Survival of Denitrifiers in Nitrate-Free, Anaerobic Environments

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Abstract
Experiments were undertaken to explain the occurrence of a high denitrification capacity in anaerobic, NO 3 - -free habitats. Deep layers of freshwater sediments that were buried more than 40 years ago and digested sludge were the habitats studied. The denitrifier populations were 3.1 × 10 3 and 3.1 × 10 5 cells cm -3 in deep sediments from a river and lake, respectively, and 5.3 × 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 predominant isolates that would grow on anaerobic medium with NO 3 - . All strict anaerobes isolated failed to denitrify. All isolates that denitrified were aerobic, gram-negative bacteria, particularly species of Pseudomonas and Alcaligenes . No detectable growth was observed when these strains were incubated with electron acceptors other than NO 3 - or O 2 . When representative isolates were added to sterile, O 2 - and NO 3 - -free porewater 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 slow formation of formazan granules when exposed to tetrazolium and by observation of motile cells. When [ 14 C]glucose was added to cell suspensions of the pseudomonads that had been starved for 3 months without electron acceptors (O 2 or NO 3 - ), 14 C-labeled products, including cell biomass, 14 CO 2 , and fermentation products, were produced. The high denitrification capacity of these anaerobic environments appears to be due to conventional respiratory denitrifiers. These organisms have the capacity for long-term survival without O 2 or NO 3 - and appear to be capable of providing for their maintenance by carrying on a low level of fermentation.

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