Membrane oxygenators: current developments in design and application

Cardiopulmonary bypass (CPB) procedures require a blood-gas exchanger (oxygenator) to temporarily replace the respiratory function of the lungs. In the past the majority of CPB procedures have been carried out with bubble oxygenators which effect gas exchange by dispersion of bubbles into the blood. Membrane oxygenators, on the other hand, utilize a hydrophobic gas permeable membrane between the blood and gas phases.Bubble oxygenators are being superseded by membrane types for CPB due to improvements in membrane technology and mass transfer efficiency. These advances are reviewed in this paper and are illustrated by reference to the gas exchange and operating characteristics of a number of clinical oxygenators designed for adult CPB.Membrane oxygenatorsare also being used for long-term support in the treatment of acute respiratory failure. Operated in a partial bypass circuit, the oxygenator may have to function for several days or weeks. In one particular treatment method, the rate of spontaneous breathing is controlled by the partial or total removal of the metabolic CO₂ production by the membrane oxygenator. For this method, known as extracorporeal CO₂ removal (ECCO₂R), the oxygenator must be optimized for CO₂ transfer at low blood flow rates. The suitability of clinical oxygenators for ECCO₂R is discussed in terms of gas exchange and functionality over a prolonged operation.     

Oxygen and CO2 transfer of a polypropylene dimpled membrane lung with variable secondary flows

Gas transfer performance is presented for one form of the Oxford membrane lung in which vortex mixing is induced in blood flow across a dimpled polypropylene membrane. Dimensional analysis has been used to define the parameters characterizing mass transfer in the device, and of three fluid mechanical parameters: Reynolds number based on peak pulsation velocity, Strouhal number, and ratio of mean to oscillatory flows, only the first has been found to affect mass transfer significantly in the ranges studied. For oxygenation, rated flows in excess of 5 l min⁻¹ m⁻² are measured. A new definition is presented for a rated flow for extracorporeal CO₂ removal and values in excess of 1.2 l min⁻¹ m⁻² are obtained.     

High-resolution suborganellar localization of Ca<Superscript>2+</Superscript>-binding protein CAS,a novel regulator of CO<Subscript>2</Subscript>-concentrating mechanism

Many aquatic algae induce a CO₂-concentrating mechanism (CCM) associated with active inorganic carbon transport to maintain high photosynthetic affinity using dissolved inorganic carbon even in low-CO₂ (LC) conditions. In the green alga Chlamydomonas reinhardtii, a Ca²⁺-binding protein CAS was identified as a novel factor regulating the expression of CCM-related proteins including bicarbonate transporters. Although previous studies revealed that CAS associates with the thylakoid membrane and changes its localization in response to CO₂ and light availability, its detailed localization in the chloroplast has not been examined in vivo. In this study, high-resolution fluorescence images of CAS fused with a Chlamydomonas-adapted fluorescence protein, Clover, were obtained by using a sensitive hybrid detector and an image deconvolution method. In high-CO₂ (5% v/v) conditions, the fluorescence signals of Clover displayed a mesh-like structure in the chloroplast and part of the signals discontinuously overlapped with chlorophyll autofluorescence. The fluorescence signals gathered inside the pyrenoid as a distinct wheel-like structure at 2 h after transfer to LC-light condition, and then localized to the center of the pyrenoid at 12 h. These results suggest that CAS could move in the chloroplast along the thylakoid membrane in response to lowering CO₂ and gather inside the pyrenoid during the operation of the CCM.     

Enhanced Chlorella vulgaris Buitenzorg growth by photon flux density alteration in serial photobioreactors

Microalgae perform oxygenic photosynthesis and are capable of taking up a large amount of CO₂, using an inducible CO₂ concentrating mechanism (CCM), and fixing CO₂ into higher compounds. These characteristics make the microalgae potentially useful for removal and utilization of CO₂ emitted from industrial plants and, generally, the usage of photosynthetic microorganisms has increased and significantly improved as a solution for CO₂ emissions. In this light and based on previous research using Anabaena cylindrica IAM M1 and Spirulina platensis IAM M 135, enhancement was sought for CO₂ fixation and biomass production by Chlorella vulgaris Buitenzorg by increasing the photon flux density concurrent with increases in culture biomass during the cellular growth phase and was compared to cultures of Chlorella grown at optimal constant illumination, with all cultures grown using Bennick basal medium, 29°C, and a flow of 1.0 atm. 10% CO₂ enriched air delivered to three in serial photobioreactors of 0.200 dm³ capacity each. The results showed that increasing illumination during culture increased biomass production of Chlorella by ∼60% as well as increased CO₂ fixation ability by ∼7.0%. It was also demonstrated that the non-competitive inhibition of [HCO₃ ⁻] as a carbon source significantly affected the cultivation in both the increasing and constant photon flux density regimes.     

CO2 in large-scale and high-density CHO cell perfusion culture

Productivity in a CHO perfusion culture reactor was maximized when pCO₂ was maintained in the range of 30–76 mm Hg. Higher levels of pCO₂ (> 150 mm Hg) resulted in CHO cell growth inhibition and dramatic reduction in productivity. We measured the oxygen utilization and CO₂ production rates for CHO cells in perfusion culture at 5.55×10^-17 mol cell^-1 sec^-1 and 5.36×10^-17 mol cell^-1 sec^-1 respectively. A simple method to directly measure the mass transfer coefficients for oxygen and carbon dioxide was also developed. For a 500 L bioreactor using pure oxygen sparge at 0.002 VVM from a microporous frit sparger, the overall apparent transfer rates (k_La+k_AA) for oxygen and carbon dioxide were 0.07264 min^-1 and 0.002962 min^-1 respectively. Thus, while a very low flow rate of pure oxygen microbubbles would be adequate to meet oxygen supply requirements for up to 2.1×10⁷ cells/mL, the low CO₂ removal efficiency would limit culture density to only 2.4×10⁶ cells/mL. An additional model was developed to predict the effect of bubble size on oxygen and CO₂ transfer rates. If pure oxygen is used in both the headspace and sparge, then the sparging rate can be minimized by the use of bubbles in the size range of 2–3 mm. For bubbles in this size range, the ratio of oxygen supply to carbon dioxide removal rates is matched to the ratio of metabolic oxygen utilization and carbon dioxide generation rates. Using this strategy in the 500 L reactor, we predict that dissolved oxygen and CO₂ levels can be maintained in the range to support maximum productivity (40% DO, 76 mm Hg pCO₂) for a culture at 10⁷ cells/mL, and with a minimum sparge rate of 0.006 vessel volumes per minute.A = volumetric agitated gas-liquid interfacial area at the top of the liquid, 1/mB = cell broth bleeding rate from the vessel, L/minCER = carbon dioxide evolution rate in the bioreactor, mol/min[CO₂] = dissolved CO₂ concentration in liquid, M[CO₂]^* = CO₂ concentration in equilibrium with sparger gas, M[CO₂]^** = CO₂ concentration in equilibrium with headspace gas, MCO₂(1) = dissolved carbon dioxide molecule in water[C_T] = total carbonic species concentration in bioreactor medium, M[C_T]_F = total carbonic species concentration in feed medium, MD = bioreactor diameter, mD_I = impeller diameter, mD_b = the initial delivered bubble diameter, mF = fresh medium feeding rate, L/minH_L = liquid height in the vessel, mk_A = carbon dioxide transfer coefficient at liquid surface, m/mink _infA ^supO = oxygen transfer coefficient at liquid surface, m/minNomenclature     

Two-tier vessel for photoautotrophic high-density cultures

Two-tier vessels, developed for culturing of microalgae and cyanobacteria at high cell density on a shaken platform, were assembled from a flat lower chamber to be filled with a CO₂ buffer and an upper flat sterile chamber for the culture that was separated from the lower chamber by a porous polypropylene membrane. Diffusive gas exchange with the atmosphere was controlled by the O₂ outlet channel. Referred to surface area, rates of CO₂ transfer to a shaken weakly alkaline buffer solution across the membrane were higher than those reached on the conventional pathway through the free upper liquid surface. Membrane-mediated CO₂ supply enabled rapid growth of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 up to ultrahigh cell density. The biomass (dry weight) concentration of Synechococcus cultures reached more than 30 g L^?1 on a buffered medium with adequate concentrations of mineral nutrients. An increase of 15 to 20 g L^?1 was observed during repeated two-day cycles. Separate pathways for CO₂ supply and oxygen outlet prevented significant loss of CO₂. Convective gas flow through the oxygen outlet channel enabled the estimation of the O₂ generation rate. The permeability of the channel for diffusive O₂/N₂ exchange limited the O₂ concentration to a moderate value. It is concluded that shaken flat cultures using CO₂ supply through a porous hydrophobic membrane and diffusive release of O₂ through a separate pathway are promising for research on microalgae and cyanobacteria.     

Simplified methods for monitoring petroleum‐contaminated ground water and soil vapor

Portable meters and simplified gas Chromatographic (GC) techniques were investigated for monitoring volatile hydrocarbon (HC), CO₂, and O₂, concentrations in groundwater, exhaust gases, and soil vapor during in situ remediation using soil vapor extraction (SVE) and air sparging (AS). Results of groundwater samples analyzed in‐house using a headspace technique compared well to split samples analyzed by a certified analytical laboratory (r² = 0.94). SVE exhaust gas HC and CO₂ concentrations measured using a GT201 portable HC/O₂ meter and a RA‐411A meter (GasTech), respectively, were highly correlated with in‐house laboratory GC analyses (r² = 0.91). O₂ concentrations fell in a small range and meter analyses were not well correlated with laboratory analyses. Results of soil gas monitoring were not as well correlated as those for exhaust gases for HC, CO₂, or O₂, perhaps due to environmental conditions such as changes in relative humidity or the wider range of soil gas values. Overall, the meters were good indicators of vapor contamination, they greatly simplified estimates of total HC mass removal, and they allowed estimates of the biological contribution to contaminant removal during the remediation process.     

Fish red blood cell carbon dioxide transport in vitro: A comparative study

Red blood cell (rbc) carbon dioxide transport was examined in vitro in three teleosts (Oncorhynchus mykiss, Anguilla anguilla, Scophthalmus maximus) and an elasmobranch (Scyliorhinus canicula) using a radioisotopic assay that measures the net conversion of plasma HCO₃⁻ to CO₂. The experiments were designed to compare the intrinsic rates of rbc CO₂ excretion and the impact of haemoglobin oxygenation/deoxygenation among the species.Under conditions simulating in vivo levels of plasma HCO₃⁻ and natural haematocrits, the rate of whole blood CO₂ excretion varied between 14.0 μmol ml⁻¹ h⁻¹ (S. canicula) and 17.6 μmol ml⁻¹ h⁻¹ (O. mykiss). The rate of CO₂ excretion in separated plasma was significantly greater in the dogfish, S. canicula. The contribution of the rbc to overall whole blood CO₂ excretion was low in the dogfish (46 ± 6%) compared to the teleosts (trout, 71 ± 4%; turbot, 64 ± 5%; eel, 55 ± 3%).To eliminate the naturally occurring differences in haematocrit and plasma [HCO₃⁻] as inter-specific variables, the rates of whole blood CO₂ excretion were determined in blood that had been resuspended to constant [HCO₃⁻] (5 mmol⁻¹) and haematocrit (20%) in appropriate teleost and elasmobranch Ringer solutions. Under such normalized conditions, the rate of whole blood CO₂ excretion was significantly higher in the turbot (22.4 ± 1.3 μmol ml⁻¹ h⁻¹) in comparison to the other species (16.4–18.4 μmol ml⁻¹ h⁻¹) and thus revealed a greater intrinsic rate of rbc CO₂ excretion in the turbot.To study the contribution of Bohr protons, the rates of whole blood CO₂ excretion were assessed in blood subjected to rapid oxygenation during the initial phase of the 3 min assay period. Rapid oxygenation significantly enhanced the rate of CO₂ excretion in the teleosts but not in the elasmobranch. The extent of the increase provided by the rapid oxygenation of haemoglobin was a linear function of the extent of the Haldane effect, as quantified in each species from in vitro CO₂ dissociation (combining) curves. Under steady-state conditions, deoxygenated blood exhibited greater rates of CO₂ excretion than oxygenated blood in the teleosts but not in the elasmobranch. As a consequence of the Haldane effect, rbc intracellular pH was increased in the teleosts by deoxygenation but was unaltered in the elasmobranch.The results, by extrapolation, suggest that the rates of CO₂ excretion in vivo are influenced by the magnitude of the Haldane effect and the extent of haemoglobin oxygenation during gill transit in addition to the intrinsic rate at which the rbc converts plasma HCO₃⁻ to CO₂.     

CO2 permeability of the rat erythrocyte membrane and its inhibition

We have studied the CO₂ permeability of the erythrocyte membrane of the rat using a mass spectrometric method that employs ¹⁸O-labelled CO₂. The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO₃⁻ permeability. For normal rat erythrocytes, we find at 37 °C a CO₂ permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10⁻³cm/s. We studied whether the rat erythrocyte membrane possesses protein CO₂ channels similar to the human red cell membrane by applying the potential CO₂ channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO₂ permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO₂ channels that are responsible for at least 50% of its CO₂ permeability.     

Biosynthesis of a 42-kD Polypeptide in the Cytoplasmic Membrane of the Cyanobacterium Anacystis nidulans Strain R2 during Adaptation to Low CO(2) Concentration

When cells of Anacystis nidulans strain R2 grown under high CO₂ conditions (3%) were transferred to low CO₂ conditions (0.05%), their ability to accumulate inorganic carbon (C_i) increased up to 8 times. Cytoplasmic membranes (plasmalemma) isolated at various stages of low CO₂ adaptation were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was a marked increase of a 42-kilodalton polypeptide in the cytoplasmic membrane during adaptation; a linear relationship existed between the amount of this polypeptide and the C_i-accumulating capability of the cells. No significant changes were observed during this process in the amount of other polypeptides in the cytoplasmic membranes or in the polypeptide profiles of the thylakoid membranes, cell walls, and soluble fractions. Spectinomycin, an inhibitor of protein biosynthesis, inhibited both the increase of the 42-kilodalton polypeptide and the induction of high C_i-accumulating capability. The incorporation of [³⁵S]sulfate into membrane proteins was greatly reduced during low CO₂ adaptation. Radioautograms of the ³⁵S-labeled membrane proteins revealed that synthesis of the 42-kilodalton polypeptide in the cytoplasmic membrane was specifically activated during the adaptation, while that of most other proteins was greatly suppressed. These results suggested that the 42-kilodalton polypeptide in the cytoplasmic membrane is involved in the active C_i transport by A. nidulans strain R2 and its synthesis under low CO₂ conditions leads to high C_i-transporting activity.     

Carbon capture and sequestration by a treatment wetland

An understanding of detailed kinetic of CO₂ removal by plants can lead to an effective design of the phytoremediation process for anthropogenic CO₂ reduction. This study examines the CO₂ removal rates of five wetland plants (Cyperus alternifolius, Dracaena fragrans, Iris ensata, Iris setosa and Thalia dealbata) by using saturation reaction and first-order reaction kinetic equations. It was determined that the elevation of CO₂ levels stimulated the plant-CO₂ uptake rate. The maximum CO₂ removal rates (k) of plants were found to range between 0.76 and 1.21 g m^?2 h^?1. The magnitude of first-order kinetic coefficient of plants (k′) had a close relationship with CO₂ level at half-velocity (K). For consistency, the same kinetics were applied to the continuous flow experiment. A saturation kinetic approach was well suited to estimate the removal rate of CO₂ in continuous flow system, while a first-order kinetic approach was limited to inflow CO₂ levels below 500 ppm.     

The effect of carbonic anhydrase inhibition on the velocity of thrombin-stimulated platelet aggregation under physiological conditions

We have studied the effect of ethoxzolamide, a specific carbonic anhydrase inhibitor, on the velocity of thrombin-stimulated platelet aggregation. After preincubation of platelet rich plasma with 10^?6 M ethoxzolamide the velocity of platelet aggregation was reduced by about 40%. Between 10^?11 M and 10^?10M ethoxzolamide was necessary to achieve a half-maximal diminution of the aggregation velocity. An identical maximal reduction of the velocity of aggregation as with ethoxzolamide could be achieved by a nearly complete removal of CO₂ from the platelet rich plasma. These results suggest that the intracellular CO₂ hydration-dehydration reaction is involved in the activation of human platelets by thrombin. It is possible that the cytosolic carbonic anhydrase of platelets provides a rapid source of the protons that are transferred across the plasma membrane during the activation process.     

Spinal cord transection inhibits HR reduction in anesthetized rats immersed in an artificial CO<Subscript>2</Subscript>-hot spring bath

Like humans, the heart rate (HR) of anesthetized rats immersed in CO₂-water is lower than that when immersed in tap water at the same temperature. To investigate the afferent signal pathway in the mechanism of HR reduction, Wistar rats were anesthetized with urethane and then the spinal cord was transected between T₄ and T₅. The animals were immersed up to the axilla in a bathtub of tap-water (CO₂ contents: 10–20 mg·l⁻¹) or of CO₂-water (965–1,400 mg·l⁻¹) at 35°C while recording HR, arterial blood pressure, and arterial blood gas parameters (PaCO₂, PaO₂, pH). Arterial blood gas parameters did not change during immersion, irrespective of CO₂ concentration of the bath water, whereas the HR was reduced in the CO₂-water bath. The inhalation of CO₂-mixed gas (5 % CO₂, 20 % O₂, 75 % N₂) resulted in increased levels of blood gases and an increased HR during immersion in all types of water tested. The HR reduction observed in sham transected control animals immersed in CO₂-water disappeared after subsequent spinal cord transection. These results show that the dominant afferent signal pathway to the brain, which is involved in inducing the reduced HR during immersion in CO₂-water, is located in the neuronal route and not in the bloodstream.     

Fish gill water boundary layer: a site of linkage between carbon dioxide and ammonia excretion

Summary Carbon dioxide excreted across fish gills is hydrated catalytically to form HCO ₃ ^– and H⁺ ions in water near the gill surface. We tested the possibility that CO₂ excretion is functionally linked to ammonia excretion through chemical reactions in the gill-water boundary layer. A bloodperfused trout head preparation was utilized in which the convective and diffusive components of branchial gas transfer were controlled. Pre-incubation of blood perfusate with the carbonic anhydrase inhibitor, acetazolamide, reduced both carbon dioxide and ammonia excretion in the blood-perfused preparation. Increasing the buffering capacity of inspired ventilatory water significantly reduced ammonia excretion, but carbon dioxide excretion was unaffected. Each of these experimental treatments significantly reduced the acidification of ventilatory water flowing over the gills. It is proposed that the catalysed conversion of excreted CO₂ to form HCO ₃ ^– and H⁺ ions provides a continual supply of H⁺ ions need for the removal of NH₃ as NH ₄ ⁺ . We suggest, therefore, that acidification of boundary layer water by CO₂ enhances blood-to-water NH₃ diffusion gradients and facilitates ammonia excretion.     

Circadian rhythms inKalanchoë: the pathway of14CO2 fixation during prolonged light

¹⁴CO₂ was applied repeatedly at 3- to 6-h intervals toKalanchoë daigremontiana leaves during continuous light of differing irradiances. The circadian rhythm in net CO₂ uptake in gasexchange measurements and its disappearance at high irradiances was confirmed by oscillating rates of¹⁴CO₂ incorporation. At 10–30 W m^-2 a markedly circadian oscillation in the¹⁴CO₂-uptake rate was measured; with increasing energy fluence rate the oscillation levelled off at a constant high uptake rate. The labelling patterns obtained during the 10 min of¹⁴CO₂ fixation indicated that the rhythm of CO₂ exchange is the consequence of a rhythmic behaviour in the C₄ pathway of CO₂ fixation. During the mininum of¹⁴CO₂ uptake no C₄ products were labelled; however, substantial amounts of label were transferred to C₄ products during the peaks of¹⁴CO₂ uptake. Metabolism of C₃ and C₄ products was also studied in pulsechase experiments at different points of the circadian cycle. In bright light (100 W m^-2), when the¹⁴CO₂ uptake was constantly high, the transfer of label into C₄ products (malic acid) was high in spite of the fact that the malate pool is known to be reduced to a permanently low level under these conditions. This led us to the conclusion that it is not the capacity of the phosphoenolpyruvatecarboxylase-mediated CO₂ fixation but rather the storage of malic acid in the vacuole that is disturbed under bright-light conditions when the circadian oscillation levelled off.Abbreviations CAM Crassulacean acid metabolism - LL continuous light - PEP phosphoenolpyruvate     

Modeling of carbon dioxide mass transfer behavior in attached cultivation photobioreactor using the analysis of the pH profiles

The CO₂ mass transfer model associated with growth kinetics of microalgal biofilm in attached cultivation photobioreactor was developed and verified by using the analysis of pH profiles which were in equilibrium with inorganic carbon components concentrations (CO₂, H₂CO₃, HCO₃ ⁻ and CO₃ ²⁻) in medium. Model simulation results showed that the model well presented the biofilm growth process. The overall volumetric mass transfer coefficient of CO₂ was more influenced by CO₂ concentration in aerated gas but less by gas aeration rate and medium circulation rate. Other bio-kinetic parameters related with the microalgal biofilm such as CO₂ diffusion coefficient in biofilm, Monod maximum utilization rate of CO₂, lag phase duration of biofilm and half-saturation CO₂ concentration in the biofilm were independent on operational conditions. The pH profiles provided a way to monitor the variations of inorganic carbon concentrations of medium and to regulate the cultivation of attached microalgal biofilm by CO₂ supplement.

     

Soil Greenhouse Gas Emissions and Carbon Dynamics of a No-Till,Corn-Based Cellulosic Ethanol Production System

Crop residues like corn (Zea mays L.) stover perform important functions that promote soil health and provide ecosystem services that influence agricultural sustainability and global biogeochemical cycles. We evaluated the effect of corn stover removal from a no-till, corn-soybean (Glycine max (L.) Merr) rotation on soil greenhouse gas (GHG; CO₂, N₂O, CH₄) fluxes, crop yields, and soil organic carbon (SOC) dynamics. We conducted a 4-year study using replicated field plots managed with two levels of corn stover removal (none; 55 % stover removal) for four complete crop cycles prior to initiation of ground surface gas flux measurements. Corn and soybean yields were not affected by stover removal with yields averaging 7.28 Mg ha^?1 for corn and 2.64 Mg ha^?1 for soybean. Corn stover removal treatment did not affect soil GHG fluxes from the corn phase; however, the treatment did significantly increase (107 %, P?=?0.037) N₂O fluxes during the soybean phase. The plots were a net source of CH₄ (～0.5 kg CH₄-C ha^?1 year^?1 average of all treatments and crops) during the generally wet study duration. Soil organic carbon stocks increased in both treatments during the 4-year study (initiated following 8 years of stover removal), with significantly higher SOC accumulation in the control plots compared to plots with corn stover removal (0–15 cm, P?=?0.048). Non-CO₂ greenhouse gas emissions (945 kg CO₂-eq ha^?1 year^?1) were roughly half of SOC (0–30 cm) gains with corn stover removal (1.841 Mg CO₂-eq ha^?1 year^?1) indicating that no-till practices greatly improve the viability of biennial corn stover harvesting under local soil-climatic conditions. Our results also show that repeated corn stover harvesting may increase N loss (as N₂O) from fields and thereby contribute to GHG production and loss of potential plant nutrients.     

Oxygen-18 Exchange as a Measure of Accessibility of CO(2) and HCO(3) to Carbonic Anhydrase in Chlorella vulgaris (UTEX 263)

We have measured the exchange of ¹⁸O between CO₂ and H₂O in stirred suspensions of Chlorella vulgaris (UTEX 263) using a membrane inlet to a mass spectrometer. The depletion of ¹⁸O from CO₂ in the fluid outside the cells provides a method to study CO₂ and HCO₃⁻ kinetics in suspensions of algae that contain carbonic anhydrase since ¹⁸O loss to H₂O is catalyzed inside the cells but not in the external fluid. Low-CO₂ cells of Chlorella vulgaris (grown with air) were added to a solution containing ¹⁸O enriched CO₂ and HCO₃⁻ with 2 to 15 millimolar total inorganic carbon. The observed depletion of ¹⁸O from CO₂ was biphasic and the resulting ¹⁸C content of CO₂ was much less than the ¹⁸O content of HCO₃⁻ in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO₃⁻ into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 × 10⁻⁴ s⁻¹ due to our experimental errors. The Fick's law rate constant for entry of CO₂ to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO₂ flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO₂ entry into the cell or both. The CO₂ hydration activity inside the cells was 160 times the uncatalyzed CO₂ hydration rate.     

Photorespiration in Air and High CO(2)-Grown Chlorella pyrenoidosa

Oxygen inhibition of photosynthesis and CO₂ evolution during photorespiration were compared in high CO₂-grown and air-grown Chlorella pyrenoidosa, using the artificial leaf technique at pH 5.0. High CO₂ cells, in contrast to air-grown cells, exhibited a marked inhibition of photosynthesis by O₂, which appeared to be competitive and similar in magnitude to that in higher C₃ plants. With increasing time after transfer to air, the photosynthetic rate in high CO₂ cells increased while the O₂ effect declined. Photorespiration, measured as the difference between ¹⁴CO₂ and ¹²CO₂ uptake, was much greater and sensitive to O₂ in high CO₂ cells. Some CO₂ evolution was also present in air-grown algae; however, it did not appear to be sensitive to O₂. True photosynthesis was not affected by O₂ in either case. The data indicate that the difference between high CO₂ and air-grown algae could be attributed to the magnitude of CO₂ evolution. This conclusion is discussed with reference to the oxygenase reaction and the control of photorespiration in algae.     

Side chain oxidation of 1,25-dihydroxyvitamin D3 in the rat: Effect of removal of the intestine

Removal of the jejunum, ileum and colon in the rat reduces the amount of ¹⁴CO₂ formed after an intravenous injection of 325 pmoles of 1,25-dihydroxy-[26,27-¹⁴C]vitamin D₃, by 65.2 ± 13.2% at 4 hours and 67.1 ± 9.12% at 8 hours. This suggests that the intestine may be one of the sites where side chain oxidation occurs. It is possible that the liver may also be involved in this process as removal of a large portion of the gut may disturb hepatic metabolism secondary to a reduction in portal blood flow. The process is not bacterial inasmuch as “germ free” animals produce at least as much ¹⁴CO₂ after the administration of 1,25-dihydroxy-[26,27-¹⁴C]vitamin D₃ as do non-germ free controls.