Abundance of nifH genes in urban, agricultural, and pristine prairie streams exposed to different levels of nitrogen loading

Ecosystem processes drive biogeochemical cycles that influence input and losses of nutrients in the biosphere. Through human activities the environment has been highly enriched with nutrients, especially nitrogen. In most ecosystems, nitrogen availability should be limited, but soils and aquatic ecosystems have been anthropogenically impacted. In streams, availability of nutrients, geochemical characteristics, hydrodynamics, and human activities influence the metabolic activities and structure of microbial communities. The aim of the current study is to contrast gene abundance and metabolic responses of N2-fixing guilds exposed to chronic nitrogen loading in three different types of Kansas prairie streams: urban, agricultural, and pristine. Nitrogen-fixation activity was expected to be negatively correlated to the level of fixed nitrogen, while nifH (nitrogenase gene) abundance would be unchanged. A combination of process-level and molecular techniques were applied to study nitrogen fixation in these small prairie streams. Nitrogen fixation activity was measured with acetylene reduction assays. Rates of acetylene reduction for urban, agricultural, and pristine prairie streams were 1.5 to 93.5, 2.1 to 112.8, and 2.9 to 81.9 fmol N2/g soil/h, respectively. The highest rates were found in leaf litter, sediments and algal biofilms. Samples of sediments and leaf litter were field-frozen for molecular analyses of the nitrogen-fixing microbial guild. Direct DNA extracts were examined by SYBR real-time PCR to determine the abundance of nifH, given a detection limit of 2 x 102 nifH gene copies/g sample. The abundance of bacterial 16S rRNA was between 1.0 x 106 to 1.0 x 1012 gene copies per gram sample. The abundance of archaeal 16S rRNA was between 1.0 x 108 to 1.0 x 10 12 gene copies per gram sample. The abundance of nifH genes ranged between 1.0 x 103 to 1.0 x 107 gene copies per gram in all streams. The assay was quantitative over at least 8 orders of magnitude, from 1 ng to 0.1 pg of nifH target. This study provides a link between the abundance of nifH genes and nitrogen-fixation activity. An understanding of the effects of nitrogen pollution on nitrogen cycling guilds in small streams will increase our ability to overcome the challenges of nutrient pollution. This work was supported by Kansas NSF EPSCoR Ecological Genomics.