MICROBIOLOGY OF AQUATIC SYSTEMS Nitrogen-Cycling Genes in Epilithic Biofilms of Oligotrophic High-Altitude Lakes (Central Pyrenees, Spain) Maria Vila-Costa & Mireia Bartrons & Jordi Catalan & Emilio O. Casamayor Received: 4 November 2013 /Accepted: 2 April 2014 # Springer Science+Business Media New York 2014 Abstract Microbial biofilms in oligotrophic environments are the most reactive component of the ecosystem. In high- altitude lakes, exposed bedrock, boulders, gravel, and sand in contact with highly oxygenated water and where a very thin epilithic biofilm develops usually dominate the littoral zone. Traditionally, these surfaces have been considered unsuitable for denitrification, but recent investigations have shown higher biological diversity than expected, including diverse anaerobic microorganisms. In this study, we explored the presence of microbial N-cycling nirS and nirK (denitrification through the conversion of NO 2 - to NO), nifH (N 2 fixation), anammox (anaerobic ammonium oxidation), and amoA (aer- obic ammonia oxidation, both bacterial and archaeal) genes in epilithic biofilms of a set of high-altitude oligotrophic lakes in the Pyrenees. The concentrations of denitrifying genes deter- mined by quantitative PCR were two orders of magnitude higher than those of ammonia-oxidizing genes. Both types of genes were significantly correlated, suggesting a potential tight coupling nitrification-denitrification in these biofilms that deserves further confirmation. The nifH gene was detected after nested PCR, and no signal was detected for the anammox- specific genes used. The taxonomic composition of denitrifying and nitrogen-fixing genes was further explored by cloning and sequencing. Interestingly, both microbial functional groups were richer and more genetically diverse than expected. The nirK gene, mostly related to Alphaproteobacteria (Bradyrhizobiaceae), dominated the denitrifying gene pool as expected for oxygen-exposed habitats, whereas Deltaproteobacteria (Geobacter like) and Cyanobacteria were the most abundant among nitrogen fixers. Overall, these results suggest an epilithic community more metabolically diverse than previously thought and with the potential to carry out an active role in the biogeochemical nitrogen cycling of high- altitude ecosystems. Measurements of activity rates should be however carried out to substantiate and further explore these findings. Introduction Industrial fixations of atmospheric nitrogen in concert with fossil-fuel combustions have altered the global nitrogen cycle in a fundamental way [1]. The global pool of reactive nitrogen (Nr), which includes all fixed bio-available forms, has at least doubled since preindustrial times [2]. Nr is sequentially trans- ferred through environment systems including the atmo- sphere. Nitrogen oxides are emitted to the atmosphere in combustions related to industrial, transport, and urban activi- ties, whereas agriculture is the main source of ammonia emis- sions. In the atmosphere, the compounds may be involved in photochemical reactions and aerosol formation. Nevertheless, most of the Nr emitted to the atmosphere is deposited back to Earth’ s surface within a short time (days) as nitrate and Electronic supplementary material The online version of this article (doi:10.1007/s00248-014-0417-2) contains supplementary material, which is available to authorized users. M. Vila-Costa : M. Bartrons : J. Catalan : E. O. Casamayor (*) Biogeodynamics&Biodiversity Group (B&B), Centre for Advanced Studies of Blanes, CEAB-CSIC, Blanes, Spain e-mail: casamayor@ceab.csic.es M. Vila-Costa e-mail: maria.vila@idaea.csic.es M. Vila-Costa Department of Ecology, Department of Environmental Chemistry, University of Barcelona, IDAEA-CSIC, Barcelona, Spain M. Bartrons Department of Environmental Sciences and Food, University of Vic, Barcelona, Spain J. Catalan B&B-CREAF, Campus UAB, Cerdanyola del Vallès, Catalunya, Spain Microb Ecol DOI 10.1007/s00248-014-0417-2