H2 Photoproduction Research Articles

Genetic disruption of both Chlamydomonas reinhardtii [FeFe]-hydrogenases: Insight into the role of HYDA2 in H2 production

Chlamydomonas reinhardtii (Chlamydomonas throughout) encodes two [FeFe]-hydrogenases, designated HYDA1 and HYDA2. While HYDA1 is considered the dominant hydrogenase, the role of HYDA2 is unclear. To study the individual functions of each hydrogenase and provide a platform for future bioengineering, we isolated the Chlamydomonas hydA1-1, hydA2-1 single mutants and the hydA1-1 hydA2-1 double mutant. A reverse genetic screen was used to identify a mutant with an insertion in HYDA2, followed by mutagenesis of the hydA2-1 strain coupled with a H2 chemosensor phenotypic screen to isolate the hydA1-1 hydA2-1 mutant. Genetic crosses of the hydA1-1 hydA2-1 mutant to wild-type cells allowed us to also isolate the single hydA1-1 mutant. Fermentative, photosynthetic, and in vitro hydrogenase activities were assayed in each of the mutant genotypes. Surprisingly, analyses of the hydA1-1 and hydA2-1 single mutants, as well as the HYDA1 and HYDA2 rescued hydA1-1 hydA2-1 mutant demonstrated that both hydrogenases are able to catalyze H2 production from either fermentative or photosynthetic pathways. The physiology of both mutant and complemented strains indicate that the contribution of HYDA2 to H2 photoproduction is approximately 25% that of HYDA1, which corresponds to similarly low levels of in vitro hydrogenase activity measured in the hydA1-1 mutant. Interestingly, enhanced in vitro and fermentative H2 production activities were observed in the hydA1-1 hydA2-1 strain complemented with HYDA1, while maximal H2-photoproduction rates did not exceed those of wild-type cells.

Control of Hydrogen Photoproduction by the Proton Gradient Generated by Cyclic Electron Flow in Chlamydomonas reinhardtii

Hydrogen photoproduction by eukaryotic microalgae results from a connection between the photosynthetic electron transport chain and a plastidial hydrogenase. Algal H₂ production is a transitory phenomenon under most natural conditions, often viewed as a safety valve protecting the photosynthetic electron transport chain from overreduction. From the colony screening of an insertion mutant library of the unicellular green alga Chlamydomonas reinhardtii based on the analysis of dark-light chlorophyll fluorescence transients, we isolated a mutant impaired in cyclic electron flow around photosystem I (CEF) due to a defect in the Proton Gradient Regulation Like1 (PGRL1) protein. Under aerobiosis, nonphotochemical quenching of fluorescence (NPQ) is strongly decreased in pgrl1. Under anaerobiosis, H₂ photoproduction is strongly enhanced in the pgrl1 mutant, both during short-term and long-term measurements (in conditions of sulfur deprivation). Based on the light dependence of NPQ and hydrogen production, as well as on the enhanced hydrogen production observed in the wild-type strain in the presence of the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, we conclude that the proton gradient generated by CEF provokes a strong inhibition of electron supply to the hydrogenase in the wild-type strain, which is released in the pgrl1 mutant. Regulation of the trans-thylakoidal proton gradient by monitoring pgrl1 expression opens new perspectives toward reprogramming the cellular metabolism of microalgae for enhanced H₂ production.

A truncated antenna mutant of Chlamydomonas reinhardtii can produce more hydrogen than the parental strain

Photoproduction of H2 gas was examined in the Chlamydomonas reinhardtii tla1 strain, CC-4169, containing a truncated light-harvesting antenna, along with its parental CC-425 strain. Although enhanced photosynthetic performance of truncated antenna algae has been demonstrated previously (Polle et al. Planta 2003; 217:49–59), improved H2 photoproduction has yet to be reported. Preliminary experiments showed that sulfur-deprived, suspension cultures of the tla1 mutant could not establish anaerobiosis in a photobioreactor, and thus, could not photoproduce H2 gas under conditions typical for the sulfur-deprived wild-type cells (Kosourov et al. Biotech Bioeng 2002; 78:731–40). However, they did produce H2 gas when deprived of sulfur and phosphorus after immobilization within thin (∼300 μm) alginate films. These films were monitored for long-term H2 photoproduction activity under light intensities ranging from 19 to 350 μE m−2 s−1 PAR. Both the tla1 mutant and the CC-425 parental strain produced H2 gas for over 250 h under all light conditions tested. Relative to the parental strain, the CC-4169 mutant had lower maximum specific rates of H2 production at low and medium light intensities (19 and 184 μE m−2 s−1), but it exhibited a 4-times higher maximum specific rate at 285 μE m−2 s−1 and an 8.5-times higher rate at 350 μE m−2 s−1 when immobilized at approximately the same cell density as the parental strain. As a result, the CC-4169 strain accumulated almost 4-times more H2 than CC-425 at 285 μE m−2 s−1 and over 6-times more at 350 μE m−2 s−1 during 250-h experiments. These results are the first demonstration that truncating light-harvesting antennae in algal cells can increase the efficiency of H2 photoproduction in mass culture at high light intensity.

Proteomic Analysis of Hydrogen Photoproduction in Sulfur-Deprived Chlamydomonas Cells

The green alga Chlamydomonas reinhardtii is a model organism to study H(2) metabolism in photosynthetic eukaryotes. To understand the molecular mechanism of H(2) metabolism, we used 2-DE coupled with MALDI-TOF and MALDI-TOF/TOF-MS to investigate proteomic changes of Chlamydomonas cells that undergo sulfur-depleted H(2) photoproduction process. In this report, we obtained 2-D PAGE soluble protein profiles of Chlamydomonas at three time points representing different phases leading to H(2) production. We found over 105 Coomassie-stained protein spots, corresponding to 82 unique gene products, changed in abundance throughout the process. Major changes included photosynthetic machinery, protein biosynthetic apparatus, molecular chaperones, and 20S proteasomal components. A number of proteins related to sulfate, nitrogen and acetate assimilation, and antioxidative reactions were also changed significantly. Other proteins showing alteration during the sulfur-depleted H(2) photoproduction process were proteins involved in cell wall and flagella metabolisms. In addition, among these differentially expressed proteins, 11 were found to be predicted proteins without functional annotation in the Chlamydomonas genome database. The results of this proteomic analysis provide new insight into molecular basis of H(2) photoproduction in Chlamydomonas under sulfur depletion.

Water Splitting by Visible Light: A Nanophotocathode for Hydrogen Production

Efficient production of solar fuels is an imperative for meeting future fossil-fuel-free energy demands. Hydrogen that is derived from the splitting of water by solar energy is clearly attractive as a clean energy vector, and there have been many attempts to construct viable molecular and biomolecular devices for photohydrogen production. A common approach in the construction of such devices is the utilization of tris(bipyridine)ruthenium, zinc porphyrin, or related molecular materials as photosensitizers in conjunction with a tethered or free electrocatalyst or enzymic system. Apart from cost, such systems suffer from having limited lifetimes, which may be attributed at least in part to the intrinsic reactivity of the organic N-donor ligands in the radical anion form of the photoexcited state and photodegradation pathways.

Towards the integration of dark- and photo-fermentative waste treatment. 2. Optimization of starch-dependent fermentative hydrogen production

Abstract Eight natural microbial consortia collected from different sites were tested for dark, hydrogen production during starch degradation. The most active consortium was from silo pit liquid under mesophilic (37 °C) conditions. The fermentation medium for this consortium was optimized (Fe, NH4+, phosphates, peptone, and starch content) for both dark fermentation and for subsequent purple photosynthetic bacterial H2 photoproduction [Laurinavichene TV, Tekucheva DN, Laurinavichius KS, Ghirardi ML, Seibert M, Tsygankov AA. Towards the integration of dark and photo fermentative waste treatment. 1. Hydrogen photoproduction by purple bacterium Rhodobacter capsulatus using potential products of starch fermentation. Int J Hydrogen Energy 2008;33(23):7020–26], in the presence of the spent dark, fermentation effluent. The addition of Zn (10 mg L−1), as a methanogenesis inhibitor that does not inhibit purple bacteria at this concentration, also did not inhibit dark, fermentative H2 production. The influence of various fermentation end products at different concentrations (up to 30 g L−1) on dark, H2 production was also examined. Added lactate stimulated, but added isobutyrate and butanol strongly inhibited gas production. Under optimal conditions the fermentation of starch (30 g L−1) resulted in 5.7 L H2 L−1 of culture (1.6 mol H2 per mole of hexose) with the co-production mainly of butyrate and acetate.

Hydrogen photoproduction by use of photosynthetic organisms and biomimetic systems

Hydrogen can be important clean fuel for future. Among different technologies for hydrogen production, oxygenic natural and artificial photosyntheses using direct photochemistry in synthetic complexes have a great potential to produce hydrogen, since both use clean and cheap sources: water and solar energy. Artificial photosynthesis is one way to produce hydrogen from water using sunlight by employing biomimetic complexes. However, splitting of water into protons and oxygen is energetically demanding and chemically difficult. In oxygenic photosynthetic microorganisms such as algae and cyanobacteria, water is split into electrons and protons, which during primary photosynthetic process are redirected by photosynthetic electron transport chain, and ferredoxin, to the hydrogen-producing enzymes hydrogenase or nitrogenase. By these enzymes, e- and H+ recombine and form gaseous hydrogen. Biohydrogen activity of hydrogenase can be very high but it is extremely sensitive to photosynthetic O2. In contrast, nitrogenase is insensitive to O2, but has lower activity. At the moment, the efficiency of biohydrogen production is low. However, theoretical expectations suggest that the rates of photon conversion efficiency for H2 bioproduction can be high enough (>10%). Our review examines the main pathways of H2 photoproduction by using of photosynthetic organisms and biomimetic photosynthetic systems.

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 microm) 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 microg Chl mL(-1) of matrix), (b) kinetics of H2 photoproduction similar to sulfur-deprived suspension cultures, (c) higher specific rates (up to 12.5 micromol 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.

Photosynthetic H2 metabolism in Chlamydomonas reinhardtii (unicellular green algae)

Unicellular green algae have the ability to operate in two distinctly different environments (aerobic and anaerobic), and to photosynthetically generate molecular hydrogen (H2). A recently developed metabolic protocol in the green alga Chlamydomonas reinhardtii permitted separation of photosynthetic O2-evolution and carbon accumulation from anaerobic consumption of cellular metabolites and concomitant photosynthetic H2-evolution. The H2 evolution process was induced upon sulfate nutrient deprivation of the cells, which reversibly inhibits photosystem-II and O2-evolution in their chloroplast. In the absence of O2, and in order to generate ATP, green algae resorted to anaerobic photosynthetic metabolism, evolved H2 in the light and consumed endogenous substrate. This study summarizes recent advances on green algal hydrogen metabolism and discusses avenues of research for the further development of this method. Included is the mechanism of a substantial tenfold starch accumulation in the cells, observed promptly upon S-deprivation, and the regulated starch and protein catabolism during the subsequent H2-evolution. Also discussed is the function of a chloroplast envelope-localized sulfate permease, and the photosynthesis-respiration relationship in green algae as potential tools by which to stabilize and enhance H2 metabolism. In addition to potential practical applications of H2, approaches discussed in this work are beginning to address the biochemistry of anaerobic H2 photoproduction, its genes, proteins, regulation, and communication with other metabolic pathways in microalgae. Photosynthetic H2 production by green algae may hold the promise of generating a renewable fuel from nature's most plentiful resources, sunlight and water. The process potentially concerns global warming and the question of energy supply and demand.

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.

Photosensitized Reduction of Water to Hydrogen Using Human Serum Albumin Complexed with Zinc−Protoporphyrin IX

We present the photophysical properties of complexes of recombinant human serum albumin (rHSA) with Zn(II)-protoporphyrin IX (ZnPP) and their activities in the photosensitized reduction of water to hydrogen (H2) using methyl viologen (MV2+) as an electron relay. The ZnPP is bound in subdomain IB of wild-type rHSA [rHSA(wt] by an axial coordination of Tyr-161 and, in the rHSA(I142H/Y161L) mutant [rHSA(His], by a His-142 coordination. Both the rHSA(wt)-ZnPP and rHSA(His)-ZnPP complexes showed a long-lived photoexcited triplet state with lifetimes (tauT) of 11 and 2.5 ms, respectively. The accommodation of ZnPP into the protein matrix efficiently eliminated the collisional triplet self-quenching process. The addition of a water-soluble electron acceptor, MV2+, resulted in a significant decrease in the triplet lifetime. The transition absorption spectrum revealed the oxidative quenching of rHSA-3ZnPP* by MV2+. The quenching rate constant (kq) and backward electron transfer rate constant (kb) were determined to be 1.4 x 10(7) and 4.7 x 10(8) M(-1) s(-1) for rHSA(wt)-ZnPP. In the presence of the colloidal PVA-Pt as a catalyst and triethanolamine (TEOA) as a sacrificial electron donor, the photosensitized reduction of water to H2 takes place. The efficiency of the photoproduction of H2 was greater than that of the system using the well-known organic chromophore, tetrakis(1-methylpyridinium-4-yl)porphinatozinc(II) (ZnTMPyP4+), under the same conditions.

Photoinduced Hydrogen Production by Direct Electron Transfer from Photosystem I Cross-Linked with Cytochrome c3 to [NiFe]-Hydrogenase

The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H2). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H2 conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP+-reductase (FNR). In the present study, to establish an H2 photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c3 (cytc3) from Desulfovibrio vulgaris. The covalent adduct of cytc3 and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc3/PSI complex. The NADPH production by the cytc3/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H2 production by the cytc3/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H2 production by the hydrogenase/cytc3/PSI complex was observed (0.30 pmol Hz/mg chlorophyll/h). These results suggest that the cytc3/PSI complex may produce H2 in vivo.

The Effect of Sulfur Re-Addition on H2 Photoproduction by Sulfur-Deprived Green Algae

Sulfur deprivation of algal cultures selectively and partially inactivates photosystem II (PSII)-catalyzed O(2) evolution, induces anaerobiosis and hydrogenase expression, and results in sustained H(2) photoproduction for several days. We show that re-addition of limiting amounts of sulfate (1-10 microM final concentration) to the cultures during the H(2)-production phase temporarily reactivates PSII photochemical and O(2)-evolution activity and re-establishes higher rates of electron transport through the photosynthetic electron transport chain. The reactivation of PSII occurs by de novo D1 protein synthesis, but does not result in the re-establishment of aerobic conditions in the reactor, detectable by dissolved-O(2) sensors. However, concomitant H(2) photoproduction is inhibited, possibly due to excessive intra-cellular levels of photosynthetically-evolved O(2). The partial recovery of electron transport rates correlates with the re-oxidation of the plastoquinone (PQ) pool, as observed by pulse-amplitude modulated (PAM) and fluorescence-induction measurements. These results show that the presence of a more oxidized PQ pool releases some of the down-regulation of electron transport caused by the anaerobic conditions.

Continuous Hydrogen Photoproduction by &lt;I&gt;Chlamydomonas reinhardtii&lt;/I&gt;: 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.

Metabolically Engineered Rhodobacter sphaeroides RV strains for Improved Biohydrogen Photoproduction Combined with Disposal of Food Wastes

Three differently metabolically engineered strains, 2 single PHA- and Hup- mutants and one double PHA-/Hup- mutant, of the purple nonsulfur photosynthetic bacterium Rhodobacter sphaeroides RV, were constructed to improve a light-driven biohydrogen production process combined with the disposal of solid food wastes. These phenotypes were designed to abolish, singly or in combination, the competition of H2 photoproduction with polyhydroxyalkanoate (PHA) accumulation by inactivating PHA synthase activity, and with H2 recycling by abolishing the uptake hydrogenase enzyme. The performance of these mutants was compared with that of the wild-type strain in laboratory tests carried out in continuously fed photobioreactors using as substrates both synthetic media containing lactic acid and media from the acidogenic fermentation of actual fruit and vegetable wastes, containing mainly lactic acid, smaller amounts of acetic acia, and traces of higher volatile acids. With the lactic acid-based synthetic medium, the single Hup- and the double PHA-/Hup- mutants, but not the single PHA- mutant, exhibited increased rates of H2 photoproduction, about one third higher than that of the wild-type strain. With the food-waste-derived growth medium, only the single Hup- mutant showed higher rates of H2 production, but all 3 mutants sustained a longer-term H2 photoproduction phase than the wild-type strain, with the double mutant exhibiting overall the largest amount of H2 evolved. This work demonstrates the feasibility of single and multiple gene engineering of microorganisms to redirect their metabolism for improving H2 photoproduction using actual waste-derived substrates.

Augmentation of H2 photoproduction in Rhodopseudomonas palustris by N-heterocyclic aromatic compounds.

Increases of 23- (5.6 mmol acetylene reduced mg dry wt(-1)) and 16- (4 mmol acetylene reduced mg dry wt(-1)) fold in nitrogenase activity and 12- (671 microl H2 mg dry wt(-1) h(-1)) and 6- (349 microl mg dry wt(-1) h(-1)) fold in H2 photoproduction in Rhodopseudomonas palustris JA1 over 24 h were achieved with pyrazine 2-carboxylate (3 mM) and 3-picoline (3 mM), respectively, and were higher than earlier reports of enhancement (1.5 to 5-fold) in biological H2 production using various alternative methods.

Reverse micelles: a novel tool for H2 production

Reverse micelles serve as a novel tool to entrap enzymes and microbial whole cells within aqueous pockets and can be of great use in enhancing the efficiency and sustainability of the biological system. Photosynthetic bacterium Rhodopseudomonas sphaeroides entrapped inside the aqueous pool of reverse micelles prepared from benzene-sodium lauryl sulphate exhibited 25-fold enhancement of H2 photoproduction rate (1.67 ml H2 [mg protein]−1 h−1) compared to cells suspended in normal aqueous medium. Hydrogen photoproduction by the bacterium was catalysed by the nitrogenase enzyme system which was supported at a low light intensity of 12 μEm−2 sec−1 photon flux energy at a wavelength of 520 nm. The optimum temperature for the process was 40 °C.

Acetylene reduction and hydrogen photoproduction by wild-type and mutant strains ofAnabaenaat different CO2and O2concentrations

Hydrogen photoproduction by growing cultures of Anabaena variabilis and A. azollae did not occur under air+CO2 or argon+CO2 atmospheres at saturating light but did take place under argon alone. It was shown that CO2 inhibited photoproduction of H2 as a result of the photosynthetic production of O2 whereas photoreduction of C2H2 by these cyanobacteria was not inhibited by O2 concentrations up to 20% in the assay gas phase. In contrast to the wild type of A. variabilis and of A. azollae, H2 photoproduction by the hydrogenase-impaired mutant A. variabilis PK84 showed only a slight dependence on O2 concentration. Thus, in the wild-type Anabaena the decrease in the observed rate of H2 evolution at elevated O2 concentrations could be the result of an increase in hydrogenase-mediated uptake of H2 via an oxyhydrogen reaction.

Challenge of new biological energy resources

Hydrogenases are enzymes that can reversibly split molecular hydrogen. Study on the structure of the active site and the mechanism of catalysis has drawn great attention because the results may be useful for the design of cheap biomimetic hydrogen catalysts for fuel cells, or as model for the photoproduction of H2. At one time the active site was generally considered to be composed of mononuclear nickel complex with ligands from the polypeptide. A breakthrough in the understanding of the structure of [NiFe] Hases occurred with the resolution crystal structure ofD. gigas [NiFe] Hases in 1995. The unexpected result challenged the previously reported spectroscopic studies and caused some academic arguments. Some methods and results used for insight into [NiFe] Hases have to be reconsidered. Different viewpoints concerning the structure of active site of [NiFe] Hases in different periods and some remaining questions will be presented.

Hydrogen photoproduction ofA. Variabilis incorporated in a photobioreactor

H2 photoproduction and nitrogenase activities in two strains ofAnabaena variabilis marked wild type ATCC 29413 and mutant PK84 exposed to thermal stress (temperature higher than the normal incubation temperature of 30°C) were studied. Cultures of both strains collected from any interval of logarithmic growth phase exhibited high H2 photoproduction and nitrogenase activities when exposed to limited time heat shock during the assay process. In contrast, the algal H2 photoproduction rate of both strains fluctuated with long term thermal stress caused by increasing the growth temperature from 30°C to 36°C.