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The inhibition of the exponential growth of Escherichia coli K-12 by different phenolic compounds was examined. Cells entrapped in calcium alginate showed a greater tolerance than cells grown in suspension. The extent of inhibition of growth of the immobilized cells depended on the period of growth in the gel matrix. After the addition of bacteriostatic concentrations of phenol or 4-chlorophenol, a dose-dependent efflux of metabolites such as ATP and of K+ ions was elicited. Provided that glucose was supplied as an energy substrate, a reaccumulation of K+ ions at low phenol concentrations was observed. The restoration of the membrane gradient for K+ always preceded the continuation of growth in the presence of the toxic compounds. Compared with free cells, those cells immobilized and grown in alginate suffered a smaller loss of cations after the addition of 4-chlorophenol. The reestablishment of gradients was observed at higher concentrations of the pollutants with entrapped cells than with free cells. Corresponding to the increase in tolerance, the membrane damage was reduced in cells grown in immobilized form for longer times. These data offer a mechanistic explanation of the protection of immobilized microorganisms from phenolic solvents. The data point to the membrane as an important cell component in the toxicity of these pollutants.

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Membranes of Escherichia coli cells grown in the presence of phenol were examined after isolation of the cytoplasmic and outer membrane fractions. Both membrane types showed reduced lipid-to-protein ratios compared to cells grown without phenol. Phenol-induced differences in the expression of individual proteins of the inner membrane were established. Different proteins of the outer membrane, probably involved in the uptake of iron, were expressed in smaller quantities after phenol addition. Growth in the presence of phenol increased the respiratory activity of the cytoplasmic membrane, whereas the direct inhibition of O2 consumption by phenol was not affected by the presence of this compound in the growth medium. E. coli cells grown entrapped in calcium alginate showed low lipid-to-protein ratios even without phenol in the growth medium. Immobilization of cells also markedly changed the protein pattern of the outer membrane.

In the presence of sublethal concentrations of phenol, 4-chlorophenol, and p-cresol in the growth medium, cells of Escherichia coli modified the fatty acid composition of their lipids. The results of these changes was an increase in the degree of saturation of lipids probably in order to compensate an increase of fluidity of the membrane induced by the phenols. Supplementation of the growth medium with saturated fatty acids could also enhance the degree of lipid saturation due to the incorporation of the acyl chains in the phospholipids. At the same time the growth of cells was less inhibited than in unsupplemented cells. The increase of tolerance of cells by manipulating the lipid composition indicates that the membrane structure plays a crucial role in the mode of action of phenols.

The restriction of oxygen transfer in Ca-alginate beads used for the immobilization of microbial cells was applied to a coupled reductive and oxidative microbial degradation of the xenobiotic 4-chloro-2-nitrophenol (CNP). The conversion of CNP by Enterobacter cloacae under anaerobic conditions led to the formation of 4-chloro-2-aminophenol (CAP, 81%) and 4-chloro-2-acetaminophenol (CAAP, 16%) after 50 h incubation. CAP, the main reduction product, was further degraded under aerobic conditions by Alcaligenes sp. TK-2, a hybrid strain isolated by conjugative in-vivo gene transfer. Whereas both degradation steps excluded one another in homogeneous systems with free cells, a coupled reductive and oxidative degradation of CNP was observed in one aerated reactor system after co-immobilization of both strains in Ca alginate. The diameter of the alginate beads used for immobilization was recognized as one main factor determining the properties of this mixed culture system.

Abbreviations: R j : formation rate of the generic product, g citric acid/(l x hr) (volumetric productivity) m p :specific citric acid productivity, g citric acid/(g biomass x hr) RT: Residence time -hrs Rs: glucose consumption rate, g/(l x hr)The effect of air saturation on citric acid production in batch, repeated batch and chemostat cultures has been studied. It was shown that, under continuous fermentation (chemostat mode), the highest concentration of citric acid equal of 98 g/l was produced at 20% of air saturation. In contrary to continuous fermentation, displaying an optimum at 20%, 80% air saturation yielded higher values in repeated batch fermentation process. 167 g/l citric acid were produced continuously with the fill and drain technique at 4.85 days, at 80% air saturation, compared with 157.6 g/l achieved within 5.4 days at 20%. Under repeated batch fermentation, the formation rate of the generic product (Rj) as well as the specific citric acid productivity (m p ) reached a maximum of 1.283 g/(l x hr) at 4.01 days and of 0.0375 g/(g x hr) at 4.58 days, respectively. The glucose consumption rate (Rx) reached a maximum value of 3.33 g/(l x hr) entering stationary phase after 2.56 days at a glucose concentration of 131.2 g/l.