed by incubation at a constant light intensity of 120 μmol m⁻² s⁻¹ and a temperature of 30°C with mechanical agitation. (A) Real time changes in dissolved O₂ levels in the culture. (B) Time course of O₂ gas changes in the headspace expressed as vol%. C) Time course of total produced H₂. The arrows indicate the time points of short N₂ flushing to remove accumulated H₂ (exceeding 5%). Presented data are average ± SEM of 3 independent experiments.

FIGURE 5

Prolonged anoxia and extended H₂ production achieved through additional N₂ flushing. Two additional N₂ flushing steps (6–9 min each) were performed at the time points marked by black dashed lines, resulting in extended H₂ production. (A) Real time changes in dissolved O₂ levels in the culture. (B) The time course of O2 gas changes in the headspace (expressed as vol%). C) Time course of total produced H₂. Presented data are average with SEM of 3 independent experiments.

FIGURE 6

Photosynthetic parameters of the pgr5 culture at various time intervals during H₂ production using the ambient and our newly developed semi‐autotrophic protocol. For employing the ambient protocol (A, B and C) sealed culture after 3 min N₂ flushing and 30 min dark incubation were exposed to constant high light (350 μmol m⁻² s⁻¹) at 30 C. For employing our newly developed method (D, E and F), sealed cultures underwent 6–9 min of N₂ flushing, followed by incubation at a constant light intensity of 120 μmol m⁻² s⁻¹ and a temperature of 30°C with mechanical agitation. Chlorophyll a fluorescence and electrochromic shift were measured as described in Methods. A), D) Maximum quantum yield of PSII photochemistry (F_v/F_m) determined in 15‐min dark‐adapted samples. B), E) Effective quantum yield of PSII photochemistry Y(II) at the growth light intensity. C), F) Total proton motive force (PMF) determined in samples illuminated for 5 min at the growth light intensity. Presented data are average with SEM of 3 independent experiments. Different letters above the bars indicate significant differences (p < 0.05).