Hajime Masukawa

Promoting R & D in Photobiological Hydrogen Production Utilizing Mariculture-Raised Cyanobacteria

This review article explores the potential of using mariculture-raised cyanobacteria as solar energy converters of hydrogen (H2). The exploitation of the sea surface for large-scale renewable energy production and the reasons for selecting the economical, nitrogenase-based systems of cyanobacteria for H2 production, are described in terms of societal benefits. Reports of cyanobacterial photobiological H2 production are summarized with respect to specific activity, efficiency of solar energy conversion, and maximum H2 concentration attainable. The need for further improvements in biological parameters such as low-light saturation properties, sustainability of H2 production, and so forth, and the means to overcome these difficulties through the identification of promising wild-type strains followed by optimization of the selected strains using genetic engineering are also discussed. Finally, a possible mechanism for the development of economical large-scale mariculture operations in conjunction with international cooperation and social acceptance is outlined.

Hydrogenases and photobiological hydrogen production utilizing nitrogenase system in cyanobacteria

Abstract We constructed three hydrogenase mutants from Anabaena sp. PCC 7120: ΔhupL (deficient in uptake hydrogenase), ΔhoxH (deficient in bidirectional hydrogenase), and ΔhupL/ΔhoxH (deficient in both genes), and showed that the Δhup and ΔhupL/ΔhoxH produced H2 at a rate 4–7 times that of wild-type under optimal conditions (Appl. Microbiol. Biotechnol. 58 (2002) 618). We have studied H2 producing activity of Δhup in more detail. H2 producing activity of Δhup cells was moderately improved in older cultures when 1% CO2 was added to the bubbling air. The efficiency of light energy conversion to H2 by the ΔhupL mutant at its highest H2 production stage was 1.0–1.6% at an actinic visible light intensity of lower than 50 W / m 2 under argon atmosphere, and the activity lasted for at least 35 min . At 250 W / m 2 , H2 producing activity gradually decreased with illumination time.

The cyanobacterium Anabaena sp. PCC 7120 has two distinct β-carotene ketolases: CrtO for echinenone and CrtW for ketomyxol synthesis

Two β‐carotene ketolases, CrtW and CrtO, are widely distributed in bacteria, although they show no significant sequence homology with each other. The cyanobacterium Anabaena sp. PCC 7120 was found to have two homologous genes. In the crtW deleted mutant, myxol 2′‐fucoside was present, but ketomyxol 2′‐fucoside was absent. In the crtO deleted mutant, β‐carotene was accumulated, and the amount of echinenone was decreased. Therefore, CrtW catalyzed myxol 2′‐fucoside to ketomyxol 2′‐fucoside, and CrtO catalyzed β‐carotene to echinenone. This cyanobacterium was the first species found to have both enzymes, which functioned in two distinct biosynthetic pathways.

Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

ABSTRACT Cyanobacteria use sunlight and water to produce hydrogen gas (H2), which is potentially useful as a clean and renewable biofuel. Photobiological H2 arises primarily as an inevitable by-product of N2 fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H2 production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N2. Most of the 49 variants examined were deficient in N2-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N2 atmosphere significantly increased their in vivo rates of H2 production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H2 compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H2 in an aerobic, nitrogen-containing environment.

Photobiological hydrogen production and nitrogenase activity in some heterocystous cyanobacteria

Publisher Summary This chapter discusses the activities of nitrogenase and H2 evolution in three cyanobacterial strains and some factors that affected these activities. Nitrogen-fixing heterocystous cyanobacteria are potential candidates for the development of photobiological hydrogen production systems. They produce H2 under aerobic conditions using water as an electron donor. For example A. variabilis IAM M-58 was most active in H2 production, and the amount of H2 produced was markedly higher than that of the other species. Hydrogen metabolism in these cyanobacteria involves at least three enzymes: nitrogenase, uptake hydrogenase, and bidirectional hydrogenase. Some researchers favor hydrogenase over nitrogenase as the hydrogen evolving system because of its high energy efficiency, but it was reported that continued production of H2 in air was mediated by nitrogenase in the heterocysts. To develop the hydrogen producing systems by cyanobacteria based on nitrogenase activity, it is important to find cyanobacterial strains which have a high activity of H2 production.

Effects of Disruption of Homocitrate Synthase Genes on Nostoc sp. Strain PCC 7120 Photobiological Hydrogen Production and Nitrogenase

ABSTRACT In the case of nitrogenase-based photobiological hydrogen production systems of cyanobacteria, the inactivation of uptake hydrogenase (Hup) leads to significant increases in hydrogen production activity. However, the high-level-activity stage of the Hup mutants lasts only a few tens of hours under air, a circumstance which seems to be caused by sufficient amounts of combined nitrogen supplied by active nitrogenase. The catalytic FeMo cofactor of nitrogenase binds homocitrate, which is required for efficient nitrogen fixation. It was reported previously that the nitrogenase from the homocitrate synthase gene (nifV) disruption mutant of Klebsiella pneumoniae shows decreased nitrogen fixation activity and increased hydrogen production activity under N2. The cyanobacterium Nostoc sp. strain PCC 7120 has two homocitrate synthase genes, nifV1 and nifV2, and with the ΔhupL variant of Nostoc sp. strain PCC 7120 as the parental strain, we have constructed two single mutants, the ΔhupL ΔnifV1 strain (with the hupL and nifV1 genes disrupted) and the ΔhupL ΔnifV2 strain, and a double mutant, the ΔhupL ΔnifV1 ΔnifV2 strain. Diazotrophic growth rates of the two nifV single mutants and the double mutant were decreased moderately and severely, respectively, compared with the rates of the parent ΔhupL strain. The hydrogen production activity of the ΔhupL ΔnifV1 mutant was sustained at higher levels than the activity of the parent ΔhupL strain after about 2 days of combined-nitrogen step down, and the activity in the culture of the former became higher than that in the culture of the latter. The presence of N2 gas inhibited hydrogen production in the ΔhupL ΔnifV1 ΔnifV2 mutant less strongly than in the parent ΔhupL strain and the ΔhupL ΔnifV1 and ΔhupL ΔnifV2 mutants. The alteration of homocitrate synthase activity can be a useful strategy for improving sustained photobiological hydrogen production in cyanobacteria.

Genetic Engineering of Cyanobacteria to Enhance Biohydrogen Production from Sunlight and Water

To mitigate global warming caused by burning fossil fuels, a renewable energy source available in large quantity is urgently required. We are proposing large-scale photobiological H2 production by mariculture-raised cyanobacteria where the microbes capture part of the huge amount of solar energy received on earth’s surface and use water as the source of electrons to reduce protons. The H2 production system is based on photosynthetic and nitrogenase activities of cyanobacteria, using uptake hydrogenase mutants that can accumulate H2 for extended periods even in the presence of evolved O2. This review summarizes our efforts to improve the rate of photobiological H2 production through genetic engineering. The challenges yet to be overcome to further increase the conversion efficiency of solar energy to H2 also are discussed.

How Close We Are to Achieving Commercially Viable Large-Scale Photobiological Hydrogen Production by Cyanobacteria: A Review of the Biological Aspects

Photobiological production of H2 by cyanobacteria is considered to be an ideal source of renewable energy because the inputs, water and sunlight, are abundant. The products of photobiological systems are H2 and O2; the H2 can be used as the energy source of fuel cells, etc., which generate electricity at high efficiencies and minimal pollution, as the waste product is H2O. Overall, production of commercially viable algal fuels in any form, including biomass and biodiesel, is challenging, and the very few systems that are operational have yet to be evaluated. In this paper we will: briefly review some of the necessary conditions for economical production, summarize the reports of photobiological H2 production by cyanobacteria, present our schemes for future production, and discuss the necessity for further progress in the research needed to achieve commercially viable large-scale H2 production.

Survey of the Distribution of Different Types of Nitrogenases and Hydrogenases in Heterocyst-Forming Cyanobactera

As a first step toward developing the methodology for screening large numbers of heterocyst-forming freshwater cyanobacteria strains for the presence of various types of nitrogenases and hydrogenases, we surveyed the distribution of these genes and their activities in 14 strains from culture collections. The nitrogenase genes include nif1 encoding a Mo-type nitrogenase expressed in heterocysts, nif2 expressed in vegetative cells and heterocysts under anaerobic conditions, and vnf encoding a V-type nitrogenase expressed in heterocysts. Two methods proved to be valuable in surveying the distribution of nitrogenase types. The first method was Southern blot hybridization of DNA digested with two different endonucleases and hybridized with nifD1, nifD2, and vnfD probes. The second method was ethane formation from acetylene to detect the presence of active V-nitrogenase. We found that all 14 strains have nifD1 genes, and eight strains also have nifD2 genes. Four of the strains have vnfD genes, in addition to nifD2 genes. It is curious that three of these four strains had similar hybridization patterns with all of the nifD1, nifD2, and vnfD probes, suggesting that there could be some bias in strains used in the present study or in strains held in culture collections. This point will need to be assessed in the future. For surveying the distribution of hydrogenases, Southern blot hybridization was an effective method. All strains surveyed had hup genes, with the majority of them also having hox genes.

A feasibility study of large-scale photobiological hydrogen production utilizing mariculture-raised cyanobacteria.

In order to decrease CO(2) emissions from the burning of fossil fuels, the development of new renewable energy sources sufficiently large in quantity is essential. To meet this need, we propose large-scale H(2) production on the sea surface utilizing cyanobacteria. Although many of the relevant technologies are in the early stage of development, this chapter briefly examines the feasibility of such H(2) production, in order to illustrate that under certain conditions large-scale photobiological H(2) production can be viable. Assuming that solar energy is converted to H(2) at 1.2% efficiency, the future cost of H(2) can be estimated to be about 11 (pipelines) and 26.4 (compression and marine transportation) cents kWh(-1), respectively.