Carlson et al (1991) demonstrated clearly that increasing concentrations of 6 alcohols inhibit anaerobic fermentation (as carried out by Saccharomyces cerevisiae); a correlation with increased partition coefficients into a hydrophobic milieu was also evident. This would tend to suggest that the action of these alcohols is primarily located at a hydrophobic site, possibly at the membrane. In this study of the effect of n-alcohols on the ATP-dependent generation of DpH and Dy across plasma membrane vesicles of Saccharomyces cerevisiae, the alcohols were shown to collapse DpH and Dy in the order C2< C3< C4< C5£C6³C7> C8> C11, i.e. that there was an optimal chain length for cytotoxicity. Inhibition of the plasma membrane H+-ATPase was insignificant (Petrov and Okorokov, 1990). Using pH-stat measurements Stevens and Hofmeyer (1993) have shown that quite modest concentrations of both octanoic and decanoic acid, in the presence of ethanol, increase the rate of passive H+ influx across the plasma membrane. It has been suggested that toxicity is a result of the increased anion and proton permeability of the plasma membrane, de-energising it and ultimately blocking the secondary transport systems (Petrov and Okorokov, 1990, Sikkema et al, 1992, Stevens and Hofmeyr, 1993). This increase in permeability may be due to the increase in the fluidity of the membrane lipids (Petrov and Okorokov, 1990), or to the solvent partitioning into the lipids causing membrane expansion (Seeman, 1972); such membrane expansion can be clearly demonstrated by fluorescence selfquenching techniques (Sikkema et al 1992) and by dielectric spectroscopy (Stoicheva et al. 1989, Salter & Kell 1992, Davey et al, 1993)). It has also been suggested that cytotoxicity is exerted mainly when a "critical", solvent-independent membrane concentration is reached (Osborne et al. 1990).
In an investigation of respiratory electron transfer activity in an asolecithin- isooctane reverse micellar system, it was suggested that in the organic medium electron transfer from NADH to O2 is arrested at the terminal oxidase step (Escobar and Escamilla, 1992). Given that respiratory electron transfer requires the diffusional motions of various membranous complexes (see e.g. Anthony 1988, Westerhoff et al. 1988), this is perhaps not surprising. However, results from other work in which the electron transport chain was replaced with artificial electron acceptors such as phenazine methosulphate ((PMS) Fukui et al 1980, Hocknull and Lilly 1987, Freeman and Lilly 1987) or menadione (Pinheiro and Cabral 1991) suggest that organic solvents can exert significant effects on the rate of electron transfer directly.
n-Alkanes (especially the larger ones) are commonly regarded as being non-cytotoxic towards a whole range of micro-organisms. It has been reported however that the growth of various yeasts on, or in the presence of these compounds results in changes in the fatty acid composition of the cell, varying according to the chain length of the alkane involved (Thorpe and Ratledge, 1972). Fractionation of the lipids from yeasts grown on various alkanes showed that although triglycerides still constituted the major fraction, their relative proportion was less than when glucose was the growth substrate. Loss of triglycerides was compensated for by a corresponding increase in the phospholipids (Thorpe and Ratledge, 1972).
In a similar investigation Vollherbst-Schneck et al (1984) showed that butanol, at sub-growth inhibitory levels had a significant fluidizing effect on the bulk lipid regions of E. coli and Clostridium acetobutylicum. When grown in the presence of butanol, C. acetobutylicum synthesised increased levels of saturated acyl chains at the expense of unsaturated chains. This has also been shown to occur in yeast (under aerobic conditions); interestingly, immobilised cells, which are known to have a higher ethanol tolerance, also have significantly more saturated fatty acyl residues than do free cells (Hilge-Rotmann and Rehm, 1991). The ratio of unsaturated to saturated fatty acids has long been known to control membrane fluidity, with increases in unsaturation leading to greater membrane fluidity, such that cells subjected to temperature changes adjust the degree of saturation to maintain a constant fluidity, a mechanism knows as homeoviscous adaptation. On this basis it would seem logical, if solvents increase the fluidity, that increasing the ratio of saturated fatty acids would reduce this effect, and restore the "normal" fluidity of the membrane. Sterols also play an important role in the regulation of membrane fluidity. Agudo (1992) showed that the presence of ergosterol was important for increased ethanol tolerance in Saccharomyces cerevisiae, along with a shift to shorter chain lengths of the fatty acids (C12-C14). Finally, Heipieper et al. (1994) summarise the evidence that in cells tolerant to organic solvents a major fraction of the cis-unsaturated is converted to the corresponding trans- fatty acids.