Simultaneous measurement of acetylene reduction and respiratory gas exchange of attached root nodules

A method was developed for the simultaneous measurement of acetylene reduction, carbon dioxide evolution and oxygen uptake by individual root nodules of intact nitrogen-fixing plants (Alnus rubra Bong.). The nodules were enclosed in a temperature-controlled leak-tight cuvette. Assay gas mixtures were passed through the cuvette at a constant, known flow rate and gas exchange was measured by the difference between inlet and outlet gas compositions. Gas concentrations were assayed by a combination of an automated gas chromatograph and a programmable electronic integrator. Carbon dioxide and ethylene evolution were determined with a coefficient of variation which was less than 2%, whereas the coefficient of variation for oxygen uptake measurements was less than 5%. Nodules subjected to repeated removal from and reinsertion into the cuvette and to long exposures of 10% v/v acetylene showed no irreversible decline in respiration or acetylene reduction. This system offers long-term stability and freedom from disturbance artifacts plus the ability to monitor continuously, rapidly and specifically the changes in root nodule activity caused by environmental perturbation.     

Responses of stomata of senescing and nonsenescing leaves of Nicotians glauca to changes in intercellular concentrations of leaf carbon dioxide

Abstract. Gas exchange measurements were performed to test the hypothesis that failure of stomata to open in senescing leaves of Nicotiana glauca is caused by elevated concentrations of carbon dioxide in the intercellular spaces of leaf mesophyll tissue (c_i). Senescing leaves selected for experiments were completely chlorotic and lacked positive rates of photosynthesis. When stomata in detached epidermis from senescing leaves were illuminated in CO2-free air, they opened to similar apertures as those in detached epidermis from nonsenescing leaves. To compare the effects of changes in c_i on stomatal responses of the two leaf types, leaf 'flags' of either nonsenescing or senescing leaves were illuminated at a photosynthetic photon flux density of 500 μmol m⁻² s⁻¹ in a gas exchange cuvette. Leaf temperatures were maintained at 23.5 ± 0.5°C, and vapour pressure differences between leaves and the air were maintained between 0.70 and 0.75kPa. C_i was adjusted by changing external concentrations of carbon dioxide in air circulating through the cuvette. Conductances and photosynthetic rates of nonsenescing leaves changed in response to changes in c_i, but neither the conductances nor the photosynthetic rates of senescing leaves were affected significantly by changes in q. We conclude that guard cells of senescing leaves of Nicotiana glauca do not lose the capacity to respond to changes in carbon dioxide concentration and that increases in c_i resulting from declining rates of mesophyll photosynthesis are not the sole cause of maintenance of stomatal closure during leaf senescence. The data suggest that factors external to guard cells may prevent them from responding to changes in carbon dioxide concentrations in intact senescing leaves.     

A field portable gas-exchange system for measuring carbon dioxide and water vapour exchange rates of leaves during fumigation with SO2

Abstract A field portable, steady-state gas-exchange system which measures both CO₂ and water vapour exchange of single intact leaves during fumigations with SO₂ is described. Within the leaf cuvette temperature, light, humidity and both CO₂ and SO₂ concentrations are controlled to preset levels. Gas flow and concentrations are controlled by mass flow controllers. Photosynthetic uptake of CO₂ can be determined either by differential depletion or null balance measurement. Water vapour exchange is measured differentially and transpiration and conductance to water vapour determined. Sulphur dioxide is measured directly within the cuvette exhaust gas line by UV-pulse fluorescence. The performance of this system under field conditions is described and the physiological measurements compared with those obtained with other systems.     

Potentiometric measurements of carbon dioxide flux of submerged aquatic macrophytes in pH-statted natural waters

SUMMARY. 1, An apparatus has been described that is suitable for potentiometric measurement of carbon dioxide flux in photosynthesizing shoots of submerged aquatic macrophytes 2. The procedure, based on methods described by Tailing (1973) for measurement of phytoplankton photosynthesis, relies upon the continuous pH-statting of the solution surrounding the tissues. The pH of the solution is monitored by electrodes from a pH meter which is linked to an auto-titrator. The rise in pH during photosynthesis is then compensated tor by controlled, small titrant additions of CO₂-ennched solution (titrant water). This replaces the CO₂ removed by the tissues without affecting the total alkalinity of the solution. If the concentration of CO₂ in the titrant water, and the volume of titrant added arc known precisely, the CO₂ flux can be calculated. 3. Total alkalinity, total CO₂ and free-CO₂ acidity of the bathing solutions and titrant waters are estimated by Gran titrations and the pH: tilre-volume data pairs are analysed by computer to provide rapid data feed-back. A modification to Tailing's equation for calculation of F₁functions has been necessary for accurate calibration of the CO₂enriched tilrant water. 4. The photosynthesis cuvette, which is surrounded by a water-jacket, is approximately I dm³ in capacity and has six compartments for the shoots. An impeller at the base of the cuvette rapidly mixes and cycles the bathing solution and flushes it over the tissues. 5. Information on temperature, light flux density, oxygen concentration. pH and titre-volume is continuously recorded into a data-logger and is fed into a computer which is programmed for data analyses. 6. Results from a typical experiment show the system to be sound and the method has considerable potential, especially in the study of aquatic plant photosynthesis in natural waters.     

Carbon dioxide injection method for enhancing hydrogenotrophic denitrification of secondary wastewater effluent in fixed bed reactor

This study presents an optimal injection method for using carbon dioxide as a carbon source for the hydrogenotrophic denitrification of secondary wastewater effluent in a laboratory-scale fixed bed reactor (FBR). The FBR was operated under three conditions: a continuous CO₂ supply, periodic CO₂ supply, and without a CO₂ supply. The continuous operation of the FBR without carbon dioxide injection resulted in an increase in pH to 10 and a noticeable level of nitrite accumulation. The continuous co-injection of carbon dioxide and hydrogen gas decreased the pH to a range of 6 ～ 8, but the denitrification efficiency decreased to 29%. The co-injection of carbon dioxide decreased the maximum dissolved hydrogen concentration and hydrogen mass transfer rate by 25 and 61%, respectively. Compared to the continuous injection method, a periodic injection of carbon dioxide increased the denitrification efficiency from 28.6 to 85% as the hydrogen flow rate and hydraulic retention time (HRT) increased. With the periodic injection of carbon dioxide, the nitrite accumulation appeared to be insignificant as the hydrogen flow rate increased.     

Acclimation to temperature of the response of photosynthesis to increased carbon dioxide concentration in Taraxacum officinale

The relative stimulation of photosynthesis by elevated carbon dioxide in C₃ species normally increases strongly with increasing temperature. This results from the kinetic characteristics of Rubisco, and has potentially important implications for responses of vegetation to increasing atmospheric carbon dioxide. It is often assumed that because Rubisco characteristics are conservative, all C₃ species have the same temperature dependence of the response of photosynthesis to elevated carbon dioxide. However, in this field study of Taraxacum officinale, there were no significant differences in the relative stimulation of photosynthesis by elevated carbon dioxide among days with temperatures ranging from 15 to 34 °C. Nevertheless, short-term measurements indicated a strong temperature dependence of the stimulation. This suggested that acclimation to temperature caused the lack of variation in the seasonal data. Experiments in controlled environments indicated that complete acclimation of the relative stimulation of photosynthesis by elevated carbon dioxide occurred for growth temperatures of 10 – 25 °C. The apparent specificity of Rubisco for carbon dioxide relative to oxygen at 15 °C, as assayed in vivo by measurements of the carbon dioxide concentration at which carboxylation equalled oxygenation, also varied with growth temperature. Changes in the apparent specificity of Rubisco accounted for the acclimation of the temperature dependence of the relative stimulation of photosynthesis by elevated carbon dioxide. It is premature to conclude that low temperatures will necessarily reduce the relative stimulation of photosynthesis caused by rising atmospheric carbon dioxide. This revised version was published online in June 2006 with corrections to the Cover Date.     

CONTINUOUS MONITORING OF CO2EXCHANGE OF LICHENS IN THE FIELD: SHORT-TERM ENCLOSURE WITH AN AUTOMATICALLY OPERATING CUVETTE

Field measurements of CO₂exchange by lichens are difficult because these poikilohydric organisms rely on direct hydration by rain, dew or fog. Continuous enclosure of thalli in conditioned, measurement cuvettes is, therefore, not experimentally reasonable. Porometric instruments with brief enclosure times, have proved useful for studies of lichen gas exchange but allow only spot measurements and recording is not easily possible for extended time periods. We describe a newly developed, automatically operating cuvette that allows long-term monitoring of lichen photosynthetic and respiratory CO₂exchange in the field. Samples, e.g. flat stone slabs covered with epilithic lichens, are positioned on the base of the cuvette where, when the cuvette is open, they are exposed to the same environmental conditions as naturally growing thalli. At regular intervals (typically 30 min), an upper lid automatically encloses the lichen forc. 3 min in a stirred cuvette; CO₂exchange is measured using IRGA techniques and microclimate data are recorded. The successful operation of the cuvette is illustrated by means of diel time courses of CO₂exchange for selected, very different weather conditions.     

Studies on the Ventilation in Submerged Fermentations

Quantitative studies on the dissolution and dissociation of carbon dioxide in a cultured system were made. The inosine fermentation and the glutamic acid fermentation were employed for this study. According to the results obtained in this experiment, the quantity of dissociated carbonic acid in cultured liquid was given by Henderson-Hasselbalch’s equation with experimental pK′. The method for the direct determination of bicarbonate ion concentration was also investigated. The Warburg direct method gave a satisfactory result for this purpose.

By using the modified Severinghaus CO₂ electrode, the relationship between partial pressure of carbon dioxide in effluent gas and that in culturing system was investigated. Partial pressure of carbon dioxide in gas phase was almost equivalent to the average value of dissolved carbon dioxide tension in liquid phase for a given short time of the fermentation. The term of r_e was introduced in order to study the dynamic characteristics of carbon dioxide evolution in submerged fermentors. The dynamic characteristics of respiration in submerged fermentation was also studied by using biological r_ab and r_e.     

Modelling non‐steady‐state isotope enrichment of leaf water in a gas‐exchange cuvette environment

The combined use of a gas‐exchange system and laser‐based isotope measurement is a tool of growing interest in plant ecophysiological studies, owing to its relevance for assessing isotopic variability in leaf water and/or transpiration under non‐steady‐state (NSS) conditions. However, the current Farquhar & Cernusak (F&C) NSS leaf water model, originally developed for open‐field scenarios, is unsuited for use in a gas‐exchange cuvette environment where isotope composition of water vapour (δ_v) is intrinsically linked to that of transpiration (δ_E). Here, we modified the F&C model to make it directly compatible with the δ_v–δ_E dynamic characteristic of a typical cuvette setting. The resultant new model suggests a role of ‘net‐flux’ (rather than ‘gross‐flux’ as suggested by the original F&C model)‐based leaf water turnover rate in controlling the time constant (τ) for the approach to steady sate. The validity of the new model was subsequently confirmed in a cuvette experiment involving cotton leaves, for which we demonstrated close agreement between τ values predicted from the model and those measured from NSS variations in isotope enrichment of transpiration. Hence, we recommend that our new model be incorporated into future isotope studies involving a cuvette condition where the transpiration flux directly influences δ_v. There is an increasing popularity among plant ecophysiologists to use a gas‐exchange system coupled to laser‐based isotope measurement for investigating non‐steady state (NSS) isotopic variability in leaf water (and/or transpiration); however, the current Farquhar & Cernusak (F&C) NSS leaf water model is unsuited for use in a gas‐exchange cuvette environment due to its implicit assumption of isotope composition of water vapor (δ_v) being constant and independent of that of transpiration (δ_E). In the present study, we modified the F&C model to make it compatible with the dynamic relationship between δ_v and δ_E as is typically associated with a cuvette setting. Using an experiment conducted on cotton leaves, we show that the modified NSS model performed well in predicting the time constant for the exponential approach of leaf water toward steady state under cuvette conditions. Such a result demonstrates the applicability of this new model to gas‐exchange cuvette conditions where the transpiration flux directly influences δ_v, and therefore suggests the need to incorporate this model into future isotope studies that employ a laser‐cuvette coupled system.     

13CO2/12CO2 exchange fluxes in a clamp‐on leaf cuvette: disentangling artefacts and flux components

Leaks and isotopic disequilibria represent potential errors and artefacts during combined measurements of gas exchange and carbon isotope discrimination (Δ). This paper presents new protocols to quantify, minimize, and correct such phenomena. We performed experiments with gradients of CO₂ concentration (up to ±250 μmol mol^?1) and δ¹³C_CO2 (34‰), between a clamp‐on leaf cuvette (LI‐6400) and surrounding air, to assess (1) leak coefficients for CO₂, ¹²CO₂, and ¹³CO₂ with the empty cuvette and with intact leaves of Holcus lanatus (C₃) or Sorghum bicolor (C₄) in the cuvette; and (2) isotopic disequilibria between net photosynthesis and dark respiration in light. Leak coefficients were virtually identical for ¹²CO₂ and ¹³CO₂, but ～8 times higher with leaves in the cuvette. Leaks generated errors on Δ up to 6‰ for H. lanatus and 2‰ for S. bicolor in full light; isotopic disequilibria produced similar variation of Δ. Leak errors in Δ in darkness were much larger due to small biological : leak flux ratios. Leak artefacts were fully corrected with leak coefficients determined on the same leaves as Δ measurements. Analysis of isotopic disequilibria enabled partitioning of net photosynthesis and dark respiration, and indicated inhibitions of dark respiration in full light (H. lanatus: 14%, S. bicolor: 58%).     

Observations on Cardiac Arrythmias during Laparoscopy

The incidence of cardiac arrhythmias occurring during laparoscopy was studied in 100 consecutive patients who received carbon dioxide to inflate the abdomen and compared with that in 45 patients in whom nitrous oxide was substituted for carbon dioxide. Seventeen patients receiving carbon dioxide and two receiving nitrous oxide developed multiple arrhythmias, the commonest variety being fusion beats due to ventricular ectopic beats. Blood gas determinations showed that carbon dioxide caused a significantly higher level of Paco₂ and a lower pH than did nitrous oxide.     

Nitrogen fixation and respiration by root nodules ofAlnus rubra Bong.: Effects of temperature and oxygen concentration

Summary Using a root nodule cuvette and a continuous flow gas exchange system, we simultaneously measured the rates of carbon dioxide evolution, oxygen uptake and acetylene reduction by nodules ofAlnus rubra. This system allowed us to measure the respiration rates of single nodules and to determine the effects of oxygen concentration and temperature on the energy cost of nitrogen fixation. Energy cost was virtually unchanged (2.8–3.5 moles of carbon dioxide or oxygen per mole of ethylene) from 16 to 26°C (pO₂=20 kPa) while respiration and nitrogenase activity were highly temperature dependent. At temperatures below 16°C, nitrogenase activity decreased more than did respiration and as a result, energy cost rose sharply. Acetylene reduction ceased below 8°C. Inhibition of nitrogenase activity at low temperatures was rapidly reversed upon return to higher temperatures. At high temperatures (above 30°C) nitrogenase activity declined irreversibly, while respiration and energy cost increased.Energy cost was nearly unchanged at oxygen partial pressures of 5 to 20 kPa (temperature of 20°C). Respiration and nitrogenase activity were strongly correlated with oxygen tension. Below 5 kPa, acetylene reduction and oxygen uptake decreased sharply while production of carbon dioxide increased, indicating fermentation. Fermentation alone was unable to support nitrogenase activity. Acetylene reduction was independent of oxygen concentration from 15 to 30 kPa. Nitrogenase activity decreased and energy cost rose above 30 kPa until nearly complete inactivation of nitrogenase at 70–80 kPa. Activity declined gradually, such that acetylene reduction at a constant oxygen concentration was stable, but showed further inactivation when oxygen concentration was once again increased. Alder nodules appear to consist of a large number of compartments that differ in the degree to which nitrogenase is protected from excess oxygen.Supported by United States Department of Agriculture Grant 78-59-2252-0-1-005-1     

Evidence for C4 photosynthesis in barley pericarp tissue

The products of photosynthetic carbon dioxide fixation were determined in isolated pericarps of immature barley grains. Of the carbon dioxide fixed after 1 min photosynthesis 84% was in the C₄ acid malic acid. The remaining label was in hexose phosphates and sucrose. By 2 min sucrose was the major labelled product and at 6 min accounted for 94% of the total carbon dioxide fixed.     

Carbon dioxide reversibly inhibits meiosis of Xenopus laevis oocyte and the appearance of the maturation promoting factor

Full-grown Xenopus laevis oocytes were incubated in NaHCO₃ buffer equilibrated with carbon dioxide (5 to 100%). Germinal vesicle breakdown never occurred in spite of the appearance of the characteristic white spot at the animal pole. The effect of carbon dioxide was analyzed during progesterone-induced maturation. Carbon dioxide did not inhibit the early steps of maturation whereas it inhibited germinal vesicle breakdown even when applied 4 hr after the initial hormonal trigger. When oocytes were treated transiently in NaHCO₃ buffer equilibrated with carbon dioxide and further incubated in Tris buffer, drastic delay in the kinetic of germinal vesicle breakdown was observed. Inhibition of progesterone-induced maturation by carbon dioxide treatment is coincident with the time of maturation promoting factor appearance (MPF). On the basis of microinjection experiments of MPF into recipient oocytes, it was also shown that MPF expression is not inhibited by carbon dioxide and thus indicates that the late phase of MPF formation and/or MPF amplification is a carbon dioxide-sensitive period.     

REDUCTION OF CARBON DIOXIDE COUPLED WITH THE OXYHYDROGEN REACTION IN ALGAE

1. Unicellular algae possessing a hydrogenase system (Scenedesmus and other species), and having been adapted by anaerobic incubation to the hydrogen metabolism, reduce oxygen to water according to the equation O₂ + 2H₂ → 2H₂O. 2. The oxyhydrogen reaction proceeds undisturbed only in the presence of carbon dioxide, which simultaneously is reduced according to the equation CO₂ + 2H₂ → H₂O + (CH₂O) = (carbohydrate). 3. The maximum yield of the induced reduction is one-half molecule of carbon dioxide reduced for each molecule of oxygen absorbed. 4. Partial reactions are recognizable in the course of the formation of water and it is with the absorption of the second equivalent of hydrogen that the carbon dioxide reduction appears to be coupled. 5. The velocity of the reaction increases in proportion to the partial pressure of oxygen, but only up to a certain point where any excess of oxygen causes the inactivation of the hydrogenase system. The reaction then ends prematurely. 6. During the oxyhydrogen reaction little or no oxygen is consumed for normal respiratory processes. 7. Small concentrations of cyanide, affecting neither photosynthesis nor photoreduction in the same cells, first inhibit the induced reduction of carbon dioxide and then lead to a complete inactivation of the hydrogenase system. 8. Hydroxylamine, added after adaptation, has either no inhibitory effect at all, or prevents solely the induced reduction of carbon dioxide without inactivating the hydrogenase system. 9. Dinitrophenol prevents the dark reduction of carbon dioxide while the reduction of oxygen continues to the formation of water. 10. Glucose diminishes the absorption of hydrogen, probably in its capacity as a competing hydrogen donor. 11. The induced reduction of carbon dioxide can be described as an oxido-reduction similar to that produced photochemically in the same cells.     

Kinetics of Carbon Dioxide Evolution in Agitation System

Kinetics of carbon dioxide evolution was investigated in agitation system. Reaction steps of carbon dioxide evolution in submerged fermentations may consist of three steps; the first, hydration of carbon dioxide liberated from living cells, the second, dehydration of bicarbonate ions and the third, formation of carbon dioxide bubbles. Taking into account the equilibrium between hydration of carbon dioxide and dehydration of bicarbonate ions at physiological pH value, the fallowings may be rate-limiting steps in mass transfer of carbon dioxide in submerged fermentations, dehydration of bicarbonate ions and the bubble formation. The overall velocity constant of these two reaction steps was determined in the agitation vessel This reaction obeyed good first-order kinetics and the term of was introduced as a velocity constant. This value was influenced by agitation speed, temperature, viscosity of the fluid and carbonic anhydrase. The value of carbon dioxide coefficient (Kd)_CO2 was higher than the oxygen absorption coefficient Kd. The driving force of mass transfer for carbon dioxide, DCO₂—pCO₂, therefore, was lower than that for oxygen, P_B—P_L. The relationship between the overall coefficient of oxygen transfer across gas-liquid interface K_La and the overall velocity constant of carbon dioxide evolution was expressed in the formula      

Sulphur metabolism in Thiorhodaceae I. Quantitative measurements on growing cells ofChromatium okenii

Growth experiments and short term experiments in a stirred cuvette showed thatChromatium okenii strain Ostrau is not able to oxidize any reduced sulphur compounds except sulphide and elementary sulphur; thiosulphate, sulphite, and thioglycolate can not be utilized as reducing agents for photosynthesis. The cells are not able to use H₂; hydrogenase could not be demonstrated. In the dark, sulphide is formed from intracellular sulphur and the carbon content of the cells decreases. Growth and turnover of sulphur compounds was followed in the light in the presence and absence of acetate as a second carbon source. Sulphide oxidation depends on the presence of CO₂ and on light intensity, i.e. sulphur metabolism is governed by the photosynthetic activity of the cells.     

The relationship between leaf and ecosystem CO<Subscript>2</Subscript> exchanges in a maize field

The relationship between leaf photosynthetic rate (A) in a vegetation canopy and the net ecosystem CO₂ exchange (NEE) over an entire ecosystem is not well understood. The aim of the present study is to assess the coordinated changes in NEE derived with eddy covariance, A measured in leaf cuvette, and their associations in a rainfed maize field. The light response-curves were estimated for the carbon assimilation rate at both the leaf and ecosystem scales. NEE and A synchronically changed throughout the day and were greater around noon and persisted longer during rapid growth periods. The leaf A had a similar pattern of daytime changes in the top, middle, and bottom leaves. Only severe leaf ageing led to a significant decline in the maximum efficiency of photosystem II (PSII) photochemistry. The greater maximum NEE was associated with a higher ecosystem quantum yield. NEE was positively and significantly correlated with the leaf A averaged based on the vertical distribution of leaf area. The finding highlights the feasibility of assessing NEE by leaf CO₂ exchange because of most of experimental data obtained with leaf cuvette methods; and also implies that simultaneously enhancing leaf photosynthetic rate, electron transport rate, net carbon assimilation at whole ecosystem might play a critical role for the enhancement of crop productivity.     

Carbon dioxide inhibition of yeast growth in biomass production

Saccharomyces cerevisiae was grown under aerobic and substrate-limiting conditions for efficient biomass production. Under these conditions, where the sugar substrate was fed incrementally, the growth pattern of the yeast cells was found to be uniform, as indicated by a constant respiratory quotient during the entire growing period. The effect of carbon dioxide was investigated by replacing portions of the nitrogen in the air stream with carbon dioxide, while maintaining the oxygen content at the normal 20% level, so that identical oxygen transfer rate and atmospheric pressure were maintained for all experiments with different partial pressures of carbon dioxide. Inhibition of yeast growth was negligible below 20% CO₂ in the aeration mixture. Slight inhibition was noted at the 40% CO₂ level and significant inhibition was noted above the 50% CO₂, level, corresponding to 1.6 × 10^?2M of dissolved CO₂ in the fermentor broth. High carbon dioxide content in the gas phase also inhibited the fermentation activity of baker's yeast.     

Characterization of a Photosynthetic Mutant Strain of Chlamydomonas reinhardi Deficient in Phosphoribulokinase Activity

A mutant strain of the unicellular green alga, Chlamydomonas reinhardi, is unable to fix carbon dioxide by photosynthesis because it is deficient in phosphoribulokinase activity. The absence of light-dependent carbon dioxide fixation in cells of the mutant strain supports the operation of the Calvin-Benson scheme of photosynthetic carbon dioxide fixation in this organism. No deficiency other than low phosphoribulokinase activity was found which would account for the inability of cells of the mutant strain to fix carbon dioxide by photosynthesis. Activities comparable to those in the wild-type strain were found for eight other enzymes of the Calvin cycle and two enzymes associated with the C₄ dicarboxylic acid pathway. The normal rates of nicotinamide adenine dinucleotide phosphate photoreduction and of photosynthetic phosphorylation observed in chloroplast fragments prepared from cells of the mutant strain indicated that the photosynthetic electron transport chain in the mutant is intact.