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

Ralstonia eutropha JMP 134 was continuously grown on phenol and 2,4-dichlor ophenoxyacetate under nutristatic conditions at elevated stationary concent rations 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 fluorescence spectra of intracellular and extracellu lar fluorophores. The latter were monitored using a modified fluorescence s pectrophotometer. The results of the nutristat experiments indicate that th e adaptation of the culture to long-term exposure to phenol and 2,4-D exhib ited dynamic characteristics of the growth pattern determined by the indivi dual substrates and their concentration, including enforced and reduced lev els of substrate conversion. This growth pattern is interpreted as an expre ssion of superimposing cellular events in order to withstand unfavorable en vironmental conditions. Finally, the growth rate attained retarded levels u nder stationary conditions, slowing down to almost zero for example in the case of about 100 mg 2,4-D/l. The growth rate profile within the various phases of adaptation was well re flected by the fluorescence signals. The NAD(P)H fluorescence was almost ex clusively emitted by the cellular pool of NADPH and behaved inversely to th e growth rate. A similar relationship was obtained for the cellular fluores cence of a flavin-containing compound. Sharply reduced growth was additiona lly accompanied by a rapid rise of the background fluorescence. These data indicate that fluorescence-derived signals provide a useful reflection of c ellular events in inhibited growth situations.