We re-evaluated PCR primers targeting nirS, nirK and nosZ genes for denaturing gradient gel electrophoresis as a tool to survey denitrifying community composition in environmental samples. New primers for both nirS and nosZ were combined with existing primers, while for nirK the previously published F1aCu:R3Cu set was chosen for denaturing electrophoresis. All three sets yielded amplicons smaller than 500 bp and amplified the correct fragment in all environmental samples. The denaturing gradient gel electrophoresis worked satisfactorily for nirK and nosZ, but not for nirS. This was probably due to the multiple melting domains in this particular nirS fragment. From the excised and sequenced bands, only sequences related to the target genes were detected and tree analysis showed that the selected primers acted as broad range primers for each of the three genes. By use of the new nirS primers it was demonstrated that agricultural soil harbours a substantial diversity of nirS denitrifiers.

The objective of this study was to explore the long-term effects of different organic and inorganic fertilizers on activity and composition of the denitrifying and total bacterial communities in arable soil. Soil from the following six treatments was analyzed in an experimental field site established in 1956: cattle manure, sewage sludge, Ca(NO 3 ) 2 , (NH 4 ) 2 SO 4 , and unfertilized and unfertilized bare fallow. All plots but the fallow were planted with corn. The activity was measured in terms of potential denitrification rate and basal soil respiration. The nosZ and narG genes were used as functional markers of the denitrifying community, and the composition was analyzed using denaturing gradient gel electrophoresis of nosZ and restriction fragment length polymorphism of narG, together with cloning and sequencing. A fingerprint of the total bacterial community was assessed by ribosomal intergenic spacer region analysis (RISA). The potential denitrification rates were higher in plots treated with organic fertilizer than in those with only mineral fertilizer. The basal soil respiration rates were positively correlated to soil carbon content, and the highest rates were found in the plots with the addition of sewage sludge. Fingerprints of the nosZ and narG genes, as well as the RISA, showed significant differences in the corresponding communities in the plots treated with (NH 4 ) 2 SO 4 and sewage sludge, which exhibited the lowest pH. In contrast, similar patterns were observed among the other four treatments, unfertilized plots with and without crops and the plots treated with Ca(NO 3 ) 2 or with manure. This study shows that the addition of different fertilizers affects both the activity and the composition of the denitrifying communities in arable soil on a long-term basis. However, the treatments in which the denitrifying and bacterial community composition differed the most did not correspond to treatments with the most different activities, showing that potential activity was uncoupled to community composition.

Knowing spatial patterns of functional microbial guilds can increase our understanding of the relationships between microbial community ecology and ecosystem functions. Using geostatistical modeling to map spatial patterns, we explored the distribution of the community structure, size, and activity of one functional group in N cycling, the denitrifiers, in relation to 23 soil parameters over a 44-ha farm divided into one organic and one integrated crop production system. The denitrifiers were targeted by the nirS and nirK genes that encode the two mutually exclusive types of nitrite reductases, the cd 1 heme-type and copper reductases, respectively. The spatial pattern of the denitrification activity genes was reflected by the maps of the abundances of nir genes. For the community structure, only the maps of the nirS community were related to the activity. The activity was correlated with nitrate and dissolved organic nitrogen and carbon, whereas the gene pools for denitrification, in terms of size and composition, were influenced by the soil structure. For the nirS community, pH and soil nutrients were also important in shaping the community. The only unique parameter related to the nirK community was the soil Cu content. However, the spatial pattern of the nirK denitrifiers corresponded to the division of the farm into the two cropping systems. The different community patterns, together with the spatial distribution of the nirS/nirK abundance ratio, suggest habitat selection on the nirS-and nirK-type denitrifiers. Our findings constitute a first step in identifying niches for denitrifiers at scales relevant to land management.

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Soil microorganisms are key players in biogeochemical cycles. Yet, there is no consistent view on the significance of microbial biodiversity for soil ecosystem functioning. According to the insurance hypothesis, declines in ecosystem functioning due to reduced biodiversity are more likely to occur under fluctuating, extreme or rapidly changing environmental conditions. Here, we compare the functional operating range, a new concept defined as the complete range of environmental conditions under which soil microbial communities are able to maintain their functions, between four naturally assembled soil communities from a long-term fertilization experiment. A functional trait approach was adopted with denitrifiers involved in nitrogen cycling as our model soil community. Using short-term temperature and salt gradients, we show that the functional operating range was broader and process rates were higher when the soil community was phylogenetically more diverse. However, key bacterial genotypes played an important role for maintaining denitrification as an ecosystem functioning under certain conditions.

Denitrifiers, a functional guild within the nitrogen cycle, are responsible for the stepwise reduction of soluble NO 3 ) to N 2 through anaerobic respiration. Despite the crucial role, denitrifiers play for nitrogen loss in agriculture and global warming by emitting the greenhouse gas nitrous oxide, we still have limited knowledge about the community ecology of denitrifiers . Easily applicable tools that could aid our understanding of the relationships between the ecology of denitrifying communities and ecosystem functioning are needed to provide knowledge-based land management strategies. T-RFLP and DGGE are rapid methods for fingerprinting microbial communities. For both T-RFLP and DGGE, assays have been developed for denitrification genes (Hallin et al. 2007), although the methods have never been compared for resolving denitrifying communities. While DGGE has potential for higher discrimination and can include sequence identification, T-RFLP allows higher throughput and avoids gel-to-gel comparison bias. Our aim was to compare the use of DGGE and T-RFLP and the subsequent statistical analysis of the fingerprints to assess the composition of the denitrifier community in soil. We used nosZ as molecular marker, because this gene encodes the nitrous oxide reductase, which catalyses the reduction of the greenhouse gas nitrous oxide to N 2 in the denitrification pathway. Of the known molecular markers for denitrifiers, the nosZ gene was recently shown to have the greatest level of congruence with 16S rRNA-based taxonomic classification (Jones et al. 2008). The obtained fingerprint data were analysed by presence ⁄ absence and relative abundancebased matrices using both semi-metric (Bray-Curtis) and metric (Euclidean) distance measures.Soil was sampled at the Swedish Ultuna Long-Term Soil Organic Matter Experiment amended with straw (S) or peat (P) and from the Lanna experimental site subject to tillage (T) or nontillage (NT) regimes. All field treatments were in triplicate and analysed separately. The Ultuna soil is a clay loam, whereas Lanna is a clay soil. DNA was extracted using the FastDNA Spin Kit for Soil (Qbiogene, Carlsbad, CA, USA) and nosZ gene fragments were amplified in triplicate 25 ll reactions using the primers nosZ-F and nosZ1622R according to Enwall et al. (2005). For T-RFLP analysis, the forward primer was labelled with hexachlorofluorescein and for DGGE, the reverse primer was GC-clamped (Throbäck et al. 2004). The triplicate hexachlorofluorescein-labelled PCR products for each sample were pooled, split in three tubes and digested in separate reactions with 10 U of the restriction enzymes BstUI, HhaI and Sau96I during 2 h according to the instructions provided by the manufacturer (New England BioLabs, Beverly, MA, USA). The enzymes were selected based on preliminary in silico restriction analysis of 79 nosZ sequences. Fluorescently labelled TRFs were separated and detected using an ABI 3730 capillary sequencer AbstractTerminal restriction fragment length polymorphism (T-RFLP) and denaturing ...

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