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Papers by John Benemann

Applied and Environmental Microbiology, 1979

Thermophilic, nitrogen-fixing, blue-green algae (cyanobacteria) were investigated for use in biop... more Thermophilic, nitrogen-fixing, blue-green algae (cyanobacteria) were investigated for use in biophotolysis. Three strains of Mastigocladus laminosus were tested and were found to be equally effective in biophotolysis as judged by nitrogenase activity. The alga, M. laminosus NZ-86-m, which was chosen for further study, grew well in the temperature range from 35 to 50°C, with optimum growth at 45°C, at which temperature acetylene reduction activity was also greatest. The maximum tolerable temperature was 55°C. Acetylene reduction activity was saturated at a light intensity of 1 × 10 4 ergs cm −2 s −1 . Atmospheric oxygen tension was found to be slightly inhibitory to acetylene reduction of both slowly growing and exponentially growing cultures. Nonsterile continuous cultures, which were conducted to test problems of culture maintenance, could be operated for 2 months without any significant decrease in nitrogenase activity or contamination by other algae. Nitrogen-starved cultures of ...

Applied and Environmental Microbiology, 1984

The mechanism of O 2 protection of nitrogenase in the heterocysts of Anabaena cylindrica was stud... more The mechanism of O 2 protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O 2 inhibition of nitrogenase activity correlated with the O 2 tension of the medium in which heterocyst formation was induced. O 2 resistance also correlated with the apparent K m for acetylene, indicating that O 2 tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O 2 that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O 2 sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O 2 but not in its absence. After 3 ...

Applied and Environmental Microbiology, 1977

Nitrogen-starved cultures of the alga Anabaena cylindrica 629 produced hydrogen and oxygen contin... more Nitrogen-starved cultures of the alga Anabaena cylindrica 629 produced hydrogen and oxygen continuously for 7 to 19 days. Hydrogen production attained a maximum level after 1 to 2 days of starvation and was followed by a slow decline. The maximum rates were 30 ml of H2 evolved per liter of culture per h or 32 mul of H2 per mg of dry weight per h. In 5 to 7 days the rate of H2 evolution by the more productive cultures fell to one-half its maximum value. The addition of 10(-4) to 5 X 10(-4) M ammonium increased the rate of oxygen evolution and the total hydrogen production of the cultures. H2-O2 ratios were 4:1 under conditions of complete nitrogen starvation and about 1.7:1 after the addition of ammonium. Thus, oxygen evolution was affected by the extent of the nitrogen starvation. Thermodynamic efficiencies of converting incident light energy to free energy of hydrogen via algal photosynthesis were 0.4%. Possible factors limiting hydrogen production were decline of reductant supply ...

Biotechnology and Bioengineering, 1979

A chemostat with cell feedback is analyzed for three kinds of limiting nutrient: a substrate diss... more A chemostat with cell feedback is analyzed for three kinds of limiting nutrient: a substrate dissolved in the inflow, a gas bubbled directly into the reactor, and light. The effects of recycle are distinct in each case, because the relationships between hydraulic detention time and nutrient inflow and outflow are different for each type of nutrient. Effluent recycle, in which the recycle stream is more dilute than the reactor, is discussed in terms of cell detention time and nutrient limitation. Results from chemostat cultures of the blue-green alga, Spirulina geitleri, demonstrate cell feedback under light limitation. Maximum productivity is fixed by the incident light intensity. At a particular dilution rate recycling increases or decreases productivity by taking cell density closer or further from the optimum density. Cell recycle with heterogeneous populations can change the outcome of species competition. Selective recycling of one species can reverse this outcome or stabilize coexistence by its selective effect on cell detention time. Experimental results from light-limited mixed cultures of S. geitleri and a Chlorella sp. verify this.

Archives of Microbiology, 1973

Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlat... more Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO2 fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.

Journal of Applied Phycology, 1992

Microalgae feeds are currently used in relatively small amounts in aquaculture, mainly for the pr... more Microalgae feeds are currently used in relatively small amounts in aquaculture, mainly for the production of larvae and juvenile shell- and finfish, as well as for raising the zooplankton required for feeding of juvenile animals. The blue-green algaSpirulina is used in substantial amounts (over 100 t y−1) as a fish and shrimp feed, and even larger markets can be projected if production costs could be reduced. Another potential large-scale application of microalgae is the cultivation ofHaematococcus for the production of the carotenoid astaxanthin, which gives salmon flesh its reddish color. In the long-term microalgae biomass high in lipids (omega-3 fatty acids) may be developed as substitutes for fish oil-based aquaculture feeds. In shrimp ponds the indigenous algal blooms supply a part of the dietary requirements of the animals, but it is difficult to maximize algal productivities. A separate algal production system could feed the shrimps and minimize the need for added feed. Bivalves feed essentially exclusively on marine microalgae throughout their life cycle. The development of cultivation technologies for such microalgae would allow the onshore production of these animals, with greatly improved product quality and safety.

Biotechnology and Bioengineering, 1979

Energy Conversion and Management, 1997

Over ten times more CO2 is fixed by plants into biomass, and annually released by decomposers and... more Over ten times more CO2 is fixed by plants into biomass, and annually released by decomposers and food chains, than is emitted to the atmosphere due to the burning of fossil fuels. Human activity is already directly and indirectly affecting almost half of the terrestrial biological C cycle. Management of even a small fraction of the biological C cycle would make a major contribution to mitigation of this greenhouse gas.Electric power generation is responsible for roughly one third of fossil CO2 emissions. Direct CO2 mitigation processes are those that reduce fossil CO2 emissions from specific power plants. Direct biological CO2 mitigation processes include the cultivation of microalgae on flue-gas or captured CO2, and the cofiring of wood with fossil fuels. Indirect biological processes, such as growing trees for C storage or for fueling dedicated biomass power plants, recapture CO2 that already has entered the atmosphere. Indirect and direct CO2 mitigation processes have the same overall effect in reducing global warming potential.Reducing global CO2 emissions from forest destruction and unsustainable agricultural and land use practices is one of the most cost-effective, and environmentally beneficial actions that can be taken now to arrest global climate change. Another near-term option is to enhance the substitution of fossil fuels with biofuels. Biofuels are a major source of fuel for the power half of mankind. Globally, biofuels could replace a substantial fraction of current fossil fuel usage.Cofiring biomass wastes and residues with coal is one of the lowest-cost, nearest-term options for reducing fossil CO2 emissions at existing power plants. Long-term demonstrations of biomass cofiring are required at full-scale coal-fired power plants to document efficiencies, ash characteristics, biomass preparation and feeding, and other technical issues. Biomass fuel resources for cofiring can be expanded in the near-term through greater recovery of wastes and residues in forestry and agriculture, and in the mid-term through systems that produce biomass specifically for use as fuels (energy crops).

Archives of Microbiology, 1984

The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under ... more 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.

Energy Conversion and Management, 1997

Microalgae present one of the few technologies for the capture and utilization of CO2 emitted by ... more Microalgae present one of the few technologies for the capture and utilization of CO2 emitted by power plants. These microscopic plants would be grown in large open ponds, into which power plant flue gas or pure CO2 (captured from power plants) is sparged, and, after harvesting, the biomass would be converted to a fossil fuel replacement, preferably a high value liquid fuel such as biodiesel. The requirements for large areas of land, favorable climate, and ample water supplies will restrict the potential of this technology. Also, even with rather favorable technical assumptions, the currently projected costs of microalgae-fuels are high, similar to most power plant CO2 capture and disposal options. However, if the technology of microalgae could achieve very high productivities, equivalent to 10% solar energy conversion, and if projected low-cost cultivation, harvesting and processing techniques could be developed, microalgae technology could become a low-cost CO2 mitigation option, particularly if prices for fossil fuels increase in the future. In the nearer-term microalgae CO2 utilization can be integrated with wastewater treatment and reclamation, providing an early application of this technology. Long-term basic and applied R&D are required to develop this technology, as one of the many options that may be required in the future to help preserve our planetary atmosphere and biosphere.

Journal of Applied Phycology, 2000

The production of H2 gas from water and sunlightusing microalgae, `biophotolysis', has been a sub... more The production of H2 gas from water and sunlightusing microalgae, `biophotolysis', has been a subjectof applied research since the early 1970s. A numberof approaches have been investigated, but most provedto have fundamental limitations or requireunpredictable research breakthroughs. Examples areprocesses based on nitrogen-fixing microalgae andthose producing H2 and O2 simultaneously fromwater (`direct biophotolysis'). The most plausibleprocesses for future applied R & D are those whichcouple separate stages of microalgal photosynthesisand fermentations (`indirect biophotolysis'). Theseinvolve fixation of CO2 into storagecarbohydrates followed by their conversion to H2by the reversible hydrogenase, both in dark andpossibly light-driven anaerobic metabolic processes. Based on a preliminary engineering and economicanalysis, biophotolysis processes must achieve closeto an overall 10% solar energy conversion efficiencyto be competitive with alternatives sources ofrenewable H2, such as photovoltaic-electrolysisprocesses. Such high solar conversion efficiencies inphotosynthetic CO2 fixation could be reached bygenetically reducing the number of light harvesting(antenna) chlorophylls and other pigments inmicroalgae. Similarly, greatly increased yields ofH2 from dark fermentation by microalgae could beobtained through application of the techniques ofmetabolic engineering. Another challenge is toscale-up biohydrogen processes with economicallyviable bioreactors.Solar energy driven microalgae processes forbiohydrogen production are potentially large-scale,but also involve long-term and economically high-riskR&D. In the nearer-term, it may be possible tocombine microalgal H2 production with wastewatertreatment.

European Journal of Biochemistry, 1979

The nitrogenase complex was isolated from nitrogen-starved cultures of Anabaena cylindrica. Sodiu... more The nitrogenase complex was isolated from nitrogen-starved cultures of Anabaena cylindrica. Sodium dithionite, photochemically reduced ferredoxin, and NADPH were found to be effective electron donors to nitrogenase in crude extracts whereas hydrogen and pyruvate were not. The K , for acetylene in vivo is ten-fold higher than the K,,, in vitro, whereas this pattern does not hold for the non-heterocystous cyanobacterium, Plectonema boryanum. This indicates that at least one mechanism of oxygen protection in vivo involves a gas diffusion barrier presented by the heterocyst cell wall. The Mo-Fe component was purified to homogeneity. Its molecular weight (220000), subunit composition, isoelectric point (4.8), Mo, Fe, and S2content (2,20 and 20 mol/mol component), and amino acid composition indicate that this component has similar properties to Mo-Fe-containing components isolated from other bacterial sources. The isolated components from A . cylindrica were found to cross-react, to varying degrees, with components isolated from Azotobacter vinelandii, Rhodospirillum rubvum, and P. boryanum.

Planta, 2000

The assembly, organization and function of the photosynthetic apparatus was investigated in the ... more 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 photon use efficiencies and maximal rates of photosynthesis in Dunaliella salina (Chlorophyta... more 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 yellowgreen, 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 (P max ) of ∼ 100 mmol O 2 (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 P max (∼ 300 mmol O 2 (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 P max to ∼ 675 mmol O 2 (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.

Photosynthesis Research, 1998

High-light (HL) grown Dunaliella salina cells exhibit lower pigment content, a highly truncated c... more 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.

This project deals with the development of biomass production technology for planktonic microalga... more This project deals with the development of biomass production technology for planktonic microalgae. A review of this subject details the use of such algae in waste treatment and the problem of harvesting the many types of microscopic algae occurring in ponds. The solution of this problem is deemed to be development of algal species control techniques which allow cultivation of

Applied and Environmental Microbiology, 1979

Thermophilic, nitrogen-fixing, blue-green algae (cyanobacteria) were investigated for use in biop... more Thermophilic, nitrogen-fixing, blue-green algae (cyanobacteria) were investigated for use in biophotolysis. Three strains of Mastigocladus laminosus were tested and were found to be equally effective in biophotolysis as judged by nitrogenase activity. The alga, M. laminosus NZ-86-m, which was chosen for further study, grew well in the temperature range from 35 to 50°C, with optimum growth at 45°C, at which temperature acetylene reduction activity was also greatest. The maximum tolerable temperature was 55°C. Acetylene reduction activity was saturated at a light intensity of 1 × 10 4 ergs cm −2 s −1 . Atmospheric oxygen tension was found to be slightly inhibitory to acetylene reduction of both slowly growing and exponentially growing cultures. Nonsterile continuous cultures, which were conducted to test problems of culture maintenance, could be operated for 2 months without any significant decrease in nitrogenase activity or contamination by other algae. Nitrogen-starved cultures of ...

Applied and Environmental Microbiology, 1984

The mechanism of O 2 protection of nitrogenase in the heterocysts of Anabaena cylindrica was stud... more The mechanism of O 2 protection of nitrogenase in the heterocysts of Anabaena cylindrica was studied in vivo. Resistance to O 2 inhibition of nitrogenase activity correlated with the O 2 tension of the medium in which heterocyst formation was induced. O 2 resistance also correlated with the apparent K m for acetylene, indicating that O 2 tension may influence the development of a gas diffusion barrier in the heterocysts. The role of respiratory activity in protecting nitrogenase from O 2 that diffuses into the heterocyst was studied using inhibitors of carbon metabolism. Reductant limitation induced by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea increased the O 2 sensitivity of in vivo acetylene reduction. Azide, at concentrations (30 mM) sufficient to completely inhibit dark nitrogenase activity (a process dependent on oxidative phosphorylation for its ATP supply), severely inhibited short-term light-dependent acetylene reduction in the presence of O 2 but not in its absence. After 3 ...

Applied and Environmental Microbiology, 1977

Nitrogen-starved cultures of the alga Anabaena cylindrica 629 produced hydrogen and oxygen contin... more Nitrogen-starved cultures of the alga Anabaena cylindrica 629 produced hydrogen and oxygen continuously for 7 to 19 days. Hydrogen production attained a maximum level after 1 to 2 days of starvation and was followed by a slow decline. The maximum rates were 30 ml of H2 evolved per liter of culture per h or 32 mul of H2 per mg of dry weight per h. In 5 to 7 days the rate of H2 evolution by the more productive cultures fell to one-half its maximum value. The addition of 10(-4) to 5 X 10(-4) M ammonium increased the rate of oxygen evolution and the total hydrogen production of the cultures. H2-O2 ratios were 4:1 under conditions of complete nitrogen starvation and about 1.7:1 after the addition of ammonium. Thus, oxygen evolution was affected by the extent of the nitrogen starvation. Thermodynamic efficiencies of converting incident light energy to free energy of hydrogen via algal photosynthesis were 0.4%. Possible factors limiting hydrogen production were decline of reductant supply ...

Biotechnology and Bioengineering, 1979

A chemostat with cell feedback is analyzed for three kinds of limiting nutrient: a substrate diss... more A chemostat with cell feedback is analyzed for three kinds of limiting nutrient: a substrate dissolved in the inflow, a gas bubbled directly into the reactor, and light. The effects of recycle are distinct in each case, because the relationships between hydraulic detention time and nutrient inflow and outflow are different for each type of nutrient. Effluent recycle, in which the recycle stream is more dilute than the reactor, is discussed in terms of cell detention time and nutrient limitation. Results from chemostat cultures of the blue-green alga, Spirulina geitleri, demonstrate cell feedback under light limitation. Maximum productivity is fixed by the incident light intensity. At a particular dilution rate recycling increases or decreases productivity by taking cell density closer or further from the optimum density. Cell recycle with heterogeneous populations can change the outcome of species competition. Selective recycling of one species can reverse this outcome or stabilize coexistence by its selective effect on cell detention time. Experimental results from light-limited mixed cultures of S. geitleri and a Chlorella sp. verify this.

Archives of Microbiology, 1973

Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlat... more Blending Anabaena cylindrica cultures results in a loss of nitrogenase activity which is correlated with the breakage of the filaments at the junctions between heterocysts and vegetative cells. Oxygen inhibition of nitrogen fixation was significant only above atmospheric concentrations. Nitrogen-fixation activities in the dark were up to 50% of those observed in the light and were dependent on oxygen (10 to 20% was optimal). Nitrogenase activity was lost in about 3 h when cells were incubated aerobically in the dark. Re-exposure to light resulted in recovery of nitrogenase activity within 2 h. Blending, oxygen, or dark pre-incubation had similar effects upon cultures grown under air or nitrogen and did not inhibit light-dependent CO2 fixation. We conclude that heterocysts are the sites of nitrogenase activity and propose a model for nitrogen fixation by Anabaena cylindrica.

Journal of Applied Phycology, 1992

Microalgae feeds are currently used in relatively small amounts in aquaculture, mainly for the pr... more Microalgae feeds are currently used in relatively small amounts in aquaculture, mainly for the production of larvae and juvenile shell- and finfish, as well as for raising the zooplankton required for feeding of juvenile animals. The blue-green algaSpirulina is used in substantial amounts (over 100 t y−1) as a fish and shrimp feed, and even larger markets can be projected if production costs could be reduced. Another potential large-scale application of microalgae is the cultivation ofHaematococcus for the production of the carotenoid astaxanthin, which gives salmon flesh its reddish color. In the long-term microalgae biomass high in lipids (omega-3 fatty acids) may be developed as substitutes for fish oil-based aquaculture feeds. In shrimp ponds the indigenous algal blooms supply a part of the dietary requirements of the animals, but it is difficult to maximize algal productivities. A separate algal production system could feed the shrimps and minimize the need for added feed. Bivalves feed essentially exclusively on marine microalgae throughout their life cycle. The development of cultivation technologies for such microalgae would allow the onshore production of these animals, with greatly improved product quality and safety.

Biotechnology and Bioengineering, 1979

Energy Conversion and Management, 1997

Over ten times more CO2 is fixed by plants into biomass, and annually released by decomposers and... more Over ten times more CO2 is fixed by plants into biomass, and annually released by decomposers and food chains, than is emitted to the atmosphere due to the burning of fossil fuels. Human activity is already directly and indirectly affecting almost half of the terrestrial biological C cycle. Management of even a small fraction of the biological C cycle would make a major contribution to mitigation of this greenhouse gas.Electric power generation is responsible for roughly one third of fossil CO2 emissions. Direct CO2 mitigation processes are those that reduce fossil CO2 emissions from specific power plants. Direct biological CO2 mitigation processes include the cultivation of microalgae on flue-gas or captured CO2, and the cofiring of wood with fossil fuels. Indirect biological processes, such as growing trees for C storage or for fueling dedicated biomass power plants, recapture CO2 that already has entered the atmosphere. Indirect and direct CO2 mitigation processes have the same overall effect in reducing global warming potential.Reducing global CO2 emissions from forest destruction and unsustainable agricultural and land use practices is one of the most cost-effective, and environmentally beneficial actions that can be taken now to arrest global climate change. Another near-term option is to enhance the substitution of fossil fuels with biofuels. Biofuels are a major source of fuel for the power half of mankind. Globally, biofuels could replace a substantial fraction of current fossil fuel usage.Cofiring biomass wastes and residues with coal is one of the lowest-cost, nearest-term options for reducing fossil CO2 emissions at existing power plants. Long-term demonstrations of biomass cofiring are required at full-scale coal-fired power plants to document efficiencies, ash characteristics, biomass preparation and feeding, and other technical issues. Biomass fuel resources for cofiring can be expanded in the near-term through greater recovery of wastes and residues in forestry and agriculture, and in the mid-term through systems that produce biomass specifically for use as fuels (energy crops).

Archives of Microbiology, 1984

The question of whether the vegetative cells of Anabaena cylindrica synthesize nitrogenase under ... more 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.

Energy Conversion and Management, 1997

Microalgae present one of the few technologies for the capture and utilization of CO2 emitted by ... more Microalgae present one of the few technologies for the capture and utilization of CO2 emitted by power plants. These microscopic plants would be grown in large open ponds, into which power plant flue gas or pure CO2 (captured from power plants) is sparged, and, after harvesting, the biomass would be converted to a fossil fuel replacement, preferably a high value liquid fuel such as biodiesel. The requirements for large areas of land, favorable climate, and ample water supplies will restrict the potential of this technology. Also, even with rather favorable technical assumptions, the currently projected costs of microalgae-fuels are high, similar to most power plant CO2 capture and disposal options. However, if the technology of microalgae could achieve very high productivities, equivalent to 10% solar energy conversion, and if projected low-cost cultivation, harvesting and processing techniques could be developed, microalgae technology could become a low-cost CO2 mitigation option, particularly if prices for fossil fuels increase in the future. In the nearer-term microalgae CO2 utilization can be integrated with wastewater treatment and reclamation, providing an early application of this technology. Long-term basic and applied R&D are required to develop this technology, as one of the many options that may be required in the future to help preserve our planetary atmosphere and biosphere.

Journal of Applied Phycology, 2000

The production of H2 gas from water and sunlightusing microalgae, `biophotolysis', has been a sub... more The production of H2 gas from water and sunlightusing microalgae, `biophotolysis', has been a subjectof applied research since the early 1970s. A numberof approaches have been investigated, but most provedto have fundamental limitations or requireunpredictable research breakthroughs. Examples areprocesses based on nitrogen-fixing microalgae andthose producing H2 and O2 simultaneously fromwater (`direct biophotolysis'). The most plausibleprocesses for future applied R & D are those whichcouple separate stages of microalgal photosynthesisand fermentations (`indirect biophotolysis'). Theseinvolve fixation of CO2 into storagecarbohydrates followed by their conversion to H2by the reversible hydrogenase, both in dark andpossibly light-driven anaerobic metabolic processes. Based on a preliminary engineering and economicanalysis, biophotolysis processes must achieve closeto an overall 10% solar energy conversion efficiencyto be competitive with alternatives sources ofrenewable H2, such as photovoltaic-electrolysisprocesses. Such high solar conversion efficiencies inphotosynthetic CO2 fixation could be reached bygenetically reducing the number of light harvesting(antenna) chlorophylls and other pigments inmicroalgae. Similarly, greatly increased yields ofH2 from dark fermentation by microalgae could beobtained through application of the techniques ofmetabolic engineering. Another challenge is toscale-up biohydrogen processes with economicallyviable bioreactors.Solar energy driven microalgae processes forbiohydrogen production are potentially large-scale,but also involve long-term and economically high-riskR&D. In the nearer-term, it may be possible tocombine microalgal H2 production with wastewatertreatment.

European Journal of Biochemistry, 1979

The nitrogenase complex was isolated from nitrogen-starved cultures of Anabaena cylindrica. Sodiu... more The nitrogenase complex was isolated from nitrogen-starved cultures of Anabaena cylindrica. Sodium dithionite, photochemically reduced ferredoxin, and NADPH were found to be effective electron donors to nitrogenase in crude extracts whereas hydrogen and pyruvate were not. The K , for acetylene in vivo is ten-fold higher than the K,,, in vitro, whereas this pattern does not hold for the non-heterocystous cyanobacterium, Plectonema boryanum. This indicates that at least one mechanism of oxygen protection in vivo involves a gas diffusion barrier presented by the heterocyst cell wall. The Mo-Fe component was purified to homogeneity. Its molecular weight (220000), subunit composition, isoelectric point (4.8), Mo, Fe, and S2content (2,20 and 20 mol/mol component), and amino acid composition indicate that this component has similar properties to Mo-Fe-containing components isolated from other bacterial sources. The isolated components from A . cylindrica were found to cross-react, to varying degrees, with components isolated from Azotobacter vinelandii, Rhodospirillum rubvum, and P. boryanum.

Planta, 2000

The assembly, organization and function of the photosynthetic apparatus was investigated in the ... more 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 photon use efficiencies and maximal rates of photosynthesis in Dunaliella salina (Chlorophyta... more 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 yellowgreen, 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 (P max ) of ∼ 100 mmol O 2 (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 P max (∼ 300 mmol O 2 (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 P max to ∼ 675 mmol O 2 (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.

Photosynthesis Research, 1998

High-light (HL) grown Dunaliella salina cells exhibit lower pigment content, a highly truncated c... more 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.

This project deals with the development of biomass production technology for planktonic microalga... more This project deals with the development of biomass production technology for planktonic microalgae. A review of this subject details the use of such algae in waste treatment and the problem of harvesting the many types of microscopic algae occurring in ponds. The solution of this problem is deemed to be development of algal species control techniques which allow cultivation of