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.     

Two simple methods for quantifying low-affinity dye-substrate binding

Binding with low-affinity ligands, such as histological dyes, can be difficult to quantitate owing to the dissociation of bound ligand with washing or the retention of nonspecifically bound ligand because of incomplete washing. The present report describes two simple, rapid methods of discriminating bound from free ligand without the need for washing steps. One method is based on the spectral changes induced in a dye ligand, Congo red, on binding to the "receptor" insulin fibrils. This method discriminates spectrophotometrically between bound and free ligand without requiring any physical separation of the two forms. No radioactive ligands are necessary, and, by using disposable cuvettes, the entire binding assay can be done in a single container without the need for transfers. The second method employs a non-traditional filtration approach that avoids the need for a washing step by measuring the decrease in concentration of the dye ligand in the filtrate rather than by applying the usual approach of measuring the absolute amount of ligand bound to the precipitated "receptor." Both methods show saturation of binding sites and give similar values for the KD and Bmax.     

Two methods of temperature control for single-molecule measurements

Modern single-molecule biophysical experiments require high numerical aperture oil-immersion objectives in close contact with the sample. We introduce two methods of high numerical aperture temperature control which can be implemented on any microscope: objective temperature control using a ring-shaped Peltier device, and stage temperature control using a fluid flow cooling chip in close thermal contact with the sample. We demonstrate the efficacy of each system by showing the change in speed with temperature of two molecular motors, the bacterial flagellar motor and skeletal muscle myosin.     

Oxygen mass transfer and oxygen respiration rate measurements utilizing fast response oxygen electrodes

Oxygen transfer coefficient and oxygen respiration rate measurements in stirred tank reactors or fermentors have been carried out utilizing currently available dissolved oxygen electrodes. Techniques based on previously derived theory¹ have been put to experimental tests and found to adequately describe the oxygen transfer phenomena observed in air–water systems and a fermenting broth.     

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.     

A simple method for recording the relative volumetric changes in cells adhering to glass

The paper describes a simple method for registering the relative volume changes of the cells adhering to a glass surface. The changes in the luminosity of cells which followed changes in the osmolarity of the medium were registered by means of microscope with a dark-field condenser and the photoelement connected through the DC amplifier to the continuous ink recorder. It was proved that changes in the cells luminosity really represented the relative cell volume changes. The method allows continuous recording of the amplitude and time course of cell volume changes throughout the experiment with many changes of the solution perfusing the cell chamber, which makes it possible to study the effects of pharmacological agents on the mechanisms of cell volume regulation. Provided that the cell membranes are destroyed by detergents the method allows measurement of the activity of the cell contractile apparatus.     

关于氧气与呼吸作用几个问题的解答

从生物能学角度、生物化学氧化还原电势角度及化学反应平衡角度,解释O2为什么会抑制无氧呼吸;同时对氧气为什么会“杀死”专性厌氧生物;厌氧生物对氧气的耐受性为何不一样做出了阐述。     

Formate-forming fungal catabolic pathway to supply electrons to nitrate respiration

Pyruvate was catabolized anaerobically by resting cells of Fusarium oxysporum to form formate and acetate. Addition of nitrate decreased the accumulation of formate in the medium with concomitant formation of nitrite and N2O. The results suggested a unique metabolic pathway that occurs in fungi mediated by pyruvate-formate lyase to supply electrons via formate to fungal denitrification.     

Suitability of the shaking flask for oxygen supply to microbiological cultures

Due of its simplicity the shaking flask is used in serial studies, e.g. in the screening for secondary metabolites or in the optimization of fermentation processes. Experimental investigations in these small bioreactors are often the first step in developing a large-scale fermentation process.Movement of the flask should produce sufficient mixing, supply of oxygen, and removal of carbon dioxide. In the case of fluids with low or moderate viscosity, gas transport is the most important aspect. This publication summarizes data necessary to calculate the gas transport. These data are derived from the consideration of the gas diffusions through the cotton plug as well as from the substance transport between the gas and liquid phases. As a result suitable fermentation conditions can be selected. Finally, the performance limits of the shaking flask are illustrated using the example of the oxygen supply in a Streptomyces tendae fermentation.List of Symbols A _s Cross section of plug - A Surface area of liquid in flask - a A/V _F specific phase interface area - c Concentration - c ^* Saturation concentration - c Plug diffusion term - D Widest diameter of flask - Diffusion coefficients in multicomponent gas mix tures - Diffusion coefficients in binary gas mixtures - Diffusion coefficient of oxygen in the liquid - d Diameter of neck of flask - e Eccentricity - G Volume-based mass flow - G _m Maximum volume-based mass flow - g Acceleration due to gravity - h Height coordinate - ¯H Mean height of plug - Hy p _i/c ^*, Henry constant - K Consistency index - k D _xy/D _xz, Ratio of diffusion coefficients in binary gas mixtures - k _M Monod constant - k _L a Mass transport coefficient: gas/liquid - M Molecular weight - m Flow exponent - n Speed of shaking - p Pressure - p _i Partial pressure of gas component i - q Area-based flow of volume - R , respiration ratio - Sc , Schmidt number - T Absolute temperature - V Flask volume - V _F Volume of liquid in flask - w Velocity of the Stefan flow - x, y, z Ratios of the partial pressures of the gases O₂, CO₂, N₂ - Rate of shear - Dynamic viscosity of the liquid - Kinematic viscosity of the liquid - Density of the liquid - _x, Density of O₂ gas - Surface tension Indices 0 State in gas volume of shaking flask - 1 State in outside air - G Gas volume - x, y, z O₂, CO₂, N₂     

The oxygen supply to thermogenic flowers

Thermogenic flowers produce heat by intense respiration, and the rates of O₂ consumption (?o₂) in some species can exceed those of all other tissues of plants and most animals. By exposing intact flowers to a range of O₂ pressures (Po₂) and measuring ?o₂, we demonstrate that the highest respiration rates exceed the capacity of the O₂ diffusive pathway and become diffusion limited in atmospheric air. The male florets on the inflorescence of Arum concinnatum have the highest known mass‐specific ?o₂ and can be severely diffusion limited. Intact spadices of Japanese skunk cabbage Symplocarpus renifolius are diffusion limited in air only when ?o₂ is maximal, but not at lower levels. True flowers of the sacred lotus Nelumbo nucifera and the appendix of Arum concinnatum are never diffusion limited in air. ?o₂ – Po₂ curves are evaluated quantitatively with the ‘Regulation Index’, a new tool to measure dependence of ?o₂ on ambient Po₂, as well as the conventional ‘Critical Po₂’. The study also includes measurements of Po₂ within thermogenic tissues with O₂‐sensitive fibre optics, and reveals that the diffusion pathway is complicated and that O₂ can be provided not only from the surface of the tissues but also from the pith of the flower's peduncle.     

First respiration estimates of cold-seep vesicomyid bivalves from in situ total oxygen uptake measurements

Vesicomyid bivalves are one of the most abundant symbiont-bearing species inhabiting deep-sea reducing ecosystems. Nevertheless, except for the hydrothermal vent clam Calyptogena magnifica, their metabolic rates have not been documented, and only assessed with ex situ experiments. In this study, gathering benthic chamber measurements and biomass estimation, we give the first in situ assessment of the respiration rate of these bivalves. The giant pockmark Regab, located at 3160m depth along the Congo-Angola margin, is a cold-seep site characterised by dense assemblages of two species of vesicomyids: Christineconcha regab and Laubiericoncha chuni with high dominance of C. regab. Two sites with dense aggregates of vesicomyids were selected to measure total oxygen uptake (TOU), and methane fluxes using IFREMER's benthic chamber CALMAR deployed by the ROV Quest 4000 (MARUM). Photographs were taken and bivalves were sampled using blade corers to estimate density and biomass. Total oxygen uptake was higher at Site 2 compared to Site 1 (respectively 492 mmol.m(-2).d(-1) and 332 mmol.m(-2).d(-1)). However, given vesicomyid densities and biomass, mean oxygen consumption rates were similar at both sites (1.9 to 2.5 μmol.g total dry mass(-1).h(-1) at the Site 1 and 1.8 to 2.3 μmol.g total dry mass(-1).h(-1) at Site 2). These respiration rates are higher than published ex situ estimates for cold-seep or hydrothermal vent bivalves. Although methane fluxes at the base of sulphide production were clearly higher at Site 2 (14.6 mmol.m(-2).d(-1)) than at Site 1 (0.3 mmol.m(-2).d(-1)), they do not seem to influence the respiration rates of these bivalves associated to sulphide-oxidizing symbionts.     

Denitrification and oxygen respiration in biofilms studied with a microsensor for nitrous oxide and oxygen

Depth distributions of O₂ respiration and denitrification activity were studied in 1- to 2-mm thick biofilms from nutrient-rich Danish streams. Acetylene was added to block the reduction of N₂O, and micro-profiles of O₂ and N₂O in the biofilm were measured simultaneously with a polarographic microsensor. The specific activities of the two respiratory processes were calculated from the microprofiles using a one-dimensional diffusion-reaction model. Denitrification only occurred in layers where O₂ was absent or present at low concentrations (of a fewM). Introduction of O₂ into deeper layers inhibited denitrification, but the process started immediately after anoxic conditions were reestablished. Denitrification activity was present at greater depth in the biofilm when the NO₃ ^– concentration in the overlying water was elevated, and the deepest occurrence of denitrification was apparently determined by the depth penetration of NO₃ ^–. The denitrification rate within each specific layer was not affected by an increase in NO₃ ^– concentration, and the half-saturation concentration (K_m) for NO₃ ^– therefore considered to be low (<25M). Addition of 0.2% yeast extract stimulated denitrification only in the uppermost 0.2 mm of the denitrification zone indicating a very efficient utilization of the dissolved organic matter within the upper layers of the biofilm.     

Kinetic study and oxygen transfer efficiency evaluation using respirometric methods in a submerged membrane bioreactor using pure oxygen to supply the aerobic conditions

The performance of a wastewater bench-scale ultrafiltration membrane bioreactor (MBR) treatment plant using pure oxygen to supply the aerobic conditions for 95 days was studied. The results showed the capacity of the MBR systems to remove organic material under a hydraulic retention time of 12 h and a sludge retention time of 39.91 days. Aeration represents its major power input; this is why the alpha-factor of the aeration and kinetic parameters (design parameters) were determined when the mixed liquid suspended solids (MLSS) was increased from 3420 to 12,600 mg/l in order to understand the system. An alpha-factor in the range 0.462-0.022 and the kinetic parameters measured with the respirometric method (K_M of 73.954-3.647 mg/l, k_d of 0.0142-0.104 day⁻¹, k_H of 0.1266-0.655 day⁻¹, and the yield mean coefficient of 0.941) were obtained. Our study suggested significant changes in the behaviour of the biological system when the concentration of MLSS was increased.     

Two specific and simple methods for genotyping of the paraoxonase/arylesterase A/B polymorphism

We developed an amplification refractory mutation system (ARMS) and mutagenically separated PCR (MS-PCR) assays for genotyping of the paraoxonase/arylesrase (PONA) A/B polymorphism as practical, reliable, and more economic alternatives to the conventional restriction fragment length polymorphism (RFLP) technique for detection of this mutation.     

General methods for enriching aldoses with oxygen isotopes

Synthetic methods are described for enriching 4-, 5-, and 6-carbon aldoses with oxygen isotopes. The general approach includes exchange between H2(1)8O and the aldehyde group of an aldose, exchange of O-1 onto C-2 of both of the 2-epimeric aldoses formed by molybdate-resin epimerization, and chain extension using cyanide addition. These methods make possible the production of all 16 aldohexoses enriched at 5 of the 6 oxygen atoms, all 8 aldopentoses enriched at 4 of the 5 oxygen atoms, and the four aldotetroses enriched at 2 of the 4 oxygen atoms. The general applicability of these methods is illustrated by the synthesis of a group of 22 different, 18O-enriched, biologically important D-aldoses having 4, 5, and 6 carbon atoms. The group includes D-[1-, 2-, 3-, 4-, O]glucose, D-[1-, 2-, 3-, 4-, and 6-18O]mannose, D-[1-, 2-, 3-, and 5-18O]arabinose, D-[1- abd 2- 18O] erythorose, and D-[1- and 2-18O]threose. The g.l.c.-m.s. characterization of these sugars with respect to the position and degree of 18O-enrichment is reported. The potential of the methods for producing aldoses having oxygen labels at multiple positions, or aldoses labeled simultaneously with oxygen, hydrogen, and carbon isotopes is discussed.     

A simple volumetric apparatus for measuring the rate of gas exchange in microorganisms

A new simple volumetric apparatus for measuring the respiration,fermentation and photosynthesis rates in microorganisms wasconstructed. The apparatus, its operation and some data obtainedwith it are presented. This apparatus 1) has an open-capillaryconstant-pressure volumeter without a compensation chamber,2) has a magnetic stirrer for stirring the reaction mixture,3) can be used to measure with a sensitivity of 0.85 µlvolume change per one mm reading without a microscope, 4) simultaneouslyuses seven volumeters, one of which is set as a thermobarometer,5) is completely immersed in a water bath to minimize the effectof ambient temperature, and 6) simplifies calculation. (Received May 9, 1977; )