Changes in the Carbonic Anhydrase Activity and the Rate of Photosynthetic O2 Evolution during the Cell Cycle of Chlorella ellipsoidea C-27

Rhythmical changes in carbonic anhydrase activity(CA) and inphotosynthesis were observed during the cell cycle of Chlorellaellipsoidea C-27 synchronized at various concentrations of dissolvedCO₂ (dCO₂ with a regime of 16 h of light and 8 h of darkness.At a constant low concentration of dCO₂ (11 {diaeresis}M), intracellularCA activity showed obvious fluctuations with a peak at 8 h afterthe initiation of illumination, while extracellular CA activity,located on the cell surface, showed only minor fluctuationsalthough the activity was as high as the maximum activity ofintracellular CA. In contrast, obvious changes in the activitiesof intra- and extracellular CA activities were not observedat a high concentration of dCO₂ (520 {diaeresis}M). The ratioof photosynthetic activity at limiting versus saturating concentrationsof dCO₂, which is indicative of the affinity of cells for CO₂,showed clear rhythmical changes during the cell cycle and theratio was higher in low-CO₂ cells than in high-CO₂ cells. Thechanges in the ratio seemed to reflect the changes in CA activity. When the cells that had been synchronized under high CO₂ conditionswere transferred to low CO₂ conditions at any given stage inthe cell cycle, CA activity was induced in every case but thecapacity for induction of CA was greater in young cells thanin mature cells. This result suggests that the capacity of cellsto induce CA over the course of the cell cycle is closely relatedto endogenous aging of the cell. (Received August 29, 1988; Accepted December 28, 1988)     

Towards a quantitative assessment of inorganic carbon cycling in photosynthetic microorganisms

Photosynthetic organisms developed various strategies to mitigate high light stress. For instance, aquatic organisms are able to spend excessive energy by exchanging dissolved CO₂ (dCO₂) and bicarbonate () with the environment. Simultaneous uptake and excretion of the two carbon species is referred to as inorganic carbon cycling. Often, inorganic carbon cycling is indicated by displacements of the extracellular dCO₂ signal from the equilibrium value after changing the light conditions. In this work, we additionally use (i) the extracellular pH signal, which requires non‐ or weakly‐buffered medium, and (ii) a dynamic model of carbonate chemistry in the aquatic environment to detect and quantitatively describe inorganic carbon cycling. Based on simulations and experiments in precisely controlled photobioreactors, we show that the magnitude of the observed dCO₂ displacement crucially depends on extracellular pH level and buffer concentration. Moreover, we find that the dCO₂ displacement can also be caused by simultaneous uptake of both dCO₂ and (no inorganic carbon cycling). In a next step, the dynamic model of carbonate chemistry allows for a quantitative assessment of cellular dCO₂, , and H⁺ exchange rates from the measured dCO₂ and pH signals. Limitations of the method are discussed.     

Real-time dissolved carbon dioxide monitoring I: Application of a novel in situ sensor for CO2 monitoring and control

Dissolved carbon dioxide (dCO₂) is a well-known critical parameter in bioprocesses due to its significant impact on cell metabolism and on product quality attributes. Processes run at small-scale faces many challenges due to limited options for modular sensors for online monitoring and control. Traditional sensors are bulky, costly, and invasive in nature and do not fit in small-scale systems. In this study, we present the implementation of a novel, rate-based technique for real-time monitoring of dCO₂ in bioprocesses. A silicone sampling probe that allows the diffusion of CO₂ through its wall was inserted inside a shake flask/bioreactor and then flushed with air to remove the CO₂ that had diffused into the probe from the culture broth (sensor was calibrated using air as zero-point calibration). The gas inside the probe was then allowed to recirculate through gas-impermeable tubing to a CO₂ monitor. We have shown that by measuring the initial diffusion rate of CO₂ into the sampling probe we were able to determine the partial pressure of the dCO₂ in the culture. This technique can be readily automated, and measurements can be made in minutes. Demonstration experiments conducted with baker's yeast and Yarrowia lipolytica yeast cells in both shake flasks and mini bioreactors showed that it can monitor dCO₂ in real-time. Using the proposed sensor, we successfully implemented a dCO₂-based control scheme, which resulted in significant improvement in process performance.     

Seasonal Variation in Schools’ Indoor Air Environments and Health Symptoms among Students in an Eastern Mediterranean Climate

The indoor air quality (IAQ) in classrooms highly affects the health and productivity of students. This article aims to clarify seasonal variation in indoor environment and sick building syndromes (SBS) symptoms in an Eastern Mediterranean climate. A series of field measurements were conducted during the fall and winter seasons from October 2011 to March 2012 in 12 naturally ventilated schools located in the Gaza Strip. Data on environmental perception and health symptoms were obtained from 724 students by using a validated questionnaire. The results showed that indoor PM₁₀ and PM_2.5 concentrations were 426.3 ± 187.6 μg/m³ and 126.6 ± 94.8 μg/m³, respectively. The CO₂ concentrations and ventilation rate widely exceeded their reference values during the winter season. The prevalence rates of general symptoms were relatively high at baseline assessment in the fall season and increased significantly during follow-up in the winter season. Significant increases in disease symptoms such as mucosal irritation and pre-existing asthma symptoms among students could be related to poor indoor air quality. Five distinct groups of SBS symptoms from factor analysis of students’ related symptoms were significantly correlated with PM₁₀ and PM_2.5, CO₂, ventilation rate, and indoor temperature. As vulnerable children, this situation negatively affects their school performance and health.     

Accelerated Death Kinetics of Aspergillus niger Spores under High-Pressure Carbonation

The death kinetics of Aspergillus niger spores under high-pressure carbonation were investigated with respect to the concentration of dissolved CO₂ (dCO₂) and treatment temperature. All of the inactivation followed first-order death kinetics. The D value (decimal reduction time, or the time required for a 1-log-cycle reduction in the microbial population) in the saline carbonated at 10 MPa was 0.16 min at 52°C. The log D values were linearly related to the treatment temperature and the concentration of dCO₂, but a significant interaction was observed between them.     

A Longitudinal Study of Sick Building Syndrome (SBS) among Pupils in Relation to SO2, NO2, O3 and PM10 in Schools in China

There are fewer longitudinal studies from China on symptoms as described for the sick building syndrome (SBS). Here, we performed a two-year prospective study and investigated associations between environmental parameters such as room temperature, relative air humidity (RH), carbon dioxide (CO₂), nitrogen dioxide (NO₂), sulphur dioxide (SO₂), ozone (O₃), particulate matter (PM₁₀), and health outcomes including prevalence, incidence and remission of SBS symptoms in junior high schools in Taiyuan, China. Totally 2134 pupils participated at baseline, and 1325 stayed in the same classrooms during the study period (2010–2012). The prevalence of mucosal symptoms, general symptoms and symptoms improved when away from school (school-related symptoms) was 22.7%, 20.4% and 39.2%, respectively, at baseline, and the prevalence increased during follow-up (P<0.001). At baseline, both indoor and outdoor SO₂ were found positively associated with prevalence of school-related symptoms. Indoor O₃ was shown to be positively associated with prevalence of skin symptoms. At follow-up, indoor PM₁₀ was found to be positively associated with new onset of skin, mucosal and general symptoms. CO₂ and RH were positively associated with new onset of mucosal, general and school-related symptoms. Outdoor SO₂ was positively associated with new onset of skin symptoms, while outdoor NO₂ was positively associated with new onset of skin, general and mucosal symptoms. Outdoor PM₁₀ was found to be positively associated with new onset of skin, general and mucosal symptoms as well as school-related symptoms. In conclusion, symptoms as described for SBS were commonly found in school children in Taiyuan City, China, and increased during the two-year follow-up period. Environmental pollution, including PM₁₀, SO₂ and NO₂, could increase the prevalence and incidence of SBS and decrease the remission rate. Moreover, parental asthma and allergy (heredity) and pollen or pet allergy (atopy) can be risk factors for SBS.     

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kLa ratio as a direct indicator of gas stripping conditions

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO₂) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO₂ was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the k_La values of O₂ (k_La[O₂]) and CO₂ [k_La(CO₂)] in the SS bioreactors as a scale-up factor for dCO₂ reduction. The k_La ratios [k_La(CO₂)/k_La(O₂)] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the k_La ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO₂ trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This k_La ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The k_La ratios for the SUBs were evaluated and conditions that provided k_La ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the k_La ratio is a powerful scale-up factor useful to control dCO₂ during fed-batch cultures.     

Real-time dissolved carbon dioxide monitoring II: Surface aeration intensification for efficient CO2 removal in shake flasks and mini-bioreactors leads to superior growth and recombinant protein yields

Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O₂ (DO) and dissolved CO₂ (dCO₂) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small-scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real-time monitoring of DO and dCO₂ in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO₂ at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results.     

Calorie restriction: A new therapeutic intervention for age-related dry eye disease in rats

A decrease in lacrimal gland secretory function is closely related to aging and leads to an increased prevalence of dry eye syndrome. Since calorie restriction (CR) is considered to prevent functional decline of various organs due to aging, we hypothesized that CR could prevent age-related lacrimal dysfunction. Six-month-old male Fischer 344 rats were randomly divided into ad libitum (AL) and CR (−35%) groups. After 6 months of CR, tear function was examined under conscious state. After euthanasia, lacrimal glands were subjected to histological examination, tear protein secretion stimulation test with Carbachol, and assessment of oxidative stress with 8-hydroxy-2 deoxyguanosine (8-OHdG) and 4-hydroxynonenal (HNE) antibodies. CR significantly improved tear volume and tended to increase tear protein secretion volume after stimulation with Carbachol compared to AL. The acinar unit density was significantly higher in the CR rats compared to AL rats. Lacrimal glands in the CR rats showed a lesser degree of interstitial fibrosis. CR reduced the concentration of 8-OHdG and the extent of staining with HNE in the lacrimal gland, compared to AL. Furthermore, our electron microscopic observations showed that mitochondrial structure of the lacrimal gland obtained from the middle-aged CR rats was preserved in comparison to the AL rats. Collectively, these results demonstrate for the first time that CR may attenuate oxidative stress related damage in the lacrimal gland with preservation of lacrimal gland functions. Although molecular mechanism(s) by which CR maintains lacrimal gland function remains to be resolved, CR might provide a novel therapeutic strategy for treating dry eye syndrome.     

Some observations on the carbon dioxide burst in chlorella and chlamydomonas

In the course of mass spectrometer studies with the algae Chlorella and Chlamydomonas, data were obtained which indicate that the CO₂ burst and gulp are sensitive to oxygen. Furthermore, the CO₂ burst was found to be strongly suppressed when wave lengths shorter than 460 nanometers were blocked at intensities adequate to saturate photosynthesis. Under appropriate conditions at 30°, the CO₂ burst was interrupted by a brief CO₂ gulp and the post illumination gulp by a brief burst of CO₂. The post illumination gulp of CO₂ could be induced during illumination by interposition of a filter blocking wave lengths shorter than 460 nanometers. These data are discussed in relation to earlier reports of the phenomenon and briefly as they affect the detection of photorespiration.     

Postillumination respiration of maize in relation to oxygen concentration and glycolic Acid metabolism

Prior illumination in CO₂-free air enhances a respiration from maize (Zea mays L.) leaves different in onset and duration from the postillumination burst of photorespiration. The course of respiration after brief illumination of attached leaves was measured as CO₂ efflux in darkness into CO₂-free atmospheres with four O₂ concentrations. The peak of CO₂ efflux following illumination was suppressed by 2.23% O₂, was completely eliminated by 0.04% O₂, and was not stimulated by 40% O₂ compared with air. Compared with air, steady dark respiration was suppressed by 0.04% O₂ but was not affected by 2.23% nor 40% O₂. Excision and subsequent uptake of distilled water through the vascular system nearly eliminated the enhanced respiration.     

Light and Temperature Regulation of Early Morning Crassulacean Acid Metabolism in Opuntia erinacea var Columbiana (Griffiths) L. Benson

During the early morning period, light and temperature exert distinctively different influences on the gas exchange patterns of the Crassulacean acid metabolism plant Opuntia erinacea through their effects on acid metabolism. An initial decrease in CO₂ uptake was triggered by illumination and was apparently due to a decreased CO₂ diffusion gradient through light-mediated decarboxylation of malate. In contrast, the morning burst of CO₂ uptake occurred at high temperature presumably in response to increases in both stomatal conductance and the CO₂ diffusion gradient, resulting from the temperature-regulated fixation of endogenous CO₂, primarily into malate. Subsequent stomatal closure, apparently due to elevated levels of internal CO₂ through rapid decarboxylation of malate at high temperature, was primarily responsible for the final termination of early morning Crassulacean acid metabolism.     

Sensory processing of ambient CO<Subscript>2</Subscript> information in the brain of the moth<Emphasis Type="Italic"> Manduca sexta</Emphasis>

Insects use information about CO₂ to perform vital tasks such as locating food sources. In certain moths, CO₂ is involved in oviposition behavior. The labial palps of adult moths that feed as adults have a pit organ containing sensory receptor cells that project into the antennal lobes, the sites of primary processing of olfactory information in the brain. In the moth Manduca sexta and certain other species of Lepidoptera, these receptor cells in the labial-palp pit organ have been shown to be tuned to CO₂, and their axons project to a single, identified glomerulus in the antennal lobe, the labial-palp pit organ glomerulus. At present, however, nothing is known about the function of this glomerulus or how CO₂ information is processed centrally. We used intracellular recording and staining to reveal projection (output) neurons in the antennal lobes that respond to CO₂ and innervate the labial-palp pit organ glomerulus. Our results demonstrate that this glomerulus is the site of first-order processing of sensory information about ambient CO₂. We found three functional types of CO₂-responsive neurons (with their cell bodies in the antennal lobe or the protocerebrum) that provide output from the antennal lobe to higher centers in the brain. Some physiological characteristics of those neurons are described.Abbreviations AL Antennal lobe - AN Antennal nerve - CMB Calyces of the mushroom body - IPSP Inhibitory postsynaptic potential - LC-I Dorsal cluster of the lateral group of AL neuronal somata - LH Lateral horn of the protocerebrum - LPN Labial-palp nerve - LPO Labial-palp pit organ - LPOG LPO glomerulus - PC Protocerebrum - PI AL neuron that projects to the PC through the inner antenno-cerebral tract - PN Projection neuron     

Environmental assessment schemes for non-domestic building refurbishment in the Malaysian context

The increase in global warming, energy consumption and greenhouse gas emissions has gained attention in various regions. In Malaysia, the government announced a voluntary commitment to reduce 40% of CO₂ emissions by 2020 and to refurbish 100 government buildings. Existing buildings make a large contribution to energy consumption and CO₂ emissions, therefore refurbishing existing buildings is an essential strategy to achieve the commitment. There is no single assessment scheme for building refurbishment in Malaysia and hence, this study aims to develop a comprehensive list of assessment themes and sub-themes for building refurbishment purposes. It examines and compares 10 assessment schemes from various countries: BREEAM, LEED, CASBEE, BEAM Plus, GBLS, Green Star, HQE, Green Mark, GBI and MyCrest. The findings revealed fourteen themes that were considered for assessment: management, sustainable site, transport, indoor environmental quality (IEQ), water, waste, material, energy, pollution, innovation, economic, social, culture and quality of services. Energy and IEQ are dominant themes in all assessment schemes. Most of the schemes are considered relatively weak in evaluating economic and social aspects, in comparison to environmental aspects. The assessment of quality of services is overlooked in most of the schemes, including GBI and MyCrest in Malaysia. Outcomes from this paper will form the baseline for a new environmental assessment scheme that aimed at non-domestic building refurbishments in Malaysia. A new model is proposed for the development of an environmental assessment scheme in the further stage.     

Effect of O2 and CO2 on net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii during dark-light-dark transitions

Net CO₂ exchange was monitored through a dark-light-dark transition, under 2% and 21% O₂ in the presence and absence of CO₂, in Chlamydomonas reinhardtii wild type and the high-CO₂-requiring mutant ca-1-12-1C. Upon illumination at 350 l/l CO₂, ca-1-12-1C cell exhibited a large decrease in net CO₂ uptake following an initial surge of CO₂ uptake. Net CO₂ uptake subsequently attained a steady-state rate substantially lower than the maximum. A large, O₂-enchanced post-illumination burst of CO₂ efflux was observed after a 10-min illumination period, corresponding to a minimum in the net CO₂ uptake rate. A smaller, but O₂-insensitive post-illumination burst was observed following a 30-min illumination period, when net CO₂ uptake was at a steady-state rate. These post-illumination bursts appeared to reflect the release of an intracellular pool of inorganic carbon, which was much larger following the initial surge of net CO₂ uptake than during the subsequent steady-state CO₂ uptake period.With the mutant in CO₂-free gas, O₂-stimulated, net CO₂ efflux was observed in the light, and a small, O₂-dependent post-illumination burst was observed. With wild-type cells no CO₂ efflux was observed in the light in CO₂-free gas under either 2% or 21% O₂, but a small, O₂-dependent post-illumination burst was observed. These results were interpreted as indicating that photorespiratory rates were similar in the mutant and wild-type cells in the absence of CO₂, but that the wild-type cells were better able to scavenge the photorespiratory CO₂.     

Conversion of photosynthetic products in the light in CO2-free O2 and N2 in leaves of Zea mays L. and Phaseolus vulgaris L.

Summary After 10 min illumination of segments of bean (Phaseolus vulgaris L.) or maize (Zea mays L.) leaves in air with ¹⁴CO₂, the atmosphere was changed to CO₂-free O₂ or N₂ and conversion of photosynthetic products in the light was investigated. The experiments have shown that after the ¹⁴CO₂ assimilation period the bean leaves contain the pool of weakly fixed ¹⁴C (WF-¹⁴C) which is converted into stable products during the subsequent period of illumination in CO₂-free N₂. In O₂ atmosphere the WF-¹⁴C pool is initially the main source of CO₂ evolved. The marked decrease in radioactivity of sucrose and starch during illumination of bean leaves in O₂ atmosphere indicates that these compounds were also the source of CO₂ evolved in the light. The total amount of previously fixed ¹⁴C remained almost on the same level during illumination of maize leaves in N₂ as well as in O₂. However, oxygen changed the distribution of ¹⁴C in photosynthetic products, which is suggested to be the consequence of the photorespiration process in maize.Abbreviation WF-¹⁴C weakly fixed ¹⁴C     

Photosynthetic Induction in a C(4) Dicot, Flaveria trinervia: I. Initial Products of CO(2) Assimilation and Levels of Whole Leaf C(4) Metabolites

Labeling patterns from ¹⁴CO₂ pulses to leaves and whole leaf metabolite contents were examined during photosynthetic induction in Flaveria trinervia, a C₄ dicot of the NADP-malic enzyme subgroup. During the first one to two minutes of illumination, malate was the primary initial product of ¹⁴CO₂ assimiltion (about 77% of total ¹⁴C incorporated). After about 5 minutes of illumination, the proportion of initial label to aspartate increased from 16 to 66%, and then gradually declined during the following 7 to 10 minutes of illumination. Nutrition experiments showed that the increase in ¹⁴CO₂ partitioning to aspartate was delayed about 2.5 minutes in plants grown with limiting N, and was highly dampened in plants previously treated 10 to 12 days with ammonia as the sole N source. Measurements of C₄ leaf metabolites revealed several transients in metabolite pools during the first few minutes of illumination, and subsequently, more gradual adjustments in pool sizes. These include a large initial decrease in malate (about 1.6 micromoles per milligram chlorophyll) and a small initial decrease in pyruvate. There was a transient increase in alanine levels after 1 minute of illumination, which was followed by a gradual, prolonged decrease during the remainder of the induction period. Total leaf aspartate decreased initially, but temporarily doubled in amount between 5 and 10 minutes of illumination (after its surge as a primary product). These results are discussed in terms of a plausible sequence of metabolic events which lead to the formation of the intercellular metabolite gradients required in C₄ photosynthesis.