Steady-state measurements of respiration rate with an oxygen electrode.

An oxygen electrode was developed which measures steady-state respiration rates in a volume of 0.25 ml and at oxygen concentrations as low as 0.1 μm. The steady state was achieved by pumping air-equilibrated buffer into the respirometer at various rates. The method is most suitable for tissue slices.     

A novel in situ probe for oxygen uptake rate measurement in mammalian cell cultures

The newly developed in situ oxygen uptake rate (in situ OUR) probe presented in this article is based on the in situ microscope technology platform. It is designed to measure the oxygen uptake rate (OUR) of mammalian cells, an important parameter for metabolic flux analysis, inside a reactor (in situ) and in real-time. The system isolates a known volume of cell culture from the bulk inside the bioreactor, monitors the oxygen consumption over time, and releases the sample again. The sample is mixed during the measurement with a new agitation system to keep the cells in suspension and prevent oxygen concentration gradients. The OUR measurement system also doubles as a standard dissolved oxygen (DO) probe for process monitoring when it is not performing OUR measurements. It can be equipped with two different types of optical sensors (i.e., DO, pH) simultaneously or a conventional polarographic DO-probe (Clark type). This new probe was successfully tested in baby hamster kidney perfusion cell cultures.     

Techniques for biochemical respiration measurements.

A small membrane-covered oxygen electrode is described. This electrode is used either as a stationary electrode in stirred solutions or as a vibrating electrode in unstirred solutions. An amplifier system for registration of the electrode current and its time derivative is also described as are two specialized reaction vessels, a miniature vessel of ca. 7-μl volume and a closed vessel for sampling during respiration measurements. The kinetics of oxygen uptake from the atmosphere of respiring solutions is investigated. The uptake follows first-order kinetics and may be calculated from simple equations. The uptake may be prevented by continuously adjusting the air space oxygen tension to the oxygen tension of the solution. This is done with the controlled gas mixer which is described. It makes possible reliable respiration measurements in open reaction vessels (e.g., photometric cuvettes). The techniques described have been developed for work with mitochondria but they have wide applicability.     

The Use of the Wide-Bore Dropping-Mercury Electrode for the Determination of Rates of Oxygen Uptake and of Oxidation of Ammonia by Micro-Organisms

S ummary : Convenient and reliable polarographic methods, using a wide-bore dropping-mercury electrode, for determining the dissolved oxygen content and the rate of respiration of sewage and activated sludge are described and verified by independent procedures. Using thiourea to inhibit Nitrosomonas spp., a technique was developed by which the uptake of oxygen by heterotrophs could be distinguished from that used in ammonia oxidation. Conditions are discussed under which accurate estimates of the rate of uptake of oxygen due to the oxidation of ammonia could be made. Typical values are given for the rates of respiration and of nitrification of activated sludge and also for the rates of respiration of a phenol-destroying pseudomonad.     

Detection of microbial growth on polycyclic aromatic hydrocarbons in microtiter plates by using the respiration indicator WST-1

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 [4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate], in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.     

Detection of the kinetics of biochemical reactions with oxygen using exchange broadening in the ESR spectra of nitroxide radicals.

To detect changes in the oxygen concentration during biochemical reactions, the exchange broadening in the ESR spectra of nitroxide radicals caused by the dissolved oxygen, has been used. The measurements have been carried out using changes in the width either of the proton hyperfine structure components or of the nitrogen hyperfine structure line with an unresolved proton structure. Detection of mitochondrial respiration in a volume of about 10(-3) cm 3 and respiration for 100 +/- 5 liver cells in a volume of about 10(-4) cm3 has been carried out.     

Integrated optical sensing of dissolved oxygen in microtiter plates: a novel tool for microbial cultivation

Microtiter plates with integrated optical sensing of dissolved oxygen were developed by immobilization of two fluorophores at the bottom of 96-well polystyrene microtiter plates. The oxygen-sensitive fluorophore responded to dissolved oxygen concentration, whereas the oxygen-insensitive one served as an internal reference. The sensor measured dissolved oxygen accurately in optically well-defined media. Oxygen transfer coefficients, k(L)a, were determined by a dynamic method in a commercial microtiter plate reader with an integrated shaker. For this purpose, the dissolved oxygen was initially depleted by the addition of sodium dithionite and, by oxygen transfer from air, it increased again after complete oxidation of dithionite. k(L)a values in one commercial reader were about 10 to 40 h(-1). k(L)a values were inversely proportional to the filling volume and increased with increasing shaking intensity. Dissolved oxygen was monitored during cultivation of Corynebacterium glutamicum in another reader that allowed much higher shaking intensity. Growth rates determined from optical density measurement were identical to those observed in shaking flasks and in a stirred fermentor. Oxygen uptake rates measured in the stirred fermentor and dissolved oxygen concentrations measured during cultivation in the microtiter plate were used to estimate k(L)a values in a 96-well microtiter plate. The resulting values were about 130 h(-1), which is in the lower range of typical stirred fermentors. The resulting maximum oxygen transfer rate was 26 mM h(-1). Simulations showed that the errors caused by the intermittent measurement method were insignificant under the prevailing conditions.     

The Dissolved Oxygen and Respiration Rates in Pellicle and Submerged Cultures of Corynebacterium diphtheriae

S ummary . The concentration of dissolved oxygen in submerged and static cultures of Corynebacterium diphtheriae was measured with a cathode ray polarograph. In a pellicle culture of the type frequently used for production of diphtheria toxin the concentration of dissolved oxygen in the medium below the pellicle fell sharply after the third or fourth day, and growth then proceeded in a medium which contained only minimal amounts of dissolved oxygen, in spite of the fact that only a cotton wool plug separated the atmosphere above the medium from the outside air.
The measurement of dissolved oxygen in submerged cultures was applied to the determination of respiration rates in growing cultures. The respiration rate for the test organisms varied at different stages in the period of a submerged batch culture. The rate was greatest at the start of the logarithmic phase, and thereafter declined until a steady state was reached.     

Detection of Microbial Growth on Polycyclic Aromatic Hydrocarbons in Microtiter Plates by Using the Respiration Indicator WST-1

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate}, in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.     

Growth and respiration of embryos and larvae of the rabbittish Siganus randalli (Pisces, Siganidae)

Growth and respiration of larval rabbitfish from Guam were examined. Larvae were reared from eggs in 2- to 10-ton tanks and were fed rotifers, Anemia , and artificial feed in succession as development proceeded through metamorphosis. Growth in length was rapid during the 12 h after hatching, then slowed until the larvae began to feed. The yolk sac was usually absorbed by 36 h after hatching. Rates of respiration of larvae and eggs were determined with a dissolved oxygen electrode at various times through development. Larval metabolism increased steadily during the embryonic stages culminating in a metabolic burst immediately after hatching. Respiration rates remained relatively stable from shortly after hatching until the onset of exogenous feeding, after which respiration rates increased with larval size. The respiration rates of post-yolk-sac larvae scaled isometrically with larval dry mass. Daily growth of feeding larvae was 27 to 28% of larval dry mass.     

Underwater survival in the dog tick Dermacentor variabilis (Acari:Ixodidae)

Ticks are blood-feeding arthropods known for their long survivability off the host. Although ticks are terrestrial, they can survive extended periods of time submerged underwater. A plastron is an alternative respiration system that can absorb oxygen from water via a thin layer of air trapped by hydrophobic hairs or other cuticular projections. The complex spiracular plate of ticks has been postulated to serve as a plastron but that function has not been verified. This study provides evidence of plastron respiration in the American dog tick, Dermacentor variabilis, and for the first time confirmed the existence of plastron respiration in Ixodidae. Longer survival rates in oxygenated water indicate that underwater respiration requires oxygen. Wetting the spiracular plate with alcohol debilitates any potential plastron function and lowers the survival rate. Survival underwater may also be enhanced by metabolic depression and possibly anaerobic respiration. This study describes the first example of plastron respiration in the Ixodidae.     

Cathodic electrochemiluminescent behavior of luminol at nafion-nano-TiO2 modified glassy carbon electrode

The electrochemiluminescence (ECL) behavior of luminol on a nafion-nano-TiO(2) modified glassy carbon electrode (nafion-nano-TiO(2)--GCE) was studied. Two ECL peaks (ECL-1 and ECL-2) were found during cathodic potential scanning. ECL-1 at ca -0.4 V (vs Ag--AgCl reference electrode) came from the reaction between luminol and active oxygen anion produced at the GCE surface directly, while ECL-2 at ca -0.9 V (vs Ag--AgCl reference electrode) came from the reaction between luminol and the active oxygen anion catalyzed by TiO(2.) The possible mechanism for the generation of both ECL peaks has been proposed. The reproducibility of the ECL intensities on nafion-nano-TiO(2)--GCE at ECL-1 and ECL-2 was good, with relative standard deviations (n = 10) of 4.3 and 1.3%, respectively. The ECL-2 generated at the nafion-nano-TiO(2)--GCE surface was further developed to detect the dissolved oxygen, and a detection limit of 0.02 mg/L was achieved. The proposed method was applied to detect dissolved oxygen in water with satisfactory result.     

Alternative respiration of fungus <Emphasis Type="Italic">Phycomyces blakesleeanus</Emphasis>

Respiratory characteristics of germinating spores, developing mycelium and mitochondria of the fungus Phycomyces blakesleeanus were investigated by means of oxygen Clark-type electrode. The effects of respiratory inhibitors and metabolic compounds on oxygen consumption were tested. It was demonstrated that P. blakesleeanus apart of cyanide-sensitive respiration, CSR, possess alternative respiration, (cyanide-resistant respiration, CRR) which is constitutive and whose capacity decreases during development. Maximum is observed for activated spores where CRR capacity is significantly greater than CSR. After treatment with antimycin A, a third type of respiration insensitive to antimycin A and low concentration of SHAM (sufficient for inhibition of CRR), but sensitive to cyanide and high concentration of SHAM, has been expressed.     

A low-volume platform for cell-respirometric screening based on quenched-luminescence oxygen sensing

Cell viability assays represent an important technology in modern cell biology, drug discovery and biotechnology, where currently there is a high demand for simple, sensitive and cost-effective screening methods. We have developed a new methodology and associated tools for cell-based screening assays, which are based on the measurement of the rates of oxygen uptake in cells by luminescence quenching. Sealable microchamber devices matching the footprint of a standard 96-well plate were developed and used in conjunction with long-decay phosphorescent oxygen probes. These devices permit cell non-invasive, real-time monitoring of cellular respiration and a rapid, one-step, kinetic assessment of multiple samples for cell viability, drug/effector action. These assays can be carried out on conventional fluorescence plate readers, they are suitable for different types of cells, including adherent and slow-respiring cells, require small sample volumes and cell numbers, and are amenable for high throughput screening. Monitoring of as little as 300 mammalian cells in 3 microl volume has been demonstrated.     

Transcriptional analysis of Shewanella oneidensis MR-1 with an electrode compared to Fe(III)citrate or oxygen as terminal electron acceptor

Shewanella oneidensis is a target of extensive research in the fields of bioelectrochemical systems and bioremediation because of its versatile metabolic capabilities, especially with regard to respiration with extracellular electron acceptors. The physiological activity of S. oneidensis to respire at electrodes is of great interest, but the growth conditions in thin-layer biofilms make physiological analyses experimentally challenging. Here, we took a global approach to evaluate physiological activity with an electrode as terminal electron acceptor for the generation of electric current. We performed expression analysis with DNA microarrays to compare the overall gene expression with an electrode to that with soluble iron(III) or oxygen as the electron acceptor and applied new hierarchical model-based statistics for the differential expression analysis. We confirmed the differential expression of many genes that have previously been reported to be involved in electrode respiration, such as the entire mtr operon. We also formulate hypotheses on other possible gene involvements in electrode respiration, for example, a role of ScyA in inter-protein electron transfer and a regulatory role of the cbb3-type cytochrome c oxidase under anaerobic conditions. Further, we hypothesize that electrode respiration imposes a significant stress on S. oneidensis, resulting in higher energetic costs for electrode respiration than for soluble iron(III) respiration, which fosters a higher metabolic turnover to cover energy needs. Our hypotheses now require experimental verification, but this expression analysis provides a fundamental platform for further studies into the molecular mechanisms of S. oneidensis electron transfer and the physiologically special situation of growth on a poised-potential surface.     

Perturbation of respiration in Candida utilis: induction of metabolic oscillations

The effects of potentially perturbating influences on the respiration of glucose-grown Candida utilis were studied using an open oxygen electrode system. Periods of anaerobiosis as short as 2 min produced an oscillation in respiration after the air supply was restored. Longer exposure to anoxia was followed by an overshoot in dissolved oxygen after switching back to a gas phase of air. Centrifugation, cold shock or nutrient starvation caused less disturbance to respiration rates than did anaerobiosis. The high frequency oscillations (period about 5 min) resulting from anaerobic-aerobic transitions are contrasted with the slow cell cycle-dependent oscillations previously observed in synchronous cultures.     

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.     

Detection of the kinetics of biochemical reactions with oxygen using exchange broadening in the ESR spectra of nitroxide radicals

To detect changes in the oxygen concentration during biochemical reactions, the exchange broadening in the ESR spectra of nitroxide radicals caused by the dissolved oxygen, has been used. The measurements have been carried out using changes in the width either of the proton hyperfine structure components or of the nitrogen hyperfine structure line with an unresolved proton structure. Detection of mitochondrial respiration in a volume of about 10^?3 cm³ and respiration for 100±5 liver cells in a volume of about 10^?4 cm³ has been carried out.     

Photochemical action spectra of CO-liganded terminal oxidases using a liquid dye laser

A method of obtaining photochemical action spectra for the relief of CO inhibition of respiration is described. Continuous readout from a membrane-covered oxygen electrode of dissolved oxygen in a stirred suspension of microorganisms under CO-oxygen gas mixtures in an open reactor enables measurement of increased respiration on illumination. Advantages presented by the use of a liquid dye laser include high intensity of emission and narrow spectral bandwidth; just two dyes (rhodamine 6G and rhodamine 110) are required to match the α-absorption maxima of the CO complexes of all known bacterial and mitochondrial terminal oxidases.     

A lab-built respirometer for plant and animal cell culture

A very simple off-line respirometer was developed to measure oxygen consumption rates of low respiring and shear-sensitive cell suspensions. The respirometer is composed of a 10 mL glass syringe in which the plunger was substituted with a polarographic dissolved oxygen probe. Mechanical agitation is provided by means of a magnetic stirring bar inside the measuring chamber and a stir plate placed below the respirometer. Abiotic oxygen fluxes occurring in the measurement chamber such as oxygen diffusion and probe oxygen consumption were investigated. The apparent oxygen uptake rate was then corrected for abiotic oxygen fluxes, leading to accurate measurements of respiration rates ranging from 0.5 to 25.0 mM x h(-1). Additionally, the effect of the stirring bar shape and of the test length on the integrity of plant (Eschschzoltzia californica) and animal (NS0) cells was evaluated. Animal cells showed a higher resistance to mechanical stirring inside the respirometer compared to plant cells (0% of broken cells and 78.1% respectively for a polygonal stirring bar and a 15 min test). For plant cells, cell damage inside the measurement chamber was reduced by optimizing the stirring bar shape and reducing the test length to 5 min or less. This very simple design was shown to provide reliable and low-cost quantification of the oxygen uptake rate of plant and animal cells and can be use even for more demanding measurements such as oxygen affinity studies.