Enhanced growth of the red algaPorphyra yezoensis Ueda in high CO2 concentrations

Leafy thalli of the red algaPorphyra yezoensis Ueda, initiated from conchospores released from free-living conchocelis, were cultured using aeration with high CO₂. It was found that the higher the CO₂ concentration, the faster the growth of the thalli. Aeration with elevated CO₂ lowered pH in dark, but raised pH remarkably in light with the thalli, because the photosynthetic conversion of HCO ₃ ^? to OH^? and CO₂ proceeded much faster than the dissociation of hydrated CO₂ releasing H⁺. Photosynthesis of the alga was found to be enhanced in the seawater of elevated dissolved inorganic carbon (DIC, CO₂ + HCO ₃ ^? + CO ₃ ^? ). It is concluded that the increased pH in the light resulted in the increase of DIC in the culture media, thus enhancing photosynthesis and growth. The relevance of the results to removal of atmospheric CO₂ by marine algae is discussed.     

The study of the effects of carbon dioxide-induced elevation of hydrogen peroxide toxicity on microbes as a novel tool for water purification

The supercritical concentration of CO₂ (SCCO₂) and a high concentration (3.0%) of molecules of hydrogen peroxide (H₂O₂) are currently being used as antiseptic and antibacterial agents. The fact that low concentrations of CO₂ have an activation effect on functional activity of microbes allows us to predict that CO₂ could elevate the toxic effect of H₂O₂ on cells. To check this hypothesis the dependency of the toxic effect of H₂O₂ on wild type of Escherichia coli K-12 on soluble concentration of CO₂ in culture media was studied. The obtained data show that culture media enriched with CO₂ leads to the increase of toxic effect of H₂O₂ on microbes at both cases when pH is constant and when it changes. So CO₂ in non-supercritical concentration could elevate the toxic effect of H₂O₂ on microbes by the activation of the metabolic processes in microbes. During the experiments we used classical microbiological methods (indirect viable cell counts or counting colony forming units (CFUs)), as well as the method of measuring hydrogen peroxide content in aqueous solution by means of enhanced chemiluminescence method in a peroxidase-luminol-p-iodophenol system. This discovery is concerning to use CO₂/H₂O₂ combination system, which could have implication in the inhibition of growth of microbes in water and the microbiological monitoring of water could provide valuable information for managing the health of exhibition of aqua ecosystems.     

Influence of carbon supply on the stimulation of light-saturated photosynthesis by blue light in Laminaria saccharina: implications for the mechanism of carbon acquisition in higher brown algae

In saturating irradiances of red light, photosynthesis of Laminaria saccharina (L.) Lamouroux was stimulated by low irradiances of continuous blue light only when the supply of dissolved inorganic carbon (DIC) was limiting. The degree of this stimulation was inversely proportional to the logarithm of the concentration of free CO₂, whether this was adjusted by varying the total DIC or the pH at a given DIC concentration. The final pH reached in a closed system was higher in blue light than in red light. Both acetazolamide and ethoxyzolamide suppressed the responses to blue light almost completely, but reduced photosynthesis in red light by only 30%. Buffering the pH of the seawater also suppressed the stimulation of photosynthesis by blue light without affecting the photosynthetic rate in red light. The transient stimulation of O₂ evolution by a blue light pulse was not accompanied by a corresponding increase in CO₂ consumption. These observations could be explained if, in analogy to the mechanism proposed for Ectocarpus (Schmid, Mills & Dring 1996, Plant Cell and Environment 19,373–382, this issue, accompanying paper), photosynthesis was supported by a blue-light-activated release of CO₂ from an internal store. We suggest that the store is located in the vacuoles of the cortical tissue of the blades. The main photosynthetic tissue, however, is in the overlying meristoderm, and blue-light-activated mobilization of the store could stimulate O₂ evolution only if periplasmic carbonic anhydrase was available to facilitate CO₂ uptake from the cortex.     

pH measurement and a rational and practical pH control strategy for high throughput cell culture system

The number of therapeutic proteins produced by cell culture in the pharmaceutical industry continues to increase. During the early stages of manufacturing process development, hundreds of clones and various cell culture conditions are evaluated to develop a robust process to identify and select cell lines with high productivity. It is highly desirable to establish a high throughput system to accelerate process development and reduce cost. Multiwell plates and shake flasks are widely used in the industry as the scale down model for large‐scale bioreactors. However, one of the limitations of these two systems is the inability to measure and control pH in a high throughput manner. As pH is an important process parameter for cell culture, this could limit the applications of these scale down model vessels. An economical, rapid, and robust pH measurement method was developed at Eli Lilly and Company by employing SNARF‐4F 5‐(‐and 6)‐carboxylic acid. The method demonstrated the ability to measure the pH values of cell culture samples in a high throughput manner. Based upon the chemical equilibrium of CO₂, HCO, and the buffer system, i.e., HEPES, we established a mathematical model to regulate pH in multiwell plates and shake flasks. The model calculates the required %CO₂ from the incubator and the amount of sodium bicarbonate to be added to adjust pH to a preset value. The model was validated by experimental data, and pH was accurately regulated by this method. The feasibility of studying the pH effect on cell culture in 96‐well plates and shake flasks was also demonstrated in this study. This work shed light on mini‐bioreactor scale down model construction and paved the way for cell culture process development to improve productivity or product quality using high throughput systems. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A method for the study of CO2 exchanges in in vitro cultured Vitis rupestris plantlets

A CO₂ assay circuit adapted to in vitro culture was designed to investigate CO₂ exchanges in test tube-grown Vitis rupestris plantlets. The CO₂ concentration of the air in culture tubes was measured by injection of samples in the open circuit. It was observed under the culture conditions used that the CO₂ content stabilized during the light phase at 3 times the CO₂ compensation point.Measurements of dark respiration under closed circuit conditions at every two-hour interval during the night did not reveal any limiting by lack of the substrate under mixotrophic culture conditions. A mathematical model of the influence of ambient CO₂ concentration on net CO₂ uptake rates under closed circuit conditions was devised and used to compare net photosynthesis at different lighting levels. Measurement of CO₂ evolution into CO₂-free air under open circuit conditions revealed a post-illumination burst characteristic of photorespiration which increased with the temperature.     

Effect of carbon dioxide and hydrostatic pressure on the pH of culture media and the growth of methanogens at elevated temperature

Summary High pressure/high temperature investigations on thermophilic methanogens require specific precautions to provide well-defined pH conditions in their culture media. Applying CO₂ as carbon source, sufficient buffering capacity of the culture medium is of crucial importance in investigations involving elevated pressures. In order to separate pressure effects on the growth and reproduction of thermophilic methanogens from pressure-induced protonation/deprotonation and increased solubility of gaseous components, direct pH measurements in common culture media in the absence and in the presence of CO₂ were performed at elevated temperature (65° C), and at pressures up to 100 MPa. Neutral phosphate buffer at high pressure shows a significant downward shift of its pH which is strongly enhanced in the presence of CO₂. In minimal media containing acetate, carbonate, formate and phosphate in 100 mM concentrations, 120 mM HEPES is found to provide optimum pH stability: near neutrality the pH change upon CO₂ saturation in the absence and in the presence of HEPES amounts to pH=2.10 and 0.41, respectively; the corresponding pressure dependences are pH/100 MPa=-0.26 and -0.07. As taken from these results, the apparent pressure dependence of the optimum growth ofMethanococcus thermolithotrophicus at 65° C in minimal medium reflects the pH shift below the cutoff point of growth (pH 5.5), rather than pressure-induced growth inhibition. At constant pH, elevated pressure up to 400 MPa is found to increase the rate and yield of growth; at the same time, alterations in the phenotype of the bacterium are observed.     

Endogenously produced CO2 induces stationary phase in tetrahymena cultures

Media concentration of total soluble CO₂ increases with culture age of Tetrahymena pyriformis. CO₂ is a weak acid and is capable of acidifying intracellular pH (pHi). Changes in pHi have been demonstrated to affect cell metabolism and growth in many systems. For these reasons, we investigated whether the concentrations of CO₂ produced in vitro were sufficient to affect cell proliferation and pHi in Tetrahymena. In this study, we used DMO to mimic the weak acid properties of CO₂. DMO is freely permeable to membranes in its uncharged form and has a pKa similar to that of CO₂/HCO. In addition, it has the advantages of being metabolically inert and non-volatile. At concentrations similar to endogenously produced CO₂, DMO acidifies pHi and arrests culture growth. In addition, procedures are described which decrease the media CO₂ concentrations in both growing and non-growing cultures. These conditions lead to increased maximum culture density at stationary phase. The data indicate that, under our conditions, accumulation of CO₂ in the culture leads to cessation of growth, probably through elimination of transmembrane pH gradients, which are necessary for regulation of metabolism and growth.     

PHYSIOLOGICAL CHARACTERISTICS OF PHOTOSYNTHESIS IN PORPHYRIDIUM CRUENTUM: EVIDENCE FOR BICARBONATE TRANSPORT IN A UNICELLULAR RED ALGA1

Various physiological characteristics of photosynthesis in the unicellular red alga Porphyridium cruentum Naegeli have been investigated. The rate of photosynthesis was optimal at 25° C and pH 7.5 and was not inhibited by 21% oxygen over a temperature range of 5 to 35° C. Kinetics of whole cell photosynthesis as a function of substrate concentration gave a K_1/2, (CO₂) of 0.3 μM. CO₂ compensation point, measured in a closed system at pH 7.5, was a constant 6.7 m?L · L^?1 over the temperature range 15 to 30° C and was unaffected by O₂ concentration. Whole cell photosynthesis, measured in a closed system at alkaline pH, showed that the rates of oxygen evolution were greatly in excess of the rate of CO₂ supply from the spontaneous dehydration of HCO₃^? in the medium. This indicates that bicarbonate is utilized by the cell to support this photosynthetic rate. These physiological characteristics of Porphyridium cruentum are consistent with the hypothesis that this alga transports bicarbonate across the plasmalemma.     

A Direct Confirmation of the Standard Method of Estimating Intercellular Partial Pressure of CO(2)

The partial pressure of CO₂ inside leaves of several species was measured directly. Small gas exchange chambers were clamped above and below the same section of an amphistomatous leaf. A flowing gas stream through one chamber allowed normal CO₂ and water vapor exchange. The other chamber was in a closed circuit consisting of the chamber, an infrared gas analyzer, and a peristaltic pump. The CO₂ in the closed system rapidly reached a steady pressure which it is believed was identical to the CO₂ pressure inside the leaf, because there was no flux of CO₂ across the epidermis. This measured partial pressure was in close agreement with that estimated from a consideration of the fluxes of CO₂ and vapor at the other surface.     

Determination of the Rate of CO(2) Evolution by Green Leaves in Light

The rate of CO₂ evolution in light by green leaves was determined by 2 methods in a closed system of gas analysis and by measuring the amount of CO₂ evolved into a CO₂ free air stream in an open system. All methods gave similar results under comparable conditions.     

Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

A method is described for growing cell suspension cultures of Chenopodium rubrum photoautotrophically for prolonged periods of time. By using a two-tier culture vessel the growth medium with the cells was separated from the CO₂ reservoir. Definite CO₂ concentrations were established by a K₂CO₃/KHCO₃ buffer. Photoautotrophic growth in C. rubrum cell suspension cultures was correlated with the CO₂ level. At 0.5% CO₂ the cell cultures contained 68 μg chlorophyll/g fresh weight and showed an increase in fresh weight of about 80% in 18 days. At 1% CO₂ an increase in fresh weight of 165% in 18 days was observed. The chlorophyll content rose up to 84 μg/g fresh weight. The photoautotrophic growth was also greatly influenced by the 2,4-D content of the medium. Cell growth was enhanced by lowering the auxin concentration. Best growth was attained (210% increase in fresh weight) at 10^?8M 2,4-D. The photosynthetic activity of the cells was measured by the light dependent ¹⁴CO₂ incorporation. At 0.5% CO₂ the cell suspensions assimilated about 100 μmol CO₂/mg chlorophyll × h. In the presence of 1% CO₂ the light driven assimilation was raised up to 185 μmol CO₂/mg chlorophyll × h. In both cases, the dark incorporation of CO₂ was merely 1.8% of the values obtained in light.     

The cultivation of animal cells at controlled dissolved oxygen partial pressure

A 3-liter culture vessel has been developed for the growth of animal cells in suspension at controlled pH and dissolved oxygen partial pressure (pO₂). The culture technique allows metabolically produced CO₂ to be measured; provision can be made to control the dissolved CO₂ partial pressure. In cultures containing a low serum concentration, gas sparging to control pO₂ was found to cause cell damage. This could be prevented by increasing the serum concentration to 10%, or by adding 0.02% of the surface-active polymer Pluronic F68. The growth of mouse LS cells in batch culture without pO₂ control was found to be limited by the availability of oxygen. Maximum viable cell populations were obtained when dissolved pO₂ was controlled at values within the range 40–100 mm Hg.     

Salinity effects on photosynthesis in isolated mesophyll cells of cowpea leaves

Mesophyll cells from leaves of cowpea (Vigna unquiculata [L.] Walp.) plants grown under saline conditions were isolated and used for the determination of photosynthetic CO₂ fixation. Maximal CO₂ fixation rate was obtained when the osmotic potential of both cell isolation and CO₂ fixation assay media were close to leaf osmotic potential, yielding a zero turgor pressure. Hypotonic and hypertonic media decreased the rate of photosynthesis regardless of the salinity level during plant growth. No decrease in photosynthesis was obtained for NaCl concentrations up to 87 moles per cubic meter in the plant growing media and only a 30% decrease was found at 130 moles per cubic meter when the osmotic potential of cell isolation and CO₂ fixation media were optimal. The inhibition was reversible when stress was relieved. At 173 moles per cubic meter NaCl, photosynthesis was severely and irreversibly inhibited. This inhibition was attributed to toxic effects caused by high Cl⁻ and Na⁺ accumulation in the leaves. Uptake of sorbitol by intact cells was insignificant, and therefore not associated with cell volume changes. The light response curve of cells from low salinity grown plants was similar to the controls. Cells from plants grown at 173 moles per cubic meter NaCl were light saturated at a lower radiant flux density than were cells from lower salinity levels.     

Soil properties differently influence estimates of soil CO2 efflux from three chamber-based measurement systems

Soil CO₂ efflux is a major component of net ecosystem productivity (NEP) of forest systems. Combining data from multiple researchers for larger-scale modeling and assessment will only be valid if their methodologies provide directly comparable results. We conducted a series of laboratory and field tests to assess the presence and magnitude of soil CO₂ efflux measurement system × environment interactions. Laboratory comparisons were made with a dynamic, steady-state CO₂ flux generation apparatus, wherein gas diffusion drove flux without creating pressure differentials through three artificial soil media of varying air-filled porosity. Under these conditions, two closed systems (Li-6400-09 and SRC-1) exhibited errors that were dependent on physical properties of the artificial media. The open system (ACES) underestimated CO₂ flux. However, unlike the two other systems, the ACES results could be corrected with a single calibration equation that was unaffected by physical differences in artificial media. Both scale and rank changes occurred among the measurement systems across four sites. Our work clearly shows that soil CO₂ efflux measurement system × environment interactions do occur and can substantially impact estimates of soil CO₂ efflux. Until reliable calibration techniques are developed and applied, such interactions make direct comparison of published rates, and C budgets estimated using such rates, difficult.     

Measurement of carbon dioxide compensation points of freshwater algae

A technique is described for the measurement of total dissolved inorganic carbon by acid release as CO₂ followed by its conversion to methane and detection by flame ionization in a modified gas chromatograph. This method was used to determine the dissolved inorganic carbon concentration reached at compensation point when algae were allowed to photosynthesize in a closed system in a buffer at known pH, and the CO₂ compensation point was calculated from this concentration. The CO₂ compensation points of 16 freshwater algae were measured at acid and alkaline pH in air-saturated medium: at acid pH the CO₂ compensation points ranged from 4.8 to 41.5 microliters per liter while at alkaline pH they ranged from 0.2 to 7.2 microliters per liter. Removal of O₂ from the medium caused a slight lowering of compensation point at acid pH but had little effect at alkaline pH. These low, O₂-insensitive compensation points are characteristic of C₄ plants. It is suggested that these low CO₂ compensation points are maintained by an active bicarbonate uptake by algae especially at alkaline pH.     

Variation of photoautotrophic fatty acid production from a highly CO2 tolerant alga,Chlorococcum littorale,with inorganic carbon over narrow ranges of pH

Photoautotrophic fatty acid production of a highly CO₂‐tolerant green alga Chlorococcum littorale in the presence of inorganic carbon at 295 K and light intensity of 170 µmol‐photon m^?2 s^?1 was investigated. CO₂ concentration in the bubbling gas was adjusted by mixing pure gas components of CO₂ and N₂ to avoid photorespiration and β‐oxidation of fatty acids under O₂ surrounding conditions. Maximum content of total fatty acid showed pH‐dependence after nitrate depletion of the culture media and increased with the corresponding inorganic carbon ratio. Namely, [HCO₃^?]/([CO₂]+n[ ]) ratio in the culture media was found to be a controlling factor for photoautotrophic fatty acid production after the nitrate limitation. At a CO₂ concentration of 5% (vol/vol) and a pH of 6.7, the fatty acid content was 47.8 wt % (dry basis) at its maximum that is comparable with land plant seed oils. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1053–1057, 2015     

Influence of carbon supply on the circadian rhythmicity of photosynthesis and its stimulation by blue light in Ectocarpus siliculosus: clues to the mechanism of inorganic carbon acquisition in lower brown algae

Stimulation or light-saturated rates of photosynthesis in Ectocarpus siliculosus (Dillwyn) Lyngb. by blue light was eliminated by increasing dissolved inorganic carbon (DIC) or by lowering pH in natural seawater. The amplitude of the circadian rhythm of photosynthesis was also diminished under these conditions, and the pH compensation points in a closed system were higher in the presence of blue light and during the circadian day. These observations suggest that blue light and the circadian clock regulate the activity of a carbon acquisition system in these plants. The inhibitor of external carbonic anhydrase, acetazolamide, reduced overall rates of photosynthesis by only about 30%, but ethoxyzolamide suppressed the circadian rhythm of photosynthesis almost completely and markedly reduced the duration of responses to blue light pulses. Similar patterns were obtained when photosynthesis was measured in strongly limiting DIC concentrations (0–0.5 mol m^?3). Since blue light stimulated photosynthesis under these conditions of strong carbon limitation, we suggest that blue light activates the release of CO₂ from an internal CO₂ store. We propose a metabolic pathway with similarities to that of CAM plants. Non-photosynthetic fixation leads to the accumulation of a storage metabolite. The circadian clock and blue light control the mobilization of CO₂ at the site of decarboxylation of this metabolite. In the presence of continuous blue light the pathway is proposed to cycle and act as a pump for CO₂ into the chloroplasts. This hypothesis helps to explain a number of previously reported peculiarities of brown algal photosynthesis.     

Isolation of Intact Chloroplasts from Dunaliella tertiolecta

Cells of Dunaliella tertiolecta from the log phase of growth were broken by rapid extrusion at low pressure through a Yeda press and the chloroplasts were isolated by centrifugation through a Percoll gradient. Osmolarity of the growth media, the suspending media, and the Percoll gradient was kept identical to minimize change in chloroplast volume and mitochondrial entrapment. The isolated intact chloroplasts were obtained in a 30 to 50% yield based on chlorophyll and were stable to washing with buffered medium. Isolated chloroplast yield and purity was dependent on cell culture condition; a cycle of 16 hours light and 8 hours dark with continuous high CO₂ was optimum. Isolated chloroplasts were about 90% intact by microscopic examination, ferricyanide-dependent O₂ evolution, and the distribution of four stromal enzymes. Enzymes associated with glycolate metabolism were not in the chloroplast fraction. The isolated chloroplasts with 10 millimolar bicarbonate evolved 24 micromoles of O₂ and fixed 21 micromoles of CO₂ per hour per milligram of chlorophyll, which rates were about one-third of those by whole cells. The inhibition of oxygen evolution by 10 millimolar phosphate was reversed by P-glycerate. Whole chloroplasts were also isolated from cells adapted to low CO₂ in air for 24 hours. On low CO₂ the cells excreted more gelatinous material, which had to be removed with additional washing of the cells, before it was possible to obtain good chloroplast preparations.     

An on-line technique for monitoring propionic acid fermentation

An on-line technique, based on measuring the increase in pressure due to CO₂ release in a closed air-tight reactor, was used to evaluate the fermentation of lactate by propionibacteria. The method was applied to batch cultures of Propionibacterium shermanii grown in yeast extract/sodium lactate medium containing lactate as a carbon source under micro-aerophilic conditions. Gas pressure evolution was compared both with substrate consumption and metabolites production and with acidification and growth. Linear relationships were found between gas pressure variation, lactate consumption and propionate and acetate production. The technique also enabled the evaluation of total CO₂ produced, by taking account of pressure, oxygen and pH measurements. These results tend to show that this simple and rapid method could be useful to monitor propionic acid bacteria growth.     

<Emphasis Type="Italic">In situ</Emphasis> carbon supplementation in large-scale cultivations of <Emphasis Type="Italic">Spirulina platensis</Emphasis> in open raceway pond

The objective of this study was to estimate the CO₂ absorptivity provided by an in situ carbon supply system using a photosynthetic culture of the cyanobacterium Spirulina platensis in an open raceway pond. The effects of initial total carbon concentrations (ranging from 0 to 0.1 mol/L), suspension depths (ranging from 5 to 20 cm) and pH values (ranging from 8.9 to 11.0) on the CO₂ absorptivity were studied. The results indicated that CO₂ absorptivity was positively correlated with pH value, negatively correlated with total carbon concentration, and only negligibly affected by the suspension depth. The optimum total carbon concentration range and pH range were 0.03 ∼ 0.09 mol/L and 9.7 ∼ 10.0, respectively. An average CO₂ absorptivity of 86.16% and average CO₂ utilization efficiency of 79.18% were achieved using this in situ carbon-supply system in large-scale cultivation of Spirulina platensis, with an initial total carbon concentration of 0.06 mol/L and pH value of 9.8. Our results demonstrated that this system could obtain a favorable CO₂ utilization efficiency in outdoor, large-scale cultivation of Spirulina platensis in open raceway ponds.