Agricultural fertilizer use has significantly increased export of bioavailable nitrogen to streams and ultimately coastal ecosystems, where eutrophication, harmful algal blooms, and hypoxia may result. Denitrification, the microbially-mediated anaerobic reduction of nitrate to dinitrogen gas, is an important N-cycle pathway for offsetting agricultural inputs to aquatic systems. Improved methods are needed for evaluating factors that influence denitrification in headwater agricultural streams and we suggest that the role of environmental factors in determining denitrification will depend on microbial community structure. As this is a bacterially mediated process, we used molecular analyses targeting the nitrous oxide reductase gene (nosZ) as part of a study seeking environmental markers of denitrification for bioassessment. As a component of a larger project examining Ohio and Indiana streams sites in the Ohio River watershed, detailed environmental surveys were conducted at a mixed use agricultural stream (an un-named stream near Wooster, OH) and two forested streams (the West Branch of the Mahoning, Ravenna, OH,  and Silver Creek, Hiram, OH). Stream sediment samples, in conjunction with chemical, physical and macroinvertebrate data, were obtained at three sites in each stream on two dates: summer and fall 2009. 

Results/Conclusions

Bacterial abundances determined with DAPI stain revealed that cell abundances varied from 6.52 x 10⁶ to 6.95 x 10⁷ cells/g dry mass across all sites. Although the agriculturally impacted stream had similar nutrient concentrations to other streams we have examined, denitrification rates (from the acetylene block method) were inexplicably lower. PCR confirmed presence of the nosZ gene in all samples on all dates including those which had very low denitrification rates (as low as 1.06 µg N/m²/h). To examine if these differences are associated with community structure variation, terminal restriction fragment polymorphism analysis of nosZ PCR products using RsaI will be conducted for all samples and assessed statistically using canonical redundancy analysis. In addition, quantitative PCR analysis of nosZ and 16S rRNA genes to compare denitrifier abundance to overall bacterial abundance is ongoing. A general trend of decreased bacterial diversity and abundance, but perhaps increased denitrifier abundance, is anticipated in the agricultural mixed use stream as compared to the forested sites.