Dynamics of nitrous oxide reductase genes (nosZ) in intertidal rocky biofilms and sediments of the Douro River estuary (Portugal), and their relation to N-biogeochemistry.

In this study, temporal variability of nosZ genotypes was evaluated in two intertidal rocky biofilms and two intertidal sediment sites of the Douro River estuary, Portugal. The results were compared to rates of key N-cycle processes and environmental variables to examine possible links between denitrifier community dynamics and N biogeochemistry. Genetic heterogeneity of the nosZ gene was evaluated by terminal restriction fragment length polymorphism analysis (T-RFLP) and by sequencing cloned nosZ gene fragments. Phylogenetic analysis showed that the majority of the nosZ genes detected were most similar to nosZ genes from isolates affiliated with alpha-subclass of the class Proteobacteria. Results revealed low nosZ genotype richness, and hierarchical cluster analysis showed significant differences in the composition of denitrifier communities that inhabit different intertidal environments of the Douro River estuary. Monthly surveys of nosZ genotypes from sandy sediments showed that, while the same T-RFLP peaks were present in all samples, shifts in the relative peak areas of the different nosZ genotypes occurred. Canonical correspondence analysis, based on data from the monthly survey, revealed a strong relationship between the relative peak areas of some T-RFLP operational taxonomic units (OTUs) with denitrification rate and NO3- availability. Results suggest that denitrifiers with specific nosZ genotypes (OTUs) have competitive advantage over others when NO3- fluctuates in the system; these fluctuations reflect, in turn, variability in denitrification rates.