Bidirectional Hydrogenase Research Articles

Powerful fermentative hydrogen evolution of photosynthate in the cyanobacterium Lyngbya aestuarii BL J mediated by a bidirectional hydrogenase.

Cyanobacteria are considered good models for biohydrogen production because they are relatively simple organisms with a demonstrable ability to generate H2 under certain physiological conditions. However, most produce only little H2, revert readily to H2 consumption, and suffer from hydrogenase sensitivity to O2. Strains of the cyanobacteria Lyngbya aestuarii and Microcoleus chthonoplastes obtained from marine intertidal cyanobacterial mats were recently found to display much better H2 production potential. Because of their ecological origin in environments that become quickly anoxic in the dark, we hypothesized that this differential ability may have evolved to serve a role in the fermentation of the photosynthate. Here we show that, when forced to ferment internal substrate, these cyanobacteria display desirable characteristics of physiological H2 production. Among them, the strain L. aestuarii BL J had the fastest specific rates and attained the highest H2 concentrations during fermentation of photosynthate, which proceeded via a mixed acid fermentation pathway to yield acetate, ethanol, lactate, H2, CO2, and pyruvate. Contrary to expectations, the H2 yield per mole of glucose was only average compared to that of other cyanobacteria. Thermodynamic analyses point to the use of electron donors more electronegative than NAD(P)H in Lyngbya hydrogenases as the basis for its strong H2 production ability. In any event, the high specific rates and H2 concentrations coupled with the lack of reversibility of the enzyme, at the expense of internal, photosynthetically generated reductants, makes L. aestuarii BL J and/or its enzymes, a potentially feasible platform for large-scale H2 production.

Improved oxygen tolerance of the Synechocystis sp. PCC 6803 bidirectional hydrogenase by site-directed mutagenesis of putative residues of the gas diffusion channel

Although of potential biotechnological interest, photobiological H2 production from microalgae and cyanobacteria is strongly limited due to the oxygen sensitivity of hydrogenases, the H2-evolving enzymes. We study here the [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 to identify structural determinants of its sensitivity to O2. Based on previous work on the hydrogenase from Desulfovibrio fructosovorans and on a structural model of the Synechocystis hydrogenase, we have created various mutants of the Synechocystis enzyme. Amino acids residues homologous to those defining the end of the intramolecular gas channel in the D. fructosovorans enzyme were specifically targeted, these residues being previously described as critical for enzyme activity and tolerance to O2. We show here that mutation I64M of the Synechocystis enzyme alters gas diffusion kinetics and improves O2 tolerance. These results constitute the first report demonstrating that an O2 tolerance-related character could be transposed from a proteobacterial hydrogenase to a cyanobacterial one, and may constitute the first published improvement of O2 tolerance of a cyanobacterial enzyme by single site-directed mutagenesis.

Characterization of ten H2 producing cyanobacteria isolated from the Baltic Sea and Finnish lakes

The genetic background and activities of the enzymes involved in H2 production were investigated from ten distinct H2 producing cyanobacteria, revealed by a recent screening. All strains are N2-fixing, filamentous and heterocystous. Southern hybridization revealed that the tested strains possess the genes encoding the conventional nitrogenase (nifHDK1), and lack the alternative nitrogenases. The high H2 production rate of these strains was shown not to be dependent on the presence of highly active nitrogenase or bidirectional hydrogenase enzymes. Moreover, most of the strains possessed a highly active uptake hydrogenase enzyme. We also examined the structure of the nif and hup operons encoding nitrogenase and uptake hydrogenase enzymes in the Calothrix 336/3 strain, the best H2 producer in the screening. We concluded that the ability of the cyanobacteria to produce high levels of H2 is not directly linked to the maximum capacities of the enzymes involved in H2 production.

Dark fermentative hydrogen production by the unicellular halotolerant cyanobacterium Aphanothece halophytica grown in seawater

Biohydrogen is an environmentally friendly alternative energy carrier that can be produced by a number of different microorganisms. The unicellular halotolerant cyanobacterium Aphanothece halophytica is one of the high potential H2 producers. Under dark fermentation, it is capable of producing H2 by the bidirectional hydrogenase activity via the catabolism of glycogen stored during photosynthesis. This work aimed to cultivate A. halophytica in natural seawater containing high salinity and minerals, with an addition of some essential nutrients, and to investigate effects of various nutritional and physical factors on its dark fermentative H2 production. A. halophytica was able to grow in natural seawater added with NaNO3. Cells grown in seawater supplemented with as little as 1.76 mM NaNO3 showed similar growth to those cultivated in normal BG11 supplemented with Turk Island salt solution. H2 production was the highest when incubating the cells in seawater without any supplementation of NaNO3. Under this condition, the highest rate of dark fermentative H2 production of 82.79 ± 3.47 nmol H2 mg-1 dry weight h−1 was found in cells incubated at 35 °C, pH 6 with the supplementation of 378 mmolC L−1 glucose, 0.25 M NaCl, and 0.4 μM Fe3+. Long-term H2 accumulation of 1,864 ± 81 nmol H2 mg−1 dry weight was observed after 8 days of dark incubation under anoxic condition, and the high yield of H2 was sustained at least up to 14 days, suggesting the possibility of utilizing natural seawater to grow A. halophytica for long-term production of H2.

The Bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 Is Reduced by Flavodoxin and Ferredoxin and Is Essential under Mixotrophic, Nitrate-limiting Conditions

Cyanobacteria are able to use solar energy for the production of hydrogen. It is generally accepted that cyanobacterial NiFe-hydrogenases are reduced by NAD(P)H. This is in conflict with thermodynamic considerations, as the midpoint potentials of NAD(P)H do not suffice to support the measured hydrogen production under physiological conditions. We show that flavodoxin and ferredoxin directly reduce the bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 in vitro. A merodiploid ferredoxin-NADP reductase mutant produced correspondingly more photohydrogen. We furthermore found that the hydrogenase receives its electrons via pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR)-flavodoxin/ferredoxin under fermentative conditions, enabling the cells to gain ATP. These results strongly support that the bidirectional NiFe-hydrogenases in cyanobacteria function as electron sinks for low potential electrons from photosystem I and as a redox balancing device under fermentative conditions. However, the selective advantage of this enzyme is not known. No strong phenotype of mutants lacking the hydrogenase has been found. Because bidirectional hydrogenases are widespread in aquatic nutrient-rich environments that are capable of triggering phytoplankton blooms, we mimicked those conditions by growing cells in the presence of increased amounts of dissolved organic carbon and dissolved organic nitrogen. Under these conditions the hydrogenase was found to be essential. As these conditions close the two most important sinks for reduced flavodoxin/ferredoxin (CO2-fixation and nitrate reduction), this discovery further substantiates the connection between flavodoxin/ferredoxin and the NiFe-hydrogenase.

The Uptake Hydrogenase in the Unicellular Diazotrophic Cyanobacterium Cyanothece sp. Strain PCC 7822 Protects Nitrogenase from Oxygen Toxicity

Cyanothece sp. strain PCC 7822 is a unicellular, diazotrophic cyanobacterium that can produce large quantities of H2 when grown diazotrophically. This strain is also capable of genetic manipulations and can represent a good model for improving H2 production from cyanobacteria. To this end, a knockout mutation was made in the hupL gene (ΔhupL), and we determined how this would affect the amount of H2 produced. The ΔhupL mutant demonstrated virtually no nitrogenase activity or H2 production when grown under N2-fixing conditions. To ensure that this mutation only affected the hupL gene, a complementation strain was constructed readily with wild-type properties; this indicated that the original insertion was only in hupL. The mutant had no uptake hydrogenase activity but had increased bidirectional hydrogenase (Hox) activity. Western blotting and immunocytochemistry under the electron microscope indicated that the mutant had neither HupL nor NifHDK, although the nif genes were transcribed. Interestingly, biochemical analysis demonstrated that both HupL and NifH could be membrane associated. The results indicated that the nif genes were transcribed but that NifHDK was either not translated or was translated but rapidly degraded. We hypothesized that the Nif proteins were made but were unusually susceptible to O2 damage. Thus, we grew the mutant cells under anaerobic conditions and found that they grew well under N2-fixing conditions. We conclude that in unicellular diazotrophs, like Cyanothece sp. strain PCC 7822, the HupLS complex helps remove oxygen from the nitrogenase, and that this is a more important function than merely oxidizing the H2 produced by the nitrogenase.

Transcriptional regulation of the bidirectional hydrogenase by oxygen and light in two Anabaena species

Anabaena variabilis ATCC 29413 and Anabaena PCC 7120 were grown in batch cultures with combined nitrogen source. The hox genes encoding the bidirectional hydrogenase were expressed at a relatively low level in the heterocyst-free filaments. Transcriptional regulation of the bidirectional hydrogenase in response to oxygen and light was found to be similar in the two closely related strains. Low oxygen tension caused c.100-fold induction of the hox genes. However, when the anaerobic cultures were incubated in darkness the hox transcript accumulation increased by one order of magnitude compared with the illuminated anaerobic cultures. In addition, darkness could also reversibly induce hox expression and was found to be a positive regulator of hox expression even in the presence of oxygen. These results imply a specific requirement for the bidirectional hydrogenase of Anabaena PCC 7120 and A. variabilis ATCC 29413 in darkness both in the presence and absence of oxygen.

Genetic Analysis of the Hox Hydrogenase in the Cyanobacterium Synechocystis sp. PCC 6803 Reveals Subunit Roles in Association, Assembly, Maturation, and Function

Hydrogenases are metalloenzymes that catalyze 2H(+) + 2e(-) ↔ H(2). A multisubunit, bidirectional [NiFe]-hydrogenase has been identified and characterized in a number of bacteria, including cyanobacteria, where it is hypothesized to function as an electron valve, balancing reductant in the cell. In cyanobacteria, this Hox hydrogenase consists of five proteins in two functional moieties: a hydrogenase moiety (HoxYH) with homology to heterodimeric [NiFe]-hydrogenases and a diaphorase moiety (HoxEFU) with homology to NuoEFG of respiratory Complex I, linking NAD(P)H ↔ NAD(P)(+) as a source/sink for electrons. Here, we present an extensive study of Hox hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803. We identify the presence of HoxEFUYH, HoxFUYH, HoxEFU, HoxFU, and HoxYH subcomplexes as well as association of the immature, unprocessed large subunit (HoxH) with other Hox subunits and unidentified factors, providing a basis for understanding Hox maturation and assembly. The analysis of mutants containing individual and combined hox gene deletions in a common parental strain reveals apparent alterations in subunit abundance and highlights an essential role for HoxF and HoxU in complex/subcomplex association. In addition, analysis of individual and combined hox mutant phenotypes in a single strain background provides a clear view of the function of each subunit in hydrogenase activity and presents evidence that its physiological function is more complicated than previously reported, with no outward defects apparent in growth or photosynthesis under various growth conditions.

Inactivation of uptake hydrogenase leads to enhanced and sustained hydrogen production with high nitrogenase activity under high light exposure in the cyanobacterium Anabaena siamensis TISTR 8012

BackgroundBiohydrogen from cyanobacteria has attracted public interest due to its potential as a renewable energy carrier produced from solar energy and water. Anabaena siamensis TISTR 8012, a novel strain isolated from rice paddy field in Thailand, has been identified as a promising cyanobacterial strain for use as a high-yield hydrogen producer attributed to the activities of two enzymes, nitrogenase and bidirectional hydrogenase. One main obstacle for high hydrogen production by A. siamensis is a light-driven hydrogen consumption catalyzed by the uptake hydrogenase. To overcome this and in order to enhance the potential for nitrogenase based hydrogen production, we engineered a hydrogen uptake deficient strain by interrupting hupS encoding the small subunit of the uptake hydrogenase.ResultsAn engineered strain lacking a functional uptake hydrogenase (∆hupS) produced about 4-folds more hydrogen than the wild type strain. Moreover, the ∆hupS strain showed long term, sustained hydrogen production under light exposure with 2–3 folds higher nitrogenase activity compared to the wild type. In addition, HupS inactivation had no major effects on cell growth and heterocyst differentiation. Gene expression analysis using RT-PCR indicates that electrons and ATP molecules required for hydrogen production in the ∆hupS strain may be obtained from the electron transport chain associated with the photosynthetic oxidation of water in the vegetative cells. The ∆hupS strain was found to compete well with the wild type up to 50 h in a mixed culture, thereafter the wild type started to grow on the relative expense of the ∆hupS strain.ConclusionsInactivation of hupS is an effective strategy for improving biohydrogen production, in rates and specifically in total yield, in nitrogen-fixing cultures of the cyanobacterium Anabaena siamensis TISTR 8012.

Factors affecting biohydrogen production by unicellular halotolerant cyanobacterium Aphanothece halophytica

The effects of several physiological parameters on H2 production rate in the unicellular halotolerant cyanobacterium Aphanothece halophytica were investigated. Under nitrogen deprivation, the growth of cells was inhibited, but H2 production rate was enhanced approximately fourfold. Interestingly, cells grown under sulfur deprivation exhibited a decrease in cell growth, H2 production rate, and bidirectional hydrogenase activity. Glucose was the preferred sugar source for H2 production by A. halophytica, but H2 production decreased at high glucose concentrations. H2 production rate was optimum when cells were grown in the presence of 0.75 M NaCl, or 0.4 μM Fe3+, or 1 μM Ni2+. The optimum light intensity and temperature for H2 production were 30 μmol photons m−2 s−1 and 35 °C, respectively. A two-stage culture of A. halophytica was performed in order to overcome the reduction of cell growth in N-free medium. In the first stage, cells were grown in normal medium to accumulate biomass, and in the second stage, H2 production by the obtained biomass was induced by growing cells in N-free medium supplemented with various chemicals for 24 h. A. halophytica grown in N-free medium containing various MgSO4 concentrations had a high H2 production rate between 11.432 and 12.767 μmol H2 mg chlorophyll a (chl a)−1 h−1, a 30-fold increase compared to cells grown in normal medium. The highest rate of 13.804 μmol H2 mg chl a −1 h−1 was obtained when the N-free growth medium contained 0.4 μM Fe3+. These results suggested the possibility of using A. halophytica and some other halotolerant cyanobacteria thriving under extreme environmental conditions in the sea as potential sources for H2 production in the future.

Diversity in hydrogen evolution from bidirectional hydrogenases in cyanobacteria from terrestrial, freshwater and marine intertidal environments

We characterized a set of 36 strains of cyanobacteria isolated from terrestrial, freshwater and marine intertidal settings to probe their potential to produce hydrogen from excess reductant, in the hope of finding novel strains with improved traits for biohydrogen production. The set was diverse with respect to origin, morphology, taxonomy and phylogeny. We found that about one half of the strains could produce hydrogen from hydrogenases in standard assays, a trait that corresponded invariably with the presence of homologues of the gene hoxH, coding for subunit H in the bidirectional Ni–Fe hydrogenase. Strains from freshwater and intertidal settings had a high incidence of hydrogen producing, hoxH containing strains, but all terrestrial isolates were negative for both. While specific rates of hydrogen production varied among strains, some novel strains displayed rates several fold higher than those previously reported. We detected two different patterns in hydrogen production. Pattern 1, corresponding to that previously known in Synechocystis PCC 6803, encompassed strains whose hydrogenase system produced hydrogen only temporarily to revert to hydrogen consumption within a short time and after reaching moderate hydrogen concentrations. Cyanobacteria displaying pattern 2, in the genera Lyngbya and Microcoleus, tended to have higher rates, did not reverse the direction of the reaction and reached much higher concentrations of hydrogen at steady state, making them of interest as potential platforms for biohydrogen production.

Characterization of LexA-mediated Transcriptional Enhancement of Bidirectional Hydrogenase in Synechocystis sp. PCC 6803 upon Exposure to Gamma Rays

Influence of gamma rays on the cyanobacterium Synechocystis sp. PCC 6803 cells was investigated in terms of a bidirectional hydrogenase, which is encoded by hoxEFUYH genes and responsible for biohydrogen production. Irradiated cells revealed a substantial change in stoichiometry of photosystems at one day after gamma irradiation at different doses. However, as evaluated by the maximal rate of photosynthetic oxygen evolution, maximal photochemical efficiency of photosystem II, and chlorophyll content, net photosynthesis or photosynthetic capacity was not significantly different between the control and irradiated cells. Instead, transcription of hoxE, hoxH, or lexA, which encodes a subunit of bidirectional hydrogenase or the only transcriptional activator, LexA, for hox genes, was commonly enhanced in the irradiated cells. This transcriptional enhancement was more conspicuously observed immediately after gamma irradiation. In contrast, hydrogenase activities were found to somewhat lower in the irradiated cells. Therefore, we propose that transcription of hox genes should be enhanced by gamma irradiation in a LexA-mediated and possibly photosynthesis-independent manner and that this enhancement might not induce a subsequent increase in hydrogenase activities, probably due to the presence of post-transcriptional and/or post-translational regulatory mechanisms.

Metabolic Pathways for Photobiological Hydrogen Production by Nitrogenase- and Hydrogenase-containing Unicellular Cyanobacteria Cyanothece

Current biotechnological interest in nitrogen-fixing cyanobacteria stems from their robust respiration and capacity to produce hydrogen. Here we quantify both dark- and light-induced H(2) effluxes by Cyanothece sp. Miami BG 043511 and establish their respective origins. Dark, anoxic H(2) production occurs via hydrogenase utilizing reductant from glycolytic catabolism of carbohydrates (autofermentation). Photo-H(2) is shown to occur via nitrogenase and requires illumination of PSI, whereas production of O(2) by co-illumination of PSII is inhibitory to nitrogenase above a threshold pO(2). Carbohydrate also serves as the major source of reductant for the PSI pathway mediated via nonphotochemical reduction of the plastoquinone pool by NADH dehydrogenases type-1 and type-2 (NDH-1 and NDH-2). Redirection of this reductant flux exclusively through the proton-coupled NDH-1 by inhibition of NDH-2 with flavone increases the photo-H(2) production rate by 2-fold (at the expense of the dark-H(2) rate), due to production of additional ATP (via the proton gradient). Comparison of photobiological hydrogen rates, yields, and energy conversion efficiencies reveals opportunities for improvement.

Construction of a chassis for hydrogen production: physiological and molecular characterization of a Synechocystis sp. PCC 6803 mutant lacking a functional bidirectional hydrogenase

Cyanobacteria are photosynthetic prokaryotes that are promising 'low-cost' microbial cell factories due to their simple nutritional requirements and metabolic plasticity, and the availability of tools for their genetic manipulation. The unicellular non-nitrogen-fixing Synechocystis sp. PCC 6803 is the best studied cyanobacterial strain and its genome was the first to be sequenced. The vast amount of physiological and molecular data available, together with a relatively small genome, makes Synechocystis suitable for computational metabolic modelling and to be used as a photoautotrophic chassis in synthetic biology applications. To prepare it for the introduction of a synthetic hydrogen producing device, a Synechocystis sp. PCC 6803 deletion mutant lacking an active bidirectional hydrogenase (ΔhoxYH) was produced and characterized at different levels: physiological, proteomic and transcriptional. The results showed that, under conditions favouring hydrogenase activity, 17 of the 210 identified proteins had significant differential fold changes in comparisons of the mutant with the wild-type. Most of these proteins are related to the redox and energy state of the cell. Transcriptional studies revealed that only six genes encoding those proteins exhibited significant differences in transcript levels. Moreover, the mutant exhibits similar growth behaviour compared with the wild-type, reflecting Synechocystis plasticity and metabolic adaptability. Overall, this study reveals that the Synechocystis ΔhoxYH mutant is robust and can be used as a photoautotrophic chassis for the integration of synthetic constructs, i.e. molecular constructs assembled from well characterized biological and/or synthetic parts (e.g. promoters, regulators, coding regions, terminators) designed for a specific purpose.

Biohydrogen production by Anabaena sp. PCC 7120 wild-type and mutants under different conditions: Light, nickel, propane, carbon dioxide and nitrogen

Abstract Increasing awareness of environmental problems caused by the current use of fossil fuel-based energy, has led to the search for alternatives. Hydrogen is a good alternative and the cyanobacterium Anabaena sp. PCC 7120 is naturally able to produce molecular hydrogen, photosynthetically from water and light. However, this H 2 is rapidly consumed by the uptake hydrogenase. This study evaluated the hydrogen production of Anabaena sp. PCC 7120 wild-type and mutants: hupL − (deficient in the uptake hydrogenase), hoxH − (deficient in the bidirectional hydrogenase) and hupL − /hoxH − (deficient in both hydrogenases) on several experimental conditions, such as gas atmosphere (argon and propane with or without N 2 and/or CO 2 addition), light intensity (54 and 152 μEm −2 s −1 ), light regime (continuous and light/dark cycles 16 h/8 h) and nickel concentrations in the culture medium. In every assay, the hupL − and hupL − /hoxH − mutants stood out over wild-type cells and the hoxH − mutant. Nevertheless, the hupL − mutant showed the best hydrogen production except in an argon atmosphere under 16 h light/8 h dark cycles at 54 μEm −2 s −1 in the light period, with 1 μM of NiCl 2 supplementation in the culture medium, and under a propane atmosphere. In all strains, higher light intensity leads to higher hydrogen production and if there is a daily 1% of CO 2 addition in the gas atmosphere, hydrogen production could increase 5.8 times, related to the great increase in heterocysts differentiation (5 times more, approximately), whereas nickel supplementation in the culture medium was not shown to increase hydrogen production. The daily incorporation of 1% of CO 2 plus 1% of N 2 did not affect positively hydrogen production rate.

Effect of culture conditions on producing and uptake hydrogen flux of biohydrogen fermentation by metabolic flux analysis method

In this work, metabolic flux analysis (MFA) method was used to estimate the effects of the culture conditions on both the producing and uptake hydrogen flux inside the cell of Klebsiella pneumoniae ECU-15. The results indicated that higher temperature could reduce the amount of the uptake hydrogen and enhance the hydrogen production from the NADH pathway. Moreover, both the producing hydrogen flux from formate and the uptake hydrogen flux were attained to the maximum at pH 7.0–7.5. The producing hydrogen flux was higher at 5g/L initial glucose than that of the other concentrations, and the uptake hydrogen flux showed the minimum value under the same condition. The apparent hydrogen generation was caused by the combined action of producing hydrogenase, uptake hydrogenase and bidirectional hydrogenase. These results were helpful to deeply understand the mechanism of the biohydrogen evolving process and establish the suitable molecular strategies for improving hydrogen production.

The role of the bidirectional hydrogenase in cyanobacteria

Cyanobacteria have tremendous potential to produce clean, renewable fuel in the form of hydrogen gas derived from solar energy and water. Of the two cyanobacterial enzymes capable of evolving hydrogen gas (nitrogenase and the bidirectional hydrogenase), the hox-encoded bidirectional Ni–Fe hydrogenase has a high theoretical potential. The physiological role of this hydrogenase is a highly debated topic and is poorly understood relative to that of the nitrogenase. Here the structure, assembly, and expression of this enzyme, as well as its probable roles in metabolism, are discussed and analyzed to gain perspective on its physiological role. It is concluded that the bidirectional hydrogenase in cyanobacteria primarily functions as a redox regulator for maintaining a proper oxidation/reduction state in the cell. Recommendations for future research to test this hypothesis are discussed.

High rates of photobiological H2 production by a cyanobacterium under aerobic conditions

Among the emerging renewable and green energy sources, biohydrogen stands out as an appealing choice. Hydrogen can be produced by certain groups of microorganisms that possess functional nitrogenase and/or bidirectional hydrogenases. In particular, the potential of photobiological hydrogen production by oxygenic photosynthetic microbes has attracted significant interest. However, nitrogenase and hydrogenase are generally oxygen sensitive, and require protective mechanisms to function in an aerobic extracellular environment. Here, we describe Cyanothece sp. ATCC 51142, a unicellular, diazotrophic cyanobacterium with the capacity to generate high levels of hydrogen under aerobic conditions. Wild-type Cyanothece 51142 can produce hydrogen at rates as high as 465 μmol per mg of chlorophyll per hour in the presence of glycerol. Hydrogen production in this strain is mediated by an efficient nitrogenase system, which can be manipulated to convert solar energy into hydrogen at rates that are several fold higher, compared with any previously described wild-type hydrogen-producing photosynthetic microbe.

Nitrogen fixation and hydrogen metabolism in cyanobacteria.

This review summarizes recent aspects of (di)nitrogen fixation and (di)hydrogen metabolism, with emphasis on cyanobacteria. These organisms possess several types of the enzyme complexes catalyzing N(2) fixation and/or H(2) formation or oxidation, namely, two Mo nitrogenases, a V nitrogenase, and two hydrogenases. The two cyanobacterial Ni hydrogenases are differentiated as either uptake or bidirectional hydrogenases. The different forms of both the nitrogenases and hydrogenases are encoded by different sets of genes, and their organization on the chromosome can vary from one cyanobacterium to another. Factors regulating the expression of these genes are emerging from recent studies. New ideas on the potential physiological and ecological roles of nitrogenases and hydrogenases are presented. There is a renewed interest in exploiting cyanobacteria in solar energy conversion programs to generate H(2) as a source of combustible energy. To enhance the rates of H(2) production, the emphasis perhaps needs not to be on more efficient hydrogenases and nitrogenases or on the transfer of foreign enzymes into cyanobacteria. A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H(2) formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.

Hydrogen Production by the Unicellular, Diazotrophic Cyanobacterium Cyanothece sp. Strain ATCC 51142 under Conditions of Continuous Light

We report on the hydrogen production properties of the unicellular, diazotrophic cyanobacterium Cyanothece sp. strain ATCC 51142. This organism has a versatile metabolism and can grow in the presence or absence of combined nitrogen and can grow photosynthetically or mixotrophically and heterotrophically in the presence of glycerol. The strain produces a bidirectional hydrogenase (encoded by the hox genes), an uptake hydrogenase (hupLS), and nitrogenase (nifHDK). We demonstrated hydrogen production by both the hydrogenase and the nitrogenase under appropriate metabolic conditions. The highest rates of hydrogen production were produced under nitrogen-fixing conditions when cells were grown and incubated under continuous light conditions, in either the presence or absence of glycerol. Under such nitrogen-fixing conditions, we have achieved rates of 300 micromol H(2)/mg chloramphenicol (Chl)/hr during the first 24 h of incubation. The levels of H(2) measured were dependent upon the incubation conditions, such as sparging with argon, which generated anaerobic conditions. We demonstrated that the same conditions led to high levels of H(2) production and N(2) fixation, indicating that low-oxygen conditions favor nitrogenase activity for both processes. The levels of hydrogen produced by the hydrogenase are much lower, typically 5 to 10 micromol H(2)/mg Chl/hr. Hydrogenase activity was dependent upon electron transport through photosystem II (PS II), whereas nitrogenase activity was more dependent on PS I, as well as on respiration. Although cells do not double under the incubation conditions when sparged with argon to provide a low-oxygen environment, the cells are metabolically active, and hydrogen production can be inhibited by the addition of chloramphenicol to inhibit protein synthesis.