The coupling of anaerobic steroid dehydrogenation to nitrate reduction in Pseudomonas N.C.I.B. 10590 and Clostridium paraputrificum.

Pseudomonas spp. capable of using sodium deoxycholate as the sole carbon source were isolated from faeces, sewage and soil by enrichment culture in a defined medium. An isolate, designated Pseudomonas N.C.I.B. 10590, oxidized deoxycholic acid during aerobic growth to yield metabolites with the 1,4-diene3-one structure; this oxidation also occurred anaerobically if nitrate was present. The nuclear dehydrogenation reactions carried out by Clostridiumparaputri$cum are of interest in the aetiology of colon cancer (Hill, 1974), but have been studied little because of the complex nutritional requirements of this organism; since the pseudomonad carries out similar reactions in a defmed medium it is more amenable to study. Steroid oxidation by the Pseudomonas was studied as a function ofnitrate concentration to demonstrate thecoupling ofthe dehydrogenation to nitrate reduction in the absence of molecular oxygen. The properties of steroid oxidation in Pseudomonas N.C.I.B. 10590 and CI. paraputriJicum were compared. Anaerobic metabolism of steroids by Pseudomonas spp. was carried out in a buffered mineral salts medium containing the required quantities of KNO, or other electron acceptor and dispensed to fill screw-capped bottles. Washed bacterial cells were added (ha1 density 5 . 0 ~ 10' cells/ml), the caps screwed tightly to exclude air and the cultures incubated at 25°C for 6 days. After acidification to pH4, the steroids were extracted into redistilled ethyl acetate, the solvent was removed by evaporation and the E244 in methanol determined. Nitrite concentrations were determined by a modified Griess-Ilosvay reaction (Pearson, 1973). Steroid products were separated by t.1.c. on silica gel GF254 by the use of two solvent systems and those with the 1,4-diene-3-one structure detected under U.V. light; other products were detected by their colour with anisaldehyde reagent (Kritchevsky et al., 1963).