Mechanisms of membrane toxicity of hydrocarbons.

Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO).

Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification

During brewery handling, production strains of yeast must respond to fluctuations in dissolved oxygen concentration, pH, osmolarity, ethanol concentration, nutrient supply and temperature. Fermentation performance of brewing yeast strains is dependent on their ability to adapt to these changes, particularly during batch brewery fermentation which involves the recycling (repitching) of a single yeast culture (slurry) over a number of fermentations (generations). Modern practices, such as the use of high-gravity worts and preparation of dried yeast for use as an inoculum, have increased the magnitude of the stresses to which the cell is subjected. The ability of yeast to respond effectively to these conditions is essential not only for beer production but also for maintaining the fermentation fitness of yeast for use in subsequent fermentations. During brewery handling, cells inhabit a complex environment and our understanding of stress responses under such conditions is limited. The advent of techniques capable of determining genomic and proteomic changes within the cell is likely vastly to improve our knowledge of yeast stress responses during industrial brewery handling.

Yeast responses to stresses associated with industrial brewery handling.

Sublethal heat and ethanol exposure induce essentially identical stress responses in yeast. These responses are characterized by the induction of heat shock proteins, proteins requiring a temperature above about 35 °C or ethanol levels above a threshold level of 4–6% (v/v) for strong induction. One induced protein, Hsp104, contributes to both thermotolerance and ethanol tolerance, while others are anti-oxidant enzymes. Heat and ethanol stress cause similar changes to plasma membrane protein composition, reducing the levels of plasma membrane H+-ATPase protein and inducing the plasma membrane-associated Hsp30. Both stresses also stimulate the activity of the fraction of H+-ATPase remaining in the plasma membrane. The resulting enhancement to catalysed proton efflux from the cell represents a considerable energy demand, yet may help to counteract the adverse effects for homeostasis of the increased membrane permeability that results from stress.

The heat shock and ethanol stress responses of yeast exhibit extensive similarity and functional overlap

Saccharomyces cerevisiae HSc was grown with ethanol at concentrations up to 10% (v/v). The immediate effects of additions of externally added ethanol on CO2 production and O2 consumption of washed organisms were studied by stopped-flow membrane inlet quadrupole mass spectrometry. Fermentative activities of organisms grown with ethanol (0-5% v/v) showed similar sensitivities to inhibition by ethanol, whereas those grown with 10% (v/v) ethanol had become protected and were markedly less sensitive. The fluidity of subcellular membrane fractions was measured by determination of the temperature dependence of the rotational order parameter of the spin label 5-doxyl stearic acid (free radical) by electron spin resonance. Mitochondria prepared from yeasts grown with 0, 7, and 9% (v/v) ethanol showed similar overall fluidity, although differences in temperature-dependent behaviour indicate altered lipid composition or lateral phase separations. On the other hand the microsomal fraction from organisms grown with 9% ethanol showed a remarkable increase in fluidity. These data suggest that the protective effects of growth with ethanol near the limit of tolerance on fermentative activities may arise from altered plasma membrane fluidity properties.

Effects of growth with ethanol on fermentation and membrane fluidity of Saccharomyces cerevisiae.

The kinetics of methane uptake by Methylococcus capsulatus (Bath) and its inhibition by ammonia were studied by stopped-flow membrane-inlet mass spectrometry. Measurements were done on suspensions of cells grown in high- and low-copper media. With both types of cells the kinetics of methane uptake are hyperbolic when oxygen is in excess. The apparent K
m and K
max for methane uptake are both higher in low-copper cells than in high-copper cells. Ammonia is a simple competitive inhibitor of methane uptake in high-copper cells when the oxygen concentration is above a few μM. The findings agree with the assumption that ammonia is a week alternative substrate for particulate methane monooxygenase. In low-copper cells the effect of ammonia is complicated and cannot be explained in terms of current assumptions on the mechanism of soluble methane monooxygenase. Our data indicate that ammonia inhibition is likely to be a more serious problem in connection with cultivation in low-copper medium than in high-copper medium.

Inhibition by ammonia of methane utilization in Methylococcus capsulatus (Bath)

SUMMARY: The immediate effects of externally added alcohols on CO2 production and O2 consumption of suspensions of washed, aerated baker's yeast were studied by stopped-flow membrane inlet mass spectrometry. Glucose-supported fermentation was progressively inhibited by increasing ethanol concentration (0-20%, v/v). The inhibition by ethanol was quite different from that observed for acetaldehyde; thus it is unlikely that toxicity of the latter can account for the observed effects. For five different alkanols (methanol, ethanol, I-propanol, 2-propanol and I-butanol) increasing inhibition of anaerobic fermentation was correlated with increased partition coefficients into a hydrophobic milieu. This suggests that the action of ethanol is primarily located at a hydrophobic site, possibly at a membrane. Results for respiratory activities were not as definite as for those for anaerobic metabolism because some alkanols act as respiratory substrates as well as giving inhibitory effects.

Helle N. Carlsen

Papers

Effects of growth with ethanol on fermentation and membrane fluidity of Saccharomyces cerevisiae.

Inhibition by ammonia of methane utilization in Methylococcus capsulatus (Bath)

Effects of alcohols on the respiration and fermentation of aerated suspensions of baker's yeast.