John R. Benemann

Biological hydrogen production; fundamentals and limiting processes

Abstract Biological hydrogen production has been known for over a century and research directed at applying this process to a practical means of hydrogen fuel production has been carried out for over a quarter century. The various approaches that have been proposed and investigated are reviewed and critical limiting factors identified. The low energy content of solar irradiation dictates that photosynthetic processes operate at high conversion efficiencies and places severe restrictions on photobioreactor economics. Conversion efficiencies for direct biophotolysis are below 1% and indirect biophotolysis remains to be demonstrated. Dark fermentation of biomass or wastes presents an alternative route to biological hydrogen production that has been little studied. In this case the critical factor is the amount of hydrogen that can be produced per mole of substrate. Known pathways and experimental evidence indicates that at most 2– 3 mol of hydrogen can be obtained from substrates such as glucose. Process economics require that means be sought to increase these yields.

Dunaliella salina (Chlorophyta) with small chlorophyll antenna sizes exhibit higher photosynthetic productivities and photon use efficiencies than normally pigmented cells

The photon use efficiencies and maximal rates of photosynthesis in Dunaliella salina (Chlorophyta) cultures acclimated to different light intensities were investigated. Batch cultures were grown to the mid-exponential phase under continuous low-light (LL: 100 μmol photon m-2 s-1) or high-light (HL: 2000 μmol photon m-2 s-1) conditions. Under LL, cells were normally pigmented (deep green) containing ∼500 chlorophyll (Chl) molecules per photosystem II (PSII) unit and ∼250 Chl molecules per photosystem I (PSI). HL-grown cells were yellow-green, contained only 60 Chl per PSII and 100 Chl per PSI and showed signs of chronic photoinhibition, i.e., accumulation of photodamaged PSII reaction centers in the chloroplast thylakoids. In LL-grown cells, photosynthesis saturated at ∼200 μmol photon m-2 s-1 with a rate (Pmax) of ∼100 mmol O2 (mol Chl)-1 s-1. In HL-grown cells, photosynthesis saturated at much higher light intensities, i.e. ∼2500 μmol photon m-2 s-1, and exhibited a three-fold higher Pmax (∼300 mmol O2 (mol Chl)-1 s-1) than the normally pigmented LL-grown cells. Recovery of the HL-grown cells from photoinhibition, occurring prior to a light-harvesting Chl antenna size increase, enhanced Pmax to ∼675 mmol O2 (mol Chl)-1 s-1. Extrapolation of these results to outdoor mass culture conditions suggested that algal strains with small Chl antenna size could exhibit 2–3 times higher productivities than currently achieved with normally pigmented cells.

Photosystem-II repair and chloroplast recovery from irradiance stress: relationship between chronic photoinhibition, light-harvesting chlorophyll antenna size and photosynthetic productivity in Dunaliella salina (green algae)

High-light (HL) grown Dunaliella salina cells exhibit lower pigment content, a highly truncated chlorophyll (Chl) antenna size, and accumulation of photodamaged PS II centers in the chloroplast thylakoids (chronic photoinhibition). In HL-grown cells, the rate of photosynthesis saturated at higher irradiances and the quantum yield was lower compared to that of normally-pigmented low-light (LL) grown cells. In spite of these deficiencies, the light-saturated rate of photosynthesis for the HL-cells, when measured on a per chlorophyll basis, was ∼3 times greater than that of the LL-grown cells. To delineate the effect of photoinhibition from the Chl antenna size on quantum yield and rate of photosynthesis, HL-acclimated cells were switched to LL-conditions. Repair of photodamaged PS II, estimated from the recovery of functional PS II centers and from the increase in the quantum yield of photosynthesis, occurred with a half-time of ∼1 h. Chlorophyll accumulation in the cells occurred with a half-time of ∼4 h. The differential kinetics in repair versus Chl accumulation provided a ‘window of opportunity’, within about 2–3 h after the HL→LL shift, when cells exhibited a high quantum yield of photosynthesis, a small Chl antenna size and a light-saturated rate that was ∼6–9 times greater than that of the normally pigmented LL-grown cells. The work provides insight on the temporal sequence of events at the chloroplast and thylakoid membrane levels, leading from a chronic photoinhibition of PS II to repair and recovery. It is suggested that it is possible to maximize photosynthetic productivity and light utilization in mass microalgal cultures by minimizing the light-harvesting Chl antenna size of the photosystems.

Photosynthetic apparatus organization and function in the wild type and a chlorophyll b-less mutant of Chlamydomonas reinhardtii. Dependence on carbon source

Abstract. The assembly, organization and function of the photosynthetic apparatus was investigated in the wild type and a chlorophyll (Chl) b-less mutant of the unicellular green alga Chlamydomonas reinhardtii, generated via DNA insertional mutagenesis. Comparative analyses were undertaken with cells grown photoheterotrophically (acetate), photomixotrophically (acetate and HCO−3) or photoautotrophically (HCO−3). It is shown that lack of Chl b diminished the photosystem-II (PSII) functional Chl antenna size from 320 Chl (a and b) to about 95 Chl a molecules. However, the functional Chl antenna size of PSI remained fairly constant at about 290 Chl molecules, independent of the presence of Chl b. Western blot and kinetic analyses suggested the presence of inner subunits of the Chl a-b light-harvesting complex of PSII (LHCII) and the entire complement of the Chl a-b light-harvesting complex of PSI (LHCI) in the mutant. It is concluded that Chl a can replace Chl b in the inner subunits of the LHCII and in the entire complement of the LHCI. Growth of cells on acetate as the sole carbon source imposes limitations in the photon-use efficiency and capacity of photosynthesis. These are manifested as a lower quantum yield and lower light-saturated rate of photosynthesis, and as lower variable to maximal (Fv/Fmax) chlorophyll fluorescence yield ratios. This adverse effect probably originates because acetate shifts the oxidation-reduction state of the plastoquinone pool, and also because it causes a decrease in the amount and/or activity of Rubisco in the chloroplast. Such limitations are fully alleviated upon inclusion of an inorganic carbon source (e.g. bicarbonate) in the cell growth medium. Further, the work provides evidence to show that transformation of green algae can be used as a tool by which to generate mutants exhibiting a permanently truncated Chl antenna size and a higher (per Chl) photosynthetic productivity of the cells.

The electron transport system in nitrogen fixation by Azotobacter. III. Requirements for NADPH-supported nitrogenase activity

Abstract Evidence has been obtained that NADPH may serve as a physiological source of reducing power for nitrogenase activity in Azotobacter vinelandii . NADH was ineffective. Electron transfer from NADPH to nitrogenase depended on four factors native to A. vinelandii cells: azotobacter ferredoxin, azotoflavin, a component replaceable by spinach ferredoxin-NADP + reductase and another soluble, heat-labile component not yet chemically characterized. The four factors probably constitute an electron transport chain between NADPH and nitrogenase.

Bioengineering aspects of biophotolysis

Abstract The practical aspects of producing hydrogen by photosynthetic microorganisms are reviewed. Various alternative concepts for hydrogen production are discussed, both single and two-stage systems. The best developed process currently is based on nitrogen-fixing heterocystous blue-green algae which can produce hydrogen and oxygen simultaneously. Solar energy conversion to hydrogen efficiencies of 0.2% averaged over several weeks have been obtained with outdoor systems. Practical systems would require a ten-fold increase in conversion efficiencies. Also, systems which produce pure hydrogen are preferred. Photosynthetic bacteria are of near-term applications. A general design for a biophotolysis system is proposed consisting of vertically arranged, thin-walled glass tubes with an inert gas recirculated through the cultures for mixing and removal of hydrogen. Gas mass transfer considerations, energy utilization, and economics favour such a system.

Production of nitrogen fertilizer with nitrogen-fixing blue - green algae

Abstract Heterocystous nitrogen-fixing blue-green algae consist of filaments containing two types of cells: the heterocysts, responsible for ammonia synthesis, and vegetative cells, which exhibit normal photosynthesis and reproductive growth. This unique biological system could be used for the conversion of solar energy into organic fertilizer, through cultivation of these algae in open ponds. The most immediately practical approach is the use of this process in conjunction with waste-water treatment. Initial experiments have involved the isolation of sewage effluents-adapted algae and their cultivation in small-scale ponds. Significant rates of biomass production and nitrogen fixation were achieved, but a substantial improvement is still needed for possible practical applications. The potential economics of such systems and the need for new sources of fertilizers are discussed.

Immunochemical evidence that nitrogenase is restricted to the heterocysts in Anabaena cylindrica

The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under anaerobic conditions was studied by immunoferritin labelling of the Fe-Mo protein (Component I). Differentiating cultures, incubated under an argon atmosphere, were treated with DCMU 12 h following initiation of induction. DCMU inhibited photosynthetic O2 production, thus insuring strict anaerobic conditions, but had no effect on nitrogenase induction. Fe-Mo protein levels, as determined by rocket immunoelectrophoresis, increased 5-fold within 24h of DCMU treatment. Immunoferritin labelling of aldehyde fixed, ultrathin cryosections of anaerobically induced filaments showed that the Fe-Mo protein was restricted to the heterocyst. Ferritin labelling was shown to be specific by the following criteria: (a) substituting preimmune goat serum for the anti-Fe-Mo protein IgG prevented ferritin labelling; (b) ferritin-conjugated, non-homologous rabbit anti-goat IgG did not bind; (c) incubation of anti-Fe-Mo protein IgG treated sections with rabbit anti-goat IgG prior to the treatment with the ferritin label also prevented labelling. The results provide direct immunochemical evidence that nitrogenase is restricted to the heterocysts even under strictly anaerobic conditions.

Oxidation of diaminobenzidine in the heterocysts ofAnabaena cylindrica

Hemoproteins were localized in the cyanobacteriumAnabaena cylindrica with diaminobenzidine (DAB). Incubation of whole cells in the light with DAB resulted in deposition of oxidized DAB on the lamellae of the vegetative cells and central heterocyst region. This reaction was greatest at pH 7.5, light-dependent, insensitive to 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea, and abolished by glutaraldehyde fixation. A light-independent oxidation of DAB was also observed with light and electron microscopy in the “honeycomb” region and periphery of heterocysts. This reaction was greatest at pH 7.5, enhanced by H2O2, and active in glutaraldehyde-fixed frozen sections. Inhibitors such as sodium cyanide, sulfide, and hydroxylamine severely reduced DAB oxidation and nitrogenase activity under aerobic but not anaerobic conditions. These results indicate that the heme proteins, localized in heterocysts by light-independent DAB oxidation, are involved in the oxygen-protection mechanism of the O2-labile nitrogenase.

Maximizing photosynthetic efficiencies and hydrogen production in microalga cultures

Publisher Summary This chapter illustrates that photosynthetic efficiencies and hydrogen production by microalgal cultures may be increased upon minimizing the number of the light-harvesting chlorophyll (Chl) antenna pigments of photosynthesis. This research employs a mutagenesis approach, based on the random insertion of tagged DNA into C. reinhardtii cells, by which to impair the Chl antenna size regulation mechanism. This procedure, along with the stringent screening employed may help to unlock the “black box” of the developmental regulation of the Chl antenna size in microalgae. Thus, it is expected that mutants with a permanently truncated Chl antenna size, as well as mutants with a permanently large Chl antenna, are isolated. The advantage of this molecular genetic approach is that it leads to the identification of genes responsible for the operation of this highly conserved regulatory mechanism.