Photoautotrophic nutrient utilization and limitation during semi‐continuous growth of Synechocystis sp. PCC6803

Microbial photosynthesis presents a valuable opportunity to capture abundant light energy to produce renewable bioenergy and biomaterials. To understand the factors that control the productivity of photosynthetic microorganisms, we conducted a series of semi‐continuous experiments using bench‐scale photobioreactor (PBR) systems, the cyanobacterium Synechocystis PCC6803 (PCC6803), and light conditions imitating actual day–night light irradiance (LI). Our results demonstrate that using normal BG‐11 medium resulted in severe phosphate (P_i) limitation for continuous operation. Mitigation of P_i‐limitation, by augmenting the P_i content of BG‐11, allowed higher biomass productivity; however, once P_i‐limitation was alleviated, limitation by inorganic carbon (C_i) or LI occurred. C_i‐limitation was detected by a low total C_i concentration (<5 mg C/L) and high and fluctuating pH. C_i‐limitation was relieved by delivering more CO₂, which led to a stable pH in the range of 7–9 and at least 5 mg/L of C_i in HCO. LI limitation, evidenced by an average LI <14 W/m² for PCC6803, was induced by a high biomass concentration of 1,300 mg/L. Thus, this work provides quantitative tools of stoichiometry and kinetics to evaluate limitation on PBRs. Biotechnol. Bioeng. 2010;106: 553–563. © 2010 Wiley Periodicals, Inc.     

RESPONSE OF LAMINARIA SACCHARINA (PHAEOPHYTA) GROWTH AND PHOTOSYNTHESIS TO SIMULTANEOUS ULTRAVIOLET RADIATION AND NITROGEN LIMITATION1

The brown macroalga Laminaria saccharina (L.) J. V. Lamour. was grown in large outdoor tanks at 50% ambient solar radiation for 3–4 weeks in July and August of 2000, 2001, and 2002, in either ambient or nitrogen (N)–enriched seawater and in either ambient light [PAR + ultraviolet radiation (UVR)] or ambient light minus UVR. Growth, N‐content, photosynthetic pigments, and RUBISCO content increased in N‐enriched seawater, indicating N‐limitation. UVR inhibited growth, but this inhibition was ameliorated by N‐enrichment. The response of growth to UVR could not be explained by changes in respiration and photosynthesis. Gross light‐saturated photosynthesis (P_max) remained unaffected by UVR but was significantly higher under N‐enrichment, as was dark respiration (R_d). UVR had no effect on pigments or N content. However, RUBISCO contents were low in the presence of UVR, reflecting the overall change in soluble cellular protein. Overall, our data indicate that the response to UVR in L. saccharina depends on other environmental factors, such as N, and these effects need to be considered when evaluating the response of macroalgae to increased UVR.     

NITROGEN LIMITATION IN DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE) LEADS TO INCREASED SUSCEPTIBILITY TO DAMAGE BY ULTRAVIOLET‐B RADIATION BUT ALSO INCREASED REPAIR CAPACITY1

Chl fluorescence was used to measure the photosynthetic capacity of the green alga Dunaliella tertiolecta in order to investigate interactions between susceptibility to acute UV‐B radiation (UVBR, 280–320 nm) exposure and decreased nitrogen availability. Under UVBR exposure the decline in the fluorescence parameters F_v/F_m (the maximum effective quantum yield Φ_PSIIe‐max) and F_v′/F_m′ (the operational quantum yield of PSII, Φ_PSIIe) were enhanced with higher UVBR fluxes, with the data well described by the Kok model, inferring that a dynamic balance existed between damage and repair with the repair proportional to the pool size of inactivated targets. When UVBR exposure was coupled with nitrogen limitation, the inhibition of photosynthesis was intensified. Under the more severely N‐limited conditions, the damage rate increased. Unexpectedly, repair rates were also stimulated under N‐limited conditions, although this was insufficient to counteract the increase in damage, so the overall effect of N limitation was an enhancement of UVBR‐induced inhibition of photosynthesis.     

Nutrient induced fluorescence transients (NIFTs) provide a rapid measure of P and C (co-)limitation in a green alga

Nutrient Induced Fluorescence Transients (NIFTs) have been shown to be a possible way of testing for the limiting nutrient in algal populations. In this study we tested the hypothesis that NIFTs can be used to detect a (co-)limitation for inorganic phosphorus (Pi) and CO₂ in the green alga Chlamydomonas acidophila and that the magnitude of the NIFTs can be related to cellular P:C ratios. We show a co-limitation response for Pi and CO₂ via traditional nutrient enrichment experiments in natural phytoplankton populations dominated by C. acidophila. We measured NIFT responses after a Pi- or a CO₂-spike in C. acidophila batch cultures at various stages of Pi and inorganic C limitation. Significant NIFTs were observed in response to spikes in both nutrients. The NIFT response to a Pi-spike showed a strong negative correlation with cellular P:C ratio that was pronounced below 3 mmol P: mol C (equivalent to 0.2 pg P cell^–1). Both cellular P and C content influenced the extent of the Pi-NIFT response. The NIFT response to a CO₂-spike correlated to low CO₂ culturing conditions and also had a negative correlation with cellular P content. A secondary response within the Pi-NIFT response was related to the CO₂ concentration and potentially reflected co-limitation. In conclusion, NIFTs provided a quick and reliable method to detect the growth-limiting nutrient in an extremophile green alga, under Pi-, CO_2- and Pi/CO₂ (co-)limited growth conditions.     

Photosynthetic and growth responses of three freshwater algae to phosphorus limitation and daylength

1. Three common species of freshwater phytoplankton, the diatom Nitzschia sp., green alga Sphaerocystis schroeteri and cyanobacterium Phormidium luridum, were grown under contrasting daylengths [18 : 6 h light : dark cycles (LD) versus 6 : 18 h LD] and phosphorus (P) regimes (P‐sufficient versus 1 μm P). The rates of growth and photosynthesis, as well as growth efficiencies and pigment concentrations, were compared among treatments. 2. The growth and photosynthetic parameters of the three species depended on both P status and daylength in a species‐specific way. The responses to P limitation depended on daylength and, conversely, the responses to daylength depended on P status. 3. Growth rates and the maximum rates of photosynthesis (P_max) of all species decreased under P limitation under both light regimes. However, the decrease of P_max because of P limitation was greater under long daylength. The P_max of the green alga S. schroeteri decreased the most (ca. sixfold) under P limitation compared with the other two species. The photosynthesis saturation parameter I_k also decreased under P limitation; the decline was significant in Nitzschia and Sphaerocystis. P‐limitation significantly increased photoinhibition (β) in Nitzschia and Sphaerocystis, but not in Phormidium. The excess photochemical capacity (the ratio of the maximum photosynthesis rate to the photosynthesis rate at the growth irradiance), characterising the ability to utilise fluctuating light, was significantly lower under P limitation. 4. The growth efficiency (growth rate normalised to daylength) declined with increasing daylength in all species. Under short daylength the cyanobacterium Phormidium had the lowest growth efficiency of the three species. 5. The cellular chlorophyll a concentration in both Nitzschia and Sphaerocystis was significantly higher under short daylength, but only under P‐sufficient conditions. In Nitzschia, under short daylength, P‐limitation significantly decreased cellular chlorophyll concentration. In contrast, P‐limitation increased cellular chlorophyll concentration in Sphaerocystis, but under long daylength only. The ratio of chlorophyll a to b in the green alga also declined under short daylength and under P‐limited conditions.     

Phosphorus recycling in photorespiration maintains high photosynthetic capacity in woody species

Leaf photosynthetic CO₂ responses can provide insight into how major nutrients, such as phosphorus (P), constrain leaf CO₂ assimilation rates (A_net). However, triose‐phosphate limitations are rarely employed in the classic photosynthesis model and it is uncertain as to what extent these limitations occur in field situations. In contrast to predictions from biochemical theory of photosynthesis, we found consistent evidence in the field of lower A_net in high [CO₂] and low [O₂] than at ambient [O₂]. For 10 species of trees and shrubs across a range of soil P availability in Australia, none of them showed a positive response of A_net at saturating [CO₂] (i.e. A_max) to 2 kPa O₂. Three species showed >20% reductions in A_max in low [O₂], a phenomenon potentially explained by orthophosphate (P_i) savings during photorespiration. These species, with largest photosynthetic capacity and P_i > 2 mmol P m^?2, rely the most on additional P_i made available from photorespiration rather than species growing in P‐impoverished soils. The results suggest that rarely used adjustments to a biochemical photosynthesis model are useful for predicting A_max and give insight into the biochemical limitations of photosynthesis rates at a range of leaf P concentrations. Phosphate limitations to photosynthetic capacity are likely more common in the field than previously considered.     

Modeling leaf CO2 assimilation and Photosystem II photochemistry from chlorophyll fluorescence and the photochemical reflectance index

We present a simple model to assess the quantum yield of photochemistry (Φ_P) and CO₂ assimilation rate from two parameters that are detectable by remote sensing: chlorophyll (chl) fluorescence and the photochemical reflectance index (PRI). Φ_P is expressed as a simple function of the chl fluorescence yield (Φ_F) and nonphotochemical quenching (NPQ): Φ_P = 1–bΦ_F(1 + NPQ). Because NPQ is known to be related with PRI, Φ_P can be remotely assessed from solar‐induced fluorescence and the PRI. The CO₂ assimilation rate can be assessed from the estimated Φ_P value with either the maximum carboxylation rate (V_cmax), the intercellular CO₂ concentration (C_i), or parameters of the stomatal conductance model. The model was applied to experimental data obtained for Chenopodium album leaves under various environmental conditions and was able to successfully predict Φ_F values and the CO₂ assimilation rate. The present model will improve the accuracy of assessments of gas exchange rates and primary productivity by remote sensing.     

Stomatal and non-stomatal limitation to photosynthesis in two trembling aspen (Populus tremuloides Michx.) clones exposed to elevated CO2 and/or O3

Leaf gas exchange parameters and the content of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) in the leaves of two 2‐year‐old aspen (Populus tremuloides Michx.) clones (no. 216, ozone tolerant and no. 259, ozone sensitive) were determined to estimate the relative stomatal and mesophyll limitations to photosynthesis and to determine how these limitations were altered by exposure to elevated CO₂ and/or O₃. The plants were exposed either to ambient air (control), elevated CO₂ (560 p.p.m.) elevated O₃ (55 p.p.b.) or a mixture of elevated CO₂ and O₃ in a free air CO₂ enrichment (FACE) facility located near Rhinelander, Wisconsin, USA. Light‐saturated photosynthesis and stomatal conductance were measured in all leaves of the current terminal and of two lateral branches (one from the upper and one from the lower canopy) to detect possible age‐related variation in relative stomatal limitation (leaf age is described as a function of leaf plastochron index). Photosynthesis was increased by elevated CO₂ and decreased by O₃ at both control and elevated CO₂. The relative stomatal limitation to photosynthesis (l_s) was in both clones about 10% under control and elevated O₃. Exposure to elevated CO₂ + O₃ in both clones and to elevated CO₂ in clone 259, decreased l_s even further – to about 5%. The corresponding changes in Rubisco content and the stability of C_i/C_a ratio suggest that the changes in photosynthesis in response to elevated CO₂ and O₃ were primarily triggered by altered mesophyll processes in the two aspen clones of contrasting O₃ tolerance. The changes in stomatal conductance seem to be a secondary response, maintaining stable C_i under the given treatment, that indicates close coupling between stomatal and mesophyll processes.     

Nitrate or ammonium: Influences of nitrogen source on the physiology of a green alga

In freshwaters, algal species are exposed to different inorganic nitrogen (N_i) sources whose incorporation varies in biochemical energy demand. We hypothesized that due to the lesser energy requirement of ammonium ()‐use, in contrast to nitrate ()‐use, more energy remains for other metabolic processes, especially under CO₂‐ and phosphorus (P_i) limiting conditions. Therefore, we tested differences in cell characteristics of the green alga Chlamydomonas acidophila grown on or under covariation of CO₂ and P_i‐supply in order to determine limitations, in a full‐factorial design. As expected, results revealed higher carbon fixation rates for ‐grown cells compared to growth with under low CO₂ conditions. ‐grown cells accumulated more of the nine analyzed amino acids, especially under P_i‐limited conditions, compared to cells provided with . This is probably due to a slower protein synthesis in cells provided with . In contrast to our expectations, compared to ‐grown cells ‐grown cells had higher photosynthetic efficiency under P_i‐limitation. In conclusion, growth on the N_i‐source did not result in a clearly enhanced C_i‐assimilation, as it was highly dependent on P_i and CO₂ conditions (replete or limited). Results are potentially connected to the fact that C. acidophila is able to use only CO₂ as its inorganic carbon (C_i) source.     

Higher electron transport rate observed at low intercellular CO2 concentration in long-term drought-acclimated leaves of Japanese mountain birch (Betula ermanii)

The influence of long‐term drought stress on photosynthesis of Japanese mountain birch (Betula ermanii Cham.) was examined using chlorophyll fluorescence and gas exchange measurements. Drought stress was imposed in potted plants by reducing irrigation frequency from daily (control) to twice‐weekly and once‐weekly. Thirty‐day‐old leaves, which had developed under fully stressed conditions, were used for the measurements. The decline in net CO₂ assimilation rate (A) observed in situ in drought‐stressed plants resulted from a lower intercellular CO₂ concentration (C_i) due to stomatal closure but the carboxylation efficiency was not affected as there was no difference in the initial slope of the A/C_i response after watering. Although there were no treatment differences in A at C_i below 270 μmol mol^?1 (with ambient air at 360 μmol mol^?1 CO₂), higher electron transport rate (ETR), photochemical quenching (q_P) and the efficiency of energy conversion of open PSII (F_v′/F_m′), and similar or even lower non‐photochemical quenching (NPQ) were observed at a given C_i in drought‐stressed plants (of both twice‐ and once‐weekly irrigation), suggesting a higher fraction of open PSII resulting from energy dissipation achieved through higher electron flow rather than through thermal dissipation in PSII antennae. The once‐weekly watered plants showed a lower ratio of gross carbon assimilation rate to ETR (A*/ETR), suggesting an enhanced alternative pathway of electron flow probably involving the Mehler‐peroxidase (MP) reaction as indicated by a higher Φ_PSII at a given Φ_CO2 under non‐photorespiratory conditions. On the other hand, plants of twice‐weekly watering exhibited almost the same A*/ETR and Φ_PSII–Φ_CO2 relationship as control plants, indicating no enhanced alternative pathways under mild drought stress.     

Limiting Factors in Photosynthesis: V. Photochemical Energy Supply Colimits Photosynthesis at Low Values of Intercellular CO(2) Concentration

Although there is now some agreement with the view that the supply of photochemical energy may influence photosynthetic rate (P) at high CO₂ pressures, it is less clear whether this limitation extends to P at low CO₂. This was investigated by measuring P per area as a function of the intercellular CO₂ concentration (C_i) at different levels of photochemical energy supply. Changes in the latter were obtained experimentally by varying the level of irradiance to normal (Fe-sufficient) leaves of Beta vulgaris L. cv F58-554H1, and by varying photosynthetic electron transport capacity using leaves from Fe-deficient and Fe-sufficient plants. P and C_i were determined for attached sugar beet leaves using open flow gas exchange. The results suggest that P/area was colimited by the supply of photochemical energy at very low as well as high values of C_i. Using the procedure developed by Perchorowicz et al. (Plant Physiol 1982 69:1165-1168), we investigated the effect of irradiance on ribulose bisphosphate carboxylase (RuBPCase) activation. The ratio of initial extractable activity to total inducible RuBPCase activity increased from 0.25 to 0.90 as leaf irradiance increased from 100 to 1500 microeinsteins photosynthetically active radiation per square meter per second. These data suggest that colimitation by photochemical energy supply at low C_i may be mediated via effects on RuBPCase activation.     

The growth of soybean under free air [CO<Subscript>2</Subscript>] enrichment (FACE) stimulates photosynthesis while decreasing in vivo Rubisco capacity

Down-regulation of light-saturated photosynthesis (A_sat) at elevated atmospheric CO₂ concentration, [CO₂], has been demonstrated for many C₃ species and is often associated with inability to utilize additional photosynthate and/or nitrogen limitation. In soybean, a nitrogen-fixing species, both limitations are less likely than in crops lacking an N-fixing symbiont. Prior studies have used controlled environment or field enclosures where the artificial environment can modify responses to [CO₂]. A soybean free air [CO₂] enrichment (FACE) facility has provided the first opportunity to analyze the effects of elevated [CO₂] on photosynthesis under fully open-air conditions. Potential ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) carboxylation (V_c,max) and electron transport through photosystem II (J_max) were determined from the responses of A_sat to intercellular [CO₂] (C_i) throughout two growing seasons. Mesophyll conductance to CO₂ (g_m) was determined from the responses of A_sat and whole chain electron transport (J) to light. Elevated [CO₂] increased A_sat by 15–20% even though there was a small, statistically significant, decrease in V_c,max. This differs from previous studies in that V_c,max/J_max decreased, inferring a shift in resource investment away from Rubisco. This raised the C_i at which the transition from Rubisco-limited to ribulose-1,5-bisphosphate regeneration-limited photosynthesis occurred. The decrease in V_c,max was not the result of a change in g_m, which was unchanged by elevated [CO₂]. This first analysis of limitations to soybean photosynthesis under fully open-air conditions reveals important differences to prior studies that have used enclosures to elevate [CO₂], most significantly a smaller response of A_sat and an apparent shift in resources away from Rubisco relative to capacity for electron transport.Abbreviations FACE Free air [CO₂] enrichment - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP Ribulose-1,5-bisphosphate - SoyFACE Soybean free air [CO₂] enrichment - VPD Vapor pressure deficit     

No photosynthetic down-regulation in sweetgum trees (Liquidambar styraciflua L.) after three years of CO2 enrichment at the Duke Forest FACE experiment

Photosynthetic capacity and leaf properties of sun and shade leaves of overstorey sweetgum trees (Liquidambar styraciflua L.) were compared over the first 3 years of growth in ambient or ambient + 200 μL L^?1 CO₂ at the Duke Forest Free Air CO₂ Enrichment (FACE) experiment. We were interested in whether photosynthetic down‐regulation to CO₂ occurred in sweetgum trees growing in a forest ecosystem, whether shade leaves down‐regulated to a greater extent than sun leaves, and if there was a seasonal component to photosynthetic down‐regulation. During June and September of each year, we measured net photosynthesis (A) versus the calculated intercellular CO₂ concentration (C_i) in situ and analysed these response curves using a biochemical model that described the limitations imposed by the amount and activity of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Vc_max) and by the rate of ribulose‐1,5‐bisphosphate (RuBP) regeneration mediated by electron transport (J_max). There was no evidence of photosynthetic down‐regulation to CO₂ in either sun or shade leaves of sweetgum trees over the 3 years of measurements. Elevated CO₂ did not significantly affect Vc_max or J_max. The ratio of Vc_max to J_max was relatively constant, averaging 2·12, and was not affected by CO₂ treatment, position in the canopy, or measurement period. Furthermore, CO₂ enrichment did not affect leaf nitrogen per unit leaf area (N_a), chlorophyll or total non‐structural carbohydrates of sun or shade leaves. We did, however, find a strong relationship between N_a and the modelled components of photosynthetic capacity, Vc_max and J_max. Our data over the first 3 years of this experiment corroborate observations that trees rooted in the ground may not exhibit symptoms of photosynthetic down‐regulation as quickly as tree seedlings growing in pots. There was a strong sustained enhancement of photosynthesis by CO₂ enrichment whereby light‐saturated net photosynthesis of sun leaves was stimulated by 63% and light‐saturated net photosynthesis of shade leaves was stimulated by 48% when averaged over the 3 years. This study suggests that this CO₂ enhancement of photosynthesis will be sustained in the Duke Forest FACE experiment as long as soil N availability keeps pace with photosynthetic and growth processes.     

PHOTOSYNTHETIC CHARACTERISTICS OF THE COCCOLITHOPHORID EMILIANIA HUXLEYI (PRYMNESIOPHYCEAE) EXPOSED TO ELEVATED CONCENTRATIONS OF DISSOLVED INORGANIC CARBON1

Light-saturated photosynthesis (P_max) of Emiliania huxleyi (Lohmann) Hay et Mohler is known to be carbonlimited at natural concentrations of dissolved inorganic carbon (DIC). In the present study, light-limited and light-saturated photosynthetic rates of E. huxleyi were studied at three concentrations of DIC (2.4, 7.4, and 12.4 mM) for high-calcite (C_in/C_tot=0.48) and low-calcite (C_in/C_tot=0.08) cells of the same strain. The photosynthetic efficiency (α) and the maximum quantum yield (θ_max)A increased by more than a factor of 2 from the lowest to the highest DIC level. P_max a, and θ_max were always higher for the high-calcite than for the low-calcite cells at identical DIC levels. This may indicate that the calcifcation process acts as an extra supplier of CO₂ for photosynthesis making the CO₂ shortage at natural DIC levels a little smaller for high-calcite than for low-calcite E. huxleyi. A dependency of θ_max on DIC has not previously been shown for marine phytoplankton. θ_max is a key parameter in recent biooptical models of phytoplankton productivity, and the results from the present study are therefore important for modeling the productivity of E. huxleyi.     

The rapid A–Ci response: photosynthesis in the phenomic era

Phenotyping for photosynthetic gas exchange parameters is limiting our ability to select plants for enhanced photosynthetic carbon gain and to assess plant function in current and future natural environments. This is due, in part, to the time required to generate estimates of the maximum rate of ribulose‐1,5‐bisphosphate carboxylase oxygenase (Rubisco) carboxylation (V_c,max) and the maximal rate of electron transport (J_max) from the response of photosynthesis (A) to the CO₂ concentration inside leaf air spaces (C_i). To relieve this bottleneck, we developed a method for rapid photosynthetic carbon assimilation CO₂ responses [rapid A–C_i response (RACiR)] utilizing non‐steady‐state measurements of gas exchange. Using high temporal resolution measurements under rapidly changing CO₂ concentrations, we show that RACiR techniques can obtain measures of V_c,max and J_max in ~5 min, and possibly even faster. This is a small fraction of the time required for even the most advanced gas exchange instrumentation. The RACiR technique, owing to its increased throughput, will allow for more rapid screening of crops, mutants and populations of plants in natural environments, bringing gas exchange into the phenomic era.     

The “one‐point method” for estimating maximum carboxylation capacity of photosynthesis: A cautionary tale

The maximum carboxylation capacity of Rubisco, V_c,max, is an important photosynthetic parameter that is key to accurate estimation of carbon assimilation. The gold‐standard technique for determining V_c,max is to derive V_c,max from the initial slope of an A–C_i curve (the response of photosynthesis, A, to intercellular CO₂ concentration, C_i). Accurate estimates of V_c,max derived from an alternative and rapid “one‐point” measurement of photosynthesis could greatly accelerate data collection and model parameterization. We evaluated the practical application of the one‐point method in six species measured under standard conditions (saturating irradiance and 400 μmol CO₂ mol^?1) and under conditions that would increase the likelihood for successful estimation of V_c,max: (a) ensuring Rubisco‐limited A by measuring at 300 μmol CO₂ mol^?1 and (b) allowing time for acclimation to saturating irradiance prior to measurement. The one‐point method significantly underestimated V_c,max in four of the six species, providing estimates 21%–32% below fitted values. We identified ribulose‐1,5‐bisphosphate‐limited A, light acclimation, and the use of an assumed respiration rate as factors that limited the effective use of the one‐point method to accurately estimate V_c,max. We conclude that the one‐point method requires a species‐specific understanding of its application, is often unsuccessful, and must be used with caution.     

On the need to incorporate sensitivity to CO2 transfer conductance into the Farquhar–von Caemmerer–Berry leaf photosynthesis model

Virtually all current estimates of the maximum carboxylation rate (V_cmax) of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) and the maximum electron transport rate (J_max) for C₃ species implicitly assume an infinite CO₂ transfer conductance (g_i). And yet, most measurements in perennial plant species or in ageing or stressed leaves show that g_i imposes a significant limitation on photosynthesis. Herein, we demonstrate that many current parameterizations of the photosynthesis model of Farquhar, von Caemmerer & Berry (Planta 149, 78–90, 1980 ) based on the leaf intercellular CO₂ concentration (C_i) are incorrect for leaves where g_i limits photosynthesis. We show how conventional A–C_i curve (net CO₂ assimilation rate of a leaf –A_n– as a function of C_i) fitting methods which rely on a rectangular hyperbola model under the assumption of infinite g_i can significantly underestimate V_cmax for such leaves. Alternative parameterizations of the conventional method based on a single, apparent Michaelis–Menten constant for CO₂ evaluated at C_i[K_m(CO₂)_i] used for all C₃ plants are also not acceptable since the relationship between V_cmax and g_i is not conserved among species. We present an alternative A–C_i curve fitting method that accounts for g_i through a non‐rectangular hyperbola version of the model of Farquhar et al. (1980 ). Simulated and real examples are used to demonstrate how this new approach eliminates the errors of the conventional A–C_i curve fitting method and provides V_cmax estimates that are virtually insensitive to g_i. Finally, we show how the new A–C_i curve fitting method can be used to estimate the value of the kinetic constants of Rubisco in vivo is presented     

CHO Cells adapted to inorganic phosphate limitation show higher growth and higher pyruvate carboxylase flux in phosphate replete conditions

Inorganic phosphate (P_i) is an essential ion involved in diverse cellular processes including metabolism. Changes in cellular metabolism upon long term adaptation to P_i limitation have been reported in E. coli. Given the essential role of P_i, adaptation to P_i limitation may also result in metabolic changes in animal cells. In this study, we have adapted CHO cells producing recombinant IgG to limiting P_i conditions for 75 days. Not surprisingly, adapted cells showed better survival under P_i limitation. Here, we report the finding that such cells also showed better growth characteristics compared to control in batch culture replete with P_i (higher peak density and integral viable cell density), accompanied by a lower specific oxygen uptake rate and cytochrome oxidase activity towards the end of exponential phase. Surprisingly, the adapted cells grew to a lower peak density under glucose limitation. This suggests long term P_i limitation may lead to selection for an altered metabolism with higher dependence on glucose availability for biomass assimilation compared to control. Steady state U‐¹³C glucose labeling experiments suggest that adapted cells have a higher pyruvate carboxylase flux. Consistent with this observation, supplementation with aspartate abolished the peak density difference whereas supplementation with serine did not abolish the difference. This supports the hypothesis that cell growth in the adapted culture might be higher due to a higher pyruvate carboxylase flux. Decreased fitness under carbon limitation and mutations in the sucABCD operon has been previously reported in E. coli upon long term adaptation to P_i limitation, suggestive of a similarity in cellular response among such diverse species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:749–758, 2017     

The role of external carbonic anhydrase in photosynthesis during growth of the marine diatom Chaetoceros muelleri

Carbon dioxide concentrating mechanisms (CCMs) act to improve the supply of CO₂ at the active site of ribulose‐1,5‐bisphosphate carboxylase/oxygenase. There is substantial evidence that in some microalgal species CCMs involve an external carbonic anhydrase (CA_ext) and that CA_ext activity is induced by low CO₂ concentrations in the growth medium. However, much of this work has been conducted on cells adapted to air‐equilibrium concentrations of CO₂, rather than to changing CO₂ conditions caused by growing microalgal populations. We investigated the role of CA_ext in inorganic carbon (C_i) acquisition and photosynthesis at three sampling points during the growth cycle of the cosmopolitan marine diatom Chaetoceros muelleri. We observed that CA_ext activity increased with decreasing C_i, particularly CO₂, concentration, supporting the idea that CA_ext is modulated by external CO₂ concentration. Additionally, we found that the contribution of CA_ext activity to carbon acquisition for photosynthesis varies over time, increasing between the first and second sampling points before decreasing at the last sampling point, where external pH was high. Lastly, decreases in maximum quantum yield of photosystem II (F_v/F_m), chlorophyll, maximum relative electron transport rate, light harvesting efficiency (α) and maximum rates of C_i‐ saturated photosynthesis (V_max) were observed over time. Despite this decrease in photosynthetic capacity an up‐regulation of CCM activity, indicated by a decreasing half‐saturation constant for CO₂ (K_0.5CO₂), occurred over time. The flexibility of the CCM during the course of growth in C. muelleri may contribute to the reported dominance and persistence of this species in phytoplankton blooms.     

Interactions between carbon dioxide and oxygen in the photosynthesis of three species of marine red macroalgae

Summary

Red algae have the highest known selectivity factor (S_rel) for CO₂ over O₂ of ribulose bisphosphate carboxylase-oxygenase (RUBISCO). This allows the prediction that a red alga relying on diffusive supply of CO₂ to RUBISCO from air-equilibrated solution should have less O₂ inhibition of photosynthesis than would an otherwise similar non-red alga with a lower S_rel of RUBISCO. Furthermore, RUBISCO shows an increased S_rel values at low temperatures. The prediction that O ₂inhibition of photosynthesis should be small for marine red algae relying on diffusive CO₂ entry growing in the North Sea with an annual temperature range of 4–16°C was tested in O₂ electrode experiments at 12°C. Phycodrys rubens and Plocamium cartilagineum, which rely on diffusive CO₂ entry showed, as predicted, only a small inhibition at lower inorganic C concentrations. Palmaria palmata, which has a CO₂ concentrating mechanism, had the expected negligible O ₂ inhibition of photosynthesis at any inorganic C concentration except (non-significantly) for saturating inorganic C.