Elias Greenbaum

Microalgae: a green source of renewable H2

This article summarizes recent advances in the field of algal hydrogen production. Two fundamental approaches are being developed. One involves the temporal separation of the usually incompatible reactions of O(2) and H(2) production in green algae, and the second involves the use of classical genetics to increase the O(2) tolerance of the reversible hydrogenase enzyme. The economic and environmental impact of a renewable source of H(2) are also discussed.

Biotechnology: Enzymatic production of biohydrogen

Although in theory the amount of hydrogen that could be generated from renewable sources of energy such as cellulose (a polymer of glucose) is vast, only 16–24% of the maximum stoichiometric yield of hydrogen from glucose (about 12 mol H2 per mol glucose) is typically achieved by biological methods. Here we show that the enzymes of the oxidative pentose phosphate cycle can be coupled to hydrogenase purified from the bacterium Pyrococcus furiosus, one of only a few hydrogenases that use NADP+ as the electron carrier, to generate 11.6 mol H2 per mol glucose-6-phosphate. Hydrogen produced by this pathway is the major product, unlike that produced by intermediate metabolic pathways of bacterial fermentation, and therefore has important practical implications for biohydrogen production.

Enzymatic production of biohydrogen.

Although in theory the amount of hydrogen that could be generated from renewable sources of energy such as cellulose (a polymer of glucose) is vast, only 16–24% of the maximum stoichiometric yield of hydrogen from glucose (about 12 mol H2 per mol glucose) is typically achieved by biological methods. Here we show that the enzymes of the oxidative pentose phosphate cycle can be coupled to hydrogenase purified from the bacterium Pyrococcus furiosus, one of only a few hydrogenases that use NADP+ as the electron carrier, to generate 11.6 mol H2 per mol glucose-6-phosphate. Hydrogen produced by this pathway is the major product, unlike that produced by intermediate metabolic pathways of bacterial fermentation, and therefore has important practical implications for biohydrogen production.

Stand-off tissue-based biosensors for the detection of chemical warfare agents using photosynthetic fluorescence induction.

Tissue biosensors made from immobilized whole-cell photosynthetic microorganisms have been developed for the detection of airborne chemical warfare agents and simulants. The sensor read-out is based on well-known principles of fluorescence induction by living photosynthetic tissue. Like the cyanobacteria and algae from which they were constructed, the sensors are robust and mobile. The fluorescence signal from the sensors was stable after 40 days, storage and they can be launched or dropped into suspected danger zones. Commercially available hand-held fluorometric detector systems were used to measure Photosystem II (PSII) photochemical efficiency of green algae and cyanobacteria entrapped on filter paper disks. Toxic agents flowing in the gas stream through the sensors can alter the characteristic fluorescence induction curves with resultant changes in photochemical yields. Tabun (GA), sarin (GB), mustard agent, tributylamine (TBA) (a sarin stabilizer), and dibutyl sulfide (DBS) (a mustard agent analog) were tested. Upper threshold limits of detectability for GA, TBA, and DBS are reported. With additional research and development, these biosensors may find application in stand-off detection of chemical and perhaps biological warfare agents under real-world conditions.

Platinized Chloroplasts: A Novel Photocatalytic Material

Colloidal platinum was prepared and precipitated directly onto photosynthetic thylakoid membranes from aqueous solution and entrapped on fiberglass filter paper. This composition of matter was capable of sustained simultaneous photoevolution of hydrogen and oxygen when irradiated at any wavelength in the chlorophyll absorption spectrum. Experimental data support the interpretation that part of the platinum metal catalyst is precipitated adjacent to the photosystem I reduction site of photosynthesis and that electron transfer occurs across the interface between photosystem I and the catalyst. Photoactivity of the material was dependent on the nature of the ionic species from which the platinum was precipitated. All photoactive samples were prepared from the hexachloroplatinate(IV) ion, whereas samples prepared by precipitation of the tetraammineplatinum(II) ion showed no hydrogen evolution activity and only transient oxygen activity. This system is among the simplest known for photosynthetically splitting water into molecular hydrogen and oxygen.

Photosynthetic Hydrogen and Oxygen Production: Kinetic Studies

Steady-state turnover times for simultaneous photosynthetic production of hydrogen and oxygen have been measured for two systems: the in vitro system comprised of isolated chloroplasts, ferredoxin, and hydrogenase, and the anaerobically adapted green alga Chlamydomonas reinhardtii [137c(+) mating type]. In both systems, the simultaneous photoproduction of hydrogen and oxygen was measured by driving the systems into the steady state with repetitive, single-turnover, flash illumination. The turnover times for production of both oxygen and hydrogen in photosynthetic water splitting are in milliseconds and are equal to or less than the turnover time for carbon dioxide reduction in intact algal cells. The oxygen and hydrogen turnover times are therefore compatible with each other and partially compatible with the excitation rate of the photosynthetic reaction centers under conditions of solar irradiation.

HYDROGEN and OXYGEN PHOTOPRODUCTION BY MARINE ALGAE

Abstract The first measurements of the simultaneous photoproduction of hydrogen and oxygen in marine green algae are reported. Eight species in the genera Chlamydomonas, Chlorella and Halochlorocococcum were tested in CO2‐free seawater. Four of the five species of Chlamydomonas were able to produce hydrogen in the light after a period of 3 ‐ 4 h of dark anaerobic adaptation. Only one of the two Chlorella species tested was able to photoproduce hydrogen, in trace amounts. Halochlorocococcum fla–9 gave positive results and Chlamydomonas species (clone f‐9) had a steady‐state rate of hydrogen and oxygen production during irradiation with a stoichiometric ratio near 2:1. The integrated yields of hydrogen and oxygen produced by this species corresponds to about 450 turnovers of the photochemical reaction centers. This number exceeds (by about a factor of 20) the electron‐carrying capacity of the electron transport chain linking Photosystems I and II. These data suggest that Chlamydomonas f‐9 makes seawater a potential substrate for solar hydrogen and oxygen production.

Nanoscale Photosynthesis: Photocatalytic Production of Hydrogen by Platinized Photosystem I Reaction Centers¶

Abstract A study of the photocatalytic production of molecular hydrogen from platinized photosystem I (PSI) reaction centers is reported. At pH 7 and room temperature metallic platinum was photoprecipitated at the reducing end of PSI according to the reaction, [PtCl6]2− + 4e− + hν → Pt↓ + 6Cl−, where it interacted with photogenerated PSI electrons and catalyzed the evolution of molecular hydrogen. The reaction mixture included purified spinach PSI reaction centers, sodium ascorbate and spinach plastocyanin. Experimental data on real-time catalytic platinum formation as measured by the onset and rates of hydrogen photoevolution as a function of time are presented. The key objective of the experiments was demonstration of functional nanoscale surface metalization at the reducing end of isolated PSI by substituting negatively charged [PtCl6]2− for negatively charged ferredoxin, the naturally occurring water-soluble electron carrier in photosynthesis. The data are interpreted in terms of electrostatic interactions between [PtCl6]2− and the positively charged surface of psaD, the ferredoxin docking site situated at the stromal interface of the photosynthetic membrane and which is presumably retained in our PSI preparation. A discussion of the rates of hydrogen evolution in terms of the structural components of the various PSI preparations as well as of those of the intact thylakoid membranes is presented.

Fabrication of dissimilar metal electrodes with nanometer interelectrode distance for molecular electronic device characterization

We report a versatile process for the fabrication of dissimilar metal electrodes with a minimum interelectrode distance of less than 6 nm using electron beam lithography and liftoff pattern transfer. This technique provides a controllable and reproducible method for creating structures suited for the electrical characterization of asymmetric molecules for molecular electronics applications. Electrode structures employing pairs of Au electrodes and non-Au electrodes were fabricated in three different patterns. Parallel electrode structures 300 μm long with interelectrode distances as low as 10 nm, 75 nm wide electrode pairs with interelectrode distances less than 6 nm, and a multiterminal electrode structure with reproducible interelectrode distances of 8 nm were realized using this technique. The processing issues associated with the fabrication of these structures are discussed along with the intended application of these devices.

The role of carbon dioxide in light-activated hydrogen production by Chlamydomonas reinhardtii.

Light-activated hydrogen and oxygen evolution as a function of CO2 concentration in helium were measured for the unicellular green alga Chlamydomonas reinhardtii. The concentrations were 58, 30, 0.8 and 0 ppm CO2. The objective of these experiments was to study the differential affinity of CO2/HCO3- for their respective Photosystem II and Calvin cycle binding sites vis-à-vis photoevolution of molecular oxygen and the competitive pathways of hydrogen photoevolution and CO2 photoassimilation. The maximum rate of hydrogen evolution occurred at 0.8 ppm CO2, whereas the maximum rate of oxygen evolution occurred at 58 ppm CO2. The key result of this work is that the rate of photosynthetic hydrogen evolution can be increased by, at least partially, satisfying the Photosystem II CO2/HCO3- binding site requirement without fully activating the Calvin-Benson CO2 reduction pathway. Data are presented which plot the rates of hydrogen and oxygen evolution as functions of atmospheric CO2 concentration in helium and light intensity. The stoichiometric ratio of hydrogen to oxygen changed from 0.1 at 58 ppm to approximately 2.5 at 0.8 ppm. A discussion of partitioning of photosynthetic reductant between the hydrogen/hydrogenase and Calvin-Benson cycle pathways is presented.