Oxygen Sensitivity of Algal H2-Production

Abstract

Photoproduction of H2 by green algae utilizes electrons originating from the photosynthetic oxidation of water and does not require metabolic intermediates. However, algal hydrogenases are extremely sensitive to O2, which limits their usefulness in future commercial H2-production systems. We designed an experimental technique for the selection of Ortolerant, H2-producing variants of Chlamydomonas reinhardtii based on the ability of wild-type cells to survive a short (20 min) exposure to metronidazole in the presence of controlled concentrations of O2, The number of survivors depends on the metronidazole concentration, light intensity, preinduction of the hydrogenase, and the presence or absence of O 2 , Finally, we demonstrate that some of the selected survivors in fact exhibit H2-production capacity that is less sensitive to O2 than the original wild-type population.Index Entries: Hydrogen; green algae; Chlamydomonas; oxygen; metronidazole.Abbreviations: ATP, adenosine triphosphate; cw15, cell wall-less strain of Chlamydomonas reinhardtii; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; Fd, ferredoxin; Iso, inhibitor concentration that decreases the rate of an enzymatic reaction to 50% of the rate measured in the absence of the inhibitor; MNZ, metronidazole, [1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole]; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced form of NADP; V o , initial rate of an enzymatic reaction; WT, wild-type.

Identification of Global Ferredoxin Interaction Networks in Chlamydomonas reinhardtii

“…Aerobic cell cultures were harvested at 16 -24 g ml Ϫ1 total chlorophyll by centrifugation and were immediately frozen in liquid nitrogen. For anaerobic induction, Chlamydomonas cultures were grown and harvested as above and resuspended in one-tenth the volume (50 ml) of anaerobic induction buffer containing 50 mM potassium phosphate, pH 7.2, and 3 mM MgCl 2 (26). The cells were then transferred to a sealed anaerobic vial in the dark and sparged with argon for 30 min.…”

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

“…Hydrogenases are described as irreversibly inactivated after exposure to O 2 , the C. reinhardtii enzyme being the most O 2 sensitive among them (Ghirardi et al, 1997;Cohen et al, 2005). In the PGRL1-deficient mutant or in the presence of glycolaldehyde or CCCP (i.e.…”

“…In this respect, at least three hypotheses have been formulated: (1) the inhibition of hydrogenase by O 2 produced by water photolysis (Ghirardi et al, 1997;Cohen et al, 2005), (2) the competition between ferredoxin-NADP + oxidoreductase (FNR) and hydrogenase activity for reduced FDX (Yacoby et al, 2011), and (3) the inhibition of electron supply to hydrogenases by the proton gradient generated by another AEF, the cyclic electron flow around PSI (PSI-CEF; Tolleter et al, 2011). First described by Arnon (1955), PSI-CEF consists in a reinjection of electrons from reduced FDX or NADPH pool in the plastoquinone (PQ) pool.…”

A Mutant in theADH1Gene ofChlamydomonas reinhardtiiElicits Metabolic Restructuring during Anaerobiosis

“…The adh1 and CC-124 (wild-type) cells were grown under mixotrophic conditions in TAP medium until they reached midlog phase (around 2 3 10 6 cells mL 21 ). Cells were then concentrated 10 times by centrifugation and resuspension in one-tenth of the initial volume of AIB buffer (Ghirardi et al, 1997). Culture concentrations were determined by counting the cells in aliquots of the culture using a hemocytometer; the cells, collected as five different aliquots from each sample, were counted and then averaged.…”

“…Chlamydomonas cultures grown on liquid TAP medium (approximately 2 3 10 6 cells mL 21 ) were concentrated 10 times by centrifugation (2,500g for 1 min) and subsequent resuspension in 0.1 volume of anaerobic induction buffer (AIB) containing 50 mM potassium phosphate (pH 7.0) and 3 mM MgCl 2 (Ghirardi et al, 1997). The AIB suspended cultures were at 2 3 10 7 cells mL 21 (1.5 3 10 7 cells mL 21 for tolerance assays).…”