Enhanced Biological Hydrogen Production from Escherichia coli with Surface Precipitated Cadmium Sulfide Nanoparticles

The precipitation of cadmium sulfide nanoparticles is induced on the surface of Escherichia coli , and the biological hydrogen production efficiency under visible light (VL) irradiation is investigated. When endogenous [Ni–Fe]‐hydrogenase is anaerobically induced, an additional 400 µmol of hydrogen gas is generated within 3 h from the hybrid system suspension (50 mL) under VL irradiation (2000 W m^?2), corresponding to an increase in hydrogen production of ≈30%. The apparent quantum efficiencies of the hybrid system under 470 and 620 nm VL irradiation are 7.93% and 9.59%, respectively, which are higher than those of many photoheterotrophic bacteria. Furthermore, the mechanism of the enhanced hydrogen evolution is investigated. The interaction between photogenerated electrons and cells of E. coli is confirmed by heat‐treatment, electron‐scavenger, and separation studies. The acceleration of pyruvate generation, inhibition of lactate fermentation, increase of formate concentration, stimulation of hydrogenase activity, and elevation of nicotinamide adenine dinucleotide (NAD)H/NAD ratio in the hybrid system are responsible for the enhanced hydrogen production. A feasibility study is also conducted using wastewater and natural sunlight for the hydrogen production by the hybrid system. An additional 120 µmol of hydrogen is generated from the hybrid system within 3 h under these conditions using natural resources.     

Utilization of flue gas for cultivation of microalgae <Emphasis Type="Italic">Chlorella</Emphasis> sp.) in an outdoor open thin-layer photobioreactor

Flue gas generated by combustion of natural gas in a boiler was used for outdoor cultivation of Chlorella sp. in a 55 m² culture area photobioreactor. A 6 mm thick layer of algal suspension continuously running down the inclined lanes of the bioreactor at 50 cm s⁻¹ was exposed to sunlight. Flue gas containing 6–8% by volume of CO₂ substituted for more costly pure CO₂ as a source of carbon for autotrophic growth of algae. The degree of CO₂ mitigation (flue gas decarbonization) in the algal suspension was 10–50% and decreased with increasing flue gas injection rate into the culture. A dissolved CO₂ partial pressure (pCO₂) higher than 0.1 kPa was maintained in the suspension at the end of the 50 m long culture area in order to prevent limitation of algal growth by CO₂. NO_X and CO gases (up to 45 mg m⁻³ NO_X and 3 mg m⁻³ CO in flue gas) had no negative influence on the growth of the alga. On summer days the following daily net productivities of algae [g (dry weight) m⁻²] were attained in comparative parallel cultures: flue gas = 19.4–22.8; pure CO₂ = 19.1–22.6. Net utilization (η) of the photosynthetically active radiant (PAR) energy was: flue gas = 5.58–6.94%; pure CO₂ = 5.49–6.88%. The mass balance of CO₂ obtained for the flue gas stream and for the algal suspension was included in a mathematical model, which permitted the calculation of optimum flue gas injection rate into the photobioreactor, dependent on the time course of irradiance and culture temperature. It was estimated that about 50% of flue gas decarbonization can be attained in the photobioreactor and 4.4 kg of CO₂ is needed for production of 1 kg (dry weight) algal biomass. A scheme of a combined process of farm unit size is proposed; this includes anaerobic digestion of organic agricultural wastes, production and combustion of biogas, and utilization of flue gas for production of microalgal biomass, which could be used in animal feeds. A preliminary quantitative assessment of the microalgae production is presented.     

Role of Light Intensity and Temperature in the Regulation of Hydrogen Photoproduction by the Marine Cyanobacterium Oscillatoria sp. Strain Miami BG7

The effects of several key environmental factors on the development and control of hydrogen production in the marine blue-green alga (cyanobacterium) Oscillatoria sp. strain Miami BG7 were studied in relation to the potential application of this strain to a bio-solar energy technology. The production of cellular biomass capable of evolving hydrogen gas was strongly affected by light intensity, temperature, and the input of ammonia as a nutrient. Depletion of combined nitrogen from the growth media was a prerequisite for the initiation of hydrogen production. Maximum hydrogen-producing capability coincided with the end of the linear phase of growth. Hydrogen production exhibited considerable flexibility to environmental extremes. The rate of production saturated at low light intensities (i.e., 15 to 30 μEinsteins/m² per s), and no photoinhibition was observed at high light intensity (i.e., 1,000 μEinsteins/m² per s). The upper temperature limit for production was 46°C. Above the light compensation point for O₂ evolution H₂ production was inhibited. However, this problem was alleviated by two related phenomena. (i) The capacity of cells to evolve oxygen deteriorated with increasing culture age and nitrogen depletion, and (ii) the ability of these cells to produce oxygen in closed anaerobic hydrogen production systems was temporally limited.     

Exploration of the hydrogen producing potential of Rhodobacter capsulatus chemostat cultures: The application of deceleration‐stat and gradient‐stat methodology

In this work, the dependency of the volumetric hydrogen production rate of ammonium‐limited Rhodobacter capsulatus chemostat cultures on their imposed biomass concentration and dilution rate was investigated. A deceleration‐stat experiment was performed by lowering the dilution rate from 1.0 d^?1 to zero aimed at a constant biomass concentration of 4.0 g L^?1 at constant incident light intensity. The results displayed a maximal volumetric hydrogen production rate of 0.6 mmol m^?3 s^?1, well below model predictions. Possibly the high cell density limited the average light availability, resulting in a sub‐optimal specific hydrogen production rate. To investigate this hypothesis, a gradient‐stat experiment was conducted at constant dilution rate of 0.4 d^?1 at constant incident light intensity. The biomass concentration was increased from 0.7 to 4.0 g L^?1 by increasing the influent ammonium concentration. Up to a biomass concentration of 1.5 g L^?1, the volumetric hydrogen production rate of the system increased according to model predictions, after which it started to decline. The results obtained provide strong evidence that the observed decline in volumetric hydrogen production rate at higher biomass concentrations was at least partly caused by a decrease in light availability. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Outdoor cultivation of Chlamydomonas reinhardtii for photobiological hydrogen production

Photobiological hydrogen production by the unicellular green alga Chlamydomonas reinhardtii has been studied under laboratory conditions to a vast extend but has not been investigated under outdoor conditions yet. Because the hydrogen-producing hydrogenase is very sensitive to oxygen, the production must be performed in a two-stage process: generation of the required algal biomass under oxygenic photosynthesis, followed by hydrogen biosynthesis under anaerobic conditions. In order to design a sustainable process, cultivation and subsequent hydrogen production under cost-free sunlight was investigated in this work for the first time. First, cells were grown in closed photobioreactors under simulated outdoor conditions according to the light intensities of an idealized summer day (up to 2,000?μmol photons m^?2?s^?1) in order to achieve results independent of varying, and therefore not reproducible, weather conditions. The following outdoor experiments showed comparable growth characteristics and similar cell densities. However, the use of cells grown under outdoor, simulated outdoor, or high light conditions generally resulted in significantly lower hydrogen yields compared to the use of cells cultivated under low and continuous irradiance. In order to lower cultivation costs during the growth phase, the use of 10% CO₂ corresponding to the CO₂ content of flue gas was investigated. By supplying additional CO₂ during growth under the light profile corresponding to an idealized summer day, no significant increase of cell densities could be achieved, but the subsequent hydrogen production increased compared to hydrogen production of cells grown under atmospheric CO₂.     

Photoproduction of hydrogen from water in hydrogenase-containing algae.

Scenedesmus obliquus and Chlorella vulgaris cells had active hydrogenase after dark anaerobic adaptation. Illumination of these algae with visible light led to an initial production of small quantities of hydrogen gas which soon ceased owing to production of oxygen by photolysis of water. The presence of oxygen-absorbing systems in a separate chamber, not in contact with the algae, gave only a slight stimulation of hydrogen production. Addition of sodium dithionite directly to the algae led to an extensive light-dependent production of hydrogen. This stimulation was due to oxygen removal by dithionite and not to its serving as an electron donor. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosystem II, abolished all hydrogen photoproduction. Hydrogen evolution was not accompanied by CO₂ production and little difference was noted between autotrophically and heterotrophically grown cells. Hydrogen was not produced in a photosystem II mutant of Scenedesmus even in the presence of dithionite, establishing that water was the source of hydrogen via photosystems II and I. Hydrogen production was stimulated by the presence of glucose and glucose oxidase as an oxygen-absorbing system. Oxygen inhibited hydrogen photoproduction, even if oxygen was undetectable in the gas phase, if the algal solution did not contain an oxygen absorber. It was demonstrated that under these conditions hydrogenase was still active and the inability to produce hydrogen was probably due to oxidation of the coupling electron carrier.     

SURVEY OF SELECTED SEA WEEDS FOR SIMULTANEOUS PHOTOPRODUCTION OF HYDROGEN AND OXYGEN1

Ten seaweed species were surveyed for simultaneous photoevolution of hydrogen and oxygen. In an attempt to induce hydrogenase activity (as measured by hydrogen photoproduction) the seaweeds were maintained under anaerobiosis in CO₂-free seawater for varying lengths of time. Although oxygen evolution was observed in every alga studied, hydrogen evolution was not observed. One conclusion of this research is that, in contrast to the microscopic algae, there is not a single example of a macroscopic alga for which the photoevolution of hydrogen has been observed, in spite of the fact that there are now at least nine macroscopic algal species known for which hydrogenase activity has been reported (either by dark hydrogen evolution or light-activated hydrogen uptake). These results are in conflict with the conventional view that algal hydrogenase can catalyze a multiplicity of reactions, one of which is the photoproduction of molecular hydrogen. Two possible explanations for the lack of hydrogen photoproduction in macroscopic algae are presented. It is postulated that electron acceptors other than carbon dioxide can take up reducing equivalents from Photosystem I to the measurable exclusion of hydrogen photoproduction. Alternatively, the hydrogenase system in macroscopic algae may be primarily a hydrogen-uptake system with respect to light-activated reactions. A simple kinetic argument based on recent measurements of the photosynthetic turnover times of simultaneous light-activated hydrogen and oxygen production is presented that supports the second explanation.     

Reinvestigation of the steady-state kinetics and physiological function of the soluble NiFe-hydrogenase I of Pyrococcus furiosus

Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H₂ evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H₂ production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low K_m for H₂ (~20 μM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 μM dissolved CO). Since the K_m for NADP⁺ is ~37 μM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.     

Effect of light intensity and thickness of culture solution on oxygen production by algae

Data from a small cylindrical culture unit with variable annular culture chambers indicate that (i) the rate of oxygen evolution by an algal culture in the linear phase of growth is a logarithmic function of light intensity, and (ii) the rate of oxygen evolution per unit volume of suspension is linearly related to the reciprocal of culture thickness. These two relationships have been combined in an empirical equation which gives the expected variation of the oxygen production rate with light intensity, culture thickness, and suspension volume. The applicability of this equation has been tested on a larger, multilight culture unit in this laboratory. The agreement between the experimental and calculated oxygen production rates was very satisfactory, suggesting that the equation is not limited to a particular culture unit but may have wide applicability. The efficiency of the culture unit from the standpoint of oxygen output (chemical energy) relative to electrical energy to supply the light source has been calculated, and the maximum value of 0.51% was obtained. The energy to run auxiliary equipment was not a factor in these calculations. The maximum efficiency in converting light energy to chemical energy was approximately 12%. An extrapolation of the experimental results suggests that approximately 2 ft³ and 30 kw would be required to provide the oxygen needs of one man.     

Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species

Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe⿿Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production.     

Interplay between light intensity,chlorophyll concentration and culture mixing on the hydrogen production in sulfur‐deprived Chlamydomonas reinhardtii cultures grown in laboratory photobioreactors

Relationships between light intensity and chlorophyll concentration on hydrogen production were investigated in a sulfur‐deprived Chlamydomonas reinhardtii culture in a laboratory scale photobioreactor (PBR) equipped with two different stirring devices. In the first case, the culture was mixed using a conventional magnetic stir bar, while in the second it was mixed using an impeller equipped with five turbines. Experiments were carried out at 70 and 140 µmol photons m^?2 s^?1 in combination with chlorophyll concentrations of 12 and 24 mg L^?1. A high light intensity (140 µmol photons m^?2 s^?1, supplied on both sides of the PBR) in combination with a low chlorophyll concentration (12 mg L^?1) inhibited the production of hydrogen, in particular in the culture mixed with the stir bar. An optimal combination for hydrogen production was found when the cultures were exposed to 140 µmol photons m^?2 s^?1 (on both sides) and 24 mg L^?1 of chlorophyll. Under these conditions, the hydrogen production output rate reached about 120 mL L^?1 in the culture mixed with the stir bar, and rose to about 170 mL L^?1 in the one mixed with the impeller. These outputs corresponded to a mean light conversion efficiency of 0.56% and 0.81%, respectively. However, the efficiency increased to 1.08% and 1.64%, respectively, when maximum hydrogen rates were considered. The better performance of the dense cultures mixed with an impeller was mainly attributed to an intermittent illumination pattern to which the cells were subjected (time cycles within 50–100 ms) which influenced the hydrogen production (1) directly, by providing the PSII with a higher production of electrons for the hydrogenase and (2) indirectly, through a higher synthesis of carbohydrates. The fluid dynamics in the PBR equipped with the impeller was characterized. The better mixing state achieved in the PBR of the new configuration makes it a useful tool for studying the hydrogen production process involving photosynthetic microorganisms, and provides a better insight into the physiology of the process. Biotechnol. Bioeng. 2009; 104: 76–90 © 2009 Wiley Periodicals, Inc.     

Mechanistic modeling of sulfur‐deprived photosynthesis and hydrogen production in suspensions of Chlamydomonas reinhardtii

The ability of unicellular green algal species such as Chlamydomonas reinhardtii to produce hydrogen gas via iron‐hydrogenase is well known. However, the oxygen‐sensitive hydrogenase is closely linked to the photosynthetic chain in such a way that hydrogen and oxygen production need to be separated temporally for sustained photo‐production. Under illumination, sulfur‐deprivation has been shown to accommodate the production of hydrogen gas by partially‐deactivating O₂ evolution activity, leading to anaerobiosis in a sealed culture. As these facets are coupled, and the system complex, mathematical approaches potentially are of significant value since they may reveal improved or even optimal schemes for maximizing hydrogen production. Here, a mechanistic model of the system is constructed from consideration of the essential pathways and processes. The role of sulfur in photosynthesis (via PSII) and the storage and catabolism of endogenous substrate, and thus growth and decay of culture density, are explicitly modeled in order to describe and explore the complex interactions that lead to H₂ production during sulfur‐deprivation. As far as possible, functional forms and parameter values are determined or estimated from experimental data. The model is compared with published experimental studies and, encouragingly, qualitative agreement for trends in hydrogen yield and initiation time are found. It is then employed to probe optimal external sulfur and illumination conditions for hydrogen production, which are found to differ depending on whether a maximum yield of gas or initial production rate is required. The model constitutes a powerful theoretical tool for investigating novel sulfur cycling regimes that may ultimately be used to improve the commercial viability of hydrogen gas production from microorganisms. Biotechnol. Bioeng. 2014;111: 320–335. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Hydrogenase activity in nitrate-grown cells of the unicellular cyanobacterium Cyanothece PCC 7822

The activity of hydrogenase in intact cells of the unicellular cyanobacterium Cyanothece PCC 7822 was investigated using a mass spectrometer with a permeable membrane inlet. A small hydrogenase-catalyzed hydrogen production was observed with nitrate-grown cells under anoxic conditions in the dark. The same cells were also capable of a much greater rate of hydrogen uptake, induced by oxygen as well as light. Light-induced hydrogen uptake was inhibited by uncoupler. In contrast, addition of uncoupler caused a four-fold stimulation of anoxic hydrogen production in the dark. It is suggested that anoxic hydrogen production is the result of fermentative metabolism.Cyanobacteria are generally considered to have at least two distinct hydrogenases (Houchins 1984). One is a membrane-bound uptake hydrogenase which appears to be associated with nitrogen fixation, removing the hydrogen produced by nitrogenase with the concomitant production of reductant or ATP (Eisbrenner et al. 1978). The second is a reversible hydrogenase located in the cytoplasm and not closely linked to nitrogen metabolism. The reversible character of this enzyme can be demonstrated in the presence of suitable electron donors or acceptors; hydrogen consumption and evolution occur at similar rates (Lambert and Smith 1980).A reversible hydrogenase capable of reducing protons with the artificial electron donor couple dithionite and methyl viologen is widely distributed amongst cyanobacteria. However its physiological role remains unclear. The enzyme appears to be sensitive to oxygen, and consequently in vivo activity can only be demonstrated under anoxic conditions (Houchins 1984).On the basis of in vivo measurements with tritium and the observed low K _m for hydrogen, the function of the reversible hydrogenase of the heterocystous cyanobacterium Anabaena has been proposed to be the uptake of hydrogen as a means of collecting additional reducing power during growth in light-limited anoxic environments (Spiller et al. 1983; Houchins 1984). However, Hallenbeck et al. (1981) reported a modest production of hydrogen by intact filaments of Anabaena.An example of a function of the reversible hydrogenase in the production of hydrogen is provided by the nonheterocystous filamentous cyanobacterium Oscillatoria limnetica. This organism is capable of shifting between oxygenic and anoxygenic photosynthesis (Oren and Padan 1978). In the latter case sulfide is the electron donor supporting photoreduction of CO₂ via photosystem I only. However when CO₂ is limiting, excess reducing equivalents are removed by a reversible hydrogenase (Belkin and Padan 1978). This hydrogen production probably enables the organism to continue photophosphorylation under these conditions.We recently reported that the unicellular cyanobacterium Cyanothece 7822 is capable of hydrogenase-catalyzed hydrogen production in vivo, without the addition of artificial reductants (Van der Oost et al. 1987). In this paper we have investigated the in vivo activity of the hydrogenase in Cyanothece by monitoring the concentrations of dissolved H₂ and O₂ in the cell suspension using a mass spectrometer with a permeable membrane inlet.Abbreviations DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU N-(3,4-dichlorophenyl) N,N-dimethylurea - FCCP carbonylcyanide-p-trifluoromethoxy phenylhydrazone - PBQ phenyl benzoquinone     

固氮蓝藻高光放氢突变种的筛选和放氢特点

作者以前报道过几种快速生长的固氮蓝藻在某种条件下能好气光放氢,其速度可以达到光合放氧速度的10—15%,但这种活性只有在不积累氢气的流动气相下或在短时间内发生。本文报道用亚硝基胍诱变所得到的Anabaena spp。Strain CA的高光放氢突变种——N9A和18A——的筛选和氢代谢特点。在达生长饱和光照以后,野生型的光放氢活性与光照强度的增加成正相关,而其吸氢活性则与之成负相关,显示高光照强度可能抑制吸氢酶的活性。无论在什么光强下,均测不到两个突变种的吸氢活性,暗示在突变种中,吸氢酶或有关系统受损伤。把细胞固相化在琼脂上,在密闭系统中,高光强下培养50个小时,两个突变种光释放和积累的氢分别为野生型的2倍(N9A)和6倍(18A),后者等于氢占气相(1%CO_2的空气)的1.8%。两个突变种在生长速度、叶绿素含量、乙炔还原活性以及光合放氧方面与野生型无明显不同。当以含50—100nM的镍离子的培养基培养时,野生型的好气净产氢活性完全丢失,其吸氢活性却增加约10倍。培养基中镍离子的存在,对两个突变种的高光放氢活性则毫无影响,而且在此情况下,仍测不出其吸氢活性。实验结果表明,这两个突变种系吸氢酶缺陷型突变种。     

Hydrogen evolution by a thermophilic blue-green alga Mastigocladus laminosus

The thermophilic blue-green alga (cyanobacterium), Mastigocladuslaminosus isolated from a hot spring, evolved hydrogen gas undernitrogen-starved conditions in light when algal cells were grownin a nitrate-free medium. Cells grown in a nitrate-medium evolvedno detectable hydrogen gas in light. The optimal temperatureand pH for hydrogen evolution were 44–49?C and 7.0–7.5.High activity of hydrogen evolution. 1.6 ml H₂/mg chl.hr, wasinduced when algal cells grown in the nitrate medium were activelyforming heterocysts in the nitrate-free medium in air. Hydrogenevolution in light was depressed by nitrogen gas and inhibitedby salicylaldoxime or DNP. This hydrogen evolution by M. laminosusis attributed to the action of nitrogenase. (Received June 20, 1979; )     

Upgrading of straw hydrolysate for production of hydrogen and phenols in a microbial electrolysis cell (MEC)

In a microbial electrolysis cell (MEC), hydrolysate produced by hydrothermal treatment of wheat straw was used for hydrogen production during selective recovery of phenols. The average H₂ production rate was 0.61 m³ H₂/m³ MEC·day and equivalent to a rate of 0.40 kg COD/m³ MEC·day. The microbial community in the anode biofilm was adapted by establishment of xylose-degrading bacteria of the Bacteriodetes phylum (16%) and Geobacter sulfurreducens (49%). During the process, 61% of the chemical oxygen demand was removed as hydrogen at 64% yield. The total energy production yield was 78% considering the energy content in the consumed compounds and the cell voltage of 0.7 V. The highest hydrogen production was equivalent to 0.8 kg COD/m³ MEC·day and was obtained at pH 7–8 and 25°C. Accumulation of 53% w/v phenolic compounds in the liquor was obtained by stepwise addition of the hydrolysate during simultaneous production of hydrogen from consumption of 95% for the hemicellulose and 100% of the fatty acids. Final calculations showed that hydrolysate produced from 1 kg wheat straw was upgraded by means of the MEC to 22 g hydrogen (266 L), 8 g xylan, and 9 g polyphenolics for potential utilization in biobased materials.     

The effect of light intensity on hydrogen production by sulfur-deprived Chlamydomonas reinhardtii

The effect of light intensity on hydrogen production by sulfur-deprived Chlamydomonas reinhardtii was studied in situ using either long- or short-term experiments, or alternatively, with samples withdrawn from the photobioreactor. Overall hydrogen production by S-deprived culture was shown to depend on the light intensity and to exhibit regions of light limitation and light inhibition. The optimal incident light intensity for hydrogen production was independent of the method of sulfur deprivation or the initial acetate concentration in the medium (12-34 mM). However, it varied with the Chl concentration and the thickness of the photobioreactor. To calculate the average light intensity in the photobioreactor under different experimental conditions, a special mathematics approach was developed. The optimal average light intensity for H(2) production appeared to be 30-40 microE m(-2)s(-1) and was independent of the Chl or acetate concentrations and the method of S deprivation. The inhibitory effect of high light intensity was related to the enhanced O(2) evolution activity during the photosynthetic stage of sulfur deprivation and to the high activity of photosystem II at the beginning of the H(2)-production phase. Data support the major role of photosystem II in supplying reductants through photosystem I to the hydrogenase throughout the H(2)-production phase.     

Inhibition of respiration and nitrate assimilation enhances photohydrogen evolution under low oxygen concentrations in Synechocystis sp. PCC 6803

In cyanobacterial membranes photosynthetic light reaction and respiration are intertwined. It was shown that the single hydrogenase of Synechocystis sp. PCC 6803 is connected to the light reaction. We conducted measurements of hydrogenase activity, fermentative hydrogen evolution and photohydrogen production of deletion mutants of respiratory electron transport complexes. All single, double and triple mutants of the three terminal respiratory oxidases and the ndhB-mutant without a functional complex I were studied. After activating the hydrogenase by applying anaerobic conditions in the dark hydrogen production was measured at the onset of light. Under these conditions respiratory capacity and amount of photohydrogen produced were found to be inversely correlated. Especially the absence of the quinol oxidase induced an increased hydrogenase activity and an increased production of hydrogen in the light compared to wild type cells. Our results support that the hydrogenase as well as the quinol oxidase function as electron valves under low oxygen concentrations. When the activities of photosystem II and I (PSII and PSI) are not in equilibrium or in case that the light reaction is working at a higher pace than the dark reaction, the hydrogenase is necessary to prevent an acceptor side limitation of PSI, and the quinol oxidase to prevent an overreduction of the plastoquinone pool (acceptor side of PSII). Besides oxygen, nitrate assimilation was found to be an important electron sink. Inhibition of nitrate reductase resulted in an increased fermentative hydrogen production as well as higher amounts of photohydrogen.     

Evidence for three distinct hydrogenase activities in Rhodospirillum rubrum

Inducer, inhibitor, and mutant studies on three hydrogenase activities of Rhodospirillum rubrum indicate that they are mediated by three distinct hydrogenase enzymes. Uptake hydrogenase mediates H2 uptake to an unknown physiological acceptor or methylene blue and is maximally synthesized during autotrophic growth in light. Formate-linked hydrogenase is synthesized primarily during growth in darkness or when light becomes limiting, and links formate oxidation to H2 production. Carbon-monoxide-linked hydrogenase is induced whenever CO is present and couples CO oxidation to H2 evolution. The enzymes can be expressed singly or conjointly depending on growth conditions, and the inhibitor or inducer added. All three hydrogenases can use methyl viologen as the mediator for both the H2 evolution and H2 uptake reactions while displaying distinct pH optima, reversibility, and sensitivity to C2H2 gas. Yet, we present evidence that the CO-linked hydrogenase, unlike the uptake hydrogenase, does not link to methylene blue as the electron acceptor. These differences allow conditions to be established to quantitatively assay each hydrogenase independently of the others both in vivo and in vitro.     

Oxygen reduction in chloroplast thylakoids results in production of hydrogen peroxide inside the membrane

Hydrogen peroxide production in isolated pea thylakoids was studied in the presence of cytochrome c to prevent disproportionation of superoxide radicals outside of the thylakoid membranes. The comparison of cytochrome c reduction with accompanying oxygen uptake revealed that hydrogen peroxide was produced within the thylakoid. The proportion of electrons from water oxidation participating in this hydrogen peroxide production increased with increasing light intensity, and at a light intensity of 630 μmol quanta m^− 2 s^− 1 it reached 60% of all electrons entering the electron transport chain. Neither the presence of a superoxide dismutase inhibitor, potassium cyanide or sodium azide, in the thylakoid suspension, nor unstacking of the thylakoids appreciably affected the partitioning of electrons to hydrogen peroxide production. Also, osmolarity-induced changes in the thylakoid lumen volume, as well as variation of the lumen pH induced by the presence of Gramicidin D, had negligible effects on such partitioning. The flow of electrons participating in lumen hydrogen peroxide production was found to be near 10% of the total electron flow from water. It is concluded that a considerable amount of hydrogen peroxide is generated inside thylakoid membranes, and a possible mechanism, as well as the significance, of this process are discussed.