SURVIVAL OF DENITRIFIERS IN NITRATE-FREE, ANAEROBIC ENVIRONMENTS

Experiments were undertaken to explain the occurrence of a high denitr ification capacity in anaerobic, NO3--free habitats. Deep layers of fr eshwater sediments that were buried more than 40 years ago and digeste d sludge were the habitats studied. The denitrifier populations were 3 .1 x 10(3) and 3.1 x 10(5) cells cm-3 in deep sediments from a river a nd lake, respectively, and 5.3 x 10(6) cells cm-3 in digested sludge. The denitrification capacities of the samples reflected the population densities. Strict anaerobic procedures were used to obtain the predom inant isolates that would grow on anaerobic medium with NO3-. All stri ct anaerobes isolated failed to denitrify. AH isolates that denitrifie d were aerobic, gram-negative bacteria, particularly species of Pseudo monas and Alcaligenes. No detectable growth was observed when these st rains were incubated with electron acceptors other than NO3- or O2. Wh en representative isolates were added to sterile, O2- and NO3--free po rewater from their original locations at their natural densities (10(5 ) cells cm-3), no change in viable population was noted over 3 months of incubation. Metabolic activity was demonstrated in these cells by s low formation of formazan granules when exposed to tetrazolium and by observation of motile cells. When [C-14]glucose was-added to cell susp ensions of the pseudomonads that had been starved for 3 months without electron acceptors (O2 or NO3-), C-14-labeled products, including cel l biomass, (CO2)-C-14, and fermentation products, were produced. The h igh denitrification capacity of these anaerobic environments appears t o be due to conventional respiratory denitrifiers. These organisms hav e the capacity for long-term survival without O2 or NO3- and appear to be capable of providing for their maintenance by carrying on a low le vel of fermentation.