Sergey Kosourov

Hydrogen photoproduction by nutrient-deprived Chlamydomonas reinhardtii cells immobilized within thin alginate films under aerobic and anaerobic conditions

A new technique for immobilizing H2‐photoproducing green algae within a thin (<400 µm) alginate film has been developed. Alginate films with entrapped sulfur/phosphorus‐deprived Chlamydomonas reinhardtii, strain cc124, cells demonstrate (a) higher cell density (up to 2,000 µg Chl mL−1 of matrix), (b) kinetics of H2 photoproduction similar to sulfur‐deprived suspension cultures, (c) higher specific rates (up to 12.5 µmol mg−1 Chl h−1) of H2 evolution, (d) light conversion efficiencies to H2 of over 1% and (e) unexpectedly high resistance of the H2‐photoproducing system to inactivation by atmospheric O2. The algal cells, entrapped in alginate and then placed in vials containing 21% O2 in the headspace, evolved up to 67% of the H2 gas produced under anaerobic conditions. The results indicate that the lower susceptibility of the immobilized algal H2‐producing system to inactivation by O2 depends on two factors: (a) the presence of acetate in the medium, which supports higher rates of respiration and (b) the capability of the alginate polymer itself to effectively separate the entrapped cells from O2 in the liquid and headspace and restrict O2 diffusion into the matrix. The strategy presented for immobilizing algal cells within thin polymeric matrices shows the potential for scale‐up and possible future applications. Biotechnol. Bioeng. 2008. Biotechnol. Bioeng. 2009;102: 50–58.

Hydrogen photoproduction under continuous illumination by sulfur-deprived, synchronous Chlamydomonas reinhardtii cultures

Unsynchronized Chlamydomonas reinhardtii cells subsequently deprived of sulfur produce H2 under continuous illumination in the laboratory for 3– 4 days. However, cultures grown outdoors will be exposed to day-and-night cycles that may synchronize their growth and cell division. While it is clear that only insigni6cant amounts of H2 can be produced by sulfur-deprived cells during the night period, little work has been done to examine the e7ects of the light=dark cycles preceding sulfur deprivation on subsequent H2 photoproduction. We show that (a) C. reinhardtii cells exhibit synchronized growth and cell division in the presence of acetate, (b) cells with the highest speci6c rates of H2 photoproduction also have the highest rates of biomass accumulation, and (c) the highest rates of starch and protein degradation coincide with the highest rates of formate and acetate accumulation, but not with H2 photoproduction. This work shows that it is possible to maximize the production of H2 by sulfur-depriving synchronized cultures at about 4 h after the beginning of the light period. ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved.

Continuous Hydrogen Photoproduction by Chlamydomonas reinhardtii Using a Novel Two-Stage, Sulfate-Limited Chemostat System

This study demonstrates, for the first time, that it is possible to couple sulfate-limited Chlamydomonas reinhardtii growth to continuous H2 photoproduction for more than 4000 h. A two-stage chemostat system physically separates photosynthetic growth from H2 production, and it incorporates two automated photobioreactors (PhBRs). In the first PhBR, the algal cultures are grown aerobically in chemostat mode under limited sulfate to obtain photosynthetically competent cells. Active cells are then continuously delivered to the second PhBR, where H2 production occurs under anaerobic conditions. The dependence of the H2 production rate on sulfate concentration in the medium, dilution rates in the PhBRs, and incident light intensity is reported.

Prolongation of H2 photoproduction by immobilized, sulfur-limited Chlamydomonas reinhardtii cultures

Two approaches to prolong the duration of hydrogen production by immobilized, sulfur-limited Chlamydomonas reinhardtii cells are examined. The results demonstrate that continuous H2 photoproduction can occur for at least 90 days under constant flow of TAP medium containing micromolar sulfate concentrations. Furthermore, it is also possible to prolong the duration of H2 production by cycling immobilized cells between minus and plus sulfate conditions.

Photoproduction of hydrogen by sulfur-deprived C. reinhardtii mutants with impaired photosystem II photochemical activity.

Photoproduction of H2 was examined in a series of sulfur-deprived Chlamydomonas reinhardtii D1-R323 mutants with progressively impaired PSII photochemical activity. In the R323H, R323D, and R323E D1 mutants, replacement of arginine affects photosystem II (PSII) function, as demonstrated by progressive decreases in O2-evolving activity and loss of PSII photochemical activity. Significant changes in PSII activity were found when the arginine residue was replaced by negatively charged amino acid residues (R323D and R323E). However, the R323H (positively charged or neutral, depending on the ambient pH) mutant had minimal changes in PSII activity. The R323H, R323D, and R323E mutants and the pseudo-wild-type (pWt) with restored PSII function were used to study the effects of sulfur deprivation on H2-production activity. All of these mutants exhibited significant changes in the normal parameters associated with the H2-photoproduction process, such as a shorter aerobic phase, lower accumulation of starch, a prolonged anaerobic phase observed before the onset of H2-production, a shorter duration of H2-production, lower H2 yields compared to the pWt control, and slightly higher production of dark fermentation products such as acetate and formate. The more compromised the PSII photochemical activity, the more dramatic was the effect of sulfur deprivation on the H2-production process, which depends both on the presence of residual PSII activity and the amount of stored starch.

Phenotypic diversity of hydrogen production in chlorophycean algae reflects distinct anaerobic metabolisms

Several species of green algae use [FeFe]-hydrogenases to oxidize and/or produce H(2) during anoxia. To further define unique aspects of algal hydrogenase activity, the well-studied anaerobic metabolisms of Chlamydomonas reinhardtii were compared with four strains of Chlamydomonas moewusii and a Lobochlamys culleus strain. In vivo and in vitro hydrogenase activity, starch accumulation/degradation, and anaerobic end product secretion were analyzed. The C. moewusii strains showed the most rapid induction of hydrogenase activity, congruent with high rates of starch catabolism, and anoxic metabolite accumulation. Intriguingly, we observed significant differences in morphology and hydrogenase activity in the C. moewusii strains examined, likely the result of long-term adaptation and/or genetic drift during culture maintenance. Of the C. moewusii strains examined, SAG 24.91 showed the highest in vitro hydrogenase activity. However, SAG 24.91 produced little H(2) under conditions of sulfur limitation, which is likely a consequence of its inability to utilize exogenous acetate. In L. culleus, hydrogenase activity was minimal unless pulsed light was used to induce significant H(2) photoproduction. Overall, our results demonstrate that unique anaerobic acclimation strategies have evolved in distinct green algae, resulting in differential levels of hydrogenase activity and species-specific patterns of NADH reoxidation during anoxia.

Heterocyst-specific flavodiiron protein Flv3B enables oxic diazotrophic growth of the filamentous cyanobacterium Anabaena sp. PCC 7120

Significance Cyanobacterial flavodiiron proteins (FDPs) comprise a protein family with unique modular structure and photoprotective functions in an oxygenic environment. It is conceivable that FDPs have made the development of oxygenic photosynthesis possible in cyanobacteria. Here, we report the ability of specific FDPs to reduce O2 directly to water in heterocyst-forming filamentous cyanobacteria, not only to support the photosynthetic machinery, but also to prevent oxidative damage of the N2-fixing enzyme nitrogenase. Whilst in the ancient environment, N2 fixation was secured from O2 inhibition, the later increase of atmospheric O2 may have initiated an important role for FDP-mediated protection of nitrogenase in maintaining the N2-fixing activity of cyanobacteria. Flavodiiron proteins are known to have crucial and specific roles in photoprotection of photosystems I and II in cyanobacteria. The filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 contains, besides the four flavodiiron proteins Flv1A, Flv2, Flv3A, and Flv4 present in vegetative cells, two heterocyst-specific flavodiiron proteins, Flv1B and Flv3B. Here, we demonstrate that Flv3B is responsible for light-induced O2 uptake in heterocysts, and that the absence of the Flv3B protein severely compromises the growth of filaments in oxic, but not in microoxic, conditions. It is further demonstrated that Flv3B-mediated photosynthetic O2 uptake has a distinct role in heterocysts which cannot be substituted by respiratory O2 uptake in the protection of nitrogenase from oxidative damage and, thus, in an efficient provision of nitrogen to filaments. In line with this conclusion, the Δflv3B strain has reduced amounts of nitrogenase NifHDK subunits and shows multiple symptoms of nitrogen deficiency in the filaments. The apparent imbalance of cytosolic redox state in Δflv3B heterocysts also has a pronounced influence on the amounts of different transcripts and proteins. Therefore, an O2-related mechanism for control of gene expression is suggested to take place in heterocysts.

Continuous Hydrogen Photoproduction by Chlamydomonas reinhardtii

This study demonstrates, for the first time, that it is possible to couple sulfate-limited Chlamydomonas reinhardtii growth to continuous H2 photoproduction for more than 4000 h. A two-stage chemostat system physically separates photosynthetic growth from H2 production, and it incorporates two automated photobioreactors (PhBRs). In the first PhBR, the algal cultures are grown aerobically in chemostat mode under limited sulfate to obtain photosynthetically competent cells. Active cells are then continuously delivered to the second PhBR, where H2 production occurs under anaerobic conditions. The dependence of the H2 production rate on sulfate concentration in the medium, dilution rates in the PhBRs, and incident light intensity is reported.

Chlamydomonas Flavodiiron Proteins Facilitate Acclimation to Anoxia During Sulfur Deprivation

The flavodiiron proteins (FDPs) are involved in the detoxification of oxidative compounds, such as nitric oxide (NO) or O2 in Archaea and Bacteria. In cyanobacteria, the FDPs Flv1 and Flv3 are essential in the light-dependent reduction of O2 downstream of PSI. Phylogenetic analysis revealed that two genes (flvA and flvB) in the genome of Chlamydomonas reinhardtii show high homology to flv1 and flv3 genes of the cyanobacterium Synechocystis sp. PCC 6803. The physiological role of these FDPs in eukaryotic green algae is not known, but it is of a special interest since these phototrophic organisms perform oxygenic photosynthesis similar to higher plants, which do not possess FDP homologs. We have analyzed the levels of flvA and flvB transcripts in C. reinhardtii cells under various environmental conditions and showed that these genes are highly expressed under ambient CO2 levels and during the early phase of acclimation to sulfur deprivation, just before the onset of anaerobiosis and the induction of efficient H2 photoproduction. Importantly, the increase in transcript levels of the flvA and flvB genes was also corroborated by protein levels. These results strongly suggest the involvement of FLVA and FLVB proteins in alternative electron transport.

Hydrogen Photoproduction by Immobilized N2-Fixing Cyanobacteria: Understanding the Role of the Uptake Hydrogenase in the Long-Term Process

ABSTRACT We have investigated two approaches to enhance and extend H2 photoproduction yields in heterocystous, N2-fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO2 supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H2 production over time. Our results demonstrated that alginate-entrapped ΔhupL cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach, Anabaena strain PCC 7120, its ΔhupL mutant, and Calothrix strain 336/3 films were supplemented with N2 by periodic treatments of air, or air plus CO2. These treatments restored the photosynthetic activity of the cells and led to a high level of H2 production in Calothrix 336/3 and ΔhupL cells (except for the treatment air plus CO2) but not in the Anabaena PCC 7120 strain (for which H2 yields did not change after air treatments). The highest H2 yield was obtained by the air treatment of ΔhupL cells. Notably, the supplementation of CO2 under an air atmosphere led to prominent symptoms of N deficiency in the ΔhupL strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O2 from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells.