Seasonal fluctuations in the population of denitrifying and N2-fixing bacteria in an acid soil of a Norway spruce forest

The seasonal fluctuations in the concentration of cultured denitrifying and N-2-fixing bacteria were followed in an ammonium fertilised and a control soil of a Norway spruce forest near Villingen/Black Forest from December 1994 to August 1998. The horizontal distribution of bacteria in three layers was determined by the MPN-method and by molecular probing (colony hybridisation) using specific 0.4-0.7 kb DNA probes for denitrification steps (narG, nirS, nirK and nosZ) and for N-2-fixation (nifH). The data showed that highest bacterial counts and higher numbers of denitrifying and N-2-fixing bacteria were generally detectable in the upper (= 5 cm) soil layer and that their amount decreased with soil depth. The concentration of these cultured bacteria showed seasonal fluctuations with highest numbers in autumn/winter/early spring and with low counts in summer. Denitrifying and N-2-fixing bacteria amounted to less than 10% of the total number of cultured bacteria determined by the MPN-method. Fertilisation with ammonium did not cause a shift in the population of these bacteria. These findings were corroborated by hybridisation experiments with genomic DNA isolated from the different layers. Strongest DNA-DNA hybridisation band intensities were obtained in the upper soil layer and their intensities decreased with soil depth. Soil samples from Villingen assayed in the laboratory produced N2O (in dependence of nitrate and C2H2 added to the vessels) and utilised this gas with higher activities in the assays with the fertilised soil. It is concluded that molecular techniques can successfully be applied for assessing seasonal fluctuations of bacterial populations in soil. Relative abundance of denitrifying and N-2-fixing bacteria can be determined from experiments with DNA isolated from soils. Attempts to transform these results to the total population of soil bacteria on a single cell basis are faced with many uncertainties.