Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode

Summary An alcohol electrode was constructed which consisted of an oxygen probe onto which alcohol oxidase was immobilized. This enzyme electrode was used, in combination with a reference oxygen electrode, to study the short-term kinetics of alcoholic fermentation by aerobic yeast suspensions after pulsing with glucose. The results demonstrate that this device is an excellent tool in obtaining quantitative data on the short-term expression of the Crabtree effect in yeasts.Samples from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae not producing ethanol, immediately (within 2 min) exhibited aerobic alcoholic fermentation after being pulsed with excess glucose. With chemostat-grown Candida utilis, however, ethanol production was not detectable even at high sugar concentrations. The Crabtree effect in S. cerevisiae was studied in more detail with commercial baker's yeast. Ethanol formation occurred only at initial glucose concentrations exceeding 150 mg·l^-1, and the rate of alcoholic fermentation increased with increasing glucose concentrations up to 1,000 mg·l^-1 glucose.Similar experiments with batch cultures of certain non-fermentative yeasts revealed that these organisms are capable of alcoholic fermentation. Thus, even under fully aerobic conditions, Hansenula nonfermentans and Candida buffonii produced ethanol after being pulsed with glucose. In C. buffonii ethanol formation was already apparent at very low glucose concentrations (10 mg·l^-1) and alcoholic fermentation even proceeded at a higher rate than in S. cerevisiae. With Rhodotorula rubra, however, the rate of ethanol formation was below the detection limit, i.e., less than 0.1 mmol·g cells^-1·h^-1.     

On the measurement of carbon dioxide uptake with a glass electrode

1. It has been shown that the glass electrode method as previously described by the author for measurements of transient rates in photosynthesis is free of systematic errors. 2. Justification has been given for the use of the steady state-gas flow method for measuring rates of assimilation under conditions in which the solution is known not to be seriously depleted of CO₂. 3. The decline of photosynthetic rates under light saturation at CO₂ pressures less than several tenths per cent of an atmosphere has been shown to be a real phenomenon. It has been suggested that there may be a real discrepancy between this finding and those of some other investigators due to a difference in the previous history of the algal suspensions.     

Continuous measurement of the cytoplasmic pH in Lactococcus lactis with a fluorescent pH indicator

The cytoplasmic pH of Lactococcus lactis was studied with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5 (and-6)-carboxyfluorescein (BCECF). A novel method was applied for loading bacterial cells with BCECF, which consists of briefly treating a dense cell suspension with acid in the presence of the probe. This results in a pH gradient, which drives accumulation of the probe in the cytoplasm. After neutralization the probe was well retained in cells stored on ice. BCECF-loaded cells were metabolically active, and were able to generate a pH gradient upon energization. The probe leaks out slowly at elevated temperatures. Efflux is stimulated upon energization of the cells, and is most likely catalyzed by an active transport system. It is a first-order process, and the rate constant could be deduced from the decrease of the fluorescence signal in periods of constant intracellular pH. This allowed a correction of the fluorescence signal for efflux of the probe. After calibration the cytoplasmic pH could be calculated from efflux-corrected fluorescence traces.     

Continuous assays of L-asparaginase by coupling with glutamic dehydrogenase and by cationic glass electrode

Continuous assays of l-asparaginase by coupling with the glutamic dehydrogenase reaction and by cationic glass electrode are described. The procedures are about equally sensitive although the latter is simpler and a more direct assay. The two methods gave identical specific activity values for a purified preparation of Escherichia colil-asparaginase.     

Continuous in vivo measurement of arterial PO2 in humans.

A system suitable for prolonged continuous in vivo measurement of human arterial PO2 is described. The system uses a polarographic electrode developed by Kimmich and Kreuzer, inserted in a specially made shunt between the radial artery and an antecubital vein. Nhe electrode surface is maintained in a fixed position parallel to the flow of blood; blood velocity dependency is small owing to the high flow rate achieved (more than 40 cm/s); clotting is prevented by the material used and the continuous instillation of heparin through the arterial end of the shunt. The system has been tested in vitro; it is stable (variation less than 0.5% in 24 h), linear and precise (plus or minus 0.2%) in a broad range of PO2 values (from about 10 mmHg to more than 700 mmHg); its response time is 0.4 s per 95% of deflection. It has been applied to 35 patients for periods ranging between 6 and 24 h; most of the patients were ventilated by an Engstrom respirator.     

Laser-Doppler measurement of tracheal mucosal blood flow: comparison with tracheal arterial flow

Blood flow in the tracheal mucosa (Qm) has been measured by laser-Doppler flowmetry in anesthetized sheep and dogs. The values have been compared with tracheal arterial inflow (Qtr) by use of an electromagnetic flow probe and with tracheal arterial perfusion pressure (Ptr) produced by mechanical perfusion. Changes in blood flow were caused by injections of methacholine, phenylephrine, and histamine into the perfusion circuit. These interventions produced a range of measurements for each animal. Correlations of Qm against Qtr were significant in two of five animals (R = 0.03-0.93); correlations of Qm against Ptr were significant in two of four animals (R = 0.56-0.96). Percent changes in Qtr were generally much larger than those of Qm, and there was considerable variability between Qm and either Qtr or Ptr. Qm reflected the same vascular changes as Ptr or Qtr in 28 interventions and showed an opposing change in 4 cases. In 11 interventions, changes measured by Ptr or Qtr were not reflected by any changes in Qm. Thus qualitative changes in tracheal perfusion measured with these methods were usually the same; quantitatively the three methods showed great differences. These differences may reflect different regulatory mechanisms in various components of the tracheal vasculature or different technical aspects of the methods used.