In comparison with fermentor-based H2 production, the use of photosynthetic organisms has received wider publicity because envisaged bioprocesses have convincing environmental credentials, that is, the ability to produce a carbonless fuel using only water, light, and air (CO2) as inputs. Hydrogen photobiology is, how­ever, highly problematic because of the incompatibility of the two essential steps:

• In the first stage, water is split to produce O2.

• In the second stage, the photoproduced electrons are combined with proteins to form H2 by either a hydrogenase or a nitrogenase — and O2 is a potent inhibitor of such an “anaerobic” system.

Nature provides two related solutions to this dilemma.64 First, filamentous cyanobacteria (e. g., Anabaena cylindrica) that compartmentalize the two reactions into different types of cell: vegetative cells for generating O2 from water and using the reducing power to fix CO2 into organic carbon compounds that then pass to specialized nitrogenase-containing heterocyst cells that evolve H2 when N2 reduction is blocked by low ambient concentrations of N2. The second scenario is that of non­heterocystous cyanobacteria that separate O2 and H2 evolution temporally (in day and night cycles), although the same overall effect could be achieved using sepa­rate light and dark reactors. With either type of nitrogen-fixing organism, however, the high energy requirement of nitrogenase would lower solar energy conversion efficiencies to unacceptably low levels.

Hydrogenase is the logical choice of biocatalyst for H2 production, and nearly 35 years have now passed since the remarkable experimental demonstration that simply mixing chloroplasts isolated from spinach leaves with hydrogenase and ferredoxin isolated by cells of Clostridium kluyveri generated a laboratory system capable of direct photolysis of water and H2 production.65 The overall reaction sequence in that “hybrid” biochemical arrangement was

H2O + light ^ /02 + 2e — + 2H+ ^ ferredoxin ^ hydrogenase ^ H2

Light-induced photolysis of water produced electrons that traveled via the photo­systems of the chloroplast preparations to reduce ferredoxin before hydrogenase catalyzing the reunion of electrons and protons to form molecular hydrogen. For over a quarter of a century, therefore, the nagging knowledge that direct photolytic H2 production is technically feasible has both tantalized and spurred on research into solar energy conversion.

Thirty years ago, the abilities of some unicellular green (chlorophyll-containing) algae, that is, microalgae, to generate H2 under unusual (02-free) conditions where hydrogenase was synthesized had already been defined.66 Such microalgae can, when illuminated at low light intensities in thin films (5-20 cellular monolayers), show conversion efficiencies of up to 24% of the photosynthetically active radiation.67

What is the biological function of hydrogenase in such highly aerobic organisms? An induction period with darkness and anaerobiosis appears to be essential.68 Photosynthetic H2 production is also enhanced if the concentration of C02 is low, suggesting that the hydrogenase pathway is competitive with the normal CO2- fixing activity of chloroplasts.69 Because the electron transport via the hydrogenase pathway is still coupled to bioenergy conservation (photosynthetic phosphorylation), hydrogenase may represent an “emergency” strategy in response to adverse environmental conditions, for example, in normally above-ground plant parts subject to water logging and anaerobiosis where essential maintenance and cellular repair reactions can still operate with a continuing source of energy. It naturally follows that reintroduction of O2 and CO2 would render such a function of hydrogenase superfluous — and explains the inhibitory effect of 02 on hydrogenase and the ability of even background levels of O2 to act as an electron acceptor in direct competi­tion with hydrogenase-mediated H2 production.70 Genetic manipulation and directed evolution of algal hydrogenases with reduced or (in the extreme case) no sensitivity to O2 is, therefore, unavoidable if maximal and sustained rates of photohydrogen production can be achieved in microalgal systems.

Progress has begun to be made on the molecular biology of microalgal hydrog — enases, including the isolation and cloning of the two genes for the homologous iron hydrogenases in the green alga Chlamydomonas reinhardtiV1 Random mutagenesis of hydrogenase genes could rapidly isolate novel forms retaining activity in the pres­ence of O2 and/or improved hydrogenase kinetics. Screening mutants of Chlamydo — monas reinhardtii has, however, revealed unexpected biochemical complexities, in particular the requirement for functional starch metabolism in H2 photoproduction.72 Several changes were indeed identified during the successful improvement of H2 photoproduction by this alga:73

• There was rational selection of mutants with altered electron transport activities with maximized electron flow to hydrogenase.

• Isolates were then screened for increased H2 production rates, leading to a mutant with reduced cellular O2 concentrations, thus having less inhibition of hydrogenase activity.

• The most productive mutant also had large starch reserves.

Using the conventional representation of electron transport inside chloroplast membrane systems, the possible interactions of photohydrogen production and other photosynthetic activities can be visualized (figure 7.7).74 Active endogenous metabolism could remove photoproduced O2 by using O2 as the terminal electron

acceptor in mitochondria; the problem of different spatial sites for O2 production and O2 utilization still, however, requires a reduced sensitivity of hydrogenase to O2 as the gas cannot be removed instantaneously — only in “test-tube” systems can O2-removing chemicals be supplied, for example, as glucose plus glucose oxidase to form gluconic acid by reaction between glucose and O2.65

The obvious implication of the redox chemistry of figure 7.7 is that the normal pro­cesses of photosynthesis, involving reduction of NADP for the subsequent reduction of CO2 to sugars, can be separated in time, with light-dependent O2 evolution and dark H2 production or if H2 production can proceed with inhibited O2 evolution, that is, “indirect biophotolysis.”64 The particular advantage of this arrangement is that the light-dependent stage can be operated in open pools to maximize productivity at mini­mal cost. Sustained H2 production could be achieved over approximately 100 hours after transfer of light-grown C. reinhardtii cells to a medium deficient in sulfur; these conditions reversibly inactivated Photosystem II and O2 evolution, whereas oxidative respiration in the continued light depleted O2, thus inducing hydrogenase.75 The subse­quent H2 production only occurred in the light and was probably a means of generating energy by Photosystem I activity (figure 7.7). Starch and protein were consumed while a small amount of acetic acid was accumulated.

This was the first reported account of a single-organism, two-stage photobiological production process for H2, although a prototype light/dark device using three stages (one light and two dark) with a marine microalga and a marine photosynthetic bacterium was tested in Japan in the 1990s.76 How much H2 could a microalgae-based approach produce? With C. reinhardtii cells given an average irradiance of 50 mol photons/m2/ day (a possible value in temperate latitudes, although highly variable on a day-to-day and seasonal basis), the maximum H2 production would be 20 g/m2/day, equivalent to 80 kg/acre/day (or 200 kg/hectare/day) — but the likely value, allowing for low yields of H2 production measured under laboratory conditions, the far from complete absorp­tion of incident light, and other factors, is only 10% of this.74 In a further refinement of this approach, the sulfate-limited microalgae were shown to form a stable process for 4000 hours: two automated photobioreactors were coupled to first grow the cells aero­bically before being continuously delivered to the second, anaerobic stage.77 Until all the biological and physical limitations can be overcome, however, large infrastructural investments in high and predictable sunlight regions would be required, and the capital costs for such solar power stations would be high, but the technical complexity may only approximate that of installing extensive photovoltaic cell banks for the direct produc­tion of electricity, an option vigorously advocated by critics of biofuels programs.78,79

Cyanobacteria (“blue-green algae”) are prokaryotes but share with higher pho­tosynthetic organisms the basic electron transport chains of Photosystems I and II (figure 7.7). The molecular biology and biochemistry of hydrogenases in cyanobac­teria is well understood, the complete genomes of several such organisms have been sequenced, and interspecies gene transfer is established.80 Much of the research has unfortunately concentrated on nitrogenase as a source of H2, but many cyanobacteria contain hydrogenases catalyzing the reversible formation of H2, a route with far more biotechnological potential for commercial H2 generation, and protein engineering has begun to reduce the O2 sensitivities of cyanobacterial hydrogenases.81 The physi­ological role of hydrogenase in cyanobacteria has been debated for decades; recent results suggest some kind of safety valve function under low O2 condition when a light-to-dark transition occurs, and inactivating quinol oxidase (an enzyme with a similar hypothetical function) and nitrate reductase (a third electron “sink”) increase photohydrogen evolution rates.82

Thermophilic cyanobacteria are known to be capable of H2 photoproduction at up to 50°C in open-air cultures maintained for more than 3 weeks.8384 If a fermentable carbon source is supplied, a sustained photoevolution of H2 can be achieved, with photolysis of water (a Photosystem II activity — see figure 7.7), whereas carbohy­drate-mediated reduction of the plastoquinone pool continues independently.85 This H2 production system has been termed “photofermentation”; in principle, relatively little light energy is required to drive the reaction because of the energy input from the fermentable substrate.64 The green alga C. reinhardtii shares this pattern of metabo­lism with cyanobacteria, behaving under photofermentative conditions much like an enteric bacterium such as E. coli, exhibiting pyruvate formate lyase activity and accu­mulating formate, ethanol, acetate, CO2, and H2 as well as glycerol and lactate.86

The overlapping molecular structures of cyanobacteria and nonphotosynthetic bacteria were exemplified by the coupling (both in vivo and in vitro) between cyano — bacterial photosynthetic electron transport components with clostridial hydrogenase; even more remarkable was the expression in a Synechococcus strain of the hydrogenase gene from C. pasteurianum, the enzyme being active in the cyanobacterial host.87 As a possible pointer to the future of designing an improved photosynthetic organism for H2 production, the “hard wiring” of a bacterial hydrogenase with a peripheral subunit of a Photosystem I subunit of the cyanobacterium Thermosynechococcus elongatus resulted in a fusion protein that could associate functionally with the rest of the Pho­tosystem I complex in the cyanobacterium and display light-driven H2 evolution.88

Photosynthetic bacteria differ from other photosynthetic organisms in using bacteriochlorophyll rather than chlorophyll as the central pigment for light-induced electron transport; they also lack Photosystem II (figure 7.7) and perform anoxy — genic photosynthesis and require electron donors more reduced than water, includ­ing reduced sulfur and organic compounds.89 Being able to fix gaseous nitrogen, the photosynthetic bacteria contain nitrogenase in addition to hydrogenase and occur globally in widely different habitats, including fresh, brackish, and sea waters, hot sulfur springs, paddy fields, wastewaters, and even in Antarctica. Hydrogen can be photoproduced in the presence of an organic substrate, sometimes with high effi­ciencies deduced from the maximum theoretical H2 production on a molar basis (table 7.3). Both free and immobilized cells have been used to produce H2 during extended periods (table 7.4). All photosynthetic bacteria can use H2 as a reductant for the fixation of CO2 into organic carbon, and considerable reengineering of the molecular biochemistry is unavoidable if the cells are to be evolved into biological H2 producers.27 Photofermentations are also known, and Rhodobacter capsulatus has been used as a test organism to evaluate photobioreactor designs potentially reaching 3.7% conversion efficiency of absorbed light energy into H2 fuel energy.90

Photosynthetic bacteria may have the additional capability of catalyzing the “water shift” reaction (chapter 6, section 6.2.1):

CO + H2O ^ CO2 + H2

Photofermentative Hydrogen Production by Photosynthetic Bacteria

TABLE 7.4

Photofermentative Hydrogen Production by Immobilized Cells

H2 Evolution rate

Species Electron donor Immobilization method (ml/hr/g dry weight)

but, unlike the thermochemical process, at moderate temperatures and without multiple passages of gases through the reaction vessel.64 A continuous process was devised for Rhodospirillum rubrum with illumination supplied by a tungsten light.91 With biomass as the substrate for gasification, a substantially (if not entirely) biological process for H2 production can be envisaged. A National Renewable Energy Laboratory report concluded that a biological reactor would be larger and slower but could achieve comparable efficiencies of heat recovery in integrated systems; the most likely niche market use would occur in facilities where the water gas shift was an option occasionally (but gainfully) employed but where the start-up time for a thermal catalytic step would be undesirable.92

Patents describing processes for H2 production using photosynthetic microbes cover at least 23 years, and include topics as diverse as their basic biology, molecular and enzymic components, and analytical methodologies. In the last 5 years, however, several patents have also appeared focusing on biohydrogen production by fermentative organisms (table 7.5).