Algal Biohydrogen Production From Water

Certain photosynthetic microbes, including algae and cyanobacteria, can produce H2 from the world's most plentiful resources in the following reactions:

2H2O + light energy -> O2 + 4H+ + 4e- -> O2 + 2H2.

The first recorded observation of this was reported over a hundred and ten years ago, when a natural bloom of Anabaena after being placed in a glass jar, started to produce H2. Hydrogen uptake and production were the first reports of H2 metabolism in a green alga (Scenedesmus obliquus) in the early 1940's.

Two distinct light-driven, H2-photoproduction pathways have been described in green algae, and there is evidence for a third, light-independent, fermentative H2 pathway coupled to starch degradation. All pathways have the reduction of ferredoxin (FD, Figure 10) in common as the primary electron-donor to a hydrogenase. Hydrogenases are enzymes that can reduce protons and release molecular H2. The major types of hydrogenases contain either iron ([FeFe]-hydrogenases, which generally are H2-evolving) or both nickel and iron ([NiFe]-hydrogenases, which are generally H2-uptake enzymes) in their active sites. Plants do not contain hydrogenase genes. (Additional information about these O2-sensitive enzymes can be found below.) The light-driven pathways can either use water as the substrate (employing both photosystems II and I) or NADH from the glycolytic breakdown of stored carbohydrate (employing only photosystem I) to product H2. Rather than utilizing the light-driven reduction of FD, the dark, fermentative pathway may also involve a pyruvate-ferredoxin-oxidoreductase (PFOR) enzyme, similar to those found in many anaerobic systems. Although PFOR-catalyzed pyruvate oxidation/FD reduction in the C. reinhardtii fermentative H2-production pathway is not proven absolutely under dark, H2-producing conditions, a PFOR gene is up-regulated in C. reinhardtii. This suggests that PFOR might provide the link between fermentative carbon dissimilation and H2 production. Up until the beginning of the 21st century, prospects for using algae to produce H2 as a fuel (in actuality H2 is classified as an energy carrier since it is not found naturally in the environment, and must be produced) were academic, despite a number of reports on the production of H2 gas using the nitrogenase function of cyanobacteria (prokaryotic, blue-green algae). This latter work, however, waned in the 1990's with the realization of limitations in the ultimate projected light conversion efficiencies obtainable by this approach.

images/Fig10.png

Figure 10. Algae exhibit 3 different pathways for H2 production. Two are driven by light and the third occurs in the dark. Either water or starch can be the electron donor. Carbon is fixed under normal photosynthesis with water as the donor, but the electron acceptor is switched at the level of ferredoxin (FD) from carbon dioxide to protons under conditions that lead to H2 production. (thanks to Prof. M. Posewitz, Colorado School of Mines for the drawing)

In 2000, a team at NREL and the University of California, Berkeley, reported that a relatively large amount of H2 could be produced for up to 4 days by the green alga, C. reinhardtii, after imposition of sulfur-deprivation stress. This technical advance rekindled interest around the world in H2-producing phototrophs and [FeFe]-hydrogenases. Since that time, the mechanism explaining the phenomenon of sulfur stress has been elucidated; and it involves the co-occurrence of aerobic photosynthesis, anaerobic fermentation, and respiration. Specifically, since hydrogenase enzymes (at least non-H2-sensing hydrogenases) are deactivated by O2 and since algae produce O2 as a by-product of photosynthesis, it is not possible at this point for them to produce H2 for long when O2 is present. However, when sulfate is removed from the medium of a mature algal culture, photosystem II (PSII) activity (see Figure 10), and consequently O2-evolution capacity in the algae, decreases because the PSII repair process is affected. When the O2-evolution rate in the culture falls below the O2-uptake rate of respiration, hydrogenase synthesis and subsequent enzyme activation occurs, and H2 starts to bubble out of the culture shortly thereafter (Figure 11). While this works in that a volumetric amount of H2 can be produced, a big price is paid with respect to the maximum energy conversion efficiency of the process. In order to evolve H2 in this manner, one has to sacrifice over 90% of the maximum rate of photosynthesis. Nevertheless, recent improvements in the rates of H2 production and the time over which H2 can be produced have been reported both in wild-type and mutant organisms. Currently, algal suspension cultures can produce up to one volume of H2 per two volumes of culture over a period of about 4 days. Furthermore, low light level, laboratory-scale energy conversion efficiencies in terms of light energy stored as H2 have surpassed 1% in wild-type C. reinhardtii cultures immobilized in alginate films (which can partially protect the O2-sensitve hydrogenase enzyme during H2 production). Furthermore, algae immobilized on glass fibers have produced H2 continuously for up to 90 days. Recent reports have also demonstrated that certain algal mutants exhibit increased rates of H2 production compared to their parental strains. Since all of these advances have been reported in different bioreactor systems, the challenge will be to demonstrate all of these improvements in one system and then to further improve the biology of that system.

images/Fig11.png

Figure 11. Laboratory scale photobioreactors located in the author's laboratory at NREL, which have been used to monitor a number of different physiological parameters in sulfur-deprived Chlamydomonas reinhardtii, a green soil alga. This was the organism of choice since more is know about this species than any other strain of green alga, and a sequenced genome is available. In this simple demonstration, H2 is being collected in an inverted graduate cylinder (upper center right) by the displacement of water. [PIX 08741]

Four biological challenges limiting biohydrogen production in algae have been identified as,

  1. the O2 sensitivity of hydrogenases,
  2. competition for photosynthetic reductant at the level of ferredoxin,
  3. regulatory issues associated with the over production of ATP, and
  4. inefficiencies in the maximum utilization of solar light energy.

Many laboratories around the world are addressing these challenges by,

  1. engineering hydrogenases to improve the enzyme's tolerance to the presence of O2,
  2. identifying metabolic pathways that compete with hydrogenases for photosynthetic reductant by genomics approaches, and engineering their down-regulation during H2 production,
  3. engineering the photosynthetic membrane to significantly decrease the efficiency of photosynthetic-electron-transport-coupled ATP production (not depicted in Figure 10; ATP is required for carbon fixation, but not for hydrogenase-coupled H2 production), and
  4. engineering the photosynthetic antenna pigment content to maximize the amount of solar light that can be used effectively in a photobioreactor.

If all of the research challenges can be over come, H2-cost projections developed by the US Department of Energy suggest that biohydrogen could compete with gasoline at about $2.50 a kg (a gallon of gasoline contains the energy equivalent of about a kg of H2).

Recently, researchers have begun to re-examining the prospects for using cyanobacteria to produce H2. These studies are making use of bidirectional, [NiFe]-hydrogenases that are found in some of these organisms. While many of the same challenges identified in eukaryotic algae are also inherent in cyanobacteria, the advantages of working with these prokaryotic organisms are that they are more easily engineered than eukaryotic algae, and have more O2-tolerant hydrogenases. On the other hand, the [FeFe]-hydrogenases, found in algae, are better catalysts than the [NiFe]-hydrogenases found in cyanobacteria.

All H2-producing systems face the same non-biological challenge of efficient storage capability. While H2 has the highest energy density in terms of weight of any fuel, it compares poorly with liquid fuels on volumetric energy density. There are many projects in the US exploring the use of chemical-hydride, metal-hydride, and sorption technologies to enhance the ability to store H2 for transportation applications, where the size of the fuel tank is critical. Notably, a recent Lawrence Livermore National Laboratory report of a high-pressure, cryogenic storage, proof-of-concept technology that can run a hybrid-electric vehicle for up to 650 miles on a tank of H2 (it can fit into the back of the vehicle) is encouraging.

Other areas of investigation for the future that researchers are starting to examine, include the application of biological knowledge of photosynthesis and hydrogenase structure/function to develope biohybrid systems (those employing biological and synthetic components), and ultimately, totally artificial photosynthetic systems that mimic the fuel-producing processes of photosynthetic organisms.