Effect of Sodium Thiosulfate on the Depletion of Photosynthetic Apparatus in Cyanobacterium Synechocystis sp. PCC 6803 Cells Grown in the Presence of Glucose

Fluorescence spectroscopy at 77 K showed that the application of glucose lead to the depletion of phycobilisomes (PBS) and photosystems (PS) 2 and 1, and that PS2 was more sensitive to glucose than PS1. The application of sodium thiosulfate, an effective scavenger of reactive oxygen intermediates, counteracted the effects of glucose. Sodium thiosulfate effectively protected photosynthetic apparatus, PS2, PS1, and PBS against glucose-induced depletion. Sodium thiosulfate showed strong capability to inhibit the disappearance of chlorophyll induced by glucose. At a relatively low concentration of glucose, the application of sodium thiosulfate can even be helpful for the assembly of photosynthetic apparatus. Hence the reactive oxygen species might be involved in the depletion of the photosynthetic apparatus in the cyanobacterium Synechocystis sp. PCC 6803 cells grown in the presence of glucose.     

Attached Growth of Sphaerotilus and Mixed Populations in a Continuous-flow Apparatus

The effects of NH(4)Cl concentration, organic nitrogen compounds, glucose concentration, dissolved oxygen concentration, and flow rate on the attached growth of pure cultures of Sphaerotilus natans and of a mixed population in a continuous-flow apparatus are described. Low concentrations of NH(4)Cl and oxygen, and high flow rates resulted in attached populations that were dominated by Sphaerotilus. The conditions that allowed maximal attached growth in pure culture did not correspond to the conditions that promoted attached growth of Sphaerotilus in a mixed population.     

Modeling of spherical fluorescent glucose microsensor systems: design of enzymatic smart tattoos

A two-substrate mathematical model of microspherical optical enzymatic glucose sensors is presented. The sensors are based on the well-known oxidation of glucose by glucose oxidase, and are constructed by the encapsulation of glucose oxidase within hydrogel microspheres coated with ultrathin polyelectrolyte multilayer films. In order to measure glucose via changes in oxygen concentration, a fluorescent oxygen indicator is co-encapsulated with the enzyme. The model was used to predict the temporal and spatial distributions of glucose and oxygen within the sphere for step increases in bulk glucose concentration. In addition, the model was used to observe the effect of varying sensor parameters, namely sphere size, film thickness, enzyme concentration, and mass transport of substrate and co-substrate within the sphere and film coatings, on the response of the sensors. A major finding was that the application of {PSS/PAH} films as thin as 12 nm can drastically improve the sensor performance over uncoated sensors based on calcium alginate microspheres. The model is proposed as an important tool for a priori design of these complex sensor structures.     

On-line characterization of a hybridoma cell culture process

The on-line determination of the physiological state of a cell culture process requires reliable on-line measurements of various parameters and calculations of specific rates from these measurements. The cell concentration of a hybridoma culture was estimated on-line by measuring optical density (OD) with a laser turbidity probe. The oxygen uptake rate (OUR) was determined by monitoring dynamically dissolved oxygen concentration profiles and closing oxygen balances in the culture. The base addition for neutralizing lactate produced by cells was also monitored on-line via a balance. Using OD and OUR measurements, the specific growth and specific oxygen consumption rates were determined on-line. By combining predetermined stoichiometric relationships among oxygen and glucose consumption and lactate production, the specific glucose consumption and lactate production rates were also calculated on-line. Using these on-line measurements and calculations, the hybridoma culture process was characterized on-line by identifying the physiological states. They will also facilitate the implementation of nutrient feeding strategies for fed-batch and perfusion cultures. (c) 1994 John Wiley & Sons, Inc.     

Production of catalase and glucose oxidase by Aspergillus niger using unconventional oxygenation of culture

A novel method for increasing dissolved oxygen concentration in culture media has been developed. It involves adding hydrogen peroxide (H2O2) to the medium which is then decomposed to oxygen and water by catalase. Some factors affecting oxygenation of culture were investigated. Maximal oxygen concentration occurred in 50 ml of the medium containing 0-2 g wet mycelium and 0.2% glucose at pH 5.0. A new apparatus for automated addition of H2O2 to the bioreactor to keep the dissolved oxygen concentration constant over the range 1-100% +/- 2% was tested. A significant increase (over sixfold) of intracellular catalase activity was obtained while the dissolved oxygen concentration remained stable (30% +/- 2%).     

Effects of Diltiazem on Bioenergetics, K+ Gradients, and Free Cytosolic Ca2+ Levels in Rat Brain Synaptosomes Submitted to Energy Metabolism Inhibition and Depolarization

Diltiazem was able to decrease the oxygen consumption rate and lactate production in synaptosomes isolated from rat forebrains, both under control and depolarized (40 microM veratridine) conditions, starting from a concentration of 250 microM. This effect was particularly evident when synaptosomes were depolarized by veratridine. This depolarization-counteracting action was evident also when transplasma membrane K+ diffusion potentials were measured after depolarization induced by veratridine and by rotenone with a glucose shortage. The concentrations of ATP, phosphocreatine, and creatine were less sensitive to diltiazem action. The concentration/response relationships were the same as those found for the oxygen consumption were the same as those found for the oxygen consumption rate, lactate production, and K+ diffusion potentials. The effects of 0.5 mM diltiazem in counteracting inhibition of energy metabolism induced by rotenone without glucose were no longer detectable when either Ca2+ or Na+ was absent from the incubation medium of synaptosomes. Diltiazem at the same concentrations (starting from 250 microM) was able to inhibit both the veratridine-induced and the rotenone-without-glucose-induced increase in intrasynaptosomal free Ca2+ levels evaluated with the fluorescent probe quin2. The results are discussed in view of a possible effect of diltiazem on voltage-dependent Na+ channels and the possibility of utilizing this approach for counteracting neuronal failure due to derangement of energy metabolism or hyperexcitation.     

A method for the control of the dissolved oxygen tension in microbial cultures

A method for the control of dissolved oxygen tension in growing microbial cultures is described. The apparatus consists of a motor-driven air sparge pipe which may be lowered or raised to give a variable point of entry of the air stream into the culture liquid and hence a variable gas dispersion and gas–liquid contact time. Control of the sparge pipe position is by means of a feedback control loop consisting of a dissolved oxygen probe, an on/off controller, and a reversing electric motor which drives the sparge pipe. The difficulty presented by the relatively slow response of the oxygen probe has been overcome by incorporating an adjustable rate of control action.     

Dynamic concentration challenges for biosensor characterization

A method and apparatus are described for characterization of the steady state and dynamic response of biosensors. The apparatus produces a steady stream of homogeneously mixed analyte whose concentration can be fixed at discrete values or varied continously. The device is ideally suited for continously operated biosensors, but is also effective for biosensors that operate in discrete sampling modes. The system permits simultaneous testing of several sensors and determination of the accuracy, precision and repeatability of sensor response. The characteristics of this testing apparatus were validated with ferrocyanide and glucose as indicators. As an example of use of the apparatus, concentration ramps were created and used to complement conventional step changes for characterizing an implantable glucose sensor. The ramp rate can be adjusted easily by scaling the apparatus to simulate the rate of concentration change anticipated during actual monitoring situations.     

The effects of glucose and oxygen on the cytochromes and metabolic activity of yeast batch cultures. II. Candida utilis

Candida utilis was grown in batch culture with and without oxygen control. The concentrations of A-, B-, and C-type cytochromes were found to vary with the initial glucose concentration, with the dissolved oxygen concentration, and with time. A-type was the most sensitive. After glucose was essentially exhausted, the yeast catabolized ethanol, if it had been growing in a relatively low initial glucose concentration, or non-glucose carbohydrate, including some of that previously accumulated within the cell, if it had been growing in a high initial glucose concentration. This difference in metabolic pattern could explain why cytochrome derepression was initiated soon after glucose uptake ceased only if initial glucose had been relatively low. The effects of glucose and dissolved oxygen concentrations on yeast cytochromes and respiratory activity are discussed.     

Verfahren und Analysenautomat zur Bestimmung von Nährlösungskomponenten und Stoffwechselprodukten

A continuous flow system with an enzyme electrode and with a new type of measuring instrument, the BIOXY-Meter, is described as an example for the determination of glucose. The BIOXY-Meter combined with various biosensors may be used in biotechnology and in other fields for the solution of a number of analytical problems. Measuring principle is the reduction of oxygen. The peak of first derivation of the current-time-curve is proportional to oxygen consumption and to decrease of substrate concentration. Enzyme fixation with common adhesive and a device for automated saturation of measuring samples with air in the sampler are also described. With the apparatus 60 samples per hour are analyzed for glucose. The linear measuring range is 0.1 mMol to 1.5 mMol with a coefficient of variation of V_k = 0.93 per cent. The described type of enzyme electrode is stable for 2 to 12 weeks or 1,000 to 5,000 measurements.     

Optimization of an Aspergillus niger glucose oxidase production process

The purpose of the present study was to ascertain the optimal concentration of dissolved oxygen in order to maximize the intracellular glucose oxidase formation in Aspergillus niger. Cultivations performed in a 3.5 l laboratory reactor showed that a dissolved oxygen concentration at 3% of saturation at a total pressure of 1.2 bar was optimal for maximizing intracellular glucose oxidase activity. Cultivations performed at higher dissolved oxygen concentrations did not produce as much glucose oxidase as those performed at 3%, although the formation rate was high. Experiments revealed that maximal intracellular glucose oxidase formation for the A. niger strain used, is accomplished by limiting the gluconic acid production rate by means of maintaining a low dissolved oxygen concentration. Several attempts to achieve higher intracellular glucose oxidase activity were also made by manipulating the glucose concentration at a 3% dissolved oxygen concentration. However, no enhancement in glucose oxidase activity was observed.     

Use of the glucose oxidase system to measure oxygen transfer rates

This investigation used the glucose oxidase system to simulate oxygen transfer rate in fermentation broths. It was demonstrated that the fungal preparation contained sufficient lactonase activity so that D -glucono-δ-lactone did not accumulate and that the rate of production of gluconic acid was proportional to the oxygen uptake rate. Enzyme concentrations of 1.5–2 g/1 were found adequate to determine oxygen absorption rates in shake flasks while maintaining the dissolved oxygen concentration of low levels. The apparent Michaelis constant for oxygen, K_m(O₂), was found to be 27% saturation with air; this value along with experimentally determined uptake rates could be used to calculate dissolved oxygen concentration in lieu of using a dissolved oxygen probe. Enzyme concentrations of 5 g/l were sufficient to give linear acid production and low dissolved oxygen concentrations in a bench-scale fermenter with no foaming or enzyme deactivation. The method is considered more valid and easier to employ than previously utilized techniques such as sulfite oxidation. Extension of the system to evaluating aeration effectiveness and scaleup of fermentation equipment is discussed.     

Whole cell immobilized amperometric biosensor based on Saccharomyces cerevisiae for selective determination of vitamin B1 (thiamine)

A new amperometric whole cell biosensor based on Saccharomyces cerevisiae immobilized in gelatin was developed for selective determination of vitamin B1 (thiamine). The biosensor was constructed by using gelatin and crosslinking agent glutaraldehyde to immobilize S. cerevisiae cells on the Teflon membrane of dissolved oxygen (DO) probe used as the basic electrode system combined with a digital oxygen meter. The cells were induced by vitamin B1 in the culture medium, and the cells used it as a carbon source in the absence of glucose. So, when the vitamin B1 solution is injected into the whole cell biosensor system, an increase in respiration activity of the cells results from the metabolic activity and causes a decrease in the DO concentration of interval surface of DO probe related to vitamin B1 concentration. The response time of the biosensor is 3 min, and the optimal working conditions of the biosensor were carried out as pH 7.0, 50mM Tris-HCl, and 30 degrees C. A linear relationship was obtained between the DO concentration decrease and vitamin B1 concentration between 5.0 x 10(-3) and 10(-1) microM. In the application studies of the biosensor, sensitive determination of vitamin B1 in the vitamin tablets was investigated.     

The oxidizing agent menadione induces an increase in the intracellular molecular oxygen concentration in K562 and A431 cells: Direct measurement using the new paramagnetic EPR probe fusinite

The intracellular molecular oxygen concentration in control and menadione-treated K562 (an erythroleukemic cell line that grows in suspension) and A431 (an epidermal carcinoma that grows in monolayer) cells was measured directly by using the new electron paramagnetic resonance (EPR) probe fusinite. Because the oxidizing agent menadione is known to damage mitochondria and the cytoplasmic membrane in other cell systems, before conducting measurements of oxygen concentration in K562 and A431 cells, it was necessary to establish injury in these systems as well. Consequently, morphological and flow cytometric analyses were conducted after menadione treatment. The data presented here show that the two cell lines are heavily damaged by menadione. Once this menadione-induced injury was demonstrated, measurements of oxygen concentration were carried out in both K562 and A431 cells. Treatment with this quinone induces a sharp increase in intracytoplasmic molecular oxygen in both cell lines (from about 1% to about 10 and 15% in K562 and A431 cells, respectively). In addition, to gain a more complete understanding of the effects of menadione on cells, the extracellular molecular oxygen concentration and the oxygen consumption rate were also measured in control and menadione-treated K562 cells. These measurements demonstrate that menadione treatment results in an increase in the extracellular oxygen concentration (from about 5% in controls to 15% in treated cells) as well as a decrease in the oxygen consumption rate (from about 10 ng O/min/10⁶ cells in controls to 3 ng O/min/10⁶ cells after menadione exposure). The importance of the new EPR probe fusinite in monitoring directly cellular functions in which oxygen is involved and the effects of menadione on cellular oxygen balance are discussed.     

Influence of Perfusate Glucose Concentration on Dialysate Lactate, Pyruvate, Aspartate, and Glutamate Levels Under Basal and Hypoxic Conditions: A Microdialysis Study in Rat Brain

Abstract: The aim of this study was to evaluate the influence of perfusion media with different glucose concentrations on dialysate levels of lactate, pyruvate, aspartate (Asp), and glutamate (Glu) under basal and hypoxic conditions in rat brain neocortex. Intracerebral microdialysis was performed with the rat under general anesthesia using bilateral probes (o.d. 0.3 mm; membrane length, 2 mm) perfused with artificial CSF containing 0.0 and 3.0 m M glucose, respectively. Basal dialysate levels were obtained 2 h after probe implantation in artificially ventilated animals. Dialysate levels of glucose were also measured for the two different perfusion fluids. The mean absolute extracellular concentration of glucose was estimated by a modification of the no-net-flux method to be 3.3 mmol/L, corresponding to an average in vivo recovery of 6% for glucose. Hypoxia was induced by lowering the inspired oxygen concentration to 3%. Hypoxia caused a disturbance of cortical electrical activity, evidenced by slower frequency and lower amplitudes on the electroencephalogram compared with prehypoxic conditions. This was associated with significant elevations of lactate, Asp, and Glu levels. There were no statistically significant differences in dialysate metabolite levels between the two perfusion fluids, during either normal or hypoxic conditions. We conclude that microdialysis with glucose-free perfusion fluid does not drain brain extracellular glucose in anesthetized rats to the extent that the dialysate lactate, pyruvate, Asp, and Glu levels during basal or hypoxic conditions are altered.     

Comparison of morphological characteristics of Streptomyces natalensis by image analysis and focused beam reflectance measurement

A morphological interpretation is presented for data collected during growth of a filamentous organism, using a focused beam reflectance measurement (FBRM) system. The morphology of the organism was also obtained using conventional semiautomatic image analysis to support the interpretation of the FBRM data. The model organism employed is the filamentous soil-borne actinomycete Streptomyces natalensis, which produces the antifungal agent pimaricin. The organism was cultivated both in shake flasks and in a bench-scale stirred tank bioreactor. It was found that FBRM could be used to track changes in the morphology of the organism throughout the course of its growth on both scales. These changes were highlighted using both the median chord length and length-weighted mean chord length obtained from the chord length distribution measured with the FBRM probe. The ability of the FBRM probe to respond to changes in both the size and morphology of mycelial aggregates was supported by standard image analysis parameters, including equivalent diameter, convex area, and compactness.     

The effects of glucose and oxygen on the cytochromes and metabolic activity of yeast batch cultures. I. Saccharomyces spp.

Saccharomyces cerevisiae and Saccharomyces carlsbergensis were grown in batch culture with and without oxygen control. The concentrations of A-, B- and C-type cytochromes of both yeasts were dependent on the oxygen concentration during growth as well as on the initial glucose concentration of the growth medium. S. cerevisiae cytochromes were maximal after growth in low glucose and low oxygen; S. carlsbergensis cytochromes were maximal after growth in low glucose and high oxygen. Except when glucose was in very low concentration, its catabolism by S. carlsbergensis was directed predominantly towards ethanolic fermentation regardless of the oxygen concentration. Growth rate, total cell mass and yield were maximal, and anabolism was closely balanced with catabolism, when glucose and oxygen of S. carlsbergensis cultures were both high. Under these conditions neither catabolism, respiratory or ethanolic, nor glucose uptake were maximal.     

Novel particulate spin probe for targeted determination of oxygen in cells and tissues

The synthesis and characterization of a new lithium octa-n-butoxy-substituted naphthalocyanine radical probe (LiNc-BuO) and its use in the determination of concentration of oxygen (oximetry) by electron paramagnetic resonance (EPR) spectroscopy are reported. The probe is synthesized as a needle-shaped microcrystalline particulate. The particulate shows a single-line EPR spectrum that is highly exchange-narrowed with a line-width of 210 mG. The EPR line-width is sensitive to molecular oxygen showing a linear relationship between the line-width and concentration of oxygen (pO(2)) with a sensitivity of 8.5 mG/mmHg. We studied a variety of physicochemical and biological properties of LiNc-BuO particulates to evaluate the suitability of the probe for in vivo oximetry. The probe is unaffected by biological oxidoreductants, stable in tissues for several months, and can be successfully internalized in cells. We used this probe to monitor changes in concentration of oxygen in the normal muscle and RIF-1 tumor tissue of mice as a function of tumor growth. The data showed a rapid decrease in the tumor pO(2) with increase of tumor volume. Human arterial smooth muscle cells, upon internalization of the LiNc-BuO probe, showed a marked oxygen gradient across the cell membrane. In summary, the newly synthesized octa-n-butoxy derivative of lithium naphthalocyanine has unique properties that are useful for determining oxygen concentration in chemical and biological systems by EPR spectroscopy and also for magnetic tagging of cells.     

An optical method for measurement of dioxygen concentration based upon quenching of phosphorescence

An optical method for measuring oxygen concentrations in aqueous solutions is described. This method is based upon the oxygen-dependent quenching of phosphorescence. Phosphorescence excitation and emission spectra and lifetimes of some of the probe molecules suitable for measurement of oxygen in aqueous solutions are given. The probes include fluorescein derivatives, 4'5'-diiodofluorescein, eosin Y, 5(and 6)-carboxyeosin, erythrosin, and 5(and 6)-carboxyerythrosin as well as the Zn(II), Y(III), Sn(IV), Lu(III), and Pd(II) derivatives of meso-tetra-(4-sulfonatophenyl)-porphine, meso-tetra-(N-methyl-4-pyridyl)-porphine and coproporphyrin. The phosphorescence lifetimes of the given probes were found to depend upon the oxygen concentration by a simple Stern-Volmer relationship with a quenching constant of approximately 10(9) M-1 S-1. Binding of the molecules to bovine serum albumin decreased the quenching constant for oxygen by approximately an order of magnitude and also inhibited probe self-quenching, indicating that at the protein binding site the probes are somewhat protected from collision with quenchers. The use of this optical method for measuring oxygen is demonstrated for reactions catalyzed by glucose oxidase and by cytochrome c oxidase. It is shown that, using this method oxygen concentrations can be measured from approximately 250 microM (air saturation) down to the nanomolar range.