Improved photosynthetic characteristics of Chlorella mutant MS700 induced by nuclear radiation

Chlorophyll a variable fluorescence and oxygen evolution were measured in microalgae Chlorella and its mutant MS700, which was mutated by nuclear radiation with an increased biomass yield. The mutant MS700 showed an improvement in photosynthetic characteristics under various CO₂ concentrations. Results showed that chlorophyll-based flash oxygen yield was 104% higher in MS700 than in the wild type, indicating more available electron acceptors in the plastoquinone pool and a higher light to chemical energy conversion efficiency. The higher chlorophyll a variable fluorescence at the end of PQ pool-regulated phase implied a more robust mechanism for consumption of electrons generated in PSII, indicating a more efficient downstream process. Monitoring of CO₂ fixation in the Calvin cycle showed that the mutant had a 27.7% higher capability of incorporating its dissolved inorganic carbon. The oxygen evolution rate was 31.4% higher in MS700, which also indicated a higher CO₂ fixation rate. These results consistently demonstrated carbon fixation efficiency in the mutant MS700, resulting in the higher biomass yield.     

Fixation of carbon dioxide by a hydrogen-oxidizing bacterium for value-added products

With rapid technology progress and cost reduction, clean hydrogen from water electrolysis driven by renewable powers becomes a potential feedstock for CO₂ fixation by hydrogen-oxidizing bacteria. Cupriavidus necator (formally Ralstonia eutropha), a representative member of the lithoautotrophic prokaryotes, is a promising producer of polyhydroxyalkanoates and single cell proteins. This paper reviews the fundamental properties of the hydrogen-oxidizing bacterium, the metabolic activities under limitation of individual gases and nutrients, and the value-added products from CO₂, including the products with large potential markets. Gas fermentation and bioreactor safety are discussed for achieving high cell density and high productivity of desired products under chemolithotrophic conditions. The review also updates the recent research activities in metabolic engineering of C. necator to produce novel metabolites from CO₂.     

Estimation of the energetic biomass yield and efficiency of oxidative phosphorylation in cell-recycle cultures of Schizosaccharomyces pombe

The energetics of growth of the fission yeast Schizosaccharomyces pombe was studied in continuous high-cell concentration cultures using a cell-recycle fermentor. Under non-O₂-limited conditions, steady-states were obtained at various specific growth rates (partial cell-recycle) with purely oxidative (glucose limitation) or respiro-fermentative (glucose excess) metabolic behaviour. The stoichiometry of biomass synthesis was established from the elemental composition of the cells and measurements of all the specific metabolic rates, i.e. consumption of glucose and O₂ and production of CO₂, ethanol and other products. The theoretical yield factor for biomass on glucose was Y_G,X = 0.85 C-mol·C-mol^–1 and maintenance requirements were negligible. Assuming a constant coupling between energy generation and biomass formation for both respirative and respiro-fermentative breakdown of glucose, the biomass yield from ATP (Y_ATP) and the efficiency of oxidative phosphorylation (P/O ratio) could be determined as 9.8 g biomass·mol ATP and 1.28 mol ATP·atom of O₂, respectively. Correspondence to: A. Pareilleux     

Enhanced biomass and oxidative stress tolerance of <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> PCC 7942 overexpressing the <Emphasis Type="Italic">DHAR</Emphasis> gene from <Emphasis Type="Italic">Brassica juncea</Emphasis>

Objectives

To improve the oxidative stress tolerance, biomass yield, and ascorbate/dehydroascorbate (AsA/DHA) ratio of Synechococcus elongatus PCC 7942 in the presence of H₂O₂, by heterologous expression of the dehydroascorbate reductase (DHAR) gene from Brassica juncea (BrDHAR).

Results

Under H₂O₂ stress, overexpression of BrDHAR in the transgenic strain (BrD) of S. elongatus greatly increased the AsA/DHA ratio. As part of the AsA recycling system, the oxidative stress response induced by reactive oxygen species was enhanced, and intracellular H₂O₂ level decreased. In addition, under H₂O₂ stress conditions, the BrD strain displayed increased growth rate and biomass, as well as higher chlorophyll content and deeper pigmentation than did wild-type and control strains.

Conclusion

By maintaining the AsA pool and redox homeostasis, the heterologous expression of BrDHAR increased S. elongatus tolerance to H₂O₂ stress, improving the biomass yield under these conditions. The results suggest that the BrD strain of S. elongatus, with its ability to attenuate the deleterious effects of ROS caused by environmental stressors, could be a promising platform for the generation of biofuels and other valuable bioproducts.

     

Effects of Irradiance on the in Vivo CO(2):O(2) Specificity Factor in Tobacco Using Simultaneous Gas Exchange and Fluorescence Techniques

The effects of gas phase O₂ concentration (1%, 20.5%, and 42.0%, v/v) on the quantum yield of net CO₂ fixation and fluorescence yield of chlorophyll a are examined in leaf tissue from Nicotiana tabacum at normal levels of CO₂ and 25 to 30°C. Detectable decreases in nonphotochemical quenching of absorbed excitation occurred at the higher O₂ levels relative to 1% O₂ when irradiance was nearly or fully saturating for photosynthesis. Photochemical quenching was increased by high O₂ levels only at saturating irradiance. Simultaneous measurements of CO₂ and H₂O exchange and fluorescence yield permit estimation of partitioning of linear photosynthetic electron transport between net CO₂ fixation and O₂-dependent, dissipative processes such as photorespiration as a function of leaf internal CO₂ concentration. Changes in the in vivo CO₂:O₂ `specificity factor' (K_sp) with increasing irradiance are examined. The magnitude K_sp was found to decline from a value of 85 at moderate irradiance to 68 at very low light, and to 72 at saturating photon flux rates. The results are discussed in terms of the applicability of the ribulose bisphosphate carboxylase/oxygenase enzyme model to photosynthesis in vivo.     

Ozone and density affect the response of biomass and seed yield to elevated CO2 in rice

Tropospheric O₃ reduces growth and yield of many crop species, whereas CO₂ ameliorates the negative effects of O₃. Thus, in a combined elevated CO₂ and O₃ atmosphere, seed yield is at least restored to that of charcoal‐filtered (CF) air at ambient CO₂. The CO₂‐induced yield increase in CF air is highly variable, suggesting other potential resource limitations. To understand such variability in response, we tested that (1) competition for resources precludes some of the CO₂ enhancement on biomass and yield; and (2) O₃ reduces competition in elevated CO₂. We grew rice (Oryza sativa L.) at five densities in CF and O₃‐fumigated (+O3) air at ambient (A) and elevated [CO₂] (+CO2) in 1997 and 1998. O₃ reduced biomass by 25% and seed yield by 13–20% in A, but had little effect in +CO2. A competition model of biomass and yield response to density based on resource availability without competition showed that fewer resources were used for biomass in +O3 than in CF (average 53% vs. 70%) in A, while in +CO2 85% of resources were used for biomass regardless of O₃ suggesting greater depletion of resources. The enhanced biomass response to CO₂ with O₃ is consistent with a 22% greater CO₂ enhancement ratio [mass in +CO2 air/mass in A air; enhancement ratio (ER)] in +O3 than in CF air. For seed yield, few resources were used (average 17% and 25% for CF in 1997 and 1998, respectively), and ER was 13% greater in +O3. With competition the rate of change of individual plant biomass to density was not affected by +CO2 in CF air in 1997 but was increased 19% with more nutrients in 1998, indicating resource limitations with +CO2. The rate of change of individual plant yield to density was reduced with CO₂ in 1997 and unchanged in 1998 showing a different response to resource limitation for reproductive biomass. The resource use in +O3‐A suggested that increased density and soil fertility might compensate for pollutant damage. Although ambient [O₃] can modulate the response to elevated CO₂, resource limitation precludes the CO₂ fertilization impact and both factors need consideration for better management and forecasts of future productivity.     

How do elevated CO2 and O3 affect the interception and utilization of radiation by a soybean canopy?

Net productivity of vegetation is determined by the product of the efficiencies with which it intercepts light (?_i) and converts that intercepted energy into biomass (?_c). Elevated carbon dioxide (CO₂) increases photosynthesis and leaf area index (LAI) of soybeans and thus may increase ?_i and ?_c; elevated O₃ may have the opposite effect. Knowing if elevated CO₂ and O₃ differentially affect physiological more than structural components of the ecosystem may reveal how these elements of global change will ultimately alter productivity. The effects of elevated CO₂ and O₃ on an intact soybean ecosystem were examined with Soybean Free Air Concentration Enrichment (SoyFACE) technology where large field plots (20‐m diameter) were exposed to elevated CO₂ (～550 μmol mol^?1) and elevated O₃ (1.2 × ambient) in a factorial design. Aboveground biomass, LAI and light interception were measured during the growing seasons of 2002, 2003 and 2004 to calculate ?_i and ?_c. A 15% increase in yield (averaged over 3 years) under elevated CO₂ was caused primarily by a 12% stimulation in ?_c , as ?_i increased by only 3%. Though accelerated canopy senescence under elevated O₃ caused a 3% decrease in ?_i, the primary effect of O₃ on biomass was through an 11% reduction in ?_c. When CO₂ and O₃ were elevated in combination, CO₂ partially reduced the negative effects of elevated O₃. Knowing that changes in productivity in elevated CO₂ and O₃ were influenced strongly by the efficiency of conversion of light energy into energy in plant biomass will aid in optimizing soybean yields in the future. Future modeling efforts that rely on ?_c for calculating regional and global plant productivity will need to accommodate the effects of global change on this important ecosystem attribute.     

Gas-Exchange Properties of Salt-Stressed Olive (Olea europea L.) Leaves

The effects of two levels of salinity on photosynthetic properties of olive (Olea europea L.) leaves were observed either in low or in high H₂O vapor pressure deficit (vpd). Under moderate salt stress, stomata were found to be less open and responsive both to light and vpd, but the predominant limitation of photosynthesis was due to the mesophyll capacity of CO₂ fixation. We elaborate a procedure to correlate mesophyll capacity and liquid phase diffusive conductance. The estimated liquid phase diffusive conductance was reduced by salt and especially by high vpd; morphological and physiological changes could be responsible for this reduction. As a result, the chloroplast CO₂ partial pressure was found to decrease both under salt and vpd stress, thus resulting in a ribulose-1,5-bisphosphate carboxylase limitation of assimilation. However, under combined salt and vpd stress, O₂ sensitivity of assimilation increased, as would be expected under conditions of limiting ribulose 1,5-bisphosphate regeneration. Fluorescence induction measurements indicated that, under these conditions, energy supply may become limiting. When Cl⁻ concentration exceeded 80 millimolar in tissue water, zero growth and 50% leaf drop was observed. Fluorescence induction showed irreversible damage at Cl⁻ levels higher than 200 millimolar and basal leaves reached this concentration earlier than the apical ones.     

O(2)-insensitive photosynthesis in c(3) plants : its occurrence and a possible explanation

Leaves of C₃ plants which exhibit a normal O₂ inhibition of CO₂ fixation at less than saturating light intensity were found to exhibit O₂-insensitive photosynthesis at high light. This behavior was observed in Phaseolus vulgaris L., Xanthium strumarium L., and Scrophularia desertorum (Shaw.) Munz. O₂-insensitive photosynthesis has been reported in nine other C₃ species and usually occurred when the intercellular CO₂ pressure was about double the normal pressure. A lack of O₂ inhibition of photosynthesis was always accompanied by a failure of increased CO₂ pressure to stimulate photosynthesis to the expected degree. O₂-insensitive photosynthesis also occurred after plants had been water stressed. Under such conditions, however, photosynthesis became O₂ and CO₂ insensitive at physiological CO₂ pressures. Postillumination CO₂ exchange kinetics showed that O₂ and CO₂ insensitivity was not the result of elimination of photorespiration.

It is proposed that O₂ and CO₂ insensitivity occurs when the concentration of phosphate in the chloroplast stroma cannot be both high enough to allow photophosphorylation and low enough to allow starch and sucrose synthesis at the rates required by the rest of the photosynthetic component processes. Under these conditions, the energy diverted to photorespiration does not adversely affect the potential for CO₂ assimilation.

     

Environmental Determinants of Transformation Efficiency in Helicobacter pylori

Helicobacter pylori uses natural competence and homologous recombination to adapt to the dynamic environment of the stomach mucosa and maintain chronic colonization. Although H. pylori competence is constitutive, its rate of transformation is variable, and little is known about factors that influence it. To examine this, we first determined the transformation efficiency of H. pylori strains under low O₂ (5% O₂, 7.6% CO₂, 7.6% H₂) and high O₂ (15% O₂, 2.9% CO₂, 2.9% H₂) conditions using DNA containing an antibiotic resistance marker. H. pylori transformation efficiency was 6- to 32-fold greater under high O₂ tension, which was robust across different H. pylori strains, genetic loci, and bacterial growth phases. Since changing the O₂ concentration for these initial experiments also changed the concentrations of CO₂ and H₂, transformations were repeated under conditions where O₂, CO₂, and H₂ were each varied individually. The results showed that the increase in transformation efficiency under high O₂ was largely due to a decrease in CO₂. An increase in pH similar to that caused by low CO₂ was also sufficient to increase transformation efficiency. These results have implications for the physiology of H. pylori in the gastric environment, and they provide optimized conditions for the laboratory construction of H. pylori mutants using natural transformation.     

Effect of pO(2) on Growth and Nodule Functioning of Symbiotic Cowpea (Vigna unguiculata L. Walp.)

Nodulated cowpea (Vigna unguiculata L. Walp. cv Vita 3:Bradyrhizobium CB 756) plants were cultured with their whole root system or crown root nodulation zone maintained for periods from 5 to 69 days after planting in atmospheres containing a range of pO₂ (1-80%, v/v) while the rest of the plant grew in normal air. Growth (dry matter yield) and N₂ fixation were largely unaffected by pO₂ from 10 to 40%. Decrease in fixation at pO₂ below 5% was due to lower nodulation and nodule mass and, at pO₂ above 60%, to a fall in specific N₂-fixing activity of nodules. Root:shoot ratios were significantly lower at pO₂ below 2.5%. The effect of pO₂ on nitrogenase activity (acetylene reduction), both of whole nodulated root systems and crown root nodulation zones, varied with plant age but was generally lower at supra- and subambient extremes of O₂. H₂ evolution showed a sharp optimum at 20% O₂ but was at most 4% of total nitrogenase activity. The ratio of CO₂ evolved to substrate (C₂H₂+H⁺) reduced by crown root nodulation zones was constant (6 moles CO₂ per mole substrate reduced) from 2.5 to 60% O₂ but at levels below 2.5 and above 80% O₂ reached values between 20 and 30 moles CO₂ per mole substrate reduced. Effects of long-term growth with nonambient pO₂ on adaptation and efficiency of functioning of nodules are discussed.     

Hydrogen utilization by Methylocystis sp. strain SC2 expands the known metabolic versatility of type IIa methanotrophs

Methane, a non-expensive natural substrate, is used by Methylocystis spp. as a sole source of carbon and energy. Here, we assessed whether Methylocystis sp. strain SC2 is able to also utilize hydrogen as an energy source. The addition of 2% H₂ to the culture headspace had the most significant positive effect on the growth yield under CH₄ (6%) and O₂ (3%) limited conditions. The SC2 biomass yield doubled from 6.41 (±0.52) to 13.82 (±0.69) mg cell dry weight per mmol CH₄, while CH₄ consumption was significantly reduced. Regardless of H₂ addition, CH₄ utilization was increasingly redirected from respiration to fermentation-based pathways with decreasing O₂/CH₄ mixing ratios. Theoretical thermodynamic calculations confirmed that hydrogen utilization under oxygen-limited conditions doubles the maximum biomass yield compared to fully aerobic conditions without H₂ addition. Hydrogen utilization was linked to significant changes in the SC2 proteome. In addition to hydrogenase accessory proteins, the production of Group 1d and Group 2b hydrogenases was significantly increased in both short- and long-term incubations. Both long-term incubation with H₂ (37 d) and treatments with chemical inhibitors revealed that SC2 growth under hydrogen-utilizing conditions does not require the activity of complex I. Apparently, strain SC2 has the metabolic capacity to channel hydrogen-derived electrons into the quinone pool, which provides a link between hydrogen oxidation and energy production. In summary, H₂ may be a promising alternative energy source in biotechnologically oriented methanotroph projects that aim to maximize biomass yield from CH_4, such as the production of high-quality feed protein.     

The Sequence of Change within the Photosynthetic Apparatus of Wheat following Short-Term Exposure to Ozone

The basis of inhibition of photosynthesis by single acute O₃ exposures was investigated in vivo using analyses based on leaf gas exchange measurements. The fully expanded second leaves of wheat plants (Triticum aestivum L. cv Avalon) were fumigated with either 200 or 400 nanomoles per mole O₃ for between 4 and 16 hours. This reduced significantly the light-saturated rate of CO₂ uptake and was accompanied by a parallel decrease in stomatal conductance. However, the stomatal limitation, estimated from the relationship between CO₂ uptake and the internal CO₂ concentration, only increased significantly during the first 8 hours of exposure to 400 nanomoles per mole O₃; no significant increase occurred for any of the other treatments. Analysis of the response of CO₂ uptake to the internal CO₂ concentration implied that the predominant factor responsible for the reduction in light-saturated CO₂ uptake was a decrease in the efficiency of carboxylation. This was 58 and 21% of the control value after 16 hours at 200 and 400 nanomoles per mole O₃, respectively. At saturating concentrations of CO₂, photosynthesis was inhibited by no more than 22% after 16 hours, indicating that the capacity for regeneration of ribulose bisphosphate was less susceptible to O₃. Ozone fumigations also had a less pronounced effect on light-limited photosynthesis. The maximum quantum yield of CO₂ uptake and the quantum yield of oxygen evolution showed no significant decline after 16 hours with 200 nanomoles per mole O₃, requiring 8 hours at 400 nanomoles per mole O₃ before a significant reduction occurred. The photochemical efficiency of photosystem II estimated from the ratio of variable to maximum chlorophyll fluorescence and the atrazine-binding capacity of isolated thylakoids demonstrated that photochemical reactions were not responsible for the initial inhibition of CO₂ uptake. The results suggest that the apparent carboxylation efficiency appears to be the initial cause of decline in photosynthesis in vivo following acute O₃ fumigation.     

Experimental determination of the respiration associated with soybean/rhizobium nitrogenase function, nodule maintenance, and total nodule nitrogen fixation

The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function.

Rates of CO₂ evolution and H₂ evolution from intact, nodulated root systems under Ar:O₂ atmospheres decreased in parallel when plants were maintained in an extended dark period. While H₂ evolution approached zero after 36 hours of darkness at 22°C, CO₂ evolution rate remained at 38° of the rate measured in light. Of the remaining CO₂ evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO₂ (per gram dry weight nodule) per hour at 22°C and 30°C, respectively.

The cost of N₂ fixation in terms of CO₂ evolved per electron pair utilized by nitrogenase was estimated from the slope of H₂ evolution rate versus CO₂ evolution rate. The cost was 2 moles CO₂ evolved per mole H₂ evolved and was independent of temperature.

In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H₂ evolution.

     

Hydrogen production by microalgae

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 H<sub>2</sub> and O<sub>2</sub> 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.     

Carbon Dioxide-Induced Oscillations in Fluorescence and Photosynthesis: Role of Thylakoid Membrane Energization in Regulation of Photosystem II Activity

The response of CO₂ fixation to a sudden increase in ambient CO₂ concentration has been investigated in intact leaf tissue from spinach (Spinacia oleracea) using a dual channel infrared gas analyzer. Simultaneous with these measurements, changes in fluorescence emission associated with a weak, modulated measuring beam were recorded. Application of brief (2-3 seconds) dark intervals enabled estimation of the dark fluorescence level (F_o) under both steady state and transient conditions. The degree of suppression of F_o level fluorescence in the light was strongly correlated with nonphotochemical quenching under all conditions. During CO₂-induced oscillations in photosynthesis under 2% O₂ the changes in nonphotochemical quenching anticipate changes in the rate of uptake of CO₂. At such low levels of O₂ and constant illumination, changes in the relative quantum efficiency of open photosystem II units were estimated as the ratio of the rate of CO₂ uptake and the photochemical quenching coefficient. Under the same conditions the relative quantum efficiency of photosystem II was found to vary inversely with the degree of nonphotochemical quenching. The relationship between changes in the rate of CO₂ uptake: photochemical quenching coefficient and nonphotochemical quenching was altered somewhat when the same experiment was conducted under 20% O₂. The results suggest that electron transport coupled to reduction of O₂ occurs to varying degrees with time during oscillations, especially when ambient O₂ concentrations are high.     

Effects of water vapor pressure deficit on photochemical and fluorescence yields in tobacco leaf tissue

The relationship between photochemical quantum yield (_s) and fluorescence yield have been investigated in leaf tissue from Nicotiana tabacum using CO₂ exchange and a modulated fluorescence measuring system. The quantum yield of CO₂ fixation at 1.6% (v/v) O₂ and limiting irradiance was reduced 20% by increasing the mean H₂O vapor pressure deficit (VPD) from 9.2 to 18.6 mbars. As [CO₂] and irradiance were varied, the intrinsic quantum yield of open photosystem II units (_s/q_Q where q_Q is the photochemical fluorescence quenching coefficient) declined linearly with the degree of nonphotochemical fluorescence quenching. The slope and y-intercept values for this function were significantly reduced when the mean VPD was 18.4 millibars relative to 8.9 millibars. Susceptibility of the leaf tissue to photoinhibition was unaffected by VPD. Elevated O₂ concentrations (20.5% v/v) reduced the intrinsic quantum yield of net CO₂ uptake due to the occurrence of O₂-reducing processes. However, the relative effect of high VPD compared to low VPD on intrinsic quantum yield was not dependent on the O₂ level. This suggests that the Mehler reaction does not mediate the response of quantum yield to elevated VPD. The results are discussed with regard to the possible role of transpiration stress in regulating dissipation of excitation by electron transport pathways other than noncyclic electron flow supporting reduction of CO₂ and/or O₂.     

Valorization of CO2 through lithoautotrophic production of sustainable chemicals in Cupriavidus necator

Coupling recent advancements in genetic engineering of diverse microbes and gas-driven fermentation provides a path towards sustainable commodity chemical production. Cupriavidus necator H16 is a suitable species for this task because it effectively utilizes H₂ and CO₂ and is genetically tractable. Here, we demonstrate the versatility of C. necator for chemical production by engineering it to produce three products from CO₂ under lithotrophic conditions: sucrose, polyhydroxyalkanoates (PHAs), and lipochitooligosaccharides (LCOs). We engineered sucrose production in a co-culture system with heterotrophic growth 30 times that of WT C. necator. We engineered PHA production (20–60% DCW) and selectively altered product composition by combining different thioesterases and phaCs to produce copolymers directly from CO₂. And, we engineered C. necator to convert CO₂ into the LCO, a plant growth enhancer, with titers of ~1.4 mg/L—equivalent to yields in its native source, Bradyrhizobium. We applied the LCOs to germinating seeds as well as corn plants and observed increases in a variety of growth parameters. Taken together, these results expand our understanding of how a gas-utilizing bacteria can promote sustainable production.     

Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H₂), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O₂, CO₂, N₂, and H₂ was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO₂ and O₂. The amount of H₂ produced per molecule of N₂ fixed was found to vary with light conditions, high light giving a greater increase in H₂ production than N₂ fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H₂ produced per molecule of N₂ fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO₂, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 μmol (mg of chlorophyll a)⁻¹ h⁻¹ to 9 μmol (mg of chlorophyll a)⁻¹ h⁻¹ after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.