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PNNL team presents new insight into H2 production by cyanobacterium Cyanothece

Researchers at the US Department of Energy’s (DOE’s) Pacific Northwest National Laboratory (PNNL) have presented a new and more complete view on the way a cyanobacterium—Cyanothece 51142—produces hydrogen.

Using genome-scale transcript and protein profiling, the team study presented and tested a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. The results, reported in an open-access paper in Nature’s Scientific Reports, show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production.

Photobiological H2 production is still a nascent technology with long-term potential for sustainable energy production with a low environmental impact. Although direct biophotolytic H2 production has been documented and studied for decades, significant challenges remain for the development of microbial strains and conditions that can directly and efficiently use sunlight and water to produce H2. Chief among them is the low production rate, which is largely due to feedback inhibition of the H2 producing enzymes by O2, an obligate byproduct of oxygenic photosynthesis. Limitations imposed by O2 sensitivity of the native hydrogenase and nitrogenase enzymes have motivated significant efforts to identify and even engineer O2 tolerant variants and multi-stage processes that temporally separate O2 and H2 evolution. However, to date, the kinetic rates and sustainability of hydrogenase-mediated H2 production are low in comparison to those reported for some diazotrophic organisms that produce H2 in oxic-environments as a byproduct of nitrogenase catalyzed N2 fixation.

Nitrogen-fixing cyanobacteria have been recognized as one of the most promising photolytic platforms for sustainable H2 production. A unicellular marine strain Cyanothece sp. ATCC 51142 (hereafter Cyanothece 51142) has emerged as a model system because of its ability to produce H2 at rates > 100 μmol-H2 hr−1 mg-Chl−1 under photosynthetic conditions associated with continuous illumination. … The current and prevailing view assumes that H2 production mediated by energetically expensive nitrogenase activity in Cyanothece 51142, and other closely related strains, is exclusively supported by ATP and reductant derived from oxidation of intracellular glycogen and/or cyclic-electron flow around photosystem (PS) I. Here we present evidence to support a new model whereby energy derived directly from oxygenic photosynthesis (i.e., linear electron flow through PS II) is an important process in funding the energy budget required for nitrogenase activity under illuminated, nitrogen-deplete conditions.

—Bernstein et al.

PNNL scientists found that the organism taps into an unexpected source of energy to create hydrogen. Researchers have known that 51142 makes hydrogen by drawing upon sugars that it has stored during growth. In this study, PNNL researchers found that the organism also draws on a second source of energy, using sunlight and water directly to make hydrogen.

Organisms such as cyanobacteria made life on the planet possible by producing the oxygen for the atmosphere 2.3 billion years ago. They also convert the abundant nitrogen in the atmosphere to a form that is essential for all plant life on the planet.

Many of these organisms are equipped with an enzyme called nitrogenase to convert inert atmospheric nitrogen to more usable forms for plants and other organisms. For a long time, scientists have known that nitrogenase produces small quantities of molecular hydrogen as a byproduct. When nitrogen is not available, the organism produces hydrogen.

The team set up Cyanothece 51142 in a bioreactor, limited the supply of nitrogen, and kept the lights on 24 hours a day for several weeks. The team used an array of high-tech equipment to yield sophisticated minute-by-minute profiles of the organism as it converted light energy to hydrogen. Scientists conducted many of their analyses using capabilities at EMSL, the Environmental Molecular Sciences Laboratory, a DOE user facility at PNNL, to “interrogate” the genes and proteins of the organism as they changed while the reactions occurred.

The team conducted a “multi-omics experiment,” studying the genomics, transcriptomics and proteomics of the organism's activity, as well as its reaction kinetics. The scientists scrutinized 5,303 genes and 1,360 proteins at eight separate times over the course of 48 hours as the bacteria, with limited nitrogen supply, switched on the activity of the nitrogenase protein.

The scientists found that in addition to drawing upon its previously stored energy, the organism captures light and uses that energy to split water to create hydrogen in real time. As one component of the organism is creating energy by collecting light energy, another part is using that energy simultaneously to create hydrogen.

This organism can make lots of hydrogen, very fast; it’s a viable catalyst for hydrogen production. The enzyme that makes the hydrogen needs a huge amount of energy. The real question is, what funds the energy budget for this important enzyme and then, how can we design and control it to create renewable fuels and to advance biotechnology?

—first author Hans Bernstein, a Linus Pauling distinguished postdoctoral fellow at PNNL

In a paper published in 2012 in mBio, Alex Beliaev, one of two scientists at the Department of Energy's Pacific Northwest National Laboratory who led the research, and colleagues raised questions about how the microbe drew upon the energy required to produce hydrogen. In the new paper, the molecular signals the team studied show that photosynthesis and the hydrogen production by nitrogenase happen hand in hand in a coordinated manner.

The team includes 11 researchers from PNNL. Beliaev began the project seven years ago as part of hydrogen production research related to biofuels, and Bernstein picked it up when he joined PNNL two years ago.

The work was funded by the Department of Energy Office of Science (Biological and Environmental Research) and by PNNL's Laboratory Directed Research and Development Program, which funds the Linus Pauling Distinguished Postdoctoral Fellowship Program.


  • Hans C. Bernstein, Moiz A. Charania, Ryan S. McClure, Natalie C. Sadler, Matthew R. Melnicki, Eric A. Hill, Lye Meng Markillie, Carrie D. Nicora, Aaron T. Wright, Margaret F. Romine and Alexander S. Beliaev (2015) “Multi-omic dynamics associate oxygenic photosynthesis with nitrogenase-mediated H2 production in Cyanothece sp. ATCC 51142,” Scientific Reports doi: 10.1038/srep16004


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