Evidence for Clostridia as Agents of Dissimilatory Reduction of Nitrate to Ammonium in Soils

Reduction of NO₃⁻ to NH₄⁺ was studied in fresh and air-dried soils which were amended with ¹⁵NO₃⁻ and glucose, acetate, or water and incubated anaerobically. Soils were either untreated or heat-shocked at 68°C for 1 hour prior to amendment. Ammonium was produced rapidly after the onset of anaerobiosis, and thereafter was incorporated into organic matter. ¹⁵NH₄⁺ plus ¹⁵N-organic matter production was observed in glucose-amended fresh soil in quantities up to 43% of added ¹⁵NO₃⁻ in untreated samples and 55% in heat-shocked samples after 5 days incubation. In soils unamended with organic carbon, NO₃⁻ reduction to NH₄⁺ was minor. Pretreatment of the soils either by air-drying or heat-shocking increased to a similar extent the amount of NO₃⁻ reduced to NH₄⁺. The activity of clostridia during the NO₃⁻ reduction was indicated by the absence of any effect exerted by heat-treatment, the production of H₂ and CO₂, and the presence of higher numbers of anaerobic sporeforming bacteria relative to denitrifying bacteria in the air-dried soil. Also, the most common isolate capable of reducing NO₃⁻ to NH₄⁺ was a Clostridium spp. The addition of washed spores of a NO₃⁻-reducing Clostridium isolated from the soil increased the formation of ¹⁵NH₄⁺-N plus ¹⁵N-organic N fourfold, an accumulation equivalent to 83% of added ¹⁵NO₃⁻-N. The reduction of NO₃⁻ to NH₄⁺ in soils was not inhibited by NH₄⁺ or glutamine, indicating that the mechanism of reduction was dissimilatory. This conclusion is supported by studies with several Clostrdium spp. isolated from the soils. The isolates were capable of reducing NO₃⁻ to NH₄⁺ and exhibited increased cell yields when NO₃⁻ was included in the growth medium. These studies suggest that the potential for significant dissimilatory NO₃⁻ reduction to NH₄⁺ exists in most soils, principally in the sporeforming genera of Clostridium and Bacillus, but that this potential is likely expressed only when soils become anoxic and are rich in C.