Measurement and interpretation of the oxygen isotope composition of carbon dioxide respired by leaves in the dark

We measured the oxygen isotope composition (delta(18)O) of CO(2) respired by Ricinus communis leaves in the dark. Experiments were conducted at low CO(2) partial pressure and at normal atmospheric CO(2) partial pressure. Across both experiments, the delta(18)O of dark-respired CO(2) (delta(R)) ranged from 44 per thousand to 324 per thousand (Vienna Standard Mean Ocean Water scale). This seemingly implausible range of values reflects the large flux of CO(2) that diffuses into leaves, equilibrates with leaf water via the catalytic activity of carbonic anhydrase, then diffuses out of the leaf, leaving the net CO(2) efflux rate unaltered. The impact of this process on delta(R) is modulated by the delta(18)O difference between CO(2) inside the leaf and in the air, and by variation in the CO(2) partial pressure inside the leaf relative to that in the air. We developed theoretical equations to calculate delta(18)O of CO(2) in leaf chloroplasts (delta(c)), the assumed location of carbonic anhydrase activity, during dark respiration. Their application led to sensible estimates of delta(c), suggesting that the theory adequately accounted for the labeling of CO(2) by leaf water in excess of that expected from the net CO(2) efflux. The delta(c) values were strongly correlated with delta(18)O of water at the evaporative sites within leaves. We estimated that approximately 80% of CO(2) in chloroplasts had completely exchanged oxygen atoms with chloroplast water during dark respiration, whereas approximately 100% had exchanged during photosynthesis. Incorporation of the delta(18)O of leaf dark respiration into ecosystem and global scale models of C(18)OO dynamics could affect model outputs and their interpretation.     

Partitioning respiration of C3-C4 mixed communities using the natural abundance 13C approach--testing assumptions in a controlled environment

Contributions of C3 and C4 plants to respiration of C3-C4 ecosystems can be estimated on the basis of their contrasting 13C discrimination. But accurate partitioning requires accurate measurements of the isotope signature of whole system respiratory CO2 (deltaR), and of its members (delta3 and delta4). Unfortunately, experimental determination of representative delta3 and delta4 values is virtually impossible in nature, generating a need for proxies (surrogates) of delta3 and delta4 values (e.g., the delta of leaf biomass). However, recent evidence indicates that there may be systematic differences among the delta of respiratory and biomass components. Thus, partitioning may be biased depending on the proxy. We tested a wide range of biomass- and respiration-based delta proxies for the partitioning of respiration of mixed Lolium perenne (C3) - Paspalum dilatatum (C4) stands growing at two temperatures inside large 13CO2/ 12CO2 gas exchange chambers. Proxy-based partitioning was compared with results of reference methods, including (i) the delta of whole plant respiratory CO2 (delta3 and delta4) or (ii) respiration rate of intact C3 and C4 plants. Results of the reference methods agreed near perfectly. Conversely, some proxies yielded erroneous partitioning results. Partitioning based on either the delta of shoot or root respiratory CO2 produced the worst bias, because shoot respiratory CO2 was enriched in 13C by several per thousand and root respiratory CO2 was depleted by several per thousand relative to whole plant respiratory CO2. Use of whole plant or whole shoot biomass delta gave satisfactory partitioning results under the constant conditions of the experiments, but their use in natural settings is cautioned if environmental conditions are variable and the time scales of respiration partitioning differ strongly from the residence time of C in biomass. Other biomass-based proxies with faster turnover (e.g., leaf growth zones) may be more useful in changing conditions.     

Effect of aeration and redox potential on the biosynthesis of the antibiotic imbricin]

The relationship between imbricin biosynthesis by Streptomyces imbricatus and the medium aeration and redox potential (Eh) was studied. The influence of the oxygen dissolution velocity within the ranges of 2.9 to 0.5 g O2/l.h was investigated and it was shown that the highest yield of the antibiotic was provided by the maximum velocity. At the background of the intensive aeration (2.9 g O2/l.h) decreasing of Eh by reducing agents such as ascorbic acid, L-thyrosin or K4Fe(CN)6 stimulated the biosynthesis whereas at the lower velocities the process proved to be inhibited. Under conditions of insufficient aeration the biosynthesis stimulation could be provided by increasing the medium Eh by acidifying agents such as K2Cr2O7, K3Fe(CN)6 or KMnO4. It was concluded that intensive synthesis of imbricin required not only efficient aeration but also definite levels of the medium redox potential.     

Determination of CO₂ sensitivity of micro-organisms in shaken bioreactors. II. Novel online monitoring method

In the present study, a new online monitoring method for the determination of the CO? sensitivity of micro-organisms, based on the values of the respiration factors [OTR (oxygen transfer rate) and CTR (carbon dioxide transfer rate)], obtained by using the RAMOS (respiratory activity monitoring system) device considering a variety of aeration rates in the measuring flask, is investigated. Based on the data of the OTR, obtained by RAMOS under a variety of specific aeration rates, the proposed new method was developed as an online monitoring method for CO? sensitivity of micro-organisms in shaken bioreactors. A maximum accumulated CO? concentration of 12% was derived in applied methods, provided that the cultivation system is carried out under optimal conditions. Additionally, to predict these conditions, an unsteady-state gas transfer model in shaken bioreactors would be very advantageous. The data of OTR obtained using the RAMOS device were analysed and recalculated by a programme considering the calibration factor (Cf). The major advantage of the new method is the possibility to determine the metabolic activity, regardless of manual sampling.     

Dynamics of dissolved oxygen isotopic ratios: a transient model to quantify primary production,community respiration,and air–water exchange in aquatic ecosystems

Dissolved O(2) is an important aquatic ecosystem health indicator. Metabolic and gas exchange (G) rates, which control O(2) concentration, are affected by nutrient loading and other environmental factors. Traditionally, aquatic metabolism has been reported as primary production:community respiration (P:R) ratios using diel measurements and interpretations of dissolved O(2) and/or CO(2) concentrations, and recently using stable isotopes (delta(18)O, Delta(17)O) and steady state assumptions. Aquatic ecosystems, such as rivers and ponds, are not at steady state and exhibit diel changes, so steady state approaches are often inappropriate. A dynamic O(2) stable isotope model (photosynthesis-respiration-gas exchange; PoRGy) is presented here, requiring a minimum of parameters to quantify daily averaged P, R, and G rates under transient field conditions. Unlike steady state approaches, PoRGy can address scenarios with 100% O(2) saturation but with delta(18)O-O(2) values that are not at air equilibrium. PoRGy successfully accounts for isotopic G when applied to an oxygen isotope equilibration laboratory experiment. PoRGy model results closely matched the diel O(2) and delta(18)O-O(2) data from three field sites with different P:R:G ratios and various P, R and G rates. PoRGy provides a new research tool to assess ecosystem health and to pose environmental impact-driven questions. Using daily averaged rates was successful and thus they can be used to compare ecosystems across seasons and landscapes.     

Estimating cell specific oxygen uptake and carbon dioxide production rates for mammalian cells in perfusion culture

We present robust methods for online estimation of cell specific oxygen uptake and carbon dioxide production rates (q(O2) and q(CO2), respectively) during perfusion cultivation of mammalian cells. Perfusion system gas and liquid phase mass balance expressions for oxygen and carbon dioxide were used to estimate q(O2), q(CO2) and the respiratory quotient (RQ) for Chinese hamster ovary (CHO) cells in perfusion culture over 12 steady states with varying dissolved oxygen (DO), pH, and temperature set points. Under standard conditions (DO = 50%, pH = 6.8, T = 36.5°C), q(O2) and q(CO2) ranges were 5.14-5.77 and 5.31-6.36 pmol/cell day, respectively, resulting in RQ values of 0.98-1.14. Changes to DO had a slight reducing effect on respiration rates with q(O2) and q(CO2) values of 4.64 and 5.47, respectively, at DO = 20% and 4.57 and 5.12 at DO = 100%. Respiration rates were lower at low pH with q(O2) and q(CO2) values of 4.07 and 4.15 pmol/cell day at pH = 6.6 and 4.98 and 5.36 pmol/cell day at pH = 7. Temperature also impacted respiration rates with respective q(O2) and q(CO2) values of 3.97 and 4.02 pmol/cell day at 30.5°C and 5.53 and 6.25 pmol/cell day at 37.5°C. Despite these changes in q(O2) and q(CO2) values, the RQ values in this study ranged from 0.98 to 1.23 suggesting that RQ was close to unity. Real-time q(O2) and q(CO2) estimates obtained using the approach presented in this study provide additional quantitative information on cell physiology both during bioprocess development and commercial biotherapeutic manufacturing.     

Large daily variation in 13C-enrichment of leaf-respired CO2 in two Quercus forest canopies

The use of the 13C : 12C isotopic ratio (delta13C) of leaf-respired CO2 to trace carbon fluxes in plants and ecosystems is limited by little information on temporal variations in delta13C of leaf dark-respired CO2 (delta13Cr) under field conditions. Here, we explored variability in delta13Cr and its relationship to key respiratory substrates from collections of leaf dark-respired CO2, carbohydrate extractions and gas exchange measurements over 24-h periods in two Quercus canopies. Throughout both canopies, delta13Cr became progressively 13C-enriched during the photoperiod, by up to 7%, then 13C-depleted at night relative to the photoperiod. This cycle could not be reconciled with delta13C of soluble sugars (delta13Css), starch (delta13Cst), lipids (delta13Cl), cellulose (delta13Cc) or with calculated photosynthetic discrimination (Delta). However, photoperiod progressive enrichment in delta13Cr was correlated with cumulative carbon assimilation (r2 = 0.91). We concluded that there is considerable short-term variation in delta13Cr in forest canopies, that it is consistent with current hypotheses for 13C fractionation during leaf respiration, that leaf carbohydrates cannot be used as surrogates for delta13Cr, and that diel changes in leaf carbohydrate status could be used to predict changes in delta13Cr empirically.     

Adaptation of the "Dynamic Method" for measuring the specific respiration rate in oxygen transfer systems through diffusion membrane

Monitoring the specific respiration rate (Q(O2)) is a valuable tool to evaluate cell growth and physiology. However, for low Q(O2) values the accuracy may depend on the measurement methodology, as it is the case in animal cell culture. The widely used "Dynamic Method" imposes serious difficulties concerning oxygen transfer cancellation, especially through membrane oxygenation. This paper presents an improved procedure to this method, through an automated control of the gas inlet composition that can minimize the residual oxygen transfer driving force during the Q(O2) measurement phase. The improved technique was applied to animal cell cultivation, particularly three recombinant S2 (Drosophila melanogaster) insect cell lines grown in a membrane aeration bioreactor. The average measurements of the proposed method reached 98% of stationary liquid phase balance method, taken as a reference, compared to 21% when the traditional method was used. Furthermore, this methodology does not require knowledge of the volumetric transfer coefficient k(L)a, which may vary during growth.     

Effect of the carbon source and aeration conditions on homoserine lysine biosynthesis in the threonine-dependent mutant Brevibacterium flavum 2T

The effect of two carbon sources (sucrose and acetate), aeration conditions and threonine concentration on the homoserine and lysine biosynthesis by the threonine-dependent mutant Brevibacterium flavum 2T was examined. It was demonstrated that acetate provided the predominant synthesis of homoserine to a far greater extent than sucrose (with the weight/weight ratio of homoserine : lysine being 2.5-5.0 and 0.8-1,2, respectively). The maximal level of homoserine and lysine was 18-21 and 3-7 g/l on the acetate containing medium and 18-22 and 12-16 g/l on the sucrose containing medium, respectively. On sucrose the total amount of amino acids and the total yield of products as related to the consumed substrate were greater than on acetate. Using the sucrose medium, the effect of aeration conditions and threonine concentration on the biosynthesis of both compounds was investigated. With an aeration increase from 1.3 to 4.6 g O2/l.hr the optimal concentration of threonine in the medium grow. The biosynthesis of homoserine was less sensitive to the inhibitory effect of excessive threonine than that of lysine. With an increase of the threonine concentration in the medium from 0.25 to 3.0 g/l the ratio homoserine : lysine grew from 1.03 to 5.20 (with the sulphite number being 4.6 g O2/l.hr). This effect was independent of the aeration conditions.     

Cultivation of microplantlets derived from the marine red alga Agardhiella subulata in a stirred tank photobioreactor

Macrophytic marine red algae are a diverse source of bioactive natural compounds. "Microplantlet" suspension cultures established from red algae are potential platforms for biosynthesis of these compounds, provided suitable bioreactor configurations for mass culture can be identified. The stirred tank bioreactor offers high rates of gas-liquid mass transfer, which may facilitate the delivery of the CO(2) in the aeration gas to the phototrophic microplantlet suspension culture. Therefore, the effects of impeller speed and CO(2) delivery on the long-term production of microplantlet biomass of the model red alga Agardhiella subulata was studied within a stirred tank photobioreactor equipped with a paddle blade impeller (D(i)/D(T) = 0.5). Nutrient medium replacement was required for sustained biomass production, and the biomass yield coefficient based on nitrate consumption was 1.08 +/- 0.09 g dry biomass per mmol N consumed. Biomass production went through two exponential phases of growth, followed by a CO(2) delivery limited growth phase. The CO(2)-limited growth phase was observed only if the specific growth rate in the second exponential phase of growth was at least 0.03 day(-)(1), the CO(2) delivery rate was less than 0.258 mmol CO(2) L(-)(1) culture h(-)(1), and the plantlet density was at least 10 g fresh mass L(-)(1). Increasing the aeration gas CO(2) partial pressure from 0.00035 to 0.0072 atm decreased the cultivation pH from 8.8 to 7.8, prolonged the second exponential phase of growth by increasing the CO(2) delivery rate, and also increased the photosynthetic oxygen evolution rate. Impeller speeds ranging from 60 to 250 rpm, which generated average shear rates of 2-10 s(-)(1), did not have a significant effect on biomass production rate. However, microplantlets cultivated in a stirred tank bioreactor ultimately assumed compact spherical shape, presumably to minimize exposure to hydrodynamic stress.     

A new measurement technique reveals temporal variation in delta18O of leaf-respired CO2

The oxygen isotope composition of CO(2) respired by Ricinus communis leaves (delta(18)O(R)) was measured under non-steady-state conditions with a temporal resolution of 3 min using a tunable diode laser (TDL) absorption spectrometer coupled to a portable gas exchange system. The SD of delta(18)O measurement by the TDL was +/- 0.2 per thousand and close to that of traditional mass spectrometers. Further, delta(18)O(R) values at isotopic steady state were comparable to those obtained using traditional flask sampling and mass spectrometric techniques for R. communis grown and measured in similar environmental conditions. As well as higher temporal resolution, the online TDL method described here has a number of advantages over mass spectrometric techniques. At isotopic steady state among plants grown at high light, the "one-way flux" model was required to accurately predict delta(18)O(R). A comparison of measurements and the model suggests that plants grown under low-light conditions have either a lower proportion of chloroplast CO(2) that isotopically equilibrates with chloroplast water, or more enriched delta(18)O of CO(2) in the chloroplast that has not equilibrated with local water. The high temporal resolution of isotopic measurements allowed the first measurements of delta(18)O(R) when stomatal conductance was rapidly changing. Under non-steady-state conditions, delta(18)O(R) varied between 50 and 220 per thousand for leaves of plants grown under different light and water environments, and varied by as much as 100 per thousand within 10 min for a single leaf. Stomatal conductance ranged from 0.001 to 1.586 mol m(-2) s(-1), and had an important influence on delta(18)O(R) under non-steady-state conditions not only via effects on leaf water H(2) (18)O enrichment, but also via effects on the rate of the one-way fluxes of CO(2) into and out of the leaf.     

Effect of aeration on lysine biosynthesis in nutrient media with different concentrations of components]

The lysine synthesis by the culture M. glutamicus T-3 on nutrient media with varying molasses concentrations was studied during cultivation under different aeration conditions. With an increase in the nutrient concentration the relationship between the lysine yield and aeration rate became very manifest. An elevation of aeration (Kv) from 1.2 to 6.3 g O2 1/hr increased the yield of lysine in the 15, 20 and 28% molasses medium by 3, 6 and 11 times, respectively. A decline in aeration decreased the biomass yield and increased the content of lactic acid and alanine in the culture liquid (to 19 and 4 g/l, respectively). The rate of respiration of the culture in the filtrate of the culture liquid measured in the Warburg apparatus depended on the cell age and molasses concentration in the nutrient medium and not on the aeration rate.     

Development of a strategy to control the dissolved concentrations of oxygen and carbon dioxide at constant shear in a plant cell bioreactor

To examine the effects of volatile components on plant cell growth, a bioreactor control system was developed to simultaneously control the dissolved concentrations of both oxygen and carbon dioxide. The first step in this work was to develop a mathematical model to account for gas-liquid mass transfer; biological utilization and production of O(2) and CO(2); and the series of chemical reactions of CO(2) in water. Using this model and dynamic measurements for dissolved O(2) and CO(2), it was observed that (1) both absorption and desorption of a volatile component could be described by a single mass transfer coefficient, K(l)a, and (2) K(l)a values for oxygen and carbon dioxide transfer were directly proportional. The second step of this work was to employ the mathematical model in an adaptive feed-forward strategy to control the dissolved O(2) and CO(2) concentrations by manipulating the inlet gas composition to the bioreactor. This strategy allowed dissolved concentrations to be controlled without the need for changing either the total gas flow rate or agitator speed. Adaptive control was required because the volumetric rates of O(2) and CO(2) consumption and production vary with time during long term operation and therefore these rates must be continually updated. As the final step, we demonstrated that this control strategy was capable of controlling the dissolved gas concentrations in both short- and long-term studies involving the cultivation of Catharanthus roseus plant cells.     

Influence of the intensity of oxygen mass transfer on the biosynthesis of amphotericin B

Influence of oxygen mass transfer intensity characterized by the rate of oxygen dissolution (S) and the agitation rate (n), as well as influence of dissolved oxygen concentration on the process of amphotericin B biosynthesis was studied. It was shown that S = 40 and 110 mg/l. min and n = 450 and 800 min-1 were respectively the lower and the upper levels of the optimal conditions by oxygen mass transfer during amphotericin B biosynthesis. When biosynthesis of amphotericin B was conducted under conditions of the optimal oxygen mass transfer, the dissolved oxygen concentration of about 12 to 15 per cent of the saturation level was critical for the culture respiration. Inhibition of the culture respiration and antibiotic synthesis was induced under conditions of increased oxygen mass transfer intensity (S greater than 110 mg/l. min and n greater than 800 min-1) by high intensity mechanical agitation of the fermentation broth. Under conditions of decreased oxygen mass transfer (S less than 40 mg/l. min and n = less than 450 min-1) it was induced by insufficient supply of oxygen to the culture. On the basis of the results it was shown possible to control the aeration and agitation conditions by the rate of oxygen uptake and dissolved oxygen concentration. The data should be considered in optimization of aeration and agitation conditions in biosynthesis of amphotericin B in large fermenters.     

Non-steady state effects in diurnal 180 discrimination by Picea sitchensis branches in the field

We report diurnal variations in 18O discrimination (18 delta) during photosynthesis (18 delta A) and respiration (18 delta R) of Picea sitchensis branches measured in branch chambers in the field. These observations were compared with predicted 18 delta (18 delta pred) based on concurrent measurements of branch gas exchange to evaluate steady state and non-steady state (NSS) models of foliage water 18O enrichment for predicting the impact of this ecosystem on the Delta 18O of atmospheric CO2. The non-steady state approach substantially improved the agreement between 18 delta pred and observed 18 delta (18 delta obs) compared with the assumption of isotopic steady state (ISS) for the Delta 18O signature of foliage water. In addition, we found direct observational evidence for NSS effects: extremely high apparent 18 delta values at dusk, dawn and during nocturnal respiration. Our experiments also show the importance of bidirectional foliage gas exchange at night (isotopic equilibration in addition to the net flux). Taken together, neglecting these effects leads to an underestimation of daily net canopy isofluxes from this forest by up to 30%. We expect NSS effects to be most pronounced in species with high specific leaf water content such as conifers and when stomata are open at night or when there is high relative humidity, and we suggest modifications to ecosystem and global models of delta 18O of CO2.     

18O pattern and biosynthesis of natural plant products

Oxygen atoms in plant products originate from CO(2), H(2)O and O(2), precursors with quite different delta18O values. Furthermore their incorporation by different reactions implies isotope effects. On this base the resulting non-statistical 18O distributions in natural compounds are discussed. The delta18O value of cellulose is correlated to that of the leaf water, and the observed 18O enrichment (approximately +27 per thousand) is generally attributed to an equilibrium isotope effect between carbonyl groups and water. However, as soluble and heterotrophically synthesised carbohydrates show other correlations, a non-statistical 18O distribution - originating from individual biosynthetic reactions - is postulated for carbohydrates. Similarly, the delta18O values of organic acids, carbonyl compounds, alcohols and esters indicate water-correlated, but individual 18O abundances (e.g. O from acyl groups approximately +19% above water), depending upon origin and biosyntheses. Alcoholic groups introduced by monooxygenase reactions, e.g. in sterols and phenols, show delta18O values near +5 per thousand, in agreement with an assumed isotope fractionation factor of approximately 1.02 on the reaction with atmospheric oxygen (delta18O=+23.5 per thousand). Correspondingly, a "thermodynamically ordered isotope distribution" is only observed for oxygen in some functional groups correlated to an origin from CO(2) and H(2)O, not from O(2). The individual isotopic increments of functional groups permit the prediction of global delta18O values of natural compounds on the basis of their biosynthesis.     

Effect of inspiratory resistive loading on control of ventilation during progressive exercise

Eight healthy volunteers performed gradational tests to exhaustion on a mechanically braked cycle ergometer, with and without the addition of an inspiratory resistive load. Mean slopes for linear ventilatory responses during loaded and unloaded exercise [change in minute ventilation per change in CO2 output (delta VE/delta VCO2)] measured below the anaerobic threshold were 24.1 +/- 1.3 (SE) = l/l of CO2 and 26.2 +/- 1.0 l/l of CO2, respectively (P greater than 0.10). During loaded exercise, decrements in VE, tidal volume, respiratory frequency, arterial O2 saturation, and increases in end-tidal CO2 tension were observed only when work loads exceeded 65% of the unloaded maximum. There was a significant correlation between the resting ventilatory response to hypercapnia delta VE/delta PCO2 and the ventilatory response to VCO2 during exercise (delta VE/delta VCO2; r = 0.88; P less than 0.05). The maximal inspiratory pressure generated during loading correlated with CO2 sensitivity at rest (r = 0.91; P less than 0.05) and with exercise ventilation (delta VE/delta VCO2; r = 0.83; P less than 0.05). Although resistive loading did not alter O2 uptake (VO2) or heart rate (HR) as a function of work load, maximal VO2, HR, and exercise tolerance were decreased to 90% of control values. We conclude that a modest inspiratory resistive load reduces maximum exercise capacity and that CO2 responsiveness may play a role in the control of breathing during exercise when airway resistance is artificially increased.     

Fractionation of the three stable oxygen isotopes by oxygen-producing and oxygen-consuming reactions in photosynthetic organisms

The triple isotope composition (delta17O and delta18O) of dissolved O2 in the ocean and in ice cores was recently used to assess the primary productivity over broad spatial and temporal scales. However, assessment of the productivity with the aid of this method must rely on accurate measurements of the 17O/16O versus 18O/16O relationship in each of the main oxygen-producing and -consuming reactions. Data obtained here showed that cleavage of water in photosystem II did not fractionate oxygen isotopes; the delta18O and delta17O of the O2 evolved were essentially identical to those of the substrate water. The fractionation slopes for the oxygenase reaction of Rubisco and respiration were identical (0.518 +/- 0.001) and that of glycolate oxidation was 0.503 +/- 0.002. There was a considerable difference in the slopes of O2 photoreduction (the Mehler reaction) in the cyanobacterium Synechocystis sp. strain PCC 6803 (0.497 +/- 0.004) and that of pea (Pisum sativum) thylakoids (0.526 +/- 0.001). These values provided clear and independent evidence that the mechanism of O2 photoreduction differs between higher plants and cyanobacteria. We used our method to assess the magnitude of O2 photoreduction in cyanobacterial cells maintained under conditions where photorespiration was negligible. It was found that electron flow to O2 can be as high as 40% that leaving photosystem II, whereas respiratory activity in the light is only 6%. The implications of our findings to the evaluation of specific O2-producing or -consuming reactions, in vivo, are discussed.     

The effect of aeration and agitation on tetracycline biosynthesis

The results of the study on relation between tetracycline biosynthesis and the specific power input for agitation in pilot plant apparatus was studied. No correlation was observed between the levels of tetracycline biosynthesis and changes in the specific power input within a range of 0.6 to 2.3 kW/m3 at the expense of changes in the mixer diameter and the agitation rate, when the aeration rate was constant. It was shown that the aeration conditions were most significant for tetracycline biosynthesis. The study provided determination of the optimal aeration conditions for biosynthesis of tetracycline.     

Discrimination in the dark. Resolving the interplay between metabolic and physical constraints to phosphoenolpyruvate carboxylase activity during the crassulacean acid metabolism cycle

A model defining carbon isotope discrimination (delta13C) for crassulacean acid metabolism (CAM) plants was experimentally validated using Kalanchoe daigremontiana. Simultaneous measurements of gas exchange and instantaneous CO2 discrimination (for 13C and 18O) were made from late photoperiod (phase IV of CAM), throughout the dark period (phase I), and into the light (phase II). Measurements of CO2 response curves throughout the dark period revealed changing phosphoenolpyruvate carboxylase (PEPC) capacity. These systematic changes in PEPC capacity were tracked by net CO2 uptake, stomatal conductance, and online delta13C signal; all declined at the start of the dark period, then increased to a maximum 2 h before dawn. Measurements of delta13C were higher than predicted from the ratio of intercellular to external CO2 (p(i)/p(a)) and fractionation associated with CO2 hydration and PEPC carboxylations alone, such that the dark period mesophyll conductance, g(i), was 0.044 mol m(-2) s(-1) bar(-1). A higher estimate of g(i) (0.085 mol m(-2) s(-1) bar(-1)) was needed to account for the modeled and measured delta18O discrimination throughout the dark period. The differences in estimates of g(i) from the two isotope measurements, and an offset of -5.5 per thousand between the 18O content of source and transpired water, suggest spatial variations in either CO2 diffusion path length and/or carbonic anhydrase activity, either within individual cells or across a succulent leaf. Our measurements support the model predictions to show that internal CO2 diffusion limitations within CAM leaves increase delta13C discrimination during nighttime CO2 fixation while reducing delta13C during phase IV. When evaluating the phylogenetic distribution of CAM, carbon isotope composition will reflect these diffusive limitations as well as relative contributions from C3 and C4 biochemistry.