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D. Hydrogen production

Min and Sherman (2010a); Bandyopadhyay et al (2010); and Sherman et al (2010)

Of all 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 such as cyanobacteria and green algae has attracted significant interest. However, nitrogenase and hydrogenase are generally oxygen sensitive, and require special protective mechanisms to function in an aerobic environment. Based on our previous work, we hypothesized that Cyanothece sp. ATCC 51142, a unicellular, diazotrophic cyanobacterial strain, could generate high levels of hydrogen under aerobic conditions. Wild type Cyanothece 51142 cells can produce hydrogen utilizing solar energy, and CO2 or glycerol. Our study indicates that hydrogen production in this strain is mediated by an efficient nitrogenase system, which can be manipulated to convert solar energy into hydrogen at rates which are several fold higher compared to any previously described wild type hydrogen producing photosynthetic microbe.

The work in the attached paper describes the use of the wild type strain of the unicellular cyanobacterium Cyanothece 51142 for highly efficient photobiological H2 production under natural aerobic conditions. In general, the oxygen sensitivity of the enzymes involved in biological H2 production makes the use of oxygenic photosynthetic organisms as a platform for H2 production an extremely challenging task. Thus, up till now, photobiological H2 production studies have largely relied on artificial interventions, which help create an anaerobic environment. It has been recognized that the use of oxygenic photosynthetic microbes that can produce H2 under aerobic conditions would be an important step forward for biological H2 production. Our study has identified Cyanothece 51142 as one such organism that has developed an effective strategy to make the best use of a diurnal cycle – synthesizing energy rich storage compounds during the day and utilizing it for nitrogen fixation at night when oxygen consuming processes renders the interior of the cell anaerobic (or suboxic), while the extracellular environment continues to be oxygen rich. This trait of Cyanothece 51142 formed the basis for the physiological perturbations that were designed for our two stage aerobic H2 production process.

Nitrogen fixation is an energy intensive process, requiring 16 molecules of ATP for every molecule of nitrogen fixed and H2 produced. However, the process is of paramount importance to diazotrophic species inhabiting ecological niches (such as deep oceans) with very low levels of nitrogenous nutrients. Consequently, photoautotrophic unicellular strains like Cyanothece 51142 are expected to have evolved effective strategies for harvesting and storing solar energy, which can be utilized at night when the energy demands are high. Our study shows that Cyanothece 51142 not only develops an intracellular environment conducive for the function of the nitrogenase enzyme, but also generates an adequate supply of ATP for this high energy requiring process. In addition, our results reveal high specific activity of the nitrogenase enzyme in this strain, a finding consistent with earlier reports which showed higher rates of nitrogen fixation in marine unicellular diazotrophs like Cyanothece compared to some filamentous strains Notably, not all unicellular cyanobacterial strains are endowed with these traits, as is evident from studies on Gloeothece, a unicellular freshwater, diazotroph, which possesses a nitrogenase enzyme with relatively low specific activity and does not have any appreciable nitrogenase mediated H2 production capacity.

Diazotrophic cyanobacteria have developed various strategies to protect their nitrogenase enzyme from the oxygen-rich environment they inhabit. However, unlike Cyanothece 51142, most diazotrophic strains are unable to exhibit nitrogenase mediated H2 production under aerobic conditions. In filamentous, heterocyst-forming diazotrophic strains, this is largely ascribed to the activities of an uptake hydrogenase enzyme system which is functionally closely associated with the nitrogenase and oxidizes the H2 produced. It has been shown that wild type Anabaena variabilis cells can generate H2 only under an argon atmosphere whereas its uptake hydrogenase mutants PK84 and AVM13 can produce H2 aerobically. A recent study also demonstrated H2 production (~25 µmoles/mg Chl.h) from the vegetative cells of wild type Anabaena variabilis under nitrogen atmosphere when strict anaerobic conditions are maintaine. The genome sequence of Cyanothece 51142 shows the presence of the hup genes for an uptake hydrogenase. The transcripts for one of these genes, hupS, were also detected under H2 producing conditions (Supplementary Fig. 1). Interestingly, the hupS transcripts in Cyanothece 51142 were present under both nitrogen sufficient and nitrogen-fixing conditions, indicating that its expression is independent of nif. The presence of hupS transcripts and the concurrent accumulation of H2 at high rates under aerobic incubation conditions are suggestive of a weak uptake hydrogenase activity in Cyanothece 51142. Such a premise is also supported by the observation that a few wild type Anabaena strains that possess uptake hydrogenase with very low specific activities can also exhibit aerobic H2 production.

The ability to utilize high concentrations of CO2 or glycerol for enhanced H2 production is an added advantage, as both of these carbon sources are abundantly available as industrial waste products, making biohydrogen production by Cyanothece 51142 an attractive option. High CO2 and glycerol provide additional carbon source and the availability of excess carbon acts as a signal for enhanced nitrogenase activity to meet the increase in nitrogen demand in these cyanobacterial cells. The rise in the glycogen level of the cells at the end of the incubation phase in glycerol supplemented cultures could be a result of cellular activities geared towards building the energy reservoirs when an external energy source is readily available.

Decades of research have unveiled various principles underlying biological H2 production. However, achieving significant increases in yield has been a major challenge. Genetic modifications of H2 yielding pathways have resulted in improvements in the production rates compared to the corresponding wild type strains. However, since the H2 production rates in these wild type strains are rather modest, even a twenty-fold increase in yield in the mutant strains is not sufficient to attain high production level. Therefore, our identification of a cyanobacterial strain exhibiting high rates of H2 production under ambient aerobic conditions offers new possibilities in photobiological hydrogen production research. Recent studies have revealed the metabolic flexibility of this cyanobacterium, and demonstrated that its robust circadian rhythm allows N2 fixation and H2 production to occur at reasonably high rates even when grown under continuous light. Prior studies have shown the robustness of other cyanobacterial systems for H2 production over a prolonged period of time, demonstrating the possibility of using high H2 yielding cyanobacterial strains for large scale production. A systems level understanding of this biological phenomenon in Cyanothece 51142 will unravel previously unknown cellular factors and regulatory mechanisms that influence the process so that they can be favorably altered to produce even higher levels of H2 as an energy carrier.

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