Ubiquinone concentrations in Athiorhodaceae grown under various environmental conditions

1. The nature and concentration of ubiquinone in six species of Athiorhodaceae have been examined after growth under aerobic and photosynthetic conditions. 2. Increase in ubiquinone concentration during adaptive synthesis of photosynthetic pigments by Rhodopseudomonas spheroides incubated under low-aeration conditions was observed. 3. The nature of the carbon source was found to have a marked effect on ubiquinone, as well as bacteriochlorophyll, concentrations.     

Fatty acid composition of Spirulina strains grown under various environmental conditions

The fatty acid distribution in 19 strains of Spirulina was studied. All but one contained γ-linolenic acid (GLA). No GLA was found in S. subsalsa, which had a very high content of palmitoleic acid. The fatty acid content of all but one of the tested strains increased with cultivation temperature and the relative amount of polyunsaturated fatty acid decreased. The highest content of GLA was found at 30–35° for most strains. High light intensities at a high temperature (38°), while not affecting the fatty acid composition, had a drastic effect on the fatty acid content, reducing it by as much as 46 %.     

Radial and axial turgor pressure measurements in individual root cells of Mesembryanthemum crystallinum grown under various saline conditions

Abstract. Radial and axial turgor pressure profiles were measured with the pressure probe in untreated and salt-treated intact roots of Mesembryanthemum crystallinum. The microcapillary of the pressure probe was inserted step-wise into the root tissue 5, 25 and 50 mm away from the root cap. For evaluation of the data, only those recordings on a given root were used in which four discontinuous increases in turgor pressure occurred. These four turgor pressure increases could be related to the rhizodermal cells and to the cells in the three cortical layers. The measurements showed that a radial turgor pressure gradient of the same magnitude (directed from the third cortical layer to the external medium) existed along the root axis. The magnitude of this turgor pressure gradient decreased with increasing salinity (up to 400 mol m^-3 NaCl) in the growth medium. Addition of 10 mol m^-3 CaCl₂ to the 400 mol m^-3 NaCl medium partly reduced the salt-induced decrease in turgor pressure, but only in cells 25–50 mm away from the root tip. Combined with this effect, a small axial turgor pressure gradient was generated, therefore, in the cortex layers which was directed to the root tip. Measurements of the volumetric elastic modulus, ?, of the wall of the individual cells showed that the presence of salt considerably reduced the magnitude of this parameter and that addition of Ca²⁺ to the strongly saline medium partially diminished this decrease. This effect was strongest in cells 50 mm away from the root tip. The magnitude of ? of rhizodermal and cortical cells increased along the root axis both in untreated and in salt-treated roots. The ? value was significantly smaller for rhizodermal cells compared to the cortical cells, with the exception of cells 50 mm from the tip. In this tissue, rhizodermal and cortical cells exhibited nearly the same values. The decrease of the ?-values with salt and the increase along the root axis under the various growth conditions could be correlated with corresponding changes in cell volume. Diurnal changes in turgor pressure could not be detected in the individual root cells, with the notable exception of the rhizodermal and cortical cells located in the region 50 mm away from the root tip of the control plants. In these cells, an increase in turgor pressure was observed during the morning hours. Determination of the average osmotic pressure in tissue sections along the roots of control and salt-treated plants revealed that at 400 mol m^-3 NaCl the osmotic pressure gradient between the tissue and the medium is exo-directed, provided that the water is not (partly) immobilized.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.     

Phosphate-uptake systems in Yarrowia lipolytica cells grown under alkaline conditions

In this study we used a newly isolated Yarrowia lipolytica strain with a unique capacity to grow over a wide pH range (3.5-10.5), which makes it an excellent model system for studying phosphate transport systems in cells grown under alkaline conditions. Phosphate uptake by Y. lipolytica yeast cells grown at pH 9.5-10 was shown to be mediated by several kinetically discrete Na+-dependent systems. One of these, a low-affinity transporter, operates at high Pi concentrations and is, to our knowledge, here kinetically characterized for the first time. The other two high-affinity systems are derepressible, come into play under conditions of Pi-starvation, and appear to be controlled by the availability of extracellular Pi. They represent the first examples of high-capacity, Na+-driven Pi transport systems in an organism belonging to neither the animal nor the bacterial kingdoms.     

Ultrastructure of intracytoplasmic membranes of Methanomonas margaritae cells grown under different conditions

The development of intracytoplasmic membranes of Methanomonas margaritae cells grown under different culture conditions was studied. Growth on methane was strongly accelerated by the addition of copper ions. Acceleration by copper, however, was not observed in the case of growth on methanol. Cells grown on methane with copper possessed intracytoplasmic membranes along the cell periphery. When the organism was grown in a medium lacking copper, intracytoplasmic membranes appeared as large vesicles surrounded by a unit membrane at the periphery of the cell. The vesicles originated from paired membranes due to the absence of copper in the medium. Cells grown on methanol with or without copper possessed a number of vesicles of different sizes arranged in a chain along the cell periphery. The possible relationship between membrane arrangement and methane oxidation is discussed.     

Distribution of thiophene derivatives in different organs of Tagetes patula seedlings grown under various conditions

An intensive accumulation of thiophene derivatives occurs during the first days of development in Tagetes seedlings. After that, the thiophene content (related to dry weight) decreases to reach low values until the twelfth day of growth. High performance liquid chromatography analyses performed on cotyledons, on hypocotyls, and on roots of normal-grown and of partly-etiolated seedlings showed that bithienylbutinen, the major compound in hypocotyls and roots, is accumulated earlier than the other thiophene derivatives. The synthesis of acetoxybutinylbithiophene seems to be light-induced. This compound is not found in cotyledons. Hydroxybutinylbithiophene is synthesized specifically in the roots, -terthiophene in cotyledons. The results obtained indicate a high intensity of thiophene metabolism in these particular organs during seedling development.     

Photosynthetic productivity of conical helical tubular photobioreactors incorporating Chlorella sp. under various culture medium flow conditions

The characteristics of the flow of culture medium significantly affects the photosynthetic productivity of bioreactors incorporating microalgae. Therefore, in order to optimize the performance of a conical helical tubular photobioreactor (CHTP) designed to be useful in practical applications, we characterized the flow pattern of the culture medium through the reactor. The effects of medium flow conditions on the photosynthetic productivity of Chlorella sp. were investigated using several different CHTP units with 0.50-m2 installation areas which were designed to vary the direction and rate of flow driven by airlift. In addition, the performance of two- and four-unit systems constructed by combining individual CHTP units was evaluated. We found that when medium flowed from the bottom to the top of the photostage, it exhibited smoother flow of culture medium than when flowing from top to bottom, which led to higher photosynthetic productivity by the former. Consistent with theoretical calculations, varying the lengths of vertical flow passages caused flow rates to vary, and higher flow rates meant smoother circulation of medium and better photosynthetic performance. Flow of medium through a four-unit CHTP system was similar to that in single units, enabling a photosynthetic productivity of 31.0 g-dry biomass per m2-installation area per day to be achieved, which corresponded to a photosynthetic efficiency of 7.50% (photosynthetically active radiation (PAR; 400-700 nm)). This high photosynthetic performance was possible because smoother medium flow attained in single units was also attained in the four-unit system.     

Performance of Chlorella sorokiniana under simulated extreme winter conditions

High annual microalgae productivities can only be achieved if solar light is efficiently used through the different seasons. During winter the productivity is low because of the light and temperature conditions. The productivity and photosynthetic efficiency of Chlorella sorokiniana were assessed under the worst-case scenario found during winter time in Huelva, south of Spain. The maximum light intensity (800?μmol photons m^-2 s^-1) and temperature (20°C) during winter were simulated in a lab-scale photobioreactor with a short light-path of 14?mm. Chemostat conditions were applied and the results were compared with a temperature-controlled situation at 38°C (optimal growth temperature for C. sorokiniana). When temperature was optimal the highest productivity was found at a dilution rate of 0.18 h^-1 (P _v?=?0.28?g Kg^-1 h^-1), and the biomass yield on light energy was high (Y _x,E?=?1.2?g?mol^-1 photons supplied). However, at suboptimal temperature, the specific growth rate of C. sorokiniana was surprisingly low, not being able to support continuous operation at a dilution rate higher than 0.02 h^-1. The slow metabolism under suboptimal temperature resulted in a decline of the light energy requirements of the cells. Consequently, the maximum winter irradiance was experienced as excessive, leading to a low photosynthetic efficiency and productivity (Y _x,E?=?0.5?g mol^-1 photons supplied, P _v?=?0.1?g Kg^-1 h^-1). At suboptimal temperature a higher carotenoid-to-chlorophyll ratio was observed indicating the activation of light-dissipating processes. We conclude that temperature control and/or light dilution during winter time will enhance the productivity.     

Changes in levels of amino acid acceptors in tRNA from Escherichia coli grown under various conditions

Active enzyme sedimentation of five asparaginase and glutaminase-asparaginase enzymes with antitumor activity was studied. The catalytically active species of each enzyme appeared to have a molecular weight greater than 100,000 g/mole. Gel filtration and disc gel electrophoresis confirmed the absence of catalytically active smaller species.     

Dark metabolism of acetate in Rhodospirillum rubrum cells, grown under photoheterotropic conditions

The mechanism of the dark assimilation of acetate in the photoheterotrophically grown nonsulfur bacterium Rhodospirillum rubrum was studied. Both in the light and in the dark, acetate assimilation in Rsp. rubrum cells, which lack the glyoxylate pathway, was accompanied by the excretion of glyoxylate into the growth medium. The assimilation of propionate was accompanied by the excretion of pyruvate. Acetate assimilation was found to be stimulated by bicarbonate, pyruvate, the C4-dicarboxylic acids of the Krebs cycle, and glyoxylate, but not by propionate. These data implied that the citramalate (CM) cycle in Rsp. rubrum cells grown aerobically in the dark can function as an anaplerotic pathway. This supposition was confirmed by respiration measurements. The respiration of cells oxidizing acetate depended on the presence of CO2 in the medium. The fact that the intermediates of the CM cycle (citramalate and mesaconate) markedly inhibited acetate assimilation but had almost no effect on cell respiration indicative that citramalate and mesaconate are intermediates of the acetate assimilation pathway. The inhibition of acetate assimilation and cell respiration by itaconate was due to its inhibitory effect on propionyl-CoA carboxylase, an enzyme of the CM cycle. The addition of 5 mM itaconate to extracts of Rsp. rubrum cells inhibited the activity of this enzyme by 85%. The data obtained suggest that the CM cycle continues to function in Rsp. rubrum cells that have been grown anaerobically in the light and then transferred to the dark and incubated aerobically.     

Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions

Summary The understanding of pathways associated with differentiated function in human epithelial cells has been enhanced by the development of methods for the short-term culture of human epithelial cells. In general these methods involve the use of serum. The subculture and maintenance of epithelial cells in long-term culture has been more problematic. A serum-free medium developed for human bronchial epithelial cells was slightly modified and found to be useful for the subculture and long-term maintenance of not only bronchial epithelial cells, but also tracheal, nasal polyp, and sweat gland epithelial cells from either normal or cystic fibrosis individuals. The cells maintained epithelial-specific characteristics after multiple subcultures. Monolayers of epithelial cells showed junctional complex formation, the presence of keratin, and micro villi. Functional studies with Ussing chambers showed short circuit current (I_sc) responses to isoproterenol, bradykinin, or calcium ionophore (A23187) in subcultured tracheal and bronchial cells. This work is supported by grants HL41928 and DK39619 (DCG), HL24136 (CBB), and HL42368 (JHW and DCG) from the National Institutes of Health, Bethesda, MD.     

Differential response in downstream processing of CHO cells grown under mild hypothermic conditions

The manufacture of complex therapeutic proteins using mammalian cells is well established, with several strategies developed to improve productivity. The application of sustained mild hypothermic conditions during culture has been associated with increases in product titer and improved product quality. However, despite associated cell physiological effects, very few studies have investigated the impact on downstream processing (DSP). Characterization of cells grown under mild hypothermic conditions demonstrated that the stationary phase was prolonged by delaying the onset of apoptosis. This enabled cells to maintain viability for extended periods and increase volumetric productivity from 0.74 to 1.02 g L^?1. However, host cell proteins, measured by ELISA, increased by ～50%, attributed to the extended time course and higher peak and harvest cell densities. The individual components making up this impurity, as determined by SELDI‐TOF MS and 2D‐PAGE, were shown to be largely comparable. Under mild hypothermic conditions, cells were less shear sensitive than those maintained at 37°C, enhancing the preliminary primary recovery step. Adaptive changes in membrane fluidity were further investigated by adopting a pronounced temperature shift immediately prior to primary recovery and the improvement observed suggests that such a strategy may be implementable when shear sensitivity is of concern. Early and late apoptotic cells were particularly susceptible to shear, at either temperature, even under the lowest shear rate investigated. These findings demonstrate the importance of considering the impact of cell culture strategies and cell physiology on DSP, by implementing a range of experimental methods for process characterization. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:688–696, 2013     

Heterotrophic production of Chlorella sp. TISTR 8990—biomass growth and composition under various production conditions

The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon‐to‐nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L^?1 glucose, 2.5 g L^?1 KNO₃) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500‐mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L^?1 was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g^?1 and 305 mg L^?1 day^?1, respectively. A repeated fed‐batch culture with four cycles of feeding with the fivefold concentrated medium in a 3‐L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L^?1 was obtained with a TFA productivity of 223 mg L^?1 day^?1. The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1589–1600, 2017     

Changes in vacuolar pH of carrot cells in suspension culture grown under saline conditions

The effect of salinity on vacuolar pH was studied in carrot (Daucus carota L.) cells grown in liquid suspension culture either in the absence or presence of 150 mM NaCl. Both vacuolar and cytoplasmic pH were determined by several independent techniques. These techniques were NMR spectrometry, distribution of radioactive probes and spectrophotometric measurement of the absorbance changes of a naturally occurring vacuolar pH indicator. There was no difference in the cytoplasmic pH between cells grown in the presence or the absence of NaCl, but the vacuolar pH of cells grown in the presence of NaCl was higher by 0.38 to 1.05 pH units (depending on the technique that was used) than the vacuolar pH of cells grown in the absence of NaCl.     

Orthophosphate influx and efflux rates of Chlorella fusca measured in a continuous turbidostat culture with 32P under various conditions

The main objective of the experiments with Chlorella fusca strain 211-8b was to measure, with adequate time resolution, the unidirectional influx rates of phosphate into non-phosphate-starved algae under different steady state conditions (light, temperature, 3-phosphoglycerate influence) or following the addition of several photosynthesis and phosphate transport inhibitors (phenylmercuric acetate, p-chloromercuribenzoate, arsenate). the algae were cultivated in a phosphate rich medium in a continuous turbidostat culture. The phosphate exchange experiments with carrier-free ³²PO ₄ ^3- were performed directly in the continuous culture. The sampling intervals after the tracer addition were 15 s.For a continuous steady state culture grown in the light (25° C) the unidirectional influx rate measured with ³²P is 260 times higher than the net uptake rate (=influx minus efflux rate) calculated from the mass balance using the data of this culture. In all experiments, except the control experiment with trichloroacetic acid killed cells, the specific activity of the intracellular inorganic orthophosphate compartment oscillates around a constant mean value which never reaches the specific activity of the nutrient medium within the duration of the short-term experiments (7.5 min). The inhibitors strongly affect the characteristics of the oscillations. The unidirectional influx rates are constant. Oscillating flushing rates with unlabelled phosphate from a storage compartment have been postulates to explain the oscillations. Oscillating rates from the individual cells are apparently synchronized by an unknown mechanism.     

Utilization of metabolic energy under saline conditions: changes in properties of ATP dependent enzymes in plant cells grown under saline conditions

The effect of growth in saline medium on the activity of two ATP utilizing enzymes was studied. Hexokinase in carrot (Daucus carota L.) cells grown in suspension culture either in the absence or presence of 150 ml NaCl, and tonoplast H⁺-ATPase in tobacco (Nicotiana tabacum L. cv. Wisconsin 38) cells grown in suspension culture either in the absence of presence of 428 mM NaCl. There was no difference in the pH profiles, NaCl sensitivity and kinetic parameters towards glucose of hexokinase activities from carrot cells grown in the presence or the absence of NaCl, but the activity from cells grown in the presence of NaCl was more resistant to inhibition by N-ethylmaleimide and to inactivation by heat. Two separate apparent Km values toward ATP were delineated in the extract from cells grown in presence of NaCl while extracts from cells grown in the absence of NaCl had only one apparent Km value. The tonoplast H⁺-ATPase from NaCl grown tobacco cells showed changed kinetic compared to this activity from cells grown in the absence of NaCl. These data may indicate that growth in NaCl results in the appearance of isozymic activity that enhances the ability of plant cells to utilize metabolic energy more efficiently.     

Stability of recombinant plasmids in transgenic microorganism cells under various environmental conditions]

The copy number of R plasmids weakly depends on the selective pressure of the respective antibiotic but does depend on the physiology of the host species and the type of plasmids and cloned genes, whose expression leads to a further load on the biosynthetic apparatus of cells. The last factor is critical in the maintenance of recombinant plasmids in transgenic microorganisms.