Diversity of denitrifying microflora and ability to reduce N sub(2)O in two soils

The ozone-depleting gas N sub(2)O is an intermediate in denitrification, the biological reduction of NO sub(3) super(-) to the gaseous products N sub(2)O and N sub(2) gas. The molar ratio of N sub(2)O produced (N sub(2)O/N sub(2)O+N sub(2)) varies temporally and spatially, and in some soils N sub(2)O may be the dominant end product of denitrification. The fraction of NO sub(3) super(-)-N emitted as N sub(2)O may be due at least in part to the abundance and activity of denitrifying bacteria which possess N sub(2)O reductase. In this study, we enumerated NO sub(3) super(-)-reducing and denitrifying bacteria, and compared and contrasted collections of denitrifying bacteria isolated from two agricultural soils, one (Auxonne, soil A) with N sub(2)O as the dominant product of denitrification, the other (Chalons, soil C) with N sub(2) gas as the dominant product. Isolates were tested for the ability to reduce N sub(2)O, and the presence of the N sub(2)O reductase (nosZ)-like gene was evaluated by polymerase chain reaction (PCR) using specific primers coupled with DNA hybridization using a specific probe. The diversity and phylogenetic relationships of members of the collections were established by PCR/restriction fragment length polymorphism of 16s rDNA. The two soils had similar numbers of bacteria which used NO sub(3) super(-) as a terminal electron acceptor anaerobically. However, the soil A had many more denitrifiers which reduced NO sub(3) super(-) to gaseous products (N sub(2)O or N sub(2)) than did soil C. Collections of 258 and 281 bacteria able to grow anaerobically in the presence of NO sub(3) super(-) were isolated from soil A and soil C, respectively. These two collections contained 66 and 12 denitrifying isolates, respectively, the others reducing NO sub(3) super(-) only as far as NO sub(2) super(-). The presence of nosZ sequences was generally a poor predictor of N sub(2)O reducing ability: there was agreement between the occurrence of nosZ sequences and the N sub(2)O reducing ability for only 42% of the isolates; 35% of the isolates (found exclusively in soil A) without detectable nosZ sequences reduced N sub(2)O whereas 21% of the isolates carrying nosZ sequences did not reduce this gas under our assay conditions. Twenty-eight different 16S rDNA restriction patterns (using two restriction endonucleases) were distinguished among the 78 denitrifying isolates. Two types of patterns appeared to be common to both soils. Twenty-three and three types of