Response of Ralstonia eutropha JMP 134 to long-term exposure to toxic substrates in nutristat cultivation as indicated by on-line fluorescence measurements

Abstract

Ralstonia eutropha JMP 134 was continuously grown on phenol and 2,4-dichlorophenoxyacetate under nutristatic conditions at elevated stationary concentrations of 90±650 mg phenol/l and 25±100 mg 2,4-D/l, respectively, in order to study the response of the bacterial population to long-term exposure to these potentially toxic substrates. The course of the cells' response over time was observed by determining distinctive growth parameters and by the on-line measurement of¯uorescence spectra of intracellular and extracellular¯uorophores. The latter were monitored using a modi®ed¯uorescence spectrophotometer. The results of the nutristat experiments indicate that the adaptation of the culture to long-term exposure to phenol and 2,4-D exhibited dynamic characteristics of the growth pattern determined by the individual substrates and their concentration, including enforced and reduced levels of substrate conversion. This growth pattern is interpreted as an expression of superimposing cellular events in order to withstand unfavorable environmental conditions. Finally, the growth rate attained retarded levels under stationary conditions, slowing down to almost zero for example in the case of about 100 mg 2,4-D/l.The growth rate pro®le within the various phases of adaptation was well re¯ected by the¯uorescence signals. The NAD(P)H¯uorescence was almost exclusively emitted by the cellular pool of NADPH and behaved inversely to the growth rate. A similar relationship was obtained for the cellular¯uorescence of a¯avin-containing compound. Sharply reduced growth was additionally accompanied by a rapid rise of the background¯uorescence. These data indicate that¯uorescence-derived signals provide a useful re¯ection of cellular events in inhibited growth situations.